T cell intrinsic role of Nod2 in promoting type 1 immunity to Toxoplasma gondii

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1 T cell intrinsic role of Nod in promoting type 1 immunity to Toxoplsm gondii Michel H Shw 1, Thornik Reimer 1, Crmen Sánchez-Vldepeñs, Neil Wrner 1, Yun-Gi Kim 1, Mnuel Fresno & Griel Nuñez 1 Nod elongs to the nucleotide-inding oligomeriztion domin receptor (NLR) fmily of proteins, which function s intrcellulr pthogen sensors in innte immune cells. Nod deficiency results in n impired immune response to cteril pthogens. However, how this protein promotes host defense ginst intrcellulr prsites is unknown. Here we found tht Nod / mice hd less clernce of Toxoplsm gondii nd lower interferon- (IFN- ) production. Reconstitution of T cell deficient mice with Nod / T cells followed y T. gondii infection demonstrted T cell intrinsic defect. Nod / CD + T cells hd poor helper T cell differentition, which ws ssocited with impired production of interleukin (IL-) nd nucler ccumultion of the trnscription fctor suunit. Our dt demonstrte T cell intrinsic role for Nod signling tht is criticl for host defense ginst T. gondii. The mmmlin nucleotide-inding oligomeriztion domin (Nod)-like receptor (NLR) proteins function s intrcellulr ptternrecognition receptors nd s regultors of host immunity y detecting microil products 1. The prototypic NLR memers, Nod1 nd Nod, sense frgments of peptidoglycn from cteri. Nod1 ctivity is triggered y γ-d-glutmyl-meso-diminopimelic cid, which is unique to peptidoglycn structures in ll Grm-negtive cteri nd certin Grm-positive cteri, including the gener Listeri nd Bcillus 1. In contrst, Nod is ctivted y murmyl dipeptide (MDP), peptidoglycn motif present in ll Grm-positive nd Grm-negtive cteri 1. The importnce of NLRs in detecting specific microil products is underscored y the susceptiility of mice with trgeted deletion of vrious NLR genes. However, the role of NLRs in sensing intrcellulr prsites is uncler t present. Toxoplsm gondii is n oligte intrcellulr protozon pthogen le to infect vrious niml species. Infection with T. gondii cn cuse severe disese, such s pneumoni nd encephlitis, in immunocompromised hosts. One mjor determinnt of the outcome of T. gondii infection is the ility of the host to elicit roust cellulr immunity to the prsite. This protective immunity is medited minly y interleukin 1 (IL-1)-induced interferon-γ () derived from nturl killer (NK) cells nd T helper type 1 (T H 1)-polrized T cells 3,. Host recognition of extrcellulr T. gondii y the Toll-like receptors (TLRs) expressed on dendritic cells (DCs) promotes secretion of the proinflmmtory cytokine IL-1. T. gondii induced IL-1 production nd optiml resistnce to infection is dependent on TLR11, which recognizes profilin-like protein from T. gondii 5. Others hve linked TLR to prsite-induced responses 6, which suggests tht the prsite expresses more thn one type of TLR lignd. In ddition to eing induced y TLRs, DC-derived IL-1 cn lso e induced y prsite-derived cyclophilin 18 y CCR5, Gα i protein coupled chemokine receptor 7. From such studies, it is evident the host is endowed with extrcellulr sensing mechnisms tht elicit n immune response to T. gondii. However, given tht T. gondii resides nd replictes in nonfusogenic vcuole in the cytoplsm, we sought to determine if the host uses n intrcellulr detection system, such s the mmmlin NLRs, to sense intrcellulr prsites. We found tht Nod provides T cell intrinsic signl tht is necessry not only for generting protective T H 1 immunity to T. gondii ut lso for driving T cell medited colitis. RESULTS Nod promotes resistnce to T. gondii To ssess whether NLRs prticipte in the host defense ginst T. gondii, we inoculted mice geneticlly deficient in vrious NLR fmily memers intrperitonelly with the virulent ME9 strin of T. gondii nd monitored the survivl of the mice. Among the mutnt mouse strins tested, mice lcking Nod1, Nlrc, ASC, Nlrp6 nd Nlrp1 showed norml resistnce to T. gondii infection; only Nod- deficient mice showed impired survivl fter T. gondii infection (Fig. 1 nd Supplementry Fig. 1). The enhnced susceptiility of Nod-null mice ws clerly distinct from tht of mice lcking IL-1p35 or (Fig. 1). Unexpectedly, mice lcking Ripk (lso clled RICK), cspse-recruitment domin (CARD)-contining kinse tht hs een linked to Nod1 nd Nod signling 8, were resistnt to prsite infection (Fig. 1). We confirmed the resistnce of the Nod-deficient mice to cute infection y exmining peritonel exudte cells (PECs) collected from the peritonel cvity on dy 7 fter infection. In oth Nod / nd wild-type mice, less thn 1% 1 Deprtment of Pthology nd Comprehensive Cncer Center, University of Michign Medicl School, Ann Aror, Michign, USA. Centro de Biologí Moleculr, Consejo Superior de Investigciones Científics, Universidd Autónom de Mdrid, Mdrid, Spin. Correspondence should e ddressed to G.N. (clx@umich.edu). Received 6 My; ccepted Septemer; pulished online 1 Novemer 9; doi:.38/ni.1816 nture immunology volume numer 1 decemer 9 167

2 Survivl (%) c (n = 18) (n = 16) RICK / (n = 15) Ifng / (n = 5) Time fter infection (d) of the cells were infected with the prsite; in contrst, greter thn % of the PECs recovered t the sme time point from Ifng / mice were infected (Fig. 1). However, exmintion of PECs collected from infected Nod-deficient mice on dy 1 showed tht lrge numers of infiltrting mononucler cells contined prsites s well s numerous extrcellulr prsites (Fig. 1c). Furthermore, in contrst to wild-type mice, which hd few (<1%) infected cells nd lower concentrtions of serum, there ws -fold greter numer of cells infected with tchyzoites nd high concentrtions of circulting in Nod / mice (Fig. 1d nd dt not shown). Together these results suggest tht the susceptiility of Nod / mice fter prsite chllenge seems to e due to delyed impirment in the ility to control prsite repliction t the site of infection. Nod / mice show impired T H 1 responses As protective immunity to