The same genomic region conditions clonal deletion and clonal deviation to the CD8 and regulatory T cell lineages in NOD versus C57BL/6 mice

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1 The sme genomi region onditions lonl deletion nd lonl devition to the nd regultory T ell lineges in NOD versus C57BL/6 mie Phillip D. Holler, Tetsuy Ymgt, Wenyu Jing, Mrkus Feuerer, Christophe Benoist*, nd Dine Mthis* Setion on Immunology nd Immunogenetis, Joslin Dietes Center; Deprtment of Mediine, Brighm nd Women s Hospitl; Hrvrd Medil Shool, Boston, MA 2215 Contriuted y Christophe Benoist, Ferury 27, 27 (sent for review Ferury 13, 27) Clonl devition is mehnism y whih immture thymoytes expressing self-retive T ell ntigen reeptor (TCR) re resued from lonl deletion y dopting n lterntive differentition pthwy resistnt to poptosis. Here, we onfirm nd generlize previous inditions tht geneti lleles in NOD mie ondition ineffetive lonl devition towrd the linege, peulir popultion of TCR lymphoytes tht eletively olonizes the intrepithelil lymphoyte pool in the gut. Thymi seletion of ells ws very ge-dependent, ourring lmost exlusively in the postntl period. Fewer ells were found in the thymus nd intrepithelil lymphoytes of BDC2.5 TCR trnsgeni mie on the NOD thn on the C57BL/6 (B6) kground; this puity extended to stndrd NOD mie, leit to lesser extent. ells resided in the BDC2.5 pnreti infiltrte, nd they were more undnt on the B6 thn the NOD kground, orrelting with ggressivity of the lesion. A (B6 g7 NOD)F 2 interross in gonist-hllenged BDC2.5 fetl thymi orgn ultures demonstrted the existene of mjor quntittive trit lous on hromosome 3, oinident with n intervl ssoited with resistne to lonl deletion. A replite linkge onfirmed these positions nd showed tht the sme region lso ontrols lonl devition towrd the CD FoxP3 regultory T ell linege. Tht lonl devition towrd the nd regultory T ell pthwys shre geneti ontrol further highlights the similrities etween these two resue lineges, onsistent with n immunoregultory role for ells. utoimmunity FoxP3 tolerne T ell ntigen reeptor (TCR) T ells onstitute peulir supopultion within the T ell linege (1, 2). They re on fide T ells, they express the lssi TCR, nd their glol gene expression profile reltes them quite losely to onventionl T ells (3), with whih they shre severl si funtionlities. On the other hnd, they hve unique loliztion, preferentilly residing in the gut, where they form sizele frtion of intrepithelil lymphoytes (IELs), nd their homodimer, rther thn performing the usul oreeptor role of the heterodimer, seems to serve primrily s lignd for the MHC-like TL moleule (), lthough this intertion is not exlusive nd its funtionl relevne remins onjeturl (5, 6). The developmentl origin of IELs ws initilly deted, with severl erly experiments suggesting tht they my hve n extrthymi origin; it now seems ler, however, tht the thymus is the primry soure of IELs (reviewed in refs. 1 nd 2). Linege tring nd reonstitution experiments in fetl thymi orgn ultures (FTOC) showed tht the immture doulepositive (DP) CD omprtment gives rise to ells (3, 7). Their seletion depends on 2-mirogloulin nd, thus, presumly on MHC lss I-like moleules, ut this is not solely restrited to the lssil K nd D moleules (, 9). The differentition into the linege is indued or enhned y enggement of the TCR on immture thymoytes y strong gonist lignd (3, 1 1). This lonl devition provides resue pthwy for self-retive T ells, euse signls from the TCR re lunted y the sene of n tively signling oreeptor. The shutdown of is lso ompnied y the tivtion of distintive gene-expression progrm, whih inludes F R through whih they signl (15) nd numer of moleules typil of ells of the innte immune system nd of memory ells (3). In this respet, ells re oneptully