P oc. Na l. Acad. Sci. USA
Vol. 96, pp. 1024–1029, Feb ua y 1999
Immunology
T cell an igen ecep o -media ed ac i a ion o he
Rasymi ogen-ac i a ed p o ein kinase pa hway
con ols in e leukin 4 ecep o unc ion and
ype-2 helpe T cell di e en ia ion
MASAKATSU YAMASHITA*, MOTOKO KIMURA†‡,MASATO KUBO‡,CHIORI SHIMIZU†,TOMIO TADA‡,
ROGER M. PERLMUTTER§,AND TOSHINORI NAKAYAMA†¶
*Depa men o De elopmen al Immunology, Chiba Uni e si y School o Medicine, and †Depa men o Molecula Immunology, G adua e School o Medicine,
Chiba Uni e si y, 1-8-1 Inohana Chuo-ku, Chiba 260-8670, Japan; ‡Resea ch Ins i u e o Biological Sciences, Science Uni e si y o Tokyo, 2669 Yamazaki,
Chiba 279-0022, Japan; and §Howa d Hughes Medical Ins i u e, and Depa men s o Immunology, Biochemis y, and Medicine, Uni e si y o Washing on,
Sea le, WA 98195
Edi ed by Tony Hun e , The Salk Ins i u e o Biological S udies, San Diego, CA, and app o ed Decembe 2, 1998 ( ecei ed o e iew
Augus 21, 1998)
ABSTRACT The cen al ole o ype-2 helpe T (Th2) cells
in he de elopmen o alle gic esponses and immune e-
sponses agains helmin hic pa asi es is well documen ed. The
di e en ia ion o Th2 cells om nai e T cells equi es bo h he
ecogni ion o an igen by T cell an igen ecep o s (TCR) and
he ac i a ion o downs eam signal- ansduc ion molecules o
he in e leukin 4 ecep o (IL-4R) pa hway, including Jak1,
Jak3, and STAT6. Li le is known, howe e , abou how hese
wo dis inc pa hways coope a e wi h each o he o induce Th2
cells. He e, we use a T cell-speci ic H-Ras-dominan -nega i e
ansgenic mouse o show ha TCR-media ed ac i a ion o he
Rasymi ogen-ac i a ed p o ein kinase pa hway al e s IL-4R
unc ion and is equi ed o Th2 cell di e en ia ion. The
enhancemen o IL-4R signaling seems o be a consequence o
bo h di ec ‘‘c oss alk’’ wi h he TCR signaling pa hway and
inc eased p o ein exp ession o downs eam signaling mole-
cules o he IL-4R pa hway. The e o e, success ul Th2 di e -
en ia ion depends on he e ec i eness o he TCR-media ed
ac i a ion o he Rasymi ogen-ac i a ed p o ein kinase pa h-
way in modi ying he IL-4R-media ed signaling pa hway.
CD4
1
helpe T cells can be di ided in o wo dis inc subpopu-
la ions: ype-1 helpe T (Th1) cells and ype-2 helpe T (Th2)
cells (1). Th1 cells p oduce in e leukin (IL)-2, in e e on
(IFN)-
g
, and umo nec osis ac o
b
, whe eas Th2 cells
p oduce IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13. The de el-
opmen o Th1 and Th2 cells is cen al o he di e si y o helpe
T cell-dependen immune esponses in in ec ious, alle gic, and
au oimmune diseases (2–4). Th1 cells media e delayed ype
hype sensi i i y and o gan-speci ic au oimmune diseases,
whe eas Th2 cells play impo an oles in alle gic and in ec-
ious diseases.
Bo h Th1 and Th2 cells di e en ia e om a common
p ecu so , and he di ec ion o di e en ia ion in o Th1 and
Th2 cells depends on he exogenous cy okines p esen du ing
p ima y an igenic s imula ion o nai e T cells (2–4). IL-12
p omo es he di e en ia ion o nai e T cells in o Th1 e ec o
cells (5, 6), whe eas IL-4 is equi ed o Th2 cell di e en ia ion
(7, 8). Fo Th2 cell di e en ia ion, he ac i a ion o signal-
ansduc ion pa hways downs eam o he IL-4 ecep o (IL-
4R) is essen ial, as e idenced by he ac ha mice ha bo ing
gene dis up ions o IL-4 (9, 10) o STAT6 (11–13) ail o
p oduce Th2 cells. Recen ly, we and o he s ha e shown ha
IL-4-induced phospho yla ion o STAT6 and Jak1 was de-
ec ed wi hin a ew minu es in Th2 cell clones and Th2 cells
di e en ia ed in i o, whe eas hei phospho yla ion was no
de ec ed in Th1 cells (14, 15). Thus, i is concei able ha he
ac i a ion le els o signaling e en s downs eam o IL-4R a e
c i ical o Th2 cell di e en ia ion and o main aining he Th2
pheno ype wi h IL-4 p oduc ion. An igen ecogni ion by T cell
an igen ecep o s (TCR) is also indispensable o bo h Th1 and
Th2 cell di e en ia ion (3). We ha e epo ed ha he e is a
p e e en ial equi emen o he ac i a ion o a p ima y y-
osine kinase, p56
LCK
(Lck), o TCR-media ed signal ans-
duc ion in Th2 cell di e en ia ion (16). Li le is known,
howe e , abou which downs eam signal- ansduc ion pa h-
ways o Lck, such as he Rasymi ogen-ac i a ed p o ein kinase
(MAPK) pa hway, a e c i ical o Th2 cell di e en ia ion and
how hese downs eam signaling molecules egula e Th2 cell
di e en ia ion.
