... PKC-u is required for TCR-induced NF-kB activation in mature but not immature T lymphocytes

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1 uffer (6 mm HEPES, 7.5 mm MgCl 2, 3 mm KCl, 1 mm ethylenedimine-tetrcetic cid, 2.5 mm dithriothreitol, 5% glycerol nd 4-(2-minoethyl)-enzenesulphonyl uoride hydrochloride) nd 2: c:p:m: of 32 P-lelled oligonucleotide proe. UN- SCAN-IT 5.1 softwre (Silk Scienti c) ws used for densitometric nlysis of the utordiogrphs. Sttisticl nlysis Hrdy±Weinerg equilirium of lleles t individul loci ws ssessed y x 2 sttistics. Hplotype frequencies for pirs of lleles were estimted using the Estimting Hplotypefrequencies (EH) softwre progrm (ftp://linkge.rockefeller.edu/softwre/eh). Linkge disequilirium coef cients D9 ˆ D=D mx nd x 2 vlues were clculted for pirs of the most common lleles t ech locus using the LINKDOS softwre progrm (distriuted with GENEPOP, ftp://ftp.cefe.cnrs-mop.fr/pu/pc/msdos/genepop/). Odds rdios with Corn eld 95% con dence intervls nd logistic regression models controlling for the effects of possile confounders were computed using STATA version 5. softwre (STATA Press). The odds rtios for hypochlorhydri were ge-djusted (ctegorized s <35, 36± 45, 46±55 nd.55 yers) ecuse of its ge-dependence 23 nd their con dence intervls were sed on roust vrince estimtes 3 which djust for within-fmily correltion, to ccount for smpling of severl memers of given fmily. Received 21 Octoer 1999; ccepted 4 Ferury Blser, M. J. Helicocter pylori nd gstric diseses. Br. Med. J. 316; 157±151 (1998). 2. El-Omr, E. et l. Helicocter pylori infection nd normlities of cid secretion in ptients with duodenl ulcer disese. Gstroenterology 19, 681±691 (1995). 3. El-Omr, E. et l. Helicocter pylori infectin nd chronic gstric cid hyposecretion. Gstroenterology 113, 15±24 (1997). 4. Corre, P. Humn gstric crcinogenesis: multistep nd multifctoril processðfirst Americn Cncer Society Awrd Lecture on Cncer Epidemiology nd Prevention. Cncer Res. 52, 6735±674 (1992). 5. Dinrello, C. A. Biologic sis for interleukin-1 in disese. Blood 87, 295±2147 (1996). 6. Wllce, J. L., Cucl, M., Mugridge, K. & Prente, L. Secretgogue-speci c effects of interleukin-1 on gstric cid secretion. Am. J. Physiol. 261, G559±G564 (1991). 7. Beles, I. L. & Clm, J. Interleukin 1 et nd tumour necrosis fctor lph inhiit cid secretion in cultured rit prietl cells y multiple pthwys. Gut 42, 227±234 (1998). 8. Prkin, D. M., Pisni, P. & Ferly, J. Estimtes of the worldwide incidence of 25 mjor cncers in 199. Int. J. Cncer 8, 827±841 (1999). 9. Sipponen, P. Gstric cncerð long-term consequence of Helicoctor pylori infection? Scnd. J. Gstroenterol. 21, (suppl.), 24±27 (1994). 1. Ymok, Y. et l. Reltionship etween Helicocter pylori icea, cga, nd vca sttus nd clinicl outcome: studies in four different countries. J. Clin. Microiol. 37, 2274±2279 (1999). 11. Hnsson, L. E. et l. The risk of stomch cncer in ptients with gstric or duodenl ulcer disese. N. Engl. J. Med. 335, 242±249 (1996). 12. Kuipers, E. J. et l. Atrophic gstritis nd Helicocter pylori infection in ptients with re ux esophgitis treted with omeprzole or fundopliction. N. Engl. J. Med. 334, 118±122 (1996). 13. Noch, L. A. et l. Mucosl tumor necrosis fctor-lph, interleukin-1 et, nd interleukin-8 production in ptients with Helicocter pylori infection. Scnd. J. Gstroenterol. 29, 425±429 (1994). 14. Bsso, D. et l. Helicocter pylori infection enhnces mucosl interleukin-1 et, interleukin-6, nd the solule receptor of interleukin-2. Int. J. Clin. L. Res. 26, 27±21 (1996). 15. Jung, H. C., Kim, J. M., Song, I. S. & Kim, C. Y. Helicocter pylori induces n rry of proin mmtory cytokines in humn gstric epithelil cells: qunti ction of mrna for interleukin-8, - 1 lph/et, grnulocyte-mcrophge colony-stimulting fctor, monocyte chemottrctnt protein-1 nd tumour necrosis fctor-lph. J. Gstroenterol. Heptol. 12, 473±48 (1997). 16. Wolfe, M. M. & Nompleggi, D. J. Cytokineinhiition of gstric cidsecretionð little goes longwy. Gstroenterology 12, 2177±2178 (1992). 17. Bidwell, J. L. et l. Cytokine gene polymorphism in humn disese: on-line dtses. hhttp:// Pociot, F. et l. ATqI polymorphisms in the humn interleukin-1 et (IL-1et) gene correlteswith IL-1 et secretion in vitro. Eur. J. Clin. Invest. 22, 396±42 (1992). 19. Snttil, S., Svininen, K. & Hurme, M. Presence of the IL-1RA llele 2 (IL1RN*2) is ssocited with enhnced IL-1et production in vitro. Scnd. J. Immunol. 47, 195±198 (1998). 2. Andus, T. et l. Imlnce of the interleukin 1 system in colonic mucosðssocition with intestinl in mmtion nd interleukin 1 receptor ntgonist genotype 2. Gut 41, 651±657 (1997). 