T. gondii is criticlly dependent on IL-1-induced production, we mesured the concentrtions of these two cytokines in the serum of Nod / mice during infection. During the course of infection, wild-type nd Nod / mice secreted similr mounts of IL-1p (Fig. ). In contrst, Nod / mice produced less thn wild-type mice did on dy 7 fter infection (Fig. ). Thus, the delyed filure to control prsite growth in the peritonel cvity of Nod / mice could e explined y insufficient production of fter infection. The min cellulr sources of during T. gondii infection re NK cells 9 nd T H 1 lymphocytes. However, we oserved no discernile difference in production y IL-1-stimulted wild-type nd Nod / NK cells (dt not shown) or y T. gondii infected mice t dy 5 (Supplementry Fig. ). Furthermore, in vivo ntiodymedited depletion of NK cells during T. gondii chllenge induced similr decrese in the serum concentrtions of on dy 5 fter infection in wild-type nd Nod / mice (Supplementry Fig. ). These experiments suggest tht Nod-deficient NK cells show no defect in production nd re consistent with reports tht NK cells re n erly source of during T. gondii infection 3. To ddress whether Nod hs role in promoting the genertion of T H 1 responses, we immunized wild-type nd Nod / mice with γ-irrdited live urcil-uxotrophic strin of T. gondii (CPS), which is nonreplictive yet le to infect cells nd induce protective immunity. This strtegy permits nlysis of T cell ctivtion nd T H 1 differentition in conditions of similr ntigen exposure nd yet Infected PECs, dy 7 (%) d Infected PECs, dy 1 (%) RICK / Ifng / Figure 1 Nod in host defense ginst T. gondii infection. Anlysis of wild-type (), Nod /, RICK-deficient (Ripk / ; clled RICK / here) nd Ifng / mice chllenged intrperitonelly with cysts of the ME9 strin of T. gondii. () Survivl of mice over d. P vlue, Mntel-Cox log-rnk test. () Tchyzoite-infected PECs on dy 7. (c) Microscopy of cytospins of PECs otined t dy 1 fter infection nd stined to visulize intrcellulr prsites (rrows). Originl mgnifiction, 5 (left) or,5 (right). (d) Infected PECs on dy 1 fter chllenge. Dt re representtive of t lest four similr experiments with 5 mice per group () or t lest three experiments with 3 5 mice per group ( d; men nd s.d. in,d). circumvents host mortlity. Using this pproch, we detected high frequency of effector CD + nd CD8 + T H 1 cells, defined y intrcellulr expression of fter in vitro restimultion, in the peritonel cvity of wild-type mice on dy 9 fter immuniztion (pek of T cell response; Fig. 3). In contrst, Nod-deficient mice fter immuniztion hd lower percentge of -producing CD + nd CD8 + T cells (Fig. 3). However, we detected no sustntil differences in the totl numer of CD + nd CD8 + cells in the peritonel cvities of wild-type nd Nod / mice (Supplementry Fig. 3). In greement with the lower frequency of + effector T cells, the secretion of y Nod / PECs ex vivo ws lso impired (Supplementry Fig. 3). DCs hve mjor role in T cell priming nd promoting IL-1-driven T H 1 immunity to T. gondii 11. Therefore, the defective T cell response seen in Nod-deficient mice could hve een due to impired DC function. To evlute whether Nod / DCs hve functionl defects in processing nd presenting T. gondii ntigen, we used T. gondii infected wild-type nd Nod-deficient one mrrow derived DCs (BMDCs) to stimulte flow cytometry purified peritonel CD + T lymphocytes derived from CPS-immunized wild-type or Nod / mice t dy 9. Both Nod / nd wild-type BMDCs efficiently induced expression in wild-type CD + T cells (Fig. ). Notly, production y Nod / CD + T cells remined ttenuted, even in the presence of wild-type DCs. Consistent with the flow cytometry nlysis, ex vivo cultures contining Nod-deficient CD + cells secreted less when stimulted with prsite-infected wild-type or Nod / BMDCs (Fig. ). To confirm tht the lunted T cell response oserved in Nod / mice ws not due to impired ntigen presenttion nd/or T cell priming y Nod / DCs, we doptively trnsferred wild-type (Thy ) CD + T cells into wild-type nd Nod / recipient mice. We then immunized these chimers with T. gondii. Similr frequencies of donor (Thy ) + CD + T cells were present in wild-type nd Nod / recipients of wild-type T cells (Fig. c). Collectively these findings indicte tht Nod / DCs re functionlly norml during Serum IL-1p (ng/ml) 8 6 Ifng / P =.578 P =.55 Serum (ng/ml) 8 6 Ifng / P = Time fter infection (d) Time fter infection (d) Figure Nod-deficient mice show impired production despite norml IL-1p production. ELISA of IL-1p () nd () in serum from T. gondii infected wild-type, Nod / nd Ifng / mice collected on dys, 3, 5 nd 7 fter inocultion. Ech symol represents n individul mouse (n = 5 mice per group); smll horizontl rs indicte the men. P vlues, unpired, two-tiled Student s t-test. Dt re representtive of t lest three independent experiments. 168 volume numer 1 decemer 9 nture immunology

3 + CD + lymphocytes (%) PEC PEC CD CD8 5 + CD8 + lymphocytes (%) P =.7 Uninfected Infected Uninfected Infected Figure 3 Nod-deficient mice show impired type 1 immune responses during T. gondii infection. () Flow cytometry nlysis of production y CD + nd CD8 + lymphocytes mong PECs otined from CPS-immunized wild-type nd Nod / mice nd stimulted for 6 h in vitro with live CPS tchyzoites, gted on CD + TCRβ + cells. Numers in qudrnts indicte percent cells in ech. () Dt pooled from ; ech dot represents one mouse nd smll horizontl rs indicte the men. P vlues, unpired, two-tiled Student s t-test. Dt re representtive of t lest three experiments with three to six mice per group (one representtive mouse per group presented in ). production y PECs from Nod +/+ Tcr / nd Nod / Tcr / mice reconstituted with Nod / CD + T cells (Fig. 5). Finlly, the resistnce of Nod / mice fter T. gondii infection ws enhnced y the presence of wild-type T cells (Supplementry Fig. ). Together these findings indicte tht the impired T cell response oserved in Nod / mice during infection is ssocited with n