He e, we show ha TCR-media ed ac i a ion o he Rasy
MAPK pa hway is equi ed o Th2 cell di e en ia ion and
ha he ac i a ion o he RasyMAPK pa hway al e s IL-4R
unc ion di ec ly, p obably by up- egula ing he kinase ac i i y
o Jak1, which enhances STAT6 y osine phospho yla ion.
Thus, Th2 cell di e en ia ion seems o be egula ed by
‘‘c oss alk’’ be ween he TCR-media ed ac i a ion o he Rasy
MAPK pa hway and he IL-4R-media ed STAT6 pa hway.
MATERIALS AND METHODS
Animals. C57BLy6 (B6) and BALByc mice we e pu chased
om CLEA Japan (Osaka). A T cell-speci ic H- as dominan -
nega i e (dnRas) ansgenic (Tg) mouse wi h he lck p oximal
p omo e has been desc ibed (17). The Tg mouse line, which
we used o gene a e subs an ial numbe s o ma u e hymocy es
and splenic T cells, had ela i ely low copy numbe s o he
ansgene. A signi ican le el o lck p oximal p omo e ac i i y
was de ec ed in ma u e T cells (18). An i-o albumin (OVA)-
speci ic TCR
ab
(DO10) Tg mice (19) we e p o ided by
Dennis Loh (Nippon Roche Resea ch Cen e , Kanagawa,
Japan). All mice used in his s udy we e main ained unde
speci ic pa hogen- ee condi ions.
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 .
PNAS is a ailable online a www.pnas.o g.
This pape was submi ed di ec ly (T ack II) o he P oceedings o ice.
Abb e ia ions: B6, C57BLy6; dnRas, H-Ras dominan nega i e;
DO10, an i-OVA-speci ic TCR
ab
; IFN, in e e on; IL, in e leukin;
IL-4R, in e leukin 4 ecep o ; Lck, lymphoid cell p o ein y osine
kinase p56
LCK
; LM, li e ma e; MAPK, mi ogen-ac i a ed p o ein
kinase; MAPKK, MAPK kinase; OVA, o albumin; PMA, pho bol
12-my is a e 13-ace a e; TCR, T cell an igen ecep o ; Tg, ansgenic;
Th1, ype-1 helpe T; Th2, ype-2 helpe T; TNP, 2,4,6- ini ophenyl.
¶To whom ep in eques s should be add essed. e-mail:
nak[email p o ec ed].
1024
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T Cell Pu i ica ion. CD4
1
T cells wi h nai e pheno ype
(CD44
low
) we e isola ed om spleens on a FACSVan age cell
so e (Bec on Dickinson) as desc ibed (16), yielding pu i y o
.98%. Whe e indica ed, nai e (CD44
low
) CD4
1
T cells om
he spleen we e p epa ed as ollows. Splenocy es we e incu-
ba ed wi h cul u e supe na an o bo h an i-CD8 (53-6.72) and
an i-CD44 (IM7) mAbs (Pha Mingen). The ea ed cells we e
washed and hen incuba ed on plas ic dishes coa ed wi h goa
an i-mouse IgGs (which c oss eac wi h a IgG, including
53-6.72 and IM7). The nonadhe en cells we e used as a
CD44
low
T cell popula ion. Con amina ion o nai e CD44
low
CD4
1
cells and CD8 T cells was less han 3%.
Immuno luo escen S aining and Flow Cy ome y Analysis.
In gene al, one million cells we e incuba ed on ice o 30 min
wi h he app op ia e s aining eagen s, acco ding o a s anda d
me hod (20). Fo IL-4R s aining, nai e CD4 T cells we e
cul u ed o 2 days wi h IL-4 (100 uni syml), immobilized
an i-TCR (H57-597; 30
m
gyml) and an i-IL-4, o immobilized
an i-TCR and IL-4. Then, he cells we e incuba ed wi h
an i-IL-4R
a
mAb (Genzyme), ollowed by an i- a Ig labeled
wi h luo escein iso hiocyana e and an i-CD4 labeled wi h
phycoe y h in (GK1.5-PE, Pha Mingen). Flow cy ome y
analysis was pe o med on FACSo and FACSVan age (Bec-
on Dickinson), and esul s we e analyzed wi h CELLQUEST
so wa e (Bec on Dickinson). The ee in acellula calcium
ion concen a ion was measu ed as desc ibed (20).
In acellula S aining o IL-4 and IFN-
g
.In acellula
s aining o IL-4 and IFN-
g
was pe o med as desc ibed (16).
