Tolerance Induced by Physiological Levels of Secreted Proteins in Transgenic Mice Expressing Human Insulin

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1 Tolernce Induced by Physiologicl Levels of Secreted Proteins in Trnsgenic Mice Expressing Humn Insulin Phyllis Jons Whiteley,* Jeffrey P. Lke,* Richrd F. Selden,* nd Judith A. Kpp* *Deprtment ofpthology, The Jewish Hospitl ofst. Louis, nd Deprtment ofpthology nd Microbiology/Immunology, Wshington University Medicl School, St. Louis, Missouri 6311; nd tdeprtment ofmoleculr Biology, Msschusetts Generl Hospitl, nd Deprtment ofgenetics, Hrvrd Medicl School, Boston, Msschusetts 2114 Abstrct We hve used trnsgenic mice to study immune tolernce to utologous, non-mhc encoded proteins tht re expressed t physiologicl levels in the circultion. The trnsgenic mice used in these studies express the humn preproinsulin gene nd synthesize humn proinsulin. Humn nd mouse insulin re secreted from the pncretic islets of trnsgenic mice in response to norml physiologicl stimuli, such s glucose. Our dt demonstrte tht the trnsgenic mice hve cquired tolernce to humn insulin. The repertoire of T cells specific for exogenous ntigens is shped by the cquired tolernce to utologous proteins since pork but not beef or sheep insulin is lso nonimmunogenic in the trnsgenic mice. We lso found tht the trnsgenic mice were tolernt to humn proinsulin, the intrcellulr precursor of insulin. Unresponsiveness to humn proinsulin most likely results from tolernce of insulin-specific nd proinsulin-specific T cells tht recognize the secreted enzymtic clevge products of proinsulin, insulin nd C-peptide. Introduction Rerrngement nd expression of T cell receptor (TcR)' genes results in the development oft cells tht recognize seemingly infinite vriety of ntigens, including utologous proteins. Tolernce normlly prevents T cells tht recognize self ntigens from cusing utoimmune diseses. For certin cell-surfce proteins, such s: MHC clss II molecules (1), the minor lymphocyte stimultory (Mls) ntigen (2), nd the mle ntigen, H-Y (3), tolernce is mintined by deletion of the relevnt clones in the thymus. Elimintion of these T cell clones is fesible becuse ll of these ntigens re present on cells tht re either locted in or trverse through the thymus during T cell ontogeny nd development. However, the mechnisms underlying tolernce to non- MHC self-proteins hve been difficult to exmine under physi- Address reprint requests to Dr. Kpp, Deprtment of Pthology, CSB-7, The Jewish Hospitl of St. Louis, 216 South Kingshighwy, St. Louis, MO Received for publiction 2 December 1988 nd in revisedform 13 July Abbrevitions used in this pper: CFA, complete Freund's djuvnt; PFC, plque-forming cell; TcR, T cell receptor. J. Clin. Invest. The Americn Society for Clinicl Investigtion, Inc /89/1 1/155/5 $2. Volume 84, November 1989, ologicl conditions. Mny experimentl models of tolernce involve neontl mice which were injected with bolus of exogenous protein (4). Although the neontlly tolerized mice remin nonresponsive to the ntigen s dults, these models cnnot directly ddress the mechnisms of tolernce induced by physiologicl levels of circulting protein ntigens. Our studies ddress the development of tolernce to self-proteins tht re not cell surfce molecules nd re not synthesized by cells in the thymus. In the present study, we hve investigted development of tolernce in trnsgenic mice tht express the humn preproinsulin gene. These mice, hve been previously shown to express mrna for humn proinsulin, the precursor form of insulin, only in pncretic islets (5). In the islets, humn proinsulin is enzymticlly cleved