An Altered Self-Peptide with Superagonist Activity Blocks a CD8-Mediated Mouse Model of Type 1 Diabetes

This information is current as Agnès Hartemann-Heurtier, Lennart T. Mars, Nadège of September 29, 2021. Bercovici, Sabine Desbois, Christophe Cambouris, Eliane Piaggio, Jacques Zappulla, Abdelhadi Saoudi and Roland S. Liblau J Immunol 2004; 172:915-922; ;

doi: 10.4049/jimmunol.172.2.915 Downloaded from http://www.jimmunol.org/content/172/2/915

References This article cites 45 articles, 16 of which you can access for free at: http://www.jimmunol.org/content/172/2/915.full#ref-list-1 http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists by guest on September 29, 2021 • Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

An Altered Self-Peptide with Superagonist Activity Blocks a CD8-Mediated Mouse Model of Type 1 Diabetes1

Agne`s Hartemann-Heurtier,*† Lennart T. Mars,*‡ Nade`ge Bercovici,2* Sabine Desbois,*‡ Christophe Cambouris,2* Eliane Piaggio,*‡ Jacques Zappulla,*‡ Abdelhadi Saoudi,‡ and Roland S. Liblau3*‡

T cell tolerance can be experimentally induced through administration of self-peptides with single amino acid substitution (altered peptide ligands or APLs). However, little is known about the effects of APLs on already differentiated autoreactive CD8؉ T cells that play a pivotal role in the pathogenesis of autoimmune diabetes. We generated a panel of APLs derived from an influenza virus -hemagglutinin peptide exhibiting in vitro functions ranging from antagonism to superagonism on specific CD8؉ T cells. A su peragonist APL was further characterized for its therapeutic activity in a transgenic mouse model of type 1 diabetes. When injected i.v. 1 day after the transfer of diabetogenic hemagglutinin-specific CD8؉ T cells into insulin promoter-hemagglutinin Downloaded from transgenic mice, the superagonist APL proved more effective than the native hemagglutinin peptide in blocking diabetes. This protective effect was associated with an inhibition of CD8؉ T cell cytotoxicity in vivo and with a decreased accumulation of these cells in the pancreas, leading to a marked reduction of intrainsulitis. In conclusion, a superagonist “self-peptide” APL was more :effective than the native peptide in treating a CD8؉ T cell-mediated diabetes model. The Journal of Immunology, 2004, 172 915–922. http://www.jimmunol.org/

ype 1 diabetes is the end result of an autoimmune disorder (10–13). APLs bound to MHC molecules can induce both quan- in which the pancreatic ␤ islet cells are selectively de- titatively and qualitatively different signals in the responding T T stroyed by the immune system (1–3). Among the thera- cells as compared with the native peptide and are categorized ac- peutic strategies aimed at specifically inhibiting the pathogenic cording to their biological activity (14). peptides induce autoimmune response without affecting the protective function of proliferation, cytokine release, and/or cytotoxicity by the activated the immune system, Ag-specific immunotherapies have shown T cells. Weak similarly elicit the full spectrum of T cell promise in animal models of type 1 diabetes. These approaches functions but at a higher concentration than the agonist peptide.

