Differential role of SLP-76 domains in T cell development and function

Di e en ial ole o SLP-76 domains in T cell de elopmen and unc ion Lali Kuma , Vadim Pi niouk, Miguel A. de la Fuen e, Dha e Laouini, and Rai S. Geha* Di ision o Immunology, Child en’s Hospi al, and Depa men o Pedia ics, Ha a d Medical School, Bos on, MA 02115 Communica ed by F ede ick W. Al , Ha a d Medical School, Bos on, MA, No embe 20, 2001 ( ecei ed o e iew Sep embe 10, 2001) The adap e SLP-76 is essen ial o hymocy e de elopmen . SLP- 76 ⴚ/ⴚ mice we e econs i u ed wi h SLP-76 dele ion mu an ans- genes o examine he ole o SLP-76 domains in T cell de elopmen and unc ion. The N- e minal domain dele ion mu an comple ely ailed o es o e hymocy e de elopmen . Mice econs i u ed wi h Gads-binding si e and SH2 domain dele ion mu an s had dec eased hymic cellula i y, impai ed ansi ion om double o single posi- i e hymocy es, and dec eased numbe s o ma u e T cells in he spleen. Calcium mobiliza ion and ex acellula signal- egula ed p o ein kinase ac i a ion we e dec eased in he Gads-binding si e mu an bu almos no mal in he SH2 domain mu an . T cells om bo h mu an s ailed o p oli e a e ollowing T cell an igen ecep o liga ion. Ne e heless, bo h mu an s moun ed pa ial cu aneous hype sensi i i y esponses and no mal T cell dependen IgG1 an ibody esponses. These esul s indica e di e en ial oles o SLP-76 domains in T cell de elopmen , p oli e a ion and e ec o unc ions. SLP-76 is an adap e p o ein p edominan ly exp essed in hema opoie ic cells (1). SLP-76 has h ee dis inc domains: an NH 2 - e minal domain (amino acids 1–155), a cen al p oline ich domain (amino acids 156–421) ha includes a Gads binding si e (amino acids 224–244), and a C- e minal SH2 domain (amino acids 422–533). Phospho yla ed y osine esidues in he NH 2 - e minal domain bind SH2-domain con aining p o eins ha include Va , Nck, and he Tec kinase I k (2–4). The SLP-76 cen al p oline ich domain associa es wi h SH3-con aining p o eins ha include Gads and PLC- ␥ 1. Upon T cell an igen ecep o (TCR) s imula ion, Gads ec ui s SLP-76 o linke o ac i a ed T cells (LAT). This ansloca es SLP-76 o glycolipid en iched mic odomains (GEM) (5, 6). LAT h ough G b2 in e ac s wi h Sos, a guanine nucleo ide exchange ac o o Ras GTPases, and may link SLP-76 o he Ras兾mi ogen-ac i a ed p o ein kinase (MAPK)兾ex acellula signal- egula ed p o ein kinase (ERK) pa hway. Finally, he SLP-76 SH2-domain in e - ac s wi h phosphop o eins such as Fyb兾SLAP130 and a 62-kDa phosphop o ein (7). SLP-76 ⫺/⫺ mice ha e a comple e block in hymocy e de el- opmen a he CD4 ⫺ CD8 ⫺ , double-nega i e s age and lack pe iphe al T cells (8, 9). SLP-76 plays an impo an ole in T cell ecep o signal ansduc ion and T cell ac i a ion. SLP-76- de icien Ju ka cells exhibi se e ely impai ed TCR兾CD3 sig- naling wi h de icien PLC- ␥ 1 ac i a ion, calcium mobiliza ion, ERK phospho yla ion, and IL-2 p oduc ion (10). O e exp es- sion o SLP-76 in a human T cell line (Ju ka ) esul s in ma ked augmen a ion o TCR media ed ac i a ion o nuclea ac o o ac i a ed T cells (NFAT) and IL-2 p oduc ion (2). Single mu- a ion o he h ee N- e minal domain y osine esidues (amino acids 113, 128, and 145), ha a e phospho yla ed a e TCR s imula