ANTIGEN PRESENTATION BY CHEMICALLY MODIFIED SPLENOCYTES INDUCES ANTIGEN-SPECIFIC T CELL UNRESPONSIVENESS IN VITRO AND IN VIVO

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1 ANTIGEN PRESENTATION BY CHEMICALLY MODIFIED SPLENOCYTES INDUCES ANTIGEN-SPECIFIC T CELL UNRESPONSIVENESS IN VITRO AND IN VIVO BY MARC K. JENKINS AND RONALD H. SCHWARTZ From the Lbortory of Immunology, Ntionl Institute of Allergy nd Infectious Diseses, Ntionl Institutes of Helth, Bethesd, Mrylnd Despite lrge body of evidence concerning the phenomenon of immune tolernce t the T cell level, the ctul mechnism of unresponsiveness is not understood. Experimentlly, T cell unresponsiveness cn be induced in dults by the intrvenous injection of high doses of soluble ntigen (1) or of ntigen coupled to syngeneic splenocytes (reviewed in references 2-6). Two mjor hypotheses hve been proposed (3-6) to explin unresponsiveness : regultion by suppressor T cells or direct inctivtion of responding T cells by ntigen. The suppression model of unresponsiveness sttes tht complex circuit of intercting suppressor T cells prevent the expression of inducer T cell function. The ntigen nd MHC specificity of suppression hs been shown in most systems to be different from tht of inducer T cells, e. g., inducer T cells recognize ntigen in ssocition with clss 11 MHC (I) molecules, while suppressor T cells often recognize ntigen in n unrestricted fshion, or in ssocition with 1-J molecules (2, 7, 8). However, severl instnces of l-restricted ntigen recognition by suppressor T cells hve been described (9, 10). As n lterntive to suppression, models of direct T cell inctivtion (clonl deletion) stte tht under certin conditions inducer T cells of the pproprite specificity re functionlly or physiclly deleted fter interction with ntigen nd l molecules (3-6). Attempts to distinguish between suppression nd clonl deletion models hve been gretly hmpered by the lck of in vitro model systems with which to study the inductive events leding to unresponsiveness. Lmb nd coworkers (11) hve reported tht clss I1-restricted humn T cell clones could be rendered unresponsive in vitro fter incubtion with free ntigen. Although this result suggested tht tolernce induction nd T cell ctivtion hd different specificities, subsequent studies by these investigtors (12) showed tht unresponsiveness induced in vitro could be blocked by nti-clss 11 ntibodies. The reltionship of these in vitro observtions to the in vivo phenomenon of T cell tolernce induction remined uncler, To ddress these issues, we exmined the specificity of tolernce induction both in vivo nd in vitro using the well-defined response of B 10.A mice to pigeon cytochrome c s model system. Previous studies (13) hve shown tht splenocytes coupled with peptide ntigens vi the chemicl crosslinker I-ethyl-3-(3-dimethyl- M. K. Jenkins is recipient of n Arthritis Foundtion postdoctorl fellowship. 302 Journl of Experimentl Medicine - Volume 165, Februry

2 JENKINS AND SCHWARTZ 303 minopropyl) crbodiimide (ECDI)' re tolerogenic in vivo. Our results show tht when ECDI-treted splenocytes were used in vitro s APCs, they filed to stimulte prolifertion by pigeon cytochrome c-specific norml T cell clones nd insted induced stte oflong-term unresponsiveness tht could not be ttributed to suppressor T cells. The induction of T cell unresponsiveness in vitro hd the sme ntigen nd l molecule specificity s T cell ctivtion. Furthermore, s predicted by the in vitro results, T cell unresponsiveness ws induced in vivo by the intrvenous injection of ntigen-coupled tretment. splenocytes prepred by ECDI The induction of this in vivo unresponsiveness lso hd the sme ntigen nd l molecule specificity s T cell ctivtion. The experiments suggested tht ECDI tretment lone inctivted n APC function, converting the splenocytes to cells tht displyed ntigen nd l molecules, but tht induced T cell tolernce insted of prolifertion. These results re most consistent with functionl clonl deletion model of T cell tolernce induction nd suggest tht ntigen/l molecule recognition under nonmitogenic conditions cn result in induction of n unresponsive stte. Mterils nd Methods Mice. B10.A/SgSn mice were purchsed from The Jckson Lbortory, Br Hrbor, ME. B10.A(4R)/SgSn, B10.A(2R)/SgSn, B10.A(3R), B10.A(5R), B10.A(18R), nd C57BL/IOSn (1110) mice were bred in our own colony from Cesrin-derived litters of breeding pirs obtined from Dr. Jck Stimpfling, McLughlin Reserch Lbortory, Gret Flls, MT. Mice were sex mtched within experiments nd used between 2-12 mo of ge. Antigens. Pigeon cytochrome c ws purchsed from Sigm Chemicl Co., St. Louis, MO. Duck cytochrome c, prepred s described (14), ws the kind gift of Dr. E. Mrgolish, Northwestern University, Evnston, IL. Pigeon cytochrome c frgments 1-65, 66-80, nd were prepred by cynogen bromide clevge nd were purified on Sephdex G50 superfine column (Phrmci Fine Chemicls, Pisctwy, NJ) (15). An nlog of moth cytochrome c frgment contining glutmine for lysine substitution t position 99 nd glutmic cid for sprtic cid substitution t position 93, moth (93E,99Q), ws synthesized by Dr. Brbr Fox (Ntionl Institute of Allergy nd Infectious Diseses, Bethesd, MD) using the Merrifield solid-phse procedure s previously described (16). Another nlog of moth cytochrome c contining glutmic cid for sprtic cid substitution t position 93 nd n lnine for leucine substitution t position 98, moth 86-89;93-103(93E,98A) 2 ws prepred by the Merrifield solid-phse procedure nd ws kindly provided by Dr. B. Singh (Dept. of Immunology, University of Albert, Cnd). PPD ws purchsed from Connught Lbortories Ltd., Willowsdle, Ontrio, Cnd.GAT ws purchsed from Veg Biotechnologies, Inc., Tucson, AZ. Preprtion ofecdi-treted Splenocytes. Erythrocyte-free splenocytes were treted with ECDI s previously described (13). Briefly, 10 8 spleen cells were incubted for 1 h on ice in 0.44 ml of 0.9% NCl contining 75 mm ECDI (Clbiochem-Behring Corp., L Joll, CA). The cells were wshed extensively in serum-free RPMI 1640 medium to stop the coupling rection. In some experiments, splenocytes were frctionted into T cellenriched or B cell nd mcrophge-enriched popultions before ECDI tretment. T cellenriched popultions were obtined by treting nylon-wool nondherent (17) B10.A splenocytes with (nti-e'# :E',, reference 18) nd (nti-as :A', ; reference 19) mabs (1 :1000 scites) for 1 h on ice, followed by 30-min incubtion in 1 :4 dilution of guine pig complement (Gibco Lbortories, Grnd Islnd, NY) t 37 C. These cells ' Abbrevition used in this pper: ECDI, 1-ethyl-3-(3-dimethylminopropyl) crbodiimide. 2 Fox, B. S., C. Chen, E. Frg, C. French, B. Singh, nd R. H. Schwrtz Evlution of the trimoleculr complex model of T cell recognition. Mnuscript in preprtion.