rely on the administration of self-Ags or self-peptides binding to Partial agonists promote some but not all T cell functions and, by guest on September 29, 2021 MHC molecules to silence potentially pathogenic T cells via ap- therefore, may alter the pattern of cytokine production (10) or lead optosis, anergy, immune deviation, and/or dominant mechanisms to anergy (15). Some APLs, which fail to induce any obvious T of immunoregulation. As a result, systemic or mucosal adminis- cell activation, can behave as TCR antagonists as they inhibit T tration of soluble self-Ags has been shown to prevent diabetes in cell responses to agonist peptides when both ligands are recog- experimental models (4–9). A related strategy is based on the ad- nized simultaneously by the T cells (16). Finally, superagonist or ministration of antigenic self-peptides with a single amino acid heteroclitic peptides stimulate T cell responses at lower concen- substitution at important TCR contact residues (referred to as al- trations than the native peptide (17, 18). tered peptide ligands or APLs4). This approach has been success- Because of their ability to modulate T cell responses, APLs have fully applied in several rodent models of autoimmune diseases an obvious potential as Ag-specific immunotherapeutic tools (19, 20). In that respect, the ability of some MHC class II-binding APLs to promote regulatory Th 2 cell populations has been used in ϩ *Institut National de la Scientifique et de la Sante´Recherche Me´dicale Unite´546, rodents to control pathogenic autoreactive CD4 T cells and Faculte´deMe´decine Pitie´-Salpeˆtrie`re, Paris, France; †Department of Diabetology and thereby prevent or treat experimental autoimmune diseases such as Metabolism, Pitie´-Salpeˆtrie`re Hospital, Paris, France; and ‡Institut National de la Scientifique et de la Sante´Recherche Me´dicale Unite´563, Purpan Hospital, Toulouse, experimental autoimmune encephalomyelitis (EAE) or diabetes in France nonobese diabetic (NOD) mice (10–13). ϩ ϩ Received for publication March 1, 2003. Accepted for publication November 4, 2003. In type 1 diabetes, both the CD4 and CD8 T cell subsets are The costs of publication of this article were defrayed in part by the payment of page involved in the destructive autoimmune process, with the autore- ϩ charges. This article must therefore be hereby marked advertisement in accordance active CD8 T cells playing a pivotal role during all phases of with 18 U.S.C. Section 1734 solely to indicate this fact. disease development (21–23). Therapeutic strategies aimed at 1 This work was supported by the French Biomedical Research Institute (Institut blocking these CD8ϩ T cell-dependent events could, therefore, National de la Scientifique et de la Sante´ Recherche Me´dicale), the French AIDS Research Agency (Agence Nationale de Recherches sur le SIDA), and the Juvenile prevent or inhibit disease progression. On this basis, MHC class Diabetes Foundation International. L.T.M. and E.P. were supported by postdoctoral I-binding self-peptides have been injected systemically to target fellowships from the Association pour la Recherche sur la Scle´rose en Plaques (The pathogenic autoreactive CD8ϩ T cells either before or during an French Multiple Sclerosis Society) and French Biomedical Research Foundation (Fondation pour la Recherche Me´dicale), respectively. ongoing autoimmune process in both NODs (23) and transgenic 2 Current address: Immuno-Designed Molecules, Paris, France. 3 Address correspondence and reprint requests to Prof. Roland S. Liblau, Institut National de la Scientific et de la Sante´Recherche Me´dicale Unite´563, Purpan Hospital, 4 Abbreviations used in this paper: APL, altered peptide ; EAE, experimental Place Dr Baylac, Toulouse 31000, France. E-mail address: rolandliblau@hotmail. autoimmune encephalomyelitis, HA, hemagglutinin; Ins, Insulin; NOD, nonobese di- com abetic; AICD, activation-induced cell death.