21. Dnis, V. A., Millington, M., Hylnd, V. J. & Grennn, D. Cytokine production y norml humn monocytes: inter-suject vrition nd reltionship to n IL-1 receptor ntgonist (IL-1R) gene polymorphism. Clin. Exp. Immunol. 99, 33±31 (1995). 22. Tounts, N. A. et l. Functionl nd ethnic ssocition of llele 2 of the interleukin-1 receptor ntgonist gene in ulcertive colitis. Gstroenterology 117, 86±813 (1999). 23. El-Omr, E. M. et l. Incresed prevlence of precncerous chnges in reltives of gstric cncer ptients: criticl role of H. pylori. Gstroenterology 118, 22±3 (2). 24. Chow, H. W. et l. Risk of stomch cncer in reltion to consumption of cigrettes, lcohol, te nd coffee in Wrsw, Polnd. Int. J. Cncer 81, 871±876 (1999). 25. Cox, A., Cmp, N. J., Nicklin, M. J., Di Giovine, F. S. & Duff, G. W. An nlysis of linkge disequilirium in the interleukin-1 gene cluster, using novel grouping method for multillelic mrkers. Am. J. Hum. Genet. 62, 118±1188 (1998). 26. Stockruegger, R. W. et l. Pernicious nemi, intrgstric cteril overgrowth, nd possile consequences. Scnd. J. Gstroenterol. 19, 355±364 (1984). 27. Ruiz, B. et l. Vitmin C concentrtion in gstric juice efore nd fter nti-helicocter pylori tretment. Am. J. Gstroenterol. 89, 533±539 (1994). 28. Crrington, M. et l. Typing of HLA-DQA1 nd DQB1 using DNA single-strnd conformtion polymorphism. Hum. Immunol. 33, 28±212 (1992). 29. Yu, C. R. et l. Differentil utiliztion of Jnus kinse-signl trnsducer ctivtor of trnscription signling pthwys in the stimultion of humn nturl killer cells y IL-2, IL-12, nd IFN-lph. J. Immunol. 157, 126±137 (1996). 3. Royll, R. M. Model roust con dence intervls using mximum likelihood estimtors. Int. Stt. Rev. 54, 221±226 (1986). Supplementry informtion is ville on Nture's World-Wide We site ( or s pper copy from the London editoril of ce of Nture. Acknowledgements We thnk A. Goldstein nd N. Chtterjee for dvice on genetic nd sttisticl issues, N. Dunlp for technicl ssistnce, D. Gillen for help with suject recruitment nd J. Goedert for suggestions. E.M.E. received Europen Helicocter pylori Study Group Reserch Fellowship from the Digestive Disorders Foundtion, UK. This project ws prtly funded y the Ntionl Cncer Institute, Ntionl Institutes of Helth, USA. Correspondence nd requests for mterils should e ddressed to E.M.E. (e-mil: elomre@mil.nih.gov).... PKC-u is required for TCR-induced NF-kB ctivtion in mture ut not immture T lymphocytes Zuoming Sun*, Christopher W. Arendt*, Wilfried Ellmeier*², Edwrd M. Scheffer³, Mry Jen Sunshine*², Leen Gndhi*, Justin Annes*, Dniel Petrzilk*², Arhm Kupferk, Pmel L. Schwrtzerg³ & Dn R. Littmn*² ² Howrd Hughes Medicl Institute nd * Moleculr Pthogenesis Progrm, Skirll Institute of Biomoleculr Medicine, New York University School of Medicine, New York, New York 116, USA ³ Ntionl Humn Genome Reserch Institute, Ntionl Institutes of Helth, Bethesd, Mrylnd 2892, USA Deprtment of Pthology, University of Chicgo, Chicgo, Illinois 6637, USA k Division of Bsic Science, Deprtment of Peditrics, Ntionl Jewish Medicl nd Reserch Center, Denver, Colordo 8262, USA... Productive interction of T lymphocyte with n ntigen-presenting cell results in the clustering of the T-cell ntigen receptor (TCR) nd the recruitment of lrge signlling complex to the site of cell±cell contct 1,2. Susequent signl trnsduction resulting in cytokine gene expression requires the ctivtion of one or more of the multiple isoenzymes of serine/threonine-speci c protein kinse C (PKC) 3. Among the severl PKC isoenzymes expressed in T cells, PKC-u is unique in eing rpidly recruited to the site of TCR clustering 4. Here we show tht PKC-u is essentil for TCRmedited T-cell ctivtion, ut is dispensle during TCR-dependent thymocyte development. TCR-initited NF-kB ctivtion ws sent from PKC-v -/- mture T lymphocytes, ut ws intct in thymocytes. Activtion of NF-kB y tumour-necrosis fctor nd interleukin-1 ws unffected in the mutnt mice. Although studies in T-cell lines hd suggested tht PKC-u regultes ctivtion of the JNK signlling pthwy 5,6, induction of JNK ws norml in T cells from mutnt mice. These results indicte tht PKC-u functions in unique pthwy tht links the TCR signlling complex to the ctivtion of NF-kB in mture T lymphocytes. We inctivted the gene encoding PKC-u y homologous recomintion in emryonic stem cells y replcing the exon encoding the ATP-inding site of the kinse domin (mino-cid residues 396± 451) with the neomycin resistnce gene. Homozygous mutnt mice seemed norml nd were fertile. Immunolot nlysis with ntiodies directed ginst sequences outside the deleted coding region of PKC-u filed to detect ny protein product, even of smller thn norml size, in thymocytes or T cells from the mutnt nimls (dt not shown). Flow cytometric nlyses of cells from thymus, spleen nd lymph nodes of the mutnt mice were indistinguishle from 42 2 Mcmilln Mgzines Ltd NATURE VOL MARCH 2