3 30 4 T CELL UNRESPONSIVENESS IN VITRO AND IN VIVO responded well to Con A in the presence of irrdited splenocytes but filed to respond to LPS (dt not shown). B cell plus mcrophge-enriched popultions were obtined by treting BIO.A splenocytes with (nti-thy-1, reference 20), (nti-ly-1, reference 21), (nti-lyt-2, reference 21), nd MAR 18.5 (mouse nti-rt Ig, reference 22) mabs (1 :10 culture superntnts) plus complement. These cells filed to respond to Con A but responded well to LPS (dt not shown). For the in vivo experiments, peptide ntigens were directly coupled to BIO.A, BI O.A(3R), BIO.A(5R), BIO.A(18R), or BI O.A(4R) splenocytes s described bove except tht the rection mixture contined 400,M ntigen nd 75 mm ECDI. Where indicted, BIO.A splenocytes were depleted of cells bering l molecules s described bove for preprtion of T cell-enriched popultions, just before ntigen coupling. Trce lbeling studies indicted tht 0.5 ug of pigeon cytochrome c ws coupled to 10' splenocytes (dt not shown). T Cell Clones. Norml T cell clones A.E7 (23) nd F1.A.2 (24) were derived from pigeon cytochrome c-immunized BIO.A nd (B10.A(3R) X B10.A)F, mice, respectively. Norml T cell clone 3R.3.11 (25) ws derived from BIO.A(3R) mice immunized with pigeon cytochrome c frgment All three norml clones express the L3T4 +, Lyt-2 - surfce phenotype. T cell clones were routinely mintined by modifiction of the Kimoto nd Fthmn (26) rest-stimultion protocol s described by Ashwell et l. (27). For the in vitro tolernce experiments, T cell clones were mintined by weekly ddition of only IL-2-contining superntnts (25-50 U/ml) for 1 mo fter the lst stimultion with ntigen nd irrdited splenocytes (3,000 rd). The few surviving splenocytes were then depleted by nti-l molecule plus complement tretment : nd S for A.E7 nd F1.A.2, or M5/114 (nti-as :A',, reference 28) for 3R Ded cells were removed on Ficoll-Hypque density grdients (Phrmci Fine Chemicls). After this tretment, T cell clones filed to respond to high doses of ntigen plus llogeneic splenocytes, indicting complete depletion of syngeneic APC. T Cell Clone Prolifertion Assy. Cloned T cells (2 X 10') were cultured in 0.2 ml of Click's medium supplemented with 10% het-inctivted FCS, 2 X 10-6 M glutmine, 5 X 10-5 M 2-ME, nd ntibiotics in 96-well microtiter pltes (Costr No ; Costr, Cmbridge, MA) with 5 X 10 5 irrdited (3,000 rd) syngeneic splenocytes nd vrying doses of ntigen or with IL-2 lone (100 U/ml). Where indicted, cultures contined 5 X 105 ECDI-treted splenocytes s APC. After 2 d of culture, 1 uci of [ sh]thymidine (6.7 mci/mm, New Englnd Nucler, Boston, MA) ws dded to ech well. 16 h lter, the cells were hrvested using PHD cell hrvester (Cmbridge Technology, Inc., Cmbridge, MA). Thymidine incorportion into DNA ws quntitted by liquid scintilltion counting. Determintions were performed in duplicte with the results expressed s A cpm, i.e., (men cpm in ntigen stimulted wells) - (men cpm in wells without ntigen). Bckground prolifertion (no ntigen) ws routinely <1,000 cpm. Induction of T Cell Clone Unresponsiveness In Vitro. T cell clones (5 X 10 5) were preincubted overnight in Costr 24-well pltes (No. 3524) in 2.0 ml of medium contining 5 X 106 ECDI-treted splenocytes with or without soluble ntigen. In some experiments, purified S or mabs (I wg/ml, kindly provided by Dr. A. Kruisbeek, NIH) were dded to the preincubtion cultures. The cloned T cells were recovered the next dy on Ficoll-Hypque density grdients, wshed extensively in RPMI 1640, nd restimulted (2 X 10') with fresh, irrdited splenocytes (5 X 10 5) nd ntigen in 96-well microtiter pltes, s described bove for the T cell clone prolifertion ssy. Recovery from preincubtion cultures ws routinely 60-80% of the input cell number. T cell clones preincubted in the bsence of ntigen with ECDI-treted or norml splenocytes responded similrly to restimultion, suggesting tht the ECDI-treted cells were not toxic (dt not shown). Kinetics nd Durtion of T Cell Clone Unresponsiveness Induced In Vitro. For the experiments shown in Fig. 6, A.E7 cells (5 X 105) were incubted with 5 X 106 ECDItreted BIO.A splenocytes in 2.0 ml medium with or without 5 UM pigeon frgment for 2, 5, or 16 h. At the indicted times, A.E7 cells were seprted from the treted APC nd ntigen on density grdients, wshed extensively, nd restimulted with norml APC nd ntigen s described bove. For the experiments shown in Fig. 7, F1.A.2 cells