into insulin nd C-peptide, which re subsequently secreted in response to norml physiologicl signls (5). The trnsgenic mice secrete physiologicl mounts of insulin nd C-peptide contining mixture of both mouse nd humn proteins. Methods Mice. C57BL/1 (H-2b) nd BALB/c (H-2d) mice were purchsed from Jckson Lbortories, Br Hrbor, ME. The H-2k/b hybrids (BlO.BR X BlO)Fl, (BALB.K X BALB.B)F1, nd the H-2d/k (BALB/c X CBA/ N)Fl mice were bred in the Animl Resources Fcility of the Jewish Hospitl. The trnsgenic mice were derived by injecting 12.5-kb frgment contining the humn preproinsulin gene into the pronuclei of ov from (C57BL/6 (H-2b) X C3H (H-2k))F1 mice tht hd been fertilized by (C57BL/6 X C3H)F1 mles (5). Mice expressing the humn insulin gene were identified by mesuring the concentrtion of humn C-peptide in the plsm of mice fter glucose chllenge (5). The trnsgenic mice were bred to (C57BL/6 X C3H)F1 from which we selected trnsgenic mice, homozygous for H-2k by H-2 typing of their erythrocytes (6). The trnsgenic mice re mintined s heterozygotes for the humn insulin gene nd bckcrossed with norml C57BL/1O (H-2b) or BALB/c (H-2d) mice to provide the H-2k/b nd H-2d/k mice used in these studies. These trnsgenic mice hve rndom ssortment of bckground genes; however, we hve previously shown there to be no detectble effect of different bckground genes on responsiveness to insulin (7). Control mice for these experiments were H-2 mtched nontrnsgenic littermtes s well s H-2 mtched F1 hybrids between inbred strins. All mice were mintined in strict ccordnce with NIH guidelines for the cre of lbortory nimls. Antigens nd immuniztions. Semisynthetic humn insulin ws gift of John Lnpher, Squibb Co., Princeton, NJ; recombinnt humn insulin, proinsulin nd C-peptide ws gift from Dr. Ron Chnce, Eli Lilly & Co., Indinpolis, IN. The immunologicl ctivities of semi-synthetic nd recombinnt humn insulin were the sme in ll our ssys, thus, they were used interchngebly in these studies. Pork insulin ws purchsed from Elnco, Indinpolis, IN nd pork proinsulin from Squibb Novo, Princeton, NJ. Mice were injected with 5 ug ntigen in 1:1 rtio with complete Freund's djuvnt (CFA) contining Mycobcterium tuberculosis H37R (Difco Lbortories, Detroit, MI). 155 P. J. Whiteley, J. P. Lke, R. F. Selden, nd J. A. Kpp

2 Antibody ssys. Serum ntibody ws mesured by n ELISA using beef insulin- or humn proinsulin-coted pltes s previously described (8). Antibodies induced by vrious insulins re extremely cross-rective nd no significnt differences were found using beef, pork, or humn insulin-coted pltes. Thus, beef insulin ws used throughout these experiments. Our ssy detects both IgG nd IgM ntibodies; however we hve previously shown tht primry immuniztion of insulin elicits predominntly insulin-specific IgG nd not IgM ntibodies (9). Insulin-specific IgG plque-forming cells (PFC) were detected with beef insulin-conjugted sheep erythrocytes s previously described (9). The results re expressed s the rithmetic men±sem PFC of three mice per group. Ech experiment hs been repeted t lest three times with very reproducible results; therefore the results of representtive experiments re shown. Results Insulin-specific tolernce in trnsgenic mice. Previous experiments hve demonstrted tht (H-2k X H-2b)Fl mice respond to pork insulin despite the fct tht the homozygous H-2k nd H-2b prentl strins do not (8-11). The bility of the Fl mice to respond to pork insulin is due to trnscomplementtion of the lph nd bet subunits of the MHC-encoded clss II molecules which form unique I proteins (12). Responses to humn insulin, which differs from pork insulin only in the terminl mino cid of the B chin (13), hve not previously been chrcterized; thus, we first determined tht norml H-2d mice responded to humn insulin, wheres H-2b nd H-2k mice did not (dt not shown). The trnsgenic H-2d/k (Fig. 1) nd H-2k/,) (Fig. 2) mice did not produce insulin-specific ntibodies when injected with humn insulin, wheres H-2 identicl, norml Fl mice nd nontrnsgenic littermtes did (Fig. 2). Trnsgenic mice were lso tolernt to pork insulin (Fig. 2). 