ion, o phenylalanine has no e ec on he abili y o SLP-76 o augmen he NFAT esponse. In con as , double o iple mu an s o hese y osine esidues and dele ion mu an s o he NH 2 - e minal egion, he p oline- ich Gads binding si e, o he SH2-domain we e inac i e (11). The ole played by a ious domains o SLP-76 in T cell de elopmen and unc ion in i o emains unknown. In his s udy, we add essed his issue by econs i u ing SLP-76 ⫺/⫺ mice wi h wild- ype (WT) SLP-76 and wi h SLP-76 unc ional do- mains dele ion mu an s. Ma e ials and Me hods SLP-76 T ansgenic Mice. The SLP-76 cDNA mu an s ⌬2–156, ⌬224–244, and ⌬421–533 (⌬SH2) we e gene a ed using he QuickChange Si e-Di ec ed Mu agenesis Ki (S a agene). WT and mu an SLP-76 cDNAs we e subcloned in he BamHI si e o he p1017 ec o ca ying he Lck p oximal p omo e (12). No I linea ized DNA was mic oinjec ed in e ilized C57BL兾6⫻SJL兾J (B6SJL) mouse oocy es. Injec ed eggs we e implan ed in o he o iduc s o pseudop egnan os e mo he s. T ansgenic ounde mice, iden i ied by PCR using ansgene- speci ic p ime s and con i med by Sou he n blo ing, we e b ed in o he SLP-76 de icien (8) backg ound by wo ounds o back-c ossing wi h SLP-76 ⫹/⫺ mice. T ansgenes and he endogenous SLP-76 locus we e de e mined by PCR. All animal expe imen s we e pe o med in compliance wi h he Na ional Ins i u es o Heal h and ins i u ional guidelines app o ed by Child en’s Hospi al in e nal animal ca e and use commi ee. An ibodies and Flow Cy ome y Analysis. S ep a idin-FITC, s ep a idin-phycoe y h in (PE), and s ep a idin-CyCh ome and mAbs (unlabeled o labeled o FITC o PE) o mouse an igens we e pu chased om Pha Mingen. Cells we e s ained and analyzed on a FACSCalibu low cy ome e (Bec on Dick- inson) as desc ibed (8). Da a on 5–20 ⫻10 5 iable, none y h oid cells, as de e mined by o wa d e sus side sca e , we e col- lec ed. FACS analysis was pe o med on cells om a leas h ee mice aged 6–8 weeks. Rabbi An i-SLP-76 An ibodies and In acellula FACS Analysis. The IgG ac ion o abbi an ise um agains GST-SLP-76 usion p o ein was en iched o e a P o ein G column and p eclea ed by GST Sepha ose. Thymocy es and splenic T cells we e pe meabilized and s ained in acellula ly, by using Cy oPe m兾 Cy oFix Ki (Pha Mingen), wi h FITC-conjuga ed an i SLP-76 an ibody. Wes e n Blo Analysis. Thymocy es (1 ⫻10 6 ) we e lysed in sample bu e con aining SDS and ␤ -me cap oe hanol and p o eins we e sepa a ed on a 10% gel by PAGE, ans e ed o ni ocel- lulose memb ane, and p obed wi h abbi an i-SLP-76 an ibody, ollowed by ho se adish pe oxidase-conjuga ed goa an i- abbi IgG (Ame sham Pha macia). P oli e a ion o Splenic T Cells. Splenocy es we e suspended in RPMI medium 1640 supplemen ed wi h L-glu amine, penicillin兾 Abb e ia ions: LAT, linke o ac i a ed T cells; TCR, T cell an igen ecep o ; SP, single- posi i e; ERK, ex acellula signal- egula ed p o ein kinase; WT, wild ype; OVA, o albu- min. *To whom ep in eques s should be add essed a : Di ision o Immunology, 300 Longwood A enue, Child en’s Hospi al, Bos on, MA 02115. E-mail: ai .geha@ ch ha a d.edu. The publica ion cos s o his a icle we e de ayed in pa by page cha ge paymen . This a icle mus he e o e be he eby ma ked “ad e isemen ” in acco dance wi h 18 U.S.C. §1734 solely o indica e his ac . 