B ie ly, he cul u ed T cells we e es imula ed wi h an i-TCR
mAb (H57-597; 30
m
gyml; e . 21) o 6hin hep esence o
2
m
M monensin, which inhibi ed he sec e ion o newly p o-
duced p o ein. Then, he cells we e ixed wi h 4% pa a o -
maldehyde o 10 min a oom empe a u e and made pe me-
able wi h 0.5% T i on X-100 (in 50 mM NaCly5 mM EDTAy
0.02% NaN
3
, pH 7.5) o 10 min on ice. A e blocking wi h 3%
BSA in PBS o 15 min, cells we e incuba ed on ice o 45 min
wi h an i-IFN-
g
labeled wi h luo escein iso hiocyana e
(XMG1.2-FITC) and an i-IL-4 labeled wi h phycoe y h in
(11B11-PE), which we e pu chased om Pha Mingen. The
s ained cells we e washed ex ensi ely wi h PBS supplemen ed
wi h 1% e al cal se um and 0.1% NaN
3
.
Immunop ecipi a ion and Immunoblo ing. In all expe i-
men s, cells we e s imula ed wi h IL-4 (100 uni syml) o 5 min
a 37°C, and he eac ions we e e mina ed by adding a 10- old
olume o ice-cold PBS. Cells we e ha es ed, washed wo
imes wi h ice-cold PBS, and made soluble in lysis bu e (50
mM T iszHCl, pH 7.4y150 mM NaCly1mMNaFy1mM
Na
3
VO
4
y1 mM EGTAy1% Nonide P-40y0.25% sodium de-
oxychola ey1 mM phenylme hylsul onyl luo idey1
m
g/ml
ap o ininy1
m
g/ml leupep iny1
m
g/ml peps a in). An ise um
was added o each sample, and samples we e incuba ed a 4°C
o e nigh . Subsequen ly, 50
m
l o p o ein G-Sepha ose was
added, and samples we e agi a ed o an addi ional2ha 4°C.
A e washing he beads h ee imes wi h lysis bu e , he
p ecipi a es we e esuspended in 50
m
lo 23Laemmli sample
bu e con aining 5% 2-me cap oe hanol, boiled o 5 min, and
unonSDSy7.5% PAGE. P o eins we e ans e ed o a
poly( inylidene di luo ide) memb ane and hen subjec ed o
immunoblo ing wi h an i-phospho y osine (RC20; T ansduc-
ion Labo a o ies, Lexing on, KY) o wi h an ise um eac i e
wi h STAT6 (R&D sys ems), Jak1, o Jak3 (Ups a e Bio ech-
nology) o a mAb eac i e wi h IL-4R
a
(Genzyme).
MAPK Assay. CD4 T cells we e ea ed wi h an i-TCR and
an i-CD4 mAb and hen c osslinked wi h goa an i-hams e Ig
an ibodies. A e s imula ion, cells we e solubilized in lysis
bu e (50 mM T iszHCl, pH 7.4y150 mM NaCly1mMNaFy10
mM Na
3
VO
4
y2 mM EDTAy1% digi oniny40
m
g/ml ap o i-
niny20
m
g/ml leupep in), a e which 0.35 310
7
cell equi a-
len s we e educed wi h 50 mM DTT and sepa a ed on an
SDSy12.5% polyac ylamide gel. P o eins we e ans e ed o a
poly( inylidene di luo ide) memb ane and hen subjec ed o
immunoblo ing by using a phospho-MAPK de ec ion ki
(9100; New England Biolabs).
P oli e a ion Assay. Nai e and cul u ed CD4 T cells we e
s imula ed o 40 h in 200
m
l o cul u e medium con aining 100
uni syml o ecombinan IL-4. Fo he las 16 h, [
3
H] hymidine
(0.5
m
Ci pe well) was added o he s imula ion cul u e, and
he inco po a ed adioac i i y was measu ed by using a
b
-pla e (16).
Measu emen o 2,4,6-T ini ophenyl (TNP)-Speci ic Igs by
ELISA. The he e ozygous dnRas Tg mice wi h (B6 3BALBy
c)F
1
backg ound we e immunized wi h 100
m
go TNP
15
-
keyhole limpe hemocyanin in comple e F eund’s adju an .
The concen a ion o an i-TNP an ibody (IgG1 and IgG2a) in
he se um was measu ed by ELISA wi h ho se adish pe oxi-
dase-conjuga ed an i-mouse IgG1 (Zymed) o an i-mouse
IgG2a (Zymed), espec i ely (16). Fo measu emen s o IgE,
OVA (20
m
g) in alum was used o immunize mice, a e which
he se um concen a ion o OVA-speci ic IgE was de e mined
wi h OVA-coa ed 96-well pla es and an i-IgE mAb (Yamasa
Shoyu, Chosin, Japan).
RESULTS AND DISCUSSION
Signal T ansduc ion Th ough TCR and IL-4R in Nai e CD4
T Cells om dnRas Tg Mice. In his pape , we assessed he ole
o he RasyMAPK pa hways on Th2 cell di e en ia ion by
using a Tg mouse model sys em ha speci ically di ec ed
exp ession o a dnRas (pRHLN17) ansgene in T cells (17).