1-1 ) Cd : ci) CD._ C'D Q U) CL e 75 S INSULIN Figure 1. Primry responses to exogenous insulin in H-2dA/ mice. Norml (BALB/c X CBA/N)Fl (htched br) nd H-2 mtched trnsgenic (dotted br) mice were injected intrperitonelly with 5 Ag of humn, beef, or sheep insulin in CFA. Ser werecollected on dy 21 nd ssyed for insulin-specific ntibodies by ELISA. The dt ws normlized to stndrd ntibody nd expressed s the reltive OD±SEM t 1: 1 dilution of serum. Preimmune ser from trnsgenic nd nontrnsgenic mice hd reltive OD..1. The numbers in prentheses represent the numbers of mice per group > 2. >. 1.5 'D < 1. C, CL.5. -S to Humn Pork Beef Sheep INSULIN Figure 2. Primry responses to exogenous insulin in H-2k/t mice. Norml (B1.BR X BIO)Fl mice (htched br), H-2 mtched nontrnsgenic (shded br), nd trnsgenic (dotted br) littermtes were injected intrperitonelly with 5 jg of humn, pork, beef, or sheep insulin in CFA. Ser were collected on dy 21 nd ssyed for insulin-specific ntibodies by ELISA. The dt ws normlized to stndrd ntibody nd expressed s the reltive OD±SEM t 1:1 dilution of serum. Preimmune ser from trnsgenic nd nontrnsgenic mice hd reltive OD..1. The numbers in prentheses represent the numbers of mice per group. However, the lck of n ntibody response in the trnsgenic mice is not due to generlized defect in the immune system, since they responded normlly to beef nd sheep insulin (Figs. 1 nd 2). The tolernce to humn insulin ws not uniquely ssocited with prticulr MHC clss II ntigen, s two different strins of trnsgenic mice gve the sme results. The kinetics of the primry ntibody response to insulin were exmined. Ser from norml nd immune mice were ssyed by ELISA for insulin-specific ntibody t vrious times fter intrperitonel injection with humn insulin. Neither nontrnsgenic or trnsgenic mice produced insulin-specific ntibody in preimmune ser or fter 7 d postinjection (Fig. 3). By dy 14 the nontrnsgenic mice hd begun to develop n immune response tht ws mximl by dy 21. By contrst, the trnsgenic mice did not produce insulin-specific ntibodies t ny time. In ddition, no insulin-specific ntibodies were induced in trnsgenic mice injected with humn insulin nd CFA in the footpd. A secondry injection of the trnsgenic mice with humn insulin lso did not elicit response. These dt demonstrte tht the kinetics nd the route of immuniztion did not effect the response. Thus, the trnsgenic mice disply complete nd long lived tolernce to humn insulin. Tolernce to humn proinsulin in trnsgenic mice. The next issue we exmined ws whether trnsgenic mice were lso tolernt to proinsulin. Proinsulin is the precursor of insulin nd is composed of single polypeptide chin contining the A nd B chins of insulin plus connecting peptide (C-peptide) (14). Proinsulin is enzymticlly cleved in clthrin-coted erly secretory vesicles (15), producing equimolr concentrtions of insulin nd C-peptide, both of which re secreted. Humn proinsulin elicited insulin-specific ntibodies in norml (H-2k"h) mice but not in the trnsgenic mice (Fig. 4 A). Similr results were lso found in H-2dfk trnsgenic mice (Fig. 4 B), gin demonstrting tht this effect ws not unique to Tolernce in Trnsgenic Mice 1551