similr to the FoxP3-dependent supopultion of CD regultory T ells (Tregs) tht lso espe lonl deletion euse of enhned resistne to poptoti signls (16 1). In reent experiments, we used the BDC2.5 TCR trnsgeni (Tg) system to tre the geneti roots of the resistne to lonl deletion in the dietes-prone NOD mouse; to our surprise, we lso oserved tht BDC2.5 NOD FTOC loes gve rise to very few ells in response to gonist peptide (19). Here, we investigted the generlity nd geneti origins of this oservtion nd found tht the sme genomi region tht ontrols lonl deletion lso onditions lonl devition to oth the nd Treg resue lineges. Results Defetive Genertion of Cells on the NOD Geneti Bkground. The BDC2.5 trnsgene enodes TCR with retivity ginst pnreti islet self-ntigen, nd thus onfers strong potentil for utoimmunity (2). Prllel FTOCs were set up with BDC2.5 dy 15 emryos on the NOD nd C57BL/6.H2 g7 kgrounds. These kgrounds shre the seleting H2 g7 hplotype t the MHC ut otherwise entil ll of the genomi differenes etween the NOD nd C57BL/6 (B6) strins. Reproduing our prior oservtions, fr fewer ells were generted in the BDC2.5/NOD ultures thn in the BDC2.5/Bg7 ultures (Fig. 1). [Note tht we hve demonstrted previously tht this differene is not just kineti one (19).] The genertion of ells prlleled the intensity of lonl deletion, refleted s the proportion of lonotype positive DP ells over the rnge of peptide doses, with 3-fold shift to higher vlues with BDC2.5/NOD (Fig. 1). In BDC2.5/NOD loes, ells were omprtively rre, even when the profile ws mthed for the intensity of lonl deletion nd when ssessed y determining their proportion mong single-positive (SP) ells or y determining their totl numer per loe. To determine whether this differene ws unique to ells displying the BDC2.5 TCR, we studied FTOCs from P1 TCR Author ontriutions: P.D.H., T.Y., W.J., M.F., C.B., nd D.M. designed reserh; P.D.H., T.Y., W.J., nd M.F. performed reserh; P.D.H., T.Y., W.J., M.F., C.B., nd D.M. nlyzed dt; nd P.D.H., T.Y., W.J., M.F., C.B., nd D.M. wrote the pper. The uthors delre no onflit of interest. Arevitions: B6, C57BL/6; DP, doule-positive; FTOC, fetl thymi orgn ulture; IEL, intrepithelil lymphoyte; LOD, logrithm of odds; QTL, quntittive trit lous; SP, single-positive; TCR, T ell ntigen reeptor; Tg, trnsgeni; Treg, regultory T ell. *To whom orrespondene should e ddressed. E-mil: dm@joslin.hrvrd.edu. 27 y The Ntionl Ademy of Sienes of the USA IMMUNOLOGY gi doi pns PNAS April 2, 27 vol. 1 no

2 %DP (BDC2.5+) %DP (V 2+) [BDCmi Peptide] (ng/ml) BDC2.5/B6g7 % out of SP (BDC2.5+) % out of SP (V 2+) [BDCmi Peptide] (ng/ml) [KAV9 Peptide] (ng/ml) [KAV9 Peptide] (ng/ml) [KAV9 Peptide] (ng/ml) Tg mie (D -restrited nd retive to the GP33 1 peptide from lymphoyti horiomeningitis virus) rossed onto the NOD-H2 nd B6 kgrounds. FTOCs were hllenged with the GP peptide. Here gin the NOD genome onferred reltive resistne to lonl deletion nd poor seletion of ells (Fig. 1). Thus, the differentil seletion of ells on the NOD nd B6 kgrounds is not quirk of the BDC2.5 TCR. In the HY nd OT-1 TCR Tg systems, the genertion of ells y gonist peptide presented y MHC lss I moleules ws ompnied y the tivtion of prtiulr luster of genes, hrteristi of ells of the innte immune system, in prtiulr NK ells (3). To see whether the ells differentiting in response to the BDC2.5 mimotope presented y MHC lss II moleules were on fide ells, we used quntittive rel-time PCR to mesure the quntities of this set of trnsripts in ells from peptide-hllenged BDC2.5/Bg7 FTOCs, reltive to onventionl T ells tht developed in unmnipulted ultures. All of these trnsripts showed the expeted differentil (Fig. 2), with overexpression rnging from 2- to 2-fold for trnsripts enoding surfe reeptors, suh s NKG2D, NK1.1, or 2B, or