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Signaling in natural killer cells

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Material Information

Title:
Signaling in natural killer cells SHIP, 2B4 and the Kinome
Physical Description:
Book
Language:
English
Creator:
Wahle, Joseph A
Publisher:
University of South Florida
Place of Publication:
Tampa, Fla
Publication Date:

Subjects

Subjects / Keywords:
NK
CD244
NSC119910
PepChip
Cytotoxicity
Dissertations, Academic -- Cancer Biology -- Doctoral -- USF   ( lcsh )
Genre:
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Abstract:
ABSTRACT: The NK cell is a large granular lymphocyte that plays a key role in protecting the body against numerous pathogens including parasites, intracellular bacteria, viral infections, as well as showing anti-tumor activity and playing a role in the rejection of allogeneic BM. Unlike other lymphocytic cell types, that utilize rearranging receptors, NK cells are regulated by a complex array of germ line encoded activating and inhibitory receptors. NK cells are often described as a front line or rapid defense given their response to stimuli can be immediate, although they also maintain functions that extend their role well into the adaptive immune system. Inhibitory receptors that recognize MHC class I molecules regulate NK cell responses and self-tolerance. Recent evidence indicates self-ligands not present in the MHC locus can also modulate NK function. We previously demonstrated that the NK receptor repertoire is disrupted by SHIP-deficiency.^ Here we show that an inhibitory receptor, 2B4, that recognizes an MHC-independent ligand is over expressed in NK cells of SHIP-/- mice at all stages of NK development and differentiation. Overexpression of 2B4 compromises key cytolytic NK functions, including killing of allogeneic, tumor and viral targets. These results demonstrate that in SHIP-/- NK cell 2B4 is the dominant inhibitory receptor. We then furthered this finding by examining the molecular basis of 2B4 dominance. We show that in SHIP-/- NK cells there is increased 2B4 expression as well as a strong bias towards the 2B4L isoform. We have also identified a greater than tenfold increase in SHP1 recruitment to 2B4.^ Consistent with this SHP1 over recruitment,both a broad and a selective SHP1 inhibitor restore SHIP-/- NK killing of complex targets.Through this study we have identified the molecular mechanism of 2B4 receptor dominance as SHP1 over-recruitment.In addition we have utilized protein array technology to explore NK signaling through the determination of the NK kinome. To this end we have been able to identify multiple pathways that may mark crucial differences between the mature and immature NK cell.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2007.
Bibliography:
Includes bibliographical references.
System Details:
System requirements: World Wide Web browser and PDF reader.
System Details:
Mode of access: World Wide Web.
Statement of Responsibility:
by Joseph A. Wahle.
General Note:
Title from PDF of title page.
General Note:
Document formatted into pages; contains 147 pages.
General Note:
Includes vita.

Record Information

Source Institution:
University of South Florida Library
Holding Location:
University of South Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 001939484
oclc - 227815843
usfldc doi - E14-SFE0002182
usfldc handle - e14.2182
System ID:
SFS0026500:00001


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S i gna l i ng i n N a t u r a l K i l l e r C e l l s : S H I P 2B 4 a nd t he K i nom e by J os e ph A W a hl e A di s s e r t a t i on s ubm i t t e d i n pa r t i a l f u l f i l l m e nt of t he r e qui r e m e nt s f or t he de g r e e of D oc t or of P hi l os ophy D e pa r t m e nt of C a nc e r B i ol ogy C ol l e ge G r a dua t e S c hool U n i ve r s i t y of S ou t h F l o r i da M a j or P r of e s s or : W i l l i a m G K e r r P h D S r i kum a r C he l l a ppa n P h D G a r y W R e ut he r P h. D N or e e n L ue t t e ke P h D D a t e of A ppr ova l : O c t obe r 19, 2007 K e yw or ds : N K C D 244, N S C 119910, P e pC hi p C yt ot oxi c i t y C opyr i ght 2007 J os e ph A W a hl e

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N ot e t o t he R e a de r T he or i gi na l of t hi s doc um e nt c ont a i ns c ol or t ha t i s ne c e s s a r y f or unde r s t a ndi ng t he da t a T he or i gi na l di s s e r t a t i on i s on f i l e w i t h t he U S F l i b r a r y i n T a m pa F l o r i da

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D e di c a t i on I w i s h t o de di c a t e t hi s t o m y f a m i l y a nd f r i e nds t ha t ha ve pl a ye d a c r uc i a l r ol e i n m e r e a c hi ng t hi s s t a ge I w oul d not ha ve be e n a bl e t o unde r t a ke s uc h a c ha l l e nge w i t hout t he i r s uppor t

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A c know l e dge m e nt s I w a nt t o t ha nk K i m P a r a i s o f or he r a bunda nt he l p a nd s uppor t f o r s o m a ny ye a r s w i t h a l l of t he N K a nd S H I P w or k I a l s o w a nt t o t ha nk A m y H a z e n f or he r he l p a nd s uppor t I a l s o w i s h t o t ha nk a l l of t he K e r r L a b m e m be r s pa s t a nd pr e s e nt w ho ha ve pr ovi de d m e w i t h s uppor t a nd a s s i s t a nc e C a r ol i ne D e s pont s A m y C os t e l l o, D a ni e l a W ood, E m i l y H ope w e l l N i c ol e G j e r t s e n, M i c he l l e C ol l a z o, a nd N a t ha n W a t t s I w a nt t o t ha nk m y c ol l a bor a t or s M a i ke l P e ppe l e n bos c h, S a nde r D i ks C ha r l e s S e nt m a n, a nd J e r r y W u f or a l l of t he i r a s s i s t a nc e t ha t pr ove d i nva l ua bl e i n c om pl e t i ng t hi s w o r k. I w a nt t o t ha nk m y c om m i t t e e m e m be r s f or a l l o f t he i r s uppor t a nd gui da nc e t hr oughout t hi s pr oc e s s F i na l l y I w a nt t o t ha nk m y m e nt or B i l l K e r r w ho ha s pr ovi de d m e w i t h i nnum e r a bl e oppor t uni t i e s a nd s uppor t t o f u r t he r m y s c i e nt i f i c g oa l s

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i T a bl e of C ont e nt s L i s t of T a bl e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v L i s t O f F i gur e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi A bs t r a c t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi i i C ha pt e r 1: I nt r oduc t i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 E a r l y N K s t udi e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 D i s c ove r y of N K c e l l s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 M i s s i ng S e l f H ypot he s i s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 N K R e c ogni t i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 S e l f M H C R e c e pt or s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 L y49 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 C D 94/ N K G 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 N on M H C R e c e pt or s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 N K G 2D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 2B 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 I nt e gr a t i on o f S i gna l i ng . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

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i i N K E f f e c t o r F unc t i ons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 C yt ot oxi c i t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 N K i n A da pt i ve I m m uni t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 N K D e ve l opm e nt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 S H I P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 T he C l o ni ng o f S H I P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 S H I P F unc t i ona l D om a i ns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 S H I P I s of o r m s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 S H I P K O M i c e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 S H I P i n N K C e l l s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 C ha pt e r 2: D om i na nc e by a n M H C I nde pe nde nt I n hi bi t or y R e c e pt or C om pr om i s e s N K K i l l i ng of C om pl e x T a r ge t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 I nt r oduc t i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 R e s ul t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 S H I P / N K R e pe r t oi r e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 2B 4 E xpr e s s i on L e ve l s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 N K G 2D M e di a t e d C yt ol ys i s i s C om pr om i s e d i n S H I P / M i c e . . . . . . . . 34 R e s t or a t i on of N K G 2D M e di a t e d C yt ol ys i s . . . . . . . . . . . . . . . . . . . . . . 35 C om pr om i s e d B M R e j e c t i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 C om pr om i s e d C yt ol ys i s of L y49H T a r ge t s . . . . . . . . . . . . . . . . . . . . . . 39 R e s t or a t i on of L y49H M e di a t e d C yt ol ys i s . . . . . . . . . . . . . . . . . . . . . . . 39 N K R P 1D M e di a t e d I nhi bi t i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

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i i i D i s c us s i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 M a t e r i a l s a nd M e t hods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 A ni m a l s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 A c ut e B one M a r r ow E ngr a f t m e nt A s s a ys . . . . . . . . . . . . . . . . . . . . . . . 46 A nt i body S t a i ni ng a nd F l ow C yt om e t r y . . . . . . . . . . . . . . . . . . . . . . . . . 46 L A K C ul t ur e s a nd C yt ol ys i s A s s a ys . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 A nt i body B l oc ki ng E xpe r i m e nt s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 S t a t i s t i c a l A na l ys i s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 C ha pt e r 3: I na ppr op r i a t e R e c r ui t m e nt a nd S H P 1 A c t i vi t y i s R e s pons i bl e f or R e c e pt or D om i na nc e i n t he S H I P de f i c i e nt N K C e l l . . . . . . . . . . . . . . . . . . . . . . . . 51 I nt r oduc t i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 R e s ul t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 2B 4 a nd S H P E xpr e s s i on i n S H I P de f i c i e nt N K C e l l s . . . . . . . . . . . . . . 53 I nc r e a s e d R e c r ui t m e nt of S H P 1 t o 2B 4 i n S H I P de f i c i e nt N K c e l l s . . . . 55 B r oa d I nhi bi t i on of T yr os i ne P hos pha t a s e A c t i vi t y R e s t or e s S H I P / N K F unc t i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 I nhi bi t i on o f S H P 1 A c t i vi t y R e s t or e s S H I P / N K C yt ol yt i c F unc t i on . . . 60 D i s c us s i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 M a t e r i a l s a nd M e t hods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 A ni m a l s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 L A K C ul t ur e s a nd C yt ol ys i s A s s a y s . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 I nhi bi t or s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

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i v W e s t e r n B l ot s a nd I m m unopr e c i pi t a t e s . . . . . . . . . . . . . . . . . . . . . . . . . 72 P T P I nhi bi t i on A s s a y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 S t a t i s t i c a l A na l ys i s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 C ha pt e r 4: T he N K K i nom e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 I nt r oduc t i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 R e s ul t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 O bt a i ni ng P ur e C e l l P opul a t i ons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 P e pt i de P hos phor yl a t i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 D i f f e r e nt i a l K i nom e s o f M a t ur e a nd I m m a t ur e N K C e l l s . . . . . . . . . . . . 82 F unc t i ona l C l a s s i f i c a t i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 D i s c us s i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 M a t e r i a l s a nd M e t hods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 A ni m a l s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 C e l l S or t i ng . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 P e pC hi p A s s a ys . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 C ha pt e r 5: D i s c us s i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 R e f e r e nc e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 A ppe ndi c e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 A bout t he A ut hor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E nd P a ge

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v L i s t of T a bl e s T a bl e 1. D i f f e r e nt i a l l y P hos phor yl a t e d P e pt i de s o f t he M a t ur e a nd I m m a t ur e K i nom e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 T a bl e A 1. N K K i nom e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

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vi L i s t of F i gur e s F i gur e 1. N K C e l l S i gna l i ng O ut c om e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 F i gur e 2. I nt e gr a t i on of A c t i va t i ng a nd I nhi bi t or y N K R S i gna l s . . . . . . . . . . . . . . . . . . 17 F i gur e 3. S H I P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 F i gur e 4. S H I P s R ol e W i t hi n P I S i gna l i ng . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 F i gur e 5. N K R R e pe r t oi r e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 F i gur e 6. 2B 4 S t a t us i s A l t e r e d i n S H I P / N K C e l l s . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 F i gur e 7. C om p r om i s e d N K G 2D M e di a t e d C yt ol ys i s of R a e 1 + T um or T a r ge t s by S H I P / N K C e l l s a nd R e s t or a t i on b y C D 48 B l oc ka de . . . . . . . . . . . . . . 36 F i gur e 8. A l l oge ne i c B M R e j e c t i on A s s a y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 F i gur e 9. C om p r om i s e d L y49H M e di a t e d C yt ol ys i s of C M V m 157 + V i r a l T a r ge t s by S H I P / N K C e l l s a nd R e s t or a t i o n by C D 48 bl oc ka de . . . . . . 40 F i gur e 10. N k r p1d a nd C l r b B l oc ka de . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 F i gur e 11. D o m i na nc e by 2B 4 C om pr om i s e s N K C yt ol ys i s of C om pl e x T a r ge t s . . . . . 44 F i gur e 12. 2B 4 O ve r E xpr e s s i on a nd I s of or m B i a s i n S H I P / N K C e l l s . . . . . . . . . . . . 54 F i gur e 13. R e c r ui t m e nt of S i gna l i ng M ol e c ul e s t o 2B 4 . . . . . . . . . . . . . . . . . . . . . . . . 56 F i gur e 14. R e c r ui t m e nt of S i gna l i ng M ol e c ul e s t o 2B 4 i n A c t i va t e d N K C e l l s . . . . . . . 58 F i gur e 15. R e s t or a t i on of S H I P / C yt ot oxi c i t y w i t h N a O V T r e a t m e nt . . . . . . . . . . . . . 60 F i gur e 16. R e s t or a t i on of C yt ot oxi c i t y w i t h S H P 1/ 2 I nhi bi t o r . . . . . . . . . . . . . . . . . . . 62

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vi i F i gur e 17. M ode l F i gur e o f 2B 4 S i gna l i ng . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 F i gur e 18. K i nom e P e pC hi p . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 F i gur e 19. D o t P l ot s S how i ng t he S or t i ng S t r a t e gy f or M a t ur e a nd I m m a t ur e N K C e l l s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 F i gur e 20. P e pC hi ps f or M a t ur e a nd I m m a t ur e N K C e l l s . . . . . . . . . . . . . . . . . . . . . 81 F i gur e 21. F unc t i ona l P i e C ha r t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

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vi i i S i gna l i ng i n N a t u r a l K i l l e r C e l l s : S H I P 2B 4 a nd t he K i nom e J os e ph A W a hl e A B S T R A C T T he N K c e l l i s a l a r ge g r a nul a r l ym phoc yt e t ha t p l a ys a ke y r ol e i n pr ot e c t i ng t he body a ga i ns t num e r ous pa t hoge ns i nc l udi ng pa r a s i t e s i nt r a c e l l ul a r ba c t e r i a vi r a l i nf e c t i ons a s w e l l a s s how i ng a nt i t um or a c t i vi t y a nd pl a yi ng a r ol e i n t he r e j e c t i on of a l l oge ne i c B M U nl i ke ot he r l ym phoc yt i c c e l l t yp e s t ha t ut i l i z e r e a r r a ngi ng r e c e pt or s N K c e l l s a r e r e gul a t e d by a c om pl e x a r r a y o f ge r m l i ne e nc ode d a c t i va t i ng a nd i nhi bi t o r y r e c e pt or s N K c e l l s a r e of t e n de s c r i be d a s a f r ont l i ne or r a pi d de f e ns e gi ve n t he i r r e s pons e t o s t i m ul i c a n be i m m e di a t e a l t hough t he y a l s o m a i nt a i n f unc t i ons t ha t e xt e nd t he i r r ol e w e l l i nt o t he a da pt i ve i m m une s ys t e m I nhi bi t or y r e c e pt or s t ha t r e c ogni z e M H C c l a s s I m ol e c ul e s r e gul a t e N K c e l l r e s pons e s a nd s e l f t ol e r a nc e R e c e nt e vi de nc e i ndi c a t e s s e l f l i ga nds not pr e s e nt i n t he M H C l oc us c a n a l s o m odul a t e N K f unc t i on. W e p r e vi ous l y de m ons t r a t e d t ha t t he N K r e c e pt or r e pe r t oi r e i s di s r upt e d by S H I P de f i c i e nc y H e r e w e s how t ha t a n i nhi bi t o r y r e c e pt or 2B 4, t ha t r e c ogni z e s a n M H C i nde pe nde nt l i ga nd i s ove r e xpr e s s e d i n N K c e l l s of S H I P / m i c e a t a l l s t a ge s of N K de ve l opm e nt a nd di f f e r e nt i a t i on. O ve r e xpr e s s i on of

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i x 2B 4 c om pr om i s e s ke y c yt ol yt i c N K f unc t i ons i nc l udi ng ki l l i ng of a l l oge ne i c t um o r a nd vi r a l t a r ge t s T he s e r e s ul t s de m ons t r a t e t ha t i n S H I P / N K c e l l 2B 4 i s t he dom i na nt i nhi bi t or y r e c e pt or W e t he n f ur t he r e d t hi s f i ndi ng by e xa m i ni ng t he m ol e c ul a r ba s i s of 2B 4 dom i na nc e W e s how t ha t i n S H I P / N K c e l l s t he r e i s i nc r e a s e d 2B 4 e xpr e s s i on a s w e l l a s a s t r ong bi a s t ow a r ds t he 2B 4L i s of or m W e ha ve a l s o i de nt i f i e d a gr e a t e r t ha n t e n f ol d i nc r e a s e i n S H P 1 r e c r u i t m e nt t o 2B 4. C ons i s t e nt w i t h t hi s S H P 1 ove r r e c r ui t m e nt bot h a br oa d a nd a s e l e c t i ve S H P 1 i nhi bi t or r e s t or e S H I P / N K k i l l i ng o f c om pl e x t a r ge t s T hr ough t h i s s t udy w e ha ve i de nt i f i e d t he m ol e c ul a r m e c ha ni s m of 2B 4 r e c e pt or dom i na nc e a s S H P 1 ove r r e c r ui t m e nt I n a ddi t i on w e ha ve ut i l i z e d p r ot e i n a r r a y t e c hnol o gy t o e xpl or e N K s i gna l i ng t hr ough t he de t e r m i na t i on o f t he N K ki nom e T o t hi s e nd w e ha ve be e n a bl e t o i de nt i f y m ul t i pl e pa t hw a ys t ha t m a y m a r k c r uc i a l di f f e r e nc e s be t w e e n t he m a t ur e a nd i m m a t ur e N K c e l l

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1 C ha pt e r 1 I nt r oduc t i on E ar l y N K S t u d i e s D i s c ov e r y of N K C e l l s T he f i r s t s t udi e s i de nt i f yi ng w ha t w oul d e ve nt ua l l y be t e r m e d t he na t ur a l ki l l e r c e l l ( N K ) w e r e pe r f o r m e d j us t ove r 20 ye a r s a go T he s e f i r s t s t udi e s w e r e f o l l ow i ng i n t he s t e ps of i nt r i gu i ng que s t i ons r a i s e d by t he c onc e pt of na t u r a l c yt ot oxi c i t y E a r l y a dop t i ve t r a ns f e r e xpe r i m e nt s i n w hi c h t he t r a ns pl a nt e d t um or s w e r e r e j e c t e d w e r e t hought t o r e l y on t he pr e s e nc e of T c e l l s ( 1 6) I n l i ne w i t h t hi s t he or y i t w a s hypot he s i z e d t ha t a t hym i c a nd/ or nude m i c e w hi c h l a c k T c e l l s w oul d ha ve a dr a m a t i c a l l y i nc r e a s e d num be r o f t um o r s a f t e r t he s e a dopt i ve t r a ns f e r s T hi s uni m pe de d gr ow t h of t um or s i n a T c e l l de f i c i e nt m ous e w a s n e ve r r e a l i z e d, i nd i c a t i ng t ha t i t w a s not t he T c e l l t ha t w a s l i m i t i ng t he e xpa ns i on of t he s e a dopt i ve l y t r a ns f e r r e d t um or c e l l s ( 7 9) T hi s l e a d pe opl e t o hypot he s i z e t ha t t he r e m us t be a n a l t e r na t e c e l l t ype a t pl a y A not he r i nt e r e s t i ng phe nom e non w a s oc c ur r i ng w i t hi n t he s e a dopt i ve t r a ns f e r e xpe r i m e nt s M uc h of t he r e a s on t ha t t he r e j e c t i on of t he t um or s w a s be i ng a t t r i but e d t o t he T c e l l w a s t ha t m a ny o f t he hos t s of t he t um o r t r a ns pl a nt s ha d be e n pr e vi ous l y i m m uni z e d or e xpos e d t o t he t um o r s T hi s a bi l i t y t o de s t r oy a p r e vi ous l y i m m uni z e d a ga i ns t a nt i ge n i s a ha l l m a r k of T c e l l s a nd t he a da pt i ve i m m un e r e s pons e ( 10, 11 ) I n

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2 c ont r a s t t o t hi s i t w a s f ound t ha t a l t hough t he s e pr e vi ous l y i m m uni z e d hos t s c oul d r e j e c t t he t um or s r e j e c t i on w a s a l s o s e e n i n non i m m uni z e d hos t s ( 12 14) T hi s a bi l i t y t o l ys e t um or t a r ge t s w i t hou t pr i or i m m uni z a t i on by t hi s y e t t o be i de nt i f i e d c e l l t ype i s w ha t w a s t e r m e d na t ur a l c yt ot ox i c i t y. F u r t he r s t udi e s w e r e a bl e t o a t t r i but e t hi s na t u r a l c yt ot oxi c i t y t o a di s t i nc t popul a t i on of l ym phoi d c e l l s ( 15 19) I n t he s e s t udi e s va r i ous gr oups i de nt i f i e d a c e l l t ha t r e m a i ne d a f t e r e l i m i na t i on of T a nd B c e l l s f r om w hol e s pl e noc yt e s T he r e m a i ni ng c e l l s w hi c h ha d a s m a l l l ym phoi d l i ke a p pe a r a nc e r e t a i ne d t he a bi l i t y, i n v i t r o, t o l ys e a l a r ge nu m be r o f vi r a l l y i nduc e d t um or s a s w e l l a s ot he r t um or s a m pl e s T he ke y t o t he s e s t udi e s w a s t ha t onc e a ga i n l i ke i n t he r e j e c t i on of a dopt i ve l y t r a ns f e r r e d t um or s pr i or i m m uni z a t i on w a s not ne c e s s a r y a nd t he r e f or e w a s na t ur a l c yt ot oxi c i t y. T hi s s m a l l l ym pho i d l i ke c e l l t ha t w a s ne i t he r T nor B c e l l w a s t he r e f or e t e r m e d a na t ur a l ki l l e r c e l l o r N K c e l l M i s s i ng Se l f H y pot he s i s A l t hough t he N K c e l l ha d be e n i de nt i f i e d, t he m a n ne r i n w hi c h i t f unc t i one d r e m a i ne d unknow n. O ne a r e a t ha t w a s puz z l i ng w a s t ha t N K c e l l s ha d va r i e d c yt ot oxi c i t y t o c e l l s o f di f f e r i ng m a j or h i s t oc om pa t i bi l i t y c om pl e x ( M H C ) ba c kgr ounds T he i ni t i a l a ns w e r t o t hi s p r obl e m w oul d c om e w h e n K a r r e a nd L j unggr e n pr opos e d t he i r m i s s i ng s e l f hypot he s i s ( 20, 21 ) T he m i s s i ng s e l f hypot he s i s ha s i t s or i gi ns i n a puz z l i ng que s t i on r a i s e d m a ny ye a r s e a r l i e r w hi c h w a s hyb r i d r e s i s t a nc e H yb r i d r e s i s t a nc e r e f e r s t o a phe nom e non w he r e a n F 1 hybr i d f r om pa r e nt s of di f f e r e nt M H C ba c kgr ound i s a bl e t o r e j e c t bone m a r r ow ( B M ) t r a ns pl a nt s f r o m bot h pa r e nt s ( 22, 23) S pe c i f i c a l l y a n H 2a / a c r os s e d w i t h a n H 2b/ b w oul d r e s ul t i n a he t e r oz ygous m o us e w i t h a n H 2a / b

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3 ha pl ot ype B M c e l l s t r a ns pl a nt e d i nt o t he F 1 hybr i d f r om e i t he r t he H 2a / a o r H 2b/ b pa r e nt w oul d be r e j e c t e d. T he s e s t udi e s pr ovi de d a n i ni t i a l f or a y i nt o t he c onc e pt o f s e l f vs non s e l f r e c ogni t i on by t he N K c e l l F ur t he r e v i de nc e f or t he m i s s i ng s e l f hypot he s i s w a s obt a i ne d t hr ough num e r ous s t udi e s i n w hi c h i t w a s f ound t ha t N K c e l l s c oul d ki l l t um or l i ne s t ha t ha d di m i ni s he d o r no M H C e xpr e s s i on ( 24 28) T hi s i de a w a s nove l a s t he t r a di t i ona l c onc e pt e s t a bl i s he d i n T c e l l s m a i nt a i ne d t ha t you ne e de d t he M H C m ol e c ul e s a s w e l l a s pe pt i de bound i n i t s c l e f t i n o r de r e l i c i t a c yt ot oxi c e f f e c t I n t he i r s t udi e s K a r r e a nd c ol l e a gue s ge ne r a t e d M H C de f i c i e nt c e l l s l i ne s i n or de r t o t e s t i n v i t r o t he a bi l i t y o f t he N K c e l l t o l ys e M H C nul l t a r ge t s ( 29) T hr ough t he s e s t udi e s t he y w e r e a bl e t o de t e r m i ne t ha t t he p r e s e nc e of a s e l f M H C i nhi bi t e d N K m e di a t e d c yt ot ox i c i t y. I n a ddi t i on t he y i nt r oduc e d H 2D d t r a ns ge ne s i nt o B l 6 H 2b m i c e t o de t e r m i ne t he e f f e c t on t he r e j e c t i on of B M t r a ns pl a nt s ( 20) I n t he s e s t udi e s t he y f ound t ha t t he i ns e r t i on of t he H 2D d t r a ns ge ne w a s a bl e t o c onf e r r e s i s t a nc e t o a l l oge ne i c B M i nt o a n H 2D d m ous e W i t h t he i r s t udi e s a nd t he ot he r i nf or m a t i o n pr e s e nt t he y pr opos e d t he c onc e pt t ha t a n N K c e l l ut i l i z e s r e c ogni t i on o f t he M H C m ol e c ul e s t o i de nt i f y s e l f vs non s e l f c e l l s a nd t he r e by pr ot e c t i ng s e l f c e l l s w hi l e r e t a i ni ng t he a bi l i t y t o l ys e non s e l f c e l l s ( 21) T he y t e r m e d t hi s t he m i s s i ng s e l f hypot he s i s

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4 F i gu r e 1. N K c e l l s i gn al i n g ou t c om e s N K e f f e c t or f unc t i ons a r e de t e r m i ne d by t he s pe c i f i c l i ga nds pr e s e nt on t he t a r ge t c e l l A ) A no r m a l s e l f c e l l e xpr e s s i ng t he a ppr opr i a t e i nhi bi t o r y s e l f l i ga nd ( M H C ) a l one t h e r e by not e l i c i t i ng a n N K r e s pons e B ) A c e l l e xpr e s s i ng a n a c t i va t i ng l i ga nd i n t he a bs e nc e of a s e l f l i ga nd w hi c h w oul d a c t i va t e t he N K c e l l C ) A m o r e c om pl e x s i t ua t i on i n w hi c h bo t h a n a c t i va t i ng l i ga nd a s w e l l a s s e l f M H C a r e pr e s e nt T he ou t c om e of t hi s s i t ua t i on w oul d de pe nd upon t he ba l a nc e of t he r e c e pt or s a s w e l l a s t he e nt i r e s i gna l i ng m i l i e u p r e s e nt D ) A s i t ua t i on t ha t w oul d oc c ur i n a B 2M / m ous e i n w hi c h ne i t he r M H C or a c t i v a t i ng l i ga nds a r e pr e s e nt on t he c e l l s ur f a c e I nhi bi t i on w oul d t he r e f or e be m e di a t e d t hr ough ot he r m e c ha ni s m s i nc l udi ng non c l a s s i c a l s e l f r e c e pt or s s uc h a s 2B 4.

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5 N K R e c ogn i t i on T he m i s s i ng s e l f hypot he s i s w a s a m a j or s t e p f or w a r d i n t he N K f i e l d A l t hough i t di d not i ndi c a t e how a n N K c e l l w oul d r e c ogni z e t he M H C i t di d p r e di c t t he pr e s e nc e of a n i nhi bi t or y t ype r e c e pt or t ha t w oul d m e di a t e M H C r e c ogni t i on. T hi s pr e di c t i on w a s m a ni f e s t e d w i t h t he di s c ove r y o f t he f i r s t N K r e c e pt or s ( N K R ) f or s e l f l i ga nds O ne of t he f i r s t N K R i de nt i f i e d w a s t he L y49 r e c e pt or ( 30 ) T hi s e a r l y s t udy i de nt i f i e d a r e c e pt or t ha t w a s f ound on 20 % o f N K 1. 1 + C D 3 N K c e l l s f r om C 57B L / 6 m i c e W he n f unc t i ona l s t udi e s w e r e pe r f o r m e d i t w a s f ound t ha t t he e xpr e s s i on of L y49 ha d a f unc t i ona l c ons e que nc e on t he a bi l i t y of N K c e l l s t o l ys e c e r t a i n t a r ge t s S pe c i f i c a l l y t he pr e s e nc e of t hi s r e c e pt or i nhi bi t e d t he l ys i s of H 2 D d pos i t i ve t a r ge t s ( 31) T hi s s t udy pr ovi de d one of t he f i r s t i ns i ght s i nt o how t he N K c e l l w oul d be a bl e t o i de nt i f y a nd l ys e t he a ppr opr i a t e t a r ge t s ba s e d on M H C e xpr e s s i on. W i t hi n t he s e f i r s t s t udi e s t he y i de nt i f i e d onl y one N K R but pr ove d a ke y c onc e pt w hi c h w a s t ha t a n i nhi bi t o r y r e c e pt or c oul d r e c ogni z e a s e l f M H C a nd t he r e by pr ot e c t a s e l f c e l l A l t hough t hi s g r e a t l y s uppor t e d t he m i s s i ng s e l f hypot he s i s i t onl y pr ovi de d pa r t of t he w hol e s t or y. A c c or di ng t o i t a ny c e l l w i t h l ow e r e d or l a c ki ng M H C e xpr e s s i on w oul d be s us c e pt i bl e t o N K a t t a c k. T hough t he pr oc e s s ha s pr ove n t o be m or e c om pl e x a s w e s e e i n m i c e l a c ki ng M H C s uc h a s t he B 2M / m ous e w he r e e ve n w i t h t he l a c k of M H C m ol e c ul e s t he r e i s not N K a ut o r e a c t i vi t y ( 32 33 ) T hi s i ndi c a t e s t ha t t he r e i s a m or e c om pl e x pr oc e s s i n pl a c e ne c e s s i t a t i ng a ba l a nc e w i t hi n t he N K c e l l o f bot h a c t i va t i ng a nd i nhi bi t o r y r e c e pt or s a s w e l l a s t he pos s i bi l i t y of p r ope r l i c e ns i ng a nd/ or e duc a t i on t ha t w i l l be di s c us s e d s ho r t l y. T o da t e a l a r ge num be r o f r e c e pt or s pr e s e nt on t he N K c e l l ha ve be e n

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6 i de nt i f i e d pr ovi di n g f o r bot h i nhi bi t i on t hr ough r e c ogni t i on of M H C a nd non M H C m ol e c ul e s a s w e l l a s a c t i va t i on t hr ough a c t i va t i ng r e c e pt or s S e l f M H C R e c e p t or s L y 49s I n t he m ous e t he m os t pr e va l e nt s e l f M H C r e c e pt or s a r e t he L y49s L y49s a r e m e m be r s of t he C t ype l e c t i n f a m i l y a nd a r e t ype I I t r a ns m e m br a ne gl yc opr o t e i ns t ha t a r e e xpr e s s e d a s di s ul f i de l i nke d hom odi m e r s on t he c e l l s ur f a c e ( 34 36 ) G e ne t i c s t udi e s of t he c hr om os om a l l oc a t i on o f t he L y49 ge ne yi e l de d t he f i ndi ng t ha t L y49 i s a c t ua l l y a m e m be r of a l a r ge r ge ne f a m i l y e nc ode d on m ous e c hr om os om e 6 i n a r e gi on t e r m e d t he N K c om pl e x ( N K C ) ( 30, 37, 38) M uc h w or k ha s be e n done s i nc e t he s e i ni t i a l s t udi e s i de nt i f yi ng a nd p r ovi di ng e xa m i na t i ons of t he f unc t i ons of t he L y49 r e c e pt or f a m i l y. T he L y49s a r e a hi gh l y r e l a t e d ge ne f a m i l y c ons i s t i ng of a t l e a s t 14 f a m i l y m e m be r s i n t he B L 6 m ous e a l l of w hi c h a r e e nc ode d w i t hi n t he N K C ( 30, 34 36, 39 45 ) M os t of t he r e c e pt or s w i t hi n t hi s f a m i l y a r e i nhi bi t o r y r e c e pt or s w i t h f e w e xc e pt i ons i e L y49 D a nd H ( 46, 47) I t w a s i de nt i f i e d e a r l y on t ha t t he di f f e r e nt r e c e pt or s w i t hi n t hi s c om pl e x ha ve a va r i e ga t e d e xpr e s s i on a nd f unc t i on O ne of t he f i r s t s t udi e s de m ons t r a t e d t hi s by i de nt i f yi ng i n a ddi t i on t o t he o r i gi na l L y49, w hi c h w a s t e r m e d L y49A t he r e w a s a l s o a L y49B a nd L y49C A n i m po r t a nt a s pe c t of t hi s s t udy w a s not onl y t ha t t he y d i s c ove r e d t he s e ne w r e c e pt or s but a l s o t ha t t he y s how e d L y4 9A a nd L y49C c oul d be e xpr e s s e d on va r yi ng s ubs e t s of N K 1. 1 + C D 3 c e l l s ( 48) T hi s p r ovi de d a c onc e pt t ha t di f f e r e nt N K c e l l s m a y ha ve di f f e r e nt c om bi na t i ons of t he s e r e c e pt or s a nd t he r e f or e t he a bi l i t y t o

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7 r e s pond di f f e r e nt l y de pe ndi ng on t he l i ga nd p r e s e nt T he a c t ua l s pe c i f i c i t y t o one or m or e M H C m ol e c ul e s de pe nds upon t he pa r t i c ul a r L y49 r e c e pt or w i t h s om e di s pl a yi ng m o r e or l e s s pr om i s c ui t y t ha n ot he r s L y49A f or i ns t a nc e ha s be e n s how n t o bi nd t o H 2D d H 2D k a nd H 2 K ( 31 49 52) a nd L y49C ha s be e n s how n t o bi nd t o H 2K b a s w e l l a s H 2 b H 2 d H 2 k H 2 s ( 35 39 53 56) S om e L y49s s how m or e s pe c i f i c i t y t o a l i m i t e d num be r o f l i ga nds s u c h a s L y49G 2 w hi c h bi nds t o H 2D d a nd H 2 L d ( 34 57) a nd L y49 I w hi c h bi n ds t o H 2K b ( 53) A n i m po r t a nt a s pe c t of a l l o f t he s e i nt e r a c t i ons i s t ha t t hi s l i s t doe s not r e pr e s e nt t he pe nul t i m a t e s pe c i f i c i t i e s of t he s e r e c e pt or s S e c ond, a nd m o r e i m por t a nt l y i t doe s not de t a i l t he r e l e va nc e a nd o r s t r e ngt h of t he s e i nt e r a c t i ons w hi c h c oul d pl a y a c r uc i a l r ol e i n t he out c om e t he y e l i c i t T he m a nne r i n w hi c h a n N K c e l l e xpr e s s e s va r yi ng L y49s on i t s c e l l s ur f a c e i s a uni qu e a nd va r i e ga t e d s ys t e m R T P C R s t udi e s ha ve s how n t ha t e a c h N K c e l l i n a popul a t i on e xpr e s s e s on a ve r a ge 1 t o 4 di f f e r e nt L y49s ( 58) T he e xpr e s s i on s ys t e m i n pl a c e f or t he L y49 s a l l ow s f or t he i r e xpr e s s i on i n a m onoa l l e l i c f a s hi on t h r ough a s ys t e m of pr oba bi l i s t i c s w i t c he s ( 59 61 ) T he pr oba bi l i s t i c s w i t c h i s m a ni f e s t e d t hr ough t he us e of a bi di r e c t i ona l pr om o t e r f or e a c h L y49 ( 61) T h e s e pr om ot e r s c a n f unc t i on i n t he f or w a r d ( O n) pos i t i on w he r e t he y f r om a vi a bl e t r a ns c r i pt or i n t he r e ve r s e ( O f f ) pos i t i on w he r e a non c odi ng t r a ns c r i pt i s f or m e d. T hr ough c om pe t i t i ve bi ndi ng o f pr o m ot e r e l e m e nt s s uc h a s C A A T / e nha nc e r bi ndi ng pr ot e i n ( C / E B P ) o r T A T A bi ndi ng pr ot e i n ( T B P ) on ove r l a ppi ng po r t i ons of t he bi di r e c t i ona l pr om ot e r s t he y a r e a bl e t o e f f e c t t he s e p r oba bi l i s t i c s w i t c he s a s w e l l a s l oc k t he pr om o t e r s i n t he e s t a bl i s he d di r e c t i on i n t he

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8 m a t ur e N K c e l l ( 61) C onf l i c t i ng r e s ul t s e xi s t a s t o w he t he r c yt oki ne s c a n i nduc e a c ha nge i n t he L y49 r e pe r t o i r e t ha t ha s be e n e s t a bl i s he d i n a m a t ur e N K c e l l S om e s t udi e s ha ve s how n t ha t onc e a n N K c e l l ha s e s t a bl i s he d i t s L y49 r e pe r t oi r e i t m a i nt a i ns i t t hr oughout i t s c l ona l pr oge ny ( 62, 63) O t he r s t udi e s ha ve s how n t ha t s om e r e c e pt or s s uc h a s L y49E a nd F c a n be i nduc e d t o c ha nge t he i r e xpr e s s i on l e ve l s t hr ough c yt oki ne s s uc h a s I L 2 a n d I L 15 ( 64) O t he r r e c e pt or s m a y a l s o unde r go a c ha nge i n s ur f a c e de ns i t y f ol l ow i ng c yt oki ne s t i m ul a t i on. A n i nt e r e s t i ng f a c e t o f t he va r i e ga t e d e xpr e s s i on of L y49s a c r os s a n e nt i r e popul a t i on of N K c e l l s i s t ha t e a c h c e l l w i l l ha ve a di f f e r e nt pot e nt i a l t o r e a c t t o a t a r ge t I n t he or y s om e N K c e l l s m a y not pos s e s s a n a ppr opr i a t e i nhi bi t o r y L y49 a t a l l T h i s ve r y s i t ua t i on ha s be e n r e a l i z e d s uc h t ha t a s m a l l popul a t i on of N K c e l l s w i t hi n t he nor m a l popul a t i on of c e l l s doe s not e xpr e s s a know n i nhi b i t o r y L y49 ( 65 66 ) B a s e d on t he know n i nf or m a t i on t hi s c oul d t he or e t i c a l l y l e a d t o a n N K c e l l t ha t i s a ut or e a c t i ve T hi s ve r y phe nom e non ha s l e d t o a de ba t e d a r e a w i t hi n t he N K f i e l d w hi c h i s t he c onc e pt of l i c e ns i ng, e duc a t i on a nd/ or t ol e r a nc e ( 65 67) I n g e ne r a l i z e d t e r m s w ha t t he s e t he or i e s s t a t e i s t ha t f or a n N K c e l l t o be c om e f ul l y f unc t i o na l i t m us t pos s e s a s e l f r e c e pt or F o r l i c e ns i ng t he pr e s e nc e of a s e l f M H C r e c e pt or c onf e r s a n a bi l i t y o r l i c e ns e upon t he N K c e l l t o be c om e f ul l y c yt ot oxi c ( 65) f or t ol e r a nc e t h e a bs e nc e of t he s e l f r e c e pt or r e nde r s t he c e l l hypor e s pons i ve ( 66) e duc a t i on s i m i l a r t o l i c e ns i ng di c t a t e s t ha t t he pr e s e nc e of t he s e l f M H C r e c e pt or i s ne c e s s a r y f or t he N K c e l l t o be c om e f ul l y c yt ot oxi c ( 57) A l t hough t he s e t he o r i e s ha ve s l i ght l y di f f e r e nt m e t hods t hr ough w hi c h a n N K c e l l

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9 a c hi e ve s t hi s f unc t i ona l s t a t e t he y a gr e e t ha t f o r a n N K c e l l t o be c om e f ul l y f unc t i ona l i t m us t pos s e s a s e l f r e c e pt or A ke y out c om e of t he s e s t udi e s i s t ha t e ve n t hough N K c e l l s do not r e a r r a nge t he i r r e c e pt or s r a t he r us i ng s uppos e dl y m or e ba s i c ge r m l i ne e nc ode d r e c e pt or s t he y s t i l l unde r go a c om pl e x pr oc e s s of r e gul a t i on t ha t a s s ur e s t he pr ope r c e l l s a nd r e c e pt or s a r e f unc t i on i ng i n t he a ppr op r i a t e m a nne r A r e m a i ni ng que s t i on f or t he L y49 r e c e pt or s i s w h a t do t he y a c t ua l l y r e c ogni z e on t he M H C m ol e c ul e a nd l i ke t he T c e l l r e c e pt or i s t he r e s pe c i f i c i t y t o t he pe pt i de be i ng pr e s e nt e d. T he L y49A r e c e pt or ha s be e n s how n t o i nt e r a c t a t t w o s i t e s on H 2D d T he f i r s t be i ng w i t hi n t he 1 a n d 2 dom a i ns a nd t he s e c ond s i t e of i nt e r a c t i on i nvo l vi ng t he 1, 2, 3 a nd 2 M dom a i ns o f t he M H C ( 68, 69 ) T hi s s pe c i f i c i t y m a y onl y hol d t r ue f or t hi s s pe c i f i c i nt e r a c t i on, a s t he L y49C r e c ogni t i on of H 2K b doe s not e xhi bi t t he s a m e i nt e r a c t i on s i t e s ( 70) T he ne c e s s i t y of a pe pt i de b e i ng pr e s e nt i s a l s o not c om pl e t e l y c l e a r I t ha s be e n s how n t ha t f o r t he L y49A r e c e pt or a pe pt i de i s ne c e s s a r y, b ut t he a c t ua l pe pt i de s e que nc e s e e m s t o ha ve no be a r i ng ( 56, 71) W he r e L y49 C a nd L y49I s e e m t o be m or e r e l i a nt on a pe pt i de be i ng pr e s e nt t o bi nd p r ope r l y ( 56, 72, 73) A l t hough t he L y49s c ons i s t of num e r ous r e c e pt or s t ha t r e c ogni z e a va r i e t y of di f f e r e nt l i ga nds t he i r c yt opl a s m i c por t i ons a s w e l l a s t he s i g na l i ng pa t hw a ys t he y a c t i va t e a r e hi ghl y c ons e r ve d. T he c yt opl a s m i c po r t i on of t he i nh i bi t or y L y49s c ont a i n i m m unor e c e pt or t y r os i ne ba s e d i nhi bi t i on m ot i f s ( I T I M ) w i t h t he c ons e ns us s e que n c e of I / S / T / L xY xxL / V ( 74 75) U pon l i ga nd e nga ge m e nt t he s e I T I M s be c om e phos phor yl a t e d a l l ow i ng f or t he r e c r ui t m e nt o f S H 2 dom a i n c ont a i ni ng pr ot e i ns T he s e phos pho

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10 t y r os i ne s a r e a bl e t o r e c r ui t t he phos pha t a s e s S H P 1, S H P 2 a nd S H I P S H P 1 a nd 2 a r e t he n a bl e t o a br oga t e t he dow ns t r e a m s i gna l i ng c a s c a de s of a c t i va t i ng r e c e pt or s t hr ough de phos phor yl a t i on of p r ot e i n t y r os i ne ki na s e s ( P T K ) ( 74 79 ) W he r e S H I P c oul d a br oga t e s i gna l s pa s s i ng t hr ough t he P I 3K pa t hw a y t hr ough t he r e m ova l of t he 5 phos pha t e of P I ( 3, 4 5) P 3 ( P I P 3) A l t hough m os t of t he L y49 r e c e pt or s ha ve be e n s how n t o f unc t i on a s i nhi bi t o r y r e c e pt or s t he r e a r e t hos e L y49s t ha t f unc t i on a s a c t i va t i ng r e c e pt or s t he s e i nc l ude L y49H a nd L y49D ( 47, 80, 81) T he ke y di f f e r e nc e w i t h t he s e r e c e pt or s w i t h r e s pe c t t o t he i r i nhi bi t or y f a m i l y m e m be r s i s t ha t t he y do not pos s e s t he c l a s s i c I T I M s e que nc e i n t he i r c yt opl a s m i c dom a i n. R a t he r t he y ha ve a pos i t i ve l y c ha r ge d a r gi ni ne i n t he t r a ns m e m br a ne por t i on o f t he c yt opl a s m i c t a i l T h i s pos i t i ve l y c ha r ge d a m i no a c i d t he n a l l ow s t he s e r e c e pt or s t o a s s oc i a t e w i t h a n i m m un or e c e pt or t yr os i ne ba s e d a c t i va t i on m ot i f ( I T A M ) c ont a i ni ng D A P 12 m ol e c ul e ( 46 82 ) T he phys i ol ogi c a l r ol e of t he s e a c t i va t i ng L y49 r e c e pt or s a nd t he i r r e c ogni t i on of s e l f M H C m ol e c ul e s ha s n ot be e n f ul l y e l uc i da t e d. L y49D ha s be e n i m pl i c a t e d i n C 57B l / 6 m i c e i n t he r e j e c t i on of H 2 D d B M a l t hough t he pr e c i s e s t r e ngt h o f t hi s i nt e r a c t i on i s not f ul l y c l e a r ( 56 83 86) I t m a y be pos s i bl e t ha t i n nor m a l phys i ol ogi c a l c ondi t i ons t h e a c t i va t i ng L y49s do not r e c ogni z e M H C m ol e c ul e s but r a t he r s t r e s s i nduc i bl e m a r ke r s s i m i l a r t o ot he r a c t i va t i ng r e c e pt or s I n t he c a s e of L y49H t he l i ga nd i s t he M C M V e nc ode d m 157 gl yc opr ot e i n ( 80 81 ) I n f a c t m i c e t ha t do not e xpr e s s t he L y49H r e c e p t or s uc h a s B a l bC do not c onf e r a n N K m e di a t e d r e s i s t a nc e t o M C M V i nf e c t i on ( 87)

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11 C D 94/ N K G 2 T he ot he r c l a s s of r e c e pt or s w i t hi n t he m ous e t ha t r e c ogni z e c l a s s I r e l a t e d m ol e c ul e s a r e t he C D 94/ N K G 2 c l a s s of r e c e pt or s ( 88 90) L i ke t he L y49 r e c e pt or s C D 94 a nd t he N K G 2A C a nd E m ol e c ul e s a r e c l o s e l y l i nke d on c hr om os om e 6 w i t hi n t he N K C ( 88, 89) U nl i ke t he L y49s t he N K G 2 r e c e pt or s f or m d i s ul f i de l i nke d he t e r odi m e r s w i t h C D 94. B ot h of t he s e r e c e pt or s a r e i n t he C t ype l e c t i n f a m i l y a nd e nc ode t ype I I t r a ns m e m br a ne r e c e pt or s ( 89, 91, 9 2) T he s e r e c e pt or s a r e c a pa bl e of r e c ogni z i ng s e l f l i ga nds t hr ough t he r e c ogni t i on of M H C c l a s s I b l i ga nds s uc h a s Q a I b i n t he m ous e ( 89, 90) O nl y t he C D 94/ N K G 2A he t e r odi m e r f unc t i ons a s a n i nhi bi t or y r e c e pt or w he r e N K G 2C a nd E f unc t i on i n a n a c t i va t i ng m a nne r ( 8 9, 91, 93 95) B ot h N K G 2C a nd E f o r m he t e r odi m e r s w i t h C D 94 but ha ve a l ys i ne r e s i due i n t he c yt opl a s m i c t a i l t ha t a l l ow s t he m t o a s s oc i a t e w i t h t he I T A M c ont a i ni ng D A P 12. I n f a c t t he pr e s e nc e of D A P 12 i s ne c e s s a r y f o r t he i r s t a bl e e xpr e s s i on on t he c e l l s ur f a c e c onf i r m i ng t ha t t he s e t w o r e c e pt or s f unc t i on i n a n a c t i va t i ng m a nn e r ( 96) T he s e r e c e pt or s a r e e xpr e s s e d i n a n or de r e d m a nne r a nd on ove r l a ppi ng s ubs e t s of c e l l s ( 97) T h i s t he r e f or e be gs t he que s t i on of w ha t i s t he pur pos e of bot h a c t i va t i ng a nd i nhi bi t or y r e c e pt or s r e c ogni z i ng t he s a m e pe pt i de s A l t hough no de f i ni t i ve e vi de nc e ha s be e n f ound i t m a y be t ha t t he r e c e pt or s a l t hough e xt r e m e l y c l os e l y r e l a t e d ha ve s l i ght va r i a nc e i n t he s t r e ngt h i n w hi c h t he y r e c ogni z e di f f e r e nt pe pt i de s a l l ow i ng d i f f e r e n t s i gna l i ng out c om e s ba s e d on ot he r s i gna l s pr e s e nt