3 ). ). Co.2 ID Q1 en.1.,.. '3 \I A) Nontrdnsgebnlc;.6 I.5 \II z \--.1.o.1 C-ft. 1 1, 1, 1/Dilution.7 - B) Trnsgenic.6 [.5 [.4 F.3 F.2 F 1 1, 1, Figure 3. Kinetics of the primry ntibody response. H-2b/k mtched nontrnsgenic (A) nd trnsgenic (B) littermtes were injected intrperitonelly with 5,g of humn insulin in CFA. Ser were collected on dys 7, 14, nd 21 nd ssyed for insulin-specific ntibodies by ELISA. The dt ws normlized to stndrd ntibody nd expressed s the reltive OD±SEM t vrious titers. Preimmune ser from trnsgenic nd nontrnsgenic mice hd reltive OD..1, which is indicted by the dshed line. The SEM for ll trnsgenic smples nd dy 7 of the nontrnsgenic smples were negligible, nd thus not included. one strin of mouse. We lso exmined these ser using proinsulin-coted ELISA pltes. No proinsulin-specific ntibodies were found in trnsgenic mice injected with humn proinsulin, wheres ser from nontrnsgenic mice bound to pltes sensitized with either insulin or proinsulin (Fig. 5). Proinsulin is composed of epitopes tht re shred with insulin nd C-peptide, rising the possibility tht tolernce to proinsulin ws ctully induced by insulin nd/or C-peptide, which re secreted rther thn the nonsecreted proinsulin. This possibility could not be investigted in the trnsgenic mice. However, we hve previously shown tht insulin-specific tolernce cn be induced in responder strins of mice by intr- CD % '7 A %I_>--; U. 1.5 C 1. >1 ~ - c..5. c) CL IF' - A) H-2k/b (7) T F (8) >.,,, r B) H-2d/k.5 H (5) (5).,,T,,. Figure 4. Primry responses to humn proinsulin. Norml (htched br) nd syngeneic trnsgenic (dotted br) mice were injected intrperitonelly with 5,ug humn proinsulin in CFA. Ser were collected on dy 21 nd ssyed for insulin-specific ntibodies by ELISA. The dt ws normlized to stndrd ntibody nd expressed s the reltive OD±SEM t 1:1 dilution of serum. The numbers in prentheses represent the numbers of mice per group. Norml H-2k/b mice re (BIO.BR X BIO)FI (A) nd H-2d/k re (BALB/c X CBA/N)Fl (B). ) c. Q -. C: CL - C) 2.1.6r o.5 ~.4 I.- D.3 Z.2 CO) *'.1 co CS I- 1 VV I I 1 Proinsulin (Mg/ml) Insulin (Mg/ml) Figure 5. Proinsulin stimultes proinsulin nd insulin-specific ntibodies. Norml BALB/c (solid circles) nd trnsgenic H-2k/l (open circles) mice were injected intrperitonelly with 5 Mg humn proinsulin in CFA. Ser were collected on dy 21 nd ssyed for ntibodies on ELISA pltes sensitized with the indicted concentrtions of humn proinsulin or humn insulin. The dt re expressed s the reltive OD±SEM for 1:1 dilution of the serum smple. venous injection of insulin (16). Tolernce is highly ntigen specific, it requires bout 1 wk to develop fully nd it lsts for - 4 wk (16). Therefore, we could use this system to determine whether the secreted proteins, insulin nd C-peptide, could induce tolernce to proinsulin in responder mice seprtely or in combintion. Norml (BlO.BR X BlO)F1 or BALB/c mice were pretreted with n intrvenous injection of humn C- peptide, humn proinsulin, humn insulin, or mixture of humn insulin nd C-peptide. 7 d lter ll mice were chllenged with humn proinsulin in CFA nd the PFC response ws mesured (Tble I). Injection of humn proinsulin tolerized mice to subsequent chllenge of proinsulin (66-82% inhibited), which confirms this model system. Insulin hd vrible effect cusing inhibition rnging from 18 to 75% nd C-peptide hd virtully no effect on responses to proinsulin. Most striking ws the finding tht pretretment with the mixture of insulin nd C-peptide inhibited the response to proinsulin s well s pretretment with proinsulin. Tble L In Vivo Tolernce to Humn Proinsulin Percent response to proinsulin in mice pretreted with: Experiment Proinsulin