the trnsription ftor Id2. Geneti Control on the Popultion in Vivo. To generlize these FTOC findings, we surveyed the tissues of intt BDC2.5 mie on the NOD versus B6.H2 g7 kgrounds. The BDC2.5 BDC2.5/NOD # (x1 3 ) # (x1 3 ) BDC.B6g7 BDC.NOD [BDCmi Peptide] (ng/ml) P1.B6 P1.NOD Fig. 1. re T ells quntittively indued y using gonist peptide in FTOC on the B6 ut not the NOD kground. () BDC2.5-Tg FTOCs on the NOD (Right) nd B6 g7 (Left) geneti kgrounds were inuted with 1 ng/ml mimotope peptide for 7 dys nd nlyzed for expression of nd (gted on BDC2.5 thymoytes). () Asolute numers of DP or SP ells in these ultures re shown ross rnge of gonist peptide onentrtions. () P1-Tg FTOCs on the NOD nd B6 g7 kgrounds were ultured with vrious onentrtions of gonist peptide nd nlyzed s ove (gted on V 2 thymoytes). Dt re representtive of three to five independent experiments. NKG2D DAP12 CD9 CCR5 Id2 FeRg NK1.1 CCL5 2B Reltive expression in αα vs αβ ells Fig. 2. BDC2.5 ells express trnsripts typil of innte immune system ells. RNA ws isolted from nd BDC2.5-Tg thymoytes purified from peptide-supplemented nd peptide-negtive FTOCs, respetively. Seleted trnsripts previously shown to e up-regulted in thymi ells (3) were quntitted y using rel-time PCR. Results re presented s the rtio of trnsript levels (normlized to hypoxnthine phosphoriosyl trnsferse) in versus thymoytes. The dt shown represent one of two experiments. TCR reognizes n ntigen speifilly expressed in pnreti islet -ells, ut erly lonl deletion is deteted in the thymus of neontl BDC2.5 mie (19), suggesting tht the BDC2.5 ntigen is etopilly expressed in thymi strom, s re numer of peripherl-tissue ntigens (21). Indeed, ells represented mjor proportion of CD CD2 lo mture SP ells in the thymus of 1-week-old BDC2.5/Bg7 mie ( % on verge), ut were rre in thymi from their BDC2.5/ NOD ounterprts (.5.% on verge) (Fig. 3 Left). We hve previously shown in the HY TCR Tg system tht the seletion of ells in HY mles ours preferentilly in the neontl period, wning y few weeks fter irth (3). This temporl differene lso ws found in the BDC2.5 TCR Tg system, in whih very few T ells were deteted in dult thymi on either geneti kground (Fig. 3 Right). In most peripherl lymphoid orgns, ells were quite rre, s illustrted in Fig. 3 for pnreti lymph node ells, ut there ws tendeny to higher representtion on the B6 g7 kground. This trend ws onfirmed nd mplified in the IEL popultions from these mie (Fig. 3). ells mde up lrge frtion of the lonotype-positive IELs in BDC2.5/Bg7 mie, ut they were essentilly sent in BDC2.5/NOD mie. Finlly, we looked for ells in the islet infiltrtes of BDC2.5/NOD versus BDC2.5/Bg7 mie. In the BDC2.5 system, reognition of the ell ntigen egins in the pnreti lymph node t 15 dys of ge, nd infiltrtion of the islets ensues quikly. Insulitis is more ggressive on the B6.H2 g7 kground, leding to dietes in 5% of BDC2.5/Bg7 nimls y 6 1 weeks of ge (22). In young mie t the onset of insulitis, ells represented lrge frtion of CD T ells in the islet-infiltrting lymphoytes (Fig. ), n unusul oservtion given tht ells re normlly onfined to the gut. ells were fr fewer in the BDC2.5/NOD pnres (Fig. ). Interestingly, ells deresed in the more stle nd respetful insulitis found t lter times in oth strins, whih very rrely progresses to overt dietes; the NOD versus B6 g7 differene ws still pprent, even fter this derese (Fig. ). Thus, the geneti ontrol tht modultes seletion in emryoni nd neontl thymi lso seems refleted in the omposition of gut- nd pnres-infiltrting ells. The BDC2.5 TCR Tg system highlights the differentition profile of T ells expressing highly self-retive TCR. To test the impt of NOD versus B6 geneti vrition on ells in the ontext of polylonl repertoire, we nlyzed tissues 71 gi doi pns Holler et l.