4 JENKINS AND SCHWARTZ ,000 60,000 20, , nm FIGURE 1. ECDI-treted splenocytes fil to stimulte prolifertion by norml T cell clone. 5 x 10 5 ECDI-treted (open circles) or 3,000 rd irrdited norml (filled circles) B10.A splenocytes were cultured with 2 X 10 4 F1.A.2 norml cloned T cells nd the indicted doses of pigeon frgment Prolifertion by F1.A.2 ws determined by ['Hlthymidine incorportion with the results expressed s 0 cpm. (5 X 10) were incubted overnight with 5 X 106 ECDI-treted B10.A splenocytes in 2.0 ml medium with or without 1 gm pigeon frgment The T cells were recovered, wshed, nd 2 X 10 4 cell were plced in 0.1 ml medium in 96-well microtiter pltes. At lter times, 5 X 105 irrdited B10.A splenocytes nd vrious doses of pigeon frgment or IL-2 lone were dded in 0.1 ml of medium. Restimultion with norml APC nd ntigen ws determined s described bove. Induction of T Cell Unresponsiveness In Vivo. Mice were injected intrvenously with 0.5 ml RPM contining either 2 X 10' or 5 X 10' ntigen- or shm-coupled splenocytes. 1 or 4 dys fter injection, recipient mice were immunized subcutneously in ech footpd nd t the bse of the til with totl of 10 nmol (in 0.2 ml) of pigeon cytochrome c or moth 86-89;93-103(93E,98A) emulsified in CFA contining Mycobcterium tuberculosis H37R (Difco Lbortories Inc., Detroit, MI). Lymph Node T Cell Prolifertion Assy. 7 d fter priming, drining lymph node T cells were prepred by pssge over nylon-wool columns (17). T cells (4 X 105 cells/well) were cultured in 96-well microtiter pltes with vrying doses of pigeon cytochrome c frgment , moth ; (93E,98A), or PPD, nd 105 irrdited (3,000 rd) syngeneic spleen cells in 0.2 ml of medium. Cultures were pulsed with ['Hlthymidine on dy 3, hrvested on dy 4, nd thymidine incorportion ws determined s described bove. Determintions were performed in triplicte nd the results re expressed s A cpm ± SEM clculted s the squre root of the sum of the squres of the individul errors. Results Antigen Presenttion by ECDI-treted Splenocytes to T Cell Clones In Vitro. Previous nlyses (13) hve reveled tht specific T cell unresponsiveness cn be induced in vivo by the intrvenous injection of syngeneic splenocytes coupled with protein ntigens by the chemicl crosslinker ECDI. To gin better understnding of this unresponsiveness, we exmined the ntigen presenttion cpcity of ECDI-treted splenocytes in vitro. Norml B10.A T cell clones proliferte in response to the COOH-terminl cynogen bromide frgment of pigeon cytochrome c (residues ) in ssocition with the E#:E l molecule expressed on APC (29). However, clone with this specificity (F1.A.2) filed to proliferte in response to B10.A splenocytes coupled with pigeon frgment by ECDI (dt not shown), or to ECDI-treted B10.A splenocytes nd soluble pigeon frgment (Fig. 1). In contrst, norml B10.A splenocytes nd pigeon frgment stimulted vigorous prolifertive response. Severl observtions suggested tht the inbility of ECDI-treted APC to

5 30 6 T CELL UNRESPONSIVENESS IN VITRO AND IN VIVO 120, , ], AM FIGURE 2. Induction of T cell unresponsiveness in vitro. Cloned T cell A.E7 (5 X 10 5 ) ws incubted overnight with vrious ECDI-treted BI O.A spleen cell popultions with or without pigeon frgment T cell-enriched nd B cell plus mcrophge-enriched popultions were prepred s described in Mterils nd Methods. The A.E7 cells were reisolted the next dy nd cultured (2 X 10") with the indicted ntigen doses nd 5 X 10 5 irrdited BIO.A spleen cells or IL-2-contining superntnts (100 U/ml) lone. Prolifertion of restimulted cells ws mesured by ['H)thymidine incorportion nd the results re expressed s 0 cpm. A.E7 preincubtion conditions : ECDI-treted B10.A unfrctionted splenocytes with (open circles) or without (filled circles) 5 jum pigeon frgment , or ECDI-treted B10.A T cells plus 5 ~um pigeon frgment (open tringles) or ECDI-treted BIO.A B cells nd mcrophges plus 5 jum frgment (open squres). stimulte prolifertion ws probbly not the result of extensive l molecule modifiction. First, ECDI-treted splenocytes hd l-restricted effects on norml T cell clones tht could be blocked with nti-l mabs (see below). Second, ECDItreted APC expressed reltively norml levels of l molecules, s detected by mabs (dt not shown). Finlly, ECDI-treted APC could wekly stimulte IL-2 production by the 2134 T cell hybridom (dt not shown) ; this cell requires only pigeon frgment nd Efl:E', molecules for ctivtion, s demonstrted by studies using purified l molecules incorported into plnr membrnes (29). These observtions re most consistent with the interprettion tht ECDI tretment inctivted n ccessory function of the APC tht is necessry to stimulte prolifertion by norml T cell clone. The Induction of T Cell Unresponsiveness In Vitro. The observtion tht ECDItreted APC expressed functionl l molecules nd yet filed to stimulte norml T cell clones to proliferte rised the possibility tht recognition of ntigen on ECDI-treted APC resulted in unresponsiveness. To test this, the norml T cell clone A.E7 (specific for pigeon frgment nd Ek :Eb) ws preincubted overnight with vrious ECDI-treted B10.A spleen cell popultions, with or without ntigen, nd then ws restimulted with ntigen nd untreted syngeneic APC (Fig. 2). A.E7 cells preincubted with pigeon frgment nd either ECDI-treted B10.A unfrctionted splenocytes or ECDI-treted B10.A B cells plus mcrophges were lmost completely unresponsive to restimultion with untreted B10.A splenocytes nd pigeon frgment These cells responded well to exogenous IL-2, indicting tht cell deth did not ccount for ntigen/l molecule unresponsiveness. In contrst, A.E7 cells preincubted with ECDItreted B l O.A splenocytes in the bsence of ntigen or with ECDI-treted Bl O.A T cells plus pigeon frgment responded well to restimultion with untreted APC nd ntigen. These results demonstrted tht ntigen presenttion in vitro by ECDI-treted splenocytes results in T cell unresponsiveness. In

6 JENKINS AND SCHWARTZ ,000 80,000 60,000 o. U 40,000-20,000-80,000 60,000 40, , T IL IL-2 [ , FM [1-651, 9M FIGURE 3. MHC specificity o T cell unresponsiveness induced in vitro. Cloned T cells (5 X 10 5 ) A.E7 () or 3R.3.11 (b) were incubted overnight with 5 X 106 ECDI-treted splenocytes with or without ntigen. The T cells were reisolted nd recultured (2 X 10 4 ) with the indicted ntigens, nd 5 X 105 B10.A (for A.E7) or B10 (for 3R.3.11) irrdited spleen cells or IL-2- contining superntnts (100 U/ml) lone. Prolifertion of restimulted cells ws mesured by [sh]thymidine incorportion. A.E7 preincubtion conditions : ECDI-treted B10.A splenocytes with (open circles) or without (filled circles) 0.5 um pigeon frgment , or ECDI-treted B10 splenocytes with (open squres) or without pigeon frgment (filled squres). (b) 3R.3.11 preincubtion conditions : ECDI-treted B10 splenocytes with (open squres) or without pigeon frgment 1-65 (filled squres), or ECDI-treted B10.A splenocytes with (open circles) or without pigeon frgment 1-65 (filled circles). ddition, the criticl popultion in spleen responsible for the induction of unresponsiveness ws ECDI-treted l' cells (B cells plus mcrophges) nd not ECDI-treted T cells, strongly rguing ginst role for suppressor T cells in this phenomenon. Specificity of the Induction of T Cell Unresponsiveness In Vitro. It hs been demonstrted (8, 29) tht inducer T cell ctivtion requires recognition of ntigen in ssocition with MHC molecules. The experiments shown in Fig. 3 were performed to determine if similr restriction governed the induction of T cell unresponsiveness in vitro. A.E7 cells (specific for frgment nd E'3:E'.) preincubted with ECDI-treted B10.A splenocytes nd pigeon frgment were lmost completely unresponsive to restimultion with norml B10.A splenocytes nd pigeon frgment (Fig. 3). Unresponsiveness ws not induced fter preincubtion with ECDI-treted BIO.A splenocytes lone or with ECDI-treted B10 splenocytes bering A#:A, clss li molecules with or without pigeon frgment In the reciprocl experiment, clone 3R.3.11 (specific for pigeon frgment 1-65 nd A'O:A'.) ws rendered unresponsive to restimultion with B10 splenocytes nd pigeon frgment 1-65 by preincubtion with ECDItreted B10 splenocytes nd frgment 1-65, but not ECDI-treted B10 splenocytes lone or ECDI-treted BIO.A splenocytes with or without ntigen (Fig. 3b). Both A.E7 nd 3R.3.11 cells tht were rendered unresponsive to ntigen nd l molecule restimultion responded to exogenous IL-2, demonstrting tht cell deth did not ccount for unresponsiveness. These results show tht the induction of T cell unresponsiveness in vitro is MHC-restricted. The specificity of unresponsiveness induced in vitro ws further exmined s shown in Fig. 4. FLA.2 cells were rendered unresponsive to restimultion with B10.A APC nd pigeon frgment fter preincubtion with ECDI-treted B10.A splenocytes nd pigeon frgment , but not with moth 93-