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 916 A SUPERAGONIST SELF-PEPTIDE BLOCKS CD8-MEDIATED DIABETES mouse models of autoimmune diabetes (24, 25). Despite this suc- Adoptive transfer of diabetes and peptide treatment cessful application of self-peptides in models of type 1 diabetes, Diabetogenic TC1 cells were generated from CL4-TCR mice as previously strategies involving MHC class I-binding APLs in vivo have not described (26). Briefly, purified naive CD8 T cells were stimulated in vitro received much attention (23). Moreover, little is known about the with HA512–520 peptide-pulsed irradiated splenocytes in the presence of effect of APLs on already differentiated CD8ϩ T cells, an impor- IL-2 (1 ng/ml) and IL-12 (20 ng/ml; a kind gift from Institute, tant parameter if the APL treatment is intended to control an on- Cambridge, MA). After 6 days of culture, the live cells were isolated using a Ficoll gradient and directly used for adoptive transfer experiments. Non- going disease in predisposed individuals or even in recently diag- irradiated 6- to 8-wk-old Ins-HA recipients received 2 ϫ 106 HA-specific nosed patients. To specifically address this question, we generated TC1 i.v. in 0.2 ml of PBS. Adoptively transferred mice were treated with d APLs from a K -restricted influenza virus hemagglutinin (HA) one i.v. injection of Cw3, HA512–520, or APL peptide 24 h posttransfer. Glycosuria was assessed three times a week for 30 days using test strips peptide (HA512–520), which were functionally characterized in ϩ (Glukotest; Roche Diagnostic, Mannheim, Germany). Diabetes was con- vitro by their ability to trigger CD8 T cells expressing a trans- firmed by measurement of blood glucose using a One Touch Ultra glu- genic TCR specific for HA512–520. Then, we set out to test the cometer (Lifescan, Milpitas, CA). Mice were considered diabetic if blood efficacy of a superagonist APL in controlling autoimmune diabetes glucose levels were above 2 g/L on two consecutive tests. For histological induced by the adoptive transfer of activated HA-specific CD8ϩ T analyses, pancreata were fixed in formol solution and processed for paraffin cells into rat insulin promoter-HA-transgenic recipient mice (26). embedding. Sections were stained with H&E and the degree of insulitis was evaluated microscopically. Materials and Methods In vivo CD8ϩ T cell tracking Mice Activated Thy1.1 diabetogenic CD8ϩ T cells were labeled for 10 min with ␤ ␣ 5 ␮M CFSE (Molecular Probes, Eugene, OR), washed, and transferred i.v. Downloaded from CL4-TCR (V 8.2, V 10.3) mice are transgenic for an HA512–520 (IYS- 6 d into Ins-HA mice (2 ϫ 10 cells/mouse). On day 1 posttransfer, Ins-HA TVASSL)-specific, H-2K -restricted TCR (27). Ins-HA mice express HA ␮ mice were injected with 0.5 g peptide (Cw3, HA512–520, or APL). Four specifically in the pancreatic ␤ islet cells (28). Hemizygous CL4-TCR and ϩ Ins-HA mice were backcrossed at least eight times onto the BALB/c ge- days posttransfer, transferred CD8 T cells were detected and enumerated netic background. In addition, CL4-TCR mice were crossed at least twice in lymphoid tissue by virtue of their Thy1.1 expression. with congenic Thy1.1 BALB/c mice. Mice were bred and manipulated in In vivo cytotoxicity assay a specific pathogen-free animal facility (Salpeˆtrie`re Medical School, Paris,