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12 N on M H C r e c e p t or s N K G 2D N K G 2D ha s be e n f ound t o be a ke y a c t i va t i ng r e c e pt or t ha t i s f ound on a l l N K c e l l s ( 98 100) N K G 2D i s a t ype I I t r a ns m e m br a n e gl yc opr ot e i n t ha t i s e xpr e s s e d a s a hom odi m e r on t he s ur f a c e of N K c e l l s ( 9 9, 101) U nl i ke ot he r N K G 2 m ol e c ul e s N K G 2D doe s not a s s oc i a t e w i t h t he C D 94 c or e c e pt or ( 102 ) W i t hi n t he m ous e N K G 2D ha s a s hor t a nd l ong i s of or m t ha t a l l ow s i t t o a s s oc i a t e w i t h e i t he r D A P 10 o r D A P 12 r e s pe c t i ve l y ( 98, 103 ) T he a bi l i t y t o ut i l i z e bot h o f t he s e a da pt e r s a l l ow s N K G 2D t o s i gna l t hr ough di f f e r e nt dow ns t r e a m pa t hw a ys i nc l udi ng P I 3K a s w e l l a s S yk a nd Z a p70 ( 102, 104 ) N K G 2D r e c ogni z e s m ol e c ul e s t ha t a r e s t r uc t ur a l l y r e l a t e d t o t he M H C b ut do not pr e s e nt pe pt i de s ( 105) T he s e m ol e c ul e s i nc l ude t he R a e 1 f a m i l y m e m be r s a s w e l l a s M ul t 1 a nd H 60 ( 98 106 108) T he R a e 1 ge ne f a m i l y i s hi ghl y hom ol ogous w he r e H 60 a nd M ul t 1 ha ve ve r y l i t t l e s e que nc e hom ol ogy ( 10 7, 109) T hi s w i de va r i e t y of l i ga nds i s a bl e t o pr e s e nt da nge r s i gna l s i n r e s pon s e t o a w i d e va r i e t y of s t r e s s e s a nd t he r e by, i n t ur n a l l ow s t he N K c e l l t o r e s pond t o a w i de va r i e t y of s t r e s s e s T he s e va r i e d i m m une c ha l l e nge s c a n i nc l ude vi r a l i nf e c t i ons w he r e e a r l y i n t he i m m une c ha l l e nge t he pr e s e nc e of one of t he s e s t r e s s i nduc i b l e m a r ke r s c a n l e a d n ot onl y t o N K m e di a t e d c yt ot ox i c i t y, but a l s o f or a n N K c e l l t o c e l l t o up r e gul a t e I F N # ( 110) T hi s i s t he n a bl e t o l e a d t o a m or e pot e nt a da pt i ve i m m une r e s pons e a s w e l l a s e f f e c t i ng t he r e gul a t i on of vi r a l t r a ns c r i pt i on e a r l y i n t he i nf e c t i on ( 111) R a ul e t e t a l ha ve s how n t ha t i n r e s pons e t o D N A da m a ge s om e c e l l s c a n upr e gul a t e N K G 2D l i ga nds t he r e by pr ov i di ng a n N K c e l l

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13 r e l i a nt m e c ha ni s m f or t he e l i m i na t i on of c e l l s w i t h a be r r a nt D N A r e pl i c a t i on a nd pos s i bl e m ut a t i ons t ha t c oul d l e a d t o t r a ns f or m a t i on ( 112) W i t hi n t um or i m m uni t y s i m i l a r s ys t e m s e xi s t w i t h s t r e s s e d c e l l s up r e gul a t i ng N K G 2D l i ga nds t o a l l ow f or bot h di r e c t l ys i s by t he N K c e l l a s w e l l a s pr oduc t i on o f c yt ok i ne s bot h t o r e gul a t e t um o r g r ow t h a s w e l l a s t o i nduc i ng a n a da pt i ve r e s pons e ( 99, 101, 107, 113 116 ) T he r e s pons e of t he N K c e l l vi a N K G 2D ha s pr ove n t o be a pot e nt e no ugh r e s pons e t ha t s om e t um or s ha ve w a ys i n w hi c h t o c om ba t N K G 2D m e di a t e d c yt ot o xi c i t y. T he s e c e l l s a r e a bl e t o pr oduc e a s ol ubl e N K G 2D l i ga nd. T hi s c a n l e a d t o t he do w n r e gul a t i on of N K G 2D on t he N K c e l l ( 117) T hi s a bi l i t y of N K G 2D t o e l i c i t a po w e r f ul a c t i va t i ng s i gna l t hr ough bot h D A P 10 a nd D A P 12 i n r e s pons e t o num e r ous s t i m u l i m a ke s i t one of t he m os t pot e nt a nd i m por t a nt a c t i va t i ng N K R 2B 4 A l t hough t he L y49 s r e c ogni t i on of M H C i s t he pr ot ot ypi c a l s ys t e m f or t he r e c ogni t i on of hos t e nc ode d l i ga nds a nd t he r e f or e t he r e c ogni t i on of s e l f t he r e a r e ot he r r e c e pt or s t ha t r e c ogni z e non M H C m ol e c ul e s e nc ode d by t he hos t one s uc h r e c e pt or i s 2B 4. 2B 4 i s a S L A M f a m i l y r e l a t e d r e c e pt or O t h e r r e c e pt or s i n t hi s f a m i l y c ons i s t of s i gna l l ym pho c yt e a c t i va t i on m ol e c ul e ( S L A M ) N K T a nd B c e l l a nt i ge n ( N T B A ) C D 2 l i ke r e c e pt or a c t i va t i ng c yt ot oxi c c e l l s ( C R A C C ) L y 9 a nd C D 84 ( 118 125) T he S L A M r e l a t e d r e c e pt or s a r e m e m be r s of t he C D 2 I g s upe r f a m i l y pos s e s s i ng t w o e xt r a c e l l ul a r I g l i ke dom a i ns T he i nt r a c e l l ul a r por t i on of t he s e r e c e pt or s c ons i s t s of one or m or e i m m unor e c e pt or t yr os i ne ba s e d s w i t c h m o t i f ( I T S M ) c ons i s t i ng of t he s e que nc e T xY xxI / V ( 126, 127) M os t o f t he r e c e pt or s i n t he S L A M f a m i l y f unc t i on t hr ough

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14 hom ot ypi c i nt e r a c t i ons a l t ho ugh 2B 4 doe s not f un c t i on i n t hi s m a nne r i ns t e a d i t r e c ogni z e s C D 48 ( 128) w hi c h i s ubi qui t ous l y e xpr e s s e d on c e l l s of t he he m a t opoi e t i c s ys t e m ( 128) 2B 4 i s e xpr e s s e d ubi qui t ous l y on N K c e l l s a s w e l l a s on s ubs e t s of T c e l l s a nd e ve n a s m a l l s ubs e t of de ndr i t i c c e l l s ( 128 12 9) 2B 4 ha s a c om pl e x r ol e i n N K c e l l f unc t i on a nd ph ys i ol ogy t ha t r e m a i ns a n a c t i ve a r e a of i nve s t i ga t i on. D e pe ndi ng on t h e c ont e xt 2 B 4 ha s be e n s how n t o a c t a s bot h a n i nhi bi t or y a nd a c t i va t i ng r e c e pt or M uc h of t he f u nc t i ona l va r i a t i on o f t hi s r e c e pt or m a y be due t o s t udyi ng i t i n bot h hum a n a nd m ous e m o de l s a s w e l l a s di f f e r e nt s t r a i ns o f m ous e ( 130 138) A m or e de f i ni t i ve r ol e f or 2B 4 i n t he B l 6 m ous e w a s de m ons t r a t e d t hr ough t he ge ne r a t i on of a 2B 4 knoc k out ( K O ) T hi s K O m ous e pr ovi de d s t r ong e vi de nc e i n t he B l 6 m ode l f or 2B 4 a s a n i nhi b i t or y r e c e pt or ( 128, 129, 139) T he a bi l i t y of t hi s r e c e pt or t o f unc t i on i n num e r ous f a c e t s a ppe a r s t o be due a t l e a s t i n pa r t t o t he m ul t i t ude of s i gna l i ng m ol e c ul e s 2B 4 c a n r e c r ui t I n bot h hu m a n a nd m ous e m ode l s unde r di f f e r e nt s i gna l i ng c ont e xt s 2B 4 ha s be e n s how n t o r e c r ui t S A P E A T 2, F ynT S H P 1, P I 3K a nd S H I P ( 133 139 142 ) H ow t he d i f f e r e nt i a l r e c r ui t m e nt o f t he s e s i gna l i ng e nt i t i e s i s c ont r ol l e d i s no t c om pl e t e l y un de r s t ood. H ow e ve r w hi c h m ol e c ul e s a r e r e c r ui t e d a nd t hus w hi c h s i gna l i s p r opa ga t e d f ol l ow i ng C D 48 e nga ge m e nt m a y be i nf l ue nc e d by t he r a t i o o f 2B 4 i s of o r m s e xpr e s s e d i n t he N K c e l l T w o 2B 4 i s of or m s ha ve be e n i de nt i f i e d i n m i c e s hor t ( 2B 4S ) a nd l on g ( 2B 4L ) t ha t w e r e p r opos e d t o ha ve a c t i va t i ng a nd i nhi bi t o r y s i gna l i ng c a pa c i t i e s r e s pe c t i ve l y ( 134) A l t hough t he e xa c t f unc t i on of t he s e t w o i s of or m s r e m a i ns t o be de f i n e d, i t i s f e a s i bl e t ha t t he di f f e r e nt i nt r a c e l l ul a r dom a i ns w i t hi n t he s e i s of or m s c oul d r e c r ui t di f f e r e nt e f f e c t or s of c e l l

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15 s i gna l i ng. 2B 4 p r e s e nt s a not he r i nt e r e s t i ng phe no m e non w i t hi n t he N K f i e l d E ve r s i nc e t he m i s s i ng s e l f hypot he s i s a nd t he di s c ove r y o f t h e f i r s t L y49s i t ha s be e n t hought t ha t i t w a s t hr ough t hi s m e c ha ni s m a l one t ha t t he N K c e l l r e c ogni z e d s e l f a nd i nh i bi t e d c yt ot oxi c i t y. I nt e r e s t i ngl y M c ne r ne y e t a l ha ve pr opos e d a not he r pos s i bi l i t y i n w hi c h 2B 4 m a y a l s o pl a y a r ol e i n s e l f r e c ogni t i on T he y ha ve pe r f or m e d e xpe r i m e nt s i n w hi c h t he y w e r e a bl e t o s how t h r ough m e c ha ni s m s non r e dunda nt t o t he L y49s t ha t 2B 4 w a s a bl e t o r e c ogni z e s e l f a nd i nhi bi t B M gr a f t r e j e c t i o n ( 131) T hi s doe s not ne c e s s a r i l y a l t e r t he c ur r e nt s ys t e m of s e l f r e c ogni t i on by t he N K c e l l ; i t m e r e l y e xt e nds t he r e c e pt or s t ha t c a n pa r t i c i pa t e i n t hi s pr oc e s s A l t hough N K G 2D a nd 2B 4 a r e c r uc i a l r e c e pt or s t h e y a r e f a r f r o m t he onl y non M H C r e c e pt or s pr e s e nt on t he N K c e l l T he r e a r e a m ul t i t ude o f ot he r s t ha t f unc t i on i n bot h a c t i va t i ng a nd i nh i bi t or y c a pa c i t i e s F o r i ns t a nc e t he na t u r a l c yt ot oxi c i t y r e c e pt or N K p46 ha s be e n s how n t o pl a y a r ol e i n bot h t um o r a nd vi r a l r e gul a t i on ( 143 145) T he l i ga nd f or t hi s r e c e pt or ha s not be e n f ul l y i de nt i f i e d but i s be l i e ve d t o be he pa r i n s ul f a t e pr ot e ogl yc a ns ( 146) N K p46 l i ke 2B 4 ha s 2 e xt r a c e l l ul a r I G dom a i ns a s w e l l a s a s hor t c yt opl a s m i c t a i l c ont a i ni ng a n a r gi ni ne t ha t a l l ow s i t t o a s s oc i a t e w i t h I T A M c ont a i ni ng m ol e c ul e s ( 147) G i ve n t he i r t he o r i z e d a bi l i t y t o b i nd t o p r ot e ogl yc a ns t hi s r e c e pt or m a y pr ove t o be a c r uc i a l r e c e pt or w i t h a n a bi l i t y t o r e c ogni z e a w i de va r i e t y of t a r ge t s

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16 I n t e gr at i on of S i gn al i n g A c om m on t he m e i n m a ny i m m une c e l l s i s t he m a nne r i n w hi c h t he y pr opa ga t e a c t i va t i ng s i gna l s t hr ough t he c e l l A s ha s be e n d i s c us s e d a l r e a dy, t he r e a r e a num be r of di f f e r e nt a c t i va t i ng l i ga nds s om e w i t h ve r y di s pa r a t e s t r uc t ur e s a nd f unc t i ons I n t he N K c e l l a nd i n m a ny ot he r i m m une c e l l s t hi s di s pa r i t y i s r e c onc i l e d by t he us e o f a da pt or m ol e c ul e s t ha t a r e a bl e t o i nt e r a c t w i t h a w i de va r i e t y of r e c e pt or s ( 98) T he s e a da pt or s c ons i s t of t he I T A M c ont a i ni ng m ol e c ul e D A P 12 or t he D A P 10 m ol e c ul e t ha t c ons i s t s of a Y xxM m ot i f ( 102) T he s e m ol e c ul e s c ont a i n a n e ga t i ve l y c ha r ge d a m i n o a c i d i n t he a m i no t e r m i nus w hi c h a l l ow s t he m t o i nt e r a c t w i t h t he pos i t i ve l y c ha r ge d a m i no a c i d i n t he t r a ns m e m br a ne dom a i n o f t he a c t i va t i ng r e c e pt or s ( 102) A n i m po r t a nt a s pe c t of ut i l i z i ng a s m a l l nu m be r o f a da pt or t ype m ol e c ul e s t o t r a ns duc e s i gna l s i s t ha t a r e l a t i ve l y s m a l l num be r of pa t hw a ys a r e ut i l i z e d ove r a nd ov e r t o t r a ns duc e t he s e s i gna l s A s um m a r y di a gr a m of t he s e s i gna l i ng pa t hw a ys i s s how n i n F i g 2. T hi s f i gur e m ode l s t he ba l a nc e t ha t e xi s t s w i t hi n t he s i gna l m o t i f s ut i l i z e d by t he N K c e l l T he a c t i va t i ng r e c e pt or s t hr ough t he i r pa i r i ng w i t h D A P 10 a nd/ or D A P 12 s i gna l t h r ough e i t he r P I 3K or P T K s r e s pe c t i ve l y. I f t he y ut i l i z e t he P T K pa t hw a y t he f i r s t l i ne o f P T K s t e nd t o be a m e m be r of t he S yk/ Z a p70 f a m i l y, w hi c h t he n a l l o w s t he s i gna l t o e m a na t e t o a num be r of pa t hw a ys T hi s r e qui r e m e nt f or t he us e of P T K or i nos i t ol phos phol i pi ds f or a c t i va t i ng s i gna l s a l l ow s t he i nhi bi t or y r e c e pt or s t h r ough t he a bi l i t y o f t he i r I T I M s t o r e c r ui t S H P a nd S H I P t o a br oga t e t he s e s i gna l s ve r y e f f i c i e nt l y.

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17 F i gu r e 2. I n t e gr at i on o f ac t i va t i n g an d i n h i b i t o r y N K R s i gn al s M ode l di a gr a m s how i ng t he us e of bot h P T K a nd P I 3K by a c t i va t i ng N K R S H P i s t he n a bl e t o a br oga t e s i gna l s e m a na t i ng t hr ough t he P T K s a nd S H I P ha s a s i m i l a r f unc t i o n w i t h i n t he P I 3K pa t hw a y. N K E f f e c t or F u n c t i on s C y t ot ox i c i t y T w o m e c ha ni s m s t he g r a nul e e xoc yt os i s m e t hod a nd t he de a t h r e c e pt or pa t hw a ys c a n a f f e c t t he d i r e c t c yt ol ys i s of t a r ge t c e l l s by N K c e l l s I n t he gr a nul e e xoc yt os i s m e t hod, w he n a n N K c e l l i s pr ope r l y a c t i va t e d, i t f or m s a s yna ps e w i t h t he t a r ge t c e l l T he f o r m a t i on o f t hi s s yna ps e t he n a l l o w s t he r e l e a s e of t he l yt i c gr a nul e s i n t he pr ope r o r i e nt a t i on t o t he t a r ge t c e l l t o i nduc e c e l l de a t h a nd/ or a popt os i s ( 148 150)

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18 T he s e l yt i c gr a nul e s i nc l ude pe r f or i n a nd t he gr a nz ym e s e a c h w i t h a uni que r ol e t ha t l e a ds t o t he t a r ge t c e l l unde r goi ng a popt os i s P e r f or i n w a s or i gi na l l y be l i e ve d t o be ne c e s s a r y t o pr oduc e hol e s i n t he t a r ge t c e l l t o a l l o w t he gr a nz ym e s t o e nt e r t he t a r ge t c e l l A l t hough t hi s r ol e ha s be e n i n que s t i on w i t h pe r f or i n pos s i bl y ha vi ng ot he r r ol e s di r e c t l y r e s ul t i ng i n a popt os i s W ha t i s c e r t a i n i s t ha t pe r f or i n i s ne c e s s a r y f or N K c e l l s t o e f f i c i e nt l y l ys e t a r ge t c e l l s ( 151, 152) I n f a c t t h e pr e s e nc e of pe r f o r i n ha s be e n s how n t o pl a y a ke y r ol e i n N K r e gul a t i on o f t um o r s ( 153 156) T he r ol e of t he gr a nz ym e s i n a popt os i s i s be t t e r unde r s t ood. I n m i c e t he r e a r e 1 0 know n gr a nz ym e s ( 157 ) G r a nz ym e B i s one of t he m or e s t udi e d of t he gr a nz ym e s a nd ha s be e n s how n t o i ni t i a t e a popt os i s i n a c a s pa s e de pe nde nt a nd i nde pe nde nt m e t hod. T h e gr a nz ym e s e l i c i t t he i r f unc t i on t hr ough c l e a va ge of c e l l ul a r pr o t e i ns a t s pe c i f i c c o ns e ns u s s e que nc e s G r a nz ym e B f unc t i ons vi a c l e a va ge a f t e r s pe c i f i c a s pa r t i c a c i d r e s i due s ( 158) one s uc h s i t e i s f ound w i t hi n c a s pa s e 3. V i a g r a nz ym e B s c l e a va ge of c a s pa s e 3 i t i s a bl e t o a c t i va t e i t a nd t he r e by t he c a s pa s e de pe nde nt a popt ot i c c a s c a de ( 1 59 161) G r a nz ym e B i s a l s o a bl e t o c l e a ve a s pa r t i c r e s i due s s i t e s on m e m be r s of t he B c l 2 f a m i l y of pr ot e i ns e s pe c i a l l y B I D T he r e by di s r upt i ng t he m i t oc hond r i a l m e m b r a ne a nd c a us i ng c yt oc hr om e C r e l e a s e a nd onc e a ga i n l e a di ng t o a popt os i s of t he c e l l ( 162, 16 3) T he de a t h r e c e pt or pa t hw a y ha s a l s o be e n s how n t o pl a y a ke y r ol e i n N K m e di a t e d c yt ot oxi c i t y ( 164 167) N K c e l l s a r e a bl e t o e xpr e s s a num be r of l i ga nds t ha t c a n i nduc e de a t h r e c e pt or pa t hw a ys i nc l udi ng F a s L T N F a nd T R A I L ( 168 171 ) S i m i l a r t o t he g r a nul e e xoc yt os i s m e t hod, upon pr ope r s t i m ul a t i on a nd s yna ps e f or m a t i on t he s e l i ga nds a r e br ought t o t he c e l l s ur f a c e a t t he s i t e of t he s yna ps e a l l ow i ng t he m t o

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19 i nt e r a c t w i t h de a t h r e c e pt or s on t he t a r ge t c e l l I nt e r e s t i ngl y t he N K c e l l i s a l s o a bl e t o a ugm e nt e xpr e s s i on of t he s e de a t h r e c e pt or s on t he t a r ge t c e l l t o m a ke t he m m or e s us c e pt i bl e t o l ys i s F or i ns t a nc e s om e t um or c e l l s e xpr e s s ve r y l i t t l e F a s o n t he i r s ur f a c e N K c e l l s t hr ough t he r e l e a s e of I F N # a r e a bl e t o up r e gul a t e F a s on t he s ur f a c e of t he s e t a r ge t c e l l s t he r e by m a ki ng t he m s us c e pt i bl e t o a c t i va t i on o f t he i r de a t h r e c e pt or pa t hw a ys ( 172) N K i n A dapt i v e i m m uni t y N K c e l l s a l t hough c l a s s i f i e d a s m e m be r s of t he i nn a t e i m m une s ys t e m a r e a l s o a bl e t o br i dge t he ga p be t w e e n t he i nna t e a nd a da p t i ve s ys t e m s I t doe s t hi s pr i m a r i l y t hr ough t he r e l e a s e of c yt oki ne s I n f a c t i t w a s s how n m a ny ye a r s a go t ha t t he r e i s di m i ni s he d C T L a c t i vi t y i n m i c e t ha t l a c k N K c e l l s ( 173, 174 ) T he m e t hods t ha t unde r l i e t hi s s t i m ul a t i on a r e br oa d a nd pr oc e e d t h r ough num e r ous pa t hw a ys F or i ns t a nc e i n r e s pons e t o a vi r a l c ha l l e nge N K c e l l s t hr ough t he s e c r e t i on o f I F N # a nd pos s i bl y ot he r c yt oki ne s c a n i nduc e m a c r opha ge s t o s t i m ul a t e T c e l l s t o di f f e r e nt i a t e i nt o T h1 a nd C D 8+ c yt ot oxi c c e l l s ( 175) N K c e l l s ha v e a l s o be e n s how n t o r e l e a s e I F N # a nd c he m oki ne s s uc h a s M I P 1 a nd R A N T E S t ha t a r e a bl e t o f unc t i on a s c he m oa t t r a c t a nt s a nd a c t i va t i ng m ol e c ul e s a t s i t e s of i nt e r a c t i on ( 176, 177) A n i nt e r e s t i ng a s pe c t of N K c e l l s a t t he i n t e r f a c e o f t he i nna t e a nd a da pt i ve i m m une s ys t e m i s t he r e l a t i ons hi p t he y c a n ha ve w i t h de ndr i t i c c e l l s ( D C ) T hi s i nt e r a c t i on c a n oc c ur i n bot h di r e c t i ons w i t h t he D C a c t i va t i ng t he N K c e l l or t he N K c e l l a i di ng i n t he a c t i va t i on a nd/ or m a t ur a t i on of t he D C a nd c a n e ve n go a s f a r a s N K c e l l s

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20 ki l l i ng t he i m m a t ur e D C O ne of t he m or e c om m o n m e t hods t hr ough w hi c h D C a r e a bl e t o a c t i va t e N K c e l l s i s t h r ough t he us e o f c yt oki n e s F or i ns t a nc e I L 12 f r om D C ha s be e n s how n t o i nc r e a s e I F N # p r oduc t i on by N K c e l l s ( 178 181) A t s i t e s of i nf l a m m a t i on D C a r e a bl e t o r e l e a s e I L 18 w hi c h i s t he n a bl e t o e f f e c t t he m i gr a t or y c a pa bi l i t y of t he N K c e l l ( 182) I L 18 a nd 12 a r e a l s o a bl e t o i nt e r a c t s yne r gi s t i c a l l y t o i nc r e a s e N K c yt ot oxi c i t y ( 183) D i f f e r e nt f or m s o f D C a r e e ve n a bl e t o, t hr ough di r e c t c e l l t o c e l l c ont a c t r e l e a s e c l a s s i c a c t i va t or s of N K c e l l s s uc h I L 2 I L 12 a nd I L 15 t o di r e c t l y a ugm e nt t he N K c e l l s a c t i va t i on s t a t us ( 1 84 189) T he e f f e c t N K c e l l s h a ve on t he D C s e e m s t o r e l y not onl y on t he c yt oki ne s a nd l i ga nds pr e s e nt but a l s o on t he r a t i o of N K : D C I f t he r a t i o i s l ow ( 1: 5 ) a nd t he pr ope r c yt oki ne s a r e pr e s e nt s uc h a s I F N # a nd T N F t h e N K c e l l c a n s t i m ul a t e i D C t o m a t ur e ( 1 90) I n t hi s m e t hod c e l l t o c e l l c ont a c t a l l ow s f o r t r i gge r i ng o f t he i D C t o m a t u r e t hr ough t he N K p30 r e c e pt or a nd pos s i bl y t hr ough t he T R E M 2 r e c e pt or ( 181 191) T he ot he r s i t ua t i on e xi s t s w he n t he r a t i o i s s w i t c he d a n d t he r e a r e m or e i D C t o N K ( 5: 1) I n t hi s s i t ua t i on t he N K c e l l i s a bl e t o l ys e t he i D C s due m os t l y t o t he i r l ow e xpr e s s i on l e ve l s of M H C O nc e a ga i n N K p30 i s a ke y r e c e pt or i n t hi s N K D C i nt e r a c t i on ( 190 192, 193) T he s e hi ghl y va r yi ng f unc t i ons s e e m t o be a t odds but bot h f unc t i ons ha ve ke y r e gul a t or y r ol e s i n a i di ng i n t he ba l a nc e of a c t i va t i on a nd t ol e r a nc e i n t he i D C N K D e ve l op m e n t T he pr oc e s s i n w hi c h a n N K c e l l p r ogr e s s e s f r om H S C t o a f ul l y f unc t i ona l m a t ur e c e l l i s f a r f r om be i ng f ul l y unde r s t ood. A t t hi s s t a ge t he pr oc e s s i s u nde r s t ood a s

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21 a l i ne a r pr og r e s s i on t ha t i s di vi de d i nt o di s t i nc t s t e ps ba s e d on t he pr e s e nc e of di s t i nc t s ur f a c e m a r ke r s l oc a t i on a nd e ve nt ua l l y t he a c qui s i t i on of f unc t i on. T he f i r s t s t a ge s of N K c e l l de ve l opm e nt i nvol ve no t t he c om m i t m e nt t o be c om i ng a nd N K c e l l a l one but r a t he r t he di f f e r e nt i a t i on of t he H S C i nt o t he c om m on l ym phoi d p r oge ni t or ( 194) A l t hough t he s e c e l l s ha ve be e n s how n t o ha ve N K pot e nt i a l i n v i v o a nd i n v i t r o t he de s i gna t i on c om m on l ym phoi d p r oge ni t or m e a ns t ha t t he s e c e l l s c a n a l s o f or m B a nd T c e l l s ( 195 197) T he ne xt s t e p t ha t ha s be e n i de nt i f i e d i s t he c om m i t m e nt t o a T N K r e s t r i c t e d pr oge ni t or ( T N K P ) A n i nt e r e s t i ng a s pe c t of t hi s c e l l i s t ha t i t ha s onl y be e n i de nt i f i e d i n t he f e t a l l i ve r a nd t hym us of t he m o us e ( 198, 199) a nd not i n t he a dul t a ni m a l T he i de nt i f i c a t i on o f t hi s c e l l i s ba s e d on a l i ne a ge ( L i n ) N K 1. 1 + a nd c ki t + phe not ype ( 200) T hi s phe not ype l i ke i t s l oc a t i on, i s ve r y i n t r i gui ng gi ve n t ha t t he pr e s e nc e of N K 1. 1 a ppe a r s a t t hi s s t a ge a nd a s w i l l be di s c us s e d i n t he ne xt s t a ge s of de ve l opm e nt i s not e xpr e s s e d a ga i n unt i l m or e di f f e r e nt i a t e d s t a ge s of t he N K c e l l S o a l t hough t hi s c e l l ha s be e n s how n t o f or m N K c e l l s i n v i v o a nd i n v i t r o i t m a y be pos s i bl e t ha t t hi s i s a n i n t e r m e di a r y w i t h a ye t t o be f ul l y e l uc i da t e d r ol e T he ge ne r a t i on o f t he N K pr oge ni t o r ( N K P ) r e pr e s e nt s t he f i r s t m a j o r s t e p t o c r e a t i ng a c e l l t ha t i s r e s t r i c t e d t o t he N K l i ne a ge r a t he r t ha n r e m a i ni ng bi pot e nt T he l i ne a r pr oc e s s f r om N K P t o m a t ur e N K c a n be di vi de d i nt o 5 di s t i nc t s t e ps t ha t onc e a ga i n a r e punc t ua t e d by t he a c qui s i t i on of di s t i nc t s ur f a c e m a r ke r s ( 201 203 ) T he s e s t a ge s of N K de ve l opm e nt oc c ur i n t he bone m a r r o w a s t he f i r s t s t e ps r e qui r e t he i nt e r a c t i on of t he N K P w i t h t he B M s t r om a T he p he not ype of t he N K P i s de s c r i be d a s L i n C D 122 + N K 1 1 D X 5 ( 203) O ne of t he e a r l i e s t ha l l m a r ks of N K c e l l

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22 de ve l opm e nt a nd t he s t e p w hi c h i s be l i e ve d t o m a r k t he t r a ns i t i on of T / N K P t o N K P i s t he a c qui s i t i on of C D 122, or t he I L 2/ 15R ( 201 2 03) T he a c qui s i t i on of C D 122 i s a c r uc i a l s t e p i n t he c om m i t m e nt t o t he N K l i ne a ge s uc h t ha t I L 15R de f i c i e nc y ( 204) a s w e l l a s I L 2R de f i c i e nc y ( 205) pr oduc e s de f e c t s i n N K c e l l de ve l opm e nt a nd f unc t i on I t ha s a l s o be e n s how n t ha t de f e c t s i n I L 15 s i gna l i ng pa t hw a ys s uc h a s J A K 3 a nd S T A T 5a / b c a n c a us e de f e c t s i n N K c e l l de ve l opm e nt ( 206) S t a ge s 2 t o 5 o f de ve l opm e nt f r om N K P t o m a t ur e N K a r e m a r ke d s e que nt i a l l y, f i r s t by t he ga i n o f M a c 1 e xpr e s s i on t he n t he C D 94/ N K G 2 r e c e pt or c om pl e x f ol l ow e d by t he L y49 r e c e pt or s I t i s i n t he f i na l s t a ge of de ve l op m e nt w i t hi n t he B M t h a t t he N K c e l l ga i ns f unc t i ona l i t y, s pe c i f i c a l l y c yt ot oxi c i t y a nd I F N # pr oduc t i on t he r e by r e s ul t i ng i n a f ul l y m a t ur e N K c e l l ( 202, 203 ) S H I P T he c l oni ng of SH I P S H I P 1 ha d a bl oom of i n t e r e s t i n 1996 w i t h nu m e r ous gr oups i nde pe nde nt l y c l oni ng i t ( 207 211) S i nc e i t s i de nt i f i c a t i on S H I P ha s be e n s how n t o hyd r ol yz e t he 5 phos pha t e of phos pha t i dyl i nos i t ol 3 4, 5 phos pha t e ( P I P 3) i n v i v o a nd i nos i t ol 1, 3 4, 5 t e t r a ki s phos pha t e ( I P 4) i n v i t r o, ( 208 210 ) T he a bi l i t y of S H I P t o hyd r ol yz e t he 5 phos pha t e of P I P 3 a l l ow s i t t o oppos e t he a c t i vi t y of P I 3K t he r e by e f f e c t i ng a w i de va r i e t y of c e l l ul a r a c t i vi t i e s i nc l udi ng pr ol i f e r a t i on di f f e r e nt i a t i on a popt os i s a nd m i gr a t i on.

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23 F i gu r e 3. S H I P A s t r uc t ur a l di a g r a m of S H I P s h ow i ng t he f unc t i ona l dom a i ns SH I P F unc t i onal D om ai ns T he s t r uc t ur e o f S H I P c ons i s t s of a n a m i no t e r m i n a l S H 2 dom a i n a 5 i nos i t ol phos pha t a s e c a r boxy t e r m i na l N pX Y m o t i f s a s w e l l a s a pol ypr ol i ne r i c h m ot i f ( F i g. 3) T he a m i no t e r m i na l S H 2 dom a i n o f S H I P ha s be e n s how n t o bi n d t o phos pho t y r os i ne s pr e s e nt i n m a ny s i gna l i ng m ol e c ul e s ( 212 214) I t m a y be t hr ough t hi s dom a i n t ha t S H I P pl a ys i t s m os t i m por t a nt r ol e i n r e ga r ds t o s i gna l i n g w i t hi n t he N K c e l l T he m a j or i nhi bi t or y r e c e p t or s w i t hi n t he N K c e l l pos s e s a n I T I M w hi c h ha s be e n s how n t o pos s e s phos pho t yr os i ne s t ha t a r e a bl e t o a s s oc i a t e w i t h t h e S H 2 dom a i n o f S H I P ( 212, 215, 216) S H I P ha s a l s o be e n s how n t o bi nd t o t he i nt r a c e l l ul a r t a i l of 2B 4 a nd K L R G 1 ( 217 ) a s w e l l a s t he F c # R I I B w he r e i t pl a ys a c r i t i c a l r ol e i n r e c e pt or l oc a l i z a t i on t o t he l i p i d r a f t ( 215 218 219 ) I nt e r e s t i ngl y S H I P s S H 2 do m a i n ha s a l s o be e n s how n t o i nt e r a c t w i t h a not he r phos pha t a s e S H P 2 a m ol e c ul e t ha t p l a ys a n e s s e nt i a l r ol e i n t he i nhi bi t or y s i gna l i ng pa t hw a y of N K c e l l s ( 220, 221) T he c a t a l yt i c dom a i n of S H I P i s i t s 5 i nos i t ol pho s pha t a s e T hi s e nz ym a t i c dom a i n i s a bl e t o hydr ol yz e t he 5 phos pha t e f r om P I P 3 a nd I P 4 ( 208 222 223 ) P I 3K c a t a l yz e s t he a ddi t i on of a phos pha t e a t t he 3 pos i t i on on P hos pha t i dyl I nos i t ol ( 4 5) P hos pha t e ( P I ( 4 5) P 2 ) c r e a t i ng P I P 3. P I P 3 pl a ys a ke y r ol e i n r e c r ui t i ng pl e c ks t r i n

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24 hom ol ogy ( P H ) dom a i n c ont a i ni ng pr o t e i ns s uc h a s A K T ( 224 228) I t i s t hr ough t he r e m ova l of t he 5 phos pha t e f r om t hi s phos phor yl a t e d P I P 3 t ha t S H I P c a n r e gul a t e pa t hw a ys t ha t f unc t i on dow ns t r e a m o f P I 3K ( 224 228) A num be r o f N pX Y m ot i f s e xi s t i n t he a m i no t e r m i nus of S H I P T he N pX Y m ot i f c a n be c om e t yr os i ne phos phor yl a t e d upo n a c t i va t i on, t hi s phos pho t y r os i ne t he n be c om e s a pos s i bl e bi ndi ng s i t e f or phos pho t yr os i ne bi nd i n g ( P T B ) dom a i n c ont a i ni ng pr o t e i ns i nc l udi ng S H C D O K 1 a nd D O K 2 ( 222 229 233 ) T he s e N pX Y m ot i f s ha ve a l s o be e n s how n t o bi nd t o t he p85 s ubuni t of P I 3K ( 234 236 ) I nt e r e s t i ngl y a l t hough S H I P c a n be phos phor yl a t e d t he f unc t i on of t he 5 i nos i t ol phos pha t a s e doe s not s e e m t o be r e l i a nt upon t hi s phos phor yl a t i on, i ndi c a t i ng t ha t t he r e c r u i t m e nt t ha t oc c ur s t h r ough S H I P s ot he r dom a i ns t o t he s i t e of i n t e r a c t i on m a y pl a y a m or e c r i t i c a l r ol e t ha n t he phos phor yl a t i on i t s e l f ( 237 ) W i t hi n t he C t e r m i nu s t he r e i s a pol yp r ol i ne r i c h m ot i f t ha t a l l ow s f or t he bi ndi ng o f p r ot e i ns t ha t c ont a i n a n S H 3 dom a i n ( 238 ) O t he r pos s i bl e r ol e s of S H I P i n s i gna l i ng ha ve r e c e nt l y be e n unc ove r e d. V a l de r r a m a C a r va j a l e t a l w e r e a bl e t o s how i nc r e a s i ng e vi de nc e t ha t S H I P doe s hydr ol yz e I P 4 i n v i v o ( 223 ) T hi s pr oc e s s c oul d e f f e c t t he f or m u l a t i on of hi ghe r num be r i nos i t ol phos pha t e s a nd t he r e f or e m a y hi nde r p r ot e i n s ynt he s i s ( 239) I t i s a l s o pos s i bl e t ha t P I 3 4 P 2 m a y a l s o a c t a s a s e c ond m e s s e nge r i n c e l l s l e a di ng t o t he a c t i va t i on o f P K B a nd A P 1 ( 24 0 242 )

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25 F i gu r e 4. S H I P s r ol e w i t h i n P I s i gn al i n g A d i a gr a m s how i ng t he pr o t e i ns i nvol ve d i n t he pr ogr e s s i on f r om P I t o P I ( 3, 4 5) P 3 a s w e l l a s t h e pos s i bl e i nt e r m e di a r i e s M a ny of t he s e di f f e r e nt P I s ha ve pos s i bl e e f f e c t on va r yi ng s i gna l i ng pa t hw a ys S om e of t he s e pos s i bl e pa t hw a ys a r e s how n i m m e di a t e l y a bove a nd be l ow t he i ndi v i dua l f o r m s I P 4 a nd ot he r I P s c a n be f o r m e d f r om t he hydr ol ys i s o f t he I P f r om t he di a c yl gl yc e r ol m ol e c ul e by a phos phol i pa s e SH I P i s of or m s S H I P 2 w a s i ni t i a l l y know n a s i nos i t ol pol yphos pha t e l i ke pr ot e i n1 ( I N P P L 1 ) ( 243) I n 1997 i t w a s i de nt i f i e d a s a 150 155kD a p r ot e i n w i t h 38% a m i no a c i d s e que nc e hom ol ogy t o S H I P 1 a nd t he r e f o r e t e r m e d S H I P 2 ( 244) T he m a j or i t y of t he 38%

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26 s e que nc e hom ol ogy e xi s t s w i t hi n t he phos pha t a s e dom a i n a s w e l l a s t he ot he r s i gna l i ng dom a i ns s o e ve n t hough t he s e que nc e hom ol ogy i s not hi gh t he f unc t i ona l hom ol ogy i s ( 238, 244 ) A l t hough t he s e t w o m ol e c ul e s s ha r e s i m i l a r dom a i ns t he y s e e m t o f unc t i on i n non ove r l a ppi ng m a nne r s e a c h s e gr e ga t i ng t o d i f f e r e nt s i gna l i ng s ys t e m s w i t h S H I P 2 ha vi ng a m or e ubi qui t ous e xpr e s s i on pa t t e r n i n c e l l s ot he r t ha n t hos e o f t he he m a t opoi e t i c s ys t e m ( 244 246) A not he r S H I P i s of or m w a s i de nt i f i e d i n hum a ns ( 210) a nd i n m i c e ( 247) t ha t w a s f ound t o l a c k t he S H 2 dom a i n T hi s i s of or m i s f o r m e d vi a a pr om o t e r t ha t e xi s t s w i t hi n i nt r on 5 / 6 ( 248 ) T hi s i s of or m ha s be e n s how n t o be e xpr e s s e d i n m u r i ne e m br yoni c s t e m c e l l s a nd H S C but not m or e di f f e r e nt i a t e d pr oge ny a nd ha s t hus be e n t e r m e d s S H I P ( 247, 24 8) SH I P K O M i c e D i f f e r e nt va r i a t i ons of t he S H I P knoc kout ( K O ) m ous e ha ve be e n c ons t r uc t e d de l e t i ng di f f e r e nt por t i ons o f t he pr ot e i n I n t he s t udi e s de t a i l e d i n t hi s di s s e r t a t i on t he S H I P K O m ous e c r e a t e d i n ou r l a b w a s ut i l i z e d ( 2 47) T hi s m ous e w a s c ons t r uc t e d by de l e t i ng t he pr om ot e r a nd pa r t o f t he f i r s t e xon of S H I P T hi s r e s ul t s i n no S H I P pr ot e i n be i ng pr e s e nt i n t he m ous e ( 216, 249, 250) S H I P i s e xpr e s s e d ve r y e a r l y i n t he de ve l opm e nt a l s t a ge s of t he e m br yo s pe c i f i c a l l y 7 5 D P C t hough t he S H I P nul l m ous e i s s t i l l vi a bl e ( 216 249 251) A l t hough t he m i c e a r e vi a bl e t he y do ha r bo r num e r ous phys i ol ogi c a l c ondi t i ons e s pe c i a l l y w i t hi n t he he m a t opoi e t i c c om pa r t m e nt P e r i phe r a l C D 8 T c e l l num be r s a r e s i gni f i c a nt l y r e duc e d w hi l e C D 4 num be r s a r e una f f e c t e d ( 249) M a s t c e l l de gr a nul a t i on i s hype r r e s pons i ve ( 252) O ne of t he m or e s e ve r e c ondi t i ons i s a m ye l opr ol i f e r a t i ve di s or de r l e a di ng t o i nc r e a s e d m ye l oi d pr oge ni t or s i n t he m a r r ow a nd

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27 m onoc yt e / m a c r opha ge s i n t he pe r i phe r y ( 249 253 ) T hi s d r a m a t i c i nc r e a s e i n m ye l oi d c e l l s l e a ds t o a n i nf i l t r a t i on o f a l ve ol a r m a c r opha g e s a nd r e s ul t s i n c ons ol i da t i on of t he l ungs w hi c h i s be l i e ve d t o be w ha t c a us e s t he m ous e s de a t h a t 8 t o 12 w e e ks of a ge ( 249) I n l i ne w i t h t hi s pr ol i f e r a t i on of m ye l oi d c e l l s t he r e i s a s i gni f i c a nt i nc r e a s e of m ye l oi d s uppr e s s or c e l l s T hi s i nc r e a s e of m ye l oi d s uppr e s s or c e l l s i s a bl e t o e f f e c t a de c r e a s e i n gr a f t ve r s us hos t di s e a s e ( 254, 255) SH I P i n N K C e l l s N um e r ous s t udi e s ha ve i ndi c a t e d a r ol e f or S H I P i n N K c e l l s T he f i r s t o f t he s e c onc e r ne d not N K c e l l s t he m s e l ve s but r a t he r t he I T I M dom a i n c ont a i ne d w i t hi n F c R f ound on B c e l l s A l t hough t he s e i ni t i a l s t udi e s di d not i de nt i f y a r ol e f or S H I P t he y m e r e l y i de nt i f i e d i t p r e s e nc e a t t he r e c e pt or ( 256) T he ne e d f or S H I P on s om e r e c e pt or s t o f unc t i on w a s l a t e r s how n w he n s t udyi ng t he r ol e of S hc i n N K c e l l m e di a t e d c yt ot oxi c i t y. I n t hi s s t udy i t w a s s how n t ha t S hc w he n bound t o L y49, i s a bl e t o r e c r ui t S H I P W he n S hc w a s m ut a t e d s o t ha t i t w a s una bl e t o be r e c r ui t e d t o t he N K R S H I P w a s a l s o no l onge r r e c r ui t e d. T hi s l a c k of S H I P r e s ul t e d i n a l os s of f unc t i on f o r t hi s r e c e pt or ( 257) O ur l a b ha s publ i s he d a s t udy i de nt i f yi ng a m or e c r uc i a l r ol e f o r S H I P i n t he N K c e l l I n t hi s s t udy i t w a s s h ow n t ha t i n t he S H I P K O m ous e t he N K R r e pe r t oi r e w a s s ke w e d w i t h L y49A a nd C / I be i ng ove r r e p r e s e nt e d. T hi s t he n l e a d t o a m uc h g r e a t e r phe nom e non i n w hi c h t he S H I P / m ous e w a s una b l e t o r e j e c t bone m a r r ow f r om a n M H C m i s m a t c he d m ous e T hi s w a s a t t r i but e d t o t he ove r r e pr e s e nt a t i on of t he s e L y49s ( 216 ) O t he r s t udi e s ha ve a l s o e xt e nde d our unde r s t a ndi ng of S H I P i n t he N K c e l l by i de nt i f yi ng a num be r of ot he r r e c e pt or s t ha t r e c r ui t S H I P w he n a c t i va t e d i nc l udi ng L y49B K L R G 1

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28 a nd 2B 4 ( 217) O t he r pos s i bl e r ol e s f or S H I P ha ve a l s o be e n hypot he s i z e d i n hum a n N K c e l l s T he a s s oc i a t i on of S H I P w i t h C D 16 ha s be e n s how n t o be ne c e s s a r y f or pr ope r r e c r ui t m e nt of t he r e c e pt or t o l i pi d r a f t s a nd t he r e f or e C D 16 m e di a t e d c yt ot oxi c i t y ( 219) I n a ddi t i on i t ha s be e n s how n t ha t a s ubs e t of hu m a n N K c e l l s ha ve e l e va t e d l e ve l s of S H I P a nd l ow e r e d pe r f o r i n l e ve l s T hi s s ubs e t of c e l l s ha s a ddi t i ona l l y be e n s how n t o be c om e f unc t i ona l l y a nne r gi c i n c hr oni c H I V i n f e c t i ons ( 258) A l t hough t he s pe c i f i c r ol e of S H I P i n m a ny of t he s e s t udi e s ha s not be e n i de n t i f i e d i t ha s s how n t ha t S H I P pl a ys a ke y r ol e w i t h num e r ous N K R a s w e l l a s r ol e s of t h e N K c e l l T h i s s t udy w i l l a t t e m pt t o bui l d of f of t he s e s t udi e s a nd de l ve de e pe r i nt o t he s pe c i f i c m ol e c ul a r r ol e S H I P pl a ys i n t he N K c e l l

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29 C H A P T E R 2 D om i na nc e by a n M H C I nde pe nde nt I nhi bi t o r y R e c e pt or C om pr om i s e s N K K i l l i ng of C om pl e x T a r ge t s I n t r od u c t i on N K c e l l s di s t i ngui s h nor m a l c e l l s f r om t hos e a l t e r e d by i nf e c t i on s t r e s s or t r a ns f or m a t i on vi a i nhi bi t or y r e c e pt or s t ha t de t e c t s e l f l i ga nds a nd a c t i va t i ng r e c e pt or s t ha t r e c ogni z e M H C l i ke l i ga nds e xpr e s s e d by t um or c e l l s vi r a l l y i n f e c t e d c e l l s or c e l l s w i t h D N A da m a ge ( 112 259 261) U nl i ke t he i r B a nd T c e l l c ount e r pa r t s N K c e l l s ge ne r a t e r e pe r t oi r e d i ve r s i t y t hr ough va r i e ga t e d a n d ove r l a ppi ng e xpr e s s i on of i nhi bi t o r y a nd a c t i va t i ng r e c e pt or s R e c ogni t i on of s e l f by N K c e l l s i s l a ye r e d a nd i nvol ve s t he r e c ogni t i on of M H C c l a s s I m o l e c ul e s ( 50) o r non M H C l i ga nds l i ke C D 48 a nd O c i l / C l r b vi a t he non c l a s s i c a l r e c e pt or s 2B 4 a nd N K R P 1 D r e s pe c t i ve l y ( 62 131 262 ) T hi s pa t t e r n of r e c e pt or di s t r i but i on c r e a t e s a n N K c om pa r t m e nt c om pos e d of d i f f e r e nt c e l l s ubs e t s e a c h pos s e s s i ng va r yi ng de gr e e s of r e s pon s i ve ne s s t o s e l f non s e l f a nd a l t e r e d s e l f I n t hi s m a nne r t he N K c om pa r t m e nt ge ne r a t e s a s uf f i c i e nt nu m be r o f c e l l s ubs e t s c a pa bl e of r e s pondi ng t o i nf e c t e d, t um or or a l l oge ne i c c e l l s w hi l e r e t a i ni ng t ol e r a nc e t o nor m a l hos t c e l l s

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30 2B 4 i s a m e m be r o f t he S L A M r e l a t e d r e c e pt or s t h a t i nc l ude s i gna l l ym phoc yt e a c t i va t i on m ol e c ul e ( S L A M ) C D 2 l i ke r e c e pt or a c t i va t i ng c yt ot oxi c c e l l ( C R A C C ) N K T B A g ( N T B A ) a nd C D 48 ( 263) M a ny o f t he s e r e c e pt or s f unc t i on t hr ough i nt e r a c t i ons w i t h ot he r f a m i l y m e m be r s i nc l udi ng 2B 4, w hi c h i nt e r a c t s w i t h C D 48. 2B 4 i s e xpr e s s e d ubi qui t ous l y on N K c e l l s a s w e l l a s ot he r c e l l s i n t he he m a t o poi e t i c s ys t e m ( 135) S t udi e s or i gi na l l y i ndi c a t e d a n a c t i va t i ng r o l e f or 2B 4, a l t hough m os t of t he s e s t udi e s w e r e pe r f or m e d i n v i t r o ut i l i z i ng a nt i body l i ga t i on e xpe r i m e nt s ( 132 133 264 ) A m or e de f i n i t i ve r o l e f o r 2B 4 w a s r e c e nt l y de m o ns t r a t e d t hr ough t he ge ne r a t i on o f a 2B 4 m ut a nt m ous e i n w hi c h a n i nhi bi t or y r ol e w a s i de nt i f i e d ( 139 ) A pot e nt i a l dua l r o l e f or 2B 4 i n N K f unc t i on c oul d be e xpl a i ne d by i t s a bi l i t y t o r e c r ui t di f f e r e nt a da pt or pr ot e i ns i nc l udi ng S A P a s w e l l a s E A T 2 ( 139 14 2) A l t houg h t he s i gna l i ng pa t hw a ys t ha t c ont r ol r e s pons e s f ol l ow i ng 2B 4 l i ga nd e nga g e m e nt ha ve ye t t o be de f i ne d i n t he i r e nt i r e t y, s e ve r a l pa r t i c i pa t i ng s i gna l i ng c om pone nt s ha ve be e n de f i ne d i nc l udi ng S A P E A T 2 F yn T S H P 1 P I 3K a nd S H I P ( 133, 139 14 2) W e ha ve pr e vi ous l y s how n t h a t S H I P i s c r i t i c a l f or m a i nt e na nc e of N K R r e pe r t oi r e di ve r s i t y i n t he pe r i phe r a l N K c om pa r t m e nt ( 216) W e ha ve f u r t he r de f i ne d t he r ol e o f S H I P i n t he N K R r e pe r t oi r e by pl a c i ng t he S H I P m ut a t i on on a de f i ne d ge ne t i c ba c kgr ound ( C 57B L 6/ J ) T he pe r i phe r a l N K c om pa r t m e nt of t he s e S H I P / m i c e ( C 57B L 6/ J ) i s di s r upt e d w i t h a p r of ound unde r r e p r e s e nt a t i on of i nhi bi t o r y N K R s pe c i f i c f or M H C c l a s s I O nl y t w o N K R 2B 4 a nd N K R P 1D a r e f ound t o be ove r e xpr e s s e d or ove r r e pr e s e nt e d i n N K c e l l s of S H I P / m i c e I nt r i g ui ngl y, bot h of t he s e r e c e pt or s a r e s pe c i f i c f or M H C i nde pe nde nt l i ga nds I n t h i s s t udy w e de m ons t r a t e t ha t S H I P

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31 de f i c i e nc y c a us e s de r e gul a t i on of 2B 4 s ur f a c e e xpr e s s i on a nd s i gna l i ng s uc h t ha t c yt ol ys i s of c om pl e x t a r ge t s i s c om p r om i s e d. R E S U L T S SH I P / N K R e pe r t oi r e W e ha ve pr e vi ous l y s how n t ha t i n t he S H I P de f i c i e nt N K c e l l t he r e i s a r e c e pt or bi a s t ow a r ds M H C s pe c i f i c i nhi bi t or y r e c e pt or s s p e c i f i c a l l y L y49 A a nd C / I T he s e i ni t i a l s t udi e s w e r e pe r f o r m e d i n a C 57B L / 6/ 129 m i xe d ba c kgr ound. S i nc e t he s e i ni t i a l s t udi e s w e ha ve c r os s e d t he S H I P / m ous e t o a f ul l y de f i ne d B l 6 ba c kgr ound W i t h t he s e m i c e w e ha ve r e a na l yz e d t he N K R r e pe r t oi r e a nd f ound a pr o f ound a nd s i gni f i c a nt dow n r e gul a t i on of a l l N K R e xc e pt 2B 4 a nd N K R P 1D ( F i g. 5 )

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32 F i gu r e 5. N K R r e p e r t oi r e A ) R e pr e s e nt a t i ve hi s t ogr a m s f or t he di f f e r e nt N K R a na l yz e d a f t e r ga t i ng on N K 1. 1 + L i n s pl e noc yt e s of 6 8 w e e k ol d S H I P / m i c e a nd t he i r W T l i t t e r m a t e s ( L i n pa ne l : I gM C D 3 T c R b G r 1 C D 11c ) R e d hi s t ogr a m s a r e f or S H I P / N K c e l l s w hi l e bl a c k hi s t ogr a m s a r e f o r W T l i t t e r m a t e c ont r ol s I n o r de r t o e s t i m a t e t he pe r c e nt a ge of N K R + c e l l s i n t he N K c om pa r t m e nt pos i t i ve N K R ga t e s w e r e s e t a t 95% of N K 1. 1 + L i n c e l l s s t a i ni ng pos i t i ve f or a n i s ot ype c ont r ol s t a i n pe r f or m e d on a n e qua l m i xt u r e of nul l a nd W T s pl e noc yt e s B ) R e pr e s e nt a t i on of i ndi vi dua l N K R i n s pl e ni c S H I P / N K c e l l s a f t e r no r m a l i z a t i on t o W T T he % o f no r m a l = ( % N K R + S H I P / / % N K R + S H I P + / + ) x 100 f or e a c h i ndi c a t e d N K R F o r 2B 4, % nor m a l w a s c a l c ul a t e d i n t he s a m e m a nne r e xc e pt t ha t M F I w a s us e d r a t he r % N K R + B l ue r e d a nd bl a c k ba r gr a phs r e p r e s e nt % no r m a l va l ue s t ha t a r e s i gni f i c a nt l y l ow e r hi ghe r a nd unc ha nge d i n t he S H I P / N K c om pa r t m e nt a s c om pa r e d t o W T r e s pe c t i ve l y. 2B 4 E x pr e s s i on L e v e l s I n or de r t o de t e r m i ne t he e f f e c t o f S H I P de f i c i e nc y on 2B 4 e xpr e s s i on, w e e xa m i ne d va r i ous s t a ge s of N K c e l l m a t u r a t i on a n d a c t i va t i on ( F i g. 6) O u r i ni t i a l a na l ys i s i nc l ude d bot h m a t ur e s pl e ni c N K c e l l s a s w e l l a s i m m a t ur e bone m a r r ow N K c e l l s A na l ys i s of N K c e l l s a t t he s e s t a ge s of m a t u r a t i on s how e d t ha t 2B 4 i s ove r e xpr e s s e d on t he s ur f a c e of S H I P / N K c e l l s a s c o m pa r e d t o N K c e l l s f r om W T l i t t e r m a t e s ( F i g 6A B ) W e f ur t he r e xa m i ne d 2B 4 s t a t us ut i l i z i ng a c t i va t e d N K c e l l s F or t he s e e xpe r i m e nt s w e a na l yz e d bot h i n v i v o po l yi nos i ni c a c i d ( pol y( I : C ) ) a c t i va t e d N K c e l l s a nd i n v i t r o a c t i va t e d N K c e l l s c ul t ur e d f or 7 da ys i n t he p r e s e nc e of I L 2.