884–889 兩 PNAS 兩 Janua y 22, 2002 兩 ol. 99 兩 no. 2 www.pnas.o g兾cgi兾doi兾10.1073兾pnas.022619199 s ep omycin, and 10% FBS and cul u ed in iplica es wi h a ious s imuli. A e 72 h, p oli e a ion was assessed by [ 3 H] hy- midine up ake assay. Measu emen o [Ca 2ⴙ ] i .Pu i ied T cells (2 ⫻10 6 兾ml) we e incuba ed wi h 4 ␮ g兾ml Fluo-4-AM (Molecula P obes) and 10 ␮ g兾ml Fu a Red (Molecula P obes) in 0.5 ml Ty ode’s bu e wi h 0.02% Plu onic F-127 (Sigma) and 1 mM P obenecid (Sigma). A e 45 min a 37°C, cells we e p eloaded wi h 2 ␮ g兾ml an i-CD3␧an ibody (Clone KT3, Se o ec) on ice o 15 min, kep a oom empe a u e o 30 min o allow clea age o he AM es e s, washed wice wi h Ty ode’s bu e , hen s imula ed wi h 5 ␮ g兾ml goa F(ab⬘) 2 an i- a IgG (ICN兾 Cappel) and analyzed by low cy ome y. In acellula Ca 2⫹ concen a ion is indica ed by Fluo-4AM兾Fu a Red luo es- cence in ensi y a io. PLC- ␥ 1 and ERK Phospho yla ion. Pu i ied T cells we e le un- s imula ed o we e s imula ed wi h 10 ␮ g兾ml an i-CD3␧(clone KT3, Se o ec) c osslinked wi h F(ab⬘) 2 goa an i- a IgG o 5 min. Fo PLC- ␥ 1, lysa es we e p epa ed in 1% Nonide P-40 lysis bu e supplemen ed wi h p o ease and phospha ase inhibi o s (13), and immunop ecipi a ed wi h an i-PLC- ␥ 1 mAb (San a C uz Bio echnology). P ecipi a ed p o eins we e esol ed by SDS兾10% PAGE, ans e ed on ni ocellulose memb ane, and p obed wi h indica ed an ibodies. Fo ERK assay, lysa es we e p epa ed in sample bu e con aining SDS and ␤ -me cap oe hanol and esol ed as abo e. Ac i a ed ERK was de ec ed using a phospho ERK1兾2-speci ic mAb ollowed by ep obing he memb ane wi h an i-ERK1 mAb (San a C uz Bio echnology). Hap en-Induced Con ac Hype sensi i i y. Mice we e sensi ized by he applica ion o 100 ␮ l o 2% oxazolone (Sigma) in e hanol o p e iously sha en abdominal skin. Fi e days la e , 10 ␮ lo 1% oxazolone we e applied o he do sal and en al su aces o he igh ea . E hanol was applied o he le ea . Ea hickness was measu ed a e 24, 48, and 72 h, using a modi ied sp ing-loaded mic ome e (Mi u oyo). An ibody Responses. Ten- o wel e-week-old mice we e immu- nized in ape i oneally wi h 100 ␮ g o o albumin (OVA) p e- cipi a ed wi h alum and bled a day 21. An i-OVA an ibodies we e de ec ed by ELISA as desc ibed (14). Fig. 1. SLP-76 mu an s and hei exp ession in hymocy es. (a) WT and SLP-76 dele ion mu an s used o econs i u e SLP-76⫺/⫺mice. (b) Exp ession o SLP-76 p o ein in 1 ⫻106 hymocy es assessed by Wes e n blo ing using abbi an i-SLP-76 an ibody. Ac in was p obed o loading con ol. Fig. 2. FACS analysis o hymocy es. Su ace exp ession o (a) CD4 s. CD8 on o al hymocy es. The pe cen age o cells ound in each quad an is indica ed. (b) TCR ␤ on o al hymocy es. (c) CD44 and CD25 on DN hymocy es. Cells we e iple s ained wi h an i-CD44-FITC, an i-CD25-phycoe y h in, and a mix u e o bio in-conjuga ed mAbs o CD3, CD4, CD8, B220, Mac1, and G -1, ollowed by s ep a idin-CyCh ome. Analysis was pe o med on ga ed CyCh ome nega i e cells. Resul s a e ep esen a i e o h ee expe imen s. Kuma e al. PNAS 兩 Janua y 22, 2002 兩 ol. 99 兩 no. 2 兩 885 IMMUNOLOGY Resul s Gene a ion o T ansgenic Mice. The cDNA cons uc s used o gene a e ansgenic mice on SLP-76 ⫺/⫺ backg ound a e