France) in keeping with the European Union legislation on animal care. To prepare target cells, BALB/c spleen cells were pulsed with either 1 ␮M http://www.jimmunol.org/ HA512–520 or Cw3 peptide (2 h at 37°C), washed, and labeled with CFSE Peptides at a final concentration of 5 ␮M for HA-loaded cells or 1 ␮M for Cw3- loaded cells. A combination of 20 ϫ 106 HA-pulsed and 10 ϫ 106 Cw3- All APLs (Ͼ70% pure) and Cw3 (RYLKNGKETL), a control Kd-binding pulsed target cells were injected i.v. into Ins-HA mice that had received peptide, were purchased from Chiron (Melbourne, Australia). For further in unlabeled 2 ϫ 106 TC1 96 h earlier and had been treated with peptide vitro and in vivo analyses, the HA , S514I, A517G, and Cw3 peptides 512–520 (Cw3, HA , or APL) 72 h earlier. Control Ins-HA mice received (Ͼ95% pure) were synthesized by the Neosystem Laboratory (Strasbourg, 512–520 CFSE-labeled splenocytes but did not receive TC1 cells nor peptide. France). Spleens from Ins-HA recipient mice were removed 18 h after the injection Measurement of peptide binding to Kd of target cells and a single-cell suspension was prepared. The number of cells with high fluorescence intensity (HA loaded) or moderate fluores- Binding of peptides to Kd was measured in a competition assay with the cence intensity (Cw3 loaded) was determined by FACS analysis (30). by guest on September 29, 2021 radiolabeled S9I peptide (SYIPSAEKI) with known affinity for the Kd molecule (29). The concentration of competitor HA peptide resulting in Results 50% inhibition of the binding of the probe peptide (IC50) was determined. Identification of altered peptide ligands The relative affinity of a given APL for Kd is the APL IC :wild-type HA 50 ϩ peptide IC50 ratio. To generate APLs able to modulate a diabetogenic CD8 T cell response, we first identified the residues of the HA peptide T cell proliferation 512–520 that are in contact with the CL4-TCR when presented in the con- Spleen single-cell suspensions from CL4-TCR-transgenic mice were stim- text of Kd. A panel of peptide analogues was generated by single ϫ 5 ulated with increasing concentrations (0–3 .10 nM) of peptide in com- amino acid substitutions at all positions except for the canonical plete RPMI 1640 supplemented with 10% FCS (Life Technologies, Pais- d ley, U.K.) for 42 h in 96-well flat-bottom microtiter plates (106 cells/well K anchor residues Y513 and L520. Each residue was substituted in 200 ␮l). Proliferation was measured by [3H]thymidine (Amersham, Ar- with either the small alanine amino acid (except for alanine at lington Heights, IL) incorporation for the last 18 h of culture. SEMs were position 517, which was changed for glycine) or the bulky isoleu- Ͻ consistently 10% of the mean. cine or leucine amino acid. Cytokine measurement and flow cytometry We then measured the ability of these peptide analogues to bind the Kd molecule. All peptides have a Kd-binding affinity similar to Cytokine production was assessed following stimulation of 105 naive CD8ϩ T cells (isolated using MACS beads to typically Ͼ98% purity) with that of the wild-type peptide, with a relative affinity ranging from 106 irradiated BALB/c spleen cells with 3 ϫ 10Ϫ1–3 ϫ 103 nM peptide in 0.4 to 2 (Table I and Fig. 1A). This allowed us to compare the 200 ␮l of complete RPMI 1640 medium. In ex vivo experiments, spleno- capacity of the wild-type and the mutated peptides to elicit the 6 cytes from Ins-HA recipient mice (10 cells/ml) were cultured with or proliferation of naive CL4-TCR CD8ϩ T cells. As shown in Table without 1 ␮⌴ peptide. Supernatants were collected after a 24-, 48-, or 72-h incubation, aliquoted, and stored at Ϫ20°C until use. Cytokine determina- I, an alanine or an isoleucine substitution at positions 516, 518, and tion was assessed by sandwich ELISA as previously described (26). The 519 moderately reduced the ability of the mutated peptide to trig- detection limit of the ELISA was 40 pg/ml for IFN-␥, 25 pg/ml for IL-4, ger T cell proliferation, indicating that these positions are not es- and 30 pg/ml for IL-10. sential TCR-contact residues (Table I and data not shown). In con- Expression of T cell activation markers was assessed after a 24-h in vitro ϫ 6 trast, substituting the threonine at position 515 for either an alanine stimulation of 5 10 /ml spleen cells from CL4-TCR mice with increas- Ͼ ing concentrations of peptide. Triple staining was performed using PE- (T515A) or an isoleucine (T515I) resulted in a drastic ( 100,000- conjugated anti-CD4 mAb, FITC-conjugated anti-CD8 mAb (Caltag Lab- fold) decrease in agonist activity. Further mutated peptides re- oratories, Burlingame, CA), and biotinylated mAbs (BD PharMingen, San vealed that among the few tolerated substitutions at position 515 Diego, CA) specific for mouse CD25 (clone 7D4), CD69 (clone HI.2F3), was the most conservative T515S change (Table I). In contrast, CD62-L (clone Mel-14), or CD90.1 (Thy1.1) followed by streptavidin-TC (Caltag Laboratories). Cells were acquired on a FACScan flow cytometer T515F and T515D induced little or no proliferative response (Fig. (BD Biosciences, Mountain View, CA) and analyzed using CellQuest (BD 1B). Albeit to a lesser extent, similar observations were made for Biosciences). position 514, at which a variable agonist activity was obtained The Journal of Immunology 917