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33 C ons i s t e nt w i t h our a na l ys i s of f r e s hl y i s ol a t e d N K c e l l s i n v i v o ( F i g 6C ) a nd i n v i t r o ( F i g. 6D ) a c t i va t e d S H I P / N K c e l l s a l s o e xhi bi t i n c r e a s e d s ur f a c e de ns i t y of 2B 4 a s c om pa r e d t o W T c ont r ol s T hus S H I P i s r e qui r e d t o m a i nt a i n no r m a l e xpr e s s i on of 2B 4 on t he c e l l s ur f a c e i ndi c a t i ng S H I P no t onl y r e gul a t e s e xpr e s s i on of N K R f or M H C l i ga nds but a l s o N K R f o r M H C i nde pe nde nt l i ga n ds M or e ove r S H I P pe r f or m s t hi s r ol e a t m ul t i pl e s t a ge s of N K de ve l opm e nt a nd di f f e r e n t i a t i on. I n a ddi t i on t o t he s e c e l l t ype s w e c r e a t e d S H I P / a nd W T c hi m e r i c m i c e i n or de r t o de t e r m i ne i f t he up r e gul a t i on of 2B 4 i n t he S H I P / N K c e l l w a s due t o c e l l e xt r i ns i c e f f e c t s ( F i g 6 E ) A s be f or e w e s e e t ha t 2B 4 s ur f a c e de ns i t y r e m a i ns e l e va t e d i n t he S H I P / m i c e F i gu r e 6. 2B 4 s t at u s i s al t e r e d i n S H I P / N K c e l l s R e pr e s e nt a t i ve ove r l a ys of 2B 4 hi s t ogr a m s a f t e r ba c k ga t i ng on N K 1 1 + L i n c e l l s B a r gr a phs r e p r e s e nt m e a n f l uor e s c e nc e i nt e ns i t y of a t l e a s t t hr e e s e pa r a t e a ni m a l s ( *p< 0. 05 n= 3 S t ude nt s t w o t a i l e d T t e s t ) S H I P / ( R e d) W T ( B l ue ) A ) I m m a t u r e B M N K c e l l s B ) M a t ur e s pl e ni c N K c e l l s C ) I n v i v o pol y I C s t i m ul a t e d N K c e l l s D ) I n v i t r o I L 2 s t i m ul a t e d L A K c e l l s E ) N K c e l l s f r om a S H I P / W T c hi m e r a

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34 N K G 2D m e di at e d c y t ol y s i s i s c om pr om i s e d i n SH I P / m i c e 2B 4 ha s r e c e nt l y be e n s how n t o f unc t i on a s a n i m p or t a nt i nhi bi t o r y r e c e pt or i n v i v o ( 131) T hus w e s pe c ul a t e d t ha t t he i nc r e a s e d s ur f a c e de ns i t y of t hi s i nh i bi t or y r e c e pt or m i ght s ke w t he ba l a nc e of i nhi bi t or y a nd a c t i va t i ng s i gna l s r e c e i ve d by S H I P / N K c e l l s a nd t he r e by a l t e r ke y c yt ol yt i c f unc t i ons T o e xa m i ne t he i m pa c t of 2B 4 ove r e xpr e s s i on on S H I P / N K f unc t i on, w e e xa m i ne d N K c yt ol ys i s of t um o r c e l l s m e di a t e d by t he a c t i va t i ng r e c e pt or N K G 2D T o e xa m i ne w he t he r c yt ol ys i s of t um or t a r ge t s t ha t e xpr e s s t he N K G 2D l i ga nd R a e 1 a r e c om pr om i s e d i n t he S H I P / N K c om pa r t m e nt w e m a gne t i c a l l y e nr i c he d s pl e ni c N K c e l l s a nd c ul t ur e d t he m f o r 7 da ys i n t he pr e s e nc e of I L 2 t o ge ne r a t e l ym phoki ne a c t i v a t e d ki l l e r ( L A K ) c e l l s I nt r i gui ngl y, w e f i nd t ha t N K c e l l r e c ove r y i n S H I P / L A K c ul t ur e s i s s i gni f i c a nt l y be t t e r t ha n t ha t of W T c ul t ur e s pr e pa r e d i n a s i m i l a r m a nne r ( p< 0 05) s ugge s t i ng t ha t S H I P de f i c i e nc y m a y e nha nc e c yt oki ne s t i m ul a t e d N K c e l l s ur vi va l a nd/ or pr ol i f e r a t i on e x v i v o ( F i g 7A ) F l ow c yt om e t r i c a na l ys i s of t he s e 7 da y L A K s r e v e a l e d t ha t s ur f a c e e xpr e s s i on of N K G 2D on a c t i va t e d N K c e l l s f r om S H I P / m i c e i s c om pa r a bl e t o t ha t of W T c ont r o l s pr e pa r e d i n a n i de nt i c a l m a nne r ( F i g 7B ) T he s ur f a c e de ns i t y of 2B 4 r e m a i ne d e l e va t e d i n S H I P / N K c e l l s a s s how n a bove ( F i g 6D ) T he c yt ol yt i c a c t i vi t y of t he a c t i va t e d N K c e l l s w a s t he n de t e r m i ne d i n a s t a nda r d 4 hou r c hr om i um r e l e a s e a s s a y a ga i ns t R M A c e l l s e xpr e s s i ng t he N K G 2D l i ga nd R a e 1. D e s pi t e e qua l s ur f a c e e xpr e s s i on of N K G 2D on S H I P / a nd W T L A K c e l l s w e f i nd t h a t c yt ol ys i s of R M A R a e 1 t r a ns f e c t a nt s by S H I P / N K c e l l s i s pr of oundl y c om pr om i s e d r e l a t i ve t o W T N K c e l l s a t a l l E : T r a t i os t e s t e d ( F i g. 7C ) I n f a c t onl y a t t he h i ghe s t E : T r a t i o, 60: 1 w a s c yt ol ys i s of R a e 1 + R M A

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35 c e l l s by S H I P / L A K c e l l s s i gni f i c a n t l y hi ghe r t ha n ba c kgr ound c yt ol ys i s obs e r ve d f or pa r e nt a l R M A c e l l s N o s i gni f i c a nt ki l l i ng of pa r e nt a l R M A c e l l s t ha t l a c k t he R a e 1 a nt i ge n, w a s s e e n f or e i t he r S H I P / or W T N K c e l l s c onf i r m i ng t he s pe c i f i c i t y of t hi s a s s a y f or t he N K G 2D l i ga nd R a e 1. T he f a c t t ha t N K G 2D e xpr e s s i on l e ve l s a r e c om pa r a bl e i n S H I P / a nd W T L A K c e l l s w hi l e N K G 2D m e di a t e d c yt ol ys i s i s pr of oundl y c om p r om i s e d i n S H I P / N K c e l l s s ugge s t s t ha t hypor e s pons i ve ne s s i n S H I P / N K c e l l s c oul d be due t o i nc r e a s e d e xpr e s s i on a nd/ or i nhi bi t o r y s i gna l s f r o m 2B 4 e nga gi ng i t s l i ga nd, C D 48, w hi c h i s e xpr e s s e d on t he s ur f a c e of bot h R M A a nd R a e 1 + R M A c e l l s ( F i g 7E ) R e s t or at i on of N K G 2D m e di at e d c y t ol y s i s T o e xa m i ne w he t he r a l t e r e d 2B 4 s i gna l i ng w a s c a us i ng t hi s hypor e s pons i ve ne s s w e t e s t e d w he t he r a nt a goni z i ng t he 2B 4 C D 48 i nt e r a c t i on r e s t or e s N K G 2D m e di a t e d ki l l i ng. I nc uba t i on of t a r ge t s w i t h a n a nt i C D 48 a nt i body w a s a bl e t o r e s t or e S H I P / L A K ki l l i ng t o W T l e ve l s ( F i g. 7D ) I n t h r e e i nde pe nde nt s t a nda r d 4 hour c hr om i um r e l e a s e a s s a ys w he r e w e t e s t e d ki l l i ng of R a e 1 + R M A t r a ns f e c t a nt s i n t he pr e s e nc e or a bs e nc e of a nt i C D 48, w e ha ve r e pe a t e dl y obs e r ve d a s t a t i s t i c a l l y s i gni f i c a nt e nha nc e m e nt of S H I P / ki l l i ng. A l t hough s om e i n c r e a s e i n W T ki l l i ng due t o C D 48 bl oc ki ng oc c ur r e d, i t w a s d r a m a t i c a l l y l e s s t ha n t h a t obs e r ve d w i t h C D 48 bl oc ka de i n S H I P / L A K c yt ol ys i s a s s a ys I n f a c t i n a l l but on e E : T r a t i o 20: 1 t he ki l l i ng by S H I P / L A K c e l l s w a s not s i gni f i c a nt l y di f f e r e nt t ha n t ha t of ki l l i ng by W T L A K pe r f o r m e d i n t he pr e s e nc e of C D 48 bl oc ka de T he a bi l i t y t o r e s t or e S H I P / L A K c yt ot oxi c i t y a ga i ns t

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36 R a e 1 + R M A t a r ge t s by bl oc ki ng t he 2B 4 C D 48 i nt e r a c t i on s ugge s t s t ha t 2B 4 ha s a dom i na nt i nhi bi t or y r ol e i n t he S H I P / N K c om pa r t m e nt F i gu r e 7. C o m p r om i s e d N K G 2D m e d i at e d c yt o l ys i s of R ae 1 + t u m or t ar ge t s b y S H I P / N K c e l l s an d r e s t or at i on b y C D 48 b l oc k ad e A ) M a gne t i c a l l y pur i f i e d N K c e l l s w e r e pl a t e d a t 2 000, 000c e l l s / m L i n t he pr e s e nc e of I L 2 f or 7 da ys C e l l s w e r e ha r ve s t e d a t da y 7 a nd t he r e s ul t i ng pe r c e nt a ge of c e l l s r e c ove r e d f r om f our s e pa r a t e e xpe r i m e nt s a r e s how n. ( *p< 0. 05 N = 4 S t ude nt s t w o t a i l e d T t e s t ) B ) N K G 2D s t a t us of N K 1. 1 + C D 3 7 da y L A K c e l l s R e pr e s e nt a t i ve hi s t ogr a m s s how i ng t he N K G 2D s t a t us of S H I P / ( bl ue ) a nd W T ( g r e e n) L A K c e l l s a r e s how n. R e d hi s t ogr a m i ndi c a t e s i s ot ype c ont r ol C ) S t a nda r d 4h r c hr om i u m r e l e a s e a s s a ys w e r e pe r f or m e d w i t h S H I P / a nd W T L A K c e l l s R M A c e l l s w i t h a nd w i t hout R a e 1 t r a n s f e c t a nt s w e r e us e d a s t a r ge t s P e r c e nt l ys i s i s i ndi c a t e d on t he l e f t a xi s a nd t he E : T r a t i os a c r os s t he bot t om a xi s *p< 0. 05 f or c yt ol ys i s of R a e 1 + R M A c e l l s by W T L A K c e l l s c om pa r e d t o S H I P / L A K c e l l s + p< 0. 05 f or c yt ol ys i s of R M A c e l l s c om pa r e d t o R a e 1 + R M A c e l l s by S H I P / L A K c e l l s

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37 D ) C yt ol ys i s of R a e 1 + R M A c e l l s w i t h a nd w i t hou t bl oc ki ng of C D 48 on t a r ge t c e l l s i n a s t a nda r d 4hr c hr om i u m r e l e a s e a s s a y. *p< 0. 05 f o r c yt ol ys i s of bl oc ke d R a e 1 + R M A c e l l s by S H I P / L A K c e l l s c om pa r e d t o W T L A K c e l l s + p< 0. 05 f or t he c yt ol ys i s of bl oc ke d R a e 1 + R M A c e l l s ve r s us unbl oc ke d R a e 1 + R M A c e l l s by S H I P / L A K c e l l s A l l c yt ot oxi c i t y e xpe r i m e nt s w e r e done i n t r i pl i c a t e a n d a r e r e p r e s e nt a t i ve of t hr e e or m or e e xpe r i m e nt s E ) C D 48 s t a t us of R M A a nd R a e 1 + R M A c e l l s C om pr om i s e d B M r e j e c t i on P r e vi ous l y w e f ound t ha t t he N K r e pe r t o i r e di s r upt i on obs e r ve d i n S H I P / m i c e on a m i xe d 129 S v/ B L 6 ba c kgr ound l e d t o a n i na bi l i t y t o r e j e c t H 2 d a nd H 2 s B M gr a f t s t ha t a r e c om pl e t e l y M H C m i s m a t c he d. W e a t t r i but e d e ngr a f t m e nt i n 129/ B L 6 S H I P / m i c e t o ove r r e pr e s e nt a t i on of L y49A a nd C ( 216) H ow e ve r L y49A a nd L y49C a r e not ove r r e pr e s e nt e d i n S H I P / m i c e on a C 57B L / 6 ba c kgr ound s ugge s t i ng t ha t r e j e c t i on of a l l oge ne i c B M gr a f t s m i ght not be c om p r om i s e d i n S H I P / m i c e on t hi s de f i ne d ba c kgr ound. A l t hough t he s e S H I P / ( B L 6) m i c e m i ght r e m a i n pe r m i s s i ve f or e ngr a f t m e nt of M H C m i s m a t c he d B M gr a f t s ow i n g t o t he c om pr om i s e d N K G 2D m e di a t e d c yt ol ys i s w e obs e r ve d a bove T hi s a ppe a r s t o be t he c a s e a s S H I P / m i c e on a C 57B L / 6 ba c kgr ound a r e s t i l l pe r m i s s i ve f or e ngr a f t m e nt of B M f r om s e ve r a l di f f e r e nt donor s w i t h f ul l M H C m i s m a t c he s ( F i g. 8) I ni t i a l l y, w e t r a ns pl a nt e d S H I P / ( B L 6) hos t s w i t h H 2 d B A L B / C B M a nd m e a s ur e d a c ut e e ngr a f t m e nt by t he s pl e ni c I U dR a s s a y us e d pr e vi ous l y ( 216) A s w a s obs e r ve d i n 129/ B L 6 S H I P / m i c e ( 216 ) w e f i nd t ha t B A L B / C H 2 d B M e ngr a f t s t he S H I P / ( B L 6) c ohor t but i s r e j e c t e d by t he i r W T l i t t e r m a t e s S ubs e que nt l y w e t e s t e d e ngr a f t m e nt o f B M f r o m a va r i e t y o f ot he r donor s w i t h f ul l M H C

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38 c l a s s I m i s m a t c he s ( H 2 p H 2 r H 2 f a nd H 2 u ) not a na l yz e d pr e vi ous l y, a nd f ound t ha t S H I P / ( B L 6 ) m i c e a r e a l s o pe r m i s s i ve f or e ngr a f t m e nt by B M f r o m t he s e donor s ( F i g. 8) F i gu r e 8. A l l oge n e i c B M r e j e c t i on as s ay S pl e ni c I U dR upt a ke i n F 9/ 10xC 57B L / 6 S H I P / a nd W T r e c i pi e nt s t r a ns pl a nt e d w i t h H 2 d d onor B M c e l l s F 6xC 57B L / 6 S H I P / a nd W T r e c i pi e nt s t r a n s pl a nt e d w i t h H 2 f H 2 p H 2 u o r H 2 r donor B M c e l l s R e d ba r gr a phs r e pr e s e nt S H I P / r e c i pi e nt s t r a ns pl a nt e d w i t h a l l oge ne i c donor B M w hi l e bl ue ba r gr a phs r e pr e s e nt W T r e c i pi e nt s t r a ns pl a nt e d w i t h a l l oge ne i c donor B M C B l a c k ba r gr a phs r e pr e s e nt B L 6 r e c i pi e nt s t r a ns pl a nt e d w i t h s ynge ne i c B L 6 donor B M C ( *p< 0 05)

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39 C om pr om i s e d c y t ol y s i s of L y 49H t ar ge t s T he l i ga nd f or L y49H ha s be e n i de nt i f i e d a s t he C M V e nc ode d m e m br a ne pr ot e i n m 157 ( 80, 81) S i nc e S H I P / N K c e l l ki l l i ng of t a r ge t s t ha t e xpr e s s l i ga nds f or N K G 2D i s c om pr om i s e d, w e pos t ul a t e d t ha t ki l l i ng of m 15 7 + t a r ge t s m i ght be s i m i l a r l y di s a bl e d. T o t e s t t hi s w e e xa m i ne d t he a bi l i t y o f L A K c e l l s f r om S H I P / a nd W T m i c e t o ki l l B a F 3 m 157 + t r a ns f e c t a nt s ( F i g 9 ) A s w i t h N K G 2D w e f i nd t ha t e x v i v o a c t i va t i on o f S H I P / N K c e l l s w i t h I L 2 r e s t or e s L y49H t o a s ur f a c e de ns i t y e s s e nt i a l l y i de nt i c a l t o t ha t i n W T c ont r ol s ( F i g 9A ) S t a nda r d 4 hour c h r om i um r e l e a s e a s s a y s w e r e t he n pe r f or m e d a t E : T r a t i os of 60, 20 a nd 6: 1 T he S H I P / L A K c ul t u r e s s how e d s e ve r e l y di m i ni s he d c yt ol ys i s of m 157 + t a r ge t s a t a l l E : T r a t i os t e s t e d ( F i g. 9B ) K i l l i ng i n t hi s a s s a y w a s c onf i r m e d a s be i ng s pe c i f i c f or t he m 15 7 a nt i ge n a s no c yt ol ys i s of B a F 3 pa r e nt a l c e l l s w a s obs e r ve d ( F i g. 9B ) R e s t or at i on of L y 49H m e di at e d c y t ol y s i s G i ve n t ha t 2B 4 s i gna l i ng c om p r om i s e s N K G 2D m e di a t e d ki l l i ng by S H I P / N K c e l l s w e a l s o e xa m i ne d w he t he r t hi s m i ght be t he c a s e f or t he ki l l i ng of vi r a l l i ga nd pos i t i ve t a r ge t s m e di a t e d by L y49H A s w a s done f or N K G 2D w e m e a s ur e d N K c yt ol ys i s us i ng a s t a nda r d 4 hour c hr om i um r e l e a s e a s s a y i n t he pr e s e nc e a nd a bs e nc e of a nt i C D 48. I n t h r e e s e pa r a t e e xpe r i m e nt s i nc uba t i on of t he m 157 + B a F t a r ge t s w i t h a nt i C D 48 pr i or t o ki l l i ng s i gn i f i c a nt l y r e s t or e d S H I P / L A K ki l l i ng ( F i g. 9C ) T he s i gni f i c a nt e nha nc e m e nt of W T ki l l i ng by bl oc ki n g of C D 48 w a s a l s o s e e n a t t he 20: 1

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40 E : T r a t i o, but i s l ow e r t ha n t ha t obs e r ve d f or a l l m 157 + c yt ol ys i s a s s a ys pe r f or m e d w i t h S H I P / N K c e l l s ( F i g. 9C ) T hus a s w i t h ki l l i ng o f t um or t a r ge t s ki l l i ng of t a r ge t s t ha t e xpr e s s a vi r a l l i ga nd f or a n N K a c t i va t i ng r e c e pt or i s a l s o c om pr om i s e d by 2B 4 i nhi bi t or y s i gna l i ng f u r t he r de m ons t r a t i ng 2B 4 i s a dom i na nt i nhi bi t o r y r e c e pt or i n t he S H I P / N K c om pa r t m e nt F i gu r e 9. C o m p r om i s e d L y49H m e d i at e d c yt ol ys i s of C M V m 157 + vi r al t ar ge t s b y S H I P / N K c e l l s an d r e s t or at i on b y C D 48 b l oc k ad e A ) L A K c e l l s w e r e i nc uba t e d w i t h a nt i N K 1. 1 C D 3, a nd L y49H H i s t ogr a m s a r e r e pr e s e nt a t i ve of t he s t a t us of L y49H i n W T ( gr e e n) a nd S H I P / ( bl ue ) L A K c e l l s a s c om pa r e d t o t he i s ot ype c ont r ol ( r e d) B ) C yt ol ys i s of B a F 3 pa r e nt a l c e l l s a nd B a F 3 t r a ns f e c t a nt s e xpr e s s i ng t he C M V m 157 l i ga nd by I L 2 a c t i va t e d L A K c ul t ur e s f r om S H I P / m i c e or W T l i t t e r m a t e s i n a s t a nda r d 4hr c hr om i um r e l e a s e a s s a y. P e r c e nt l ys i s i s on t h e l e f t a xi s a nd E : T r a t i os a c r os s t he bot t om a xi s *p< 0 05 f o r c yt ol ys i s of m 157 + B a F t a r ge t c e l l s by W T L A K c om pa r e d t o S H I P / L A K + p< 0. 05 f or c yt ol ys i s of m 157 + B a F c e l l s c om pa r e d t o B a F c e l l s a l one by S H I P / L A K C ) C yt ol ys i s of m 157 + B a F c e l l s w i t h a nd w i t hout bl oc ki ng of C D 48 on

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41 t a r ge t c e l l s i n a s t a nda r d 4hr c hr om i um r e l e a s e a s s a y. + p< 0. 05 F o r c yt ol ys i s of bl oc ke d ve r s us unbl oc ke d m 157 + B a F c e l l s by S H I P / L A K c e l l s *p< 0 05 F o r c yt ol ys i s of bl oc k e d m 157 + B a F c e l l s by W T L A K c e l l s c om pa r e d t o S H I P / L A K c e l l s A l l c yt ot oxi c i t y e xpe r i m e nt s w e r e done i n t r i pl i c a t e a n d a r e r e p r e s e nt a t i ve of t w o o r m o r e e xpe r i m e nt s N K R P 1D m e di at e d i nhi b i t i on O ur r e s t or a t i on o f c yt ot oxi c i t y w i t h a C D 48 bl oc k a de pr ovi de s s t r ong e vi de nc e t ha t 2B 4 i s t he dom i na nt i nhi bi t o r y r e c e pt or A l t h ough gi ve n t ha t N K R P 1D w a s a l s o ove r e xpr e s s e d on S H I P / N K c e l l s w e pe r f o r m e d C l r b a nt i body bl oc ki ng e xpe r i m e nt s t o de t e r m i ne i f t hi s c oul d r e s t or e S H I P / N K c yt ot oxi c i t y. I n t he s e e xpe r i m e nt s w e s e e no i m pr ove m e nt w i t h C l r b bl oc ka de a l one a s w e l l a s no i m pr ove m e nt w he n 2B 4 a nd C l r b w e r e bl oc ke d t oge t he r ( F i g. 10) F i gu r e 10. N K R P 1D C l r b b l oc k ad e C yt ol ys i s of R M A or R M A R a e 1 + c e l l by S H I P / L A K c e l l s a t a r a t i o o f 20: 1 C yt ol ys i s w a s pe r f o r m e d e i t he r w i t h L A K c e l l s a l one i n

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42 t he pr e s e nc e of C D 48 bl oc ka de C l r b bl oc ka de or C D 48 a nd C l r b bl oc ka de pe r f o r m e d i n c om bi na t i on. D I S C U S S I O N I n t hi s s t udy w e ha ve s how n t ha t S H I P / N K c e l l s e xhi bi t ove r e xpr e s s i on of 2B 4 t hr oughout a l l s t a ge s of N K m a t ur a t i on a nd a c t i va t i on. W e ha ve a l s o s how n t ha t 2B 4 i nhi bi t or y s i gna l i ng i n S H I P / N K c e l l s r e p r e s s e s k i l l i ng of c om pl e x t a r ge t s w he r e e f f e c t i ve r e c ogni t i on a nd ki l l i ng r e qui r e s i nt e g r a t i o n of bot h a c t i va t i ng a nd i n hi bi t or y s i gna l s O ur f unc t i ona l s t udi e s i ndi c a t e i nc r e a s e d 2B 4 i nhi bi t or y s i gna l i ng di s r upt s t hi s ba l a nc e s uc h t ha t ke y a c t i va t i ng r e c e pt or s l i ke N K G 2D a nd L y49H a r e una bl e t o e f f e c t i ve l y pr om ot e c yt ol ys i s A l t hough t he m ol e c ul a r m e c ha ni s m s r e s pons i bl e f or t hi s i m ba l a nc e r e m a i n t o be de f i ne d, w e pr opos e t w o h ypot he s e s t o e xpl a i n t hi s a l t e r e d f unc t i on. I n our f i r s t hypot he s i s w e pr opos e t ha t t he qua nt i t a t i ve di f f e r e nc e i n 2B 4 e xpr e s s i on be t w e e n S H I P / a nd W T N K c e l l s l e a ds t o a n i nc r e a s e i n t he ba s a l l e ve l of 2B 4 i nhi bi t or y s i gna l s r e c e i ve d by a n N K c e l l a nd t hus a l t e r s t he ba l a nc e of a c t i va t i ng a nd i nhi bi t or y s i gna l s f or c i ng t he c e l l t ow a r ds hyp or e s pons i ve ne s s ( F i g. 11B ) A n N K c e l l r e c e i ve s a va r i e t y of a c t i va t i ng a nd i nhi bi t or y i nput s f r om e xt e r na l t a r ge t s A ba l a nc e of t he s e i nhi bi t or y a nd a c t i va t i ng s i gna l s m us t be a c hi e ve d i n or de r f or t he c e l l t o m a i nt a i n t ol e r a nc e t o s e l f I n o r de r f or a n N K c e l l t o c a r r y out c yt ol ys i s t he a c t i va t i ng r e c e pt or s m us t be i nt e gr a t e d t o ove r c om e i nhi bi t or y s i gna l s T hus i nc r e a s e d ba s a l i nhi bi t or y s i gna l s f r om ubi qui t ous 2B 4 C D 48 i nt e r a c t i ons c oul d c om pr om i s e t he a bi l i t y

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43 of a S H I P / N K c e l l t o r e s pond e f f i c i e nt l y t o a c t i va t i ng s i gna l s f r om e i t he r N K G 2D o r L y49H I n t ha t c a s e t he r ol e o f S H I P i s t o l i m i t t h e s ur f a c e e xpr e s s i on of 2B 4 t o a l e ve l t ha t doe s not i nt e r f e r e w i t h nor m a l a c t i va t i ng r e c e p t or s e f f e c t i ng N K c yt ol yt i c f unc t i on. A l t e r na t i ve l y, w e p r opos e t ha t t he r e c oul d a l s o be a qua l i t a t i ve c ha nge i n t he 2B 4 i nhi bi t or y s i gna l i ng i n S H I P / N K c e l l s s uc h t ha t e a c h 2B 4 r e c e pt or de l i ve r s a m or e pot e nt ne ga t i ve s i gna l ( F i g 11C ) A l t hough t he pr e c i s e m ol e c ul a r m e c ha ni s m s of 2B 4 s i gna l i ng ha ve not be e n f u l l y e l uc i da t e d, ke y c om p one nt s i n t hi s s i gna l i ng pa t hw a y ha ve be e n i de nt i f i e d. O f r e l e va nc e t o t hi s s t udy i s t ha t S H I P ha s be e n s how n t o be r e c r ui t e d t o 2B 4, s ugge s t i ng i t c a n i n f l ue nc e 2B 4 s i gna l i ng ( 14 0, 141 265) S H I P m a y i n f a c t be r e c r ui t e d t o 2B 4 t o oppos e t he a c t i ons of P I 3K C ons i s t e nt w i t h t hi s P I 3K i s a l s o r e c r ui t e d t o 2B 4 w he r e i t c a n t r i gge r t he a c t i va t i on of dow ns t r e a m e f f e c t or s i nc l udi ng A K T a nd P L C # ( 140, 266) T h i s P I 3K pa t hw a y m a y pl a y a r ol e dow ns t r e a m of E A T 2, w hi c h ha s be e n s how n t o be a ke y a da pt or p r ot e i n f o r s i gna l s e m a na t i ng f r om 2B 4 ( 139) T hus a l a c k of S H I P m a y l e a d t o unoppos e d P I 3K s i gna l i n g a t 2B 4 a nd t hus a qua l i t a t i ve di f f e r e nc e i n 2B 4 s i gna l i ng ( F i g. 11C ) W e pos t ul a t e t ha t i n t he a bs e nc e of S H I P P I 3K s i gna l i ng m a y r un unc he c ke d i n N K c e l l s a nd t ha t t hi s m a y not onl y i nc r e a s e t he i nhi bi t or y s i gna l e m a na t i ng f r om i ndi vi dua l 2B 4 r e c e pt or s but a l s o i nc r e a s e t r a n s c r i pt i on of 2B 4 a nd pos s i bl y ot he r dow ns t r e a m i nhi bi t or y s i gna l i ng c om pone nt s l i ke E A T 2

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44 F i gu r e 11. D o m i n an c e b y 2B 4 c om p r om i s e s N K c yt ol ys i s of c o m p l e x t ar ge t s A ) S H I P c om pe t e nt c e l l w i t h no r m a l l e ve l s of 2B 4 a n d a pr ope r ba l a nc e of i nhi bi t or y s i gna l s f r om 2B 4, a nd a c t i va t or s l i ke N K G 2D or L y49H i s m a i nt a i ne d unt i l a c t i va t i on ove r c om e s i nhi bi t i on. B ) I nc r e a s e d 2B 4 s ur f a c e d e ns i t y i n t he S H I P / c e l l i nc r e a s e s ba s a l l e ve l s of 2B 4 i nhi bi t o r y s i gna l i ng a nd t hi s ove r r i de s a c t i va t i ng r e c e pt or s C ) 2B 4 i nhi bi t or y s i gna l s i n t he S H I P de f i c i e nt c e l l a r e qu a l i t a t i ve l y a l t e r e d i n a w a y t ha t l e a ds t o t he a c t i va t i ng s i gna l s be i ng ove r r i dde n. D ) M ode l f or how unoppos e d 2B 4 s i gna l s i n t he S H I P de f i c i e nt c e l l m i ght f e e d f o r w a r d t o: ( i ) i n c r e a s e i t s e xpr e s s i on a nd t hus ba s a l l e ve l s of i nhi bi t o r y s i gna l i ng a s i n ( B ) or ( i i ) i nc r e a s e e xpr e s s i on of a ne ga t i ve s i gna l i ng

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45 c om pone nt ( e g E A T 2) ut i l i z e d by 2B 4 t o e f f e c t bot h a qua l i t a t i ve a nd qua nt i t a t i ve c ha nge i n 2B 4 i nhi bi t o r y s i gna l i ng. I t i s a l s o qui t e pr o ba bl e t ha t t he a bove pot e nt i a l m e c ha ni s m s c oul d a c t i n c onc e r t t o di s r upt t he f unc t i on of t he S H I P de f i c i e nt N K c e l l T ha t i s a qua l i t a t i ve c ha nge c a us e d by a l a c k of S H I P s i gna l i ng a t 2B 4 m a y i ni t i a t e a q ua nt i t a t i ve c ha nge by de r e gul a t i ng 2B 4 s ur f a c e e xp r e s s i on. T h e i n i t i a l qua l i t a t i ve c ha nge c oul d a l s o e f f e c t c ha nge s i n e xpr e s s i on ot he r s i gna l i ng m ol e c ul e s t ha t pa r t i c i pa t e i n 2B 4 s i gna l i ng a nd t hus f u r t he r a l t e r i ng i nhi bi t o r y s i gna l s e m a na t i ng f r om 2B 4 ( F i g. 11D ) I n t hi s m a nne r t he S H I P / N K c e l l be c o m e s l oc ke d i nt o a f e e d f or w a r d 2B 4 i nhi bi t or y s i gna l i ng m ode r e nde r i ng t he c e l l hypor e s pons i ve i n t he pr e s e nc e of i t s l i ga nd C D 48. O ur f i ndi ngs e xt e nd S H I P s r e gul a t i on of t he N K r e c e pt or r e pe r t oi r e t o M H C i nde pe nde nt i nhi bi t or y r e c e pt or s but a l s o de m o ns t r a t e how t hi s s e e m i ngl y m i nor c om po ne nt of N K R r e gul a t i on i s a bs ol ut e l y c r i t i c a l t o t he nor m a l c yt ol yt i c f unc t i on by t he N K c om pa r t m e nt M A T E R I A L S A N D M E T H O D S A ni m al s T he S H I P / m i c e us e d i n t h i s s t udy w e r e pr e vi ous l y c r e a t e d i n ou r l a b ( S e e C hp 1) M i c e w e r e m a i nt a i ne d by i nt e r c r os s i ng S H I P + / m i c e ( F 10 t o t he C 57B L 6/ J ba c kgr ound) t he r e by a l l ow i ng f o r s uf f i c i e nt num be r s of bot h S H I P / a s w e l l a s W T l i t t e r m a t e s f or a l l e xpe r i m e nt s A l l e xpe r i m e nt s w e r e pe r f or m e d w i t h S H I P / a nd W T l i t t e r m a t e s be t w e e n 6 9 w e e ks of a ge

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46 A c ut e bone m ar r ow e ngr af t m e nt as s ay s B one m a r r ow e ngr a f t m e nt w a s a s s a ye d t hr ough t h e us e of t he s pl e ni c I U dR upt a ke a s s a y. F or t he s e e xpe r i m e nt s w hol e B M w a s i s ol a t e d f r om m i c e of t he not e d M H C ha pl ot ype ( i e H 2D ) S H I P + / + S H I P / a nd s ynge ne i c B L 6 hos t s w e r e l e t ha l l y i r r a di a t e d w i t h 950 R a ds w i t h a ga m m a i r r a di a t or ( P e r ki n E l m e r ) I r r a di a t i on w a s gi ve n i n t w o dos e s w i t h 2 or m or e hou r s of r e s t i n be t w e e n dos e s P os t i r r a di a t i on t he hos t m i c e r e c e i ve d 5x10 6 w hol e B M ( W B M ) c e l l s i n a t ot a l vol um e of 200 L f r om a l l oge ne i c or s ynge ne i c donor s a s i ndi c a t e d i n t he r e s ul t s vi a a n i nt r a ve nous i nj e c t i on. F our da ys pos t B M T 3 m C i of 1 2 5 I dU r d w a s i nj e c t e d i nt r a ve n ous l y t o a l l ow f or t he m e a s ur e m e nt of e ngr a f t m e nt of t he B M T O n t he f i f t h da y pos t B M T s pl e e ns of t he hos t a ni m a l s w e r e r e m ove d. E ngr a f t m e nt w a s t he n a s s e s s e d t hr ough t he m e a s ur e m e nt of 1 2 5 I upt a ke i n t he w hol e s pl e e n w i t h a ga m m a c ount e r ( P e r ki n E l m e r W i z a r d1470) A nt i body s t ai ni ng and f l ow c y t om e t r y F or a l l f l ow c yt om e t r y e xpe r i m e nt s w hol e s pl e noc yt e s W B M or L A K c ul t ur e s w e r e ha r ve s t e d a nd pr e pa r e d i n t o a s i ngl e c e l l s us pe ns i on. W hol e s pl e noc yt e s a nd W B M w e r e r e d bl ood c e l l l ys e d f o r 5 m i nut e s a t r oom t e m pe r a t ur e i n R B C l ys i s buf f e r c ons i s t i ng of 0. 15 M N H 4 C l 1 0m M K H C O 3 a nd 0. 1m M E D T A C e l l s w e r e s pun dow n a t 300xG f o r 5 m i nut e s a nd t he n r e s us pe nde d a t 1x10 6 c e l l s / 50 L i n s t a i ni ng m e di a S t a i ni ng m e di a c ons i s t s of P B S w i t h 3 % F B S a nd H E P E S C e l l s w e r e t he n F c bl oc ke d f or 15 m i nut e s on i c e w i t h a nt i C D 16/ 32 a nt i body. T h e c e l l s w e r e t he n s t a i ne d w i t h t he a ppr opr i a t e a nt i body c om bi na t i on. A nt i bodi e s us e d f or s t a i ni ng i nc l ude d: N K 1 1; 2B 4;

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47 A 1 ( m ous e I gG 2a k ) ; 5 E 6 ( m ous e I gG 2a k ) ; 4 E 5 ( r a t I gG 2a k) ; H B F 719 ( m ous e I gG 1, k) ; 4D 11 ( r a t I gG 2a k) ; Y L I 90 ( m ous e I gG 1 k) ; C D 94 ( r a t I gG 2a k ) w e r e ob t a i ne d f r om B D P ha r m i nge n ( S a n J os e C A ) C 7 ( ha m s t e r I gG 1) w a s pu r c ha s e d f r om e B i oS c i e nc e ( S a n D i e go, C A ) 3D 10 ( r I gG 1) w a s c onj uga t e d t o bi ot i n a nd us e d f or s t a i ni ng L y49H a s pr e vi ous l y de s c r i be d ( 81) T he a nt i K L R E 1 ( 7E 8 ) w a s pr e vi ous l y de s c r i be d a nd w a s c onj uga t e d t o bi ot i n a nd r e ve a l e d w i t h S A A P C a s de s c r i be d he r e ( 267) S a m pl e s w e r e a c qui r e d on a F A C S C a l i bur a nd a na l yz e d us i ng F l ow J o6. 3. D e a d c e l l s w e r e e xc l ude d f r om t he a na l ys i s ba s e d o n e xc l us i on of t he 7A A D dye L A K c ul t ur e s and c y t ol y s i s as s ay s S pl e e ns w e r e ha r ve s t e d a nd pr e pa r e d i n t o a s i ngl e c e l l s us pe ns i on f r om S H I P / a nd W T m i c e W hol e s pl e noc yt e s w e r e r e d bl ood c e l l l ys e d f or 5 m i nut e s w i t h R B C l ys i s buf f e r C e l l s w e r e t he n s pun dow n a t 300xG f or 5 m i nut e s a nd r e s us pe nde d a t 2. 5x10 8 c e l l s / m L i n m i l t e nyi bu f f e r w hi c h c ons i s t s of P B S 0 5% F B S 0. 5 % H E P E S a nd E D T A C e l l s w e r e t he n pr e pa r e d f or e nr i c hm e nt w i t h t he M i l t e nyi M ous e N K c e l l e nr i c hm e nt ki t T he l i ne a ge s pe c i f i c a n t i body c oc kt a i l c ons i s t i ng of C D 4, C D 5, C D 8, C D 19, G r 1 a nd T e r 119 w a s a dde d t o t he w hol e s pl e noc yt e s a t a c onc e nt r a t i on of 5 L / 1x10 7 c e l l s f or 10 m i nu t e s i n a r e f r i ge r a t o r A n a ddi t i ona l 30 L o f m e di a pe r 1x10 7 c e l l s w a s t he n a dde d t o e a c h s a m pl e A nt i bi ot i n m i c r obe a ds w e r e t he n a dde d a t a c onc e nt r a t i on of 10 L / 1x10 7 c e l l s a nd i nc uba t e d i n t he r e f r i ge r a t or f o r 15 m i nut e s C e l l s w e r e t he n w a s he d a nd r e s us pe nde d i n m i l t e nyi buf f e r a t a c onc e nt r a t i on o f 200x10 6 c e l l s / m L T he c e l l s w e r e t he n r un on t he A ut oM a c ( M i l t e nyi ) on t he D e pl e t e S pr ogr a m

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48 P e r c e nt of c e l l s r e c ove r e d va r i e d f r om 5 t o 25% be t w e e n S H I P / a nd W T N K c e l l s w i t h a pur i t y of 10 60 % E nr i c he d N K c e l l s w e r e pl a t e d a t a de ns i t y of 2x10 6 c e l l s pe r m L i n 6 w e l l di s he s C e l l s w e r e c ul t ur e d f o r s e ve n da ys i n a c t i va t i on m e di a c ons i s t i ng of R P M I 10% F B S L gl ut P e ni c i l l i n/ S t r e pt om yc i n N a py r uva t e non e s s e nt i a l a m i no a c i ds a nd 2000uni t s / m L hum a n r I L 2 ( P r ol e uki n) O n da y 1 of c ul t ur e c e l l s w e r e s uppl e m e nt e d w i t h 1m L o f f r e s h a c t i va t i on m e d i a O n da y 3 non a dhe r e nt c e l l s w e r e r e m ove d t h r ough a de m i de pl e t i on of 1 2m L a nd f r e s h m e di a w a s a dde d t o t he c ul t u r e s O n da y 4 6 m e di a w a s a dde d a s ne c e s s a r y. O n da y 7 a s t a nda r d 4 hou r c hr o m i um r e l e a s e a s s a y w a s pe r f or m e d. O n t he da y of t he e xpe r i m e nt t a r ge t c e l l s w e r e r e s us pe nde d a t 1x10 6 c e l l s / m L i n c h r om i um r e l e a s e m e di a c ons i s t i ng of R P M I w i t h 3% F B S a nd H E P E S 100 C i of 5 1 C r w a s a dde d t o 1x10 6 t a r ge t c e l l s a nd t he n i nc uba t e d f o r 60 m i n ut e s a t 37 C w i t h ge nt l e a gi t a t i on e ve r y 15 m i nut e s A f t e r i nc uba t i on t a r ge t c e l l s w e r e s pun dow n a t 300xG f o r 5 m i nut e s a nd w a s he d t w i c e w i t h 3m L of c hr om i um m e di a T he t a r ge t c e l l s w e r e t he n c ount e d a nd r e s us pe nde d a t 3000 c e l l s / 100 L i n c hr om i um m e di a I n o r de r t o r e m ove t he L A K c e l l s f r om c ul t u r e t he y m us t be l i f t e d t hr ough t he us e o f P B S w i t h E D T A T he f i r s t s t e p i s t o r e m ove t he non a dhe r e nt a nd de a d c e l l s f r om t he t op of t he c ul t ur e t hr ough r e m ova l o f t he t op 1 2 m L o f m e di a T he r e m a i ni ng m e di a i s t he n pi pe t t e d up a nd dow n t o f r e e l oos e c e l l s T he m e di a c ont a i ni ng t he f r e e d L A K c e l l s i s r e m ove d a nd pl a c e d on i c e 2m L of c ol d P B S E D T A w a s t he n a dde d t o e a c h w e l l a nd pl a c e d a t 4 C f or 10 15 m i nut e s O ve r e xpos ur e t o t he E D T A or e xpos ur e a t w a r m e r t e m pe r a t ur e s c a n pr ove ha z a r dous t o t he L A K c e l l s s o c a r e w a s t a ke n t o a s s ur e t he c e l l s r e m a i ne d a t 4 C A f t e r t he 10 15 m i nu t e