2 c PKC +/- PKC -/- PKC +/- PKC -/- PKC +/- PKC -/ Gte Gte Totl Cells T3.7 T3.7 T Gte Gte T3.7 Figure 1 Thymic mturtion in PKC-v-null mice., Positive selection of the HY trnsgenic TCR, s reveled y stining with T3.7, in + thymocytes from femle mice heterozygous or homozygous for the trgeted PKC-u muttion. nd gtes re the sme s those shown in the dot plots, where the percentges of totl thymocytes re indicted., Expression of the clonotypic TCR in +, ut not +, lymph node cells T3.7 from femle mice. c, Negtive selection of doule-positive thymocytes in mle mice expressing the HY trnsgenic TCR. Totl thymocyte numers were: nd in heterozygous nd null femles, respectively, nd nd in the heterozygous nd null mles. those of wild-type littermte controls with regrd to cell numers nd levels of surfce proteins (dt not shown). Further exmintion of thymocyte differentition ws done y mting to mice expressing trnsgenic TCR speci c for the mle ntigen, HY, in the context of the clss I molecule H-2D (ref. 7). In femle mice, T cells tht express the trnsgenic TCR re preferentilly selected to ecome + single-positive cells, nd cn e detected with the clonotypic ntiody T3.7. In the sence of PKC-u, positive selection in femle nimls proceeded normlly (Fig. 1, ). In mle mice, negtive selection, re ected s depletion of doulepositive thymocytes, ws lso unffected y the loss of PKC-u (Fig. 1c). The signlling function of PKC-u therefore does not seem to e required for the key selection processes during thymocyte differentition. Becuse the expression of PKC-u is restricted to lymphocytes of the T-cell linege 8, we sought to determine whether the TCR signlling pthwy in peripherl T cells ws ffected y its sence. Prolifertion of puri ed T lymphocytes ws mesured fter stimultion with plte-ound nti- monoclonl ntiody (ma) with or without nti-cd28 stimultion. In response to either of these stimuli, prolifertion of T cells from the homozygous mutnt mice ws reduced y more thn 2-fold reltive to tht of wild-type T cells (Fig. 2). A mrked decrese in prolifertion ws lso oserved in PKC-u-de cient T cells stimulted with nti-cd28 plus 12-O-tetrdecnoylphorol-13-cette (TPA). Reduced prolifertion of PKC-v -/- T cells ws ccompnied y signi cnt reduction in the level of secreted interleukin-2 (IL-2) (Fig. 2). The ddition of IL-2 t 2 units ml -1 modestly restored the nti-- induced prolifertive response of the mutnt T cells (Fig. 2), indicting tht signlling events downstrem of the cytokine receptor remined intct. Prolifertion of T cells from PKC-v -/- mice ws lso sustntilly reduced in mixed lymphocyte rections (Fig. 2c). To study the role of PKC-u in the priming of T cells in vivo, we exmined the responses of wild-type nd mutnt mice to T-cell-dependent ntigen, keyhole limpet hemocynin (KLH). The speci c response of splenic T cells to KLH ws evluted two weeks fter injection, s shown in Fig. 2d. As expected, T cells recovered from wild-type mice responded to KLH in dose-dependent mnner. However, T-cell prolifertion in mutnt mice remined t seline level identicl to tht in wild-type control mice injected with crrier lone. These results re consistent with requirement for PKC-u in T-dependent responses in vivo. Prolifertion of T cells fter crosslinking of the TCR is consequence of upregulted trnscription of the IL-2 gene nd of the IL- 2R chin gene, whose product contriutes to high-f nity receptor for IL-2 (ref. 9). Expression of IL-2R (CD25) nd of CD69, cell-surfce mrker on ctivted T cells, cn lso e induced y treting T cells with phorol esters 1. In response to nti-/ CD28 or tretment with TPA, the induction of oth CD25 nd CD69 ws reduced or sent in the PKC-u-de cient T cells (Fig. 2e). The reduced prolifertive responses of T cells lcking PKC-u re therefore due to decresed levels of oth IL-2 nd the CD25- contining high-f nity IL-2 receptor. The induction of cytokine gene expression nd T-cell prolifertion fter the inding of TCR nd CD28 to their cognte lignds requires proximl signlling events initited y TCR/-ssocited protein tyrosine kinses 11. After nti- crosslinking, there were no pprent differences in erly tyrosine phosphoryltion events etween T cells from mutnt nd control nimls (dt not shown). Becuse previous studies in cell lines implicted PKC-u in the ctivtion of vrious MAP kinses, prticulrly JNK (Jun mino-terminl kinse), we next exmined these pthwys 5,6.In PKC-u-de cient T cells stimulted with nti- ma, ctivtion of ERK (extrcellulr signl-regulted kinse) exhiited similr pro le to tht of wild-type cells (Fig. 3). JNK nd p38 MAP kinse (MAPK) ctivtion in response to nti-/cd28 seemed norml in mutnt T cells stimulted immeditely ex vivo (dt not shown) or prestimulted with nti- to upregulte JNK protein expression 12 (Fig. 3). The diverse signlling pthwys downstrem of the TCR converge on severl key trnscription fctors, including NF-AT, AP-1 nd NFkB, which hve importnt roles in the expression of IL-2 nd CD25 fter stimultion with nti-tcr nd nti-cd28 (refs 13, 14). We NATURE VOL MARCH Mcmilln Mgzines Ltd 43