Insulin C-Peptide Insulin plus C-peptide 1* 34±11 82±1 1 N.D. 2$ 18±12 25±5 7±6 7±3 3* 18±4 44± ±4 Norml (BALB.K X BALB.B)F1 (experiment 1) or BALB/c (experiments 2 nd 3) mice were injected with PBS (control) or 1,g humn proinsulin, insulin, C-peptide, or both insulin nd C-peptide. After 7 d, ll mice were chllenged in the footpd with 5 tig of humn proinsulin in CFA. 14 d lter, lymph nodes were ssyed for insulin-specific PFC responses. The dt is expressed s the percentge of the control response±sem to proinsulin fter PBS pretretment. Control responses rnge from 559 to 1,238 PFC per 16 cells. * Three mice per group. * Five mice per group. q IF!F 1552 P. J. Whiteley, J. P. Lke, R. F. Selden, nd J. A. Kpp

4 Discussion The dt in this report demonstrte tht trnsgenic mice, which hve circulting physiologicl concentrtions of humn insulin, re tolernt to humn insulin; wheres H-2 identicl, inbred strins nd nontrnsgenic littermtes re not. The lck of n ntibody response in the trnsgenic mice is not due to generlized defect in the immune system, since they responded normlly to beef nd sheep insulin. These observtions provide direct evidence to support the hypothesis tht under physiologicl conditions, tolernce to circulting self-proteins is cquired. Trnsgenic mice were lso tolernt to pork insulin, suggesting tht the repertoire of T cells specific for exogenous ntigens is shped by cquired tolernce to utologous proteins. Furthermore, cquired tolernce ws not uniquely ssocited with prticulr MHC clss II ntigen, s two different strins of trnsgenic mice gve the sme results. Becuse the strins of mice used s recipients of the trnsgene normlly produce insulin-specific ntibodies in response to humn insulin, the trnsgenic mice clerly express l ntigens tht cn properly ssocite with humn insulin. The trnsgenic mice lso hve the pproprite genes to produce T cell receptors expressing V nd VJ segments, which cn bind complex of processed humn insulin in ssocition with I ntigens becuse the recipients of the insulin gene hve T cells tht do so. Thus, cquired tolernce involves functionl ttenution of the T cells specific for humn insulin. Furthermore, only the T cells nd not the B cells from trnsgenic mice re tolernt to humn insulin (mnuscript in preprtion). Our experiments lso were designed to exmine immune responsiveness to humn proinsulin. Development of n ntibody response to n utologous orgn-specific, intrcellulr protein such s proinsulin would be expected if sequestered ntigens fil to tolerize T cells with the pproprite receptors. If this were true, then the trnsgenic mice should develop norml immune response to humn proinsulin, but they did not. Tolernce to proinsulin might be induced by the very smll mount of proinsulin tht reches the circultion which hs been estimted t 1-5% of the level of insulin (17). However, proinsulin is composed of epitopes tht re shred with insulin nd C-peptide, both of which re secreted proteins rising the possibility tht tolernce to proinsulin ws induced by the secreted frgments of proinsulin. Our experiments demonstrted tht neither C-peptide nor insulin lone could induce the sme degree of tolernce to humn proinsulin s chieved by pretretment with proinsulin in nontrnsgenic mice. However, when both were dministered together, tolernce ws virtully complete. The simplest interprettion of these results is tht proinsulin stimultes insulin-specific nd C-peptide specific helper T cells, either one of which cn interct with B cells in the development of n ntibody response to proinsulin. Ifthe helper T cells re in excess, then loss ofone or the other popultion of helper T cells would not significntly