7 30 8 T CELL UNRESPONSIVENESS IN VITRO AND IN VIVO 260,000 b ], nm ], nm FIGURE 4. Antigen nd l molecule specificity of T cell unresponsiveness induced in vitro. T cell clone FI.A.2 ws preincubted with ECDI-treted splenocytes with or without ntigen s described in the legend to Fig. 2. Prolifertion of restimulted cells ws mesured by [ sh]- thymidine incorportion. F1.A.2 preincubtion conditions : ECDI-treted B10.A splenocytes with (open circles) or without (filled circles) 1.0 jam pigeon frgment ( nd b), or ECDI-treted B10.A splenocytes with 1.0 AM moth peptide (93E,99Q) (, open tringles), or ECDI-treted BI O.A(4R) splenocytes with (b, open squres) or without (b,filled squres) 1.0,iiM pigeon frgment (93E,99Q), nonstimultory peptide nlog contining the moth cytochrome c sequence with glutmine for lysine substitution t residue 99 (29) (Fig. 4). Similrly, preincubtion of FI.A.2 with pigeon frgment nd ECDItreted B10.A(4R) splenocytes, which do not express E,3:E, molecules, did not result in unresponsiveness (Fig. 4b). Furthermore, preincubtion of A.E7 with ECDI-treted BIO.A splenocytes nd pigeon frgment , in the presence of nti-e,:e. but not nti-a#:a«mabs, completely blocked the induction of unresponsiveness in vitro (Tble I), demonstrting tht the l molecules present on ECDI-treted splenocytes re required for the induction of the unresponsive stte. In ll cses, unresponsiveness could not be ccounted for by cell deth, s the clones proliferted well in response to exogenous IL-2 fter preincubtion with the pproprite ntigen nd ECDI-treted APC. These results demonstrte tht the induction of T cell unresponsiveness in vitro hs the identicl ntigen nd l molecule specificity s the induction of the T cell clone's prolifertive response. Dependence of the Induction of Unresponsiveness on the Antigen Concentrtion nd the Number of ECDI-Treted APC. The quntittive spects of the induction of unresponsiveness re shown in Fig. 5. A.E7 preincubted with fixed number of ECDI-treted B10.A splenocytes nd incresing concentrtions of pigeon frgment (Fig. 5), or with fixed concentrtion of pigeon frgment nd incresing numbers of ECDI-treted BIO.A splenocytes (Fig. 5b), were progressively less responsive to restimultion. Mximl effects were observed with 1-10 AM of ntigen nd 5 x 106 ECDI-treted splenocytes. Kinetics of Induction of T Cell Unresponsiveness In Vitro. A.E7 cells were

8 JENKINS AND SCHWARTZ 309 Group TABLE I Effect of Anti-l mabs on the Induction of T Cell Unresponsiveness In Vitro Preincubtion conditions* Restimultion response (A cpm) to : T cell APC Antigen Antibody BIO.A APC + clone A A.E7 B10.A-ECDI , ,000 B A.E7 B10.A-ECDI ,500 (97) 125,000 C A.E7 B10.A-ECDI $ (nti-ep :E.) 87,500(8) 105,000 D A.E7 B10.A-ECDI (nti-ab :A) 3,000 (97) 155,000 * A.E7 cells (5 X 10 5 ) were preincubted overnight with 5 X 106 ECDI-treted BIO.A splenocytes without ntigen (group A) or with 5 X 106 ECDI-treted BIO.A splenocytes nd 5 JAM pigeon frgment without (group B) or with 1 jug/ml $ (group C) or (group D) mabs. *After preincubtion, A.E7 cells were reisolted nd cultured (2 X 10') with 5 X 10 5 B10.A irrdited splenocytes with or without 0.01 um pigeon frgment or 100 U of IL-2 lone. The results re expressed s A cpm. Numbers in prentheses show percent inhibition of the prolifertive response compred with group A. FIGURE 5. Dose dependency of T cell unresponsiveness on ntigen concentrtion nd APC number. T cell clone A.E7 (5 X 10 5 ) ws preincubted overnight with 5 X 106 ECDI-treted BIO.A splenocytes nd the indicted concentrtions (AM) of pigeon frgment () or with 1 um pigeon frgment nd the indicted numbers of ECDI-treted B10.A splenocytes (b). The T cells were reisolted nd restimulted s described in the legend to Fig. 2. The results re expressed s A cpm. IL-2 preincubted with ECDI-treted BlO.A splenocytes with or without pigeon frgment for vrious periods of time to determine the time course for the induction of unresponsiveness in vitro (Fig. 6). Unresponsiveness to restimultion with norml APC nd ntigen ws pprent fter only 2 h, ws prtilly induced fter 5 h, nd ws completely induced fter 16 h of preincubtion. Thus, unresponsiveness ws not totlly induced immeditely, but required >5 h to chieve mximl effect. Durtion of T Cell Unresponsiveness Induced In Vitro. The durtion of in vitroinduced unresponsiveness ws tested by preincubting FLA.2 cells overnight with ECDI-treted BIO.A splenocytes with or without pigeon frgment , followed by restimultion 2, 4, 6, or 8 d lter (Fig. 7). At ll time points, FLA.2 cells were completely unresponsive to restimultion with untreted BIO.A splenocytes nd ntigen, lthough they remined responsive to IL-2 (dt not shown). Thus, s reported for unresponsiveness induced in vivo by ntigen-coupled cells