We used a Tg mouse line ha exp essed ela i ely low copy
numbe s o he ansgene, and only a weak inhibi ion o
posi i e selec ion o T cells in he hymus was obse ed (da a
no shown). In addi ion, sligh ly dec eased bu subs an ial
numbe s o splenic CD4 T cells we e de ec ed in he dnRas Tg
mice. In he spleen o dnRas Tg mice, nai e (CD44
low
) CD4
1
T cells show a no mal ange o su ace pheno ypes, such as
usage o he a iable egion o he TCR
b
chain and exp ession
le els o TCR
ab
, CD3«, CD4, CD25, and CD69 (da a no
shown). Fi s , we assessed he TCR-media ed signal-
ansduc ion pa hways in T cells om dnRas Tg mice. As
shown in Fig. 1a, eshly isola ed splenic CD4 T cells e-
sponded no mally o TCR c osslinking and mobilized in a-
cellula calcium. An i-TCR-mAb-induced TCR-
z
phospho y-
la ion was no impai ed (da a no shown). In ma ked con as ,
he ac i a ion o MAPK kinase (MAPKK) a e an i-TCR
s imula ion, as de e mined by MAPK (E k1 and E k2) phos-
pho yla ion, was comp omised se e ely (Fig. 1b). An i-TCR-
induced p oli e a i e esponses and IL-2 p oduc ion o nai e
CD4 T cells o dnRas Tg mice we e dec eased sligh ly, whe eas
he le els o IL-4 and IFN-
g
p oduc ion we e no dec eased bu
inc eased (da a no shown).
Nex , we in es iga ed bo h p oximal and dis al signaling
e en s media ed by he IL-4R in nai e CD4 T cells om dnRas
Tg mice. Nai e CD4 T cells exp essed equi alen le els o
IL-4R
a
chain, and i s exp ession was up- egula ed no mally
wi h IL-4 s imula ion (Fig. 1c). Simila ly, no signi ican di -
e ence in IL-4R
a
exp ession was de ec ed a e s imula ion
wi h ei he an i-TCR and an i-IL-4 o an i-TCR and IL-4. The
amoun s o STAT6 p o ein (da a no shown) and IL-4-induced
STAT6 phospho yla ion we e no mal (Fig. 1d). IL-4-induced
p oli e a ion was also equi alen (Fig. 1e). In addi ion, an i-
TCR-induced phospho yla ion o JNK and SAPK was no
impai ed in dnRas Tg nai e CD4 T cells (da a no shown).
Thus, downs eam signal- ansduc ion pa hways o TCR and
IL-4R in nai e CD4 T cells o dnRas Tg mice seemed o be
no mal, excep he TCR-media ed RasyMAPK pa hway.
Requi emen o Ac i a ion o RasyMAPK Pa hway in Th2
Cell Di e en ia ion in Vi o and in Vi o.The equi emen o
ac i a ion o RasyMAPK signaling pa hways in Th1yTh2 cell
di e en ia ion was examined h ough he use o in i o
Immunology: Yamashi a e al.P oc. Na l. Acad. Sci. USA 96 (1999) 1025
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induc ion cul u e sys ems (16). Nai e CD4 T cells om dnRas
Tg mice c ossed wi h DO10 Tg mice (19) we e s imula ed wi h
an igenic OVA pep ide in he p esence o an igen-p esen ing
cells o 5 days. T cells om DO10 Tg mice ha a e no dnRas
Tg LM p e e en ially di e en ia ed in o IL-4-p oducing Th2
cells in an an igen-dose-dependen ashion (Fig. 2a Uppe ).
Howe e , in dnRas 3DO10 double-Tg mice, de elopmen o
Th2 cells was comp omised se e ely, and a signi ican inc ease
in he numbe o Th1 T cells was obse ed (Fig. 2a Lowe ). The
impai men o Th2 cell di e en ia ion was con i med by
measu ing ano he Th2 cy okine, IL-5, by ELISA (da a no
shown). Th2 cell di e en ia ion induced by a minimal dose o
an igenic pep ide and exogenous IL-4 was also inhibi ed in
dnRas 3DO10 double-Tg mice (Fig. 2b Le ). Because he
ac i a ion o RasyMAPK does no seem o be in ol ed in he
IL-4R-media ed signal- ansduc ion pa hway (22) and IL-4R
unc ion is no mal in dnRas Tg T cells (Fig. 1), he obse ed
blocking e ec seems o be caused by a consequence o
TCR-media ed RasyMAPK ac i a ion. In con as , IL-12-
dependen induc ion o Th1 cell di e en ia ion emained
in ac (Fig. 2b Righ ). Bo h Th1 and Th2 cells we e gene a ed
when nai e CD4 T cells om non-TCR Tg (B6 3BALByc)F
1
LM mice we e s imula ed wi h an i-TCR mAb in he p esence
o exogenous IL-2 and IL-4 (Fig. 2c). In his condi ion, he
gene a ion o Th2 cells was inhibi ed signi ican ly in dnRas Tg
cul u e, whe eas ha o Th1 cells was enhanced signi ican ly
(31.8% s. 71.7%). To exclude he possibili y ha he obse ed
blocking e ec on Th2 cell di e en ia ion is caused by a
skewed de elopmen and an accumula ion o Th1 cell p ecu -
so s in dnRas Tg mice, we ex ended he analysis by using
no mal (B6 3BALByc)F
1
mice and a speci ic inhibi o o
MEK (MAPKK), PD98059 (23, 24). As we expec ed, he
addi ion o 10
m
M and 30
m
M PD98059 o he induc ion
cul u e blocked Th2 cell di e en ia ion in a dose-dependen
manne (Fig. 2c Lowe ). Thus, hese esul s clea ly show ha
he TCR-media ed ac i a ion o RasyMAPK pa hway was
equi ed o Th2 di e en ia ion in i o. The enhanced gen-
e a ion o Th1 cells obse ed in dnRas Tg T cell cul u es o
no mal T cell cul u es ea ed wi h PD98059 could be a
consequence o a ‘‘de aul ’’ pa hway o Th1 cell di e en ia ion
as p oposed (25). Wo mannin (a phospha idylinosi ol 3-ki-
nase inhibi o ) and SB203580 (a p38 MAPK inhibi o ) had no
e ec on Th2 cell di e en ia ion (da a no shown). Howe e ,
i is s ill possible ha o he Ras-induced signaling pa hways
may in ol ed in he di e en ia ion p ocess o Th2 cells in
addi ion o he E k MAPK pa hway.