shown in Fig. 1a. Non- ansgene-bea ing li e ma es we e designa ed SLP-76 ⫹/⫹ and SLP-76 ⫺/⫺ . T ansgenic lines ha exp essed SLP-76 p o ein in hymocy es in amoun s compa able o hose exp essed in SLP-76 ⫹/⫹ mice, as assessed by Wes e n blo ing (Fig. 1b) and FACS (see Fig. 8, which is published as suppo ing in o ma ion on he PNAS web si e, www.pnas.o g), we e ex- panded o s udy. Simila esul s we e ob ained when indepen- den lines o SLP-76 WT (n⫽2), SLP-76 ⌬2–156 (n⫽2), SLP-76 ⌬224–244 (n⫽3), and SLP-76 ⌬SH2 (n⫽4) mice we e examined. Thymocy e De elopmen . Thymic cellula i y in SLP-76 ⫺/⫺ mice was ⬇1% ha o SLP-76 ⫹/⫹ mice (1.6 ⫻10 6 ,n⫽6). SLP-76 ⫺/⫺ mice had no de ec able CD4 ⫹ CD8 ⫹ DP, CD4 ⫹ CD8 ⫺ single- posi i e (SP) o CD4 ⫺ CD8 ⫹ SP hymocy es (Fig. 2a) and no de ec able TCR ␤ ⫹ cells (Fig. 2b). The SLP-76 WT ansgene es o ed hymic cellula i y (112 ⫻10 6 ,n⫽4) and hymocy e de elopmen . The pe cen ages o DN, DP, and SP cells and o TCR ␤ ⫹ cells in SLP-76 WT mice we e simila o SLP-76 ⫹/⫹ li e ma es (Fig. 2 aand b). SLP-76 ⌬2–156 comple ely ailed o escue hymocy e cellula i y (1.9 ⫻10 6 ,n⫽4; Fig. 2 aand b). SLP-76 ⌬224–244 and SLP-76 ⌬SH2 es o ed hymic cellula i y o app oxima ely one hi d (35 ⫻10 6 ,n⫽7) and one hal no mal (55 ⫻10 6 ,n⫽8), espec i ely. P og ession om DN o DP cells was pa ially impai ed in SLP-76 ⌬224–244 and SLP-76 ⌬SH2 mice, as e idenced by an inc ease in he pe cen age o DN cells and a dec ease in he pe cen age o DP cells (Fig. 2a). The e was also a ela i e inc ease in he pe cen age o SP CD8 ⫹ cells. The pe cen age o TCR ␤ ⫹ cells and he densi y o TCR ␤ on hy- mocy es we e dec eased compa ed wi h SLP-76 ⫹/⫹ and SLP-76 WT mice (Fig. 2b). In pa icula , pe cen ages o TCR hi and TCR in e media e hymocy es we e dec eased in hese mice. The sequence o de elopmen o DN hymocy es is CD25 ⫺ CD44 ⫹ 3CD25 ⫹ CD44 ⫹ 3CD25 ⫹ CD44 ⫺ 3 CD25 ⫺ CD44 ⫺ cells (15). SLP-76 ⫺/⫺ mice show a ela i e in- c ease o he CD25 ⫹ CD44 ⫺ popula ion, whe eas CD25 ⫺ CD44 ⫺ cells, he mos ma u e among DN T cell p ogeni o s, a e almos unde ec able (8). This block was comple ely o e come by e- cons i u ion wi h WT SLP-76 ansgene (Fig. 2c). DN cells om SLP-76 ⌬2–156 mice exhibi ed a block iden ical o ha o SLP-76 ⫺/⫺ mice (Fig. 2c). Recons i u ion wi h SLP-76 ⌬224–244 and SLP-76 ⌬SH2 pa ially o e came he block in he ansi ion om he CD25 ⫹ CD44 ⫺ o he CD25 ⫺ CD44 ⫺ s age. Exp ession o TCR ␤ and he CD69 Ac i a ion Ma ke s on DP and SP Thymocy es. Posi i e selec ion o hymocy es in o SP cells is accompanied by up- egula ion o TCR兾CD3 and CD69 exp es- sion (16, 17). In bo h SLP-76 ⫹/⫹ and SLP-76 WT mice, exp es- sion o TCR ␤ and CD69 was up- egula ed in SP cells (Fig. 3 and Fig. 9, which is published as suppo ing in o ma ion on he PNAS web si e). Exp ession o TCR ␤ and CD69 was also up- egula ed on SP cells o SLP-76 ⌬224–244 and SLP-76 ⌬SH2 mice, bu he pe cen ages o TCR ␤ ⫹ and CD69 ⫹ SP cells and densi y (MFI) o TCR ␤ and CD69 we e lowe han in SLP-76 ⫹/⫹ and SLP-76 WT con ols. Pe iphe al T Cells. Spleens o SLP-76 ⫺/⫺ con ain no de ec able CD3 ⫹ , CD4 ⫹ ,o CD8 ⫹ cells (Fig. 4a). In oduc ion o he SLP-76 WT ansgene es o ed splenic CD3 ⫹ , CD4 ⫹ , and CD8 ⫹ cells o no mal. The SLP-76 ⌬2–156 ansgene comple ely