3 therefore focused our nlysis on the effect of the PKC-v muttion on ctivtion of these fctors. We oserved no differences in the trnsloction of NF-AT1 nd NF-AT2 to the nucleus fter the ctivtion of mutnt nd wild-type T cells with nti-/cd28 or with TPA plus ionomycin (Fig. 4). In contrst, there ws mrked decrese in AP-1 ctivtion, upon stimultion either with nti- or with TPA, in PKC-u-de cient T cells in comprison with wild-type cells (Fig. 4). AP-1 consists of Fos nd Jun fmily memers, which re trnscriptionlly induced erly fter crosslinking with nti-tcr or the tretment of T cells with phorol esters. Becuse JNK ctivity seemed norml under conditions of TCR nd phorol stimultion tht filed to ctivte AP-1 (Fig x Prolifertion (c.p.m.) IL-2 (units/ml) PKC-θ +/+ PKC-θ +/- PKC-θ -/- med med + IL-2 CD28 TPA CD28 TPA Iono CD28 [ 3 H]thymidine incoportion c 3, 25, 2, 15, 1, 5, PKC-θ +/+ PKC-θ +/- PKC-θ -/- 1:2 1:4 1:8 2:2 2:4 2:8 4:2 4:4 4: x Responders: stimultors KLH (µg) d [ 3 H]thymidine incoportion 25, 2, 15, 1, 5, PKC+/control PKC+/KLH PKC-/control PKC-/KLH e /CD28 TPA /CD / /- +/ CD69 Figure 2 Impired T-cell ctivtion in PKC-v -/- mice., Impired prolifertive responses. Left pnel: puri ed lymph-node T cells stimulted with medium lone or plte-ound nti- e (1 mg ml -1 ), with or without recominnt murine IL-2 (2 units ml -1 ). Middle pnel: splenic T cells stimulted with nti-e (1 mgml -1 ) in the presence or sence of solule nti-cd28 (1 mgml -1 ). Right pnel: lymph-node T cells stimulted with TPA (1 ng ml -1 ) nd solule nti-cd28 (1 mg ml -1 ) or ionomycin (Iono) (125 ng ml -1 )., IL-2 production in superntnts of splenic T cells stimulted with plte-ound nti-e (1 mgml -1 ) nd solule nti-cd28 (1 mgml -1 ) s mesured on HT-2 responders. c, Mixed lymphocyte rections of T cells from littermte mice nd mitomycin C-treted BALB/c CD25 stimultors. All prolifertion ssys were performed in triplicte nd verged, with error rs denoting s.d. d, Impired T-dependent response to ntigen in PKC-v -/- mice. KLH or crrier lone ws injected intrperitonelly; fter 2 weeks, splenocytes were restimulted with KLH in vitro nd T-cell prolifertion ws mesured. Results were verged for four mice in ech group. e, Surfce expression of CD25 nd CD69, ssyed y ow cytometry 14 h fter stimultion with 1 mgml -1 plte-ound nti-e, with 1 mgml -1 nti-e plus 1 mgml -1 plte-ound nti-cd28, or with 1 ng ml -1 TPA. Dotted lines represent expression on unstimulted control cells Mcmilln Mgzines Ltd NATURE VOL MARCH 2

4 nd dt not shown), the defect in AP-1 ctivtion might re ect decresed expression of Fos nd/or Jun in the sence of PKC-u. NF-kB/Rel fmily memers hve een implicted s downstrem trgets of TCR nd CD28 signlling events, prticulrly in the ctivtion of the IL-2 promoter 13,15,16. The mechnism of NF-kB ctivtion fter enggement of these cell-surfce receptors hs remined elusive. Phorol esters induce NF-kB in mny different cell types nd, in comintion with C 2+ ionophore, trigger the prolifertion of T cells. In ddition, phorol ester-induced depletion of PKC rogtes TCR-medited T-cell prolifertion nd NFkB ctivtion 17. On the sis of these results, it hs een suggested tht stimultion with TCR results in PKC-medited ctivtion of NF-kB. Stimultion of T cells from PKC-v -/- nd wild-type mice with tumour-necrosis fctor- (TNF-) or IL-1 resulted in strong ctivtion of NF-kB in nucler extrcts (Fig. 5). The speci city of the electrophoretic moility shift ws con rmed y supershifting with ntiodies ginst p5 or p65 (Fig. 5, nd dt not shown). In contrst, on crosslinking with nti- or nti- plus nti- CD28, there ws striking defect in NF-kB ctivtion in PKC-u -/- T cells (Fig. 5,, nd dt not shown). T cells from the mutnt mice lso filed to ctivte NF-kB in response to tretment with TPA. Although stimultion with TPA plus ionomycin did not restore full prolifertive response in mutnt T cells, this tretment resulted in the ctivtion of NF-kB in the sence of PKC-u (Fig. 5). This nding suggests tht recruitment of conventionl PKCs (responsive to oth dicylglycerol nd cytoplsmic C 2+ ) cn compenste for the sence of PKC-u to restore t lest prt of the iologicl response downstrem of the