Th2 cells play an impo an ole in s imula ing B cells o
p oduce high le els o an igen-speci ic IgG1 and IgE in i o,
whe eas he IgG2a iso ype seems o be a consequence o Th1
cell di e en ia ion (26). Consequen ly, we wished o assess he
ole o Ras ac i a ion in Th2 cell di e en ia ion in i o by
using dnRas Tg mice and assessing he iso ype o an igen-
speci ic an ibodies. Con ol LM mice and dnRas Tg mice we e
immunized wi h TNP-keyhole limpe hemocyanin, and se um
concen a ions o TNP-speci ic IgG1 and IgG2a we e mea-
su ed. The se um concen a ions o Th2 cell-dependen iso-
ype IgG1 we e signi ican ly lowe in dnRas Tg mice (Fig. 2d
Le ). Th1-dependen IgG2a le els we e no dec eased bu
inc eased abou 2- old (Fig. 2d Cen e ). In addi ion, he se um
concen a ion o OVA-speci ic IgE was dec eased signi ican ly
in dnRas Tg mice (Fig. 2d Righ ). These esul s a e consis en
wi h subs an ial impai men o an igen-speci ic Th2 cell di -
e en ia ion in i o in dnRas Tg mice, whe eas Th1 cell
di e en ia ion emained in ac and was enhanced.
Imp o emen o IL-4R Func ion A e TCR S imula ion o
2 Days. Fo Th2 cell di e en ia ion, TCR-media ed ac i a ion
o he RasyMAPK pa hway and IL-4-media ed ac i a ion o
STAT6 a e equi ed. The IL-4 ea men o nai e T cells
wi hou TCR liga ion does no induce Th2 cell di e en ia ion
(3),andIL-4 ecep o unc iondi e s be ween es ablished Th1
and Th2 clones (14, 15). The e o e, TCR-media ed ac i a ion
o he RasyMAPK pa hway may al e IL-4R-media ed signal-
ing. Consequen ly, we in es iga ed whe he TCR s imula ion
induced changes in IL-4R-media ed signaling. Fig. 3ashows
he gene a ion o Th1 and Th2 cells om nai e T cells 2 days
o 5 days a e TCR s imula ion in he p esence o IL-2 and
IL-4. A 2 days, only a ew T cells exp ess Th1 o Th2
pheno ype. Howe e , IL-4-induced phospho yla ion o STAT6
p o ein was enhanced d ama ically (1.0% s. 17.5%) in he
cells s imula ed wi h an i-TCR o 2 days (induc ion cul u e;
Fig. 3b Le ). The exp ession le els o STAT6 p o ein also we e
inc eased wi h TCR s imula ion in he induc ion cul u e.
In e es ingly, he enhancemen was no a ec ed by he deple-
ion o IL-4 om he induc ion cul u e caused by he neu al-
izing an ibody (Fig. 3b Righ ), indica ing ha he changes we e
caused by TCR-media ed consequences. Consis en wi h he
phospho yla ion le els o STAT6, IL-4-induced p oli e a ion
was imp o ed g ea ly when nai e T cells ecei ed TCR-
media ed s imula ion (Fig. 3c). Al hough he ea men wi h
IL-4 alone inc eased cell-su ace exp ession le els o IL-4R
(Fig. 1c), IL-4R unc ion was no imp o ed in e ms o
IL-4-dependen p oli e a ion (Fig. 3c).
FIG. 1. Signal ansduc ion h ough TCR and IL-4R in nai e CD4
T cells om dnRas Tg mice. (a) In acellula ee-calcium ion le els
a e TCR c osslinking (a whi e gap) we e measu ed by low cy o-
me ic analysis o Indo-1-labeled nai e CD4 T cells o dnRas Tg mice
and li e ma e (LM) con ols. The mean a io o iole o blue
luo escence o Indo-1 is plo ed e sus ime a e s imula ion. Shown
a e da a ob ained by ga ing elec onically on CD4 T cells. The
pe cen ages o esponding cells and mean pe cen ages a e also shown.