3 Fig. 3. ells re preferentilly seleted on the B6 versus NOD kgrounds in vivo. () Thymoytes from BDC/Bg7 nd BDC/NOD mie were isolted t 1 week (Left) nd 5 weeks (Right) of ge nd nlyzed y using ytometry (gted on BDC2.5 CD thymoytes). Representtive dt of t lest two thymi per group re shown. () Pnreti lymph node ells from 7- to 9-week-old BDC/Bg7 nd BDC/NOD mie. (Left) The plots re gted on live ells. (Right) The plots re further gted on BDC2.5 B22 CD ells. () IELs were isolted from 9- to 1-week-old BDC/Bg7 nd BDC/NOD mie nd nlyzed y using flow ytometry (gted on BDC2.5 ells). Representtive dt from two experiments re shown. from non-tg NOD nd B6.H2 g7 mie. ells were sent from spleens of oth strins (Fig. ). In gut lymphoytes, higher proportion of ells were oserved mong IELs from B6.H2 g7 thn from NOD mie (35.2 vs. 21.5%, respetively BDC/B6g7 BDC/NOD CD B6g7 NOD Pnres, 3- weeks.%.1% 5% 2.5% Gted on + ells d CD Pnres, weeks % mong TCR IELs 5 13%.5 % B6g7 NOD Fig.. Fewer ells on the NOD geneti kground. ( nd ) Lymphoid ells isolted from the pnreti tissue from BDC2.5/B g7 or BDC2.5/ NOD mie t 3 weeks () nd weeks () of ge. ( Left nd Left) The plots re gted on live ells. ( Right nd Right) The plots re further gted on BDC2.5 B22 CD ells (representtive of two to four experiments). () Splenoytes from 7-week-old NOD nd B6 g7 mie (gted on TCR CD ells, representtive of four individul mie/strins). (d) (Left) IELs from - to 1-week-old B6 g7 nd NOD mie (gted on TCR IELs). (Right) Compiltion of the perentge of IELs mong TCR IELs in B6 g7 (n 7) versus NOD (n 7) mie. on verge; P.2) (Fig. d). Thus the differentil propensity for B6 nd NOD mie to selet ells when triggered y the strongly self-retive BDC2.5 TCR is lso mnifest, leit in more muted fshion, in norml polylonl repertoires. Geneti Mpping of Clonl Devition. Beuse the geneti differenes in lonl devition to the linege oserved in FTOC proved to hve strong orrelte in vivo, it ws of interest to mp the lotion of the lous (or loi) tht underlie(s) this differene. FTOCs were performed with loes from prentl, F 1, nd F 2 interrosses etween BDC2.5/NOD nd BDC2.5/Bg7 mie, ll supplemented with low dose of peptide to indue ells. The primry quntittive trits ssessed were the extent of lonl deletion (est refleted s the perentge of DP ells) (19) nd of lonl devition (est refleted s the perentge of within lonotype-positive SP ells). Loes from F 1 nd F 2 emryos showed n intermedite phenotype, skewed towrd the NOD phenotype (Fig. 5). The rnge of phenotypes in the F 2 offspring suggested multigeni ontrol with dominne of the NOD llele(s). A whole-genome sn ws performed with 12 SNP mrkers, sped t n verge of 19. megses (M); dt were proessed y using simple mrker/trit ssoition in R/qtl (23). A mjor quntittive trit lous (QTL) ws found on hromosome 3, entered t 5 M, with strongly signifint logrithm of odds (LOD) sore of 6.67 (permuttion nlysis showed threshold LOD sore of 3.5 for genomi-wise orreted signifine of P.5) (Fig. 5). A few weker QTLs were oserved in the suggestive rnge, not quite rehing full sttistil signifine, in prtiulr on distl hromosome 1. Interestingly, these were the regions tht showed the strongest ssoition with resistne to lonl deletion in our previous nlysis (19), nd strong QTL for lonl deletion (ssoition