intrinsic defect in Nod-deficient T cells. T. gondii infection nd suggest tht Nod functions in T cell intrinsic mnner to promote optiml T H 1 immunity to T. gondii. T cell intrinsic role for Nod To directly ssess the function of Nod in T cells, we doptively trnsferred wild-type nd Nod / CD + T cells into T cell ntigen receptor-β deficient (Tcr / ) or Nod / Tcr / recipients. We then immunized the reconstituted mice with CPS nd ssessed the frequency of effector T cells 9 d lter. Nod +/+ Tcr / nd Nod / Tcr / recipient mice reconstituted with wild-type CD + T cells hd high frequencies of + CD + T lymphocytes fter prsite chllenge (Fig. 5). However, there ws significntly lower percentge of + CD + T lymphocytes in mice reconstituted with Nod-deficient CD + T cells regrdless of the genotype of the recipient mice (Fig. 5). The impired CD + T cell response ws consistent with the lower CD + CD CD Toxo-DC + Toxo-DC + Toxo-DC Figure DC function during T. gondii infection is unffected y the sence of Nod. () Flow cytometry nlysis of production y CD + T cells otined from the peritonel cvities of CPS-immunized wild-type nd Nod / mice t dy 9, sorted y flow cytometry nd then left unstimulted ( Toxo-DC) or stimulted for 6 h with T. gondii infected BMDCs from wild-type mice (+ Toxo-DC) or Nod / mice (+ Nod / Toxo-DC); plots re % + CD + lymphocytes c Nod in helper T cell differentition nd IL- production To etter understnd the function of Nod in n in vitro ntigenspecific T cell response, we stimulted nive Nod / ovlumin (OVA)-specific OT-II T cell ntigen receptor (TCR) trnsgenic cells with OVA-pulsed BMDCs. Wild-type nd Nod / OT-II cells stimulted with OVA expressed similr mounts of erly ctivtion mrkers, such s CD5 (Fig. 6) nd CD69 (dt not shown). This unimpired T cell ctivtion ws lso reflected in the similr frequency of ctivted CD hi CD6L lo CD + lymphocytes (Fig. 6). However, unexpectedly, Nod / OT-II cells stimulted in the presence of wild-type or Nod / DCs showed impired IL- production (Fig. 6). IL- hs criticl role in T cell priming for the production of oth IL- nd 11. To investigte whether the impired IL- production y Nod / T cells resulted in ttenuted T H 1 or T H cell development, we cultured nive wild-type OT-II cells nd Nod / OT-II CD + CD + P =.51 P = in vitro (ng/ml) + Thy CD + lymphocytes (%) CD + CD + P =.3 P =.7 Toxo-DC Toxo-DC Toxo-DC Toxo-DC Donor: (Thy ) (Thy ) (Thy-1. + ) (Thy-1. + ) CD (Thy ) Donor: (Thy ) (Thy-1. + ) (Thy ) (Thy-1. + ) gted on CD + TCRβ + cells (left). Numers in qudrnts indicte percent cells in ech. Right, pooled dt; ech symol represents one mouse nd smll horizontl rs indicte the men. P vlues, unpired, two-tiled Student s t-test. Dt re representtive of three experiments with three to four mice per group (one representtive mouse per group presented t left). () secretion y peritonel CD + cells ssessed s descried in, ut restimulted for h in vitro. P vlues, unpired, two-tiled Student s t-test. Dt re representtive of three experiments. (c) production y donor CD + cells mong PECs isolted from wild-type (Thy-1.) or Nod / (Thy-1.) recipients of intrvenous injection of splenic CD + cells sorted y mgnetic-ctivted cell sorting from nive wild-type (Thy-1.1) mice; recipient mice were chllenged intrperitonelly with irrdited CPS prsites nd ws ssessed y intrcellulr cytokine stining on dy 9 fter chllenge (left; gted on CD + TCRβ + Thy cells). Numers in qudrnts indicte percent cells in ech. Right, pooled dt; ech symol represents one mouse nd smll horizontl rs indicte the men. Dt re representtive of three experiments. nture immunology volume numer 1 decemer 9 169

4 Figure 5 Nod / CD cells hve n intrinsic defect in production. () production y PECs isolted from Nod +/+ Tcr / (Thy-1.) or Nod / Tcr / (Thy-1.) recipients of intrvenous injection of splenic CD + cells sorted y mgnetic-ctivted cell sorting from wild-type (Thy-1.1) nd Nod / (Thy-1.1) mice; PECs collected on dy 9 fter recipient mice were chllenged with irrdited CPS prsites nd ws ssessed y intrcellulr cytokine stining fter in vitro stimultion of PECs with live prsites (left; gted on CD + TCRβ + Thy cells). Numers in qudrnts indicte percent cells in ech. Right, pooled dt; ech symol represents one mouse nd smll horizontl rs indicte the men. () ELISA of in superntnts of PECs from reconstituted mice descried in, cultured for h together with live prsites. P vlues, unpired, two-tiled Student s t-test. Dt re representtive of three experiments with four to eight mice per group. cells in T H 1- or T H -polrizing conditions. In T H 1 conditions, the wild-type T cells produced high concentrtions of. In contrst, the differentition of nive Nod / T cells into T H 1 cells ws severely impired (Fig. 6c). IL- production y Nod-deficient cells cultured in T H -polrizing conditions ws lso significntly lower (Fig. 6c). To determine whether the lower IL- production y Nod / T cells ws ccountle for the impired helper T cell differentition, we dded exogenous IL- to the cultures; exogenous IL- ws sufficient to rescue the impired T H 1 nd T H differentition of Nod / T cells (Fig. 6d). Generl role of Nod in T cell responses Our finding tht Nod functions in T cell utonomous wy rised the question of whether other T cell responses would e dversely ffected y Nod deficiency. To ddress this possiility, we used vrious doptive-trnsfer systems in which the innte immune cells in recipient mice were Nod sufficient, wheres the donor T cells were either Nod sufficient or Nod deficient. First we proed whether Nod / T cells were le to undergo homeosttic prolifertion or lymphopeniinduced prolifertion, spce-driven expnsion of T cell popultions tht cts s compenstory mechnism to restore the peripherl OT-II OT-II CD5 Dy 1.6 Dy CD6L Figure 6 Impired helper T cell differentition nd IL- production y Nod / T cells. () Flow cytometry nlysis of the expression of CD5, CD nd CD6L y purified wild-type OT-II nd Nod / OT-II cells incuted together in vitro with OVA-loded BMDCs. Left, CD5 expression on dy (thin 9 c in vitro (ng/ml) Dy CD 6 P = T H 1 T H ril- Nod +/+ Tcr / Tcr / in vitro (ng/ml) Dy 1 Dy IL- in vitro (ng/ml) Donor: CD (Thy ) Donor: P =. 