(b) Phospho yla ion s a us o MAPK (E k1 and E k2) was examined
2 min and 5 min a e TCR c osslinking by using a phospho-MAPK
de ec ion ki . Shown is he ela i e inc ease ( old) in phospho yla ion
o E k1 and E k2 wi h espec o uns imula ed con ol cells ( ime 0).
Densi ome ic measu emen was used o he quan i ica ion; h ee
independen expe imen s we e done, and simila esul s we e ob-
ained. (c) Cell-su ace exp ession o IL-4R
a
chain on nai e CD4 T
cells om Tg
2
LM mice (do ed line) and om dnRas Tg mice (solid
line) was de e mined a e a 2-day induc ion cul u e wi h indica ed
s imulan s. Backg ound s aining is shown (ha ched a eas). (d) Ty-
osine phospho yla ion o STAT6 in esponse o IL-4 in nai e CD4 T
cells om dnRas Tg mice. F eshly p epa ed CD44
low
T cells we e
s imula ed wi h IL-4 (100 uni syml) o 5 min, and he y osine
phospho yla ion s a us o STAT6 was assessed by immunoblo ing
wi h an i-phospho y osine mAb (RC20). A bi a y densi ome ic uni s
a e shown unde each band. (e) No mal p oli e a i e esponse o IL-4
(100 uni syml o 40 h) by nai e CD4 T cells om dnRas Tg mice.
1026 Immunology: Yamashi a e al.P oc. Na l. Acad. Sci. USA 96 (1999)
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In ol emen o RasyMAPK Pa hway in he Imp o emen o
IL-4R Func ion. Consequen ly, we wished o assess he in-
ol emen o TCR-media ed RasyMAPK ac i a ion in he
imp o emen o IL-4R unc ion. Nai e CD4 T cells we e
p epa ed om dnRas Tg mice wi h panning and cul u ed wi h
an i-TCR mAb in he p esence o IL-2 and IL-4 o 2 days, and
hen he phospho yla ion s a us o STAT6, IL-4R
a
, Jak1, and
Jak3 was assessed a e 5 min o IL-4 s imula ion. As shown in
Fig. 4a, all o hese signaling molecules we e y osine phos-
pho yla ed a e 5 min o IL-4 s imula ion in Tg
2
LM mice.
Howe e , hei phospho yla ion was dec eased subs an ially in
dnRas Tg mice. The dec ease was mos d ama ic in IL-4R
a
and Jak1 wi h espec o he amoun o p o ein p esen in he
p ecipi a es. Du ing he i s 2 days o he induc ion cul u e
wi h an i-TCR, p o ein exp ession le els o STAT6 (Fig. 3b),
IL-4R
a
, Jak1, and Jak3 (da a no shown) we e inc eased.
The e o e, TCR-media ed signals induced wo di e en e -
ec s on IL-4R-media ed signal ansduc ion: an inc ease in he
amoun o STAT6, Jak1, and Jak3 p o ein and inc eased
suscep ibili y in he phospho yla ion o hese molecules. The
la e seemed o be a consequence o he ac i a ion o he
RasyMAPK signaling pa hway.
To in es iga e u he he molecula mechanism egula ing
IL-4R-media ed signal ansduc ion by he RasyMAPK pa h-
way, we used PMA as an ac i a ing agen on he RasyMAPK
pa hway. Fi s , he e ec o PMA on Th1yTh2 cell di e en-
ia ion was assessed. Nai e CD4 T cells om no mal (B6 3
BALByc)F
1
mice we e s imula ed wi h a ious concen a ions
o PMA in he p esence o 300 nM ionomycin (Fig. 4b).
Consis en wi h he esul s ob ained hus a , a PMA dose-
dependen inc ease in he gene a ion o Th2 cells and a
dec ease in Th1 cell di e en ia ion we e obse ed. Fig. 4c
shows ha he PMA-induced Th2 cell di e en ia ion was
blocked subs an ially by PD98059. Again, he enhancemen o
Th1 cell di e en ia ion was obse ed. These esul s sugges
ha he obse ed e ec o PMA was a MEK (MAPKK)-
dependen phenomenon. Nex , nai e CD4 T cells we e ea ed
wi h PMA o 4 h, and IL-4-media ed phospho yla ion o
STAT6, Jak1, and Jak3 was assessed (Fig. 4d). The le els o
phospho yla ion o STAT6 and Jak1 clea ly we e inc eased
when nai e T cells we e ea ed wi h PMA. The inc ease was
de ec ed e en 15 min a e he ea men (da a no shown) and
was ep oducible in h ee independen expe imen s. Because
p o ein exp ession le els o STAT6, Jak1, and Jak3 we e no
changed by he 4-h ea men wi h PMA (Fig. 4d Righ ), he
enhancemen o y osine phospho yla ion o hese molecules
was no a esul o up- egula ion o ansc ip ion o ansla ion.