TCR/CD28 signl. TCR-medited signls required for selection events in the thymus nd for the ctivtion of peripherl T cells re thought to e fundmentlly different, s they hve different outcomes 18. For exmple, ntigens tht stimulte the production of cytokines nd the prolifertion of mture T cells induce thymocyte poptosis, wheres those tht induce positive selection fil to ctivte in the periphery. Becuse T-cell development seemed unffected in the sence of PKC-u, we compred NF-kB ctivtion in thymocytes nd splenic T cells. Surprisingly, ctivtion of NF-kB ws found to e norml in PKC-v -/- thymocytes treted with nti- or TPA (Fig. 5c). This result suggests tht PKC isoenzyme distinct from PKC-u is involved in TCR-initited signlling events in thymocytes. Before ctivtion, NF-kB suunits re sequestered in the cytosol y the negtive regultor IkB. To determine whether signlling to IkB is defective in PKC-v -/- T cells, we exmined its degrdtion in mture T cells nd thymocytes stimulted with nti- ma or TNF-. Consistent with the ndshift nlysis, on crosslinking, IkB- ws degrded in wild-type T cells ut not in mture T cells lcking PKC-u (Fig. 5d). As predicted, -induced IkB- degrdtion ws norml in thymocytes from wild-type nd mutnt mice, nd no defect ws oserved in response to TNF- in ny of the cell types exmined. PKC-u is memer of the `novel' suclss of PKC fmily memers, which lso includes the d, e nd h isoenzymes 19. These kinses re ctivted y dicylglycerol, wheres the conventionl suclss of PKCs, which include the, 1, 2 nd g isoenzymes, require oth dicylglycerol nd C 2+ for ctivtion. The genertion of dicylglycerol in response to vriety of signls results in plsm memrne recruitment nd ctivtion of PKCs. In ddition, speci c serine nd threonine phosphoryltion of conventionl PKCs is required for them to cquire competence to recruitment nd ctivtion 2. It is not yet known whether PKC-u ctivtion is lso dependent on similr phosphoryltion events. However, recruitment of PKC-u, ut not other PKC isoenzymes, to the site of TCR interction with mjor histocomptiility complex/peptide on ntigen-presenting cells suggests tht the locl genertion of dicylglycerol is not suf cient for memrne recruitment of PKC in T cells 4. Speci c signls re likely to engge nd ctivte PKC-u differentilly fter stimultion with nti-tcr, nd the rchitecture of preformed signlling complexes proly ccounts for the selective ility of this PKC isoenzyme to ctivte NF-kB, AP-1 None Anti- TPA + Iono Stimultion NF-AT1/2 A PKC-θ PKC-θ +/+ PKC-θ / min ERK P ERK PKC-θ +/+ PKC-θ / min p38 MAPK P J F J F Supershift NF-AT None Anti- TPA TPA + Iono Stimultion J F J F J F J F Fos/Jun A PKC-θ c-jun supershift c-fos supershift AP-1 p54 JNK P p46 JNK P p46 JNK Figure 3 Norml MAP kinse ctivtion in PKC-v -/- T cells., Phospho-ERK immunolot of T cells stimulted for the indicted durtions with plte-ound nti-e. As control for loding, the lot ws stripped nd re-proed with nti-erk ntiserum., Activtion of p38 MAPK nd JNK in puri ed T cells prectivted with nti- nd then rested nd restimulted with comintion of nti- nd nti-cd28. The sme lot ws proed with nti-phospho-p38 MAPK, nti-phospho-jnk nd nti-jnk ntiodies. Figure 4 EMSA nlysis of NF-AT nd AP-1 in PKC-v -/- T cells. Puri ed T cells were stimulted for 8 h with 1 mg ml -1 plte-ound nti- or 5 ng ml -1 TPA nd 2 ng ml -1 ionomycin (Iono), nd nucler extrcts were sujected to ndshift nlysis., Norml trnsloction of NF-AT in ctivted PKC-v -/- T cells. Antiodies (A) speci c for NF-AT1 nd NF-AT2 were employed in supershift ssys s indicted., Defective ctivtion of AP-1 in T cells lcking PKC-u. Antiodies speci c for c-jun nd c-fos were used for supershift nlysis. NATURE VOL MARCH Mcmilln Mgzines Ltd 45

5 Figure 5 Bndshift nlysis of NF-kB ctivtion in mture T cells nd thymocytes., Lck of NF-kB ctivtion in PKC-v -/- T cells in response to TCR triggering or tretment with TPA. Nucler extrcts were prepred from T cells stimulted for 8 h with 1 mgml -1 plteound nti-, 5 ng ml -1 TPA, 2 ng ml -1 ionomycin (Iono), or TPA nd ionomycin (T + I) together. EMSA ws conducted in the presence or sence of ntiodies (A) ginst p5., Norml NF-kB ctivtion in response to TNF- nd IL-1. T cells were incuted for 3 min with 2 ng ml -1 TNF-,1ngml -1 IL-1 or plte-ound nti- s positive control. c, Norml ctivtion of NF-kB in thymocytes from PKC-v -/- mice. Thymocytes nd mture T cells were compred for the ility to ctivte NF-kB, s in. d, PKC-u is required for IkB- degrdtion in mture T cells ut not in thymocytes. Thymocytes or puri ed splenic T cells (2 1 6 ) were stimulted with 1 mgml -1 plteound nti- or 1 ng ml -1 TNF- for different durtions s indicted. IkB- in the cytoplsmic frction of the