6 CPS +CPS Nod +/+ Tcr / recipients + Thy CD + lymphocytes (%) Donor: + Thy CD + lymphocytes (%) P <.5 T lymphocyte pool in conditions of lymphopeni 1. Nod / donor T cells, when trnsferred lone, persisted in lymphopenic host (Supplementry Fig. 5). However, when trnsferred together with wild-type CD + T cells into the sme hosts, Nod-deficient T cells underwent little lymphopeni-induced prolifertion (Supplementry Fig. 5). This result suggests tht Nod / cells re unsuccessful t competing for the limited resources required for such prolifertion. As n dditionl test to evlute the role of Nod in regulting T cell function, we used mouse model of colitis in which the trnsfer of purified CD + CD5 CD5RB hi T cells into recomintionctivting gene 1 deficient (Rg1 / ) mice leds to colitis driven y T H 1 cytokines 13,1. Wild-type CD + CD5 CD5RB hi T cells induced P =.9 T H 1 T H ril- OT-II OT-II IL- in vitro (ng/ml) d in vitro (ng/ml) 3 1 in vitro (ng/ml) Donor: CPS BMDC + OVA (µg/ml) 6 P =.1 P =.61 (Thy ) (Thy ) Nod +/+ Tcr / Nod +/+ Tcr / (Thy ) (Thy ) Tcr / Tcr / P =.95 +CPS Tcr / recipients IL- in vitro (ng/ml) 3 1 IL- in vitro (ng/ml) P =.168 BMDC + OVA (µg/ml) P =. P =.33 T H 1 T H T H 1 T H + ril- ( ng/ml) + ril- ( ng/ml) lines) nd on dy 1 or (thick lines); numers ove rcketed lines (left) indicte percent cells in gte. Right, numers in qudrnts indicte percent cells in ech. () ELISA of IL- in superntnts of wild-type OT-II cells nd Nod / OT-II cells cultured together for 3 d with wild-type or Nod / BMDCs loded with vrious doses (horizontl xes) of OVA. (c,d) Polriztion efficiency of splenic wild-type OT-II cells nd Nod / OT-II cells purified nd cultured with OVAloded wild-type BMDCs in T H 1- or T H -polrizing conditions in the sence ( ril-; c) or presence (+ ril-; d) of exogenous IL-, ssessed on dy 6 on the sis of the production of (T H 1) nd IL- (T H ) fter restimultion for h with nti-cd3. P vlues, unpired, two-tiled Student s t-test. Dt re representtive of t lest three experiments with three to four mice per group (men nd s.d. of three mice per group in d). 17 volume numer 1 decemer 9 nture immunology

5 Weight (% of initil) * ** *** Time fter trnsfer (weeks) c Sm int L int Histologic score d in vitro (ng/ml) P =.98 CD3 + CD3 Figure 7 Role of Nod in T cell induced colitis. () Body weights of Rg1 / recipients of purified CD + CD5 CD5RB hi T cells from Nod / or wildtype mice, presented s percent of initil weight (t dy ). *P <.5, **P <.5, ***P <.5 nd (unpired, two-tiled Student s t-test). () Gross morphology of colons of the recipient mice in t 8 weeks fter reconstitution. (c) Histology of smll intestine nd colonic tissues from the mice in (left); rrowheds indicte inflmmtory cell infiltrtion. Originl mgnifiction, 1,. Right, severity of intestinl inflmmtion in the mice in, ssessed y histologicl scoring in three mjor ctegories: extent of epithelil dmge, level of involvement, nd inflmmtory cell infiltrtion. (d) ELISA of concentrtions in culture superntnts of mesenteric lymph nodes otined from the mice in nd restimulted for h with nti-cd3. P vlues, unpired, two-tiled Student s t-test. Dt re representtive of two experiments with five mice per group. wsting disese, s predicted (Fig. 7). In contrst, Rg1 / mice reconstituted with Nod / CD + CD5 CD5RB hi T cells remined helthy nd none developed wsting disese, s shown y their increse in ody weight (Fig. 7). By gross exmintion, the lrge intestine ws thickened in Rg1 / recipients of wild-type CD + CD5 CD5RB hi cells ut not in Rg1 / recipients of Nod / CD + CD5 CD5RB hi cells (Fig. 7). Microscopic exmintion of the smll nd lrge intestines isolted from Rg1 / mice reconstituted with wild-type CD + CD5 CD5RB hi cells showed greter ccumultion of inflmmtory cells (Fig. 7c). In contrst, sections from the intestines of Rg1 / recipients of Nod / c HA-Nod Myc IP: HA Lyste IP: HA Lyste IP: Lyste IP: Lyste Flg-Nod Flg-CARMA1 Myc-Bcl Myc- Myc-RICK IP: Flg Lyste c-myc c-myc RICK Bcl- RICK Bcl- CD8RE AP-1 ctivity ( 3 ) Nod CD + CD5 CD5RB hi cells showed sustntilly less severe histologicl lesions (Fig. 7c). Consistent with the microscopic exmintion, Rg1 / recipients of Nod-deficient T cells hd lower histologicl scores (Fig. 7c), nd T cells in the mesenteric lymph nodes of these mice produced less (Fig. 7d). Together these dt support the ide of direct role for Nod in regulting T cell function. Nod inds nd enhnces Il trnscription The lunted lymphopeni-induced prolifertion, T cell driven colitis nd IL- secretion y Nod / T cells suggested tht Nod P <.5 P < Time fter CD3+CD8 stim (h): GAPDH β-ctin p-p65 GAPDH β-ctin NE KO CE KO Figure 8 Nod intercts with nd enhnces Il trnscription. () Immunolot nlysis (IB) of whole-cell lystes of HEK93T cells trnsiently trnsfected for 8 h with hemgglutinin (HA)-tgged, Myc-tgged or Flg-tgged expression plsmid(s) (1 µg ech; ove lnes) efore (Lyste) nd fter immunoprecipittion (IP) of proteins with nti- or nti-hemgglutinin (Nod; left) or with nti-flg (Nod or CARMA1; right). Bcl- serves s specificity control. Results re representtive of three experiments. () Luciferse ctivity in Jurkt cells trnsiently trnsfected with luciferse reporter driven y CD8RE, long with equivlent mounts of Nod, nd expression constructs (elow rs), then stimulted with nti-cd3 nd nti-cd8; results re normlized to those of renill luciferse reporter construct (to normlize for trnsfection efficiency). P vlues, unpired, two-tiled Student s t-test. Dt re representtive of three independent experiments (verge nd s.d.). (c) Immunolot nlysis of nucler (NE) nd cytosolic (CE) frctions from purified T cells stimulted for 1 9 h with plte-ound nti-cd3 nd nti-cd8 (CD3+CD8 stim). GAPDH (glycerldehyde phosphte dehydrogense) nd β-ctin serve s loding controls. KO, Nod / ; p-, phosphorylted. Results re representtive of three similr experiments. nture immunology volume numer 1 decemer 9 171