diminish the ntibody response, wheres loss of both sets would brogte the response. These results suggest tht tolernce to proinsulin in trnsgenic mice is most likely induced by the combined effects of the secreted insulin nd C- peptide. Tolernce hs been reported for cell-surfce ntigen in trnsgenic mice bering n llogeneic clss II MHC ntigen fused to the rt insulin promotor (18). Although the trnsgenic I-E ntigen ws synthesized only in the bet cells of the pncres nd the kidney, the recipient mice were tolernt to the I-E ntigens of the donor hplotype s mesured in mixed lymphocyte response. This dt nd ours demonstrte tht ntigens tht re not expressed by cells in the thymus cn, nevertheless, induce tolernce; however, the mechnism responsible remins to be determined. Like tolernce to MHC clss II, Mls, nd H-Y ntigens, tolernce to these ntigens lso could be medited by clonl deletion in the thymus; but if so, it is very sensitive process s only very smll quntities ofthese proteins enter the circultion nd ultimtely rech the thymus. Alterntively, the clones of T cells which recognize these ntigens might not be deleted in the thymus s we hve shown to be the cse for mouse insulin-specific helper T cells (19, 2). Autoimmune disese could still be verted if such utorective T cells re rendered nergic (21-23) or held in check by suppressor T cells (1 1, 19, 2, 24, 25). The trnsgenic mice expressing humn insulin provide model system for determining the physiologicl mechnisms responsible for induction nd mintennce of self-tolernce. If gene therpy is going to be used to correct genetic bnormlities, it will be importnt to hve such models to study the immunologicl consequences of expressing foreign genes before such therpy is tried in humns. Acknowledgments We thnk Dr. Ron E. Chnce, Eli Lilly, for the generous gifts of purified recombinnt humn insulin, proinsulin, nd C-peptide s well s his continued interest in our work; Mr. John Lnpher, Squibb Novo, for the generous gift of semi-synthetic humn insulin; Dr. Ron Gingerich, Wshington University, for humn C-peptide determintions; Drs. Ver Huptfeld, Miro Huptfeld, nd Donld Shreffier, Wshington University Medicl School, for H-2 typing regents. This work ws supported by the U. S. Public Helth Service grnts AI nd 2 P6 DK2 S79-12, from the Ntionl Institutes of Helth. References 1. Kppler, J. W., N. Roehm, nd P. Mrrck T cell tolernce by clonl elimintion in the thymus. Cell. 49: Kppler, J. W., U. Sterz, J. White, nd P. Mrrck Self-tolernce elimintes T cells specific for Mls-modified products of the mjor histocomptibility complex. Nture (Lond.). 332: Kisielow, P., H. Bluthmnn, U. D. Sterz, M. Steinmetz, nd H. von Boehmer Tolernce in T-cell-receptor trnsgenic mice involves deletion of nonmture CD4+8' thymocytes. Nture (Lond.). 333: Siskind, G Immunogic Tolernce. In Fundmentl Immunology. W. E. Pul, editor. Rven Press, New York Selden, R. F., M. J. Skoskiewicz, K. B. Howie, P. S. Russell, nd H. M. Goodmn Regultion of humn insulin gene expression in trnsgenic mice. Nture (Lond.). 321: Stimpfling, J. H The use of PVP s developing gent in mouse hemgglutintion tests. Trnsplnt. Bull. 27: Kpp, J. A., nd D. S. Stryer H-2 linked Ir gene control of ntibody responses to porcine insulin. I. Development of insulinspecific ntibodies in some but not ll nonresponder strins injected with proinsulin. J. Immunol. 121: Jensen, P. E., C. W. Pierce, nd J. A. Kpp Regultory mechnisms in immune responses to heterologous insulins. II. Suppressor T cell ctivtion ssocited with nonresponsiveness in H-2b mice. J. Exp. Med. 16: Jensen, P. E., nd J. A. 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