cells ws detected y Western lot nlysis. As control for loding, the sme lots were lso proed with n ntiody ginst Lck. nd other signlling pthwys. PKC-u hs een reported to e ssocited with Vv, Rc-1 gunine nucleotide exchnge fctor tht hs key functions in clustering of the TCR signlling complex nd in TCR-medited signlling 21±23. PKCs hve lso een implicted in the ctivtion of Itk, protein tyrosine kinse required for TCR-induced prolifertion 24. The interction of PKC-u with these signlling proteins might e involved in its selective recruitment to the suprmoleculr ctivtion clusters, where it co-loclizes centrlly, djcent to TCR ggregtes 1. The function of PKC-u in these signlling complexes remins uncler, lthough our results indicte tht it must e involved in the ctivtion of the NF-kB pthwy s well s other signlling pthwys tht cnnot e completely restored y tretment with TPA plus C 2+ ionophore in the sence of PKC-u. The ctivtion of NF-kB y TCR triggering involves PKC-udependent upstrem signlling pthwy tht results in the degrdtion of IkB- ut is distinct from pthwys used y TNF nd IL-1 receptor fmily memers. It remins to e determined whether the distinct pthwys converge in the ctivtion of components of the IkB kinse complex, which is required for ctivtion of NF-kB y TNF nd IL-1 (ref. 25). Severl studies hve suggested tht NF-kB hs n importnt function in TCR-medited thymocyte selection 26±28. The ctivtion of different PKC isoenzymes might thus contriute to the different responses to TCR ligtion etween immture nd mture T cells. M Methods Genertion of PKC-u -/- mice A 129Sv/J mouse genomic clone contining the exon encoding the ATP-inding site of the kinse domin, which corresponds to exon 11 in the humn PKC-v gene, ws used to prepre the trgeting vector. A 1.2-kilose (k) genomic frgment locted 59 to the exon ws mpli ed y the polymerse chin rection nd cloned etween the ClI nd XI sites in the PL2-Neo plsmid polylinker ( gift from H. Gu nd K. Rjewsky). At the other end of the Neo selection mrker, 6-k 39 BmHI frgment, with nking HSV-TK gene, ws inserted. E14 ES cells were trnsfected with linerized trgeting vector nd, fter the selection of colonies with G418 nd gncyclovir, homologous recomintion events were con rmed y Southern lot nlysis of DNA digested with KpnI nd BmHI. Positive clones were used to generte chimeric founder mice y microinjection into C57BL/6J lstocysts. Antiodies nd regents Antiodies ginst murine e (145-2C11), CD28 (37.51), CD69 nd CD25 nd ntihumn JNK were otined from PhrMingen. Antiodies ginst phospho-jnk nd p38 MAPK were purchsed from NEB, nd nti-erk1/2 ws purchsed from Promeg. Antiodies speci c for phospho-erk (E-4), NF-kB p5 (C-19), NF-AT1 (7A6), NF-AT2 (G1-D1), c-jun (H-79), c-fos (K-25) nd IkB- (C-21) were otined from Snt Cruz Biotechnology. An Lck-speci c ma ws purchsed from Trnsduction Ls nd the T3.7 ma ws gift from Hung-Si Teh. TNF-, IL-1 nd IL-2 were otined from Roche. Prolifertion ssys T cells isolted from lymph node or spleen were enriched to.8% purity with mouse T- cell enrichment columns (R&D Systems). For nti- stimultions, T cells (5 1 4 )in 16 ml prolifertion medium (RPMI supplemented with 1% FCS, 5 mm -mercptoethnol, 2 mm L-glutmine nd 5 units ml -1 penicillin/streptomycin) were dded in triplicte to 96-well pltes precoted with nti-e ntiody (either 1. or 1. mgml -1 ). Where indicted, IL-2 ( nl concentrtion 2 units ml -1 ) or nti-cd28 (1 mgml -1 ) were lso dded. TPA (1 ng ml -1 ) ws dded either with ionomycin (125 ng ml -1 ) or with solule nti-cd28 (1 mg ml -1 ). The T cells were incuted for 48 h nd 1 ml of superntnt ws removed for IL-2 ssys, efore pulsing with 1 mci of [ 3 H]thymidine for 16±2 h. For the MLR ssys, T cells were puri ed from littermte mice (B6/129 ckground) nd mixed in triplicte t vrious densities with mitomycin C-treted splenocytes from BABL/c mice, or with syngeneic splenocytes prepred in n identicl mnner. After 4 d of growth, the cells were pulsed for 12 h with 1 mci [ 3 H]thymidine. Speci c prolifertion ws clculted s the men rdioctive count in triplicte wells fter sutrction of the seline prolifertion of syngeneic cultures. Mesurement of IL-2 IL-2 produced from the cultures ws mesured y quntifying the prolifertion of the IL- 2-dependent cell line HT-2. A stndrd curve ws estlished y using recominnt IL-2. MAP kinse ssys Puri ed T cells were ctivted ex vivo on 96-well pltes coted with nti- (1 mgml -1 ). For the ssy shown in Fig. 3, T cells were rst incuted in 1% FCS in RPMI on nti Mcmilln Mgzines Ltd NATURE VOL MARCH 2