6 my function downstrem of either TCR or costimultory signls. As TCR-driven phosphoryltion of the mitogen-ctivted protein kinses Jnk, p38 nd Erk ws similr in wild-type nd Nod / T cells (Supplementry Fig. 6), we investigted whether Nod is involved in the CD8 costimultory signling pthwy. The production of IL- in response to CD8 costimultion depends on ctivtion of the trnscription fctor NF-κB suunit, which intercts with the NF-κB-inding sites s well s CD8-responsive elements (CD8REs) in the proximl Il promoter region 15,16. The relevnce of for IL- production is underscored y the oservtion tht T cells from deficient mice show profound deficiency in IL- production 17. The ility of to interct with CD8REs to induce IL- production is regulted y NF-κB-inducing kinse (; lso clled MAP3KI) 18. T cells from lymphoplsi (ly/ly) mice tht er muttion in the gene encoding lso show defects in IL- secretion in response stimultion with ntiody to CD3 (nti-cd3) nd nti-cd8 (ref. 19). Given those findings, we hypothesized tht Nod my interct with nd/or in T lymphocytes to promote IL- production. To determine if Nod cn interct with or, we trnsiently trnsfected humn emryonic kidney (HEK93T) cells with expression vectors encoding, nd/or Nod. As reported efore, immunoprecipitted together with Nod (Fig. 8). Notly, ws lso present in Nod immunoprecipittes (Fig. 8). When Nod, nd were coexpressed simultneously, oth nd immunoprecipitted together with Nod (Fig. 8), which suggests tht these three proteins cn form tertiry complex. As specificity control, Bcl-, which is known to hve role in T cell receptor signling, ssocited with dptor CARMA1, s reported efore 1, ; however, we oserved no interction etween Bcl- nd Nod (Fig. 8) Hving estlished the existence of n interction mong Nod, nd, we next ssessed whether Nod hs function in regulting IL- trnscription induced y CD8 costimultion in Jurkt T cells. We trnsiently trnsfected cells with CD8RE AP-1 reporter construct long with vrious comintions of, nd/or Nod expression vectors. As expected, trnsfection of sustntilly upregulted trnscription of the CD8RE AP-1 reporter, nd the presence of synergisticlly enhnced the ility of to upregulte this trnscription 18. Notly, Nod synergisticlly enhnced -driven CD8RE AP-1 reporter ctivity, nd coexpression of, nd Nod resulted in mximum ctivtion of the CD8RE AP-1 element, despite the finding tht Nod y itself or in the presence of did not induce ny significnt reporter ctivity (Fig. 8). These results re consistent with positive role for Nod in the trnscriptionl regultion of Il. Finlly, the finding tht Nod intercted with oth nd prompted us to ssess whether Nod deficiency could ffect nucler ccumultion of, n event criticl for Il trnscription. We prepred nucler extrcts from CD8-costimulted wild-type nd Nod / T cells nd ssessed the presence of nucler. Consistent with pulished studies 16, we oserved sustntil nucler ccumultion of in wild-type cells fter 6 9 h of stimultion (Fig. 8c). In contrst, Nod / cells hd less thn hlf tht mount of nucler t the sme time points fter stimultion. The impired nucler ccumultion of ws not generl defect ffecting ll NF-κB suunits induced fter costimultion, s we oserved similr mounts of phosphorylted p65 (nother NF-κB suunit) in the nuclei of wildtype nd Nod / cells (Fig. 8c). Together these dt suggest tht the Nod is required for optiml nucler ccumultion of nd trnscription of Il. DISCUSSION Our study hs demonstrted tht Nod is required for host resistnce to T. gondii. The greter susceptiility of Nod / mice fter T. gondii infection ws unexpected, given tht Nod ws initilly identified s sensor for intrcellulr cteri. To our knowledge, T. gondii does not contin motifs nlogous to the Nod gonist MDP. It is possile tht Nod promotes host immune responses ginst T. gondii y sensing other prsite-derived proteins injected into the host cytosol during the invsion process 3,. However, this seems unlikely, given tht T. gondii infected Nod / DCs nd mcrophges hd unimpired cytokine secretion, s well s ctivtion of mitogen-ctivted protein kinses (dt not shown). Thus, the lower resistnce of Nod / mice fter T. gondii infection is not ssocited with defects in pthogen recognition. Our in vivo trnsfer experiments estlished tht Nod hs T cell intrinsic role in the genertion of n effective T H 1 response. This work is the first to our knowledge to demonstrte tht Nod is required for optiml IL- production y ctivted T cells. IL- hs multifceted role in T cell iology, including T cell differentition 5 7. Accordingly, the ttenuted IL- secretion y Nod-deficient T cells mnifests s profound impirment in helper T cell differentition. The ddition of exogenous IL- during in vitro polriztion ws sufficient to fully rescue the impired T H differentition of nive Nod / CD + T cells nd to prtilly (>65%) rescue their impired T H 1 differentition. Tht finding suggests tht Nod promotes T H 1 differentition through IL--dependent nd IL--independent pthwys. Furthermore, the presence of wild-type CD + T cells in vivo ws le to prtilly rescue Nod / mice fter prsite chllenge, presumly ecuse of the prcrine ction of IL- produced y wild-type CD + T lymphocytes 8. Thus, the impired IL- production y Nod / T cells during ctivtion is sufficient to lter helper T cell differentition, therey resulting in lower resistnce of Nod / mice to T. gondii, pthogen known to induce vigorous T H 1 response. The oserved impired T cell function in the sence of Nod ws not limited to production fter T. gondii infection ut lso included lymphopeni-induced T cell popultion expnsion nd T cell driven colitis, two models systems tht re positively driven y interction of the TCR nd the coreceptor CD8 with complexes of peptide nd mjor histocomptiility complex nd with B7 molecules, respectively, on ccessory cells Given tht TCR signling seemed to e lrgely intct in the sence of Nod, we hypothesized tht Nod my ffect the