ailed Fig. 3. Exp ession o TCR ␤ on DP and SP hymocy es. Ga ed DP and SP cells we e analyzed o exp ession o TCR ␤ a e s aining wi h an i-CD4- phycoe y h in, an i-CD8-CyCh ome, and an i-TCR ␤ -FITC. Resul s a e ep esen- a i e o h ee expe imen s. Fig. 4. FACS analysis and SLP-76 exp ession in spleen cells. (a) Su ace exp ession o CD3 and o CD4 and CD8 on spleen cells. Resul s a e ep esen a i e o h ee expe imen s. (b) Exp ession o SLP-76 p o ein in pu i ied T cells by in acellula s aining wi h FITC-conjuga ed abbi an i-SLP-76 an ibody (c) Exp ession o SLP-76 p o ein in 1 ⫻106splenocy es assessed by Wes e n blo ing. 886 兩 www.pnas.o g兾cgi兾doi兾10.1073兾pnas.022619199 Kuma e al. o epopula e spleens wi h T cells. Bo h SLP-76 ⌬224–244 and SLP-76 ⌬SH2 es o ed splenic CD3 ⫹ T cells o ⬇50% o no mal. In bo h cases, he densi y o CD3 exp ession was abou hal o ha o SLP-76 ⫹/⫹ T cells, and he e was a ela i e inc ease in CD8 ⫹ cells (Fig. 4a). Simila pheno ypes we e ob ained wi h blood mononuclea cells (da a no shown). In acellula FACS s aining e ealed ha T cells om spleens o mice econs i u ed wi h SLP-76 ansgenes exp essed SLP-76 p o eins a compa able in ensi ies (Fig. 4b). Wes e n blo ing e ealed ha he exp essed SLP-76 p o eins in splenocy es we e o he expec ed sizes (Fig. 4c). These esul s indica e ha he lck p oximal p omo e is d i ing sus ained SLP-76 exp ession in ma u e T-cells. T Cell P oli e a ion and IL-2 Recep o Exp ession. Splenocy es om SLP-76 WT mice p oli e a ed no mally o an i-CD3. In con as , splenocy es om bo h SLP-76 ⌬224–244 and SLP-76 ⌬SH2 mice ailed o p oli e a e o an i-CD3, o e a wide ange o concen- a ions (0.01 o 10 ␮ g兾ml; see Fig. 10, which is published as suppo ing in o ma ion on he PNAS web si e). The same T cells p oli e a ed no mally o PMA ⫹ionomycin. In e ac ions be- ween IL-2 and IL-2R play an impo an ole in T cell p oli e - a ion and op imal p oduc ion o IL-2 by T cells, and equi e co-s imula ion ia CD28 (18). IL-2, bu no an i-CD28 mAb, co ec ed he ailu e o T cells om SLP-76 ⌬224–244 and SLP-76 ⌬SH2 mice o p oli e a e in esponse o immobilized an i-CD3 (Fig. 5). T cells om SLP-76 ⌬224–244 and SLP-76 ⌬SH2 mice poo ly up- egula ed IL-2R ␣ chain (CD25) exp es- sion ollowing an i-CD3 s imula ion (35% and 30% CD25 ⫹ cells, espec i ely) compa ed wi h T cells om SLP-76 ⫹/⫹ mice and SLP-76 WT mice (93% and 83% CD25 ⫹ cells, espec i ely). CD25 exp ession on hese cells was enhanced by an i-CD28 (54% CD25 ⫹ cells o SLP-76 ⌬224–244 and 43% CD25 ⫹ cells o SLP-76 ⌬SH2) and IL-2 (59% CD25 ⫹ cells o SLP-76 ⌬224–244 and 52% CD25 ⫹ cells o SLP-76 ⌬SH2) (see Fig. 11, which is published as suppo ing in o ma ion on he PNAS web si e). Simila esul s we e ob ained o CD69 exp ession (da a no shown). In acellula Calcium Mobiliza ion, PLC- ␥ 1, and ERK Phospho yla ion. SLP-76 WT mice mobilized calcium no mally ollowing an i- CD3 liga ion. Calcium mobiliza ion was educed in T cells om SLP-76 ⌬224–244 mice, and minimally a ec ed in T cells om SLP-76 ⌬SH2 mice (Fig. 6a). Fig. 5. An i CD3 induced p oli e a ion o splenic T cells. P oli e a ion o pu i ied T cells o an i-CD3 (coa ed a 5 ␮ g兾ml) in he p esence o absence o an i-CD28 (coa ed a 5 ␮ g兾ml) and mouse IL-2 (20 ng兾ml), and o PMA (50 