6 -coted tissue culture dishes. After 16 h, cells were gently detched, rested for 3±5 h on norml tissue-culture dishes, nd then ctivted for vrious durtions on 96-well pltes contining co-immoilized nti- nd nti-cd28 (5 mg ml -1 ech). Proteins were extrcted y the ddition of n equl volume of 2 reducing smple uffer nd, fter eing oiled, (.5±1.) 1 6 cell equivlents were dded to the lnes of 1% polycrylmide gels. Proteins were trnsferred to Immoilon-P (Millipore) nd proed with phospho-speci c MAP kinse ntiodies for detection y chemiluminescence (Pierce). Nucler extrct preprtion nd electrophoretic moility-shift nlysis Puri ed T cells (1 6 ) were wshed in PBS nd resuspended in 1 ml of 1 mm HEPES ph 7.9, 1 mm KCl,.1 mm EDTA nd Complete protese inhiitors (Roche). The cells were incuted on ice for 15 min. Nonidet P4 ws dded to nl concentrtion of.5%, the cells were vortex-mixed vigorously, nd the mixture ws centrifuged for 3 s. The nucler pellets were resuspended in 4 ml of 2 mm HEPES ph 7.9,.4 M NCl, 1 mm EDTA nd protese inhiitors, nd the tue ws rocked for 15 min t 4 8C. After centrifugtion for 5 min, the superntnt ws collected nd glycerol ws dded to nl concentrtion of 5%; 2 ml of ech extrct ws sujected to ndshift nlysis. Electrophoretic moility-shift nlyses (EMSAs) were performed with the following oligonucleotides nd inding uffers: NFAT oligonucleotide: 59-GCCCAAAGAGGAAAA TTTGTTTCATACAG-39; NFAT 5 inding uffer: 5 mm Tris-HCl ph 7.5, 5 mm KCl, 2.5 mm MgCl 2,.5 mm EDTA, 5% glycerol, 25 mg ml -1 poly (di:dc), 5 mm dithiothreitol nd 1 mg ml -1 BSA; NF-kB oligonucleotide: 59-ACCAAGAGGGATTTCACCT AAATC-39;NF-kB5 inding uffer: 25 mm Tris-HCl ph 7.5, 5 mm EDTA, 25 mgml -1 poly (di:dc), 5 mm dithiothreitol nd 1 mg ml -1 BSA; AP-1 oligonucleotide: 59-CGCTT GATGACTCAGCCGGAA-39; AP-15 inding uffer: sme s tht for NFAT c.p.m. of lelled proe ws used in ech rection, nd ndshifts were resolved on 5% polycrylmide gels in.5 TBE running uffer. 23. Holsinger, L. J. et l. Defects in ctin-cp formtion in Vv-de cient mice implicte n ctin requirement for lymphocyte signl trnsduction. Curr. Biol. 8, 563±572 (1998). 24. Kwkmi, Y. et l. Activtion nd interction with protein kinse C of cytoplsmic tyrosine kinse, Itk/Tsk/Emt, on FceRI cross-linking on mst cells. J. Immunol. 155, 3556±3562 (1995). 25. Zndi, E. & Krin, M. Bridging the gp: composition, regultion, nd physiologicl function of the IkB kinse complex. Mol. Cell. Biol. 19, 4547±4551 (1999). 26. Esslinger, C. W., Jongeneel, C. V. & McDonld, H. R. Survivl-independent function of NF-kB/Rel during lte stges of thymocyte differentition. Mol. Immunol. 35, 847±852 (1998). 27. Boothy, M. R., Mor, A. L., Scherer, D. C., Brockmn, J. A. & Bllrd, D. W. Perturtion of the T lymphocyte linege in trnsgenic miceexpressing constitutive repressor of nucler fctor (NF)-kB. J. Exp. Med. 185, 1897±197 (1997). 28. Hettmnn, T., DiDonto, J., Krin, M. & Leiden, J. M. An essentil role for nucler fctor kb in promoting doule positive thymocyte poptosis. J. Exp. Med. 189, 145±158 (1999). Acknowledgements We thnk S. Mrmon for prepring fusion proteins used to generte ntiser; M. Brown for nlysis of the ntiser; B. Alfonso nd W. O'Brien for genotyping; D. Alpert, M. Rincon nd R. Dvis for dvice on the JNK ssys; nd E. Skolnik, A. Zychlinsky, Y. Zou nd D. Unutmz for their criticl reding of the mnuscript. C.W.A. is supported y fellowship wrd from the Medicl Reserch Council of Cnd. Z.S. is supported y n NIH Moleculr Oncology nd Immunology trining grnt. E.M.S. is Howrd Hughes Medicl Institute±NIH Reserch Scholr. P.L.S. is supported in prt y Serle Scholr wrd, nd A.K is supported y grnt from the NIH. W.E. is n Associte nd D.R.L. is n Investigtor of the Howrd Hughes Medicl Institute. Correspondence nd requests for mterils should e ddressed to D.R.L. (e-mil: littmn@sturn.med.nyu.edu). Immuniztions KLH (2 mgml -1 ) ws llowed to dsor on 1 mg of luminium hydroxide for 1 h t room temperture. After wshing undsored KLH with PBS, the KLH±luminium hydroxide mixture ws resuspended in 2 ml of PBS nd injected intrperitonelly. Splenocytes recovered 2 weeks lter were stimulted t cells per well (in triplicte) with,.1, 1 or 1 mg ml -1 KLH for 3 d. Cells were then pulsed with 1 mci of [ 3 H]thymidine for 1 d. Received 4 Jnury; ccepted 8 Ferury Monks, C. R., Freierg, B. A., Kupfer, H., Sciky, N. & Kupfer, A. Three-dimensionl segregtion of suprmoleculr ctivtion clusters in T cells. Nture 395, 82±86 (1998). 2. Grkoui, A. et l. The immunologicl synpse: moleculr mchine controlling T cell ctivtion. Science 285, 221±227 (1999). 3. Vlge, V. E., Wong, J. G., Dtlof, B. M., Sinskey, A. J. & Ro, A. Protein kinse C is required for responses to T cell receptor lignds ut not to interleukin-2 in T cells. Cell 55, 11±112 (1988). 