9 31 0 T CELL UNRESPONSIVENESS IN VITRO AND IN VIVO 1 o, ooo 1 zoooo, -~ 100, h 5h 140,000 1zoooo, -I /\ 100,000 U [81-104], gm [81-104], pm 16h 200, , , , , ,000 80,000-60, ,000-20,000-0 ~ i r IL-2 [81-104], gm FIGURE 6. Time course of the induction of T cell unresponsiveness in vitro. T cell clone A.E7 (5 x 10 5) ws preincubted with 5 x 10 6 ECDI-treted BIO.A splenocytes with (open circles) or without (filled circles) 5 AM pigeon frgment for the indicted times, fter which the T cells were reisolted nd restimulted s described in the legend to Fig. 2. The results re expressed s 0 cpm. (30), unresponsiveness induced in vitro ppers to lst for reltively long period of time. Specificity of T Cell Unresponsiveness Induced In Vivo. Our studies demonstrte tht ECDI-treted splenocytes induce T cell unresponsiveness in vitro. In ddition, others hve shown (3-5, 13) tht intrvenous injection of ntigen-coupled splenocytes results in T cell unresponsiveness in vivo. Bsed on these observtions, we exmined the fine specificity of T cell unresponsiveness to pigeon cytochrome c induced in vivo. B10.A mice were injected intrvenously with BIO.A splenocytes coupled with ntigen vi ECDI. 4 d lter, recipient mice were immunized in the footpds with pigeon cytochrome c emulsified in CFA. As shown in Fig. 8, T cells from recipients of splenocytes coupled vi ECDI with intct pigeon cytochrome c or pigeon frgment proliferted very poorly in response to frgment (Fig. 8), lthough these cells responded normlly to PPD present in the djuvnt used for immuniztion (Fig. 8c). Control T cells from mice receiving splenocytes treted with ECDI in the bsence of ntigen, proliferted strongly in response to pigeon frgment (Fig. 8). The intct molecule ws better tolerogen thn the frgment, possibly becuse there lso exist B10.A T cells tht re specific for pigeon frgment 1-65 in ssocition with A'6:A (25) ; these my hve fcilitted the survivl nd/or expnsion of T cells specific for frgment nd E13:E.

10 JENKINS AND SCHWARTZ 31 1 Dy 2 Dy 4 60,000 50,000 50,000 40,000 2 U 10,000 10, _ j -v [ , um [ , NM 40,000 -, 30,000 20,000 10,000 Dy 6 Dy 8 50,000 ss 40, ,000N `, 40,000-30,000-20,000-10, n r--, [ , pm [81-104), pm FIGURE 7. Durtion of T cell unresponsiveness induced in vitro. T cell clone FLA.2 (5 X 105/well) ws preincubted with ECDI-treted B10.A splenocytes (5 X 106/well) with (open circles) or without (filled circles) 1.0 pm pigeon frgment The T cells were reisolted, plced in microtiter pltes (2 X 10'), nd restimulted by the ddition of vrious ntigen concentrtions nd 5 X 10 5 irrdited BIO.A splenocytes 2, 4, 6, or 8 d lter. The results re expressed s 0 cpm. The ntigen specificity of the tolernce induction ws similr to tht of prolifertion becuse B10.A splenocytes coupled with pigeon frgment or intct duck cytochrome c (neither of which stimulte prolifertive response in B 10.A mice, reference 31) filed to induce hyporesponsiveness in vivo (Fig. 8 ). A requirement for l molecules ws lso demonstrted, s B10.A splenocytes tht were depleted of cells bering l molecules nd then coupled with pigeon cytochrome c were poor tolerogens in vivo (Fig. 8b). Furthermore, pigeon coupled B10.A(4R) splenocytes (Kk, A#, A, Es, E., Db), which do not express E,9:E, molecules, filed to induce frgment specific T cell unresponsiveness in pigeon cytochrome c-primed B10.A (Kk, As, A., E0, E, Dd) mice (Fig. 9, -c). This suggests tht the Ek:E l molecule is necessry for the tolernce induction. An lterntive explntion for this lst result, however, is tht llogeneic effects in vivo due to the H-2D region disprity between B10.A nd B10.A(4R) obscured the tolernce induction. To eliminte this possibility the experiment ws repeted using BIO.A(2R) (K'`, A#, A, Efl, E., Db ) recipients nd frgment , ECDI-coupled B10.A(2R) (tolerogenic) vs. B10.A(4R) (non-

11 312 T CELL UNRESPONSIVENESS IN VITRO AND IN VIVO b 200,000 30,000 20,000.1 c 20,000 16,000-12,000-8,0M 160, ,000 U 10,000-4,000-80,000 40, [81-104], gm [81-104], ILM [PPDL 4hd Antigen specificity of unresponsiveness induced in vivo by ECDI-treted spleno- FIGURE 8. cytes. B10.A mice (two per group) were injected intrvenously with 2 x 107 ECDI-treted B10.A splenocytes coupled with pigeon cytochrome c (closed squres in -c), pigeon frgment (open circles in nd c), pigeon frgment (open squres in nd c), duck cytochrome c (open tringles in nd c), nothing (filled circles in -c), or 2 x 10 7 B10.A splenocytes depleted of cells bering l molecules nd then coupled with pigeon cytochrome c (filled tringles in b). 4 d lter, recipient mice were immunized with pigeon cytochrome c in CFA. 7 d fter priming, drining lymph node T cell prolifertion in response to pigeon frgment ( nd b) or PPD (c) ws determined in ['Hlthymidine uptke ssy. The results re expressed s 0 cpm. cpm vlues for no ntigen controls rnged from 1,779 ± 304 to 3,343 ± 637. tolerogenic) splenocytes. The results were the sme (dt not shown). It should be noted tht B10.A(4R) splenocytes, ECDI-coupled with GAT, n ntigen recognized in ssocition with AO:A' (32), induced specific unresponsiveness to GAT in syngeneic recipients (dt not shown). This control showed tht ntigencoupled B10.A(4R) splenocytes could be tolerogenic when their expressed l molecule ws involved in ctivtion. Finlly, to rule out role for I-J in this prticulr form of tolernce induction, B10.A(3R) mice (A#, A., Es, E, I- )were injected intrvenously with B10.A(5R) splenocytes (A#, A, Efl, E, I Jk) coupled vi ECDI with synthetic nlog of moth cytochrome c, 86-89;93-103(93E,98A). As shown in Fig. 10 this I+ incomptible combintion induced the mximum level of unresponsiveness ttinble with the syngeneic combintion. In contrst, B10.A(18R) splenocytes (AQ, A, Eb6, E«, If b ), which re IJ-comptible with B10.A(3R) but do not express E,3:E molecules, filed to induce mximl unresponsiveness. The prtil unresponsiveness observed in this cse my be due to the existence of AO:A.-restricted, 86-89;93-103(93E,98A)-specific clones in BIO.A(3R) mice. Tken together, these experiments demonstrte tht the induction of T cell unresponsiveness in vivo to pigeon cytochrome c hs the sme ntigen nd l molecule specificity (frgment nd E,3:E,) s does the induction of T cell unresponsiveness in vitro. We conclude from these experiments tht this form of tolernce induction is MHC-restricted. Discussion In this report we demonstrte tht ntigen presenttion by chemiclly modified APC cn induce unresponsiveness in vitro for T cell clones nd in vivo for lymph node T cells. The induction of this unresponsiveness hd the sme ntigen nd MHC specificity s T cell ctivtion, i. e., it depended on both the E# :E. l