ng兾ml) ⫹ionomycin (500 ␮ M). Resul s a e ep esen a i e o h ee expe imen s. Fig. 6. Calcium lux, phospho yla ion o PLC- ␥ 1, and ERK ollowing TCR兾CD3 liga ion in splenic T cells. (a)Ca 2⫹mobiliza ion: T cells p eloaded wi h Fluo-4-AM and Fu a Red we e p eincuba ed wi h an i-CD3 (2 ␮ g兾ml), washed, hen s imula ed wi h 5 ␮ g兾ml goa F(ab⬘)2an i- a IgG (GAR) and analyzed by low cy ome y. In acellula Ca2⫹concen a ion is indica ed by he Fluo-4AM兾Fu a Red luo escence in ensi y a io. Io, ionomycin. (b) PLC- ␥ 1 y osine phospho yla ion: PLC- ␥ 1 immunop ecipi a es om pu i ied T cells s imula ed o 0–5 min wi h c osslinked an i-CD3 we e Wes e n blo ed wi h an iphosphop y osine mAb 4G10 (Uppe ), and ep obed wi h an i- PLC- ␥ 1(Lowe ). (c) ERK phospho yla ion. Lysa es om pu i ied T cells s imula ed as abo e we e p obed wi h phospho-ERK-speci ic an ibody (Uppe ) and ep obed wi h ERK-speci ic an ibody (Lowe ). Resul s shown a e ep esen a i e o h ee expe imen s. Kuma e al. PNAS 兩 Janua y 22, 2002 兩 ol. 99 兩 no. 2 兩 887 IMMUNOLOGY Ty osine phospho yla ion o PLC- ␥ 1 ollowing TCR兾CD3 liga ion was no mal in T cells om WT SLP-76 mice (Fig. 6b), bu was dec eased in T cells om SLP-76 ⌬224–244 and SLP-76 ⌬SH2 mice (Fig. 6b). This dec ease was speci ic, because y- osine phospho yla ion o ZAP-70, which is ups eam o SLP-76, was no mal in hese cells (see Fig. 12, which is published as suppo ing in o ma ion on he PNAS web si e). ERK ac i a ion is impai ed in SLP-76 de icien Ju ka cells (10). Phospho yla ion o ERK1兾2 ollowing TCR兾CD3 s imu- la ion in T cells om SLP-76 WT mice was compa able o SLP- 76 ⫹/⫹ mice. Baseline ERK1兾2 phospho yla ion was inc eased in T cells om mice econs i u ed wi h mu an ansgenes. Induced ERK1兾2 phospho yla ion was ⬇4- old dec eased in T cells om SLP-76 ⌬224–244 mice (n⫽3) and was less sus ained (⬇1.5- old dec eased) in T cells om SLP-76 ⌬SH2 (n⫽3; Fig. 6c). In Vi o T Cell E ec o Responses. Oxazolone sensi ized SLP-76 ⫺/⫺ mice comple ely ailed o exhibi ea swelling up o 72 h a e hap en challenge. Ea swelling in SLP-76 WT mice was simila o ha o SLP 76 ⫹/⫹ con ols. The e was pa ial ea swelling in bo h SLP-76 ⌬224–244 and SLP-76 ⌬SH2 mice (Fig. 7a). To de e mine he T helpe esponses, we measu ed he an ibody esponse o OVA. All mice made an IgM an ibody esponse compa able o ha o SLP-76 ⫹/⫹ mice li e ma es (Fig. 7b). SLP-76 ⫺/⫺ and SLP-76 ⌬2–156 mice made no IgG1 an ibody o IgE an ibody esponses o OVA. SLP-76 WT mice made IgG1 and IgE esponses compa able o SLP-76 ⫹/⫹ con ols. SLP-76 ⌬224–244 made a no mal IgG1 esponse, bu had a signi ican ly dec eased IgE esponse o OVA. SLP-76 ⌬SH2 mice made IgG1 and IgE esponses o OVA compa able o SLP-76 ⫹/⫹ con ols (Fig. 7b). Discussion The da a p esen ed demons a e ha SLP-76 domains play di e en ial oles in media ing hymopoiesis and ac i a ion o ma u e T cells in i o. The ailu e o he SLP-76 N- e minal domain mu an o es o e hymocy e de elopmen in SLP-76 ⫺/⫺ mice sugges s ha his domain is c i ical o signaling by he p eTCR, which is equi ed o he ma u a ion o DN cells and hei ansi ion o DP cells. The block in SLP-76 ⌬2–156 mice could be due o he inabili y o he SLP-76 mu an o ec ui membe s o he Va , I k and兾o Nck amilies, and兾o addi ional p o eins ha may