4. Monks, C. R., Kupfer, H., Tmir, I., Brlow, A. & Kupfer, A. Selectivemodultion of protein kinse C-u during T-cell ctivtion. Nture 385, 83±86 (1997). 5. Werlen, G., Jcinto, E., Xi, Y. & Krin, M. Clcineurin preferentilly synergizes with PKC-u to ctivte JNK nd IL-2 promoter in T lymphocytes. EMBO J. 17, 311±3111 (1998). 6. Ghffri-Trizi, N. et l. Protein kinse Cu, selective upstrem regultor of JNK/SAPK nd IL-2 promoter ctivtion in Jurkt T cells. Eur. J. Immunol. 29, 132±142 (1999). 7. Kisielow, P., Bluthmnn, H., Sterz, U. D., Steinmetz, M. & von Boehmer, H. Tolernce in T-cellreceptor trnsgenic mice involves deletion of nonmture thymocytes. Nture 333, 742±746 (1988). 8. Meller, N., Altmn, A. & Iskov, N. New perspectives on PKCu, memer of the novel sufmily of protein kinse C. Stem Cells 16, 178±192 (1998). 9. Smith, K. A. The interleukin 2 receptor. Annu. Rev. Cell Biol. 5, 397±425 (1989). 1. Genot, E. M., Prker, P. J. & Cntrell, D. A. Anlysis of the role of protein kinse C-,-e, nd -z in T cell ctivtion. J. Biol. Chem. 27, 9833±9839 (1995). 11. Weiss, A. & Littmn, D. R. Signl trnsduction y lymphocyte ntigen receptors. Cell 76, 263±274 (1994). 12. Weiss, L. et l. Regultion of c-jun NH(2)-terminl kinse (jnk) gene expression during T cell ctivtion. J. Exp. Med. 191, 139±146 (2). 13. Shpiro, V. S., Truitt, K. E., Imoden, J. B. & Weiss, A. CD28 medites trnscriptionl upregultion of the interleukin-2 (IL-2) promoter through composite element contining the CD28RE nd NF-IL- 2B AP-1 sites. Mol. Cell. Biol. 17, 451±458 (1997). 14. Northrop, J. P. et l. NF-AT components de ne fmily of trnscription fctors trgeted in T-cell ctivtion. Nture 369, 497±52 (1994). 15. Jin, J., Loh, C. & Ro, A. Trnscriptionl regultion of the IL-2 gene. Curr. Opin. Immunol. 7, 333± 342 (1995). 16. Jmieson, C., McCffrey, P. G., Ro, A. & Sen, R. Physiologic ctivtion of T cells vi the T cell receptor induces NF-kB. J. Immunol. 147, 416±42 (1991). 17. Jin, J., Vlge-Archer, V. E., Sinskey, A. J. & Ro, A. The AP-1 site t -15 p, ut not the NF-kB site, is likely to represent the mjor trget of protein kinse C in the interleukin 2 promoter. J. Exp. Med. 175, 853±862 (1992). 18. Slon-Lncster, J. & Allen, P. M. Altered peptide lignd-induced prtil T cell ctivtion: moleculr mechnisms nd role in T cell iology. Annu. Rev. Immunol. 14, 1±27 (1996). 19. Newton, A. C. Regultion of protein kinse C. Curr. Opin. Cell Biol. 9, 161±167 (1997). 2. Edwrds, A. S., Fux, M. C., Scott, J. D. & Newton, A. C. Croxyl-terminl phosphoryltion regultes the function nd sucellulr locliztion of protein kinse C II. J. Biol. Chem. 274, 6461±6468 (1999). 21. Kong, Y. Y. et l. Vv regultes peptide-speci c poptosis in thymocytes. J. Exp. Med. 188, 299±2111 (1998). 22. Fischer, K. D. et l. Vv is regultor of cytoskeletl reorgniztion medited y the T-cell receptor. Curr. Biol. 8, 554±562 (1998).... Control of T H 2 polriztion y the chemokine monocyte chemottrctnt protein-1 Long Gu, Susn Tseng, ReneÂe M. Horner, Crmen Tm, Mssimo Lod & Brrett J. Rollins Deprtment of Adult Oncology, Dn-Frer Cncer Institute, Brighm & Women's Hospitl, nd Hrvrd Medicl School, 44 Binney Street, Boston, Msschusetts 2115, USA... Activted T lymphocytes differentite into effector cells tilored to meet disprte chllenges to host integrity 1. For exmple, type 1 nd type 2 helper (T H 1 nd T H 2) cells secrete cytokines tht enhnce cell-medited nd humorl immunity, respectively. The chemokine monocyte chemottrctnt protein-1 (MCP-1) cn stimulte interleukin-4 production 2 nd its overexpression is ssocited with defects in cell-medited immunity 3, indicting tht it might e involved in T H 2 polriztion. Here we show tht MCP-1-de cient mice re unle to mount T H 2 responses. Lymph node cells from immunized MCP-1 -/- mice synthesize extremely low levels of interleukin-4, interleukin-5 nd interleukin-1, ut norml mounts of interferon-g nd interleukin-2. Consequently, these mice do not ccomplish the immunogloulin suclss switch tht is chrcteristic of T H 2 responses nd re resistnt to Leishmni mjor. These effects re direct rther thn due to norml cell migrtion, ecuse the trf cking of nive T cells is undistured in MCP-1 -/- mice despite the presence of MCP-1- expressing cells in secondry lymphoid orgns of wild-type mice. Thus, MCP-1 in uences oth innte immunity, through effects on monocytes, nd dptive immunity, through control of T helper cell polriztion. MCP-1 is CC chemokine tht ttrcts monocytes, memory T lymphocytes nd nturl killer cells in vitro 4±8. Severl models of trnsgenic expression in the mouse hve vlidted it s predominntly monocytic chemottrctnt in vivo 3,9±11. Despite the existence of other CC chemokines tht ttrct monocytes in vitro with NATURE VOL MARCH Mcmilln Mgzines Ltd 47