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
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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
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兩
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兩
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兩
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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
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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. Fu he mo e, he
esul s ob ained sugges di e en app oaches o selec i ely in-
e e e wi h T cell ac i a ion in immune-media ed diseases.
We hank She yl F eeman o echnical assis ance. This wo k was
suppo ed by U.S. Public Heal h Se ice G an AI-35714. V.P. is a
Cha les A. King T us ellow. M.A.F. is suppo ed by a Spanish Minis y
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Kuma e al. PNAS
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ol. 99
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IMMUNOLOGY