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49 i nc uba t i on t he r e m a i ni ng c e l l s w e r e f r e e d f r o m t he pl a t e t hr ough pi pe t t i ng L A K c e l l s w e r e t he n c ount e d a nd s pun dow n a t 300xG f or 5 m i nut e s a nd r e s us pe nde d i n c hr om i um m e di a s u c h t ha t t he a ppr op r i a t e num be r of e f f e c t or c e l l s w e r e i n 100 L of m e di a N K c e l l s a nd t a r ge t c e l l s w e r e t he n c om bi ne d i n 3 r a t i o s 60: 1, 20: 1 a nd 6. 33: 1 i n 96 w e l l pl a t e s a t a t ot a l vol u m e of 200 L T a r ge t c e l l num be r s w e r e m a i nt a i ne d a t 3000 c e l l s f or a l l e xpe r i m e nt s P l a t e s w e r e s pun dow n a t 200xG f or 1 m i nut e T he c e l l s w e r e t he n i nc uba t e d a t 37 C f or 4 hou r s A f t e r i nc uba t i on t h e t op 100 L of s upe r na t a nt w a s c ol l e c t e d a nd m e a s ur e d f or r a di oa c t i vi t y on a ga m m a c ount e r ( P e r ki n E l m e r W i z a r d1470) S po nt a ne ous r e l e a s e c ont r ol s w e r e pe r f or m e d i n t he a bs e nc e of e f f e c t or c e l l s M a xi m a l r e l e a s e w a s m e a s ur e d by a ddi ng 1 0% T r i t on X t o t he t a r ge t c e l l s a l one P e r c e nt l ys i s w a s c a l c ul a t e d by t he f ol l ow i ng f o r m ul a 100 X ( e xpe r i m e nt a l C P M s pont a ne ous r e l e a s e C P M ) / ( m a xi m um r e l e a s e C P M s pont a ne ous r e l e a s e C P M ) A l l e xpe r i m e nt s w e r e pe r f or m e d i n t r i pl i c a t e a nd r e s ul t s w e r e ve r i f i e d w i t h a s e pa r a t e e xpe r i m e nt pe r f or m e d on a di f f e r e nt da t e A nt i body bl oc k i ng e x pe r i m e nt s A nt i body bl oc ki ng e xpe r i m e nt s w e r e pe r f o r m e d i n t he s a m e m a nne r a s s t a nda r d c yt ot oxi c i t y e xpe r i m e nt s e xc e pt f o r t he a ddi t i on o f t he bl oc ki ng a nt i body. T a r ge t c e l l s w e r e l oa de d nor m a l l y a nd pl a c e d a t 3000 c e l l s / 100 L i n c hr om i um m e di a T he t a r ge t c e l l s w e r e t he n i nc uba t e d w i t h a nt i C D 16/ 32 f o r 1 5 m i nut e s t o bl oc k F c r e c e pt or s B ot h t he t a r ge t c e l l s t ha t w e r e t o be bl oc ke d w i t h a nt i C D 48 a s w e l l a s t he unbl oc ke d c ont r ol s unde r w e nt F c bl oc ki ng T a r ge t c e l l s w e r e t he n i nc uba t e d w i t h a nt i C D 48 ( B C M 1 c l one f r om e B i os c i e nc e ) a t 1 g pe r m i l l i on c e l l s f or 15 m i nut e s A l l a nt i body i nc uba t i ons w e r e

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50 done a t r oom t e m pe r a t ur e due t o t he f a c t t ha t t he c ool i ng of t a r ge t c e l l s t o 4 C s e e m e d t o ha ve a de l e t e r i ous e f f e c t t o c hr om i um l oa di ng T h e a nt i 16/ 32 a nd C D 48 a nt i body w a s not w a s he d of f p r i or t o a ddi t i on of t he e f f e c t or c e l l s A no r m a l c yt ot ox i c i t y a s s a y de s c r i be d a bove w a s t he n pe r f or m e d St at i s t i c al anal y s i s S t a t i s t i c a l a na l ys i s w a s done us i ng G r a phpa d P r i s m T he s t a t i s t i c a l t e s t t ha t w a s pe r f or m e d t o c om pa r e r e c e pt or e xpr e s s i on l e ve l s s pl e ni c I U dR upt a ke a s w e l l a s pe r c e nt l ys i s i n c hr om i um r e l e a s e a s s a y s w a s a S t ude nt s t w o t a i l e d T t e s t N = 3 e xc e pt w he r e a gr e a t e r N i s i nd i c a t e d. R e s ul t s w e r e c ons i de r e d s i gni f i c a nt w i t h a p< 0. 05

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51 C ha pt e r 3 I na ppr opr i a t e R e c r ui t m e nt a nd S H P 1 A c t i vi t y i s R e s pons i bl e f or R e c e pt or D om i na nc e i n t he S H I P de f i c i e nt N K C e l l I n t r od u c t i on T he pr oc e s s by w hi c h a n N K c e l l r e c ogni z e s a t a r g e t c e l l a nd de l i ve r s a s uf f i c i e nt s i gna l t o t r i gge r t a r ge t l ys i s i s de t e r m i ne d by a n a r r a y of i nhi bi t o r y a nd a c t i va t i ng r e c e pt or s on t he c e l l s ur f a c e N K di s c r i m i na t i on o f s e l f f r om a l t e r e d s e l f i nvol ve s i nhi bi t or y r e c e pt or r e c ogni t i on of M H C I m ol e c ul e s ( 50) a nd non M H C l i ga nds l i ke C D 48 a nd C l r 1b ( 131 262 268 269 ) N K r e c ogni t i on of i nf e c t e d o r da m a ge d c e l l s ( a l t e r e d s e l f ) i s c oor di na t e d t h r ough s t r e s s i nduc e d l i ga nds ( e g M I C A M I C B R a e 1, H 60, M ul t 1) or v i r a l l y e nc ode d l i ga nds ( e g m 157 he m a gl ut t i ni n ) r e c ogni z e d by va r i ous a c t i va t i ng r e c e pt or s i nc l udi ng N K G 2D L y49H a n d N K p46/ N c r 1 ( 112 145 259 260 270) T he pr oc e s s of i ni t i a l t a r ge t c e l l r e c ogni t i on a nd t h e r e c r ui t m e nt o f a p pr op r i a t e dow ns t r e a m s i gna l i ng m ol e c ul e s t o t he N K s yna ps e i s c a r e f ul l y c oor d i na t e d i n o r de r f or t he N K c e l l t o e f f e c t i ve l y ki l l t he t a r ge t A l t hough m a ny o f t he ke y pl a ye r s i n t he pr oc e s s a r e know n, t he m a nne r i n w hi c h t he s e di s pa r a t e s t e ps a nd pa t hw a ys a r e c oor di na t e d i s l e s s w e l l unde r s t ood. ( 271 273) N K a c t i va t i ng r e c e pt or s s uc h a s N K G 2D a nd L y49H upon l i ga nd e nga ge m e nt a r e

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52 a bl e t o bi nd D A P 10 or D A P 12 m o l e c ul e s t ha t c ont a i n a n I T A M or Y xxM m o t i f ( 102, 274) T hi s t he n a l l ow s f or t he r e c r ui t m e nt of va r i ous e f f e c t or s of c e l l s i gna l i ng, i nc l udi ng t he S r c a nd S yk r e l a t e d pr ot e i n t yr os i ne ki na s e s t ha t s ubs e que nt l y l e a d t o t he a c t i va t i on of m or e di s t a l e f f e c t or pa t hw a ys s uc h a s t he P I 3K a nd M A P / E R K pa t hw a ys ( 271, 275 277) I nhi bi t o r y r e c e pt or s t ha t e nga ge s e l f l i ga nds c a n oppos e a c t i va t i on of t he s e pa t hw a ys t hr ough t he r e c r u i t m e nt o f va r i ous S H 2 dom a i n c ont a i ni ng phos pha t a s e s t o t he i r I T I M T he s e i nc l ude S H P 1 a nd S H P 2, w hi c h a r e r e s pons i bl e f or t he r e m ova l of t yr os i ne phos pha t e s ( 76, 77 278 279 ) a nd t he i nos i t ol phos pha t a s e S H I P ( 216) w hi c h i s r e s pons i bl e f or t he r e m ova l of t he 5 phos pha t e f r om P I ( 3, 4 5) P 3 ( 208, 209) 2B 4 i s a m e m be r o f t he S L A M r e l a t e d r e c e pt or s ( 1 21, 133) I t f unc t i ons t h r ough t he r e c ogni t i on o f a not he r S L A M f a m i l y m e m be r C D 48, t ha t i s ubi qui t ous l y e xpr e s s e d on c e l l s of t he he m a t opoi e t i c s ys t e m ( 128, 129) 2 B 4 ha s a c om pl e x r ol e i n N K c e l l f unc t i on a nd phys i ol ogy t ha t r e m a i ns a n a c t i ve a r e a of i nve s t i ga t i on. D e pe ndi ng on t he c ont e xt 2B 4 ha s be e n s how n t o a c t a s bot h a n i nhi bi t or y a nd a c t i va t i ng r e c e pt or ( 130 138) T hi s i s l i ke l y due a t l e a s t i n pa r t t o t he a bi l i t y of 2B 4 t o di f f e r e nt i a l l y r e c r ui t va r i ous dow ns t r e a m e f f e c t or s of c e l l s i gna l i ng. U nde r di f f e r e nt s i gna l i ng c ont e xt s a nd i n di f f e r e nt s pe c i e s 2B 4 c a n r e c r ui t S A P E A T 2 F yn T S H P 1, P I 3K a nd S H I P ( 133, 139 142) H ow t he di f f e r e nt i a l r e c r ui t m e nt of t he s e s i gna l i ng e nt i t i e s i s c ont r ol l e d i s not c om pl e t e l y unde r s t ood. H ow e ve r w hi c h m ol e c ul e s a r e r e c r ui t e d a nd t hus w hi c h s i gna l i s pr opa ga t e d f ol l ow i ng C D 48 e nga ge m e nt m a y be i nf l ue nc e d by t he r a t i o o f 2B 4 i s of o r m s e xpr e s s e d i n t he N K c e l l T w o 2B 4 i s of or m s ha ve be e n i de nt i f i e d i n m i c e s hor t ( 2B 4S ) a nd l ong ( 2B 4L ) t ha t w e r e pr opos e d t o ha ve a c t i v a t i ng a nd i nhi bi t o r y s i gna l i ng

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53 c a pa c i t i e s r e s pe c t i ve l y ( 134) A l t hough t he e xa c t f unc t i on of t he s e t w o i s of or m s r e m a i ns t o be de f i ne d i t i s f e a s i bl e t ha t t he di f f e r e nt i nt r a c e l l ul a r dom a i ns w i t hi n t he s e i s of or m s c oul d r e c r ui t d i f f e r e nt e f f e c t or s o f c e l l s i gna l i ng. 2 B 4 c oul d a l s o m e di a t e va r i ous s i gna l i ng out c om e s t hr ough c h a nge s i n t he a va i l a bi l i t y or r e c r ui t m e nt of di f f e r e nt s i gna l i ng m ol e c ul e s F o r i ns t a nc e i t ha s be e n s ho w n t ha t t he r e a r e di m i ni s he d l e ve l s o f t he S A P p r ot e i n i n i m m a t ur e hum a n N K c e l l s T h e l a c k of t h i s ke y a c t i va t i ng m o l e c ul e i n t he c e l l a ppe a r s t o l oc k 2B 4 i nt o a n i nhi bi t or y s i gna l i ng m ode ( 138 ) I n ot he r S L A M f a m i l y m e m be r s n a m e l y C D 150, t he r e i s e vi de nc e t ha t t he p r e s e nc e or a bs e nc e of S A P c a n r e gul a t e t he bi ndi ng of bot h S H P 1 a nd S H I P t o t he i m m uno r e c e pt or ba s e d t yr os i ne s w i t c h m ot i f s ( I T S M ) o f t h i s r e c e pt or ( 127) W e ha ve pr e vi ous l y de m ons t r a t e d t ha t t he N K r e c e pt or r e pe r t o i r e i s hi gh l y di s r upt e d by S H I P de f i c i e nc y ( 216, 280) T hi s r e pe r t oi r e di s r upt i on l e a ds t o r e c e pt or dom i na nc e by 2B 4 s uc h t ha t i nhi b i t or y s i gna l s f r o m 2B 4 r e pr e s s ki l l i ng of c om p l e x t a r ge t s ( 280) I n t h i s s t udy w e de f i ne t he m ol e c ul a r ba s i s f or 2B 4 s dom i na nc e of ke y N K a c t i va t i ng r e c e pt or s f o r bot h s t r e s s i nduc e d a nd vi r a l l y e nc ode d N K a c t i va t i ng l i ga nds R E S U L T S 2B 4 and SH P e x pr e s s i on i n SH I P de f i c i e nt N K c e l l s W e pr e vi ous l y s how e d t ha t 2B 4 l e ve l s a r e i nc r e a s e d on t he s ur f a c e of S H I P / N K c e l l s T o de t e r m i ne i f t hi s i nc r e a s e i s due t o i nc r e a s e d e xpr e s s i on of 2B 4, r a t he r t ha n i nc r e a s e d s ur f a c e de pos i t i on, w e bl ot t e d w hol e c e l l l ys a t e s pr e pa r e d f r om s or t e d S H I P /

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5 4 a nd W T N K c e l l s f or t he pr e s e nc e of 2B 4 ( F i g 12 A ) T hi s a na l ys i s r e ve a l s c ons i s t e nt w i t h our p r e vi ous F A C S a na l ys i s t ha t s t e a dy s t a t e l e ve l s of 2B 4 a r e i nc r e a s e d i n S H I P / N K c e l l s W e a l s o f i nd t ha t t he r a t i o of 2B 4S t o 2 B 4L i s s ke w e d t ow a r ds t he l ong i s of or m ( 2B 4L ) i n t he S H I P / N K c e l l r e l a t i ve t o W T N K c e l l s I n a ddi t i on t o 2B 4, w hol e c e l l l ys a t e s ( W C L ) w e r e bl ot t e d f or S H P 1 a nd S H P 2 ( F i g. 12B C ) T hi s r e ve a l e d t ha t l i ke 2B 4, S H P 1 i s ove r e xpr e s s e d i n S H I P / N K c e l l s a s c om pa r e d t o W T I n c ont r a s t S H P 2 l e ve l s a r e c ons i s t e nt l y c om pa r a bl e be t w e e n S H I P / a nd W T N K c e l l s F i gu r e 12. E xp r e s s i on of s i gn al i n g m ol e c u l e s i n N K WC L N K 1. 1 + C D 3 N K c e l l s w e r e s or t e d f r o m s pl e e ns of S H I P / ( ) a nd W T ( + ) m i c e W C L w e r e pr e pa r e d a nd W e s t e r n bl ot s pe r f or m e d f o r t he i ndi c a t e d p r ot e i n, bl ot s w e r e s ubs e que nt l y s t r i ppe d a nd r e pr obe d f o r a c t i n a s a l oa di ng c ont r ol A ) B l ot s w e r e pr obe d f o r 2B 4 r e ve a l i ng a n i s of or m bi a s i n t he S H I P / N K c e l l B ) S H P 1 C ) S H P 2. T he s e W e s t e r n bl ot s a r e r e pr e s e nt a t i ve of t hr e e i nde pe nde nt e xpe r i m e nt s

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55 I nc r e as e d r e c r ui t m e nt of SH P 1 t o 2B 4 i n SH I P de f i c i e nt N K c e l l s D ue t o t he ove r e xp r e s s i on of 2B 4, t he bi a s t ow a r ds t he 2B 4L i s of or m a nd S H P 1 ove r e xpr e s s i on w e hypot he s i z e d t ha t t he r e m i ght b e a qua l i t a t i ve c ha nge i n s i gna l s e m a na t i ng f r om 2B 4 i n S H I P / N K c e l l s T o e xa m i ne t hi s pos s i bi l i t y, w e pr e pa r e d 2B 4 I P s f r om s or t e d S H I P / a nd W T N K c e l l s ( F i g. 13) G i ve n t he i nc r e a s e of S H P 1 i n S H I P / N K c e l l s w e e xpl or e d t he r e c r ui t m e nt of i t a s w e l l a s S H P 2 t o 2B 4 ( F i g. 13A ) I n t he s e bl ot s w e s e e a s ubs t a nt i a l i nc r e a s e i n t he c o I P of S H P 1 t o 2B 4 i n t he S H I P / N K c e l l a s c om pa r e d t o W T N K c e l l s H ow e ve r no c ha nge i s s e e n i n S H P 2 r e c r ui t m e nt t o 2B 4 be t w e e n S H I P / a nd W T N K c e l l s T he s e bl ot s w e r e s ubs e que nt l y s t r i ppe d a nd r e p r obe d f or 2B 4. W e w e r e t he n a bl e t o qua nt i t a t e t he a m ou nt of S H P 1, S H P 2 a nd 2B 4 pr e s e nt i n t he s e I P s T hi s a l l ow e d us t o c om pa r e t he r e l a t i ve a m ount of e a c h o f t he s e pr ot e i ns pr e s e nt i n t he I P s ( F i g 13B ) T h r ough t h i s c om pa r i s on w e w e r e a bl e t o s how t ha t t he r e i s a ppr oxi m a t e l y 2 f o l d m o r e 2B 4 i n t he S H I P / 2B 4 I P s c om pa r e d t o W T T hi s 2 f ol d gr e a t e r a m ount o f 2B 4 i n t he S H I P / N K c e l l w a s e xpe c t e d due t o t he f a c t t ha t a s w e ha ve pr e vi ous l y s how n, t he r e i s a ppr oxi m a t e l y 2 f ol d i nc r e a s e i n t he a m ount of 2B 4 on t he s ur f a c e of S H I P / N K c e l l s a s m e a s ur e d by f l o w c yt om e t r y ( 280) T he r e f o r e i f e qua l c e l l e qui va l e nt s w e r e l oa de d w e w oul d e xpe c t ~ 2 f ol d m or e 2B 4 i n t he I P s o f S H I P / N K c e l l s a s c om pa r e d t o W T I P s W e w e r e a l s o a bl e t o s how t ha t i n t he S H I P / I P s t he r e i s a t l e a s t a 10 f ol d i nc r e a s e i n S H P 1 r e c r ui t m e nt s o a l t hough t he r e i s m o r e 2B 4 i n S H I P / I P s t he r e i s d r a m a t i c a l l y m or e S H P 1 W e a l s o pe r f or m e d t hi s s a m e a na l ys i s of S H P 1 r e c r ui t m e nt t o 2B 4 us i ng a c he m i l um i ne s c e nt s e c o nda r y a nd a L i c or O dys s e y i m a ge r T hi s a l l ow e d us onc e a ga i n t o qua nt i t a t e t he a m ou nt of S H P 1 r e c r ui t e d t o 2B 4. T h r ough t hi s t e c hni que w e w e r e a bl e t o r e c onf i r m ou r S H P 1 f i ndi ng s how i ng t ha t t he r e i s

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56 a ppr oxi m a t e l y 1 6 t i m e s m or e S H P 1 r e c r ui t e d t o 2 B 4 i n t he S H I P / N K c om pa r e d t o t he W T G i ve n t he ke y r o l e of S H I P i n oppos i ng P I 3K s i gna l i ng, w e bl ot t e d f o r t he P I 3K s ubuni t s p110 a nd p85 ( F i g. 13D E ) I n bot h i ns t a nc e s w e s e e a s m a l l but c ons i s t e nt i nc r e a s e i n t he a s s oc i a t i on of 2B 4 w i t h bot h P I 3K s ubuni t s i n S H I P / N K c e l l s T hi s c ha nge l i ke l y r e f l e c t s i nc r e a s e d 2B 4 e xpr e s s i on i n S H I P de f i c i e nt N K c e l l s r a t he r t ha n pr e f e r e nt i a l r e c r u i t m e nt o f t he s e P I 3K s ubuni t s E A T 2 ha s be e n p r opos e d t o be a ke y m e di a t or of t he 2B 4 i nhi b i t or y pa t hw a y ( 139) a nd t he r e f or e w e a l s o bl ot t e d 2B 4 I P s f or E A T 2 w he r e w e s e e no a pp r e c i a bl e di f f e r e nc e ( F i g. 13F )

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57 F i gu r e 13. R e c r u i t m e n t of s i gn al i n g m ol e c u l e s t o 2B 4. N K 1. 1 + C D 3 N K c e l l s w e r e s or t e d f r om s pl e e ns of S H I P / ( ) a nd W T ( + ) m i c e W C L w e r e t he n pr e pa r e d f r om t he pur i f i e d N K c e l l s a nd 2B 4 I P s p r e pa r e d. A ) 2B 4 a nd i t s i s ot ype c ont r ol w e r e I P i n pa r a l l e l T he I P s w e r e t he n bl ot t e d f or S H P 1 a nd S H P 2. 2B 4 w a s bl ot t e d i n t he I P s t o de t e r m i ne t he t ot a l a m ount o f r e c e pt or t ha t w a s pr e c i pi t a t e d i n or de r t o nor m a l i z e t he s a m pl e s ( W C L c ont r ol ) B ) T he l e ve l s o f S H P 1, S H P 2 a nd 2B 4 p r e s e nt i n t he I P s w e r e qua nt i f i e d by I m a ge qua nt s of t w a r e T he s e r a t i os w e r e t he n c om pa r e d i n t he ba r gr a p h s how i ng t ha t a l t hough t he r e i s a n i nc r e a s e i n 2B 4 i n t he nul l I P t he r e i s a m uc h gr e a t e r i nc r e a s e i n S H P 1 C ) S H P 1 w a s pr obe d f o r i n 2B 4 I P s us i ng a f l uo r oc hr om e t a gge d s e c onda r y a nd de ve l ope d on a L i c or O dys s e y i m a ge r a l l ow i ng t he i nt e ns i t y o f t he S H P 1 ba nds t o be qua nt i t a t e d. T he r e s ul t i ng va l ue s a r e s how n be l ow e a c h ba nd i n a r bi t r a r y f l uor e s c e nc e uni t s ( F U ) D ) p110 s ubuni t of P I 3 K i na s e W B on 2B 4 I P s E ) p85 s ubuni t of P I 3 K i na s e W B on 2B 4 I P s F ) E A T 2 W B on 2B 4 I P s C e l l s w e r e pool e d f r om m u l t i pl e a ni m a l s t o obt a i n s uf f i c i e nt num be r s T he s e I P a nd W e s t e r n bl ot s a r e r e pr e s e nt a t i ve of t hr e e i nde pe nde nt e xpe r i m e nt s pe r f or m e d w i t h s e pa r a t e s a m pl e s on di f f e r e nt da t e s G i ve n t ha t our f unc t i ona l a s s a ys of 2B 4 s i m pa c t o n N K c yt ol yt i c f unc t i on a r e pe r f or m e d w i t h L A K c e l l s w e a l s o e xa m i ne d S H P 1 a nd S H P 2 r e c r ui t m e nt t o 2B 4 r e c e pt or c om pl e xe s i n S H I P / a nd W T L A K c e l l s A s w a s obs e r ve d w i t h f r e s hl y i s ol a t e d N K c e l l s t he r e i s a d r a m a t i c i nc r e a s e i n t he r e c r ui t m e nt of S H P 1 t o 2B 4 i n a c t i va t e d S H I P / N K c e l l s c om pa r e d t o W T w he r e S H P 2 r e m a i ns e qua l i n t he s a m e c e l l s ( F i g. 14A ) O nc e a ga i n w e w e r e a bl e t o qua nt i t a t e t he a m ount of S H P 1, S H P 2 a nd 2B 4

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58 pr e s e nt i n t he 2B 4 I P s T h i s f i ndi ng a gr e e d w i t h t h e f i ndi ng i n r e s t i ng N K c e l l s t ha t e ve n t hough t he r e i s a 2 f ol d i nc r e a s e i n 2B 4 e xpr e s s i on i n t he S H I P / N K c e l l t he i nc r e a s e d r e c r ui t m e nt of S H P 1 i s m uc h g r e a t e r T a ke n t oge t he r t he a na l ys i s of bot h r e s t i ng a nd a c t i va t e d N K c e l l s s ugge s t s t ha t 2B 4 dom i na nc e of a c t i va t i ng r e c e pt or s a nd t he hypor e s pons i ve ne s s of S H I P / N K c e l l s c oul d be d ue t o a n i na pp r opr i a t e de gr e e of S H P 1 r e c r ui t m e nt t o 2B 4 r e c e pt or c om pl e xe s F i gu r e 14. R e c r u i t m e n t of s i gn al i n g m ol e c u l e s t o 2B 4 i n ac t i vat e d N K c e l l s 2B 4 a nd i t s i s ot ype c ont r ol w e r e i m m unopr e c i pi t a t e d i n pa r a l l e l f r om W C L o f S H I P / ( ) a nd W T ( + ) L A K c e l l s I P s w e r e r e s ol ve d by S D S P A G E a nd t he n W e s t e r n bl ot t e d A ) S H P 1 a nd S H P 2. 2B 4 w a s bl ot t e d i n t he 2B 4 I P s i n or de r t o no r m a l i z e t he a m ount of r e c e pt or pr e s e nt i n t he I P ( *W C L c ont r ol ) B ) S H P 1, S H P 2 a n d 2B 4 l e ve l s w e r e qua nt i f i e d us i ng I m a ge qua nt s of t w a r e a nd c om pa r e d i n a ba r gr a ph, s how i ng t ha t S H P 1 i s dr a m a t i c a l l y ove r r e c r ui t e d t o 2B 4 i n t he S H I P / L A K c e l l s c om pa r e d t o t he W T T he s e I P a nd W e s t e r n bl o t s a r e r e p r e s e nt a t i ve of t h r e e i n de pe nde nt e xpe r i m e nt s B r oad i nhi bi t i on of t y r os i ne phos phat as e ac t i v i t y r e s t or e s SH I P / N K c y t ol y t i c f unc t i on

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59 G i ve n t he i na ppr opr i a t e de gr e e of S H P 1 r e c r ui t m e nt t o 2B 4 i n S H I P / N K c e l l s w e e xpl or e d t he pos s i bi l i t y o f us i ng c he m i c a l i nhi b i t or s t o bl oc k i t s t yr os i ne phos pha t a s e a c t i vi t y t o de t e r m i ne i f t hi s c oul d r e s t or e ki l l i ng of c om pl e x t a r ge t s by S H I P de f i c i e nt N K c e l l s W e f i r s t us e d N a O V a br oa dl y a c t i ng t y r os i ne phos pha t a s e i nhi bi t or t o c ount e r a c t t he e f f e c t s of t he S H P 1 ove r r e c r ui t m e n t t o 2B 4 ( F i g 14A ) W e f i nd t ha t t he a ddi t i on of 1 00 M s odi um or t hova na da t e t o S H I P / N K c yt ol ys i s a s s a y s r e s t or e s t he i r a bi l i t y t o m e di a t e e f f i c i e nt ki l l i ng ( F i g 15A ) I m p or t a nt l y, w e c ons i s t e nt l y obs e r ve no i nc r e a s e d ki l l i ng by W T L A K c e l l s a ga i ns t e i t he r R M A or R M A R a e 1 + t a r ge t s f ol l ow i ng t he a ddi t i on of N a O V H ow e ve r t o ou r s ur pr i s e w e obs e r ve d t ha t N a O V t r e a t m e nt i nc r e a s e d t he c a pa c i t y of S H I P / N K c e l l s t o k i l l R M A pa r e nt a l c e l l s t ha t do no t e xpr e s s t he N K G 2D l i ga nd, R a e 1. I n f a c t S H I P de f i c i e nt N K c yt ol ys i s of R M A pa r e nt a l t a r ge t s e xc e e ds t ha t of W T L A K c e l l s W e ha ve c ons i s t e nt l y obs e r ve d t hi s s upe r nor m a l ki l l i ng of R M A t a r ge t s i n t h r e e s e pa r a t e s t udi e s w i t h N a O V t r e a t e d S H I P / L A K c e l l s T hi s f i ndi ng i ndi c a t e s phos pha t a s e i nhi bi t i on c a n r e s t or e t he a bi l i t y o f S H I P / N K c e l l s t o ki l l c om pl e x t a r ge t s vi a N K G 2D w hi l e a l s o e xpa ndi ng t he c a pa c i t y of S H I P de f i c i e nt N K c e l l s t o ki l l t u m or c e l l s i n t he a bs e nc e of l i ga nds f o r N K G 2D W e a l s o t e s t e d t he a bi l i t y of N a O V t o i nc r e a s e c yt ot oxi c i t y w i t h B a F 3 a nd m 157 + B a F 3 t a r ge t s ( F i g. 15B ) O nc e a ga i n w e obs e r ve t ha t N a O V i s a bl e t o i nc r e a s e t he c a pa c i t y of S H I P / N K c e l l s t o ki l l bot h t he pa r e nt a l B a F 3 c e l l s a s w e l l a s t he a c t i va t i ng l i ga nd pos i t i ve m 157 + B a F 3 c e l l s A l t hough t he s e di f f e r e nc e s a r e not a s dr a m a t i c a s i s s e e n i n N K G 2D ki l l i ng. N one t he l e s s w e c ons i s t e nt l y obs e r ve i nc r e a s e d ki l l i ng w i t h bot h B a F 3 pa r e nt a l a nd m 157 + t a r ge t s T a ke n t oge t he r w i t h our pr e vi ous f i ndi ngs de m ons t r a t i ng 2B 4 dom i na t e s N K G 2D a nd L y49H i n S H I P de f i c i e nt N K c e l l s ( 280) t he a bi l i t y of N a O V t o r e s t or e k i l l i ng by S H I P /

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60 N K c e l l s a ga i ns t m ul t i pl e t a r ge t s i ndi c a t e s i nc r e a s e d t yr os i ne phos pha t a s e a c t i vi t y i s l oc ki ng t he S H I P / N K c e l l i n t o a hypo r e s pons i ve s t a t e F i gu r e 15. R e s t or at i on of S H I P / c yt o t oxi c i t y w i t h N aO V t r e at m e n t S t a nda r d f ou r hour 5 1 C r r e l e a s e a s s a ys w e r e pe r f or m e d w i t h S H I P / ( / ) o r W T ( + / + ) L A K c e l l s A r a t i o of 30: 1 a nd 3000 t a r ge t c e l l s w e r e us e d f o r a l l c ondi t i ons A l l e xpe r i m e nt s w e r e pe r f or m e d i n t r i p l i c a t e A s s a ys w e r e pe r f or m e d i n t he pr e s e nc e of 100 M N a O V ( N a O V ) or w i t h m e di a a l one ( ) A l l gr a phs a r e r e pr e s e nt a t i ve of 3 o r m o r e i nde pe nde nt e xpe r i m e nt s ( *= p< 0. 05) A ) R M A or R a e 1 + R M A t r a ns f e c t a nt s w e r e us e d a s t a r ge t s B ) B a F or M 157 + B A F t r a ns f e c t a nt s w e r e us e d a s t a r ge t s I nhi bi t i on of SH P 1 ac t i v i t y r e s t or e s SH I P / N K c y t ol y t i c f unc t i on T o f u r t he r t e s t t he hypot he s i s t ha t i na ppr op r i a t e r e c r ui t m e nt of S H P 1 t o 2B 4 i s l oc ki ng S H I P / N K c e l l s i nt o a hypo r e s pons i ve s t a t e w e t e s t e d s e ve r a l nove l l ow M W c om pounds t ha t ha ve t he a bi l i t y t o i nhi bi t t he phos pha t a s e a c t i vi t y of S H P 1 a t M l e ve l s

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61 T he s e c om pounds w e r e i de nt i f i e d du r i ng a s c r e e n f or S H P 2 i nhi bi t or s ( 281) W e s c r e e ne d 6 c om pounds w i t h pr e di c t e d M a c t i vi t y a ga i ns t S H P 1 a nd 2 O f t he s e 6 c om pounds w e i de nt i f i e d one N S C 119910 ( F i g. 1 6A ) w hi c h w a s e f f e c t i ve i n r e s t or i ng t he c yt ol yt i c c a pa c i t y of S H I P / N K c e l l s T he s e l e c t i vi t y of t hi s c om pound w a s t e s t e d i n v i t r o a ga i ns t S H P 1, S H P 2 a nd P T P 1b ( F i g. 16B ) I n t he s e e xpe r i m e nt s w e w e r e a bl e t o s how t ha t N S C 119910 i s a ppr oxi m a t e l y 10 f ol d m or e s e l e c t i ve t o S H P 1 a nd a ppr oxi m a t e l y 100 f o l d m o r e s e l e c t i ve t o S H P 2 t h a n a ve r y c l os e l y r e l a t e d t yr os i ne phos pha t a s e P T P 1b. W e ne xt t e s t e d t he a bi l i t y of N S C 119910 t o r e s t or e ki l l i ng i n t he S H I P / N K c e l l T he e f f e c t i ve i n v i t r o dos e a t w hi c h N S C 119910 w a s a bl e t o r e s t or e S H I P / c yt ot oxi c i t y w a s de t e r m i ne d i n a dos e t i t r a t i on e xpe r i m e nt T hr ough t hi s 67. 32 M w a s i de nt i f i e d a s t he e f f e c t i ve dos e ( F i g. 16C ) T hi s c onc e nt r a t i on, 67. 32 M of N S C 119910, w a s us e d f o r a l l s ubs e que nt s t a nda r d 5 1 C r r e l e a s e a s s a y s T he a ddi t i on of N S C 119910 s i gni f i c a nt l y r e s t or e d ki l l i ng o f R a e 1 + R M A a s w e l l a s pa r e nt a l R M A t a r ge t s by S H I P / N K c e l l s w hi l e i t ha d no e f f e c t on t he c yt ol yt i c a c t i vi t y of W T N K c e l l s a ga i ns t R a e 1 + t a r ge t s ( F i g. 16D ) T he a ddi t i on of N S C 119910 t o L A K c e l l s h a d no e f f e c t on t he e xp r e s s i on l e ve l s of N K G 2D ( F i g. 16F ) W e ha ve a l s o pe r f or m e d t he s e e xpe r i m e nt s w i t h m 157 + B a F 3 t a r ge t s A s s how n i n f i gur e 16 t he a ddi t i on of N S C 119910 a l s o i nc r e a s e d S H I P / N K ki l l i ng of m 157 + t a r ge t s A l t hough t he i nc r e a s e i s not a s dr a m a t i c a s w e obs e r ve w i t h N K G 2D m e di a t e d c yt ol ys i s t hi s i nc r e a s e ha s be e n obs e r ve d c ons i s t e nt l y i n m ul t i pl e c yt ol ys i s a s s a ys w i t h t he m 157 + B a F 3 t a r ge t s T he s e t yr os i ne p hos pha t a s e i nhi bi t i on s t udi e s w he n pa i r e d w i t h our bi oc he m i c a l de t e r m i na t i on of i na pp r opr i a t e S H P 1

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62 r e c r ui t m e nt t o 2B 4 i n S H I P / N K c e l l s p r ovi de s a m e c ha ni s t i c r a t i ona l e f or t he hypor e s pons i ve ne s s of S H I P / N K c e l l s

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63 F i gu r e 16. R e s t or at i on of c yt ot o x i c i t y w i t h S H P 1/ 2 i n h i b i t or N S C 119910 A ) M ol e c ul a r s t r uc t ur e of N S C 119910. T he s t r uc t ur e of t hi s m ol e c ul e w a s c onf i r m e d by pr ot on N M R ( s e e M a t e r i a l s a nd M e t hods ) B ) N S C 119910 w a s t e s t e d f or i t s a bi l i t y t o i nhi bi t t he phos pha t a s e a c t i vi t y of pur i f i e d S H P 1 S H P 2 a nd P T P 1b. C ) T he i n v i v o e f f e c t i ve dos e of N S C 119910 w a s de t e r m i ne d t hr o ugh a dos e t i t r a t i on e xpe r i m e nt 10, 25 a nd 50X t he i n v i t r o I C 50 o f 2 7 M w a s us e d. D E ) S t a nda r d 4 hou r 5 1 C r r e l e a s e a s s a ys w e r e pe r f or m e d w i t h S H I P / ( / ) or W T ( + / + ) L A K c e l l s A r a t i o of 30 : 1 a nd 3000 t a r ge t c e l l s w e r e us e d f o r a l l c ondi t i ons A l l c ondi t i ons w e r e pe r f o r m e d i n t r i pl i c a t e A s s a y s w e r e pe r f or m e d i n t he pr e s e nc e of 67 32 M N C I 119910 or m e di a a l one ( ) T he s e c yt ol ys i s s t udi e s w i t h N S C 119901 a r e r e pr e s e nt a t i ve of t hr e e i nde pe nde nt e xpe r i m e nt s ( *= p< 0. 05) D ) R a e 1 + R M A t r a ns f e c t a nt s w e r e us e d a s t a r ge t s E ) M 157 + B a F 3 t r a ns f e c t a nt s w e r e us e d a s t a r ge t s F ) S H I P / ( ) o r W T ( + + ) c e l l s w e r e i nc uba t e d w i t h 67. 32 M N S C 119910 ( 119910) or m e di a a l o ne ( C N T ) f or 4 hou r s T he c e l l s w e r e t he n s t a i ne d f or N K 1. 1, C D 3 a nd N K G 2D a nd a na l yz e d f or N K G 2D e xpr e s s i on. D i s c u s s i on P r e vi ous l y w e ha ve s how n t ha t S H I P de f i c i e nc y l e a ds t o a n N K r e c e pt or r e pe r t oi r e di s r upt i on s uc h t ha t 2B 4 a c t s a s a dom i n a nt i nhi bi t or y r e c e pt or ( 280) I n t hi s s t udy w e ha ve e xt e nde d t he s e f i ndi ngs t o i de nt i f y a m ol e c ul a r m e c ha ni s m r e s pons i bl e f or 2B 4 r e c e pt or dom i na nc e i n S H I P / N K c e l l s W e ha ve pr e vi ous l y s how n t ha t t he r e i s s i gni f i c a nt ove r r e p r e s e nt a t i on of 2B 4 on t he s ur f a c e of S H I P / N K c e l l s W e ha ve e xt e nde d t hi s by de m ons t r a t i ng t ha t i n t he S H I P de f i c i e nt N K c e l l t he r e i s not onl y m or e s ur f a c e de pos i t i on of 2B 4, but a l s o s i gni f i c a nt l y m or e 2B 4 pr ot e i n e xpr e s s e d by S H I P /

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64 N K c e l l s W e ha ve a l s o de t e r m i ne d t ha t w he n c o m pa r e d t o t he W T N K c e l l t he r e i s a bi a s i n t he S H I P / N K c e l l t ow a r ds t he 2B 4L i s of o r m W e e xa m i ne d t he va r i ous s i gna l i ng m ol e c ul e s t ha t a r e r e c r ui t e d t o 2B 4 i n S H I P / N K c e l l s W e f ound t ha t t he r e i s a s m a l l i nc r e a s e i n t he P I 3K s ubuni t s p110 a nd p8 5 t ha t i s m os t l i ke l y a t t r i but a bl e t o i nc r e a s e d 2B 4 e xpr e s s i on. W e ha ve a l s o i de nt i f i e d t ha t t he r e i s no de m ons t r a bl e di f f e r e nc e i n e i t he r S H P 2 or E A T 2 r e c r ui t m e nt t o 2B 4. F u r t he r m or e w e ha ve i de nt i f i e d t ha t t he r e i s a ppr ox i m a t e l y 10 t o16 t i m e s m or e S H P 1 r e c r ui t e d t o 2B 4 i n S H I P / N K c e l l s a s c om pa r e d t o W T W e w e r e a bl e t o r e ve r s e t he e f f e c t of t he S H P 1 ove r r e c r ui t m e nt by i nhi bi t i ng i t s e nz ym a t i c a c t i vi t y us i ng e i t he r a b r oa d a c t i ng t yr os i ne p hos pha t a s e i nhi bi t or ( N a O V ) or a m or e s e l e c t i ve S H P i nhi bi t or ( N S C 11 9910) T he s e r e s ul t s ha ve l e d us t o hypot he s i z e t ha t S H I P de f i c i e nc y l e a ds not onl y t o 2B 4 r e c e pt or do m i na nc e but 2B 4L bi a s a s w e l l a s a l t e r e d i nhi bi t or y s i gna l i ng w i t hi n t he S H I P / N K c e l l W e ha ve de ve l ope d a m ode l i nc or po r a t i ng t he ke y di f f e r e nc e s t ha t e xi s t w i t hi n 2B 4 s i gna l i ng i n t he S H I P / a nd W T e nvi r onm e nt ( F i g. 17)

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65 F i gu r e 17. M od e l f i g u r e of 2B 4 s i gn al i n g. A ) 2 B 4 s i gna l i ng i n a W T N K c e l l B ot h s hor t a nd l ong i s of or m s of 2B 4 a r e p r e s e nt a s w e l l a s pos s i bl e a c t i va t i ng a nd i nhi bi t or y s i gna l i ng m ol e c ul e s T he s i gna l t ha t i s de l i ve r e d, e i t he r a c t i va t i on or i nhi bi t i on, w i l l de pe nd upon t he l i ga nd pr e s e nt a nd t he c ont e xt of t he s i gna l B ) 2B 4 s i gna l i ng i n a S H I P / N K c e l l T he r e i s a l a c k o f S H I P e xp r e s s i on a nd bi a s t ow a r ds t he 2B 4L i s of o r m T he s e 2 f a c t or s l e a d t o a pr o f ound i nc r e a s e i n S H P 1 r e c r u i t m e nt a nd t he r e f or e t i p t he ba l a nc e t ow a r ds c ons t i t ut i ve i nhi bi t or y s i gna l i ng. T he l a c k of S H I P m a y a l s o a l l ow unoppos e d P I 3K a c t i vi t y a t 2B 4 t ha t m a y i n t ur n pr om ot e i nc r e a s e d 2B 4 e xpr e s s i on. G i ve n t ha t S H I P i s a ke y i nhi bi t o r of t he P I 3K pa t hw a y, w e i ni t i a l l y c ons i de r e d t he pos s i bl e ove r r e c r ui t m e nt of t he P I 3K s ubuni t s p110 a nd p85 t o 2B 4 m i ght be r e s pons i bl e f or t he q ua l i t a t i ve c ha nge i n 2B 4 f unc t i on i n S H I P de f i c i e nt N K c e l l s G i ve n t ha t t he i nhi bi t i on of S H P 1 w a s a bl e t o r e s t or e ki l l i ng i n S H I P / N K c e l l s t o W T l e ve l s i t s t a nds t o r e a s on t ha t P I 3K doe s not pl a y a m a j or r o l e i n r e nde r i ng S H I P / N K c e l l s

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66 hypor e s pons i ve P I 3K c oul d s t i l l pl a y a s ubt l e a nd i ndi r e c t r ol e i n 2B 4 r e c e pt or dom i na nc e A s i s de t a i l e d i n f i gur e 17 i n t he a bs e nc e of S H I P P I 3K a c t i vi t y m a y not ne e d t o be i nc r e a s e d, but r a t he r i t s unoppos e d a c t i vi t y c oul d pot e nt i a l l y a l t e r 2B 4 e xpr e s s i on a n d c ont r i but e t o r e c e pt or dom i na nc e P r e vi ous s t udi e s ha ve i de nt i f i e d A P 1 bi ndi ng s i t e s i n t he p r om ot e r of 2B 4 ( 282) P I 3K c a n t r i gge r nuc l e a r t r a ns l oc a t i on o f A P 1 vi a a c t i va t i on o f P K C $ ( 283 ) a nd t hus unoppos e d P I 3K a c t i vi t y a t 2B 4 c oul d pot e nt i a l l y i nc r e a s e 2B 4 e xpr e s s i on a nd/ or bi a s i s o f or m us a ge t ow a r ds 2B 4L 2B 4 ha s pr ove n t o ha ve a s om e w ha t c om pl e x r ol e i n N K bi ol ogy w i t h i n v i t r o a nd i n v i v o e xpe r i m e nt s i ndi c a t i ng bot h a c t i va t i ng a nd i nhi bi t or y r ol e s i n N K f unc t i on ( 1 30 138) T hi s d i s pa r i t y ha s be e n a t t r i but e d, t o s om e e xt e nt t o t he va r i ous s i gna l i ng a da pt or s t ha t c a n pot e nt i a l l y a s s oc i a t e w i t h t he I T S M of 2B 4 ( 133, 139 142 ) B ot h S H P 1 a nd S H P 2 ha ve be e n s how n t o be r e c r ui t e d t o 2B 4 i n c e r t a i n c ont e xt s ( 137, 140, 142) a nd a r e a l s o ke y r e gul a t or s of i nhi bi t o r y s i gna l i ng f or M H C I r e c e pt or s on N K c e l l s I n t hi s s t udy w e i de nt i f i e d a ~ 10 t o 16 f ol d i nc r e a s e i n S H P 1 r e c r ui t m e nt t o 2B 4 i n t he S H I P / N K c e l l T hi s i s a ke y f i ndi ng g i ve n t ha t w e ha ve pr e vi ous l y s how n t ha t t he s ur f a c e e xpr e s s i on of 2B 4 i s i nc r e a s e d onl y ~ 2 f ol d i n t he S H I P / N K c e l l c om pa r e d t o t he W T N K c e l l ( 280) T he r e i s c l e a r l y a qua l i t a t i ve c ha nge i n t he 2B 4 r e c e pt or c om pl e xe s s uc h t ha t a m uc h l a r ge r pr opo r t i on o f 2B 4 m ol e c ul e s a s s oc i a t e w i t h S H P 1 i n t he a bs e nc e of S H I P e xpr e s s i on. 2B 4 ha s up t o 4 t y r os i ne r e s i due s i n i t s c yt opl a s m i c t a i l t ha t c a n be phos phor yl a t e d a nd r e c r ui t dow ns t r e a m s i gna l i ng m ol e c ul e s ( 140) B ot h S H I P a nd S H P 1 ha ve S H 2 dom a i ns t ha t c a n b i nd ove r l a p pi ng phos phot yr os i ne s i n 2B 4 ( 140) T hus i n t he S H I P de f i c i e nt N K c e l l t he r e i s l i ke l y gr e a t e r a c c e s s t o 2B 4 by S H P 1. T hi s dr a m a t i c

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67 i nc r e a s e of S H P 1 a t 2B 4 r e c e pt or c om pl e xe s c oul d a l t e r t he ba l a nc e of s i gna l i ng i n t he S H I P de f i c i e nt N K c e l l I m por t a nt l y 2B 4 ha s be e n s how n t o be r e c r ui t e d t o t he N K s yna ps e ( 284, 285) T he r e f o r e t he i nc r e a s e d pr e s e nc e of S H P 1 a t t he N K s yna ps e i n S H I P / N K c e l l s i s l i ke l y t o t e r m i na t e a c t i va t i ng s i gna l s be f or e t he y p r opa ga t e t o m o r e di s t a l e f f e c t or s r e qui r e d f or N K f unc t i on. I n t hi s s t udy w e ut i l i z e d t w o phos pha t a s e i nhi bi t or s ; f i r s t a br oa d a c t i ng phos pha t a s e i nhi bi t or N a O V s e c ond, a m or e s pe c i f i c S H P i nhi bi t or N S C 119910. W e ut i l i z e d bot h o f t he s e c om pounds i n a n a t t e m pt t o c ount e r a c t S H P 1 ove r r e c r ui t m e nt a nd t he r e by r e s t or e ki l l i ng by t he S H I P / N K c e l l s t o W T l e ve l s N a O V w a s a bl e t o s uc c e s s f ul l y r e s t or e ki l l i ng by S H I P / N K c e l l s of R a e 1 + c e l l s t o W T l e ve l s I nt e r e s t i ngl y t he ki l l i ng o f R M A pa r e nt a l c e l l s by S H I P / N K c e l l s w a s a l s o s i gni f i c a nt l y i nc r e a s e d. W e pr opos e t ha t t hi s i nc r e a s e i n S H I P / ki l l i ng, i n t he a bs e nc e of a s t r ong a c t i va t i ng l i ga nd, r e s ul t s f r om t he unde r r e pr e s e nt a t i on of M H C s pe c i f i c i nhi bi t or y r e c e pt or s on S H I P / N K c e l l s t ha t w oul d no r m a l l y p r e ve nt W T ki l l i ng o f M H C I + t a r ge t s l a c ki ng s t r ong a c t i va t i ng l i ga nds T he r e f or e w he n i nhi b i t or y dom i na nc e of 2B 4 i s r e l e a s e d by phos pha t a s e bl oc ka de t hi s pr e s um a bl y e na b l e s s up e r nor m a l ki l l i ng of M H C I + t a r ge t s t ha t l a c k a c t i va t i ng l i ga nds O u r r e s ul t s w i t h t he B a F 3 t a r ge t s a r e l e s s c l e a r but none t he l e s s pr ovoc a t i ve M os t i m por t a nt l y w e s e e a c ons i s t e nt i nc r e a s e i n S H I P / c yt ot oxi c i t y i n t he pr e s e nc e of N a O V r e c onf i r m i ng t he a bi l i t y of phos pha t a s e bl oc ka de t o i nc r e a s e c yt ot oxi c i t y of t he hypor e s pons i ve S H I P / N K c e l l a ga i ns t B a F 3 a nd m 157 + c e l l s I m po r t a nt l y w he n w e l a t e r t e s t e d t he m or e s pe c i f i c N S C 119910 w e do no t s e e t he