in e ac wi h he SLP-76 N- e minal domain (2–4). Nei he dis up ion o indi idual o dual combina ions o he Va p o eins Va 1 and Va 2 (19–22), no o he Tec kinases I k, Tec, and Rlk兾Txk (23, 24) blocks T cell de elopmen a he DN s age. SLP-76 ⌬224–244, and SLP-76 ⌬SH2 beha ed simila ly in hei abili y o econs i u e T cell de elopmen and unc ion in SLP-76 ⫺/⫺ mice. Bo h mu an s es o ed DN o DP and DP o SP ansi ion in he hymus. This es o a ion was incomple e be- cause hymic cellula i y, CD25 ⫺ CD44 ⫺ ma u e DN cells, and DP cells we e all dec eased (Fig. 2). Fu he mo e, hymic selec ion in o SP cells was a ec ed, because hese cells had dec eased exp ession o TCR ␤ and o CD69, which a e no mally up- egula ed upon ma u a ion o DP in o SP cells (Fig. 3). These da a sugges ha bo h Gads binding and SH2 domain in e ac- ions a e necessa y o op imal p eTCR and TCR signaling in hymocy es. The skewing o SP hymocy es in hese mice owa d CD8 ⫹ cells is consis en wi h de ec i e TCR signaling, because he s eng h o TCR signaling in luences lineage commi men o CD4 and CD8 wi h o me a o ed by TCR ac i a ion o ERK kinase (25). Al hough ERK ac i a ion was no measu ed in hymocy es, i was dec eased in he splenic T cells o hese mice. Ma u e T cells we e p esen in he blood, spleen, and lymph nodes o SLP-76 ⌬224–244 and SLP-76 ⌬SH2 mice and ex- p essed he SLP-76 ansgenes. This inding sugges s ha he lck p omo e d i ing he ansgene was ac i e in ma u e T cells, as has been epo ed in o he s udies (26, 27). Howe e , hei pe cen age was dec eased, and hey had dec eased exp ession o TCR ␤ and inc eased p opo ion o CD8 ⫹ cells (Fig. 4). T cells om SLP-76 ⌬224–244 and SLP-76 ⌬SH2 mice had dec eased CD25 and CD69 exp ession ollowing liga ion o TCR兾CD3 and comple ely ailed o p oli e a e o an i-CD3 (Fig. 5). Up- egula ion o CD25 exp ession and co ec ion o he p oli e a i e de ec by IL-2 sugges ha esidual signaling ia TCR兾CD3 occu s in hese cells. Calcium mobiliza ion was dec eased in T cells om SLP-76 ⌬224–244 mice, bu minimally a ec ed in SLP-76 ⌬SH2 mice (Fig. 6a). This inding is consis en wi h indings in SLP-76 de icien Ju ka T cells ans ec ed wi h SLP-76 mu an s (13). The dec eased esponse o SLP-76 ⌬224–244 mice canno simply be explained by lowe le el o exp ession o TCR兾CD3, because Fig. 7. Hap en Induced Con ac Hype sensi i i y and an ibody esponse o OVA. (a) G oups o ou mice we e sensi ized wi h oxazolone and challenged wi h hap en ( igh ea ) o e hanol (le ea ). Resul s ep esen he di e ence in hickness be ween hap en- and ehicle-challenged ea s. (b) An i-o a IgM, IgG1, and IgE an ibodies in he se a o mice ( h ee mice o SLP-76⫺/⫺and SLP-76 ⌬2–156, ou mice o he o he s) immunized in ape i oneally wi h 100 ␮ g OVA. Numbe s indica es he P alues in compa ison o SLP-76⫹/⫹mice (*indica es P⬍0.01 by pai ed es wi h Welch’s co ec ion; n.s. ⫽no signi ican ). 