Table I. Altered peptide ligands for the CL4-TCRa

Peptide Sequence Relative MHC Binding EC50

HA512–520 IYSTVASSL 1 410 pM I512A A------ND 2 ␮M I512V V------1.1 220 nM S514Q --Q------0.9 415 pM S514G --G------1.1 450 pM S514V --V------2.0 30 nM S514T --T------ND 35 nM S514A --A------1.2 310 nM S514L --L------1 Ͼ300 ␮M S514P --P------1.2 Ͼ300 ␮M S514D --D------1.1 None S514I --I------1.1 None T515P ---P----- 1 405 pM T515S ---S----- 0.8 2 nM T515F ---F----- 0.4 Ͼ300 ␮M T515A ---A----- 1.1 Ͼ300 ␮M T515I ---I----- 0.9 None T515V ---V----- 1.2 None T515L ---L----- 1.3 None Downloaded from T515D ---D----- 0.7 None T515Q ---Q----- 1.2 None V516A ----A---- 1 215 nM V516T ----T---- ND 42 nM V516I ----I---- 1.1 2 nM A517G -----G--- 0.9 15 pM

A517V -----V--- 1.1 2.5 ␮M http://www.jimmunol.org/ A517S -----S--- ND 3 ␮M S518A ------A-- 0.8 250 nM S518T ------T-- ND 20 nM S519A ------A- 13nM S519T ------T- 0.8 30 nM S519I ------I- 1.1 190 nM

a The relative affinity of various peptides for the Kd molecule was determined by competition with binding of the 125I-labeled S9I peptide to Kd. The relative affinity is calculated based on the concentration of the tested peptide resulting in 50% inhibition d of the fixation of the tracer S9I peptide to K (IC50) and is expressed as the APL IC50:wild-type HA peptide IC50 ratio (the higher the ratio, the lower the affinity of the tested peptide for the Kd molecule). The agonist activity of the peptides was determined by guest on September 29, 2021 in vitro by stimulating splenocytes from CL4-TCR mice with increasing concentrations of peptide (0–3 ϫ 105 nM) and is expressed as the EC50, i.e. the concentration of peptide capable of stimulating T cell proliferation at a level equivalent to 50% of the maximum level obtained with the HA512–520 peptide. b None, no proliferation with 300 ␮M peptide concentration. depending on the nature of the substituting amino acid: S514A is without altering the maximum proliferative response (Fig. 2A). a weak agonist whereas S514I is unable to induce proliferation Since the A517G peptide has a nearly identical affinity for the Kd (Table I). Fig. 1B shows representative T cell proliferative re- molecule to that of the wild-type peptide (relative affinity: 0.9) the sponses with 514 or 515 mutated peptides. Taken together, these superagonist activity is attributable to improved interactions of the results suggest that these residues are in direct contact with the Kd-peptide complex with the CL4-TCR following removal of a CL4-TCR; position 515 fulfils the criteria for a primary TCR con- methyl group. tact residue (14), while position 514 is likely an important sec- To further investigate the superagonist properties of the A517G ondary TCR contact residue. We therefore investigated whether peptide, the expression of activation markers on CD8ϩ T cells was the nonagonist peptide mutants at position 514 or 515 exhibited assessed after a 24-h in vitro stimulation with peptide. As shown antagonist properties. A reproducible antagonist activity was only in Fig. 2B, 0.3 nM A517G induced the expression of CD69 and observed for peptide S514I (data not shown). CD25 on most CD8ϩ T cells, whereas Ͻ50% of the CD8ϩ T cells Interestingly, substituting the alanine at position 517 with a expressed these molecules when stimulated with a 10-fold higher bulky residue such as valine (A517V) or serine (A517S) reduced concentration of the native HA peptide. The CD62-L down-regu- the agonist activity, while shortening its side chain by introducing lation induced by either peptide was, however, similar (data not a glycine (A517G) significantly enhanced the agonist capacity (Ta- shown). ble I). This would suggest that, in a similar manner to positions To test whether cytokine production would be influenced by the 514 and 515, the side chain of residue 517 is available to contact HA peptide analogue, we measured the release of IFN-␥, IL-4, and the TCR. However, this contact appears to inhibit, rather than sup- IL-10 by CD8ϩ T cells activated by HA peptide- or A517G-pulsed port, the productive interaction of the Kd-peptide complex with the APCs. As shown in Fig. 2C, a 10-fold higher concentration of CL4-TCR, giving rise to enhanced proliferation when the 517 side A517G (30 nM) compared with HA (3 nM) was required to induce chain is trimmed. an optimal production of IFN-␥, suggesting that the superagonist A517G elicits a modified signal. Nevertheless, the cytokine profile A517G is a superagonist peptide of the CD8ϩ T cells was not shifted toward a TC2 type since The A517G peptide has a higher agonistic activity than the wild- neither IL-10 nor IL-4 production was detected (data not shown). type HA peptide on the CL4-TCR CD8ϩ T cells, shifting the pro- The in vitro response of preactivated HA-specific TC1 cells to liferation dose-response curve to the left by a factor of 10–30, but the A517G superagonist and to the wild-type HA peptide was also 918 A SUPERAGONIST SELF-PEPTIDE BLOCKS CD8-MEDIATED DIABETES