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68 s a m e i nc r e a s e of ki l l i ng o f m 157 + c e l l s by W T L A K c e l l s i ndi c a t i ng t ha t t he i nc r e a s e w e s e e w i t h N a O V i s due t o non S H P 1 r e l a t e d e f f e c t s I n a ddi t i on t o r e s t or a t i on of S H I P / ki l l i ng w e s e e a n i nc r e a s e i n t he ki l l i ng of R M A c e l l s by W T N K c e l l s T h i s i s not onl y a pl a us i bl e r e s ul t i t i s a n e xpe c t e d a nd pos i t i ve r e s ul t I n t he W T N K c e l l t he nor m a l M H C i nhi bi t or y r e c e pt or s a r e p r e s e nt a nd f unc t i oni ng, i n oppos i t i on t o t he S H I P / N K c e l l w he r e t he s e r e c e pt or s a r e dow n r e gul a t e d. S H P 1 a nd S H P 2, ke y m ol e c ul e s t o i nhi bi t or y s i gna l i ng, a r e a l s o p r e s e nt a nd f unc t i ona l i n t he W T N K c e l l T he r e f or e i n t he pr e s e nc e of a pot e nt S H P i nhi bi t o r t he r e w oul d be a pa uc i t y of i nh i bi t or y s i gna l i ng. T he ke y i s t he r e f or e t ha t w he n i nhi bi t or y s i gna l s m e di a t e d t hr ough S H P a r e r e m ove d, be t h a t of S H P r e c r ui t e d t o L y49 or 2B 4 c yt ot oxi c i t y i s i nc r e a s e d. I m por t a nt l y i n t he c a s e o f t he S H I P / c yt ot oxi c i t y i t w a s not j us t i nc r e a s e d but r e s t or e d t o t he s a m e l e ve l o f t he W T unde r goi ng S H P i nh i bi t i on. F or unde t e r m i ne d r e a s ons t he r e s t or a t i on o f B a F m 157 + c e l l s i s not a s d r a m a t i c W ha t t hi s doe s i ndi c a t e i s t ha t t ha t t he r e a r e qua l i t a t i ve di f f e r e nc e s t ha t e xi s t be t w e e n t he s i gna l i ng m i l i e us a t pl a y be t w e e n R M A B a F a nd t he L A K c e l l s S uc h t ha t i n N K G 2D m e di a t e d c yt ot oxi c i t y 2B 4 i s t he no t onl y a dom i na nt i nhi bi t or y r e c e pt or i t i s l i ke l y t he s ol e i nhi bi t or y r e c e pt or r e s t r a i ni ng ki l l i ng T he s e r e s ul t s s t r ongl y r e i nf o r c e t ha t 2B 4 i s t he dom i na nt i nhi bi t or y r e c e pt or i n t he S H I P / N K c e l l s a s w e l l a s pr ovi di ng s t r ong pr oo f t ha t S H P 1 ove r r e c r ui t m e nt i s t he m ol e c ul a r m e c h a ni s m be hi nd t hi s dom i na nc e

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69 M at e r i al s A n d M e t h od s A ni m al s T he S H I P / m i c e us e d i n t hi s s t udy w e r e pr e vi ous l y c r e a t e d i n ou r l a b ( S e e C hp. 1) M i c e w e r e m a i nt a i ne d by i nt e r c r os s i ng S H I P + / m i c e ( F 10 t o t he C 57B L 6/ J ba c kgr ound) t he r e by a l l ow i ng f o r s uf f i c i e nt num be r s of bot h S H I P / a s w e l l a s W T l i t t e r m a t e s f or a l l e xpe r i m e nt s A l l e xpe r i m e nt s w e r e pe r f or m e d w i t h S H I P / a nd W T l i t t e r m a t e s be t w e e n 6 9 w e e ks of a ge L A K c ul t ur e s and c y t ol y s i s as s ay s S pl e e ns w e r e ha r ve s t e d a nd pr e pa r e d i n t o a s i ngl e c e l l s us pe ns i on f r om S H I P / a nd W T m i c e W hol e s pl e noc yt e s w e r e r e d bl ood c e l l l ys e d f or 5 m i nut e s w i t h R B C l ys i s buf f e r C e l l s w e r e t he n s pun dow n a t 300xG f or 5 m i nut e s a nd r e s us pe nde d a t 2. 5x10 8 c e l l s / m L i n m i l t e nyi bu f f e r w hi c h c ons i s t s of P B S 0 5% F B S 0. 5 % H E P E S a nd E D T A C e l l s w e r e t he n pr e pa r e d f or e nr i c hm e nt w i t h t he M i l t e nyi M ous e N K c e l l e nr i c hm e nt ki t T he l i ne a ge s pe c i f i c a nt i body c oc kt a i l c ons i s t i ng of C D 4, C D 5, C D 8, C D 19, G r 1 a nd T e r 119 w a s a dde d t o t he w hol e s pl e noc yt e s a t a c onc e nt r a t i on of 5 L / 1x10 7 c e l l s f or 10 m i nu t e s i n a r e f r i ge r a t o r A n a ddi t i ona l 30 L o f m e di a pe r 1x10 7 c e l l s w a s t he n a dde d t o e a c h s a m pl e A nt i bi ot i n m i c r obe a ds w e r e t he n a dde d a t a c onc e nt r a t i on of 10 L / 1x10 7 c e l l s a nd i nc uba t e d i n t he r e f r i ge r a t or f o r 15 m i nut e s C e l l s w e r e t he n w a s he d a nd r e s us pe nde d i n m i l t e nyi buf f e r a t a c onc e nt r a t i on o f 200x10 6 c e l l s / m L T he c e l l s w e r e t he n r un on t he A ut oM a c ( M i l t e nyi ) on t he D e pl e t e S pr ogr a m P e r c e nt of c e l l s r e c ove r e d va r i e d f r om 5 t o 25% be t w e e n S H I P / a nd W T N K c e l l s w i t h a pur i t y of 10 60 %

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70 E nr i c he d N K c e l l s w e r e pl a t e d a t a de ns i t y of 2x10 6 c e l l s pe r m L i n 6 w e l l di s he s C e l l s w e r e c ul t ur e d f o r s e ve n da ys i n a c t i va t i on m e di a c ons i s t i ng of R P M I 10% F B S L gl ut P e ni c i l l i n/ S t r e pt om yc i n N a py r uv a t e non e s s e nt i a l a m i no a c i ds a nd 2000uni t s / m L hum a n r I L 2 ( P r ol e uki n) O n da y 1 of c ul t ur e c e l l s w e r e s uppl e m e nt e d w i t h 1m L o f f r e s h a c t i va t i on m e di a O n da y 3 non a dhe r e nt c e l l s w e r e r e m ove d t h r ough a de m i de pl e t i on of 1 2m L a nd f r e s h m e di a w a s a dde d t o t he c ul t u r e s O n da y 4 6 m e di a w a s a dde d a s ne c e s s a r y. O n da y 6 7 a s t a nda r d 4 hour c hr om i u m r e l e a s e a s s a y w a s pe r f or m e d. O n t he da y of t he e xpe r i m e nt t a r ge t c e l l s w e r e r e s us pe nde d a t 1x10 6 c e l l s / 100 L i n c hr om i um r e l e a s e m e di a c ons i s t i ng of R P M I w i t h 3% F B S a nd H E P E S 100 C i of 5 1 C r w a s a dde d t o 1x10 6 t a r ge t c e l l s a nd t he n i nc uba t e d f o r 60 m i n ut e s a t 37 C w i t h ge nt l e a gi t a t i on e ve r y 15 m i nut e s A f t e r i nc uba t i on t a r ge t c e l l s w e r e s pun dow n a t 300xG f o r 5 m i nut e s a nd w a s he d t w i c e w i t h 3m L of c hr om i u m m e di a T he t a r ge t c e l l s w e r e t he n c ount e d a nd r e s us pe nde d a t 3000 c e l l s / 100 L i n c hr om i um m e di a I n o r de r t o r e m ove t he L A K c e l l s f r om c ul t u r e t he y m us t be l i f t e d t hr ough t he us e o f P B S w i t h E D T A T he f i r s t s t e p i s t o r e m ove t he non a dhe r e nt a nd de a d c e l l s f r om t he t op of t he c ul t ur e t hr ough r e m ova l o f t he t op 1 2 m L o f m e di a T he r e m a i ni ng m e di a i s t he n pi pe t t e d up a nd dow n t o f r e e l oos e c e l l s T he m e di a c ont a i ni ng t he f r e e d L A K c e l l s i s r e m ove d a nd pl a c e d on i c e 2m L of c ol d P B S E D T A w a s t he n a dd e d t o e a c h w e l l a nd pl a c e d a t 4 C f or 10 15 m i nut e s O ve r e xpos ur e t o t he E D T A or e xpos ur e a t w a r m e r t e m pe r a t ur e s c a n pr ove ha z a r dous t o t he L A K c e l l s s o c a r e w a s t a ke n t o a s s ur e t he c e l l s r e m a i ne d a t 4 C A f t e r t he 10 15 m i nu t e i nc uba t i on t he r e m a i ni n g c e l l s w e r e f r e e d f r o m t he pl a t e t hr ough pi pe t t i ng L A K c e l l s w e r e t he n c ount e d a nd s pun dow n a t 300xG f or 5 m i nut e s a nd r e s us pe nde d i n c hr om i um

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71 m e di a s uc h t ha t t he a ppr op r i a t e num be r of e f f e c t or c e l l w e r e i n 100 L of m e di a N K c e l l s a nd t a r ge t c e l l s w e r e t he n c om bi ne d a t a r a t i o of 20: 1 i n 96 w e l l pl a t e s a t a t ot a l vol um e of 200 L T a r ge t c e l l num be r s w e r e m a i n t a i ne d a t 3000 c e l l s f or a l l e xpe r i m e nt s P l a t e s w e r e s pun dow n a t 200xG f o r 1 m i nut e T he c e l l s w e r e t he n i nc uba t e d t oge t he r a t 37 C f or 4 ho u r s A f t e r i nc u ba t i on s upe r na t a nt s w e r e c ol l e c t e d a nd m e a s ur e d f or r a di oa c t i vi t y on a ga m m a c ount e r ( P e r ki n E l m e r W i z a r d1470) S pont a ne ous r e l e a s e c ont r ol s w e r e pe r f or m e d i n t h e a bs e nc e of e f f e c t or c e l l s M a xi m a l r e l e a s e w a s m e a s ur e d by a ddi ng 10% T r i t o n X t o t he t a r ge t c e l l s a l one P e r c e nt l ys i s w a s c a l c ul a t e d by t he f ol l ow i ng f o r m ul a 100 X ( e xpe r i m e nt a l C P M s pont a ne ous r e l e a s e C P M ) / ( m a xi m um r e l e a s e C P M s pont a ne ous r e l e a s e C P M ) A l l e xpe r i m e nt s w e r e pe r f or m e d i n t r i p l i c a t e a nd r e s ul t s w e r e ve r i f i e d w i t h a s e pa r a t e e xpe r i m e nt pe r f or m e d on a di f f e r e nt da t e I nhi bi t or s A l l e xpe r i m e nt s us i ng s odi um or t hova na da t e ( N a O V ) w e r e pe r f or m e d w i t h 100 M a c t i va t e d N a O V N a O V w a s a c t i va t e d by a dj us t i ng t he pH of a 200m M s t oc k t o pH 10. 0 by t he a ddi t i on N a O H or H C l a s ne c e s s a r y f ol l ow e d by boi l i ng unt i l t he s ol ut i on be c om e s c ol or l e s s a nd t he n c ool i ng t o r oom t e m p e r a t ur e T hi s pr oc e s s i s t he n r e pe a t e d unt i l t he pH of t he N a O V s t a bi l i z e s a t 10. 0 ( 286) F or a l l N a O V c yt ot oxi c e xpe r i m e nt s L A K c e l l s a nd t a r ge t s c e l l s w e r e pr e pa r e d a s de s c r i be d a bove P r i or t o i nc uba t i on w i t h t a r ge t s c e l l s N a O V w a s a dde d t o t he L A K c e l l s a nd a l l ow e d t o i nc uba t e f or 15 t o 30 m i nut e s a t r oom t e m pe r a t ur e T a r ge t c e l l s w e r e t ha n a dde d t o t he w e l l s a nd a nor m a l c yt ot oxi c a s s a y w a s pe r f or m e d. C ont r ol s f or N a O V i nhi bi t i on a s s a ys c on s i s t e d of n or m a l

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72 c yt ot oxi c a s s a ys i n t he a b s e nc e of N a O V t o m e a s ur e nor m a l l ys i s a s w e l l a s s pont a ne ou s r e l e a s e c ont r ol s i n t he p r e s e nc e of N a O V N S C 119910 w a s obt a i ne d f r om t he D r ug S ynt he s i s a nd C he m i s t r y B r a nc h, D e ve l opm e nt a l T he r a pe ut i c s P r ogr a m D i vi s i on of C a n c e r T r e a t m e nt a nd D i a gnos i s N C I T he s t r uc t ur e of N S C 119910 w a s c onf i r m e d by pr ot on N M R us i ng a V a r i a n M e r c ur y P l us O xf or d A S 400 s pe c t r om e t e r T he 1 H N M R s pe c t r um w a s r e c or de d a t 400 M H z us i ng D M S O d 6 a s s ol ve nt a nd t e t r a m e t hyl s i l a ne ( T M S ) a s a n i nt e r na l s t a nda r d. C he m i c a l s hi f t va l ue s a r e r e por t e d i n pa r t s pe r m i l l i on ( $ ) T he c om pound s how s c ha r a c t e r i s t i c s i gna l s a s f ol l ow s : $ 12. 643 ( s 1 H O H di s a ppe a r e d on D 2 O s ha ke ) 12. 432 ( s 1H O H di s a ppe a r e d on D 2 O s ha ke ) 10. 103 ( br s 1H di s a ppe a r e d on D 2 O s ha ke ) 7. 486 ( d, J = 9. 2 H z 1H A r ) 7. 411 ( d, J = 9. 2 H z 1H A r ) 6. 438 ( d, J = 8. 8 H z 1H A r ) 6. 403 ( d, J = 9. 2 H z 1H A r ) 2. 024 0. 7 96 ( m c yc l ohe xyl m oi e t y ) F or c hr om i um r e l e a s e a s s a ys N S C 119910 w a s r e c ons t i t ut e d i n D M S O a t 27m M N S C 11 9910 w a s t he n a dde d t o L A K c e l l s i m m e di a t e l y be f or e t he a ddi t i on of t a r ge t c e l l s on i c e f or a f i na l c onc e nt r a t i on of 67. 32 M C ont r ol s w e r e pe r f or m e d i n pa r a l l e l w i t h D M S O a l one a s w e l l a s no i nhi bi t o r S pont a ne ou s r e l e a s e c ont r ol s w e r e a l s o pe r f or m e d i n t he pr e s e nc e of N S C 119910. W e s t e r n B l ot s and I m m unopr e c i pi t at e s A l l w e s t e r n bl ot a nd I P s t udi e s w e r e pe r f o r m e d w i t h pur e c e l l popul a t i ons ( > 95% pur e ) F or f r e s hl y i s ol a t e d N K c e l l s s pl e e ns w e r e r e m ove d f r om S H I P / a nd W T

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73 l i t t e r m a t e s a nd m a de i n t o a s i ngl e c e l l s us pe ns i on. W hol e s pl e noc yt e s a nd W B M w e r e r e d bl ood c e l l l ys e d f or 5 m i nut e s a t r oom t e m pe r a t ur e i n R B C l ys i s buf f e r c ons i s t i ng of 0. 15 M N H 4 C l 10m M K H C O 3 a nd 0. 1m M E D T A C e l l s w e r e s pun dow n a t 300xG f o r 5 m i nut e s a nd t he n r e s us pe nde d a t 1x10 6 c e l l s / 50 L i n s t a i ni ng m e di a S t a i ni ng m e di a c ons i s t s of P B S w i t h 3 % F B S a nd H E P E S C e l l s w e r e t he n F c b l oc ke d f or 15 m i nut e s on i c e w i t h a nt i C D 16/ 32 a nt i body C e l l s w e r e t he n s t a i ne d f or s or t i ng w i t h N K 1. 1 F I T C C D 3 P E a nd D A P I C e l l s w e r e s t a i ne d f or 15 m i n ut e s on i c e a nd t he n w a s he d t w i c e w i t h s t a i ni ng m e di a C e l l s w e r e r e s us pe nde d i n s t a i ni ng m e di a w i t h 1 % F B S f o r s or t i ng A l l c e l l s or t i ng w a s pe r f o r m e d on a F A C S A r i a ( B e c k on D i c ks on) S or t s w e r e pe r f o r m e d w i t h a 70 M noz z l e a t a r a t e o f 1000 5000 c e l l s / s e c ond. A l l s a m pl e s w e r e ke pt a t 4 C f or t he dur a t i on of t he s or t i nc l udi ng t he s or t e d c e l l s w hi c h w e r e s or t e d i nt o s t a i ni ng m e di a c ont a i ni ng 3% F B S A f t e r s or t i ng c e l l s w e r e s pun dow n a t 300xG f or 5 m i nut e s a t 4 C a nd t he n l ys e d f or 30 m i nut e s on i c e i n a m odi f i e d T N E buf f e r c o ns i s t i ng of 50m M T r i s H C l 1 % N P 40, 150m M N a C l 1m M E D T A 1m M P M S F 1m M N a O V 1m M N a F a nd 10 L / m L of P r ot e a s e i nhi bi t or c oc kt a i l m i x ( S i gm a A l dr i c h, C a t #p8340) C e l l de br i s w a s t he n s pun dow n a t 15, 000xG f or 15 m i nut e s a t 4 C a nd t he s upe r na t a nt c ol l e c t e d. F or W e s t e r n bl ot s e qua l c e l l e qui va l e nt s f or S H I P / a nd W T l ys a t e s w e r e br ought up t o a vol um e o f 20 L w i t h 4X L D S bu f f e r ( I nvi t r oge n) c ons i s t i ng of 250m M T r i s H C l 20% gl yc e r o l 8% L D S S e r va B l ue D T T w a s t he n a dde d t o t he s a m pl e f or a f i na l c onc e nt r a t i on o f 50m M a nd t he s a m pl e s w e r e he a t e d f or 10 m i nut e s a t 90 C S a m pl e s w e r e t he n r e s ol ve d on a 4 12% B i s T r i s ge l ( I nvi t r oge n) a nd t r a ns f e r r e d t o a n E C L m e m br a ne ( A m e r s ha m ) B l ot s

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74 w e r e bl oc ke d w i t h 5 % N F M P B S T f or 1 hou r a t r oom t e m pe r a t ur e P r i m a r y a nt i bodi e s w e r e us e d a t va r yi ng c onc e nt r a t i ons ; p110( C e l l S i gna l i ng, 1: 1000 ) p85( C e l l s i gna l i ng, 1: 1000) E a t 2 ( a ki nd gi f t of A nd r e V e i l l e t t e 0. 5 g/ m L ) S H P 1 ( B D T r a ns duc t i on L a bor a t or i e s 1: 500) S H P 2 ( 1: 1000 C e l l S i gna l i ng ) 2B 4 ( R & D 0. 2 g/ m L ) P r i m a r y a nt i bodi e s w e r e i nc uba t e d w i t h t he m e m br a ne i n 5 % N F M P B S T f or 1 hour a t r oom t e m pe r a t ur e or a t 4 C f or 12 hour s P r i m a r y a nt i b odi e s w e r e w a s he d of f w i t h a m i ni m u m of t hr e e 15 m i nut e w a s he s w i t h P B S T T he a pp r o pr i a t e a nt i I gG H R P s e c onda r y f o r t he s pe c i f i c pr i m a r y w a s us e d. S e c onda r i e s w e r e i nc u ba t e d w i t h t he m e m b r a ne i n 5 % N F M P B S T f or 1 hour a t r oom t e m pe r a t ur e M e m br a ne s w e r e t he n w a s he d a m i ni m um of t hr e e t i m e s f or 15 m i nut e s e a c h w a s h. S upe r S i gn a l H R P de t e c t i on s ys t e m ( P i e r c e ) w a s t he n a ppl i e d t o t he m e m br a ne t ha t w a s t he n e xpos e d t o f i l m w hi c h w a s s ubs e que nt l y de ve l ope d. F o r bl ot s w i t h hi gh ba c kgr ound a ddi t i ona l w a s he s w e r e pe r f or m e d a s ne e de d a t r oom t e m pe r a t ur e Q ua nt i f i c a t i on w a s pe r f or m e d us i ng I m a ge qua nt s of t w a r e ( G E H e a l t hc a r e ) I n o r de r t o us e t hi s s of t w a r e t he bl ot i s f i r s t s c a nne d a s a hi gh r e s ol ut i on t i f f T he ba nds of i nt e r e s t a r e t he n de l i ne a t e d by t he us e r a s w e l l a s t he a r e a f r om w hi c h ba c kgr ound w i l l be c a l c ul a t e d. T he s of t w a r e t he n c a l c ul a t e d t he i nt e gr a t e d de ns i t y va l ue ( I D V ) T he I D V i s c a l c ul a t e d by a r e a x ( m e a n D e ns i t y ba c kgr ound) T o a s s ur e t ha t a r e a s of di f f e r i ng s i z e di d not s ke w qua nt i t a t i on ba nds w e r e de l i ne a t e d by boxe s of t he s a m e a r e a be t w e e n s a m pl e s t ha t w oul d be di r e c t l y c om pa r e d ( i e : S H P 1 be t w e e n S H I P / a nd W T ) F or a l l bl ot s t ha t us e d a f l uo r e s c e nt l y t a gge d s e c onda r y a l l s a m pl e s a nd w e s t e r n bl ot t i ng t e c hni que s w e r e i de nt i c a l t o a bov e e xc e pt t he bl oc ki ng a s w e l l a s a nt i body i nc uba t i on w a s done i n L i c or bl oc ki ng bu f f e r ( L i c or ) S e c onda r i e s o f t he a ppr opr i a t e a nt i I gG t ha t w e r e c onj uga t e d t o a n A l e xa f l uor 488 o r 680 ( I nvi t r oge n) w e r e

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75 ut i l i z e d. A f t e r t he s e c onda r y a nt i body w a s w a s he d of bl o t s w e r e s c a nne d w i t h a L i c or O dys s e y i m a ge r r a t he r t ha n us i ng f i l m O dys s e y s of t w a r e w a s t he n us e d t o qua nt i t a t e t he pr ot e i n l e ve l s O nl y t he w hol e l a ne s w e r e us e r i de nt i f i e d a nd t he s of t w a r e de l i ne a t e d t he i ndi vi dua l ba nds a s w e l l a s c a l c ul a t i ng ba c kgr ound R e s ul t s w e r e gi ve n i n a n a r bi t r a r y f l uor e s c e nt uni t ( F U ) F or I P s a f t e r s or t i ng c e l l s w e r e s pun dow n a t 300x G f or 5 m i nut e s a t 4 C a nd t he n l ys e d f or 30 m i nut e s on i c e i n a m odi f i e d T N E buf f e r c ons i s t i ng of 50m M T r i s H C l 1 % N P 40, 150 m M N a C l 1m M E D T A 1m M P M S F 1m M N a O V 1m M N a F a nd 10 L / m L of P r ot e a s e i nhi bi t o r c oc kt a i l m i x ( S i gm a A l d r i c h, p8340) C e l l de br i s w a s t he n s pun dow n a t 15, 000xG f or 15 m i nut e s a t 4 C a nd t he s upe r na t a nt c ol l e c t e d. E qua l c e l l e qui va l e nt s w e r e br ought up t o 500 L i n i c e c ol d T N E bu f f e r B ot h i s ot ype a nd 2B 4 I P s w e r e pe r f or m e d i n pa r a l l e l unde r t he s a m e c ondi t i o ns w i t h a n e qua l num be r of c e l l e qui va l e nt s A l l s t e ps w e r e pe r f or m e d a t 4 C a l l buf f e r s w e r e i c e c ol d a nd a l l c e nt r i f uga t i on s t e ps w e r e pe r f o r m e d i n a p r e c hi l l e d c e nt r i f uge P r e c l e a r i ng of W C L w a s pe r f or m e d by a ddi ng 50 L a nt i m ous e I G I P be a ds ( e B i os c i e nc e ) t o W C L f or 60 m i nut e s w i t h c ons t a nt m i xi ng on a r ot a t i ng m i xe r T he be a ds w e r e t he n s pun dow n a t 10, 000xG f o r 10 m i nut e s a nd s upe r na t a nt s w e r e c o l l e c t e d a nd pr e c l e a r e d i n t he s a m e m a nne r one a ddi t i ona l t i m e C l e a r e d l ys a t e s w e r e t he n i nc uba t e d w i t h a n a nt i 2B 4 a nt i body ( B D ) o r i s ot ype c ont r ol ( M s I gG 2A B D ) f or 90 m i nut e s 50 L of a nt i m ous e I G I P be a ds w e r e t he n a dde d t o t he l ys a t e s f or 90 m i nut e s w hi l e m i xi ng on a r ot a t i ng m i xe r A f t e r i nc uba t i on t he be a ds w e r e w a s he d 6 t i m e s by s pi nni ng dow n a t 10, 000xG f or 30 s e c onds a nd r e m ovi ng s upe r na t a nt a nd r e pl a c i ng w i t h f r e s h T N E l ys i s buf f e r

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76 A f t e r t he l a s t w a s h 50 L o f L D S s a m pl e bu f f e r w a s a dde d t o t he be a ds a nd t he y w e r e he a t e d a t 90 C f or 10 m i nut e s T he y s a m pl e s w e r e t he n s pun a t 10 000X G f or 5 m i nut e s a nd t he s upe r na t a nt s w e r e c ol l e c t e d a nd r e s ol ve d b y S D S P A G E a nd b l ot t e d a s de s c r i be d. 1x10 6 c e l l e qui va l e nt s w e r e l oa de d i nt o e a c h w e l l f or w e s t e r n a na l ys i s P T P I nhi bi t i on A s s ay P T P a c t i vi t y w a s m e a s ur e d us i ng t he f l uor oge ni c 6 8 di f l uo r o 4 m e t hyl um be l l i f e r yl phos pha t e ( D i F M U P ; I nv i t r og e n, C a r l s ba d, C A ) a s t he s ubs t r a t e E a c h r e a c t i on c ont a i ne d 25m M H E P E S 50m M N a C l 0. 05 % T r i t on 1m M di t hi ot hr e i t ol 20 M D i F M U P 10nM M i c r oc ys t i n L R 20nM G S T P T P a nd 10 l of t e s t c om pound o r di m e t hyl s ul f oxi de ( s ol ve nt ) i n a t ot a l r e a c t i on vol um e of 100 l i n bl a c k 96 w e l l pl a t e s R e a c t i on w a s i ni t i a t e d by a ddi t i on o f D i F M U P a n d t he i nc uba t i on t i m e w a s 30 m i nut e s a t r oom t e m pe r a t ur e D i F M U P f l uo r e s c e nc e s i gna l w a s m e a s ur e d a t a n e xc i t a t i on o f 355 nm a nd a n e m i s s i on of 460 nm w i t h a pl a t e r e a de r ( V i c t or 2 1420; P e r ki n E l m e r W a l l a c G a i t he r s bur g, M D ) I C 50 w a s de f i ne d a s t he c onc e nt r a t i on of a n i nhi b i t or t ha t c a us e d a 50% de c r e a s e i n t he P T P a c t i vi t y. F or I C 50 de t e r m i na t i o n, e i ght c onc e nt r a t i ons of N S C 119910 a t one t hi r d di l ut i on ( ~ 0 5 l og ) w e r e t e s t e d. T he r a nge s of N S C 119910 c onc e nt r a t i ons us e d i n e a c h P T P a s s a y w e r e de t e r m i ne d f r om pr e l i m i na r y t r i a l s E a c h e xpe r i m e nt w a s pe r f or m e d e i t he r i n t r i pl i c a t e or du pl i c a t e a nd I C 50 da t a w e r e de r i ve d f r om a t l e a s t t w o i nde pe nde nt e xpe r i m e nt s T he c u r ve f i t t i ng pr og r a m P r i s m 4 ( G r a phP a d S of t w a r e S a n D i e go, C A ) w a s us e d t o c a l c ul a t e I C 50 va l ue s

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77 St at i s t i c al A nal y s i s S t a t i s t i c a l a na l ys i s w a s done us i ng G r a phpa d P r i s m T he s t a t i s t i c a l t e s t t ha t w a s ut i l i z e d w a s a S t ude nt s t w o t a i l e d T t e s t N = 3 e xc e pt w he r e a gr e a t e r N i s i ndi c a t e d. R e s ul t s w e r e c ons i de r e d s i gni f i c a nt w i t h a p< 0. 05

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78 C H A P T E R 4 T he N K K i nom e I n t r od u c t i on O ve r t he l a s t f e w ye a r s a r r a y a n d m a s s s pe c t r om e t r y t e c hnol ogi e s ha ve e na bl e d a na l ys i s of t he t r a ns c r i pt om e ( 287, 288) a nd pr ot e o m e of m a ny c e l l t ype s i nc l udi ng t he N K c e l l ( 289) T hi s i nf or m a t i on ha s pr ove n t o be a nd w i l l c ont i nue t o be o f s i gni f i c a nt va l ue t o t he e l uc i da t i on of m ol e c ul a r m e c ha ni s m s t ha t gove r n not onl y ba s a l c e l l ul a r f unc t i ons but a l s o s om e of t he m o r e uni que t a s ks of i ndi vi dua l c e l l t ype s i nc l udi ng t he N K c e l l A n e qua l l y, i f not m or e i m po r t a nt goa l i s t o de f i ne not j us t t hos e pr o t e i ns t ha t a r e pr e s e nt but r a t he r t ha t a r e a c t i ve a nd pa r t i c ul a r l y t he s i gna l i ng pa t hw a ys t he s e f unc t i oni ng pr ot e i ns a r e i nvol ve d i n. E nz ym e s t ha t phos phor yl a t e t y r os i ne s e r i ne a nd t h r e oni ne r e s i due s pl a y a m a j or r ol e i n s i gna l i ng c a s c a de s t ha t de t e r m i ne c e l l c yc l e e nt r y, s ur vi va l a nd t he di f f e r e nt i a t i on of c e l l s i n t he m a m m a l i a n body i nc l udi ng t he he m a t opoi e t i c s ys t e m K now i ng t he di f f e r e nc e s t ha t e xi s t w i t hi n i ndi vi dua l pr ot e i ns a s w e l l a s c om pl e t e s i gna l i ng pa t hw a ys t ha t a r e a c t i ve i n N K c e l l s a nd t he i r i m m a t ur e pr e c ur s or s w i l l pr ov i de c r i t i c a l i nf or m a t i on f or unde r s t a ndi ng t he i r bi o l ogy. T ow a r ds t hi s e nd, w e ha ve ut i l i z e d k i nom e a na l ys i s t e c hni que s t o e xpl or e t he N K l i ne a ge A r r a y t e c hnol ogy ha s be e n de ve l ope d t o m e a s ur e

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79 e nz ym a t i c a c t i vi t y be t w e e n w hol e c e l l l ys a t e s a nd pr ot e i n s ubs t r a t e s ( 98, 290 293) S pe c i f i c a l l y, a r r a ys c ont a i ni ng m ul t i pl e t a r ge t c ons e ns us s e que n c e s f or pr ot e i n ki na s e s ha ve be e n a s s e m bl e d ( 294) W i t hi n t he s e a r r a ys 1 176 9 12 a m i no a c i d pe pt i de s ha ve be e n a r r a ye d i n dup l i c a t e ( F i g 18 ) T he pe pt i de s t ha t ha ve be e n a r r a ye d a r e ba s e d upon know n or p r e di c t e d phos phor yl a t i on s i t e s a c r os s t h e m a m m a l i a n ki nom e T hi s a l l ow s f o r a w i de r a ngi ng de t e c t i on of s e r i ne t h r e oni ne a nd t yr os i ne phos phor yl a t i on e ve nt s t ha t a r e m e di a t e d vi a ki na s e s pr e s e nt i n w hol e c e l l l ys a t e s T h i s t e c hnol ogy ha s be e n va l i da t e d by a c om pr e he ns i ve de s c r i pt i on o f t he t e m por a l ki ne t i c s of phos phor yl a t i on e ve nt s i nduc e d by l i popol ys a c c ha r i de s t i m ul a t i on ( 295) C onf i de nc e i n t he us e f ul ne s s of t hi s t e c hnol ogy f o r s t udyi ng s i gna l t r a ns duc t i on ha s c om e f r om W e s t e r n bl ot a na l ys i s of l i popol ys a c c ha r i de s t i m ul a t e d c e l l s w hi c h w a s c or r obor a t e d w i t h t he de m ons t r a t i on t ha t ki na s e i nhi bi t or s e f f e c t e d pe pt i de a r r a y phos phor yl a t i on pa t t e r ns c ons i s t e nt w i t h t he e xpe c t e d a c t i on of t he s e i nhi bi t o r s ( 295)

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80 F i gu r e 18. K i n om e P e p C h i p D i a gr a m s how i ng t he l a yout o f pe pt i de s on t he a r r a y. E a c h a r r a y ha s t he 1176 pe pt i de s a r r a ye d i n dup l i c a t e T he di f f e r e nt i a t i on o f t he N K c e l l f r om N K P t o f ul l y f unc t i ona l m a t ur e N K c e l l pr ogr e s s e s i n a l i ne a r pr oc e s s t ha t c a n be f ol l ow e d by t he ga i n a nd/ o r l os s of s ur f a c e m a r ke r s ( 201 203 ) T he s e s t a ge s of N K de ve l opm e nt oc c ur i n t he bone m a r r ow a s t he s e f i r s t s t e ps r e qui r e t he i nt e r a c t i on of t he N K P w i t h t he B M s t r om a T he f i r s t ke y s ur f a c e m a r ke r i s C D 122 o r t he I L 2/ 15R ( 201 203 ) T he s e r e c e pt or s h a ve pr ove n t o be e s s e nt i a l t o N K de ve l opm e nt a s de f e c t s i n t he i r s i g na l i ng l e a d t o de f e c t s i n N K de ve l opm e nt ( 204, 205, 296) I t ha s a l s o be e n s how n t ha t de f e c t s i n I L 15 s i gna l i ng pa t hw a ys s uc h a s J a k3 a nd S t a t 5a / b c a n c a us e de f e c t s i n N K c e l l de ve l opm e nt ( 206) T he ne xt m a j o r s t e p w he n a c e l l m ove s f r om N K P t o i m m a t ur e N K c e l l i s m a r ke d by t he l os s of M a c 1 e xpr e s s i on. R e s ul t i ng i n a phe not ype of N K 1 1 + C D 3 M a c 1 / l o T he ne xt s t e ps a r e m a r ke d by t he a c qui s i t i on of t he N K R C D 94/ N K G 2 a nd t he n t he L y49s a nd f i na l l y f ul l f unc t i ona l m a t ur i t y ( 202 203 ) T he i m m a t ur e N K c e l l s w e w i l l be e xa m i ni ng i n t hi s s t udy a r e f r om t he B M a nd w e r e i de nt i f i e d on t he ba s i s of N K 1 1 + C D 3 a nd M a c 1 / l o W he r e t he m a t u r e N K c e l l s w e r e N K 1 1 + C D 3 f r om t he s pl e e n. I n t h i s s t udy w e ut i l i z e ki nom e p r of i l i ng i n o r de r t o i de nt i f y ke y s i gna l i ng di f f e r e nc e s t ha t e xi s t be t w e e n t he s e t w o c e l l t ype s

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81 R e s u l t s O bt ai ni ng pur e c e l l popul at i ons T hi s f i r s t s t a ge of t hi s s t udy w a s t o obt a i n pur e W C L f r om t he i m m a t ur e a nd m a t ur e N K c e l l s T o t hi s e nd s or t e d p opul a t i ons o f N K 1. 1 + C D 3 M a c 1 l o / i m m a t ur e N K c e l l s f r om t he B M a nd N K 1. 1 + C D 3 m a t ur e N K c e l l s f r om t he s pl e e n w e r e obt a i ne d ( F i gur e 19 ) T hr e e i nde pe nde nt s or t s w e r e pe r f o r m e d f or e a c h c e l l t ype c ons i s t i ng of a m i ni m um of 250, 000 c e l l s f r om w hi c h 3 s e t s of W C L w e r e pr e pa r e d F i gu r e 19. K i n om e s or t i n g s t r at e gy. D ot pl ot s s how i ng t he s or t i ng s t r a t e gy f o r m a t ur e a nd i m m a t ur e N K c e l l s . I ni t i a l ga t i ng s t r a t e gy i s s how n i n t he l e f t pa ne l P os t s or t pur i t y i s s how n i n t he r i ght pa ne l A l l pl ot s a r e a f t e r ba c k ga t i ng on s c a t t e r a nd l i ve c e l l s A ) N K 1 1 + L i n m a t u r e N K c e l l s B ) N K 1. 1 + L i n M a c 1 / l o i m m a t ur e N K c e l l s

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82 P e pt i de P hos phor y l at i on I n or de r t o m e a s ur e phos phor yl a t i on of i ndi vi dua l pe pt i de s pr e s e nt on t he P e pC hi p W C L f r o m t he pu r e c e l l popu l a t i ons w e r e a ppl i e d t o t he P e pC hi p i n t he pr e s e nc e of 3 3 P A T P a nd a c t i va t i on m i x. A f t e r i nc uba t i on of t he W C L w i t h t he pe pt i de s on t he a r r a y a phos pho i m a gi ng c a s s e t t e w a s e xpos e d t o t he P e pC hi ps T hr e e s e t s of c hi ps w e r e ut i l i z e d f o r e a c h c e l l t ype gi v i ng a t o t a l of s i x r e pl i c a t e s i n t ot a l f o r e a c h pe pt i de on t he a r r a y f o r e a c h c e l l t ype F i gu r e 20 s how s t he r e s ul t i ng i m a ge s of t he s i x c hi ps a f t e r de ve l opi ng t he phos pho i m a gi ng c a s s e t t e T he s e r e s ul t i ng i m a ge s w e r e a na l yz e d us i ng S c a na l yz e s of t w a r e T hi s a l l ow e d us t o obt a i n a num e r i c a l va l ue f or t he phos phor yl a t i on of e a c h i nd i vi dua l pe pt i de T he s e 6 r e pl i c a t e s w e r e t he n nor m a l i z e d a c r os s a l l r e pl i c a t e s pr ovi di ng us w i t h a c om pl e t e ki nom e f or bot h m a t u r e a nd i m m a t ur e N K c e l l s t ha t c oul d be c om pa r e d be t w e e n c e l l t ype s ( S e e a ppe ndi x 1 f or t he t ot a l ki nom e )

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83 F i gu r e 20. P e p C h i p s f or m a t u r e an d i m m at u r e N K c e l l s T he r e s ul t i ng i m a ge s a f t e r phos phor i m a gi ng s c r e e ns a r e e xpos e d t o t he P e pC hi ps a nd t he n de ve l ope d. T he s e i m a ge s w e r e us e d t o de t e r m i ne t he phos phor yl a t i on s i gna t ur e f o r A ) I m m a t ur e N K c e l l s a nd B ) M a t ur e N K c e l l s D i f f e r e nt i al k i nom e s of m at ur e and i m m at ur e N K c e l l s I n or de r t o i de nt i f y t hos e pe pt i de s t ha t a r e di f f e r e nt i a l l y phos phor yl a t e d be t w e e n t he i m m a t ur e a nd m a t ur e N K c e l l s t he i r k i nom e s w e r e c om pa r e d t hr ough a W i l c oxon r a nk s um a na l ys i s O f t he 1176 pe pt i de s pr e s e nt o n t he a r r a ys onl y 3. 9 % o f t he pe pt i de s or 46 s pot s i n t ot a l w e r e di f f e r e nt i a l l y phos phor yl a t e d be t w e e n t he i m m a t ur e a nd m a t ur e N K c e l l s I ndi c a t i ng a s w oul d be e xpe c t e d t ha t t he s e c e l l t ype s ut i l i z e s i m i l a r s i gna l i ng pa t hw a ys O f t he s e 46 pe pt i de s 11 ha ve a hi ghe r p hos phor yl a t i on s i gna t ur e i n t he m a t ur e N K c e l l a s c om pa r e d t o t he i m m a t u r e c e l l l e a vi ng 35 t o ha ve a hi ghe r phos phor yl a t i on s i gna t ur e i n t he i m m a t ur e N K c e l l s ( T a bl e 1 ) S p o t B i o l o g i c a l f u n c t i o n P r o t e i n S e q u e n c e 6 7 6 C e l l M o r p h o g e n e s i s / D i f f F i b r o b l a s t g r o w t h f a c t o r r e c e p t o r 3 S T D E Y L D L S 5 2 7 S i g n a l i n g H e m a t o p o i e t i c c e l l s p e c i f i c L Y N s u b s t r a t e P E G D Y E E V L 4 3 4 N A m e t a b o l i s m C h r o m o d o m a i n h e l i c a s e D N A b i n d i n g 1 P S E K S E E I T 4 1 3 S i g n a l i n g F o c a l a d h e s i o n k i n a s e 1 E T D D Y A E I I 8 5 5 S i g n a l i n g G a m m a a m i n o b u t y r i c a c i d r e c e p t o r R D E E Y G Y E A 4 4 4 N A m e t a b o l i s m Z i n c f i n g e r p r o t e i n R l f E E E L Y L E P L 1 0 2 4 S i g n a l i n g T y r o s i n e p r o t e i n k i n a s e J A K 3 K R P S F R A K A 2 3 3 M e t a b o l i s m P y r u v a t e k i n a s e L R R A S V A Q L 2 2 4 M e t a b o l i s m P y r u v a t e k i n a s e L R R A S L 7 1 7 S i g n a l i n g F i b r o b l a s t g r o w t h f a c t o r r e c e p t o r 4 V S E E Y L D L R

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84 7 5 5 S i g n a l i n g R e c e p t o r t y r o s i n e p r o t e i n k i n a s e E r b B 1 D N P D Y Q Q D F 9 4 1 p r o t e i n m e t a b o l i s m e I F 2 b e t a D P T M S K K K K 6 3 7 M e t a b o l i s m H M G C o A l y a s e K A A Q I S V R G L 7 3 5 C e l l g r o w t h / M a i n t e n a n c e T r o p o n i n T Q K A Q T E R K S 2 6 3 T r a n s c r i p t i o n R u n t r e l a t e d t r a n s c r i p t i o n f a c t o r 3 S G R G K 5 6 2 D N A r e p a i r M u t S a l p h a R K A S R K E 9 3 1 C e l l g r o w t h / M a i n t e n a n c e T r o p o n i n I Q H L K S V M L Q 5 6 1 C e l l g r o w t h / M a i n t e n a n c e L a m i n A / C R L R L S P S P T 8 8 6 S i g n a l i n g N a c h a n n e l p r o t e i n t y p e I I a l p h a s u b u n i t E R R P S N V S Q 5 7 0 S i g n a l i n g 1 4 3 3 p r o t e i n b e t a / a l p h a W T S D T Q G D E 7 0 2 C e l l g r o w t h / M a i n t e n a n c e M i c r o t u b u l e a s s o c i a t e d p r o t e i n t a u S K A G S L G N I 9 1 0 S i g n a l i n g M e m b r a n e p r o g e s t i n r e c e p t o r b e t a K S R R T I 8 9 6 S i g n a l i n g P r o t e i n t y r o s i n e p h o s p h a t a s e G 1 E R N L S F E I K 8 5 0 I m m u n e r e s p o n s e H U S S Y 1 8 Q E K E S E R L A 9 0 3 T r a n s c r i p t i o n N u c l e a r f a c t o r N F k a p p a B p 1 0 5 s u b u n i t F R K L S F T E S 8 3 4 S i g n a l i n g C a d e p e n d e n t p r o t e i n k i n a s e t y p e I I g a m m a H R Q E T V E A L 9 4 7 S i g n a l i n g M E K 2 S M A N S F V G T 1 0 8 5 T r a n s c r i p t i o n R e t i n o b l a s t o m a a s s o c i a t e d p r o t e i n P Y K F P S S P L R I P G Z 9 5 4 T r a n s c r i p t i o n H i s t o n e H 1 2 A S G S F K L 7 0 6 C e l l g r o w t h / M a i n t e n a n c e C a l d e s m o n D K V T S P T K V 9 5 3 m e t a b o l i s m N e u t r o p h i l c y t o s o l f a c t o r 1 R K R L S Q D A Y 9 6 8 I m m u n e r e s p o n s e M y e l i n b a s i c p r o t e i n P K R G S G K D G 7 0 7 S i g n a l i n g N a c h a n n e l p r o t e i n t y p e X a l p h a s u b u n i t F R R F T P D S L 1 1 2 8 T r a n s p o r t K e l l b l o o d g r o u p g l y c o p r o t e i n I S I T S R K A Q 1 1 2 9 U n k n o w n P D Z d o m a i n c o n t a i n i n g p r o t e i n 3 N F L K T S A G S 5 5 8 C e l l g r o w t h / M a i n t e n a n c e M A R C k i n a s e s u b s t r a t e A V A S S P S K A 9 6 0 I m m u n e r e s p o n s e C o m p l e m e n t f a c t o r B p r e c u r s o r T E S Q S L T L T 8 8 3 S i g n a l i n g P K A C a l p h a I G R F S E P H A 1 0 8 4 S i g n a l i n g M C S F I r e c e p t o r p r e c u r s o r N D S N Y V V K G 9 7 5 S i g n a l i n g I G F b i n d i n g p r o t e i n 1 L M A P S E E D H 1 1 7 0 p r o t e i n m e t a b o l i s m P h o s p h o r y l c h o l i n e t r a n s f e r a s e A K Q S P S S S P T 5 6 9 S i g n a l i n g O p s i n 2 T V S K T E T S Q 9 2 3 S i g n a l i n g C e n t a u r i n d e l t a 2 P G G S T P V S S 9 6 3 I m m u n e r e s p o n s e M y e l i n b a s i c p r o t e i n R H R D T G I L D 9 5 7 S i g n a l i n g P r o t e i n p h o s p h a t a s e i n h i b i t o r 1 S L A M S P R Q R 7 4 2 T r a n s c r i p t i o n R e t i n o b l a s t o m a a s s o c i a t e d p r o t e i n P Y K F P S S P L R I P G Z T ab l e 1. T he 46 s pot s t ha t a r e di f f e r e nt i a l l y phos p hor yl a t e d be t w e e n m a t ur e a nd i m m a t ur e N K c e l l s T he pe pt i de s a bove t he doubl e l i ne ha ve a hi ghe r l e ve l o f phos phor yl a t i on i n t he m a t ur e N K c e l l s a nd t hos e be l ow a r e hi ghe r i n i m m a t ur e N K c e l l s T he a m i no a c i d s e que nc e f or e a c h pe pt i de i s s how n.