CD8 signling pthwy, which results in the ctivtion of genes tht depend on or NF-κB p5-p65 complexes 33. Genes tht elong to the former ctegory include IL- (ref. 17). Our results demonstrting tht Nod cn interct with nd nd tht the presence of Nod oosts Il reporter element trnscription induced y nd support our hypothesis tht Nod cts downstrem of the CD8 signling pthwy. Furthermore, nucler ccumultion of ws lower in the sence of Nod. Together our dt suggest not only tht Nod cn interct with ut lso tht this interction fcilittes the nucler entry of required for Il trnscription. It is conceivle tht Nod, which contins two mino-terminl CARDs, functions s scffolding protein tht promotes interction with other CARD-contining proteins, such s Bcl- nd CARD11, which hve een demonstrted to promote NF-κB signling fter T cell ctivtion 3. However, this seems unlikely, given tht interction etween Nod nd Bcl- ws undetectle nd tht Nod / T cells re phenotypiclly distinct from T cells lcking Bcl- or CARD11 (refs. 35,36). In ddition to contining CARDs, Nod contins series 17 volume numer 1 decemer 9 nture immunology

7 of croxy-terminl leucine-rich repets tht my medite proteinprotein interctions 37. Thus, given our findings, we propose tht Nod, in ddition to sensing microil products in innte immune cells such s mcrophges nd DCs, cn lso operte s moleculr scffold tht integrtes costimultory signls necessry for proper T cell function. Nod ctivtion in mcrophges fter MDP stimultion results in the recruitment nd ctivtion of RICK, serine-threonine kinse essentil for the ctivtion of NF-κB nd mitogen-ctivted protein kinses, resulting in the relese of proinflmmtory cytokines nd chemokines 8. In our studies, Nod ctivtion in T cells seemed to trigger the induction of distinct signling pthwy not shred with MDP-stimulted mcrophges. During T. gondii infection, RICK ws dispensle for the induction of n effective type 1 immune response; this finding is consistent with pulished results demonstrting tht type 1 immune responses re norml in the sence of RICK 38,39. Together our dt suggest tht, t lest in T cells, Nod hs function independent of MDP. Therefore, the two divergent signling pthwys induced y Nod my e dependent on the context of the stimulus, s well s the responding cell type. The role of Nod in regulting T cell function hs een the focus of recent studies. The results of these studies suggest differences in the requirements for Nod in promoting optiml ntiody responses in vrious experimentl conditions. For exmple, Nod-deficient mice show norml OVA-specific T H responses fter immuniztion with Nod1 gonist nd norml humn serum lumin specific production of immunogloulin G fter tretment with TLR7 gonist 1. In contrst, ntigen-specific ntiody production is lower in response to incomplete Freund s djuvnt nd ntigen (without the ddition of either TLR or NLR gonists) in the sence of Nod (ref. ). The lst finding would e consistent with our oservtion tht Nod regultes dptive immunity independently of its innte role in sensing MDP. The discrepncy in the requirements for Nod in generting effective dptive immunity in these studies my e due to the use of different djuvnts, which could differentilly influence the expression of other B7-relted costimultory molecules 3 nd/or tumor necrosis fctor receptor superfmily molecules (which re involved in T cell ctivtion) on ntigen-presenting cells. Thus, the influence of Nod on dptive immune responses in vivo my e determined y whether or not CD8 signling is preferentilly required. Although the precise mechnistic role of Nod in ridging the nucler ccumultion of nd trnscription of Il in T cells remins to e fully elucidted, the in vivo relevnce of Nod function in T cells ws demonstrted y the chllenge of Nod / mice with T. gondii. Our results re in ccord with pulished reports demonstrting the requirement for IL- (ref. 5) nd T cell intrinsic expression of 6 in host resistnce to T. gondii. Furthermore, the impired ility of Nod / T cells to proliferte homeostticlly nd drive colitis suggests more glol impirment in the ility of the host to mount n effective immune response in the sence of Nod. Finlly, our finding tht Nod directly prticiptes in the genertion of dptive immune responses underscores the importnce of this protein in regulting oth innte nd dptive immunity. Methods Methods nd ny ssocited references re ville in the online version of the pper t Note: Supplementry informtion is ville on the Nture Immunology wesite. Acknowledgments We thnk A. Weiss (University of Cliforni, Sn Frncisco) for the pcd8re/ap-1 luc reporter plsmid; G.Y. Chen for criticlly reding the mnuscript; S. Koonse for ssistnce with mnging mice colonies; the University of Michign Flow Cytometry nd the immunology core fcilities for ssistnce with flow cytometry nd enzyme-linked immunosorent ssy (ELISA); nd G.S. Yp for discussions. Supported y the US Ntionl Institutes of Helth (R1 DK6177; nd T3- HL7517 to M.H.S.), the Swiss Ntionl Science Foundtion (T.R.), the University of Michign Comprehensive Cncer Center (Y.-G.K.), the Spnish Ministerio de Cienci e Innovcion (SAF to M.F.), Europen Union (Eicosnox, for M.F.), Comunidd de Mdrid (S-SAL-159/6 to M.F.) nd L Red Temátic de Investigción Coopertiv en Enfermeddes Crdiovsculres (RD6/1/13 to M.F.). AUTHOR CONTRIBUTIONS M.H.S. developed the concept nd designed experiments; C.S.-V. nd M.F. designed nd did Jurkt nd CD8RE AP-1 ssys; T.R., N.W. nd Y.-G.K. provided technicl support nd conceptul dvice; M.H.S. nd G.N. prepred the mnuscript; G.N. directed the reserch; nd ll uthors discussed the results nd implictions nd commented on the mnuscript t ll stges. Pulished online t Reprints nd permissions informtion is ville online t reprintsndpermissions/. 1. Shw, M.H., Reimer, T., Kim, Y.G. & Nuñez, G. NOD-like receptors (NLRs): on fide intrcellulr microil sensors. Curr. Opin. 