assessed. As shown in Fig. 2D, the spontaneous proliferation of TC1 cells is abrogated by stimulation with the HA peptides or an agonistic anti-CD3 mAb, most likely through induction of activa- tion-induced cell death. Here again, the dose-response curve in response to the A517G peptide is clearly shifted to the left as compared with that elicited by the wild-type HA peptide. How- ever, the plateau reached at high peptide concentrations is similar.

A517G is more effective than the native HA peptide in blocking diabetes in Ins-HA mice

We have previously shown that i.v. injection of HA512–520 peptide ϫ prevents or blocks diabetes in (Ins-HA CL4-TCR)F1 mice (25). Since the A517G peptide appeared more potent than the wild-type HA peptide in vitro, we tested its in vivo capacity to modulate diabetes induced by transfer of 2 ϫ 106 HA-specific TC1 cells into adult, nonirradiated Ins-HA-transgenic recipients. This protocol reproducibly induces a lethal diabetes with a 100% incidence (26). One day after T cell transfer, the recipient mice received a single Downloaded from i.v. dose of either Cw3, HA512–520, or A517G peptide. High ␮ ϭ HA512–520 peptide doses (25 g, n 7 mice, data not shown) or 2.5 ␮g (Fig. 3A) completely blocked diabetes (lethal diabetes in all 9 Cw3-treated mice vs 0 of 10 HA512–520-treated mice; p ϭ Ϫ4 FIGURE 1. Properties of selected APLs with substitutions at position 10 , Fisher’s exact test). A similar protective effect was observed 514 or 515. A, The affinity of individual APLs for the Kd molecule was with a single i.v. dose of 25 ␮g(n ϭ 7 mice, data not shown) or determined as described in Materials and Methods. Two independent ex- 2.5 ␮g (Fig. 3A) of A517G, demonstrating that high doses of both http://www.jimmunol.org/ periments were performed with similar results. B, Proliferation of splenic peptides are similarly potent in preventing diabetes. T cells from CL4-TCR mice that were stimulated with increasing concen- Strikingly, with a dose of 0.5 ␮g the effect was quite different trations of HA or APLs. Results are expressed as the mean of 512–520 between HA and A517G peptides. Indeed, 11 of 12 mice [3H]thymidine uptake obtained in triplicate. Two independent experiments 512–520 injected with 0.5 ␮gHA peptide developed persistent dia- were performed with similar results. 512–520 betes, which proved lethal in four cases (Fig. 3B). In contrast, only 1 of 12 mice injected with the superagonist peptide developed diabetes during the 30-day follow-up period (Fig. 3B, p ϭ 10Ϫ4). by guest on September 29, 2021