FIG. 2. Requi emen o ac i a ion o RasyMAPK pa hway in Th2 cell di e en ia ion in i o and in i o.(a) Nai e CD4 T cells om dnRas 3
DO10 double-Tg mice we e s imula ed wi h an igenic pep ide (OVA; 323-339) and i adia ed BALByc (H-2
d
) an igen-p esen ing cells o 5 days,
and in acellula p oduc ion o IFN-
g
and IL-4 was de ec ed. (b) Nai e T cells we e s imula ed wi h ei he a minimal dose (0.3
m
M) o an igenic
pep ide and exogenous IL-4 (100 uni syml; Le )o wi h1
m
M an igenic pep ide in he p esence o an i-IL-4 mAb and exogenous IL-12 (0.1 uni yml;
Righ ). (c) Th1yTh2 cell di e en ia ion o nai e T cells om dnRas Tg mice wi h (B6 3BALByc)F
1
backg ound and he e ec o a speci ic inhibi o
o MEK (MAPKK), PD 98059. Nai e CD4 T cells om no mal and dnRas Tg mice we e s imula ed wi h immobilized an i-TCR in he p esence
o IL-2 (30 uni syml) and IL-4 (100 uni syml) o 2 days and hen cul u ed in he medium wi h he same concen a ions o IL-2 and IL-4 o ano he
3 days. The cul u ed cells we e subjec ed o in acellula s aining wi h an i-IL-4 and an i-IFN-
g
.(d) E ec o o e exp ession o dnRas on helpe
T cell di e en ia ion in i o.Tg
2
LM mice and dnRas Tg mice we e p imed on day 0 and boos ed on day 21; 2 weeks a e each immuniza ion,
se um concen a ions o an igen-speci ic an ibodies we e de e mined by ELISA. Mice wi h B6 backg ounds we e immunized wi h TNP-keyhole
limpe hemocyanin (100
m
g pe mouse) in comple e F eund’s adju an , and se um concen a ions o TNP-speci ic IgG1 and IgG2a we e measu ed
(Le and Cen e ). DO10 3dnRas Tg
2
con ol (D10
1y2
) and DO10 3dnRas double-Tg (D10
1y2
3dnRas) mice we e immunized wi h OVA (100
m
g pe mouse) in alum, and se um concen a ions o OVA-speci ic IgE we e measu ed (Righ ). Ba s depic mean alues o ou animals wi h s anda d
de ia ions exp essed as e o ba s.
Immunology: Yamashi a e al.P oc. Na l. Acad. Sci. USA 96 (1999) 1027
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Thus, Jak1 andyo STAT6 seemed o be a ge s o pos ans-
la ional egula ion by he RasyMAPK signaling pa hway.
In his epo , we show ha he TCR-media ed ac i a ion o
RasyMAPK pa hway is equi ed o Th2 cell di e en ia ion
and ha Th1 and Th2 cell di e en ia ion exhibi di e en ial
dependence on ac i a ion o he RasyMAPK pa hway. Th2 cell
di e en ia ion was impai ed in he dnRas Tg T cells; howe e ,
Th1 cell di e en ia ion emained in ac , and some imes i was
e en enhanced (Fig. 2). Se e al ecen epo s ha e sugges ed
ha low-dose in ec ion o low an igen concen a ion a o s
induc ion o Th1 esponses, whe eas high-dose an igenic s im-
ula ion p e e en ially induces Th2 esponses (27–29). Taken
oge he wi h ou indings, hese esul s sugges ha weak
an igenic s imula ion may no ac i a e he RasyMAPK pa h-
FIG. 4. IL-4-induced phospho yla ion on STAT6 and Jak1 in nai e
T cells was egula ed by he RasyMAPK signaling pa hway. (a) Nai e
CD4 T cells om dnRas Tg mice we e cul u ed o 2 days wi h
immobilized an i-TCR mAb and IL-4 and cul u ed wi hou IL-4 o
8 h. Then, IL-4-induced phospho yla ion on STAT6, IL-4R
a
, Jak1,
and Jak3 was assessed by immunop ecipi a ion wi h speci ic mAb o
each p o ein and, a e immunoblo ing, wi h an i-phospho y osine
mAb. A bi a y densi ome ic uni s a e shown unde each band. The
amoun o p o ein was de e mined also by eblo ing he same
memb ane wi h speci ic mAbs. (b) Pho bol 12-my is a e 13-ace a e
(PMA) dose-dependen inc ease in he gene a ion o Th2 cells in i o.
Nai e T cells om (B6 3BALByc)F
1
mice we e ea ed wi h indica ed
doses o PMA o 2 days in he p esence o ionomycin (300 nM), IL-2
(30 uni syml), and IL-4 (100 uni syml). (c) E ec o PD98059 on he
PMA-induced Th1yTh2 cell di e en ia ion. (d) Nai e CD4 T cells
we e ea ed wi h PMA (30 ngyml) o 4 h, and IL-4-induced phos-
pho yla ion on STAT6, Jak1, and Jak3 was assessed by he same
me hod used in a. The amoun o each p o ein exis ing in he cells wi h
o wi hou PMA ea men was also de e mined (Righ ).