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Sher, A., Oswld, I.P., Hieny, S. & Gzzinelli, R.T. Toxoplsm gondii induces T-independent response in nturl killer cells tht requires oth dherent ccessory cells nd tumor necrosis fctor-α. J. Immunol. 15, (1993).. Shw, M.H. et l. Tyk negtively regultes dptive Th1 immunity y mediting IL- signling nd promoting -dependent IL- rectivtion. J. Immunol. 176, (6). 11. Reis e Sous, C. et l. In vivo microil stimultion induces rpid CD ligndindependent production of interleukin 1 y dendritic cells nd their redistriution to T cell res. J. Exp. Med. 186, (1997). 1. Jmeson, S.C. Mintining the norm: T-cell homeostsis. Nt. Rev. Immunol., (). 13. Morrissey, P.J., Chrrier, K., Brddy, S., Liggitt, D. & Wtson, J.D. CD + T cells tht express high levels of CD5RB induce wsting disese when trnsferred into congenic severe comined immunodeficient mice. Disese development is prevented y cotrnsfer of purified CD + T cells. J. Exp. Med. 178, 37 (1993). 1. Powrie, F., Lech, M.W., Muze, S., Cddle, L.B. & Coffmn, R.L. Phenotypiclly distinct susets of CD + T cells induce or protect from chronic intestinl inflmmtion in C. B-17 scid mice. Int. Immunol. 5, (1993). 15. Frser, J.D., Irving, B.A., Crtree, G.R. & Weiss, A. Regultion of interleukin- gene enhncer ctivity y the T cell ccessory molecule CD8. Science 51, (1991). 16. Zhou, X.Y. et l. Moleculr mechnisms underlying differentil contriution of CD8 versus non-cd8 costimultory molecules to IL- promoter ctivtion. J. Immunol. 168, (). 17. Kontgen, F. et l. Mice lcking the c-rel proto-oncogene exhiit defects in lymphocyte prolifertion, humorl immunity, nd interleukin- expression. Genes Dev. 9, (1995). 18. Snchez-Vldepens, C., Mrtin, A.G., Rmkrishnn, P., Wllch, D. & Fresno, M. NF-κB-inducing kinse is involved in the ctivtion of the CD8 responsive element through phosphoryltion of nd regultion of its trnsctivting ctivity. J. Immunol. 176, (6). 19. Ymd, T. et l. Anorml immune function of hemopoietic cells from lymphoplsi (ly) mice, nturl strin with mutnt NF-κB-inducing kinse. J. Immunol. 165, 8 81 ().. Pn, Q. et l. NF-κB-inducing kinse regultes selected gene expression in the Nod signling pthwy. Infect. Immun. 7, (6). 1. Gide, O. et l. CARMA1 is criticl lipid rft-ssocited regultor of TCR-induced NF-κB ctivtion. Nt. Immunol. 3, ().. Wng, D. et l. A requirement for CARMA1 in TCR-induced NF-κB ctivtion. Nt. Immunol. 3, (). nture immunology volume numer 1 decemer 9 173

8 3. Brdley, P.J. & Siley, L.D. Rhoptries: n rsenl of secreted virulence fctors. Curr. Opin. Microiol., (7).. Crruthers, V.B. & Siley, L.D. Sequentil protein secretion from three distinct orgnelles of Toxoplsm gondii ccompnies invsion of humn firolsts. Eur. J. Cell Biol. 73, (1997). 5. Mlek, T.R. & Byer, A.L. Tolernce, not immunity, crucilly depends on IL-. Nt. Rev. Immunol., (). 6. Brem, J.H. et l. A distl region in the interferon-γ gene is site of epigenetic remodeling nd trnscriptionl regultion y interleukin-. J. Biol. Chem. 79, (). 7. Lio, W. et l. Priming for T helper type differentition y interleukin -medited induction of interleukin receptor α-chin expression. Nt. Immunol. 9, (8). 8. D Souz, W.N., Schluns, K.S., Msopust, D. & Lefrncois, L. Essentil role for IL- in the regultion of ntivirl extrlymphoid CD8 T cell responses. J. Immunol. 168, (). 9. Liu, Z. et l. B7 interctions with CD8 nd CTLA- control tolernce or induction of mucosl inflmmtion in chronic experimentl colitis. J. Immunol. 167, (1).. Gudmundsdottir, H. & Turk, L.A. A closer look t homeosttic prolifertion of CD + T cells: costimultory requirements nd role in memory formtion. J. Immunol. 167, (1). 31. Hgen, K.A. et l. A role for CD8 in lymphopeni-induced prolifertion of CD T cells. J. Immunol. 173, (). 3. Yen, M.H., Lepk, N. & Swin, S.L. Induction of CD T cell chnges in murine AIDS is dependent on costimultion nd involves dysregultion of homeostsis. J. Immunol. 169, (). 33. Civil, A., Rensink, I., Arden, L.A. & Verweij, C.L. Functionl disprity of distinct CD8 response elements towrd mitogenic responses. J. Biol. Chem. 7, (1999). 3. Sun, S.C. & Ley, S.C. New insights into NF-κB regultion nd function. Trends Immunol. 9, (8). 35. Rulnd, J. et l. Bcl is positive regultor of ntigen receptor-induced ctivtion of NF-κB nd neurl tue closure. Cell, 33 (1). 36. Hr, H. et l. The MAGUK fmily protein CARD11 is essentil for lymphocyte ctivtion. Immunity 18, (3). 37. Koe, B. & Kjv, A.V. The leucine-rich repet s protein recognition motif. Curr. Opin. Struct. Biol. 11, (1). 38. Hll, H.T. et l. RIP contriutes to Nod signling ut is not essentil for T cell prolifertion, T helper differentition or TLR responses. Eur. J. Immunol. 38, 6 7 (8). 39. Nemrini, C., Reissmnn, R., Kopf, M. & Mrslnd, B.J. Effective T-cell immune responses in the sence of the serine/threonine kinse RIP. Microes Infect., 5 5 (8).. Mglhes, J.G. et l. Nod-dependent Th polriztion of ntigen-specific immunity. J. Immunol. 181, (8). 1. Koyshi, K.S. et l. Nod-dependent regultion of innte nd dptive immunity in the intestinl trct. Science 7, (5).. Moreir, L.O. et l. Modultion of dptive immunity y different djuvnt-ntigen comintions in mice lcking Nod. Vccine 6, (8). 3. Greenwld, R.J., Freemn, G.J. & Shrpe, A.H. The B7 fmily revisited. Annu. Rev. Immunol. 3, (5).. Croft, M. The role of TNF superfmily memers in T-cell function nd diseses. Nt. Rev. Immunol. 9, (9). 5. Villegs, E.N., Lieermn, L.A., Crding, S.R. & Hunter, C.A. Susceptiility of interleukin--deficient mice to Toxoplsm gondii is ssocited with defect in the production of γ interferon. Infect. Immun. 7, (). 6. Mson, N.J., Liou, H.C. & Hunter, C.A. T cell-intrinsic expression of regultes Th1 cell responses essentil for resistnce to Toxoplsm gondii. J. Immunol. 17, (). 17 volume numer 1 decemer 9 nture immunology