FIGURE 2. Functional characteristics of A517G, a superagonist HA peptide analogue. A, Proliferation of splenic T cells from CL4-TCR mice that were 3 stimulated with increasing concentrations of HA512–520 or A517G peptide. Results are expressed as the mean of [ H]thymidine uptake obtained in triplicate and are representative of four independent experiments. B, Cell surface phenotype of CD8ϩ T cells. Spleen cells from CL4-TCR mice were stimulated with the indicated peptide concentration for 24 h, then labeled with fluorescent mAbs and analyzed by flow cytometry. The histograms, gated on the CD8ϩ T ␥ ϩ cell population, are representative of three independent experiments. C, IFN- production of CD8 T cells from CL4-TCR mice stimulated with HA512–520 or A517G peptide. CD8ϩ T cells were purified from CL4-TCR mice and cultured for 48 h in the presence of syngeneic irradiated APCs without or with increasing concentrations of peptide. IFN-␥ concentration in the supernatants was determined by ELISA. The data are representative of two independent experiments. Results are expressed as the mean of duplicates with a SEM of Ͻ10%. D, Proliferation of TC1 cells, isolated through a Ficoll gradient at day

6 of culture, following restimulated in vitro for 48 h in the presence of irradiated splenocytes pulsed with increasing concentrations of HA512–520 or A517G peptide or in the presence of 0.5 ␮g/ml anti-CD3 mAb. The Journal of Immunology 919

protect against CD8ϩ T cell-mediated diabetes. However, the su- peragonist is significantly more effective at low doses. Mechanisms underlying the therapeutic effect of low-dose superagonist peptide To investigate the mechanisms by which the superagonist peptide achieved its superior therapeutic effect, we assessed the survival, expansion, and functional properties of the transferred ␤ cell-spe- cific CD8ϩ T cells following superagonist treatment. T cell survival and expansion were investigated using CFSE- labeled, Thy1.1 HA-specific TC1 transferred i.v. into congenic Thy1.2 Ins-HA mice (2 ϫ 106 TC1/mouse). Mice were treated ␮ 24 h later with a single dose of 0.5 g of Cw3, HA512–520,or A517G peptide. Four days posttransfer, the proportion of CD8ϩ T cells from donor origin (Thy1.1ϩ) in the pancreatic lymph nodes of HA-treated (n ϭ 4) or A517G-treated mice (n ϭ 5) was reduced to 42.4 Ϯ 12.0% and 36.7 Ϯ 5.4% of the value of Cw3-treated mice, respectively (Fig. 5A). This reduction was not different be-