with the perentge of DP) lso ws found in the sme region in the present ohort. Not surprisingly, the twoprmeter plot of Fig. 5 revels strong orreltion etween these two metris mong the F 2 mie. Thus, the QTLs for resistne to lonl deletion nd for ineffetive lonl devition to the linege re very tightly linked, if not identil. These QTLs mpped quite losely to the position of the idd3 lous, n importnt omponent of suseptiility to utoimmune dietes in the NOD mouse, one omponent of whih is loted etween 36.3 nd 37.2 M on hromosome 3 (2, 25). We tested whether the idd3 region might ontin the lonl deletion/ devition QTL y rossing the BDC2.5 trnsgene onto the NOD.B6idd3R5 line nd nlyzing FTOC ultures prepred IMMUNOLOGY Holler et l. PNAS April 2, 27 vol. 1 no

4 % mong SP 6 2 B6g7 NOD F1 F2 LOD sore 6 2 %D P n=91 %C D LOD=3.5 p=.5 Chromosome %DP % mong SP BDC2.5/NOD BDC2.5/NOD.idd3 BDC2.5/B6g7 d % DP Fig. 5. The sme geneti regions ondition lonl deletion nd lonl devition to.() BDC2.5-Tg FTOCs on the B6 g7, NOD, nd F 1 or F 2 interrosses were inuted with 1 ng/ml peptide for 7 dys nd nlyzed y using flow ytometry. Mens re indited y horizontl r. () LOD plots for the F 2 ohort with the perentge of DP nd ells out of the totl numer of SP ells s quntittive trits (n 91). () Two-prmeter stter plot of the perentge of DP nd ells (of the totl numer of SP ells) for the F 2 ohort. (d) Comprison of BDC2.5-Tg FTOCs on the NOD, NOD.Idd3R5, nd B6 g7 kgrounds (supplemented with 1 ng/ml mimotope peptide). The two-prmeter stter plots show the perentge of DP (inditing lonl deletion) nd the perentge of ells mong SP ells (lonl devition). from suh mie. As illustrted in Fig. 5d, loes from NOD.idd3 mie were s resistnt s those from NOD mie, inditing tht the lonl deletion/devition QTL mps outside of the M intervl nd is distint from the most tightly defined idd3 region, lthough it might orrespond to other idd3 suloi. As disussed ove, the Treg supopultion of CD ells onstitutes, s do ells, resue linege for self-retive thymoytes. Whether Treg ell differentition is tully indued y self-reognition, s suggested in some systems (17, 26), or simply results from stronger resistne to lonl deletion (1, 27) my well e funtion of the prtiulr TCR nd its ffinity/vidity for self-mhc/peptide lignds. We hve reently reported tht the indution of Treg ells is lso under strong geneti ontrol, with ler differene in responsiveness to indution y ognte peptide on the B6 nd NOD kgrounds (M.F., unpulished oservtions). We sked whether the sme regions might ontrol diversion into the nd Treg pthwys, employing seond ohort of FTOCs from BDC2.5/ (B6 g7 NOD) F 2 emryos. This time, the output of nd of FoxP3 Treg ells were oth reorded for the sme thymi loes in response to fixed dose of peptide. As expeted, tight orreltion ws gin oserved etween the degree of lonl deletion (inversely proportionl to the perentge of DP) nd the lonl devition towrd ells (Fig. 6 Left). This orreltion lso extended to the proportion of FoxP3 Treg ells mong CD SP ells (Fig. 6 Center nd Right). The wholegenome SNP sn performed on these mie gin rought up the min QTL on hromosome 3, essentilly oinident for ll three prmeters (Fig. 6). As might e expeted from the dt in Fig. 5d, the min pek is telomeri to the idd3 region ut does % % DP % FoxP3+ Tregs % DP % FoxP3+ Tregs % LOD Foxp3+CD25+ / CDSP DP Idd3 Idd17 Idd1 Chromosome Chr3 mp position (M) Fig. 6. The sme geneti regions ondition lonl devition to nd Treg ells. () BDC2.5-Tg F 2 FTOCs (n 56) were inuted with 1 ng/ml mimotope peptide for 7 dys nd nlyzed y using flow ytometry for the perentge of (of totl SP ells), perentge of DP ells, nd perentge of FoxP3 ells (of totl CD SP ells). () LOD plot for the whole-genome sn in this F 2 ohort, with the perentge of ells mong SP ells, the perentge of DP ells, nd the perentge of FoxP3 mong CD SP ells s quntittive trits. () Higher resolution view of the hromosome 3 region for the sme sn s gi doi pns Holler et l.

5 enompss idd1 nd idd17. Thus, the differentil effiy of poptosis indution, genertion, nd Treg indution enoded in the B6 nd NOD genomes ll mp to the sme hromosome nd region thereof. Disussion The retive potentil nd tul funtion of ells remins ontroversil (1, 2). Whether IELs mount protetive responses to gut ntigens or re primrily immunomodultory is n open question. They do possess strong ytotoxi tivity, suggesting tive effetor funtion (2, 29), ut two reports indite tht they my lso hve regultory roles, t lest in olitis models (3, 31). The present results pper to weigh in fvor of the ltter funtion euse they show oth ells nd FoxP3 Treg ells to e resue lineges tht shre n importnt geneti ontrol element. On the other hnd, ells re not Treg look-likes euse they do not express FoxP3, nor do they overexpress mny of the genes of the well hrterized Treg signture (dt not shown). The origin of T ells ws long deted, ut the onsensus is tht they re of thymi origin (1, 2). Reggregtion himer experiments (3) showed tht ells originte from immture DP when triggered y ognte ntigen presented y MHC lss I moleules. Our results onfirm tht seletion to the linege is lrgely independent of the restriting moleule nd n e indued y ny strong gonist signl, irrespetive of MHC lss restrition. There is n interesting temporl spet to the seletion of ells. Our previous work (19) determined tht their genertion in the mle HY TCR Tg system ourred predominntly in the fetl/neontl stges nd wned rpidly therefter. The sme kineti ehvior ws oserved with the BDC2.5 system. Beuse key elements distinguish the HY nd BDC2.5 systems (restrited y MHC lss I versus MHC lss II moleules nd y uiquitous versus tissue-speifi ntigen), it is resonle to speulte tht this temporl vrition my e generl phenomenon: ells re seleted mong the utoretive T ells present in the erly stges of life, nd this wve of resued ells olonizes the gut. This view is onsistent with the oservtion tht thymetomy ffets the IEL pool only when performed in the neontl period (32). The origin of this temporl differene is fully onjeturl t this point: Different modes of ntigen presenttion in the perintl thymus, differentil tivity of ostimultory pthwys, or different responsiveness of perintl thymoytes ould ll e involved. The ge-dependene of seletion does not mirror tht of Treg ells, however, euse the ltter tully inrese in frequeny with the ge of the individul (ref. 33 nd our unpulished dt). Cells nd Dietes. Do ells ontriute to utoimmune dietes, nd is their reltive puity omponent of suseptiility in NOD mie? The very presene of ells in the lymphoyti infiltrte