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85 F unc t i onal C l as s i f i c at i on T he goa l o f t hi s s t udy w a s not onl y t o i de nt i f y t hos e pe pt i de s t ha t a r e di f f e r e nt i a l l y phos phor yl a t e d be t w e e n t he s e c e l l t y pe s but a l s o t o i de nt i f y ke y s i gna l i ng pa t hw a ys t ha t a r e ut i l i z e d di f f e r e nt l y be t w e e n t he s e t w o de ve l opm e nt a l s t a ge s I n t he de s i gn of t he P e pC hi p pe pt i de s e que nc e s w e r e c hos e n t ha t c or r e s ponde d t o know n or pr e di c t e d phos phor yl a t i on s i t e s a c r os s t he m a m m a l i a n ki nom e T he r e f or e a l l s i t e s ha ve a pr e di c t e d pr ot e i n t ha t t he pe pt i de r e pr e s e nt s a l t ho ugh s om e ove r l a p m a y e xi s t be t w e e n pr ot e i ns w i t h s i m i l a r a m i no a c i d r e s i due s I n or de r t o a s s ur e t ha t w e ha ve t he m os t a c c ur a t e r e pr e s e nt a t i on of a s pe c i f i c pr ot e i n w e r e c onf i r m e d t he i de nt i t y o f e a c h o f t he 46 s pot s t ha t a r e di f f e r e nt i a l l y phos phor yl a t e d T he 9 12 a m i no a c i d s e que nc e w a s e nt e r e d i nt o pr ot e i n s e que nc e a l i gnm e nt t ool s T he pr ot e i n w i t h t he s t r onge s t a l i gnm e nt w a s ut i l i z e d i n m os t i ns t a nc e s t a ki ng i nt o a c c ount t he a c t ua l s e r i ne t hr e oni ne o r t y r os i ne t ha t i s be i ng phos phor yl a t e d a s w e l l a s t hos e a m i no a c i ds i m m e di a t e l y a dj a c e nt t o i t W i t h t he s e pr ot e i ns w e t he n ut i l i z e d t he hum a n p r ot e i n r e f e r e nc e da t a ba s e ( H P R D ) t o a s s i gn a bi ol ogi c a l f unc t i on f or e a c h pe pt i de ( T a bl e 1) W e t he n s e gr e ga t e d t he s e f unc t i ons t o t he i m m a t ur e or m a t ur e N K c e l l ba s e d on w hi c h c e l l t ype ha d a hi ghe r phos phor yl a t i on f or t he pr ot e i n ha vi ng t he gi ve n f unc t i on ( F i gu r e 21) I n doi ng t hi s w e w e r e a bl e t o i m m e di a t e l y s e e t ha t t he r e a r e dr a m a t i c di f f e r e nc e s t ha t e xi s t w i t hi n t he p r oc e s s e s t ha t a r e be i ng unde r t a ke n by t he c e l l s a s i de nt i f i e d by t he s e 46 pr ot e i ns I n t he m a t ur e N K c e l l w e s e e t ha t a s w oul d be e xpe c t e d, t he l a r ge s t a m o unt of t he f unc t i on i s de vot e d t o s i gna l i ng. W i t hi n t hi s f unc t i ona l c a t e gor y w e ha ve i nc l ude d F G F R J A K 3, a nd F A K 1. W he r e i n t he i m m a t ur e N K c e l l w e s e e t he l a r ge s t f unc t i ona l c a t e gor y i s c e l l

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86 gr ow t h/ m a i nt e na nc e T hr ough l ooki ng a t t he b r oa d f unc t i ona l di f f e r e nc e s w e c a n s e e t he di f f e r e nc e s i n t he f unc t i ons t he s e t w o c e l l t ype s ut i l i z e t o c a r r y out t he i r uni que f unc t i ons F i gu r e 21. F u n c t i on al p i e c h ar t s f or t h e p e p t i d e s d i f f e r e n t i a l l y p h os p h or yl a t e d b e t w e e n m at u r e an d i m m a t u r e N K c e l l s A ) M a t ur e N K c e l l s B ) I m m a t ur e N K c e l l s D i s c u s s i on I n t hi s s t udy t h r ough t he us e of t he P e pC hi p w e ha ve ge ne r a t e d a ki nom e f or bot h t he i m m a t u r e a nd m a t u r e N K c e l l W e ha ve t he n c om pa r e d t he s e ki nom e s a nd i de nt i f i e d 46 pe pt i de s t ha t a r e di f f e r e nt i a l l y phos phor yl a t e d be t w e e n t he s e 2 c e l l t ype s W e ha ve t he n e xpl or e d t he bi ol ogi c a l f unc t i on t ha t t he s e 46 pr ot e i ns a r e i nvol ve d i n a n a t t e m pt t o i de nt i f y pos s i bl e s i gna l i ng di f f e r e nc e s t ha t e xi s t s be t w e e n t he s e de ve l opm e nt a l s t a ge s

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87 C e r t a i n pe pt i de s ha ve pr ove n t o be e xt r e m e l y i nt e r e s t i ng a nd m a y l e nd t o a be t t e r unde r s t a ndi ng of t he pa t hw a ys t ha t a r e ke y t o t he s e t w o c e l l t ype s T he i nc r e a s e d phos phor yl a t i on of J A K 3 i n t he m a t u r e N K c e l l i s a ve r y pos i t i ve f i ndi ng. T he ne e d f or I L 15/ 2R i n N K c e l l a c t i va t i on i s w e l l de f i ne d ( 2 05) N K c e l l s s pe c i f i c a l l y e xpr e s s t he # c ha i n of t he I L 2/ 15R I t ha s be e n s how n t ha t m i c e de f i c i e nt i n t he # c ha i n do not pr oduc e m a t ur e N K c e l l s ( 204, 205) J A K 3 ha s be e n s how n t o a s s oc i a t e w i t h t he # c ha i n i n N K c e l l s I t ha s a l s o be e n s how n t ha t m i c e l a c ki ng J A K 3 do not pr oduc e m a t ur e N K c e l l s r a t he r t he i r N K c e l l s a r e s t uc k i n a n i m m a t ur e s t a t e ( 206) T hi s s how s t he pi vot a l r ol e of J A K 3 i n N K c e l l s p r ovi di ng a s t r ong p r oof of c onc e pt of t hi s s ys t e m a s J A K 3 i s f ound t o ha ve a n ove r a l l hi gh l e ve l of phos phor yl a t i on a s w e l l a s be i ng s i gni f i c a nt l y m or e phos phor yl a t e d i n t he m a t u r e N K c e l l I n t he m a t u r e N K c e l l w e s e e t ha t t he r e i s t he i nc r e a s e d phos phor yl a t i on of t w o gr ow t h f a c t or r e c e pt or s F G F R 3 a nd 4 B e t w e e n t he s e 2 r e c e pt or s t he r e i s t he a bi l i t y t o bi nd a ppr oxi m a t e l y 10 of t he 22 di f f e r e nt F G F ( 29 7) F G F R ha ve be e n s how n t o pl a y r ol e s f r om e m br yoni c de ve l opm e nt t o t h r oughout t he a dul t a ni m a l W i t hi n t he s e va r i ous s t a ge s t he F G F ha ve be e n s how n t o pl a y a r o l e i n n um e r ous f unc t i ons i nc l udi ng pr ol i f e r a t i on di f f e r e nt i a t i on a nd m i gr a t i on ( 297) W i t hi n t he c ur r e nt l i t e r a t ur e t he r e ha s ye t t o be a ny de s c r i pt i on of a r ol e of F G F i n i m m a t ur e or m a t ur e N K c e l l s T hi s t he r e f o r e m a y r e pr e s e nt a nove l f i ndi ng i de nt i f yi ng a pos s i bl e r ol e f o r F G F R i n t he r e gul a t i on of N K c e l l s

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88 T he N F % B pa t hw a y ha s be e n s how n t o pl a y a va r i e t y of r ol e s i n t he i m m une s ys t e m i nc l udi ng N F % B m i c e ha vi ng a de c r e a s e d i m m une r e s pons e ( 298) T he p50 pr e c ur s or p105 w hi c h ha s a n i nc r e a s e d phos phor yl a t i on i n i m m a t ur e N K c e l l s ha s be e n s how n t o ha ve a num be r of uni que r o l e s w i t hi n t he i m m une s ys t e m ( 298 ) p105 t hr ough i t s a s s oc i a t i on w i t h ot he r s i gna l i ng m ol e c ul e s ha s be e n s how n t o f unc t i on i n t he M A P / E R K pa t hw a y, s pe c i f i c a l l y ups t r e a m o f M E K ( 2 99) M E K a s w e l l a s t he p105 s ubuni t ha ve be e n f ound i n t hi s s t udy t o ha ve a hi ghe r l e ve l of phos phor yl a t i on i n t he i m m a t ur e N K c e l l c om pa r e d t o t he m a t ur e c e l l T hi s pa t hw a y of p105 t o M E K a nd t he n f ur t he r dow ns t r e a m ha s be e n s how n t o e f f e c t num e r ous di f f e r e nt out c om e s i n i m m une c e l l s i nc l udi ng gr a nul e r e l e a s e i n t he N K c e l l ( 300) A l t hough w e c a n no t de t e r m i ne e xa c t l y w ha t out c om e w oul d be e l i c i t e d a t t hi s s t a ge i t i s a n e xt r e m e l y pos i t i ve f i ndi ng t o i de nt i f y t w o pr ot e i ns t ha t c a n f unc t i on up or dow n s t r e a m of e a c h ot he r i n t he s a m e phos phor yl a t i on s t a t e M a t ur e N K c e l l s a r e be l i e ve d t o be i n a hom e os t a t i c s t a t e m os t of t he t i m e i n t e r m s of t he r e pr ol i f e r a t i ve pot e nt i a l w he r e t he i m m a t ur e N K c e l l w oul d m os t l i ke l y be c yc l i ng m or e of t e n i n or de r t o e f f e c t i t s m a t u r a t i on a nd e xpa ns i on ( 301) T he i nc r e a s e d phos phor yl a t i on of 14 3 3 f i t s i n w i t h t hi s c yc l i ng s t a t e of t he i m m a t u r e N K c e l l 14 3 3 w hi c h c a n be up r e gul a t e d by p53 c a n r e gul a t e t he G 2/ M pr ogr e s s i on of t he c e l l c yc l e ( 302, 303 ) I t doe s t hi s by s e que s t e r i ng C D C 2/ c yc l i n B c om pl e xe s i n t he c yt op l a s m t hus i nhi bi t i n g t he c e l l f r om p r ogr e s s i ng t hr ough t he r e m a i nde r of t he c e l l c yc l e ( 302, 303) T hi s m a y t he r e f o r e be a ke y r e g ul a t o r of t he N K c e l l s pr ol i f e r a t i ve s t a t e

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89 A l t hough t he f unc t i on of m a ny o f t he s e pr ot e i ns a n d/ or pa t hw a ys a r e w e l l know n i n ot he r c e l l t ype s t he i r r ol e s w i t hi n N K c e l l s i s r e l a t i ve l y unde t e r m i ne d. T hi s s t udy ha s t he r e f or e a l l ow e d us t o i de nt i f y s o m e pos s i bl e pa t h w a ys t ha t m a y be f unc t i on i ng w i t hi n N K c e l l s I t i s a l s o i m por t a nt t o not e t ha t a l t hough w e ha ve c onc e nt r a t e d on t he s ubs t r a t e s t ha t a r e be i ng phos phor yl a t e d i n t hi s s t u dy i t i s i m por t a nt t o not e t ha t t hi s i s not t he onl y m a nne r i n w hi c h t he ki nom e c hi ps c a n be a na l yz e d. I t i s a l s o pos s i bl e t o e xpl o r e di f f e r e nt i a l k i na s e a c t i vi t y r a t he r t ha n di f f e r e nt i a l phos phor yl a t i on. I n t e r m s of t hi s pr oc e s s w e w oul d be a bl e t o de f i ne or a t l e a s t m a k e a pr oba bl y e s t i m a t i on of t he ki na s e phos phor yl a t i n g e a c h pe pt i de W e c oul d t he n gr ou p t he pe pt i de s vi a t he i r ki na s e a nd de t e r m i ne i f c e r t a i n ki na s e s a r e m or e or l e s s a c t i ve T hi s m a y pr ove us e f ul i n a t t e m pt i ng t o be t t e r e l uc i da t e pa t hw a ys a s oppos e d t o i ndi vi du a l pr ot e i ns M at e r i al s an d M e t h od s A ni m al s A l l m i c e us e d i n t hi s s t udy w e r e C 57B L 6/ J m i c e o f 8 10 w e e ks of a ge O n t he da y of c e l l i s ol a t i on t he s pl e e n a nd B M w e r e r e m o ve d f r om s uf f i c i e nt a ni m a l s t o obt a i n a m i ni m um of 250, 000 pur e c e l l s C e l l Sor t i ng F or m a t ur e N K c e l l s s pl e e ns w e r e r e m ov e d a nd m a de i nt o a s i ngl e c e l l s us pe n s i on. W hol e s pl e noc yt e s w e r e r e d bl ood c e l l l ys e d f or 5 m i nut e s a t r oom t e m pe r a t ur e i n R B C l ys i s buf f e r c ons i s t i ng of 0. 15 M N H 4 C l 10m M K H C O 3 a nd 0. 1m M E D T A C e l l s w e r e s pun dow n a t 300xG f o r 5 m i nut e s a nd t he n r e s u s pe nde d a t

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90 1x10 6 c e l l s / 50 L i n s t a i ni ng m e di a S t a i ni ng m e di a c ons i s t s of P B S w i t h 3% F B S a nd H E P E S C e l l s w e r e t he n F c bl oc ke d f or 15 m i nu t e s on i c e w i t h a nt i C D 16/ 32 a nt i body C e l l s w e r e t he n s t a i ne d f or s or t i ng w i t h N K 1. 1 + C D 3 G r 1 I gM a nd D A P I C e l l s w e r e s t a i ne d f or 15 m i nut e s on i c e a nd t he n w a s he d t w i c e w i t h s t a i ni ng m e di a C e l l s w e r e r e s us pe nde d i n s t a i ni ng m e di a w i t h 1 % F B S f or s or t i ng. A l l c e l l s or t i ng w a s pe r f or m e d on a F A C S A r i a ( B e c kt on D i c ks on) S or t s w e r e p e r f or m e d w i t h a 70 M noz z l e a t a r a t e of 1000 5000 c e l l s / s e c ond. A l l s a m pl e s w e r e ke pt a t 4 C f o r t he dur a t i on of t he s or t i nc l udi ng t he s or t e d c e l l s w hi c h w e r e s or t e d i n t o s t a i ni ng m e di a c ont a i ni ng 3 % F B S F or i m m a t u r e N K c e l l s B M w a s f l us he d f r om f e m ur s of m i c e a nd pr e pa r e d t he s a m e a s s pl e e n c e l l s I m m a t u r e N K c e l l s s or t e d on t he ba s i s of N K 1. 1 + C D 3 G r 1 I gM M a c 1 / l o A l l s or t i ng w a s pe r f or m e d on a F A C S A r i a ( B e c kt on D i c ks on) P e pC hi p as s ay s F or ki nom e a r r a y s a m pl e s a m i n i m um o f 250 000 c e l l s w e r e s or t e d f o r e a c h of t he t hr e e c e l l ki nom e r e pl i c a t e s C e l l s w e r e l ys e d i n c e l l l ys i s buf f e r c ons i s t i ng of 20m M T r i s H C l ( pH 7. 5 ) 150m M N a C l 1m M N a 2 E D T A 1m M E G T A 1% T r i t on X 100, 2. 5m M s odi um pyr ophos pha t e 1m M M gC l 2 1 m M gl yc e r ophos pha t e 1m M N a 3 V O 4 1m M N a F 1 g/ m l l e upe pt i n ( S i gm a A l d r i c h) 1 g/ m l a pr ot i ni n ( S i gm a A l dr i c h ) 1m M P M S F V ol um e s of t he c e l l l ys a t e s w e r e e qua l i z e d w i t h di H 2 O T he c e l l l ys a t e s w e r e t he n pa s s e d t hr ough a 0. 22 m l ow pr ot e i n bi ndi n g f i l t e r 10 L of a c t i va t i on bu f f e r w a s t he n a dd e d t o t he f i l t e r e d W C L A c t i va t i on m i x c o ns i s t s of 50% gl yc e r ol 50 M A T P 0. 05% v/ v B r i j 35, 0. 25 m g/ m l bovi ne s e r um a l bu m i n, 3 3 P # A T P ( 1000 kB q) T he pe pt i de a r r a y m i x w a s t he n a dde d t o t he P e pC hi p a nd i t w a s t he n i nc uba t e d a t 37C i n

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91 a hum i di f i e d s t ove f or 90 m i nut e s T he pe pt i de a r r a y w a s w a s he d t w i c e w i t h T r i s buf f e r e d s a l i ne w i t h 1% T w e e n 20 t he n t w i c e i n 2 M N a C l a nd t he n t w i c e i n de m i ne r a l i z e d H 2 O a nd f i na l l y a i r dr i e d. T he c hi p s w e r e e xpos e d i n a phos pho i m a gi ng c a s s e t t e f or 72 hour s A f t e r t he 72 hour s t he phos p ho i m a gi ng c a s s e t t e s w e r e s c a nne d us i ng a S t or m P hos pho i m a ge r ( G E H e a l t hc a r e ) F r om t he S t or m i m a ge r w e obt a i ne d hi gh r e s ol ut i on i m a ge f i l e s t ha t w e r e i m por t e d i nt o t he S c a na l yz e s of t w a r e p r ogr a m ( L a w r e nc e B e r kl e y N a t i ona l L a bor a t or y C A ) f or a na l ys i s T o obt a i n m e di a n s pot de ns i t y a 28 X 42 gr i d w a s ove r l a i d ont o t he P e pC hi p t ha t de l i ne a t e d e a c h i ndi vi dua l pe pt i de s pot T he S c a na l yz e pr ogr a m t he n c a l c ul a t e s t he m e di a n s pot de ns i t y f o r e a c h s qua r e w i t hi n t he g r i d t he r e by pr ovi di ng a va l ue f o r t he phos phor yl a t i on l e ve l f or e a c h pe pt i de A f t e r t he m e di a n s pot de ns i t i e s w e r e obt a i ne d f or e a c h s pot on t he c h i p t he da t a w a s nor m a l i z e d a c r os s e a c h c hi p. N o r m a l i z a t i on w a s a c hi e ve d by c or r e c t i on of t he s pot de ns i t y f or t he i ndi vi dua l ba c kgr ound t o di m i ni s h i nt e r a r r a y va r i a nc e s t he r e by nor m a l i z i ng t he t o t a l phos phor yl a t i on of t he P e pC hi p & t o be e qua l be t w e e n a l l s a m pl e s T hi s w a s done by t a ki ng t he phos phor yl a t i on va l ue f or a n i ndi vi dua l s pot di vi di ng i t by t he s um of t he t ot a l phos phor yl a t i on of a l l s pot s on t he c hi p a nd t he n m ul t i pl yi ng t ha t by t he t ot a l num be r of pe pt i de s ( 1176) I n or de r t o be i nc l ude d i n t he ki nom e a na l ys i s t he m e a n phos phor yl a t i on be t w e e n t he t w o r e pl i c a t e s on one P e pC hi p & ha d t o ha ve a c or r e l a t i on va l ue o f > + 0. 85 I n a ddi t i on a ny da t a t ha t w a s i nc ons i s t e nt ( i e S E M be t w e e n da t a poi nt s > 1. 96) w e r e e xc l ude d f r om f u r t he r a na l ys i s B ot h t he c or r e l a t i on a nd s t a nda r d de vi a t i on w e r e c a l c ul a t e d us i ng M i c r os of t E xc e l us i ng t he f ol l ow i ng f o r m ul a s ;

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92 f or t he c or r e l a t i on c oe f f i c i e nt a nd f o r s t a nda r d de vi a t i on of t he m e a n ( S E M ) W he r e x o r y i s t he va l ue on t he a r r a y be i ng c om pa r e d a nd n i s t he nu m be r of r e pl i c a t e s us e d i n t he c a l c ul a t i on

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93 C ha pt e r 5 D i s c us s i on I n t he s e s t udi e s w e ha ve a t t e m pt e d t o be t t e r e l uc i d a t e t he s i gna l i ng m e c ha ni s m s us e d by t he N K c e l l T o t hi s e nd w e ha ve ut i l i z e d a br oa dl y s w e e pi ng a ppr oa c h t h r ough t he e xa m i na t i on of t he ki nom e of bo t h m a t ur e a nd i m m a t ur e N K c e l l s W e ha ve a l s o e xa m i ne d N K c e l l s i gna l i ng w i t hi n t he m o r e s pe c i f i c s i gna l i ng c ont e xt of S H I P i n t he N K c e l l T hr ough t he s e m e t hods w e ha ve i de nt i f i e d po s s i bl e nove l s i gna l i ng m e c ha ni s m s ut i l i z e d by t he N K c e l l f r om t he s t udy of t he ki no m e I n t he s t udy of S H I P i n t he N K c e l l w e ha ve be e n a bl e t o t a ke a n i ni t i a l obs e r va t i on of 2B 4 ove r e xpr e s s i on i n t he S H I P / N K c e l l de t e r m i ne t he f unc t i ona l c ons e que nc e of t hi s r e c e pt or ove r e xpr e s s i on a s N K hypor e s pons i ve ne s s a nd f i na l l y de t e r m i ne t he m ol e c ul a r m e c ha ni s m be hi nd t hi s hypor e s pons i ve ne s s a s S H P 1 ove r r e c r ui t m e nt T he r e s ul t s w e ha ve obt a i ne d f r om our s t udy of S H I P i n t he N K c e l l ha ve l e d us t o f or m a hypot he s i s w he r e N K c e l l ba s e d t r e a t m e nt s c oul d pos s i bl y be de ve l ope d. W he n w e l ook a t t he r ol e o f N K c e l l s w i t hi n t he r e a l m o f t um or r e gul a t i on w e s e e t ha t t u m or c e l l s ha ve de ve l ope d a num be r o f hur d l e s i n or de r t o c i r c um ve nt s ur ve i l l a nc e vi a t he N K c e l l W ha t e ve r m e t hod a t um or ut i l i z e s t o e s c a pe s ur ve i l l a nc e by a n N K c e l l i n t he e nd i t a t t e m pt s t o t i p t he ba l a nc e of a c t i va t i ng a nd i nh i bi t or y s i gna l s t o t he i nhi bi t or y s i de T he

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94 ke y t he n i s t o i de nt i f y a m e t hod t ha t w i l l a l l ow a n N K c e l l t o ove r c om e t he s e ba r r i e r s a nd t i p t he ba l a nc e ba c k t o a c t i va t i on. O ne pos s i bi l i t y i s t o ut i l i z e a t e m por a r y S H I P a bl a t i on i n or de r t o a l t e r t he N K R r e pe r t oi r e A s w e ha ve s how n i n F i g 5 t hi s c oul d l e a d t o a s i gni f i c a nt dow n r e gul a t i on of M H C s pe c i f i c i nhi bi t or y r e c e pt or s T hi s w oul d t he n a l l ow t he ba l a nc e of i nhi bi t or y a nd a c t i va t i ng s i gna l s t o be s ke w e d s uc h t ha t t he a c t i va t i ng s i gna l s m a y be a bl e t o p r e dom i na t e I n t hi s s t udy w e ha ve ut i l i z e d a S H I P 1 K O m ous e i n or de r t o e f f e c t t he a l t e r a t i on of N K r e c e pt or r e pe r t oi r e I n a c l i ni c a l s e t t i ng w e w oul d e nvi s i on t he us e of a s m a l l m ol e c ul e i nhi bi t o r t ha t w oul d be a bl e t o bl oc k S H I P 1. I n doi ng s o w e w oul d be a bl e t o r e ve r s i bl y a l t e r t he N K r e c e pt or r e pe r t oi r e T hi s ha s gr e a t pot e nt i a l e ve n i n t he p r e s e nc e of di m i ni s he d a c t i va t i ng or i nc r e a s e d i nhi bi t or y l i ga nds due t o t he f a c t t ha t w i t h l ow e r e d M H C s pe c i f i c i nhi bi t o r y r e c e pt or s e ve n r e l a t i ve l y l ow a m ount s of a c t i va t i ng l i ga nds w oul d pr e dom i na t e A c onc e r n of t hi s m e t hod w oul d be t ha t t he l os s of i n hi bi t or y r e c e pt or s w oul d a l l ow a ut or e a c t i vi t y, a l t hough t hi s i s not t he l i ke l y out c o m e A s ha s be e n di s c us s e d s i gna l i ng w i t hi n a n N K c e l l i s t w o pa r t p r oc e s s c ons i s t i ng of bot h a c t i va t i ng a nd i nhi bi t o r y s i gna l s T he r e f or e t he l a c k of i nhi bi t o r y s i gna l a l one doe s not e l i c i t c e l l l ys i s a n a c t i va t i ng s i gna l a l t hough pos s i bl y a ve r y m i nut e s i gna l i s s t i l l ne e de d, t he r e f o r e nor m a l c e l l s w i t hout s t r e s s i nduc i bl e l i ga nds pr e s e nt w oul d s t i l l be p r ot e c t e d. T he us e of S H I P i nhi bi t i on a l one m a y w e l l not be s uf f i c i e nt t o e f f e c t t he de s i r e d r e s pons e A s our w o r k ha s s how n ot he r i nhi bi t o r y r e c e pt or s m a y be a bl e t o c om pe ns a t e f or t he l os s of L y49 e xpr e s s i on, i n our s t udi e s 2B 4 ha s pr ove n ve r y a de pt a t t hi s r ol e

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95 W i t hi n t he hum a n s ys t e m i t m a y not be t he s a m e s i t ua t i on a s i n t he hum a n 2B 4 us ua l l y f unc t i ons a s a n a c t i va t i ng r e c e pt or T hi s i s due t o t he r e c r ui t m e nt o f S A P t o 2B 4. A l t hough i t i s i m por t a nt t o not e t ha t w i t hi n t he hu m a n s ys t e m 2B 4 c a n f unc t i on a s a n i nhi bi t or y r e c e pt or f or i ns t a nc e w i t hi n l ym ph nod e de r i ve d N K c e l l s I n a ddi t i on a s w e ha ve s how n i n t hi s s t udy t he i nhi bi t i on of S H I P i s a bl e t o not on l y a l t e r t he N K r e pe r t oi r e but i t i s a l s o a bl e t o qua l i t a t i ve l y a l t e r t he 2B 4 r e c e pt or c om pl e x s uc h t ha t 2B 4, e ve n i n t he hum a n, c oul d be l oc ke d i n t o a do m i na nt i nhi bi t or y r ol e T he r e f or e w i t hi n t he hum a n s ys t e m e i t he r 2B 4 or a n a l t e r na t e i nhi bi t or y r e c e pt or m a y l oc k c e l l s i nt o a hypo r e s pons i ve s t a t e l uc ki l y a s ha s be e n di s c us s e d N K c e l l s ut i l i z e ve r y r e dunda nt s i gna l i ng pa t hw a ys e s pe c i a l l y t o e l i c i t a n i nh i bi t or y s i gna l T he r e f or e w e pr opos e t ha t dua l or t a nde m i nhi bi t i on o f S H I P 1 a nd S H P 1 m i ght be us e d t o t e m por a r i l y i nc r e a s e N K c l e a r a nc e of t um or c e l l s T he f i r s t s t e p w oul d be t o i nhi bi t S H I P t o c r e a t e a n N K c om pa r t m e nt t ha t i s ove r l y de pe nde nt on one or l i m i t e d num be r o f i nh i bi t or y r e c e pt or s t ha t w oul d l i m i t t um o r ki l l i ng. T hi s c oul d t he n be f ol l ow e d by t r e a t m e nt w i t h a n S H P 1 i nhi bi t or t o unl e a s h t he ki l l i ng c a pa c i t y of t he N K c om pa r t m e nt a ga i ns t t um or c e l l s A l t hough i n t hi s s t udy w e us e d a S H I P / N K c e l l s a nd c he m i c a l i nhi bi t i on o f S H P 1 a c t i vi t y, i t m a y be pos s i bl e t o r e ve r s i bl y i nhi bi t S H I P a nd S H P 1 us i ng R N A i n t e r f e r e nc e a nd/ or c he m i c a l i nhi bi t o r s T h r ough t hi s i t m a y be pos s i bl e t o r e ve r s i bl y i nh i bi t bot h of t he s e pr ot e i ns t he r e by unl e a s hi n g t he s t r ong c yt ot o xi c pot e nt i a l of t he N K c e l l on t r a ns f or m e d c e l l s

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96 R e f e r e n c e s 1. C ol l a vo, D A C ol om ba t t i a nd L C hi e c o B i a nc hi 1974 T l ym phoc yt e r e qui r e m e nt f o r M S V t u m our p r e ve nt i on or r e gr e s s i on. N at ur e 249: 169 170 2. G or c z yns ki R M 1974. I m m uni t y t o m ur i ne s a r c om a vi r us i nduc t e d t um o r s I I S uppr e s s i on of T c e l l m e di a t e d i m m uni t y by c e l l s f r om p r ogr e s s or a ni m a l s J I m m unol 112: 1826 1838 3. G or c z yns ki R M 1974. I m m uni t y t o m ur i ne s a r c om a vi r us i nduc t e d t um o r s I S pe c i f i c T l ym phoc yt e s a c t i ve i n m a c r opha ge m i gr a t i on i nhi bi t i on a nd l ym phoc yt e t r a ns f or m a t i on J I m m unol 112 : 1815 1825 4. G or c z yns ki R M a nd C N or b ur y. 1974. I m m un i t y t o m u r i ne s a r c om a vi r us i nduc e d t um our s 3 A na l ys i s of t he c e l l popul a t i on s i nvol ve d i n p r ot e c t i on f r om l e t ha l t um our pr ogr e s s i on of s ubl e t ha l l y i r r a di a t e d, M S V i noc ul a t e d, m i c e B r J C anc e r 30: 118 128 5. B e r e ns on, J R A B E i ns t e i n, J r a nd A F e f e r 1 975. S ynge ne i c a dopt i ve i m m unot he r a py a nd c he m oi m m unot he r a py of a F r i e nd l e uke m i a : r e qui r e m e nt f or T c e l l s J I m m unol 115 : 234 238 6. G l a s e r M D H L a vr i n a nd R B H e r be r m a n. 19 76. I n vi vo p r ot e c t i on a ga i ns t s ynge ne i c G r os s vi r us i nduc e d l ym phom a i n r a t s : c om pa r i s on w i t h i n vi t r o s t udi e s of c e l l m e di a t e d i m m uni t y. J I m m unol 116: 1507 1 511 7. M ol l e r R A N i e l s e n, a nd F R e ym a nn. 1975 M u l t i pl e ba s a l c e l l c a r c i nom a a nd i nt e r na l m a l i gna nt t um or s A r c h D e r m at ol 111: 584 585 8. O ut z e n, H C R P C us t e r G J E a t on, a nd R T P r e hn. 1975 S pont a ne ous a nd i nduc e d t um or i nc i de nc e i n ge r m f r e e nude m i c e J R e t i c ul oe ndot he l Soc 17: 1 9 9. S c hw a r t z R S 1975. A not he r l ook a t i m m unol ogi c s ur ve i l l a nc e N E ng l J M e d 293: 181 184 10. H e l l s t r om K E a nd I H e l l s t r om 1974. L ym phoc yt e m e di a t e d c yt ot oxi c i t y a nd bl oc ki ng s e r um a c t i vi t y t o t um o r a nt i ge ns A dv I m m unol 18: 209 277

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97 11. H e r be r m a n, R B J L M c C oy, a nd P H L e vi ne 1974. I m m unol ogi c a l r e a c t i ons t o t um or a s s oc i a t e d a nt i ge ns i n B ur ki t t s l ym phom a a nd ot he r l ym phom a s C anc e r R e s 34: 1222 1227 12. O l dha m R K a nd R B H e r be r m a n. 1973. E va l u a t i on of c e l l m e di a t e d c yt ot oxi c r e a c t i vi t y a ga i ns t t um or a s s oc i a t e d a nt i ge ns w i t h 125I i odode oxyur i di ne l a be l e d t a r ge t c e l l s J I m m unol 111 : 862 871 13. R os e nbe r g, E B R B H e r be r m a n P H L e vi ne R H H a l t e r m a n, J L M c C oy, a nd J R W unde r l i c h. 1972. L ym phoc yt e c yt ot ox i c i t y r e a c t i ons t o l e uke m i a a s s oc i a t e d a nt i ge n s i n i de nt i c a l t w i ns I nt J C anc e r 9: 648 658 14. T a ka s ugi M M R M i c ke y, a nd P I T e r a s a ki 19 73. R e a c t i vi t y of l ym phoc yt e s f r om no r m a l pe r s ons o n c ul t ur e d t um o r c e l l s C anc e r R e s 33: 2898 2902 15. H e r be r m a n, R B M E N unn, H T H ol de n a nd D H L a vr i n. 1975. N a t ur a l c yt ot oxi c r e a c t i vi t y o f m ous e l ym phoi d c e l l s a ga i n s t s ynge ne i c a nd a l l oge ne i c t um or s I I C ha r a c t e r i z a t i on of e f f e c t or c e l l s I nt J C anc e r 16: 230 239 16. H e r be r m a n, R B M E N unn, a nd D H L a vr i n. 1975. N a t ur a l c yt ot oxi c r e a c t i vi t y of m ous e l ym phoi d c e l l s a ga i ns t s ynge n e i c a c i d a l l oge ne i c t um or s I D i s t r i but i on of r e a c t i vi t y a nd s pe c i f i c i t y. I nt J C anc e r 16: 216 229 17. K i e s s l i ng, R E K l e i n, a nd H W i gz e l l 1975. N a t ur a l ki l l e r c e l l s i n t he m ous e I C yt ot oxi c c e l l s w i t h s pe c i f i c i t y f or m ous e M ol one y l e uke m i a c e l l s S pe c i f i c i t y a nd di s t r i but i on a c c or di ng t o ge not ype E ur J I m m unol 5: 112 117 18. K i e s s l i ng, R E K l e i n, H P r os s a nd H W i gz e l l 1975. N a t ur a l ki l l e r c e l l s i n t he m ous e I I C yt ot oxi c c e l l s w i t h s pe c i f i c i t y f or m ous e M ol one y l e uke m i a c e l l s C ha r a c t e r i s t i c s of t he ki l l e r c e l l E ur J I m m unol 5 : 117 121 19. S e ndo, F T A oki E A B oys e a nd C K B u a f o 1975. N a t ur a l oc c ur r e nc e of l ym phoc yt e s s how i ng c yt ot oxi c a c t i vi t y t o B A L B / c r a di a t i on i nduc e d l e uke m i a R L m a l e 1 c e l l s J N at l C anc e r I ns t 55: 603 609 20. O hl e n, C G K l i ng P H ogl und M H a ns s on, G S c a ngos C B i e be r i c h, G J a y, a nd K K a r r e 1 989 P r e ve nt i on o f a l l oge ne i c bone m a r r ow gr a f t r e j e c t i on by H 2 t r a ns ge ne i n donor m i c e Sc i e nc e 246 : 666 668 21. L j unggr e n, H G a nd K K a r r e 1990. I n s e a r c h o f t he 'm i s s i ng s e l f ': M H C m ol e c ul e s a nd N K c e l l r e c ogni t i on I m m unol T oda y 11: 237 244 22. C udkow i c z G a nd M B e nne t t 1971 P e c ul i a r i m m unobi ol ogy of bone m a r r ow a l l ogr a f t s I I R e j e c t i on of pa r e nt a l g r a f t s by r e s i s t a nt F 1 hybr i d m i c e J E x p M e d 134: 1513 1528

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98 23. C udkow i c z G a nd M B e nne t t 1971 P e c ul i a r i m m unobi ol ogy of bone m a r r ow a l l ogr a f t s I G r a f t r e j e c t i on by i r r a di a t e d r e s ponde r m i c e J E x p M e d 134 : 83 102 24. S a nt ol i D G T r i nc hi e r i L M or e t t a C M Z m i j e w s ki a nd H K opr ow s ki 1978. S pont a ne ous c e l l m e di a t e d c yt ot oxi c i t y i n hum a ns D i s t r i but i on a nd c ha r a c t e r i z a t i on of t he e f f e c t or c e l l C l i n E x p I m m unol 33: 309 318 25. T r i nc hi e r i G D S a nt ol i a nd H K opr ow s ki 1978 S pont a ne ous c e l l m e di a t e d c yt ot oxi c i t y i n hum a ns : r ol e o f i nt e r f e r on a nd i m m unogl obul i ns J I m m unol 120: 1849 1855 26. B e c ke r S R K i e s s l i ng, N L e e a nd G K l e i n 197 8. M odul a t i on o f s e ns i t i vi t y t o na t ur a l ki l l e r c e l l l ys i s a f t e r i n v i t r o e xpl a nt a t i on of a m ous e l ym phom a J N at l C anc e r I ns t 61: 1495 1498 27. K unke l L A a nd R M W e l s h. 1981 M e t a bol i c i nhi bi t or s r e nde r r e s i s t a nt t a r ge t c e l l s s e ns i t i ve t o na t ur a l k i l l e r c e l l m e di a t e d l ys i s I nt J C anc e r 27: 73 79 28. S t e r n, P M G i dl und, A O r n a nd H W i gz e l l 198 0. N a t ur a l ki l l e r c e l l s m e di a t e l ys i s of e m br yona l c a r c i nom a c e l l s l a c ki ng M H C N at ur e 285: 341 342 29. H ogl und, P H G L j ung gr e n C O hl e n, L A hr l un d R i c ht e r G S c a ngos C B i e be r i c h, G J a y, G K l e i n, a nd K K a r r e 1988 N a t ur a l r e s i s t a nc e a ga i ns t l ym phom a gr a f t s c onve ye d by H 2D d t r a ns ge ne t o C 57B L m i c e J E x p M e d 168: 1469 1474 30. Y okoya m a W M P J K e hn D I C ohe n a nd E M S he va c h. 1990. C hr om os om a l l oc a t i on of t he L y 49 ( A 1, Y E 1/ 48 ) m ul t i ge ne f a m i l y G e ne t i c a s s oc i a t i on w i t h t he N K 1. 1 a nt i ge n. J I m m unol 14 5: 2353 2358 31. K a r l hof e r F M R K R i ba udo a nd W M Y oko ya m a 1992. M H C c l a s s I a l l oa nt i ge n s pe c i f i c i t y of L y 49+ I L 2 a c t i va t e d na t ur a l ki l l e r c e l l s N at ur e 358: 66 70 32. B i x, M N S L i a o, M Z i j l s t r a J L or i ng, R J a e ni s c h, a nd D R a ul e t 1991 R e j e c t i on of c l a s s I M H C de f i c i e nt ha e m opoi e t i c c e l l s by i r r a di a t e d M H C m a t c he d m i c e N at ur e 349: 329 331 33. L j unggr e n, H G L V a n K a e r H L P l oe gh a nd S T one ga w a 1994 A l t e r e d na t ur a l ki l l e r c e l l r e pe r t o i r e i n T a p 1 m ut a nt m i c e P r oc N at l A c ad Sc i U S A 91: 6520 6524 34. M a s on, L H J R O r t a l do H A Y oung V K um a r M B e nne t t a nd S K A nde r s on. 1995. C l oni ng a nd f unc t i ona l c ha r a c t e r i s t i c s of m ur i ne l a r ge gr a nul a r

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99 l ym phoc yt e 1: a m e m be r of t he L y 49 ge ne f a m i l y ( L y 49G 2) J E x p M e d 182: 293 303 35. S t one m a n, E R M B e nne t t J A n K A C he s nut E K W a ke l a nd, J B S c he e r e r M J S i c i l i a no, V K um a r a nd P A M a t he w 1995. C l oni ng a nd c ha r a c t e r i z a t i on of 5 E 6( L y 49C ) a r e c e pt or m ol e c ul e e xpr e s s e d on a s ubs e t of m ur i ne na t ur a l ki l l e r c e l l s J E x p M e d 182: 305 31 3 36. Y okoya m a W M L B J a c obs O K a na ga w a E M S he va c h, a nd D I C ohe n. 1989. A m ur i ne T l ym phoc yt e a nt i ge n be l ongs t o a s upe r ge ne f a m i l y o f t ype I I i nt e gr a l m e m br a ne p r ot e i ns J I m m unol 143 : 1379 1386 37. G i or da R a nd M T r uc c o. 1991. M ous e N K R P 1. A f a m i l y of ge ne s s e l e c t i ve l y c oe xpr e s s e d i n a dhe r e nt l ym phok i ne a c t i va t e d ki l l e r c e l l s J I m m unol 147 : 1701 1708 38. Y okoya m a W M J C R ya n, J J H unt e r H R S m i t h, M S t a r k, a nd W E S e a m a n. 1991. c D N A c l oni ng o f m ous e N K R P 1 a nd ge ne t i c l i nka ge w i t h L Y 49 I de nt i f i c a t i on o f a na t u r a l ki l l e r c e l l ge ne c o m pl e x on m ous e c hr om os om e 6. J I m m unol 147: 3229 3236 39. B r e nna n, J S L e m i e ux J D F r e e m a n, D L M a g e r a nd F T a ke i 1996. H e t e r oge ne i t y a m ong L y 49C na t ur a l k i l l e r ( N K ) c e l l s : c ha r a c t e r i z a t i on of hi gh l y r e l a t e d r e c e pt or s w i t h d i f f e r i ng f unc t i ons a nd e xpr e s s i on pa t t e r ns J E x p M e d 184: 2085 2090 40. B r ow n, M G S F ul m e k K M a t s um ot o, R C ho, P A L yons E R L e vy, A A S c a l z o, a nd W M Y okoya m a 1997 A 2 M b Y A C c ont i g a nd phys i c a l m a p o f t he na t ur a l ki l l e r ge ne c om pl e x on m ous e c hr om os om e 6. G e nom i c s 42: 16 25 41. B r ow n, M G A A S c a l z o, K M a t s um ot o, a nd W M Y okoya m a 1997 T he na t ur a l ki l l e r ge ne c om pl e x: a ge ne t i c ba s i s f or un de r s t a ndi ng na t ur a l ki l l e r c e l l f unc t i on a nd i nna t e i m m uni t y I m m unol R e v 155 : 5 3 65 42. C ha n, P Y a n d F T a ke i 1989. M ol e c ul a r c l oni n g a nd c ha r a c t e r i z a t i on of a nove l m ur i ne T c e l l s ur f a c e a nt i ge n, Y E 1/ 48 J I m m unol 142: 1727 1736 43. W ong, S J D F r e e m a n, C K e l l e he r D M a ge r a nd F T a ke i 1991. L y 49 m ul t i ge ne f a m i l y N e w m e m be r s o f a s upe r f a m i l y of t ype I I m e m br a ne pr ot e i ns w i t h l e c t i n l i ke do m a i ns J I m m unol 147: 1417 142 3 44. Y okoya m a W M a nd B F P l ouga s t e l 2003 I m m une f unc t i ons e nc ode d by t he na t ur a l ki l l e r ge ne c om pl e x. N at R e v I m m unol 3 : 3 04 316

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100 45. Y okoya m a W M a nd W E S e a m a n. 1993. T he L y 49 a nd N K R P 1 ge ne f a m i l i e s e nc odi ng l e c t i n l i ke r e c e pt or s on na t u r a l k i l l e r c e l l s : t he N K ge ne c om pl e x. A nnu R e v I m m unol 11 : 613 635 46. S m i t h, K M J W u, A B B a kke r J H P hi l l i ps a nd L L L a ni e r 1998. L y 49D a nd L y 49H a s s oc i a t e w i t h m ous e D A P 12 a nd f o r m a c t i va t i ng r e c e pt or s J I m m unol 161: 7 10 47. M a s on, L H S K A nde r s on, W M Y okoya m a H R S m i t h R W i nkl e r P i c ke t t a nd J R O r t a l do. 1996. T he L y 49D r e c e p t or a c t i va t e s m ur i ne na t u r a l ki l l e r c e l l s J E x p M e d 184 : 21 19 2128 48. B r e nna n, J D M a ge r W J e f f e r i e s a nd F T a ke i 1994. E xp r e s s i on of di f f e r e nt m e m be r s of t he L y 49 ge ne f a m i l y de f i ne s di s t i nc t na t ur a l ki l l e r c e l l s ubs e t s a nd c e l l a dhe s i on pr ope r t i e s J E x p M e d 180: 2287 229 5 49. T a ke i F J B r e nna n a nd D L M a ge r 1997 T he L y 49 f a m i l y: ge ne s pr ot e i ns a nd r e c ogni t i on of c l a s s I M H C I m m unol R e v 155 : 67 77 50. D a ni e l s B F F M K a r l hof e r W E S e a m a n, a n d W M Y okoya m a 1994. A na t ur a l ki l l e r c e l l r e c e pt or s pe c i f i c f o r a m a j o r hi s t oc om pa t i bi l i t y c om pl e x c l a s s I m ol e c ul e J E x p M e d 180: 687 692 51. S e nt m a n, C L M Y O l s s on, M S a l c e do, P H og l und, U L e nda hl a nd K K a r e 1994. H 2 a l l e l e s pe c i f i c pr ot e c t i on f r om N K c e l l l ys i s i n vi t r o f or l ym phobl a s t s but not t um o r t a r ge t s P r ot e c t i on m e di a t e d by a l ph a 1/ a l pha 2 do m a i ns J I m m unol 153: 5482 5490 52. K a ne K P 1994. L y 49 m e di a t e s E L 4 l ym pho m a a dhe s i on t o i s ol a t e d c l a s s I m a j or hi s t oc om pa t i bi l i t y c om pl e x m ol e c ul e s J E x p M e d 179 : 1011 1015 53. Y u, Y Y T G e or ge J R D or f m a n, J R o l a nd, V K um a r a nd M B e nne t t 1996 T he r ol e of L y49A a nd 5E 6( L y49C ) m o l e c ul e s i n hybr i d r e s i s t a nc e m e di a t e d by m ur i ne na t ur a l ki l l e r c e l l s a ga i ns t nor m a l T c e l l bl a s t s I m m uni t y 4: 67 76 54. B r e nna n, J G M a hon, D L M a ge r W A J e f f e r i e s a nd F T a ke i 1996 R e c ogni t i on of c l a s s I m a j o r hi s t oc om pa t i bi l i t y c o m pl e x m ol e c ul e s by L y 49: s pe c i f i c i t i e s a nd dom a i n i nt e r a c t i ons J E x p M e d 1 83: 1553 1559 55. L i a n, R H Y L i S K ubot a D L M a ge r a nd F T a ke i 1999 R e c ogni t i on o f c l a s s I M H C by N K r e c e pt or L y 49C : i de nt i f i c a t i o n of c r i t i c a l r e s i due s J I m m unol 162: 7271 7276 56. H a nke T H T a ki z a w a C W M c M a hon, D H B us c h, E G P a m e r J D M i l l e r J D A l t m a n, Y L i u D C a do, F A L e m o nni e r P J B j or km a n a nd D

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101 H R a ul e t 1999 D i r e c t a s s e s s m e nt of M H C c l a s s I bi ndi ng by s e ve n L y49 i nhi bi t or y N K c e l l r e c e pt or s I m m uni t y 11 : 67 77 57. J oha ns s on, M H E H ogl und, M C N a ka m ur a J C R ya n, a nd P H ogl und 1998. A l pha 1/ a l pha 2 do m a i ns of H 2D ( d) but not H 2L ( d) i nduc e m i s s i ng s e l f r e a c t i vi t y i n vi vo no e f f e c t o f H 2 L ( d) on p r ot e c t i on a ga i ns t N K c e l l s e xpr e s s i ng t he i nhi bi t or y r e c e pt or L y49G 2. E ur J I m m unol 28 : 4198 4206 58. K ubot a A S K ubot a S L ohw a s s e r D L M a ge r a nd F T a ke i 1999. D i ve r s i t y of N K c e l l r e c e pt or r e pe r t oi r e i n a dul t a nd ne ona t a l m i c e J I m m unol 163: 212 216 59. H e l d, W a nd D H R a ul e t 1997. E xp r e s s i on of t h e L y49A ge ne i n m ur i ne na t ur a l ki l l e r c e l l c l one s i s pr e dom i na nt l y but not e xc l us i ve l y m ono a l l e l i c E ur J I m m unol 27: 2876 2884 60. H e l d, W J R ol a nd, a nd D H R a ul e t 1995. A l l e l i c e xc l us i on of L y49 f a m i l y ge ne s e nc odi ng c l a s s I M H C s pe c i f i c r e c e pt or s on N K c e l l s N at ur e 376: 355 358 61. S a l e h, A G E D a vi e s V P a s c a l P W W r i ght D L H odge E H C ho S J L oc ke t t M A bs ha r i a nd S K A nde r s on. 2004. I d e nt i f i c a t i on of pr oba bi l i s t i c t r a ns c r i pt i ona l s w i t c he s i n t he L y49 ge ne c l us t e r : a e uka r yot i c m e c ha ni s m f o r s e l e c t i ve ge ne a c t i va t i on. I m m uni t y 21 : 55 66 62. R a ul e t D H R E V a nc e a nd C W M c M a hon. 2001. R e gul a t i on of t he na t u r a l ki l l e r c e l l r e c e pt or r e pe r t oi r e A nnu R e v I m m unol 1 9: 291 330 63. V e i not t e L L B T W i l he l m D L M a ge r a nd F T a ke i 2003 A c qui s i t i on o f M H C s pe c i f i c r e c e pt or s on m ur i ne na t ur a l ki l l e r c e l l s C r i t R e v I m m unol 23: 251 266 64. G a ys F K M a r t i n, R K e ne f e c k, J G A us t a nd C G B r ooks 2005 M ul t i pl e c yt oki ne s r e gul a t e t he N K ge ne c om pl e x e nc ode d r e c e pt or r e pe r t oi r e of m a t ur e N K c e l l s a nd T c e l l s J I m m unol 175: 2938 2947 65. K i m S J P our s i ne L a ur e nt S M T r us c ot t L L y ba r ge r Y J S ong L Y a ng, A R F r e nc h, J B S unw oo S L e m i e ux, T H H a ns e n, a nd W M Y okoya m a 2005 L i c e ns i ng of na t ur a l ki l l e r c e l l s by hos t m a j or hi s t o c om pa t i bi l i t y c om pl e x c l a s s I m ol e c ul e s N at ur e 436: 709 713 66. F e r na nde z N C E T r e i ne r R E V a nc e A M J a m i e s on, S L e m i e ux, a nd D H R a ul e t 2005 A s ubs e t of na t ur a l ki l l e r c e l l s a c hi e ve s s e l f t ol e r a nc e w i t hout e xpr e s s i ng i nhi bi t or y r e c e pt or s s pe c i f i c f or s e l f M H C m ol e c ul e s B l ood 105: 4416 4423