FIG. 3. Imp o emen o IL-4R-media ed signal ansduc ion a e
TCR s imula ion. (a) An i-TCR-induced Th1yTh2 cell di e en ia ion
on day 2 and day 5. An i-TCR-s imula ed nai e CD4 T cells om
(B6 3BALByc)F
1
mice we e ha es ed on day 0, day 2, and day 5. The
pe cen ages o cells in each a ea a e shown. (b) Nai e CD4 T cells om
(B6 3BALByc)F
1
mice we e cul u ed o 2 days wi h medium,
immobilized an i-TCR mAb, IL-4 alone, immobilized an i-TCR mAb
and IL-4, o immobilized an i-TCR and an i-IL-4 mAb (induc ion
cul u e). The pe cen ages o eco e ed li e cells in hese cul u es we e
92%, 123%, 89%, 120%, and 135%. The induced cells we e cul u ed
wi hou IL-4 o an i-IL-4 o ano he 8ha 37°C, and hen he cells
we e s imula ed wi h IL-4 (100 uni syml) o 5 min. The phospho y-
la ion s a us o STAT6 and he amoun o STAT6 p o ein we e
assessed by immunop ecipi a ion wi h an i-STAT6 mAb and, a e
immunoblo ing, wi h an i-phospho y osine o an i-STAT6 mAb. The
equi alen o 20 million cells was loaded in each lane. (c)In i o
cul u ed cells like hose in bwe e s imula ed wi h IL-4 (100 uni syml)
o 40 h, and [
3
H] hymidine inco po a ion (o e he las 16 h) was
measu ed.
1028 Immunology: Yamashi a e al.P oc. Na l. Acad. Sci. USA 96 (1999)
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way e icien ly and hus may a o Th1 cell di e en ia ion,
whe eas s ong s imula ion wi h highe concen a ions o
an igenic pep ide may be equi ed o ac i a e he RasyMAPK
pa hway su icien ly o induce Th2 cell di e en ia ion. This
explana ion is consis en wi h he esul s ob ained om ou
p e ious analysis o Th1yTh2 di e en ia ion in he dominan -
nega i e Lck Tg mice (16). Thus, he ex en o which he
RasyMAPK pa hway is ac i a ed seems o de e mine he
lineage (Th1 o Th2 cells) in o which nai e T helpe cells will
di e en ia e. Because a ious alle gic diseases a e closely
ela ed o Th2 dominan immune esponses (2, 3), i is
concei able ha he down- egula ion o ac i a ion in he
RasyMAPK pa hway could esul in dec eased suscep ibili y o
alle gic diseases. Recen ly, he equi emen o he JNK
MAPK pa hway in Th1 cell di e en ia ion has been epo ed
(30). Thus, he de e mina ion o Th1 o Th2 cell di e en ia-
ion in nai e CD4 T cells in he pe iphe y seems o depend on
he ac i a ion o dis inc MAPK signaling pa hways.
Second, IL-4R unc ion on nai e T cells is ound o be
up- egula ed by TCR-media ed ac i a ion o he RasyMAPK
pa hway. F om ou esul s wi h sho - e m PMA ea men
(Fig. 4d), he quali a i e imp o emen in IL-4R-media ed
signal ansduc ion is caused in pa by pos ansla ional
egula ion o Jak1 andyo STAT6 molecules by he Rasy
MAPK signaling pa hway. Al hough he p ecise molecula
mechanism by which he RasyMAPK pa hway egula es he
Jak1ySTAT6 pa hway emains unclea , one possibili y is ha
ac i a ed MEK (MAPKK) phospho yla es and up- egula es
Jak1 ac i i y. In ac , he e is a po en ial y osine phospho -
yla ion si e o MEK (MAPKK), ‘‘TEY’’ (31), and a h eonine
esidue as pa o he XPXSyTP mo i (32) in mouse Jak1. Ou
p elimina y expe imen s wi h an i-phospho y osine mAb spe-
ci ic o TEY sugges ed ha sho - e m PMA s imula ion
caused phospho yla ion o TEY on Jak1 molecules (da a no
shown). Recen ly, Pe icoin e al. (33) epo ed ha he
an ip oli e a i e ac ion o IFN-
a
equi ed componen s o he
TCR signaling cascade, such as Lck and ZAP-70. He e, we
p esen e idence ha he TCR-media ed RasyMAPK ac i a-
ion con ols IL-4R-media ed signaling and de e mines he
di ec ion o helpe T cell di e en ia ion. Indeed, ou da a
sugges ha he lineage choices made by nai e Th cells a e
an igen s imula ion a e in luenced clea ly by c oss alk be ween
he an igen ecep o - and cy okine ecep o -media ed signal-
ing cascades.
We hank D s. Da id Wies and Ralph T. Kubo o help ul
commen s du ing he p epa a ion o he manusc ip . We a e also
g a e ul o D . Masa u Taniguchi o wa m-hea ed suppo . This wo k
was suppo ed by g an s om he Minis y o Educa ion, Science, and
Cul u e (Japan), by he Minis y o Heal h and Wel a e (Japan), by he
Ueha a Memo ial Founda ion, and by he Kanagawa Academy o
Science and Technology.
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