9 ONLINE METHODS Mice. All mice were from the Jckson Lortory. Nod / mice hve een descried 1. All mice (C57BL/6 ckground) were red nd mintined in specific pthogen free conditions t the University of Michign niml fcility. Animl studies were done ccording to pproved protocols y the University of Michign Committee on Use nd Cre of Animls. T. gondii infection. Cysts of the virulent ME9 strin of T. gondii were otined from the rins of chroniclly infected mice. Mice were chllenged y intrperitonel injection of T. gondii cysts. For ssessment of prsite urden, cytospin preprtions were mde from peritonel lvge fluid collected on dys 7 nd 1 fter infection nd infected cells were counted y microscopy. Vccintion consisted of single intrperitonel dose of 1 6 prsites of the CPS strin tht hd een irrdited t 15 krds (dose rte of Gy/min). Cell culture nd cytokine ssys. Single-cell suspensions were prepred from individul peritoneum from ech mouse fter infection. Cell suspensions were cultured for h in complete RPMI-16 medium (Invitrogen Life Technologies) with live CPS tchyzoites. For in vitro restimultion of peritonel exudte CD + T cells with T. gondii infected DCs, peritonel CD + T lymphocytes from CPS-primed wild-type or Nod / mice t dy 9 were sorted y flow cytometry nd 1 5 cells were susequently incuted together with 5 T. gondii infected DCs in 96-well pltes; culture superntnts were collected t h for cytokine nlysis. For in vitro T cell ctivtion nd IL- production, splenic CD + T cells from wild-type OT-II or Nod / OT-II trnsgenic mice were purified with MACS MicroBeds (Miltenyi Biotec). Purified CD + T cells (>95% purity) were then cultured together with OVA-loded wild-type or Nod / BMDCs (rtio, 1:1). At vrious time points, culture superntnts were collected nd cells were nlyzed y flow cytometry for vrious T cell ctivtion mrkers. For T cell polriztion experiments, CD + cells were incuted with n equl numer of OVA-loded BMDCs with or without exogenous IL- ( ng/ml). For T H 1 conditions, µg/ml of nti-il- (11B11) nd 5 ng/ml of exogenous IL-1 were dded. For T H conditions, µg/ml of nti-il-1 (C17.8), µg/ml of nti- (XMG1.) nd ng/ml of exogenous IL- were dded (ll from ebiosciences). Cells were cultured for 3 d nd llowed to rest for n dditionl 3 d. Polrized T H 1 or T H cells were collected fter 6 d of culture, then were counted nd restimulted for h with plte-ound nti-cd3 (1 µg/ml; 15-C11; BD Biosciences) nd, nd IL- secretion ws mesured y ELISA. The Jurkt humn leukemi T cell line ws cultured in RPMI-16 medium supplemented with 5% (vol/vol) FBS, mm L-glutmine nd ntiiotics. Flow cytometry. PECs from CPS-primed mice t dy 9 were cultured for 6 h in RPMI-16 medium in the presence or sence of live CPS tchyzoites, with the ddition of refeldin A ( µg/ml) during the finl h. Directly conjugted monoclonl ntiodies from ebiosciences were used for surfce stining. After surfce stining, cells were fixed nd mde permele with Cytofix/Cytoperm nd Perm/Wsh (BD Biosciences). For stining of intrcellulr cytokine, llophycocynin-conjugted nti- (XMG1.; BD Biosciences) ws dded to permele cells. For detection of intrcellulr in cocultures of T. gondii infected DCs nd CD + T cells, peritonel CD + T lymphocytes from individul CPS-primed mice t dy 9 were sorted y flow cytometry nd incuted for h with T. gondii infected DCs t rtio of 1:1, with the ddition of refeldin A during the finl h; stining ws done s descried ove. A FACSCliur ws used for flow cytometry of 1 5 cells. Dt were nlyzed with FlowJo softwre (Tree Str). Preprtion of APCs. For preprtion of T. gondii infected DCs, mouse BMDCs from wild-type or Nod / mice were otined s descried 7 nd were infected for 1 h with irrdited T. gondii tchyzoites t dose of four prsites per DC. After incution, free prsites were removed y low-speed centrifugtion for min. For preprtion of OVA-DCs, BMDCs were incuted for h with CpG oligonucleotide (.5 µg/ml; InvivoGen) in the presence or sence of ovlumin ( µg/ml; Worthington). After incution, DCs were wshed three times in complete medi nd were used to stimulte wild-type OT-II or Nod / OT-II trnsgenic CD + T cells. Adoptive trnsfer. Totl splenic CD + T cells from wild-type Thy nd Nod / Thy mice were isolted s descried ove. CD + T cells (5 5 ) were injected intrvenously through the til veins of Tcr / or Nod / Tcr / recipient mice. Reconstituted mice were then chllenged on the sme dy with 1 6 irrdited CPS prsites. PECs t dy 9 were then isolted for nlysis s descried ove. For reconstitution of Rg1 / mice, CD + CD5 CD5RB hi cells purified y flow cytometry were injected intrvenously into Rg1 / recipients. In vitro iochemicl ssys. Purified CD + cells ( 6 ) were stimulted for vrious times in -well pltes (Corning) coted with nti-cd3 (1 µg/ml; BD Biosciences) nd nti-cd8 ( µg/ml; 37.51; BD Biosciences). Nucler nd cytoplsmic frctions from stimulted cells were extrcted with NE-PER regents (Pierce Biotechology). For immunolot nlysis, nucler nd cytoplsmic extrcts (1 µg totl) were proed with ntiodies specific for (sc-71; Snt Cruz Biotechology) nd p65 (33; Cell Signling). Trnsfection nd luciferse ssys. Trnscriptionl ctivity in Jurkt cells ws mesured with the pcd8re AP-1 reporter plsmid (gift from A. Weiss) fter trnsient trnsfection of exponentilly growing cells ( 6 cells/ml in Opti-MEM) with Lipofectmine PLUS (Life Technologies). The totl mount of DNA in ech trnsfection ws kept constnt y use of the corresponding empty expression vectors. After h of incution, RPMI medium contining 5% (vol/vol) FBS ws dded to cells nd the incution ws continued for 16 h to complete trnsfection. Cells were collected nd lysed nd luminescence ws mesured for s in luminometer with the Dul-Luciferse Assy System kit (Promeg). CD8RE AP-1 reporter ctivity ws normlized to the renill luciferse ctivity otined for control smples of ech trnsfection. Sttistics. Prism softwre (GrphPd) ws used to determine the sttisticl significnce of differences in the mens of experimentl groups with unpired, two-tiled Student s t-tests, unless otherwise specified. P vlues of less thn.5 were considered significnt. 7. Lutz, M.B. et l. An dvnced culture method for generting lrge quntities of highly pure dendritic cells from mouse one mrrow. J. Immunol. Methods 3, 77 9 (1999). doi:.38/ni.1816 nture immunology