12 60,000 50,000 40,000 U 30, ,000 JENKINS AND SCHWARTZ 31 3 b 10, [81-104], ~M [81-104], ~M c 100,000 80,000 60,000 40,000 20,000-0 [PPO],N9/ml FIGURE 9. Frgment , ECDI-coupled B10.A(4R) splenocytes do not induce unresponsiveness in B10.A. B10.A mice (two per group) were injected intrvenously with 5 X 10' ECDI-treted BIO.A splenocytes () coupled with nothing (filled circles) or pigeon frgment (open circles), or with 5 X 10' BIO.A(4R) ECDI-treted splenocytes (b) coupled with nothing (filled squres) or frgment (open squres). 1 d lter, mice were primed with pigeon cytochrome c nd 7 d lter, T cell prolifertion ws determined s described in the legend to Fig. 8. The results re expressed s 0 cpm. cpm vlues for no ntigen controls rnged from 657 ± 117 to 1,098 ± 103. ( nd b) the response to pigeon frgment ; (c) the response to PPD. molecule nd on residues contined within the pigeon cytochrome c frgment Furthermore, the filure of moth (93E,99Q) to induce unresponsiveness in vitro demonstrted the requirement for recognition of the lysine t position 99, residue tht hs been suggested (29) to contct the T cell receptor of clones specific for pigeon frgment Our findings re consistent with recent report (33) tht demonstrted tht the specificity of the induction of neontl tolernce in vivo ws identicl to tht of T cell ctivtion nd other reports (4, 34-37) tht hve demonstrted tht tolernce induction in vivo nd in vitro is MHC-restricted. Previous work hs shown (2, 38) tht T suppressors contribute to unresponsiveness by inhibiting the function of helper nd delyed-type hypersensitivity T cells. Severl points suggest tht the in vivo unresponsiveness exmined in this pper is not medited by suppression. T suppressors generlly do not inhibit T cell prolifertion (13, 38), lthough some exmples hve been reported (39, 40). Furthermore, in severl instnces prolifertive T cells nd T suppressors hve been shown to recognize distinct portions of the sme ntigen (8). Oki nd Sercrz (1) hve shown tht removl of suppressor determinnt destroys the

13 314 T CELL UNRESPONSIVENESS IN VITRO AND IN VIVO 50,000 [86-89 ;93-103(93E,98A)], gm FIGURE 10. I,J comptibility is not required for the induction of T cell unresponsiveness in vivo. BIO.A(3R) mice (two per group) were injected intrvenously with 5 X 10 7 ECDI-treted BI O.A(3R) splenocytes coupled with nothing (filled circles) or 5 X 107 ECDI-treted B10.A(3R) (open circles), B10.A(5R) (filled squres), or B10.A(18R) (open squres) splenocytes coupled with moth 86-89;93-103(93E,98A). I d lter, mice were primed with 10 nmol of moth ;93-103(93E,98A) nd 7 d lter, T cell prolifertion ws determined in ['H]thymidine uptke ssy. The results re expressed s 0 cpm. cpm vlues for no ntigen controls rnged from 524 ± 29 to 1,140 ± 735. tolerogenicity of hen egg white lysozyme. In contrst, pigeon cytochrome c frgment , which contins the ntigenic determinnt recognized by prolifertive T cells, induced unresponsiveness when ssocited with the E"k:Ek l molecule, suggesting direct effect of the tolerogen on the responding T cells. However, the filure of frgment , ECDI-coupled B10.A(4R) splenocytes (I Jb) to tolerize the pigeon cytochrome c response of BIO.A or B10.A(2R) recipients (I Jk) could lso be ttributed to the requirement for I-J comptibility between the tolerogen nd suppressor T cells (2, 4). Tht this is not the cse is indicted by experiments in which moth cytochrome c, ECDI-coupled BIO.A(5R) splenocytes (E,9, E., I Jk) induced mximl moth cytochrome c-specific T cell unresponsiveness in IJ-incomptible BIO.A(3R) recipients (E', E, b IJ ), wheres moth cytochrome c, ECDI-coupled IJ-comptible B10.A(18R) splenocytes (Eb, E,, If b ) did not. Thus, I-E comptibility ppers to be necessry nd sufficient for this form of rpid tolernce induction. Our observtion tht cloned T cells could be rendered unresponsive in vitro suggests direct effect of ntigen nd ECDI-treted APC on the specific T cells. Becuse ECDI-treted B cells plus mcrophges, essentilly depleted of T cells, could induce unresponsiveness in the presence of ntigen, n indirect mechnism involving suppressor T cell induction would seem to be unlikely. Furthermore, studies by Quill nd Schwrtz' using plnr lipid membrnes to induce similr nonresponsive stte in vitro, hve demonstrted tht only l molecules nd ' Quill, H. nd R. H. Schwrtz, Stimultion of norml inducer T cell clones with ntigen presented by purified l molecules in plnr lipid membrnes: specific induction of long-lived stte of prolifertive nonresponsiveness. Submitted for publiction.