888 兩 www.pnas.o g兾cgi兾doi兾10.1073兾pnas.022619199 Kuma e al. SLP-76 ⌬SH2 mice had a compa able low exp ession o TCR兾 CD3, ye luxed calcium almos no mally. PLC- ␥ 1 phospho yla ion was dec eased in T cells om SLP-76 ⌬224–244 and SLP-76 ⌬SH2 mice (Fig. 6b). Residual PLC- ␥ 1 phospho yla ion in SLP-76 ⌬224–244 T cells sugges s ha phospho yla ion o PLC- ␥ 1 may occu in he absence o Gads media ed ec ui men o SLP-76 o LAT. This phospho yla ion may in ol e he ecen ly demons a ed di ec in e ac ion o SLP-76 wi h PLC- ␥ 1 (13). Dec eased PLC- ␥ 1 phospho yla ion wi h almos no mal calcium luxes in SLP-76 ⌬SH2 mice sugges s ha he emaining phospho yla ion is su icien o PLC- ␥ 1 ac i a ion leading o no mal calcium lux. A simila inding has been epo ed in SLP-76 de icien Ju ka cells econs i u ed wi h a SLP-76 SH2 domain R448K poin mu an (13). Ac i a ion o he MAP kinase ERK was dec eased in SLP-76 ⌬224–244 mice (Fig. 6c). Dec eased gene a ion o DAG subse- quen o dec eased ac i a ion o PLC- ␥ may ha e esul ed in impai ed ac i a ion o he Ras exchange ac o RasGRP, and impai ed ac i a ion o he Ras兾Ra 兾ERK pa hway (28, 29). E k ac i a ion was less a ec ed in T cells om SLP-76 ⌬SH2 mice, which mobilized calcium no mally. Baseline phospho yla ion o ERK was inc eased in mu an T cells, pa icula ly in SLP-76 ⌬SH2 T cells. I should be no ed ha dele ion o he SH2 domain, in con as o he R448K mu a ion ha inac i a es his domain, may no only p e en a achmen o SLP-76 ia SH2 o a ge p o eins, bu may also ac i a e he adap e o signaling elemen s which a e inhibi ed by he s uc u e o he in ac p o ein. T cells om SLP-76 ⌬224–244 and SLP-76 ⌬SH2 mice moun ed pa ial esponses o speci ic an igen. These esponses included pa ial CHS esponse o oxazolone, and no mal IgG1 an ibody esponse o he TD an igen OVA, wi h no mal IgE esponse o OVA in SLP-76 ⌬SH2 mice (Fig. 7). CHS in ol es he cy okines IFN- ␥ , IL-2, IL-4, and IL-10 (30). IgG1 and IgE esponses o TD an igens equi es IL-4 and CD40L:CD40 in- e ac ions be ween T and B cells (31). Ou indings sugges ha he Gads binding si e and he SH2 domain o SLP-76 may be dispensable, a leas pa ially, o he p oduc ion o T cell cy okines in ol ed in hese T cell e ec o esponses. The pheno ype o SLP-76 ⌬224–244 mice is simila o ha o Gads ⫺/⫺ mice, suppo ing he impo ance o he SLP-76-Gads- LAT complex in p eTCR and TCR signaling. The de ec in SLP-76 ⌬224–244 mice is less se e e han ha obse ed in LAT ⫺/⫺ mice (32). This inding sugges s ha TCR signaling may p oceed, albei subop imally, in he absence o SLP-76 ec ui - men o a s. This may occu ia o he LAT-associa ed p o eins, as sugges ed by he obse a ion ha a LAT mu an ha ails o ec ui SLP-76, pa ially es o es T cell signaling in LAT de i- cien Ju ka cells (33). T cells om SLP-76 ⌬SH2 mice ailed o p oli e a e ollowing TCR liga ion. This inding was unexpec ed gi en he abili y o SLP-76 ⌬SH2 o es o e TCR signaling in SLP-76 de icien Ju ka T cells. This esul sugges s ha p oli e a ion o p ima y T cells has mo e complex equi emen s han ac i a ion o biochemical pa hways and epo e gene exp ession in T cell lines. The SH2 domain o SLP-76 couples he TCR o FYB兾 SLAP130. FYB兾SLAP130 has been implica ed in ac in cy oskel- e al eo ganiza ion (34), which is c i ical o IL-2 p oduc ion and T cell p oli e a ion (35). Molecules o he han FYB兾SLAP130 ha in e ac wi h he SH2 domain o SLP-76 may also be impo an o TCR signaling. The di e en ial ole o SLP-76 domains in T cell de elopmen and unc ion sugges s ha hymocy e ma u a ion, and ac i a ion o ma u e T cells is egula ed di e en ly. 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