of BDC2.5 mie ws quite surprising nd suggestive euse they were thought to e exlusive to IEL popultions. It is quite striking tht ells re more undnt in the ggressive infiltrte, erly in the ourse of disese, thn in the more estlished nd stle insuliti lesion fter weeks of ge. This oservtion might e tken s n indition of pthogeni role for ells, ut ounter interprettion in whih they ply dmpening role in the islets is eqully vlid. Their reltive puity in dietes-prone NOD mie my lso e tken s n rgument for protetive role, ut suh lues n e misleding: There is ler preedent in the prdoxil pthogeni role of NK ells, whih re underrepresented in NOD mie, llowing the nimls to survive their utoimmune potentil, t lest until reeding ge (3). Whether nd how ells re plyers in dietes pthogenesis ertinly wrrnts further explortion. Wht Is the Lous (Loi)? The sme geneti region ppers to ondition the defetive hndling of T ells displying strongly self-retive TCRs y using three different modes of tolerne indution. At the level of resolution fforded y the present geneti nlysis, one nnot stte tht the very sme gene is involved in ll three defets, ut this does mke n ttrtive hypothesis. For instne, signling defet downstrem of the TCR in NOD thymoytes might result in glolly weker signls nd thus dmpen the tivtion of poptosis nd of lterntive modes of differentition. The NOD phenotype ppers mostly dominnt in F 1 mie, whih might suggest tht the NOD llele enodes n overtive inhiitor, suh s n inhiitory phosphtse. These defets mp rodly to the idd3 idd17 region nd my thus orrespond to QTL for generl suseptiility to dietes ut not to the idd3 suregion entered round the Il2 nd Il21 genes. This oinidentl mpping supports the notion tht the seletion defets do prtke in the suseptiility to utoimmune dietes in NOD mie. Whtever the nture of the lous (or loi) turns out to e, the present results indite tht the T ell repertoire in NOD mie must hve peulirly ised omposition. Cells whose retivity to self would destine them to deletion or to lonl devition into hrmless Treg or phenotypes on the B6 g7 kground re llowed to mture nd populte lymphoid omprtments in the NOD mouse. It is oneivle tht some dpttion or tuning tkes ple nd tht the geneti vrition tht ffets thymoyte mturtion lso influenes signls in peripherl T ells nd thus prevents or inhiits their tivtion. Nevertheless, dngerous sitution is reted, one tht my promote the emergene of fully utoretive ells should this dmper eome intivted. One ould drw here prllel etween NOD mie nd the surfy mutnt. The FoxP3 defiieny in the ltter prevents the sequestrtion of utoretive TCRs into the Treg omprtment, nd these TCRs insted pper on stndrd T ells, where they generte unontrolled lymphoprolifertion nd utoimmunity (35). In summry, we show tht the defetive tolerne indution in the NOD thymus enompsses three distint spets of the dpttion of the T ell repertoire to self under the ontrol of the sme geneti region if not of the sme gene. It will e importnt to further explore the impt of this shift in repertoires. The prtil defiit of ells in the NOD mouse dds nother potentil plyer in the st of hrters tht medite utoimmune dietes. Mterils nd Methods Mie. NOD/LtDOI (NOD), C57BL/6.H2 g7 (B6 g7 ), nd BDC2.5 TCR Tg mie (2) on the NOD nd B6.H2 g7 kgrounds (BDC2.5/NOD nd BDC2.5/Bg7, respetively) were red in our speifi-pthogen-free fility t the Joslin Dietes Center. F 2 emryos were generted y interrossing F 1 mie (BDC2.5 Tg non-tg) nd genotyping for the BDC2.5 trnsgene nd H2 g7 hplotype (19). NOD.Idd3R5 ongeni mie (Toni Frms, Germntown, NY) were interrossed with BDC2.5/NOD mie, genotyped t the Idd3 intervl with D3Mit21. P1 TCR Tg mie on the NOD nd B6 kgrounds were from The Jkson Lortory (Br Hror, ME). FTOCs. Fetl thymus loes were disseted from emryoni-dy emryos nd ultured s desried (19). When indited, they were supplemented with BDC2.5-speifi peptide mimotope (BDCmi, peptide 1-63) (36) or P1-speifi peptide, p33 (KAVYNFATM) (37). F 2 FTOCs were rried out in the presene of 1 ng/ml BDCmi peptide. Medium nd peptide were hnged every other dy. For most nlyses of BDC2.5 ells, thymoytes were stined with nti-bdc2.5 lonotype (3), IMMUNOLOGY Holler et l. PNAS April 2, 27 vol. 1 no

6 nti-cd, nti-, nd nti- [H , whih reognizes oth the NOD (.1) nd B6 (.2) lleli forms of ] (BD Phrmingen, Sn Diego, CA). Anlysis of Lymphoid Popultions. Single-ell suspensions of lymph node, spleen, or pnres were generted y using mehnil disruption with frosted glss slides. For IELs, the smll intestine ws ut into smll piees ( 5 mm) nd extensively wshed in old PBS; then IEL ws extrted y using shking in 1 Hnks s lned slt solution/1% FCS/1 mm EDTA t 37 C for 2 min (three repets). Relesed IEL were purified y using Peroll grdient entrifugtion (6 g) t the interphse etween 67% nd % frtions. V CD or thymoytes were sorted from peptide-supplemented or ontrol FTOCs, RNA ws isolted y using TRIzol, nd DNAs were quntitted y using SYBR Green quntittive rel-time PCR s desried (3). Geneti Mpping. DNA ws isolted from BDC2.5 (B6 g7 NOD) F 2 emryos nd ws genotyped for SNP mrkers distinguishing B6 nd NOD lleles y using fluorogeni PCR. SNP mrkers overed ll 19 utosomes nd Idd loi with n verge sping of 19. M (see full list of primer/proe sets nd genomi lotions in ref. 19). Anlyses used vrious mpping nd sttistil pkges (S, Mpmker/Exp, Mpmker/QTL, nd R/qtl) (39). Dt were heked for genotyping errors y using R/qtl. Linkge peks were identified y using simple mrker regression (S ) or intervl mpping (R/qtl), with normlized quntittive trits (divided y the experimentl men to ontrol for etweenexperiment vriility). Empiril P vlues for experiments were estlished y using permuttion tests (1, permuttions, R/qtl). Tests for dominnt/dditive/reessive effets nd lultions of vrine explined were performed y using Mpmker/QTL nd S. We thnk E. Hytt nd K. Httori for ssistne with mie, J. LVehio nd G. Buruzl for ytometry, nd W. Besse nd C. Cmpell for SNP genotyping. This work ws supported y Juvenile Dietes Reserh Foundtion Grnt -2-36, Ntionl Institutes of Helth Grnt DK627, Young Chir funds, nd the Joslin Dietes Center s Ntionl Institute of Dietes nd Digestive nd Kidney Diseses/Dietes nd Endorinology Reserh Center ore filities. P.D.H., T.Y., W.J., nd M.F. reeived postdotorl fellowships from the Cner Reserh Institute, from the Uehr Memoril Foundtion nd the Io Foundtion, from the Amerin Dietes Assoition ( mentor-sed fellowship), nd from the Germn Reserh Foundtion (Emmy Noether Fellowship FE 1/1-1), respetively. 1. 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