14 JENKINS AND SCHWARTZ 31 5 ntigen re required for this effect. The identicl specificity of T cell unresponsiveness induced in vitro nd in vivo with tht required to stimulte T cell prolifertion in vitro suggests tht the sme receptor occupncy event occurs in ech instnce. These results strongly rgue tht functionl clonl deletion mechnism underlies the in vivo unresponsiveness we hve observed in our system. Thus, it should be possible for the first time to dissect out the biochemicl bsis of T cell unresponsiveness using the in vitro correlte of n in vivo model system. One model for functionl clonl deletion involves the blockde of T cell receptors by ntigen/mhc molecules (5, 30). Our results suggest tht T cell receptor blockde or modultion does not ccount for T cell inctivtion since the in vitro-induced unresponsiveness lsted for t lest 8 d in the bsence of tolerogen. Others hve demonstrted (41) significnt cell surfce reexpression of T cell receptors modulted by mabs only 48 h fter ntibody tretment. Furthermore, Quill nd Schwrtz' hve shown tht T cell clones express norml levels of T cell receptor molecules t times when the cells re completely unresponsive. However, it is still formlly possible tht ntigen/l molecules derived from the tolerogen remin ssocited with the T cell clones for long periods of time without interfering with the binding of n nti-v,3 mab. We would propose tht the T cells recognize ntigen in ssocition with l molecules on the surfce of ECDI-treted APC in mnner similr to tht which occurs on norml APC. However, the ECDI tretment ppers to impir n dditionl APC signl necessry to induce IL-2 production nd T cell prolifertion. This interprettion is consistent with the recent results of Weiss et l. (42), which demonstrte tht certin T cells require T cell receptor perturbtion plus dditionl signls to produce IL-2. Our results extend this by suggesting tht incomplete signling in combintion with T cell receptor occupncy by ntigen/l molecules results in n unresponsive stte. This stte ppers not to involve inhibition of the IL-2-R pthwy, s T cell clones unble to respond to ntigen/l molecule restimultion responded normlly to exogenous IL-2. In ddition, this unresponsive stte is induced in the bsence of T cell prolifertion, distinguishing it from unresponsiveness induced by exposure of T cells to high doses of IL-2 (43). Thus, the model first proposed for B cells by Bretscher nd Cohn (44) nd lter modified by others (3, 6), in which receptor occupncy in the bsence of other signls results in tolernce induction, could be pplied to T cells. If so, T nd B cells would use similr mechnisms of tolernce induction. In this regrd, it should be noted tht the in vitro T cell unresponsiveness reported here nd the in vitro B cell unresponsiveness reported by others (45) hve similr time courses of induction. Preliminry evidence from our lbortory suggests tht the T cells re prtilly ctivted fter incubtion with ntigen nd ECDI-treted APC nd tht this prtil ctivtion renders the T cell incpble of producing IL-2 for t lest 8 d in vitro. This interprettion differs from previous conclusions on the cellulr bsis for ntigen-coupled, cell-induced unresponsiveness. It hs been proposed by others (3, 4) tht the tolerogenicity of ntigen- or hpten-coupled cells is relted to the high ntigen density on these cells nd the intrvenous route of their dministrtion. Our results suggest tht crucil function of ECDI is not

15 31 6 T CELL UNRESPONSIVENESS IN VITRO AND IN VIVO only to couple ntigen to the splenocytes but lso to inctivte the APC ccessory signl(s) necessry to fully ctivte T cells. Lmb et l. (11) hve lso reported the in vitro induction of unresponsiveness fter incubtion of humn T cell clones with ntigen frgments in the bsence of APC. Since subsequent studies (12) showed tht the induction of unresponsiveness could be blocked with nti-clss 11 MHC ntibodies, it is possible tht the clss II MHC-positive humn T cells were presenting ntigen to ech other. Therefore, in both murine nd humn systems, nonmitogenic ntigen presenttion in vitro ppers to result in unresponsiveness. A prediction of this type of model would be tht other cells tht express surfce clss II MHC molecules but do not support T cell prolifertion, e. g., UV-irrdited splenocytes (46), gmmirrdited (3,000 rd) resting B cells (47), neontl splenocytes (48), nd possibly thymic epithelil cells (49), might induce unresponsiveness. Recently published experiments (50) hve shown tht even certin nonstimultory, nti-t cell receptor mabs induced stte of unresponsiveness, further supporting the ide tht receptor occupncy under nonmitogenic conditions cn result in T cell inctivtion. In summry, we hve shown tht nonmitogenic, ntigen presenttion by ECDItreted APC induces inhibition of T cell clones in vitro nd in vivo, suggesting tht functionl clonl deletion is one mechnism of tolernce induction. Future experiments will be imed t identifying the biochemicl events resulting in unresponsiveness, nd the nture of the ECDI-sensitive molecule(s) on the APC. Finlly, the possible ppliction of this procedure to inducing specific unresponsiveness in ongoing utoimmune disese s well s before orgn trnsplnttion is currently under investigtion. Summry We investigted the ntigen specificity nd presenttion requirements for inctivtion of T lymphocytes in vitro nd in vivo. In vitro studies reveled tht splenocytes treted with the crosslinker 1-ethyl-3-(3-dimethylminopropyl)- crbodiimide (ECDI) nd soluble ntigen frgments filed to stimulte significnt prolifertion by norml pigeon cytochrome c-specific T cell clones, suggesting tht the chemicl tretment inctivted full ntigen presenttion function. However, T cell clones exposed to ECDI-treted splenocytes nd ntigen in vitro were rendered unresponsive for t lest 8 d to subsequent ntigen stimultion with norml presenting cells. As predicted by the in vitro results, specific T cell unresponsiveness ws lso induced in vivo in B10.A mice injected intrvenously with B10.A, but not B10.A(4R), splenocytes coupled with pigeon cytochrome c vi ECDI. The ntigen nd MHC specificity of the induction of this T cell unresponsiveness in vitro nd in vivo ws identicl to tht required for T cell ctivtion. These results suggest tht nonmitogenic T cell recognition of ntigen/mhc on ECDI-modified APCs results in the functionl inctivtion of T cell clones. We thnk Ms. S. Strnes nd Ms. J. Eccrd for preprtion of this mnuscript ; Drs. J. Ashwell, D. Cohen, B. Fox, R. Germin, K. McCoy, D. Prdoll, H. Quill, nd A. Singer for criticl reding of the mnuscript ; nd Ms. Chun Chen for expert technicl ssistnce. Receivedfor publiction 16 September 1986.