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102 67. J oha ns s on, S M J oha ns s on, E R os m a r a ki G V a hl ne R M e hr M S a l m on D i von, F L e m onni e r K K a r r e a nd P H ogl und. 2 005. N a t ur a l ki l l e r c e l l e duc a t i on i n m i c e w i t h s i ngl e or m ul t i pl e m a j or h i s t oc om pa t i bi l i t y c om pl e x c l a s s I m ol e c ul e s J E x p M e d 201: 1145 1155 68. T or m o, J K N a t a r a j a n, D H M a r gul i e s a nd R A M a r i uz z a 1999 C r ys t a l s t r uc t ur e of a l e c t i n l i ke na t ur a l ki l l e r c e l l r e c e pt or bound t o i t s M H C c l a s s I l i ga nd. N at ur e 402: 623 631 69. W a ng, J M C W hi t m a n, K N a t a r a j a n, J T o r m o, R A M a r i uz z a a nd D H M a r gul i e s 2002. B i ndi ng of t he na t ur a l ki l l e r c e l l i nhi bi t or y r e c e pt or L y49A t o i t s m a j or hi s t oc om pa t i bi l i t y c om pl e x c l a s s I l i ga nd C r uc i a l c ont a c t s i nc l ude bot h H 2D d A N D be t a 2 m i c r og l obul i n. J B i ol C he m 277 : 1433 1442 70. D a m J R G ua n, K N a t a r a j a n, N D i m a s i L K C hl e w i c ki D M K r a nz P S c huc k, D H M a r gul i e s a nd R A M a r i uz z a 200 3. V a r i a bl e M H C c l a s s I e nga ge m e nt by L y49 na t u r a l ki l l e r c e l l r e c e pt or s d e m ons t r a t e d by t he c r ys t a l s t r uc t ur e of L y49C bound t o H 2K ( b) N at I m m unol 4: 1213 1222 71. C or r e a I a nd D H R a ul e t 1995 B i ndi ng of di ve r s e pe pt i de s t o M H C c l a s s I m ol e c ul e s i nhi bi t s t a r ge t c e l l l ys i s by a c t i va t e d na t ur a l ki l l e r c e l l s I m m uni t y 2: 61 71 72. M i c ha e l s s on, J A A c hour M S a l c e do, A K a s e S j os t r om J S undba c k, R A H a r r i s a nd K K a r r e 2000. V i s ua l i z a t i on of i nh i bi t or y L y49 r e c e pt or s pe c i f i c i t y w i t h s ol ubl e m a j or hi s t oc om pa t i bi l i t y c om pl e x c l a s s I t e t r a m e r s E ur J I m m unol 30: 300 307 73. F r a nks s on, L J S undba c k, A A c hour J B e r nl i nd R G l a s a nd K K a r r e 1999 P e pt i de de pe nde nc y a nd s e l e c t i vi t y of t he N K c e l l i nhi bi t or y r e c e pt or L y 49C E ur J I m m unol 29: 2748 2758 74. V e l y, F a nd E V i vi e r 1997. C ons e r va t i on of s t r u c t ur a l f e a t ur e s r e ve a l s t he e xi s t e nc e of a l a r ge f a m i l y o f i nhi bi t or y c e l l s ur f a c e r e c e pt or s a nd noni nhi bi t or y/ a c t i va t or y c ount e r pa r t s J I m m unol 1 59: 2075 2077 75. B ur s ht yn, D N A S L a m M W e s t on, N G upt a P A W a r m e r da m a nd E O L ong. 1999 C ons e r ve d r e s i due s a m i no t e r m i na l of c yt opl a s m i c t yr os i ne s c ont r i but e t o t he S H P 1 m e di a t e d i nhi bi t o r y f unc t i on of ki l l e r c e l l I g l i ke r e c e pt or s J I m m unol 162: 897 902 76. F r y, A M L L L a ni e r a nd A W e i s s 1996. P hos phot yr os i ne s i n t he ki l l e r c e l l i nhi bi t or y r e c e pt or m ot i f o f N K B 1 a r e r e qui r e d f or ne ga t i ve s i gna l i ng a nd f or a s s oc i a t i on w i t h pr ot e i n t yr os i ne ph os pha t a s e 1C J E x p M e d 184: 295 3 00

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103 77. N a ka m ur a M C E C N i e m i M J F i s he r L D S hul t z W E S e a m a n, a nd J C R ya n. 1997 M ous e L y 49A i nt e r r up t s e a r l y s i g na l i ng e ve nt s i n na t ur a l ki l l e r c e l l c yt ot oxi c i t y a nd f unc t i ona l l y a s s oc i a t e s w i t h t he S H P 1 t yr os i ne phos pha t a s e J E x p M e d 185: 673 6 84 78. D a e r on, M S L a t our O M a l be c E E s pi nos a P P i na S P a s m a ns a nd W H F r i dm a n. 1995. T he s a m e t y r os i ne ba s e d i nhi bi t i on m ot i f i n t he i nt r a c yt opl a s m i c dom a i n of F c ga m m a R I I B r e gul a t e s ne ga t i ve l y B C R T C R a nd F c R de pe nde nt c e l l a c t i va t i on. I m m uni t y 3: 635 646 79. P a ni G K D F i s c he r I M l i na r i c R a s c a n, a nd K A S i m i novi t c h. 1996. S i gna l i ng c a pa c i t y of t he T c e l l a nt i ge n r e c e pt or i s ne ga t i ve l y r e gul a t e d by t he P T P 1C t yr os i ne phos pha t a s e J E x p M e d 184: 839 852 80. A r a s e H E S M oc a r s ki A E C a m pbe l l A B H i l l a nd L L L a ni e r 2002 D i r e c t r e c ogni t i on o f c yt om e ga l ovi r us by a c t i va t i n g a nd i nhi bi t o r y N K c e l l r e c e pt or s Sc i e nc e 296: 1323 1326 81. S m i t h, H R J W H e us e l I K M e ht a S K i m B G D or ne r O V N a i de nko, K I i z uka H F ur uka w a D L B e c km a n, J T P i ng e l A A S c a l z o, D H F r e m ont a nd W M Y okoya m a 2002. R e c ogni t i o n of a vi r us e nc ode d l i ga nd by a na t ur a l ki l l e r c e l l a c t i va t i on r e c e pt or P r oc N at l A c ad S c i U S A 99: 8826 8831 82. M a s on, L H J W i l l e t t e B r ow n, S K A nde r s on, P G os s e l i n, E W S hor e s P E L ove J R O r t a l do, a nd D W M c V i c a r 1998 C h a r a c t e r i z a t i on of a n a s s oc i a t e d 16 kD a t yr os i ne phos phopr ot e i n r e qui r e d f or L y 4 9D s i gna l t r a ns duc t i on. J I m m unol 160: 4148 4152 83. G e or ge T C L H M a s on, J R O r t a l do V K um a r a nd M B e nne t t 1999. P os i t i ve r e c ogni t i on o f M H C c l a s s I m ol e c ul e s by t he L y49D r e c e pt or of m ur i ne N K c e l l s J I m m unol 162: 2035 2043 84. G e or ge T C J R O r t a l d o, S L e m i e ux, V K um a r a nd M B e nne t t 1999. T ol e r a nc e a nd a l l or e a c t i vi t y of t he L y49D s ubs e t of m ur i ne N K c e l l s J I m m unol 163: 1859 1867 85. N a ka m ur a M C P A L i nne m e ye r E C N i e m i L H M a s on, J R O r t a l do, J C R ya n, a nd W E S e a m a n. 1999 M ous e L y 49D r e c ogni z e s H 2D d a nd a c t i va t e s na t ur a l ki l l e r c e l l c yt ot oxi c i t y. J E x p M e d 189: 493 500 86. F ur uka w a H K I i z uka J P ou r s i ne L a ur e nt N S ha s t r i a nd W M Y okoya m a 2002. A l i ga nd f o r t he m ur i ne N K a c t i va t i on r e c e p t or L y 49D : a c t i va t i o n of t ol e r i z e d N K c e l l s f r o m be t a 2 m i c r ogl obul i n de f i c i e nt m i c e J I m m unol 169 : 126 136

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104 87. L e e S H A Z a f e r Y de R e pe nt i gny, R K ot ha r y M L T r e m bl a y, P G r os P D upl a y, J R W e bb, a nd S M V i da l 2003. T r a ns ge ni c e xpr e s s i on of t he a c t i va t i ng na t ur a l ki l l e r r e c e pt or L y49H c onf e r s r e s i s t a nc e t o c yt om e ga l ovi r us i n ge ne t i c a l l y s us c e pt i bl e m i c e J E x p M e d 197 : 515 5 26 88. L ohw a s s e r S P H a nde D L M a ge r a nd F T a k e i 1999. C l oni ng o f m u r i ne N K G 2A B a nd C : s e c ond f a m i l y of C t ype l e c t i n r e c e pt or s on m ur i ne N K c e l l s E ur J I m m unol 29: 755 761 89. V a nc e R E A M J a m i e s on, a nd D H R a ul e t 1 999. R e c ogni t i on of t he c l a s s I b m ol e c ul e Q a 1( b) by put a t i ve a c t i va t i ng r e c e pt or s C D 94/ N K G 2C a nd C D 94/ N K G 2E on m ous e na t ur a l ki l l e r c e l l s J E x p M e d 190 : 1801 1812 90. V a nc e R E J R K r a f t J D A l t m a n P E J e ns e n, a nd D H R a ul e t 1998. M ous e C D 94/ N K G 2A i s a na t ur a l ki l l e r c e l l r e c e pt or f o r t he nonc l a s s i c a l m a j or hi s t oc om pa t i bi l i t y c om pl e x ( M H C ) c l a s s I m ol e c ul e Q a 1( b) J E x p M e d 188: 1841 1848 91. B r ooks A G P E P os c h, C J S c o r z e l l i F B or r e go, a nd J E C ol i ga n. 1997. N K G 2A c om pl e xe d w i t h C D 94 de f i ne s a nove l i n hi bi t or y na t ur a l ki l l e r c e l l r e c e pt or J E x p M e d 185: 795 800 92. L a z e t i c S C C ha ng, J P H ouc hi ns L L L a ni e r a nd J H P h i l l i ps 1996 H um a n na t ur a l ki l l e r c e l l r e c e pt or s i nvol ve d i n M H C c l a s s I r e c ogni t i on a r e di s ul f i de l i nke d he t e r odi m e r s o f C D 94 a nd N K G 2 s ubuni t s J I m m unol 157 : 4741 4745 93. H ouc hi ns J P L L L a ni e r E C N i e m i J H P h i l l i ps a nd J C R ya n. 1997 N a t ur a l ki l l e r c e l l c yt ol yt i c a c t i vi t y i s i nhi b i t e d by N K G 2 A a nd a c t i va t e d by N K G 2 C J I m m unol 158: 3603 3609 94. C a r r e t e r o, M C C a nt oni T B e l l on, C B ot t i no R B i a s s oni A R odr i gue z J J P e r e z V i l l a r L M or e t t a A M or e t t a a n d M L ope z B ot e t 1997. T he C D 94 a nd N K G 2 A C t ype l e c t i ns c ova l e nt l y a s s e m bl e t o f o r m a na t ur a l ki l l e r c e l l i nhi bi t or y r e c e pt or f o r H L A c l a s s I m ol e c ul e s E ur J I m m unol 27: 563 567 95. L e D r e a n, E F V e l y, L O l c e s e A C a m bi a ggi S G ui a G K r ys t a l N G e r voi s A M or e t t a F J ot e r e a u, a nd E V i vi e r 1998 I nhi b i t i on of a nt i ge n i nduc e d T c e l l r e s pons e a nd a nt i body i nduc e d N K c e l l c yt ot oxi c i t y by N K G 2A : a s s oc i a t i on of N K G 2A w i t h S H P 1 a nd S H P 2 p r ot e i n t yr os i ne p hos pha t a s e s E ur J I m m unol 28: 264 276 96. L a ni e r L L B C or l i s s J W u, a nd J H P hi l l i ps 1998. A s s oc i a t i on of D A P 12 w i t h a c t i va t i ng C D 94/ N K G 2C N K c e l l r e c e pt or s I m m uni t y 8: 693 701

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116 210. K a va na ugh, W M D A P ot S M C hi n, M D e u t e r R e i nha r d, A B J e f f e r s on, F A N or r i s F R M a s i a r z L S C ous e ns P W M a j e r us a nd L T W i l l i a m s 1996. M ul t i pl e f or m s o f a n i nos i t ol pol yphos pha t e 5 phos pha t a s e f or m s i gna l i ng c om pl e xe s w i t h S hc a nd G r b2 C ur r B i ol 6: 438 44 5 211. K e r r W G M H e l l e r a nd L A H e r z e nbe r g. 199 6. A na l ys i s of l i popol ys a c c ha r i de r e s pons e ge ne s i n B l i ne a ge c e l l s de m ons t r a t e s t ha t t he y c a n ha ve di f f e r e nt i a t i on s t a ge r e s t r i c t e d e xpr e s s i on a nd c ont a i n S H 2 do m a i ns P r oc N at l A c ad Sc i U S A 93 : 3947 3952 212. O s bor ne M A G Z e nne r M L ubi nus X Z ha ng Z S ongya ng, L C C a nt l e y, P M a j e r us P B ur n, a nd J P K oc ha n. 1996 T he i nos i t ol 5' phos pha t a s e S H I P bi nds t o i m m unor e c e pt or s i gna l i ng m ot i f s a nd r e s p onds t o hi gh a f f i ni t y I gE r e c e pt or a ggr e ga t i on. J B i ol C he m 271 : 29271 292 78 213. M or a n, M F C A K oc h, D A nde r s on, C E l l i s L E ngl a nd, G S M a r t i n, a nd T P a w s on. 1990. S r c hom ol ogy r e gi on 2 dom a i ns di r e c t pr ot e i n p r ot e i n i nt e r a c t i ons i n s i gna l t r a ns duc t i on. P r oc N at l A c ad Sc i U S A 87: 8622 8626 214. E l l i s C M M or a n, F M c C or m i c k, a nd T P a w s o n. 1990 P hos phor yl a t i on of G A P a nd G A P a s s o c i a t e d pr ot e i ns by t r a ns f or m i n g a nd m i t oge ni c t yr os i ne ki na s e s N at ur e 343: 377 381 215. O no, M S B ol l a nd, P T e m ps t a nd J V R a ve t c h. 1996. R ol e of t he i nos i t ol phos pha t a s e S H I P i n ne ga t i ve r e gul a t i on of t he i m m une s ys t e m by t he r e c e pt or F c ( ga m m a ) R I I B N at ur e 383: 263 266 216. W a ng, J W J M H ow s on, T G ha ns a h, C D e s pont s J M N i nos S L M a y, K H N guye n, N T oya m a S o r i m a c hi a nd W G K e r r 2002 I nf l ue nc e of S H I P on t he N K r e pe r t oi r e a nd a l l oge ne i c bone m a r r ow t r a n s pl a nt a t i on. Sc i e nc e 29 5: 2094 2097 217. T e s s m e r M S C F uge r e F S t e ve na e r t O V N a i de nko, H J C hong G L e c l e r c q, a nd L B r os s a y. 2007. K L R G 1 bi nds c a d he r i ns a nd pr e f e r e nt i a l l y a s s oc i a t e s w i t h S H I P 1 I nt I m m unol 19: 391 400 218. T r i da nda pa ni S T K e l l e y, M P r a d ha n D C oone y, L B J us t e m e nt a nd K M C ogge s ha l l 1997. R e c r ui t m e nt a nd phos phor yl a t i o n of S H 2 c ont a i ni ng i nos i t ol phos pha t a s e a nd S hc t o t he B c e l l F c ga m m a i m m unor e c e pt or t yr os i ne ba s e d i nhi bi t i on m ot i f pe pt i de m ot i f M ol C e l l B i ol 17: 43 05 4311 21 9. G a l a ndr i ni R I T a s s i G M a t t i a L L e nt i M P i c c ol i L F r a t i a nd A S a nt on i 2002. S H 2 c ont a i ni ng i nos i t ol phos pha t a s e ( S H I P 1) t r a ns i e nt l y t r a ns l oc a t e s t o r a f t dom a i ns a nd m odul a t e s C D 16 m e di a t e d c yt ot o xi c i t y i n hum a n N K c e l l s B l ood 100: 4581 4 589

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127 A ppe ndi x A T ab l e A 1. N K k i n om e T he t ot a l ki nom e f or bot h m a t ur e N K ( N K ) a nd i m m a t ur e N K ( i N K ) w i t h a l l 6 r e pl i c a t e s pr e s e nt V a l ue s ha ve b e e n nor m a l i z e d. S p o t S E Q U E N CE N K 1 N K 2 N K 3 N K 4 N K 5 N K 6 i N K 1 i N K 2 i N K 3 i N K 4 i N K 5 i N K 6 1 H E Y IY V D P M 1 3 5 0 5 0 0 7 6 0 5 0 1 1 4 0 7 0 0 6 4 0 5 9 0 5 3 0 7 3 0 7 1 0 8 6 2 K RP S V RA K A 2 9 5 1 5 7 2 5 0 2 1 9 1 8 6 0 8 6 1 4 7 1 5 0 1 4 9 1 6 6 1 7 0 1 1 8 3 E D N E Y T A RQ 1 6 8 0 4 9 1 1 4 0 3 9 1 5 8 0 3 5 0 6 1 0 4 2 0 7 8 0 5 3 1 1 5 0 4 7 4 E N K L Y G M S D 0 7 1 0 3 8 0 5 3 0 2 4 1 0 5 0 3 6 0 4 5 0 3 5 0 3 5 0 3 6 0 8 1 0 4 4 5 H RL L T L D P V 0 6 4 0 2 9 0 4 4 0 2 2 1 2 3 0 2 6 0 6 3 0 3 2 0 5 6 0 4 4 0 6 1 0 6 6 6 L E K K Y V RR D 0 6 7 0 3 4 0 3 6 0 2 9 1 1 3 0 4 5 0 6 1 0 2 7 0 5 5 0 4 5 0 4 6 0 3 5 7 P Y K F P S S P L RI P G Z 0 6 2 0 2 7 0 3 7 0 3 8 1 3 5 0 3 6 0 5 6 0 4 1 0 4 3 0 3 7 0 4 4 0 2 6 8 G K RQ T E R E K 1 3 3 0 5 8 0 3 7 0 4 1 0 9 5 0 6 4 0 9 0 0 5 6 0 6 1 0 7 2 0 6 0 0 6 0 9 A P A T S P K A E 0 9 2 0 2 4 0 5 4 0 2 8 1 3 6 0 3 8 0 6 0 0 3 4 0 2 9 0 3 4 0 6 4 0 5 5 1 0 D E E E S E Q G A 1 6 3 0 3 3 0 9 9 0 2 7 1 3 3 0 4 4 0 5 9 0 3 7 0 9 0 0 4 5 0 6 9 0 5 3 1 1 F F R RS K IA V 1 0 9 0 4 7 0 7 0 0 3 8 1 3 8 0 4 5 0 7 1 0 6 1 0 7 1 0 7 3 0 7 4 0 4 6 1 2 G M T E Y V A T R 0 8 1 0 3 8 0 4 7 0 2 3 0 9 6 0 2 5 0 6 9 0 3 5 0 4 3 0 4 3 0 5 0 0 4 5 1 3 E A A L Y K N L L 0 6 6 0 3 6 0 5 5 0 1 7 1 0 2 0 4 4 0 4 9 0 4 0 0 4 6 0 5 0 0 4 7 0 3 3 1 4 E L IL S P R S K 1 1 8 0 4 2 0 6 7 0 5 1 1 1 7 0 3 7 0 8 2 0 7 0 0 8 1 1 0 9 0 6 3 0 4 0 1 5 RRA V S E Q D A 0 6 3 0 2 4 0 4 5 0 2 3 0 7 3 0 3 0 0 5 8 0 2 2 0 2 3 0 3 2 0 3 1 0 4 6 1 6 S E D N S E D E I 1 7 8 0 5 1 1 1 8 0 3 4 1 1 2 0 3 3 0 7 3 0 3 8 0 5 2 0 6 4 0 8 1 0 4 7 1 7 P A S L S RA K A 1 6 0 0 7 9 0 7 0 0 5 0 1 1 7 0 5 4 0 8 7 0 8 0 0 7 3 0 8 0 0 9 4 0 8 4 1 8 RRA S L 2 5 1 1 2 6 1 8 4 1 0 9 1 9 9 0 5 7 1 4 3 1 4 3 1 1 9 1 2 7 2 7 2 0 6 5 1 9 A K K M S T Y N V 1 3 3 0 8 1 1 0 1 0 9 4 1 1 3 0 5 6 0 9 6 0 8 7 1 3 9 1 2 6 0 7 8 0 7 2 2 0 D D IN S Y E A W 1 7 1 0 6 1 1 1 7 0 6 3 1 5 7 0 3 5 0 5 5 0 6 6 1 6 1 0 9 0 1 0 5 0 5 6 2 1 E T RF T D T RK 1 2 6 0 4 8 0 8 1 0 5 6 1 3 5 0 4 6 0 9 2 0 6 5 0 9 4 1 0 1 0 8 6 0 4 3 2 2 Q L S T S E E N S 0 7 0 0 2 7 0 3 9 0 3 6 0 4 8 0 3 4 0 5 9 0 3 2 0 3 9 0 4 0 0 4 1 0 4 0 2 3 S P RK S P K K S 2 1 5 1 3 0 1 2 5 1 0 2 0 8 8 0 7 6 1 3 5 1 8 1 1 1 0 1 2 0 0 9 8 0 9 5 2 4 RRK A S G P 1 6 9 1 0 0 0 9 9 0 8 9 0 9 2 0 6 4 1 1 8 1 0 1 1 1 2 0 9 7 0 9 7 0 8 3 2 5 RQ L RS P RR T 2 5 7 1 2 7 1 9 3 1 9 5 2 4 2 1 0 8 1 5 5 1 6 5 2 3 4 1 6 1 2 9 7 1 2 2 2 6 S A V A S N M RD 0 8 9 0 4 3 0 4 6 0 4 1 0 8 4 0 3 6 0 6 0 0 4 7 0 4 9 0 3 8 0 6 4 0 3 3 2 7 RT P P P S G 1 1 3 0 5 4 0 4 9 0 3 7 0 8 1 0 3 4 0 7 6 0 4 6 0 4 7 0 6 0 0 5 6 0 3 6 2 8 L RRA S 1 5 1 0 7 3 1 1 0 0 9 3 1 3 1 0 6 0 1 0 0 0 7 6 0 8 3 1 0 7 1 1 7 0 5 7 2 9 S S T G S ID M V 0 9 2 0 3 4 0 7 1 0 3 7 0 4 5 0 2 7 0 6 3 0 3 3 0 3 7 0 4 3 0 2 6 0 4 3 3 0 T L A S S F K RR 3 6 2 2 6 6 2 9 5 2 4 1 0 9 9 1 0 0 1 7 5 1 8 4 1 5 8 1 7 5 1 4 4 1 0 4 3 1 V G A F S T V K G 0 9 9 0 7 9 0 8 0 0 6 5 0 5 4 0 4 0 0 7 6 0 7 0 0 7 2 0 9 9 0 6 8 0 5 2 3 2 Y S G H S M S D P 0 5 9 0 3 0 0 5 2 0 3 0 0 5 3 0 3 8 0 6 3 0 3 5 0 5 5 0 4 1 0 4 8 0 3 9 3 3 S P G E Y V N IE 0 9 2 0 3 9 0 7 4 0 3 0 0 7 8 0 3 0 0 7 4 0 3 4 0 6 0 0 5 9 0 4 4 0 4 0 3 4 RK RS A K E 1 3 4 0 8 8 1 0 7 0 8 9 1 1 8 1 0 2 1 0 4 1 3 7 1 4 3 1 2 0 1 1 0 0 8 9 3 5 V IN E T S Q H H 0 6 5 0 4 2 0 5 4 0 3 3 0 6 3 0 4 1 0 6 4 0 3 3 0 4 3 0 4 8 0 6 3 0 3 0 3 6 S P V V S G D T S 0 6 1 0 3 4 0 5 7 0 3 5 0 5 1 0 3 3 0 6 3 0 2 5 0 8 6 0 2 7 0 3 3 0 5 3

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128 3 7 L RRF S L A T M 2 3 6 1 6 6 1 9 4 1 2 9 1 1 8 0 7 9 1 2 1 1 2 7 0 9 8 1 1 5 1 1 4 1 0 4 3 8 P RP A S V P P S 0 8 7 0 5 1 0 5 5 0 4 4 0 5 7 0 5 9 0 7 2 0 4 8 0 6 4 0 7 7 0 4 5 0 5 4 3 9 S S R P S S N R S 2 1 5 1 0 5 1 3 0 1 0 8 1 3 6 1 1 1 1 1 8 0 9 6 0 8 9 1 0 6 1 3 8 1 1 7 4 0 T K F A S D D E H 0 7 2 0 3 4 0 6 2 0 3 1 0 6 4 0 2 8 0 6 6 0 3 5 0 5 9 0 4 0 0 4 7 0 4 7 4 1 L S D D S F I E D 1 6 6 0 7 1 1 5 9 0 9 5 1 0 3 0 3 8 0 9 3 0 6 4 0 8 0 0 9 6 0 9 1 0 7 1 4 2 P S P K T P P G S 0 7 3 0 4 1 0 8 0 0 4 4 0 7 4 0 4 0 0 6 0 0 4 3 0 6 0 0 3 7 0 9 4 0 2 8 4 3 K D IG S E S T E 1 3 6 0 3 9 0 9 9 0 3 5 0 5 7 0 4 7 0 7 1 0 4 1 0 5 9 0 4 9 0 4 7 0 5 7 4 4 K RRG S V P IL 2 4 2 1 8 6 1 9 4 1 7 9 1 4 4 1 6 6 1 4 2 1 6 2 2 1 7 1 8 8 1 4 5 1 6 7 4 5 K RA K A K T A K K R 2 5 0 2 5 4 2 7 5 2 8 0 2 0 7 2 4 5 2 8 1 3 3 7 3 6 9 2 9 1 2 2 4 2 0 2 4 6 Q S Y S S S Q RV 0 7 7 1 0 5 0 6 2 0 5 3 0 6 1 0 4 7 0 8 8 0 7 2 0 6 3 0 5 8 0 7 1 0 5 7 4 7 K G T G Y IK T E 1 3 1 0 8 9 0 6 3 0 6 8 0 9 7 1 1 4 0 9 3 0 7 5 0 8 3 0 1 3 0 9 2 1 0 0 4 8 K RS L S E M E I 0 6 5 0 3 1 0 5 3 0 4 2 0 8 1 0 4 4 0 6 0 0 3 5 0 5 0 0 3 7 0 5 5 0 4 8 4 9 K RK Q G S V RG L 2 7 1 1 6 3 2 1 6 1 8 8 1 6 1 1 0 5 1 4 4 1 5 9 1 8 3 2 0 3 1 0 7 0 9 3 5 0 H T RD S E A Q R 0 6 4 0 3 3 0 3 9 0 2 9 1 3 9 0 3 1 0 5 4 0 6 1 0 5 4 0 2 9 0 4 8 0 2 2 5 1 M A E V S W K V L 0 4 5 0 3 2 0 4 6 0 2 5 1 1 9 0 3 3 0 5 3 0 6 3 0 5 0 0 3 9 0 5 6 0 2 5 5 2 E E G IS Q E S S 0 6 1 0 4 1 0 4 0 0 2 1 1 0 5 0 5 1 0 6 0 0 4 2 0 6 5 0 3 5 0 5 8 0 2 1 5 3 E Q Q Q T E D E L 0 7 8 0 3 1 0 5 5 0 3 1 0 8 0 0 3 7 0 5 3 0 2 7 0 9 0 0 3 2 0 5 4 0 2 7 5 4 IG E G T Y G V V 0 8 7 0 3 4 0 7 2 0 3 9 0 8 2 0 2 8 0 6 5 0 4 7 0 8 4 0 4 4 0 6 3 0 2 9 5 5 M M T P Y V V T R 0 9 6 0 2 8 0 7 0 0 3 1 1 0 8 0 2 6 0 8 2 0 5 4 0 7 4 0 6 2 0 7 8 0 2 9 5 6 P Y K F P S S P L RI P G Z 0 5 3 0 1 7 0 6 0 0 1 6 1 1 2 0 2 6 0 6 4 0 5 3 0 5 5 0 1 8 0 7 8 0 2 8 5 7 G Q E V Y V K K T 0 9 6 0 4 0 0 5 3 0 4 1 0 9 5 0 2 7 0 7 7 1 1 2 0 7 4 0 7 5 0 4 1 0 2 8 5 8 A Q E T S G E E I 0 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1 1 1 0 0 6 0 6 5 0 3 7 0 7 9 0 2 4 0 5 7 0 3 8 1 0 6 G RT G RRN S I 3 5 2 1 3 2 3 5 2 2 3 9 5 5 6 0 6 0 3 4 3 2 7 3 2 8 8 2 4 5 5 5 0 1 2 1 1 0 7 A T P IY L D IL 0 9 7 0 3 1 1 2 6 0 7 3 1 3 4 0 2 7 0 8 9 0 5 1 0 6 9 0 6 0 1 2 0 0 5 4 1 0 8 D M RQ T V A V G 0 4 5 0 2 3 0 7 6 0 3 8 0 6 9 0 2 3 0 6 1 0 4 3 0 5 3 0 2 1 1 1 4 0 3 5 1 0 9 G D RF T D E E V 1 3 5 0 4 1 1 8 4 1 0 7 1 1 0 0 2 3 0 8 3 0 5 7 1 2 8 1 0 5 1 2 2 0 4 0 1 1 0 G S G S S V T S R 0 8 4 0 1 9 0 8 6 0 8 6 1 0 6 0 3 4 1 0 1 0 7 3 0 8 0 0 4 6 1 3 9 0 4 9 1 1 1 E E P Q Y E E I P 1 7 9 0 3 9 1 9 3 1 2 9 1 8 0 0 1 7 0 9 2 0 7 3 1 0 6 0 8 8 1 5 4 0 4 2 1 1 2 E RRL S L V P D 1 0 2 0 2 3 1 0 9 0 8 5 1 1 6 0 1 9 0 8 0 0 4 2 0 8 5 0 3 1 0 6 9 0 3 3 1 1 3 RRS S S R P IR 2 6 0 0 9 3 2 3 8 1 4 4 3 9 9 0 6 8 2 3 0 2 1 2 2 0 0 1 5 5 4 7 5 1 5 1 1 1 4 S K IG S T E N L 0 5 6 0 2 1 0 5 7 0 3 4 0 7 0 0 3 0 0 7 6 0 5 1 0 4 9 0 3 1 0 8 3 0 4 7 1 1 5 N D M T S L 0 4 9 0 1 5 0 9 2 0 2 6 0 3 8 0 2 1 0 6 3 0 2 5 0 6 1 0 2 4 0 9 3 0 3 5 1 1 6 A A RL S L T D P 0 4 0 0 2 1 0 6 4 0 5 0 0 3 7 0 2 4 0 6 8 0 2 6 0 4 9 0 3 7 0 7 4 0 3 6 1 1 7 A RRS T T D A G 0 4 3 0 1 5 0 5 4 0 4 5 0 3 7 0 2 8 0 6 7 0 3 7 0 5 9 0 3 2 1 0 5 0 3 8 1 1 8 D L K D T K Y K L 0 4 7 0 1 6 0 5 6 0 6 6 0 5 3 0 2 4 0 6 6 0 2 6 0 6 0 0 4 1 0 8 3 0 4 8 1 1 9 G A F S T V K G V 0 9 7 0 4 6 0 9 1 0 8 1 0 8 1 0 3 0 1 0 8 0 8 1 0 9 7 0 4 8 0 7 8 0 5 4 1 2 0 RG RA S S H S S 1 6 4 0 6 9 1 2 6 0 8 3 1 9 2 0 8 4 1 3 1 1 3 1 1 0 2 1 0 3 2 1 3 1 0 1 1 2 1 RK RS RA E F 1 3 1 0 4 7 1 0 0 0 5 6 0 6 6 0 4 6 1 1 4 0 9 0 0 9 5 0 8 4 1 3 2 0 6 9 1 2 2 L RA A S L G 1 0 4 0 4 2 1 1 8 0 4 5 0 8 5 0 2 2 1 0 8 0 8 2 0 8 4 0 8 5 1 1 6 0 5 6 1 2 3 RRRQ S V L N L 1 3 3 0 5 8 1 8 3 0 8 0 1 6 3 0 4 9 1 6 3 1 6 4 1 2 7 0 9 4 1 6 7 0 9 2 1 2 4 S ID E Y F S E Q 1 0 9 0 3 5 1 2 2 0 7 4 0 5 9 0 3 0 0 7 8 0 5 0 0 9 2 0 6 1 0 9 6 0 4 1 1 2 5 E E S E S D 0 8 5 0 2 2 1 5 0 0 8 5 0 9 9 0 2 7 0 8 0 0 5 4 1 1 0 0 6 9 0 8 1 0 5 0 1 2 6 RRRS 2 1 4 0 9 2 2 1 3 1 7 5 2 9 3 1 1 2 2 3 2 2 5 6 2 4 9 1 5 9 2 8 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131 2 1 1 RRA V S E L D A 0 5 2 0 3 1 0 3 8 0 3 6 0 6 6 0 3 0 0 6 2 0 3 3 0 3 8 0 3 0 0 4 3 0 4 3 2 1 2 S D G G Y M D M S 0 8 5 0 5 0 0 5 1 0 3 2 0 6 8 0 3 9 0 6 2 0 5 7 0 5 0 0 3 8 0 7 0 0 4 8 2 1 3 N W H M T P P RK 1 0 8 0 9 1 0 6 7 0 5 1 0 9 6 1 2 1 0 9 8 0 8 0 0 7 7 0 6 7 1 2 5 1 0 6 2 1 4 RRA S I 2 7 9 1 8 7 2 0 6 1 5 7 1 8 5 2 1 1 1 6 6 1 3 3 1 6 2 1 3 4 2 9 5 1 7 0 2 1 5 A K K G S E Q E S 0 6 1 0 4 4 0 4 8 0 4 1 0 6 1 0 4 7 0 5 7 0 4 3 0 6 5 0 3 5 0 5 9 0 5 1 2 1 6 D D E M T G Y V A 0 9 4 0 5 8 0 7 6 0 6 9 0 9 8 0 3 7 0 6 2 0 5 1 0 7 3 0 6 0 0 6 9 0 6 7 2 1 7 E T D Y Y RK G G 0 7 3 0 4 6 0 6 7 0 6 3 0 7 0 0 3 1 0 6 4 0 6 4 0 7 0 0 5 2 0 4 6 0 4 6 2 1 8 Y V T T S T RT Y 1 3 2 0 9 0 0 6 8 0 4 4 0 7 0 0 6 3 1 0 2 0 9 9 0 5 6 0 7 6 0 9 9 0 8 1 2 1 9 S P Q P S R RG S 2 8 8 2 2 0 1 3 4 1 0 5 1 9 7 1 9 2 1 3 7 1 7 2 1 3 6 1 4 7 2 6 5 1 8 6 2 2 0 RRA A S V A 2 3 1 1 5 5 1 1 2 0 8 5 1 1 5 1 5 3 1 2 1 1 2 3 0 9 1 1 0 6 1 6 8 1 0 7 2 2 1 RP S E S N G Q P 0 8 2 0 4 2 0 4 4 0 3 6 0 3 7 0 3 0 0 5 5 0 2 9 0 4 0 0 2 5 0 3 8 0 3 5 2 2 2 S A S T T P V K K 1 1 4 0 8 5 0 6 9 0 6 1 0 7 6 1 0 1 0 8 5 0 7 9 0 6 8 0 8 1 0 7 9 1 0 9 2 2 3 N S Y G S R RG N 2 9 9 2 5 4 1 6 6 2 0 7 2 9 9 2 4 3 1 7 3 1 7 1 1 3 7 1 5 5 2 2 1 3 2 0 2 2 4 L RRA S L 1 3 8 1 2 7 1 1 9 1 4 4 1 1 7 0 8 0 0 8 0 0 8 3 0 7 6 1 1 6 0 8 9 0 9 0 2 2 5 S S T D S A D S G 0 7 3 0 4 1 0 6 2 0 3 0 0 3 8 0 3 3 0 6 0 0 3 8 0 4 1 0 3 2 0 4 0 0 4 3 2 2 6 T K S G S T T K N 1 2 6 1 1 2 0 7 6 0 9 4 0 8 4 0 8 4 0 9 7 0 9 1 1 1 3 0 8 8 1 1 0 0 8 2 2 2 7 V E T T Y A D F I 0 8 8 0 6 5 0 7 4 0 4 5 0 9 0 0 4 8 0 6 7 0 3 5 0 5 3 0 4 0 0 6 6 0 5 0 2 2 8 Y S F T T T A E R 0 6 6 0 4 6 0 3 7 0 2 7 0 6 1 0 3 8 0 6 0 0 3 5 1 1 9 0 2 3 0 4 3 0 4 5 2 2 9 S P F K Y Q S L L 0 6 1 0 4 7 0 5 0 0 3 7 0 6 2 0 4 5 0 4 7 0 3 9 0 6 3 0 4 3 0 3 8 0 4 7 2 3 0 RK RS A A E 1 1 0 0 9 9 0 5 9 0 7 2 1 0 5 0 7 7 0 7 5 0 8 7 0 6 9 0 9 3 0 7 4 0 9 1 2 3 1 V IK RS P RK R 1 9 3 1 7 4 2 0 2 2 0 4 2 6 3 2 7 2 1 7 0 2 6 9 2 4 2 2 4 1 2 0 4 2 4 9 2 3 2 S P V K S P E A K 0 6 5 0 4 2 0 5 5 0 4 2 0 5 4 0 4 1 0 5 0 0 3 6 0 4 6 0 5 0 0 5 4 0 5 7 2 3 3 L RRA S V A Q L 2 4 5 2 3 2 2 4 1 1 7 7 1 8 4 1 2 3 1 3 2 1 3 3 1 1 1 1 6 6 1 5 8 1 3 8 2 3 4 P RM P S L S V P 0 6 5 0 4 5 0 5 3 0 3 4 0 5 8 0 4 4 0 6 2 0 2 7 0 3 2 0 4 0 0 3 7 0 3 6 2 3 5 S S P V Y Q D A V 0 8 8 0 6 3 0 7 3 0 8 2 0 9 3 0 4 8 0 7 0 0 4 5 0 7 2 0 5 0 0 6 6 0 4 7 2 3 6 T K D T Y D A L H 0 7 0 0 4 7 0 5 2 0 4 9 0 6 6 0 3 5 0 5 0 0 4 1 0 6 3 0 4 4 0 4 7 0 4 7 2 3 7 L RS P S W E P F 0 7 3 0 6 5 0 6 4 0 6 1 0 8 0 0 4 1 0 4 4 0 3 9 0 5 1 0 5 9 0 6 9 0 5 2 2 3 8 P S L P T P P T R 1 1 7 1 0 0 0 7 2 0 7 9 1 1 0 0 8 2 0 7 8 0 6 6 0 8 3 1 0 1 1 2 9 1 0 1 2 3 9 K A S A S P RRK 2 1 8 1 9 7 2 0 6 1 9 4 2 7 0 2 0 1 1 2 0 1 7 8 1 8 9 2 2 8 2 5 5 2 2 6 2 4 0 K RRD Y L D L A 0 8 4 0 5 7 0 8 5 0 5 3 0 8 9 0 4 8 0 6 2 0 4 2 0 5 5 0 5 0 0 5 7 0 8 3 2 4 1 P A P A V RA S D R A 0 7 7 0 4 2 0 5 7 0 2 9 0 7 3 0 3 5 0 4 8 0 3 5 0 5 4 0 3 5 0 4 7 0 4 2 2 4 2 Q S RA S D K Q T 0 5 5 0 4 5 0 5 0 0 3 2 0 6 7 0 4 8 0 5 9 0 3 1 0 4 2 0 3 7 0 6 2 0 5 6 2 4 3 K G Q E S F K K Q 1 2 9 1 2 9 0 9 6 1 2 0 1 4 0 1 5 2 1 3 6 1 2 2 0 9 8 1 2 2 1 6 2 1 3 9 2 4 4 K RS G S V Y E P 1 6 3 2 2 8 1 7 4 2 2 2 1 3 1 1 3 2 1 1 9 1 6 5 1 2 0 1 7 6 1 6 6 1 9 9 2 4 5 K RA Q IS V RG L 1 7 0 1 7 5 1 8 3 2 1 4 2 0 1 2 0 4 1 3 9 1 7 8 1 7 6 1 7 7 2 3 1 2 0 7 2 4 6 H S S Q S Q G G G 0 6 1 0 2 9 0 3 8 0 3 8 0 5 9 0 3 3 0 7 7 0 4 1 0 4 7 0 3 3 0 5 4 0 3 7 2 4 7 M A E A Y S E IG 1 1 3 0 6 5 1 1 4 0 9 3 1 1 6 0 4 4 0 7 7 0 4 5 0 9 7 1 0 2 1 0 2 0 5 0 2 4 8 E E E S S Y S Y E 3 4 9 2 4 9 2 7 8 3 1 0 3 3 2 0 7 9 1 1 9 0 9 4 1 6 8 2 7 1 2 0 6 1 4 3 2 4 9 E Q P G S D D E D 1 0 0 0 5 9 1 5 4 1 0 2 0 6 7 0 4 0 0 7 2 0 6 2 1 1 5 1 0 4 0 6 3 0 3 9 2 5 0 IE Q F S T V K G 0 3 9 0 2 6 0 6 8 0 7 6 0 4 6 0 3 7 0 6 0 0 4 1 0 4 6 0 4 6 0 4 2 0 3 4 2 5 1 M L D H S E S T K 0 4 0 0 2 6 0 6 3 0 4 6 0 4 7 0 4 2 0 5 7 0 4 7 0 4 4 0 4 3 0 6 8 0 4 4 2 5 2 P Y K F P S S P L RI P G Z 0 4 5 0 2 8 0 6 3 0 4 1 0 4 4 0 4 4 0 6 2 0 3 9 0 5 1 0 4 5 0 7 1 0 4 7 2 5 3 G P RT T RA Q G 1 7 3 1 4 6 0 9 7 1 1 8 1 2 7 1 0 6 1 3 6 1 1 7 1 1 9 1 4 4 1 4 3 1 2 3 2 5 4 A Q D T Y L V L D 0 7 6 0 5 1 0 8 2 0 7 5 0 9 1 0 4 5 0 6 2 0 4 9 0 7 2 0 6 0 0 8 6 0 6 8 2 5 5 D IP E S Q M E E 0 6 9 0 5 7 0 7 0 0 7 1 0 7 2 0 3 9 0 6 3 0 5 2 0 7 5 0 5 9 0 7 4 0 5 4 2 5 6 F P RA S F G S R 1 6 9 1 2 7 2 2 1 1 5 2 2 2 8 1 6 3 1 6 2 1 6 2 1 2 0 1 5 3 2 6 2 1 6 5 2 5 7 G RK A S G S S P 0 9 1 0 6 6 1 0 5 0 9 3 1 0 4 1 0 8 1 1 6 1 0 7 0 9 3 1 0 3 1 0 8 0 8 8 2 5 8 E D V G S D E E E 1 2 3 0 5 1 1 8 2 1 2 1 0 9 9 0 3 9 0 8 3 0 6 2 1 3 0 1 3 1 1 5 4 0 5 8 2 5 9 E P A V S P L L P 0 5 6 0 4 6 0 9 0 0 7 9 0 6 8 0 4 9 0 6 8 0 5 2 0 7 3 0 5 9 1 1 4 0 9 0 2 6 0 RRRA S Q L K I 1 0 3 0 8 0 0 7 0 0 9 2 0 8 7 0 4 8 0 8 2 0 9 2 0 7 4 0 8 5 0 7 7 0 5 5 2 6 1 S F T T T A E RE 0 4 7 0 3 7 0 3 9 0 4 1 0 6 2 0 3 8 0 6 2 0 4 5 0 6 6 0 3 8 0 5 7 0 4 3 2 6 2 P L S RT L 0 4 4 0 4 7 0 7 4 0 5 5 0 6 9 0 5 1 0 5 4 0 4 7 0 7 6 0 5 4 0 7 3 0 4 6 2 6 3 S G RG K 0 8 8 0 6 7 0 8 7 0 9 0 1 1 1 1 0 3 1 0 3 1 2 8 0 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1 5 8 1 5 3 1 4 9 1 0 9 0 9 4 2 8 0 V RRV S D D V R 0 4 7 0 3 9 0 7 6 0 6 8 0 6 3 0 5 3 0 6 5 0 3 9 0 6 3 0 5 9 0 5 9 0 5 6 2 8 1 S RK M S IQ E Y 0 5 9 0 5 4 0 5 3 0 8 6 0 9 5 0 3 9 0 5 9 0 4 3 0 5 6 0 6 5 1 0 4 0 7 7 2 8 2 L S G F S F K K S 1 2 4 1 2 8 1 1 7 1 1 5 1 4 6 1 2 2 1 1 4 1 1 9 1 2 8 1 4 1 1 4 2 1 2 5 2 8 3 P S S T S S S S I 0 4 9 0 4 8 0 5 9 0 6 0 0 7 4 0 6 2 0 5 2 0 3 5 0 5 5 0 5 3 0 9 3 0 7 1 2 8 4 S T G IY E A L E 1 4 7 1 0 9 1 6 1 1 7 1 1 5 4 1 0 4 0 6 4 0 5 9 0 9 4 1 1 2 1 4 9 1 1 9 2 8 5 T P P K S P S A S 0 4 9 0 3 9 0 6 4 0 5 2 0 6 4 0 4 1 0 5 8 0 6 9 0 6 7 0 4 6 0 7 2 0 5 4 2 8 6 L Y S S S P G G A 0 4 9 0 4 5 0 7 0 0 5 6 0 6 1 0 5 8 0 5 9 0 3 6 0 5 8 0 4 7 0 5 8 0 4 8 2 8 7 P V P K S P V E E 0 5 1 0 4 1 0 7 9 0 5 6 0 6 0 0 5 2 0 5 8 0 2 9 0 6 6 0 5 0 0 5 1 0 3 9 2 8 8 K K A S F K A K K 1 7 3 1 8 7 2 2 8 2 3 6 2 4 3 2 0 9 1 3 7 2 3 7 3 2 3 2 6 8 2 0 2 1 8 1 2 8 9 K S P A K T P V K 1 3 1 1 1 7 1 1 0 1 2 5 1 5 8 1 4 8 1 2 3 1 3 8 1 4 0 1 7 0 2 0 7 1 6 2 2 9 0 P L S K T 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RP S I RA K A 2 8 2 1 2 7 3 5 0 2 2 1 3 8 4 1 1 0 1 6 9 1 9 9 1 9 7 1 5 5 1 8 4 1 3 7 3 4 9 N P G F Y V E A N 0 9 0 0 4 9 1 1 0 0 8 5 2 4 4 0 5 0 0 8 3 0 9 7 0 8 7 0 6 3 0 9 1 0 6 0 3 5 0 P Y K F P S S P L RI P G Z 0 4 5 0 2 3 0 5 2 0 7 2 0 9 6 0 3 8 0 5 8 0 7 1 0 5 8 0 1 8 0 4 2 0 3 9 3 5 1 G S RG S G S S V 0 6 1 0 4 3 0 6 7 0 3 7 0 9 8 0 5 6 1 0 9 0 8 9 0 5 2 0 5 5 0 8 9 0 8 1 3 5 2 D A G A S P V E K 0 3 8 0 1 5 0 6 8 0 4 9 0 7 3 0 2 3 0 5 8 0 3 8 0 6 7 0 5 1 0 4 3 0 6 8 3 5 3 D RRV S V A A E 0 3 9 0 2 2 0 4 2 0 2 3 1 0 3 0 2 0 0 6 5 0 2 8 0 7 0 0 3 4 0 4 2 0 4 5 3 5 4 G G RA S D Y K S 0 7 7 0 4 2 0 7 1 0 3 4 1 8 6 0 4 0 1 0 5 1 0 3 0 8 5 0 6 8 0 7 5 0 6 7 3 5 5 G V D T Y V E M R 0 4 5 0 2 6 0 8 7 0 4 7 2 5 1 0 2 8 0 5 9 0 5 5 0 6 9 0 5 0 0 6 4 0 3 8 3 5 6 E G T H S T K RG 0 9 6 0 6 3 0 8 4 0 7 0 1 9 7 0 6 7 1 0 3 1 2 4 0 9 7 0 6 4 0 7 1 0 6 3 3 5 7 E S L E S Y E IN 0 8 2 0 3 6 1 0 5 0 8 1 1 6 7 0 4 8 0 7 7 0 8 7 0 8 5 0 5 7 1 0 5 0 5 1 3 5 8 RV RK T K G K Y 1 8 4 1 0 0 2 3 8 1 6 5 2 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S K S 1 5 8 1 2 9 1 8 7 1 1 4 1 8 1 0 9 9 1 4 6 1 5 8 1 0 7 0 9 3 1 3 1 1 3 5 3 7 0 V RK IS G L 0 9 1 0 6 1 0 7 3 0 4 5 0 9 6 0 4 3 1 0 6 1 3 4 0 9 4 0 7 7 1 0 1 0 5 7 3 7 1 A D G V Y A A S G 0 6 1 0 2 4 0 9 6 0 5 7 0 9 9 0 3 1 0 7 7 0 7 7 0 9 7 0 5 6 0 5 1 0 3 6 3 7 2 T G F L T E Y V A 0 7 4 0 2 8 1 2 0 0 7 5 0 9 2 0 5 7 0 6 0 0 6 9 0 9 2 0 9 0 0 7 9 0 5 7 3 7 3 T V S T S L G H S 0 4 8 0 2 8 0 5 7 0 4 8 0 6 8 0 5 7 0 6 8 0 4 9 0 6 4 0 7 8 0 4 8 0 3 8 3 7 4 Y D K E Y Y S V H 0 8 3 0 4 7 0 9 6 0 6 6 1 5 9 0 6 5 1 0 9 0 9 9 0 9 8 0 8 4 1 2 7 2 1 1 3 7 5 RK L K S Q G T R 1 2 2 0 8 0 1 0 5 0 6 0 1 6 3 1 1 9 1 2 8 1 4 7 1 0 9 1 0 6 1 4 3 1 4 0 3 7 6 N RK P S K D K D 0 8 0 0 7 1 0 9 6 0 5 3 1 8 5 1 4 7 1 2 7 1 5 1 0 9 4 0 9 2 1 7 4 1 7 6 3 7 7 L RRA S P G 0 5 8 0 5 8 0 7 3 0 4 6 1 4 7 0 8 2 1 2 8 1 0 4 0 6 6 0 6 9 1 1 4 1 1 7 3 7 8 Y M A P Y D N Y V 1 0 8 0 4 8 1 1 4 0 7 7 1 3 5 0 4 2 0 9 4 1 1 3 1 0 9 0 8 6 1 0 5 0 7 0 3 7 9 S S K A Y G N G Y 0 5 4 0 3 3 0 8 5 0 4 4 0 7 2 0 4 3 0 6 8 0 6 2 0 9 3 0 9 1 0 5 0 0 5 2 3 8 0 P L T P S G E A P 0 4 0 0 4 0 0 7 4 0 4 2 0 8 8 0 8 3 0 5 5 0 6 9 0 8 8 0 6 1 0 7 6 0 6 9 3 8 1 RRRRP T P A 1 9 7 2 3 3 3 4 5 3 6 0 5 3 4 4 9 3 2 3 7 3 2 4 4 2 1 3 3 4 5 3 5 5 1 6 3 8 2 T E G Q Y Q P Q P 0 3 7 0 3 6 0 5 8 0 4 6 0 9 0 0 6 8 0 7 6 0 5 5 1 1 2 0 6 4 0 8 0 0 6 6 3 8 3 T S S S S IF D I 0 5 7 0 3 9 0 8 2 0 4 6 1 1 4 0 5 9 0 6 2 0 6 4 0 9 4 0 6 4 0 8 9 0 8 0 3 8 4 P P S P S L S RH 2 4 2 1 8 7 1 9 2 1 6 4 2 8 8 1 8 9 1 9 3 1 9 5 2 1 0 1 7 3 2 2 7 2 2 6