16 JENKINS AND SCHWARTZ References 1. Oki, A., nd E. Sercrz T cell tolernce studied t the level of ntigenic determinnts. I. Ltent rectivity to lysozyme peptides tht lck suppressogenic epitopes cn be reveled in lysozyme-tolernt mice. J. Exp. Med. 161 : Germin, R. N., nd B. Bencerrf A single mjor pthwy of T lymphocyte interctions in ntigen-specific immune suppression. Scnd. J. Immunol. 13 :1. 3. Clmn, H. N., S. D. Miller, P. J. Conlon, nd J.W. Moorhed Control of experimentl contct sensitivity. Adv. Immunol. 30: Monroe, J. G., A Lowy, R. D. Grnstein, nd M. 1. Greene Studie s of immune responsiveness nd unresponsiveness to the p-zobenzenersonte (ABA) hpten. Immunol. Rev. 80: Borel, Y Hptens bound to self IgG induce immunologic tolernce, while when coupled to syngeneic spleen cells they induce immune suppression. Immunol. Rev. 50 : Nossl, G. J. V Cellulr mechnisms of immunologic tolernce. Annu. Rev. Immunol. 1 : Tniguchi, M., nd J. F. A. P. Miller Enrichmen t ofspecific suppressor T cells nd chrcteriztion of their surfce mrkers. J. Exp. Med. 146: Goodmn, J W., nd E. E. Sercrz The complexity of structures involved in T-cell ctivtion. Annu. Rev. Immunol. 1 : Asno, Y., nd R. J. Hodes T cell regultion of B cell ctivtion. Cloned Lyt T suppressor cells inhibit the mjor histocomptibility complex-restricted interction of T helpers with B cells nd/or ccessory cells. J. Exp. Med. 158: Jenkins, M. K., nd S. D. Miller Immunoregultor y pthwys in dult responder mice Estblishment of GAT-specific suppressor T cell clone from GAT-tolernt responders which fferently regultes DTH responses. J Mol. Cell. Immunol. 2 : Lmb, J. R., B. J. Skidmore, N. Green, J. M. Chiller, nd M. Feldmnn Induction of tolernce in influenz virus-immune T lymphocyte clones with synthetic peptides of influenz hemgglutinin. J. Exp. Med. 157: Lmb, J. R., nd M. Feldmnn Essentil requirement for mjor histocomptibility complex recognition in T-cell tolernce induction. Nture (Lond. ). 308: Miller, S.D., R. P. Wetzig, nd H. N. Clmn The induction of cell-medited immunity nd tolernce with protein ntigens coupled to syngeneic lymphoid cells. J. Exp. Med. 149: Brutigen, D. L., S. Ferguson-Miller, nd E. Mrgolish Mitochondril cytochrome c: preprtion nd ctivity of ntive nd chemiclly modified cytochromes c. Methods Enzymol. 53 : Corrdin, G., nd H. A. Hrbury Clevg e of cytochrome c with cynogen bromide. Biochim. Biophys. Act. 221 : Schwrtz, R. H., B. S. Fox, E. Frg, C. Chen, nd B. Singh The T lymphocyte response to cytochrome c. V. Determintion of the miniml peptide size required for stimultion oft cell clones nd ssessment ofthe contribution ofech residue beyond this size to ntigenic potency. J. Immunol. 135 : Julius, M. H., E. Simpson, nd L. A. Herzenberg A rpid method for the isoltion of functionl thymus-derived murine lymphocytes. Eur. J. Immunol. 3: Ozto, K., N. Myer, nd D. H. Schs Hybridom cell lines secreting monoclonl ntibodies to mouse H-2 nd l ntigens. J. Immunol. 124: Oi, V. T., P. P. Jones, J. W. Goding, nd L. A. Herzenberg Properties of monoclonl ntibodies to mouse Ig llotypes, H-2, nd l ntigens. Curr. Top. Microbiol. Immunol. 81 :115.

17 31 8 T CELL UNRESPONSIVENESS IN VITRO AND IN VIVO 20. McGrth, M. S., E. Pillemer, nd I. L. Weissmn Murine leukemogenesis : monoclonl ntibodies to T cell determinnts rrest T-lymphom cell prolifertion. Nture (Lond. ). 285: Ledbetter, J. A., nd L. A. Herzenberg Xenogeneic monoclonl ntibodies to mouse lymphoid differentition ntigens. Immunol. Rev. 47 : Lnier, L. L.,G. A. Gutmn, D. E. Lewis, S. T. Griswold, nd N. L. Wrner Monoclon l ntibodies ginst rt immunoglobulin kpp chins. Hybridom. 1 : Mtis, L. A., D. L. Longo, S. M. Hedrick, C. Hnnum, E. Mrgolish, nd R. H. Schwrtz Clon l nlysis of the mjor histocomptibility complex-restriction nd the fine specificity of ntigen recognition in the T cell prolifertive response to cytochrome c. J. Immunol. 130: Ashwell, J. D., B. S. Fox, nd R. H. Schwrtz Function l nlysis of the interction of the ntigen-specific T cell receptor with its lignds.j. Immunol. 136: Suzuki, G., nd R. H. Schwrtz The pigeon cytochrome c-specific T cell response of low responder mice. I. Identifiction of ntigenic determinnts on frgment 1-65.J. Immunol. 136 : Kimoto, M., nd C. G. Fthmn Antigen-rectiv e T cell clones. 1. Trnscomplementing hybrid I-A region gene products function effectively in ntigen presenttion. J. Exp. Med. 152: Ashwell, J. D., C. Chen, nd R. H. Schwrtz High frequency nd nonrndom distribution of llorectivity in T cell clones selected for recognition of foreign ntigen in ssocition with self clss II molecules.j. Immunol. 136: Bhttchry, A., M. E. Dorf, nd T. A. Springer A shred llontigenic determinnt on l ntigens encoded by the I-A nd I-E subregions : evidence for I region gene dupliction. J. Immunol. 127: Schwrtz, R. H T-Lymphocyte recognition ofntigen in ssocition with gene products of the mjor histocomptibility complex. Annu. Rev. Immunol. 3: Quill, H., L. Crlson, B. S. Fox,J. Weinstein, nd K. H. Schwrtz Optimiztion of ntigen presenttion to T cell hybridoms by purified l molecules in plnr membrnes. l molecule polymorphism determines the ntigenic fine specificity of the response to cytochrome c peptides. J. Immunol. Methods. In press. 30. Miller, S. D., M. S. Sy, nd H. N. Clmn The induction of hpten-specific T cell tolernce using hpten-modified lymphoid cells. II. Reltive roles of suppressor T cells nd clone inhibition in the tolernt stte. Eur. J. Immunol. 7: Solinger, A. M., M. E. Ultee, E. Mrgolish, nd R. H. Schwrtz T lymphocyte response to cytochrome c. 1. Demonstrtion of T-cell heteroclitic prolifertive response nd identifiction of topogrphic ntigenic determinnt on pigeon cyto chrome c whose immune recognition requires two complementing mjor histocomptibility complex-linked immune response genes. J. Exp. Med. 150: Schwrtz, R. H., nd W. E. Pul T-Lymphocyte-enriched murine peritonel exudte cells. II. Genetic control of ntigen-induced T-lymphocyte prolifertion. J. Exp. Med. 143: Gmmon, G., K. Dunn, N. Shstri, A. Oki, S. Wilbur, nd E. E. Sercrz Neontl T-cell tolernce to miniml immunogenic peptides is cused by clonl inctivtion. Nture (Lond. ). 319: Groves, E. S., nd A. Singer Role of the H-2 complex in the induction of T cell tolernce to self minor histocomptibility ntigens. J. Exp. Med. 158: Mtzinger, P., R. Zmoysk, nd H. Wldmnn Self-tolernce is H-2 restricted. Nture (Lnd.). 308: Rm mensee, H., nd M. J. Bevn Evidence from in vitro studies tht tolernce to self ntigens is MHC-restricted. Nture (Lond.). 308 :741.