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3 0 3 1 0 8 6 0 7 1 1 0 5 1 1 0 0 3 7 0 3 7 3 9 6 E N A F S P S RS 0 6 6 0 5 8 0 6 2 0 4 5 0 6 3 0 3 2 0 5 0 0 1 4 0 4 7 0 3 6 0 2 4 0 3 2 3 9 7 H K S G Y L S S E 0 6 7 0 4 4 0 4 3 0 4 4 0 8 5 0 2 8 0 6 6 0 3 4 0 4 5 0 4 7 0 3 2 0 3 5 3 9 8 L A Y E S H E S M 0 4 8 0 3 6 0 6 8 0 5 9 1 0 1 0 5 3 0 5 1 0 1 5 0 5 6 0 3 4 0 4 3 0 3 7 3 9 9 P Y K F P S S P L RI P G Z 0 5 7 0 3 7 0 6 1 0 6 2 1 1 4 0 4 0 0 5 3 0 3 2 0 9 1 0 4 3 0 6 3 0 5 8 4 0 0 G H Q G T V P S D 0 5 2 0 3 7 0 3 7 0 3 3 0 9 1 0 6 0 0 5 8 0 4 9 0 4 0 0 2 1 0 3 3 0 4 7 4 0 1 A N D E Y F IR K 0 5 7 0 3 4 0 4 9 0 4 6 0 6 2 0 3 1 0 5 8 0 3 2 0 3 9 0 2 4 0 3 2 0 3 4 4 0 2 D E A S T T V S K 0 5 4 0 4 4 0 5 5 0 5 3 0 6 7 0 3 5 0 5 4 0 4 4 0 6 0 0 3 1 0 2 6 0 3 9 4 0 3 F E A RY Q Q P F 0 7 6 0 6 7 0 7 1 0 6 1 0 6 6 0 2 8 0 6 4 0 4 2 0 7 2 0 5 0 0 3 1 0 3 3 4 0 4 G L L R S W N D P 0 8 2 0 6 0 0 7 4 0 6 0 0 8 5 0 3 6 0 7 0 0 4 5 0 6 1 0 5 4 0 5 4 0 3 3 4 0 5 D T V T S P Q RA 3 0 4 2 2 3 2 6 5 2 3 9 0 9 4 0 3 7 1 3 2 1 1 5 1 5 0 1 7 4 0 4 1 0 2 6 4 0 6 E K H H S ID A Q 0 5 8 0 5 6 0 6 4 0 5 5 0 9 6 0 3 9 0 8 8 0 5 3 0 6 1 0 3 9 0 7 6 0 3 9 4 0 7 RRA S T IE M P 1 3 4 1 2 3 0 8 3 0 7 7 0 5 4 0 3 0 1 0 0 1 7 1 0 5 0 0 6 2 0 3 4 0 4 1 4 0 8 S D E E S N D D S 1 4 9 1 4 8 1 6 1 0 7 0 0 7 1 0 3 8 0 7 9 0 5 5 0 8 9 0 6 5 0 3 7 0 4 9 4 0 9 N V F S S P G G T 0 5 7 0 4 6 0 8 3 0 5 2 0 6 0 0 3 1 0 6 2 0 3 2 0 5 8 0 3 1 0 3 1 0 4 9 4 1 0 RRA S F 2 6 7 2 5 1 2 0 2 2 0 2 0 5 4 0 3 4 1 8 1 1 9 2 1 1 2 1 5 1 0 3 1 0 4 0 4 1 1 A K G G T V K A A 1 1 4 1 0 6 0 6 0 0 6 6 0 5 8 0 3 4 1 2 4 0 8 3 0 7 6 0 8 3 0 3 9 0 4 1 4 1 2 D D E IT Q D E N 0 9 8 0 8 8 0 8 9 0 7 2 0 9 3 0 3 0 0 7 6 0 4 9 0 8 2 0 6 5 0 3 4 0 4 2 4 1 3 E T D D Y A E I I 0 9 7 0 7 8 1 0 0 0 8 7 1 0 0 0 5 8 0 6 3 0 3 2 0 7 0 0 5 1 0 4 4 0 4 7 4 1 4 Y T T N S P S K I 0 7 4 0 9 4 0 4 4 0 4 9 0 4 0 0 4 3 0 6 8 0 3 1 0 4 5 0 3 6 0 2 5 0 4 2 4 1 5 S P L K S P Y K I 1 1 7 1 2 1 0 8 6 0 6 4 0 4 3 0 2 8 1 0 1 0 8 7 0 5 7 0 8 0 0 3 0 0 3 1 4 1 6 RK RT RK E 1 7 2 1 6 8 1 6 0 1 2 5 0 5 4 0 5 4 1 3 9 1 4 6 1 9 0 1 4 0 0 5 2 0 5 1 4 1 7 RP P G F T P F R 1 6 6 1 8 0 0 9 7 1 0 3 0 5 1 0 4 1 1 3 1 1 2 9 0 8 3 1 0 4 0 3 7 0 4 4 4 1 8 S A S G T P N K E 0 5 0 0 5 2 0 3 8 0 4 3 0 6 1 0 5 3 0 4 6 0 3 9 0 5 2 0 3 5 0 2 7 0 3 6 4 1 9 N S V D T S S L S 0 6 4 0 5 4 0 7 7 0 6 4 0 6 1 0 4 6 0 6 8 0 3 6 0 5 9 0 5 2 0 3 5 0 4 5 4 2 0 L RA N S I 0 7 0 0 6 8 0 6 7 0 7 3 0 7 2 0 6 1 0 8 6 0 3 2 0 6 5 0 6 6 0 4 7 0 3 9 4 2 1 S S S S S P S RR 2 8 4 3 2 2 1 4 1 1 1 9 0 4 6 0 7 1 1 5 9 1 4 4 0 8 6 1 5 1 0 7 1 0 6 8 4 2 2 T K S A S F L K G 1 5 6 1 5 6 1 2 8 1 0 7 0 3 3 0 3 6 1 1 5 0 8 2 0 6 3 0 8 1 0 3 0 0 5 3 4 2 3 V E S L S S S E E 1 1 7 1 1 0 1 0 2 0 9 5 0 5 4 0 3 5 0 8 0 0 5 8 0 7 7 0 6 2 0 3 8 0 4 3 4 2 4 Y RRN S V R F L 1 8 1 2 3 4 1 8 9 1 9 2 0 4 9 0 5 2 2 0 6 2 3 3 1 1 6 1 5 0 0 4 6 0 6 5 4 2 5 S P A L T G D E A 0 8 4 0 6 0 0 6 9 0 6 9 0 4 6 0 3 4 0 6 5 0 4 1 0 8 2 0 5 1 0 3 4 0 5 0 4 2 6 RK Q IT V R 2 0 1 1 9 2 1 6 8 1 7 2 0 7 3 0 7 9 1 8 6 2 4 8 1 9 1 1 6 5 0 5 3 0 6 3 4 2 7 V H N RS K IN L 1 0 9 1 1 0 1 0 2 1 1 4 0 6 8 0 6 7 0 9 8 0 9 6 1 0 9 1 0 6 0 5 3 0 5 4 4 2 8 S P V H S IA D E 0 9 4 0 6 0 0 7 2 0 5 0 0 5 2 0 3 7 0 6 0 0 3 3 0 4 4 0 5 4 0 2 8 0 4 6 4 2 9 L RRA S L G A F 2 5 9 2 9 1 2 5 6 2 1 8 0 6 7 0 7 4 1 7 6 1 3 6 1 4 7 1 6 8 0 6 6 0 7 6 4 3 0 P RK G S P RK G 1 7 9 1 7 0 1 1 1 1 0 0 0 5 2 0 4 1 1 2 1 0 9 7 1 1 3 0 9 6 0 3 6 0 5 0 4 3 1 S S P G S P G T P 0 6 7 0 5 1 0 6 9 0 6 2 0 4 9 0 4 2 0 7 9 0 4 7 0 4 5 0 4 8 0 3 4 0 4 6 4 3 2 T K A A S E K K T 0 8 4 0 5 8 0 8 9 0 9 3 0 6 1 0 3 5 0 8 6 0 5 3 0 7 8 0 6 4 0 3 6 0 4 2 4 3 3 L RRS S S V G Y 4 1 6 4 0 7 4 2 1 4 3 3 1 0 9 0 9 2 3 3 5 2 7 5 2 5 4 3 8 2 0 9 8 1 0 7 4 3 4 P S E K S E E IT 0 8 9 0 8 3 0 8 8 1 0 0 0 6 3 0 5 6 0 7 6 0 5 0 0 5 3 0 5 5 0 4 2 0 4 4 4 3 5 K A Q E Y F N IK 0 6 8 0 5 3 0 3 9 0 4 9 0 4 7 0 5 0 0 8 1 0 3 8 0 6 6 0 5 1 0 4 5 0 4 6 4 3 6 K RQ S S T S N A 1 2 7 1 5 8 0 9 2 0 8 1 0 8 1 0 6 8 1 1 9 1 0 0 1 1 7 0 9 4 0 5 9 0 6 9 4 3 7 Q K RP S Q RS K Y L 2 8 2 3 2 1 2 6 4 2 4 1 1 1 7 0 7 4 1 9 2 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9 2 1 2 8 4 6 4 D D E E S E S D 1 6 8 1 2 7 2 1 7 1 8 8 1 1 7 1 0 6 1 0 8 1 0 6 1 2 8 1 4 8 1 0 8 0 7 9 4 6 5 V K RIS G L 1 0 8 0 9 7 0 9 0 1 0 2 0 9 5 1 0 3 1 2 2 1 2 9 0 8 7 1 1 5 0 9 4 0 9 5 4 6 6 RRK D Y P A L H 0 6 9 0 5 8 0 6 9 0 7 5 1 7 2 0 9 8 1 0 9 0 6 5 0 7 2 0 9 2 0 9 1 0 8 2 4 6 7 S E K K S K G L G 0 6 5 0 3 7 0 5 8 0 6 3 0 6 4 0 5 8 0 8 4 0 6 3 0 6 3 0 7 6 0 7 4 0 7 6 4 6 8 P P S A Y G S V K 0 8 7 0 7 6 0 9 5 1 1 4 1 0 0 1 0 1 1 0 4 0 7 4 0 8 5 1 2 5 1 2 3 1 0 6 4 6 9 RRF S V 1 3 9 1 4 0 2 5 9 1 9 3 2 0 1 2 3 3 1 7 3 1 3 9 1 3 0 1 7 3 2 5 1 2 0 6 4 7 0 S T RR S V RG S 3 7 7 3 8 9 3 4 2 3 8 5 3 9 2 4 7 6 2 3 0 3 2 0 2 3 6 3 2 3 4 8 7 4 9 7 4 7 1 T Q S T S G RR R 2 5 6 3 0 4 2 7 5 2 7 6 2 6 7 3 2 5 2 0 2 2 9 3 2 8 7 2 8 7 3 3 0 3 5 9 4 7 2 V N E L S K D IG 0 3 7 0 4 8 0 5 4 0 6 0 0 7 3 0 6 5 0 7 2 0 5 2 0 6 7 0 5 1 0 6 7 0 6 8 4 7 3 RA G E T R F T D 0 3 6 0 4 1 0 5 9 0 5 8 0 6 2 0 6 2 0 6 6 0 2 7 0 6 7 0 4 1 0 5 9 0 5 1 4 7 4 S P S P S F R W P 0 5 1 0 5 4 0 7 2 0 7 9 1 1 4 1 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N Y ID L D 0 9 3 0 8 6 1 0 1 1 6 5 1 3 9 1 0 7 0 7 9 0 5 9 0 9 0 1 2 2 1 0 5 0 9 7 4 8 6 K RK Q IS V RG L 1 8 4 1 9 7 1 8 4 2 1 9 1 8 7 1 5 9 1 7 9 1 4 0 2 0 7 1 8 7 1 4 8 1 7 3 4 8 7 S RG D Y M T M Q 0 4 5 0 5 1 0 3 9 0 5 1 0 6 0 0 7 8 0 5 6 0 4 1 0 5 8 0 3 7 0 5 9 0 5 4 4 8 8 K K K G S G E D D 0 4 3 0 4 1 0 5 8 0 6 2 0 8 5 1 0 3 0 8 0 0 4 8 0 7 6 0 6 5 1 0 2 0 9 5 4 8 9 K T S P S S S P A 0 3 5 0 4 9 0 4 9 0 6 0 0 7 2 0 9 1 0 7 4 0 3 8 0 6 7 0 5 7 0 8 9 0 8 8 4 9 0 P RRR S S F G I 0 6 8 0 7 9 0 8 0 0 8 5 0 5 1 0 6 4 0 6 2 0 5 4 0 7 8 0 6 5 0 5 6 0 5 1 4 9 1 IG S V S E D N S 1 6 5 1 5 7 1 8 7 1 5 6 0 4 5 0 6 2 1 7 1 2 3 2 1 5 0 1 9 1 0 4 5 0 3 3 4 9 2 N A P V S A L G E 0 4 7 0 3 7 0 6 6 0 4 7 0 6 1 0 5 4 0 2 9 0 3 6 0 6 9 0 6 7 0 4 5 0 3 9 4 9 3 E E T P Y S Y P T 1 4 1 1 1 4 1 9 8 1 8 1 0 8 5 0 7 0 1 1 5 1 5 2 1 2 4 1 9 3 0 7 3 0 4 8 4 9 4 E RS P S P S F R 1 5 2 1 2 3 1 8 8 1 4 7 0 6 8 0 6 4 1 9 7 2 0 1 1 2 2 1 8 0 0 7 1 0 6 0 4 9 5 IRK Y T M RRL 1 5 2 1 0 9 2 3 3 1 8 0 0 9 6 0 9 2 2 2 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0 4 6 1 0 7 0 8 2 0 7 0 1 1 4 0 3 9 0 3 8 5 1 2 RG A IS A E V Y 0 6 7 0 6 8 0 9 2 0 8 8 0 5 3 0 6 1 0 8 7 0 7 1 1 0 0 1 1 6 0 5 0 0 4 6 5 1 3 RK IS A S E F 0 4 4 0 5 7 0 6 7 0 7 4 0 3 8 0 5 5 0 9 3 0 8 6 0 7 0 0 7 8 0 3 6 0 4 2 5 1 4 L H RA S L G 0 2 7 0 6 3 0 9 7 1 0 3 0 4 6 0 5 9 0 7 4 0 7 3 0 7 1 1 2 0 0 3 0 0 4 4 5 1 5 RRRP T P A M L 0 9 6 1 1 4 1 0 9 1 2 4 0 4 9 0 6 1 1 0 5 1 4 3 0 8 7 1 4 6 0 4 7 0 6 0 5 1 6 S G Y S S P G S P 0 3 4 0 3 2 0 6 7 0 5 7 0 3 0 0 5 2 0 5 4 0 4 4 0 5 7 0 6 3 0 3 6 0 3 9 5 1 7 L G E G T P 0 4 7 0 4 4 0 7 8 0 5 7 0 5 2 0 4 1 0 5 5 0 5 0 0 5 9 0 7 3 0 3 6 0 3 6 5 1 8 RRL S S L R A 3 2 6 3 5 0 4 0 8 3 2 2 1 4 9 1 6 8 3 6 3 4 4 9 1 8 8 3 6 0 1 4 6 1 4 0 5 1 9 T A E S S Q A E E 0 3 8 0 5 6 0 9 4 0 7 8 0 3 8 0 7 1 0 7 7 0 4 1 1 1 2 0 7 1 0 3 7 0 4 8 5 2 0 T RRA S F S A Q 1 5 9 1 5 3 2 2 0 2 8 4 0 5 4 0 6 8 1 4 5 1 7 4 1 1 7 1 7 4 0 5 6 0 4 2 5 2 1 V S RT S A V P T 0 4 3 0 4 7 0 6 4 0 7 3 0 4 4 0 5 6 0 8 3 0 4 8 0 4 3 0 7 1 0 4 0 0 3 9 5 2 2 RE Q L S T S E E 0 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0 4 6 0 4 4 5 3 3 K L IN S IA D T 0 3 4 0 5 0 0 6 8 0 6 3 0 4 1 0 6 3 0 6 3 0 3 4 0 6 6 0 7 4 0 4 7 0 4 8 5 3 4 L D D Q Y T S S S 1 3 6 1 2 3 1 5 0 1 8 9 0 5 7 0 7 0 1 1 0 0 9 8 1 4 1 1 5 5 0 5 2 0 4 4 5 3 5 P Y D N Y V P S A 0 9 4 0 8 7 1 3 3 1 2 2 0 5 2 0 5 3 0 8 7 0 8 1 1 1 6 1 4 3 0 3 9 0 3 7 5 3 6 S RQ L S S G V S 0 3 1 0 4 3 0 5 2 0 4 1 0 5 5 0 6 7 0 7 1 0 4 4 0 7 0 0 6 2 0 3 7 0 5 3 5 3 7 K N IV T P R T P 0 8 6 1 4 7 0 9 8 1 2 3 0 7 2 0 7 7 1 2 3 1 3 1 1 4 2 1 6 7 0 5 8 0 6 3 5 3 8 L K L A S P E L E 0 2 6 0 4 0 0 6 8 0 6 0 0 4 2 0 5 3 0 6 2 0 2 9 1 0 1 0 9 5 0 2 4 0 3 5 5 3 9 Q L ID S M A N S 0 3 8 0 3 6 0 6 2 0 4 4 0 5 3 0 4 9 0 5 8 0 3 0 0 6 0 0 6 8 0 3 4 0 2 8 5 4 0 IV Y K S P V V S 0 5 0 0 3 6 0 7 4 0 6 1 2 0 2 1 2 3 0 7 8 0 7 7 0 9 6 0 9 2 1 0 6 0 8 2 5 4 1 N G Y IS A A E L 0 5 4 0 4 5 1 7 5 1 6 2 0 9 4 0 5 2 0 8 7 0 9 2 1 5 3 1 9 4 0 6 6 0 4 0 5 4 2 E IL N S P E K A 0 2 0 0 2 8 0 3 6 0 3 6 0 5 3 0 4 3 0 5 1 0 3 7 0 5 7 0 4 9 0 4 2 0 3 9 5 4 3 E S P E S T E I T 0 3 6 0 2 6 0 8 0 0 3 2 0 7 6 0 3 2 0 5 6 0 4 3 0 6 1 0 6 4 0 3 8 0 4 6 5 4 4 K RP S H RA K A 0 9 9 0 9 7 1 1 4 0 7 1 2 0 9 1 4 2 1 2 3 1 2 5 1 2 9 1 0 9 1 3 8 1 6 1 5 4 5 N N Y V Y ID P T 0 5 8 0 4 2 0 9 1 0 5 0 1 2 7 0 5 8 0 7 2 0 6 5 0 7 9 0 7 0 1 0 0 0 7 9 5 4 6 P Y K F P S S P L RI P G Z 0 2 7 0 2 6 0 3 8 0 2 9 0 5 6 0 3 8 0 6 6 0 5 0 0 4 9 0 3 7 0 3 2 0 3 2 5 4 7 G S P R T P RRG 1 3 7 1 5 5 2 2 0 1 5 1 3 6 2 3 9 7 1 9 1 2 0 6 2 1 4 1 8 9 2 8 9 3 1 9 5 4 8 D A D E Y L I P Q 0 8 0 0 6 3 1 1 6 1 0 2 1 7 5 1 1 4 0 7 1 0 5 8 0 9 7 1 0 9 1 4 3 1 0 8 5 4 9 D RL V S A RS V 0 7 8 0 7 2 0 7 6 0 6 4 1 2 4 0 9 8 1 1 3 1 2 3 0 9 8 1 3 3 1 0 8 0 9 7 5 5 0 G G L T S P G L S 0 2 8 0 2 6 0 5 7 0 2 7 0 5 9 0 2 6 0 5 2 0 4 0 0 7 1 0 4 4 0 4 5 0 4 6 5 5 1 G T V P S D N ID 0 3 4 0 1 6 0 8 3 0 3 5 0 6 5 0 2 6 0 6 5 0 4 5 0 7 5 0 6 9 0 3 4 0 3 6 5 5 2 E G S A Y E E V P 0 4 8 0 4 8 0 7 3 0 5 2 1 6 3 0 8 7 0 9 9 1 1 3 0 7 3 0 7 8 1 3 0 1 0 2 5 5 3 E S II S Q E T Y 0 9 3 0 5 3 0 9 5 0 7 4 1 3 7 0 9 6 0 7 8 0 8 0 0 9 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E T S Q V A P 0 5 0 0 6 1 0 3 7 0 4 7 0 4 5 0 8 7 0 6 4 0 9 5 0 4 5 0 4 5 0 5 6 0 8 5 9 8 7 P Y K F P S S P L RI P G Z 0 5 4 0 8 5 0 4 0 0 4 8 0 5 6 0 8 0 0 5 9 0 7 9 0 5 7 0 5 1 0 6 8 0 6 5 9 8 8 G G S V T K K R K 2 1 6 2 3 7 1 4 1 1 9 7 0 8 3 1 1 5 1 9 0 2 2 6 1 7 5 1 5 8 0 9 8 0 6 7 9 8 9 A K RIS G K M A 2 0 5 2 4 2 0 7 8 1 2 1 0 7 6 1 4 4 1 2 7 1 4 0 1 0 2 0 9 7 1 0 5 0 9 8 9 9 0 D D P S Y V N V Q 0 9 6 1 1 2 0 4 1 0 6 9 0 4 4 1 2 2 0 6 7 0 7 2 0 6 5 0 5 9 0 9 5 0 7 0 9 9 1 E V E K S P V K S 0 6 1 0 6 9 0 3 4 0 4 9 0 2 0 0 8 8 0 5 3 0 8 2 0 6 5 0 3 3 0 8 5 0 7 5 9 9 2 G K S S S Y S K Q 1 2 4 1 6 8 0 6 1 0 8 1 0 3 7 1 1 7 1 1 6 1 2 8 0 9 0 0 8 9 0 9 0 1 0 1 9 9 3 D S RS S L I RK 1 5 8 2 2 3 0 8 9 1 6 0 0 4 7 1 4 9 1 2 4 1 4 9 0 9 2 0 9 2 0 8 5 1 1 1 9 9 4 E K A K S P V P K 0 8 4 1 4 0 0 5 4 0 7 3 0 5 6 1 3 1 0 9 1 1 3 2 0 8 7 0 7 4 0 9 0 0 8 3 9 9 5 RQ RK S R RT I 2 2 9 2 8 6 1 2 9 1 6 6 0 9 6 1 8 0 1 7 0 2 1 1 1 3 9 1 3 0 1 5 0 1 5 0 9 9 6 S A Y A T V K A Y 0 9 7 0 9 2 0 2 7 0 6 0 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146 1 0 8 1 E E P V Y E A E P 1 7 8 1 8 0 1 2 1 2 1 3 0 8 0 2 0 8 0 9 9 1 2 9 1 4 0 1 9 7 1 2 1 1 5 3 1 0 8 2 E RRN S IL T E 0 5 8 1 0 1 0 5 1 1 0 5 0 5 9 0 9 8 0 7 6 1 0 6 0 6 4 1 0 5 0 7 2 0 8 4 1 0 8 3 IN E T S Q H H D 0 2 4 0 3 6 0 3 1 0 4 4 0 7 2 0 7 2 0 6 0 0 8 2 0 4 3 0 7 3 0 4 7 0 8 3 1 0 8 4 N D S N Y V V K G 0 3 0 0 3 7 0 3 3 0 4 8 0 5 4 0 6 1 0 6 1 0 8 7 0 5 5 0 8 7 0 5 0 0 8 1 1 0 8 5 P Y K F P S S P L RI P G Z 0 3 1 0 4 4 0 3 3 0 5 2 0 5 4 0 7 0 0 6 4 0 8 6 0 6 2 0 7 0 0 6 2 0 6 7 1 0 8 6 G RN A S T N D S 0 5 3 0 5 9 0 4 8 0 5 5 0 6 5 1 0 3 0 7 8 0 9 2 0 6 3 0 5 5 0 7 1 0 8 9 1 0 8 7 A RS G S S T Y S 1 1 6 2 0 6 0 6 9 1 0 1 0 7 3 2 4 5 1 4 0 1 5 7 0 7 2 1 1 2 1 7 0 2 6 7 1 0 8 8 D L L T S P D V G 0 4 8 0 7 3 0 9 8 0 7 9 0 7 0 1 1 6 0 6 7 0 9 3 0 7 9 1 0 9 0 6 6 1 1 9 1 0 8 9 G A G N S L R T A 0 7 0 1 1 8 0 4 4 0 7 1 0 7 2 1 2 3 1 2 8 1 1 9 0 7 3 1 0 1 0 9 7 1 1 9 1 0 9 0 G RT W T L A G T 0 5 3 1 0 2 0 2 9 0 5 3 0 5 6 1 0 6 1 1 6 1 2 9 0 6 4 0 9 1 0 7 4 0 7 9 1 0 9 1 E E H V Y S F P N 0 5 2 0 7 7 0 4 7 0 7 2 0 6 4 1 1 8 0 7 7 1 0 2 0 7 5 0 9 7 0 7 3 0 7 4 1 0 9 2 E RH H S ID A Q 0 5 0 0 5 5 0 5 2 0 4 9 0 7 1 1 1 5 0 7 5 0 9 7 0 5 3 0 7 1 0 7 2 0 7 1 1 0 9 3 RRRRA A S V A 2 6 0 3 4 5 2 3 6 2 2 5 0 9 8 3 3 2 2 1 2 2 4 5 2 4 7 2 0 2 2 2 5 2 6 7 1 0 9 4 S IRD T P A K N 0 4 6 0 7 5 0 4 9 0 6 2 0 4 1 1 2 2 0 7 9 0 8 7 0 7 0 0 6 2 0 6 4 1 3 2 1 0 9 5 G G Y S L G 0 4 6 0 7 7 0 5 9 0 6 5 0 5 0 1 1 3 0 8 6 1 0 4 0 7 0 0 9 4 0 6 3 1 1 2 1 0 9 6 A A A S F K A K K 0 8 3 1 3 6 0 7 6 1 1 9 0 7 8 1 3 9 1 2 4 1 4 6 1 0 9 1 2 9 1 1 1 1 3 6 1 0 9 7 A RD IY K N D Y 0 6 1 0 7 9 0 5 1 0 9 0 0 4 9 1 2 2 0 6 9 1 0 3 0 9 9 1 1 9 0 8 7 1 1 1 1 0 9 8 D K E V S D D E A 0 5 1 0 7 1 0 7 2 0 9 4 0 7 5 1 5 4 0 6 1 0 9 7 0 8 7 1 3 0 0 8 4 0 8 9 1 0 9 9 F RK F T K S E R 1 4 9 2 3 6 1 5 8 1 9 7 1 2 7 2 5 9 1 5 6 1 9 6 2 1 9 1 9 2 1 6 9 1 5 4 1 1 0 0 RF F G S D RG A 1 0 5 2 1 8 1 0 5 1 5 2 0 6 1 1 8 8 1 3 2 1 5 5 0 9 4 1 2 2 1 2 3 2 0 1 1 1 0 1 P RT P G G RR 0 6 7 1 3 6 0 8 3 0 8 7 0 3 9 1 0 6 1 0 2 1 2 7 0 9 5 1 2 3 0 5 7 1 2 7 1 1 0 2 K RRV S E V 0 4 6 0 9 8 0 3 8 0 7 0 1 3 5 2 0 0 0 9 8 1 1 6 0 7 4 1 0 3 1 6 7 1 9 9 1 1 0 3 RRRG S S IP Q 1 2 0 1 9 5 0 7 9 1 3 2 0 8 5 1 5 0 1 3 2 1 6 7 1 1 2 1 2 5 1 3 3 1 9 2 1 1 0 4 S G D T S P RH L 0 4 0 0 5 2 0 4 1 0 6 3 0 5 1 0 9 9 0 7 4 0 9 0 0 5 8 0 8 0 0 6 0 1 0 5 1 1 0 5 K Q IT V R 0 9 0 1 3 7 0 8 3 0 9 7 2 6 4 1 7 9 1 2 3 1 5 3 1 1 4 1 4 0 1 2 3 1 2 3 1 1 0 6 S S K RA 1 0 6 1 9 0 1 3 7 1 0 5 1 3 4 2 0 6 1 3 2 1 6 4 3 7 4 1 4 7 1 5 7 1 3 7 1 1 0 7 S V S S S S Y RR 3 0 6 4 1 2 2 3 4 3 8 0 1 0 0 4 3 4 2 2 9 3 1 8 2 1 0 2 8 1 2 7 9 4 7 7 1 1 0 8 T RQ A S I S G P 0 6 8 1 4 8 0 5 5 1 0 6 0 4 9 1 5 6 1 1 0 1 2 4 0 7 7 1 0 0 0 8 1 2 3 3 1 1 0 9 V RT Y T H E V V 0 5 2 0 9 0 0 4 9 0 7 8 0 7 9 1 4 3 0 8 0 1 1 4 0 9 0 2 0 5 0 7 3 1 4 9 1 1 1 0 RE IL S RR P S 2 5 9 3 4 5 1 3 3 2 9 6 1 3 7 3 7 9 1 8 3 2 6 5 1 3 7 1 4 9 2 4 1 4 1 1 1 1 1 1 G A S G S F K L 0 4 5 0 9 7 0 5 0 1 1 0 0 7 9 1 0 0 1 0 1 1 1 2 0 8 1 1 0 2 0 6 6 1 6 0 1 1 1 2 V RRS D RA 0 9 0 1 8 3 0 8 1 1 1 0 0 7 4 1 4 7 1 1 3 1 5 3 1 1 8 2 0 0 1 2 7 1 5 8 1 1 1 3 V T RRT L S M D 1 0 6 1 7 6 1 0 2 1 7 2 0 6 9 1 5 5 1 1 5 1 6 6 1 6 3 1 3 4 1 2 9 1 4 8 1 1 1 4 S RRG S E S S E 0 7 9 1 2 9 0 6 7 1 1 3 0 6 9 2 4 3 0 8 0 1 2 4 0 8 9 0 8 4 0 9 3 1 9 6 1 1 1 5 P A S A Y G S V K 0 4 4 0 8 4 0 4 3 0 7 5 0 3 7 1 6 3 0 6 8 1 2 8 0 5 3 0 6 3 0 6 6 1 5 3 1 1 1 6 P W Q V S L R T R 0 4 4 0 7 9 0 4 2 0 8 5 0 3 4 1 5 3 0 6 4 1 1 0 0 7 9 0 9 4 0 7 5 1 3 2 1 1 1 7 S T S RS L Y S S 1 5 4 3 9 8 1 2 6 3 0 9 1 3 8 4 0 0 1 8 1 2 4 2 1 8 6 1 7 8 3 0 7 4 4 2 1 1 1 8 T RG G S L E R S 0 4 8 1 3 4 0 5 3 1 2 8 0 6 4 1 3 4 1 0 5 1 2 1 0 7 7 1 2 3 1 1 2 2 4 9 1 1 1 9 P F K L S G L S F 0 2 3 0 6 3 0 2 9 0 8 5 0 5 4 1 1 4 0 6 3 0 8 2 0 6 9 0 9 2 0 6 2 0 8 6 1 1 2 0 Q E Q E Y V Q A V 0 3 7 0 3 8 0 2 9 0 6 2 0 4 4 1 3 0 0 5 5 0 8 9 0 7 6 1 0 5 0 9 7 1 0 6 1 1 2 1 K K RF S F K K S 1 7 0 2 4 6 2 5 4 3 2 3 1 3 7 3 3 7 1 9 2 3 0 0 4 3 3 3 7 6 3 3 2 3 0 8 1 1 2 2 K Y L A S A S T M 0 6 0 1 2 2 0 6 5 1 0 4 0 5 2 2 0 0 0 8 4 1 1 9 1 0 4 0 9 9 1 0 0 1 8 2 1 1 2 3 P RRV S RRR R 0 3 7 0 8 7 0 7 6 0 8 6 0 2 7 1 5 5 0 8 0 1 0 9 1 0 4 0 9 6 0 6 2 1 3 4 1 1 2 4 S RK R S G E A T 0 4 1 1 0 6 0 3 3 0 7 7 0 5 4 2 1 4 0 7 3 1 0 3 0 6 6 0 9 1 1 0 2 1 5 1 1 1 2 5 K M K D T D S E E 0 2 0 0 6 5 0 2 9 1 0 6 0 5 7 1 0 6 0 7 2 0 9 8 0 6 4 1 0 3 0 5 0 1 1 8 1 1 2 6 L F RL S E H S S 0 1 8 0 4 8 0 2 2 0 7 7 0 3 2 1 0 4 0 5 6 0 9 0 0 4 4 0 7 5 0 5 1 0 6 4 1 1 2 7 Q D E N T V S T S 0 3 1 0 6 6 0 2 2 1 0 9 0 2 6 1 0 0 0 5 8 0 9 9 0 6 0 0 9 9 0 4 1 0 6 6 1 1 2 8 IS IT S RK A Q 0 4 8 0 9 6 0 4 8 0 5 6 0 8 4 1 0 1 0 9 4 1 3 0 0 7 5 1 2 1 1 0 9 1 1 0 1 1 2 9 N F L K T S A G S 0 4 1 0 5 9 0 4 2 0 5 2 0 5 7 0 7 9 0 9 9 1 0 7 0 5 5 0 8 7 0 8 3 0 8 7 1 1 3 0 E H IP Y T H M N 0 5 0 0 9 1 0 5 0 0 5 4 0 5 1 1 1 9 0 8 7 1 1 4 0 9 0 1 1 2 0 7 4 0 8 7 1 1 3 1 E S M E S Y E L N 1 3 8 1 4 8 1 2 7 1 1 5 0 6 6 2 0 9 1 1 3 1 2 2 0 9 8 1 2 5 1 6 5 1 2 5 1 1 3 2 K K RF S F K K S 2 5 7 2 3 9 1 8 4 2 2 9 1 3 6 2 6 8 2 8 8 2 5 2 3 0 3 2 7 4 2 0 0 1 8 8 1 1 3 3 N K Q G Y K A RQ 0 8 4 1 5 8 1 3 2 0 5 7 1 3 1 1 9 3 1 2 6 1 4 7 0 6 4 1 1 8 1 7 7 1 5 0 1 1 3 4 P Y K F P S S P L RI P G Z 0 4 9 0 6 3 0 7 0 0 4 4 0 5 0 0 8 7 0 7 6 0 9 7 0 4 2 0 8 1 0 6 2 0 7 6 1 1 3 5 G S G T S S R P S 0 6 7 1 1 8 0 6 7 0 5 0 0 5 3 1 0 8 1 0 6 1 2 6 0 6 2 0 9 4 0 6 3 0 7 0 1 1 3 6 A V M V S H Y IH 0 9 7 1 2 8 1 0 4 1 0 7 0 6 4 1 5 5 1 1 3 1 3 9 1 2 4 1 3 4 1 6 0 1 1 7 1 1 3 7 D P P G T E S F V 0 9 0 1 3 9 1 2 5 1 2 4 0 6 0 1 7 3 1 0 0 1 4 5 1 1 4 1 4 7 1 2 3 1 0 6 1 1 3 8 G F K RS Y E E H 0 7 1 1 0 1 1 8 7 0 6 3 0 6 0 1 4 1 1 0 6 1 2 1 0 7 7 1 1 4 0 7 7 0 7 5

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147 1 1 3 9 G S RRR RRRY 1 6 7 1 8 8 1 8 5 1 0 0 2 2 6 3 8 6 2 2 5 2 1 2 1 5 6 1 7 1 3 6 0 2 7 0 1 1 4 0 E F P L S P P K K 0 7 5 1 0 1 0 8 3 0 4 9 0 8 1 1 4 7 1 0 0 1 2 2 1 2 3 1 1 0 1 2 5 1 3 8 1 1 4 1 E S E K T K T K E 0 4 8 0 6 0 0 8 6 0 4 9 0 6 1 1 0 1 0 7 2 0 9 8 0 5 7 0 8 0 0 6 2 0 7 1 1 1 4 2 RS S M S G L H L 0 8 3 1 3 3 2 1 3 0 9 0 0 5 7 1 4 3 0 9 0 1 3 8 0 9 2 1 1 2 1 0 2 1 0 5 1 1 4 3 S L V N S RA Q E 0 4 5 0 5 7 1 7 3 0 5 0 0 5 6 1 2 5 0 6 8 1 0 3 0 5 8 0 9 2 0 7 8 0 5 6 1 1 4 4 RRA T V A 1 3 9 2 3 9 1 3 5 0 9 3 1 0 2 2 5 8 1 4 9 1 7 6 1 1 1 1 2 9 1 5 4 1 3 5 1 1 4 5 A D S F S L H D A 0 7 8 1 0 8 0 9 5 0 7 0 0 7 2 1 9 1 0 9 2 1 3 9 0 8 6 0 9 6 1 2 5 0 9 6 1 1 4 6 A T RRS Y V S S 1 6 2 2 1 2 1 5 5 1 2 7 1 5 2 3 4 2 1 7 8 1 7 7 1 0 0 1 2 7 2 6 1 1 9 5 1 1 4 7 D N ID S Q G RN 0 5 7 0 6 8 0 9 5 0 4 7 0 7 3 1 2 4 0 8 9 1 0 1 2 2 0 0 8 5 0 7 6 0 9 1 1 1 4 8 G D S E S G E E E 0 8 6 1 3 1 1 0 1 1 4 0 0 5 6 1 0 2 1 0 0 1 3 9 1 2 3 1 2 4 0 6 2 0 4 8 1 1 4 9 RK RK S S Q A L 1 0 1 1 6 0 1 7 9 0 9 2 0 8 1 1 7 3 1 3 2 1 7 3 2 1 2 1 4 6 1 0 7 1 1 6 1 1 5 0 A K RS RK E 0 8 5 1 1 1 1 2 0 0 8 7 0 5 5 1 2 7 1 1 5 1 4 9 1 0 9 1 2 4 0 6 5 0 8 0 1 1 5 1 L RRA T L G 1 1 5 1 7 0 1 3 5 1 2 0 1 4 7 2 4 2 1 4 3 1 5 6 0 9 1 1 2 4 1 4 5 1 9 0 1 1 5 2 RRS S S V G Y I 1 1 2 2 0 0 1 5 1 1 3 6 1 6 5 2 1 9 1 5 7 1 8 6 1 1 5 1 2 1 1 8 7 1 8 2 1 1 5 3 S K V T S K A G S 0 8 1 1 2 3 0 8 3 0 6 0 0 7 4 1 9 3 1 1 9 1 4 5 0 8 5 0 8 8 1 3 0 1 2 1 1 1 5 4 RG Y S L G 1 1 2 1 7 6 0 9 2 0 7 8 0 6 5 1 4 3 1 4 3 1 5 9 0 7 8 0 8 9 1 2 1 1 0 7 1 1 5 5 A A RT P G G RR 1 1 2 2 1 9 1 6 1 1 1 8 1 0 0 1 7 7 2 0 0 2 0 0 1 6 7 1 4 5 1 8 4 1 2 4 1 1 5 6 T E P Q Y Q P G E 0 5 0 0 7 6 0 9 5 0 7 2 0 6 4 1 0 0 0 7 0 0 9 5 1 2 6 1 2 6 0 6 6 0 6 9 1 1 5 7 T T RR S A S K T 1 7 2 2 6 8 1 2 7 1 2 9 1 1 0 2 1 4 1 4 0 1 7 5 1 2 7 1 2 8 1 7 3 2 1 7 1 1 5 8 W L T K T P E G N 0 3 9 0 6 3 0 6 9 0 5 5 0 7 0 1 3 7 0 7 1 1 1 5 1 0 6 0 6 5 0 7 0 1 0 1 1 1 5 9 RG RS S V Y S A 0 5 1 0 8 8 1 1 9 0 6 2 1 8 6 4 2 7 0 8 2 1 2 5 1 8 6 0 6 1 3 8 4 5 2 8 1 1 6 0 RK RT L R RL 1 6 6 2 6 8 2 2 5 1 5 5 1 2 4 2 5 4 1 8 0 2 4 5 1 9 4 1 2 4 2 8 4 3 3 6 1 1 6 1 L RH A S L G 0 8 1 1 1 6 1 0 7 0 6 6 0 8 3 1 4 4 1 0 5 1 5 7 0 7 0 0 8 6 0 8 6 1 1 5 1 1 6 2 Y G N G Y S S N S 0 5 6 0 9 9 0 5 7 0 6 0 0 8 2 1 5 5 1 0 1 1 2 4 1 2 5 0 9 2 0 8 0 0 7 5 1 1 6 3 S RT P S L P T P 0 4 6 0 6 5 0 3 6 0 7 8 0 5 5 0 9 1 0 8 2 1 2 0 0 8 4 0 7 4 0 7 9 0 7 1 1 1 6 4 P K K G S K K A V 0 4 2 0 8 3 0 6 5 0 8 5 0 6 4 1 2 7 0 7 3 1 0 0 0 8 4 1 2 4 1 0 3 1 0 1 1 1 6 5 L E L S D D D D 0 6 0 1 1 8 0 9 7 1 2 6 0 7 2 1 4 5 0 7 9 1 3 2 1 1 8 1 3 0 1 1 8 1 0 6 1 1 6 6 T D D G Y M P M S 0 5 6 0 7 3 0 7 0 0 6 5 1 2 6 2 5 6 0 7 7 1 2 4 1 3 6 0 8 9 0 8 7 1 3 3 1 1 6 7 T RRL T G F L P 1 0 3 1 3 9 0 5 6 0 7 5 1 1 3 2 0 7 1 2 5 1 7 0 1 3 4 1 1 2 1 4 3 1 4 3 1 1 6 8 P P E K T E E E E 0 7 1 0 8 4 0 4 5 0 5 9 0 6 4 1 2 8 0 9 3 1 2 1 0 6 8 0 9 9 0 7 2 0 8 9 1 1 6 9 RT G RS G S V 1 7 2 3 9 1 1 5 2 1 9 0 1 4 2 3 4 7 2 8 8 3 2 9 1 3 2 1 4 6 3 4 0 2 6 0 1 1 7 0 K Q S P S S S P T 0 4 1 0 5 5 0 3 2 0 5 8 0 7 1 1 0 9 0 7 8 1 1 4 0 6 1 0 9 2 1 4 4 1 0 9 1 1 7 1 L L RP S RR V R 1 2 4 1 8 5 0 8 5 2 2 2 2 2 5 3 8 4 1 5 6 2 5 5 1 6 5 1 7 5 5 4 4 5 3 4 1 1 7 2 Q M A L T P V V V 0 4 3 0 6 8 0 3 3 0 6 3 0 5 8 1 2 7 0 8 6 1 1 8 0 6 0 0 8 7 1 0 5 1 7 2 1 1 7 3 S RS RS P G RP 1 4 9 2 9 2 1 8 3 2 5 9 1 0 4 2 7 0 1 9 1 2 6 2 1 0 4 1 5 9 2 7 1 3 0 1 1 1 7 4 K RK RS RK E S 1 3 6 1 7 9 2 1 2 1 9 0 1 1 5 2 0 3 1 9 3 2 3 7 2 1 4 1 9 6 2 0 5 2 0 0 1 1 7 5 L Q A IS P K Q S 0 5 0 0 7 4 0 4 4 0 4 8 0 7 3 1 1 5 1 0 3 1 2 7 1 0 4 0 6 7 0 9 3 0 9 3 1 1 7 6 Q RRRS L E P P 0 5 8 1 1 6 0 4 2 0 5 5 1 4 9 3 0 6 1 0 4 1 2 2 0 5 8 0 7 4 1 0 8 0 9 5

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A bout t he A ut hor J os e ph W a hl e gr a dua t e d f r om E c ke r d C ol l e ge i n S t P e t e r s bur g F L i n 2002 w i t h a B S i n M a r i ne B i ol ogy. D ur i ng t hi s t i m e he be ga n w or ki ng i n t he l a bor a t or y of G a r y L i t m a n, P h. D w he r e he pa r t i c i pa t e d i n r e s e a r c h i nvol vi ng t he i de nt i f i c a t i on o f i m m une t ype r e c e pt or s i n l ow e r ve r t e br a t e s F r om t he r e J os e ph j oi ne d t he C a nc e r B i ol ogy P h D pr ogr a m a t H L e e M of f i t t C a nc e r C e nt e r a t t he U n i ve r s i t y of S out h F l or i da w he r e he j oi ne d t he l a bor a t o r y of W i l l i a m G K e r r P h. D I n hi s t e nur e i n t he K e r r L a b J os e ph w or ke d on a va r i e t y of pr oj e c t s f oc us i ng p r i m a r i l y on t he N K c e l l D u r i ng t hi s t i m e he ha s pr e s e nt e d hi s w or k a t t h r e e i nt e r na t i ona l m e e t i ngs obt a i ne d a p r e doc t or a l gr a nt f r om t he A m e r i c a n H e a r t A s s oc i a t i on, a s w e l l a s publ i s hi ng 2 pa pe r s i n t he J our na l of I m m unol ogy, one of w hi c h w a s a C ut t i ng E dge pa pe r


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Wahle, Joseph A.
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Signaling in natural killer cells :
b SHIP, 2B4 and the Kinome
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by Joseph A. Wahle.
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[Tampa, Fla] :
University of South Florida,
2007.
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ABSTRACT: The NK cell is a large granular lymphocyte that plays a key role in protecting the body against numerous pathogens including parasites, intracellular bacteria, viral infections, as well as showing anti-tumor activity and playing a role in the rejection of allogeneic BM. Unlike other lymphocytic cell types, that utilize rearranging receptors, NK cells are regulated by a complex array of germ line encoded activating and inhibitory receptors. NK cells are often described as a front line or rapid defense given their response to stimuli can be immediate, although they also maintain functions that extend their role well into the adaptive immune system. Inhibitory receptors that recognize MHC class I molecules regulate NK cell responses and self-tolerance. Recent evidence indicates self-ligands not present in the MHC locus can also modulate NK function. We previously demonstrated that the NK receptor repertoire is disrupted by SHIP-deficiency.^ Here we show that an inhibitory receptor, 2B4, that recognizes an MHC-independent ligand is over expressed in NK cells of SHIP-/- mice at all stages of NK development and differentiation. Overexpression of 2B4 compromises key cytolytic NK functions, including killing of allogeneic, tumor and viral targets. These results demonstrate that in SHIP-/- NK cell 2B4 is the dominant inhibitory receptor. We then furthered this finding by examining the molecular basis of 2B4 dominance. We show that in SHIP-/- NK cells there is increased 2B4 expression as well as a strong bias towards the 2B4L isoform. We have also identified a greater than tenfold increase in SHP1 recruitment to 2B4.^ Consistent with this SHP1 over recruitment,both a broad and a selective SHP1 inhibitor restore SHIP-/- NK killing of complex targets.Through this study we have identified the molecular mechanism of 2B4 receptor dominance as SHP1 over-recruitment.In addition we have utilized protein array technology to explore NK signaling through the determination of the NK kinome. To this end we have been able to identify multiple pathways that may mark crucial differences between the mature and immature NK cell.
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Dissertation (Ph.D.)--University of South Florida, 2007.
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Adviser: William G. Kerr, Ph.D.
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NK.
CD244.
NSC119910.
PepChip.
Cytotoxicity.
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Dissertations, Academic
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x Cancer Biology
Doctoral.
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FTS
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RC254.6 (ONLINE)
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