tween the HA- and A517G peptide-treated groups ( p ϭ 0.65, un- Downloaded from paired Student’s t test). The reduction was even more pronounced in the spleen of HA-treated (32.2 Ϯ 4.7%) and A517G-treated (22.5 Ϯ 2.7%) animals ( p ϭ 0.12, comparing HA-treated and FIGURE 3. Blockade of adoptive transfer of diabetes in superagonist or A517G-treated groups). As expected, the preactivated TC1 cells agonist peptide-treated mice. Nonirradiated adult Ins-HA recipient mice proliferated vigorously following transfer into Ins-HA mice. Be- 6 ϩ were injected i.v. with 2 ϫ 10 activated HA-specific CD8 T cells and fore transfer, the activated TC1 cells were brightly labeled with http://www.jimmunol.org/ 24 h later received the indicated quantity of peptide. Glycosuria was as- CSFE, whereas 4 days after transfer the vast majority of Thy1.1 sessed three times a week for 30 days and diabetes was confirmed by blood donor cells had completely diluted their CFSE fluorescence in all glucose measurements. A, The animals received 2.5 ␮g of either Cw3 (n ϭ groups (mean percentage of splenic Thy1.1 TC1 cells negative for ϭ ϭ 9), HA512–520 (n 10), or A517G peptide (n 10). The comparison of the CFSE fluorescence: 99.3% in two Cw3-, 98.4% in two HA-, and protective effect of HA and A517G was not significant, p ϭ 0.80, 512–520 98.1% in two A517G-treated mice), indicating a high rate of cell Fisher’s exact test. B, The animals received a single injection of 0.5 ␮g ϭ Ͻ Ϫ4 ␮ division. Collectively, these results suggest that both the wild-type (n 12 mice per group; p 10 )or0.1 g of either HA512–520 or A517G ϩ peptide (n ϭ 8 mice/group; p ϭ 0.46). and the superagonist peptide deleted most transferred CD8 T cells; however, a substantial population of HA-specific Thy1.1 CD8ϩ T cells persisted in both groups. by guest on September 29, 2021 To evaluate the functional properties of these remaining T cells, Moreover, pancreata from additional groups of mice, treated with splenocytes from peptide-treated Ins-HA mice were recovered on 0.5 ␮g of either Cw3, HA , or A517G peptide, were ana- 512–520 day 4 after TC1 cell transfer and cultured with or without the lyzed histologically on day 4 after TC1 transfer. A significantly native peptide for 72 h. A comparable production of IFN-␥ was greater number of islets remained free of infiltration in mice detected in splenocyte cultures from HA512–520- or A517G-treated treated with A517G compared with HA512–520, whereas a lower Ϫ mice, both spontaneously (0.36 Ϯ 0.31 and 0.39 Ϯ 0.37 ng/ml, number of islets exhibited an invading insulitis (Fig. 4, p Ͻ 10 4). respectively; mean Ϯ SEM of three independent experiments) and Thirty days posttransfer, 85% of islets were still free of insulitis in upon 1 ␮MHA peptide restimulation (1.8 Ϯ 1.4 and 1.7 Ϯ A517G-treated animals (data not shown). When the dose of pep- 512–520 1.6 ng/ml, respectively), whereas higher, although not statistically tide was reduced even further, to 0.1 ␮g, the superagonist peptide significantly so, IFN-␥ production was detected in Cw3-treated showed partial therapeutic properties; however, these proved not to animals (0.72 Ϯ 0.88 spontaneously and 3.11 Ϯ 1.68 ng/ml upon be statistically significant (Fig. 3B). Taken together, these data HA peptide restimulation). No IFN-␥ was detected in cultures show that both the native and the superagonist peptide are able to from unmanipulated Ins-HA mice. Neither IL-4 nor IL-10 was

detected spontaneously or upon HA512–520 restimulation (data not shown). These results suggest that the superior protective effect of the superagonist peptide does not rely on a reduced IFN-␥ pro- duction (31) or a switch in the cytokine profile (10–13). We next investigated the in vivo cytotoxic capacity of the trans- ferred TC1 cells after peptide treatment. Ins-HA mice received 2 ϫ 106 preactivated TC1 and were treated on day 1 with 0.5 (Fig. 5B) ␮ or 25 (Fig. 5C) g Cw3, HA512–520, or A517G peptide i.v. On day 4 after TC1 transfer, the mice additionally received a mixture of highly fluorescent (CFSEhigh) target cells loaded with HA peptide and moderately fluorescent (CFSEdull) target cells loaded with Cw3 control peptide (Fig. 5, B and C). The ratio of CFSEhigh to FIGURE 4. Inhibition of insulitis in superagonist peptide-treated dull Ins-HA mice. Ins-HA mice were transferred with 2 ϫ 106 TC1 and treated CFSE cells was determined in the spleen 18 h later. As shown ␮ ϭ ϭ in Fig. 5, B and C, the transferred HA-loaded target cells were on day 1 with 0.5 g Cw3 (n 4; 532 islets), HA512–520 (n 4; 445 islets), or A517G peptide (n ϭ 4; 522 islets). Pancreata were removed on day 4, largely eliminated in TC1 recipients treated with the control pep- and histological analysis was performed after H&E staining. Islets were tide, whereas the Cw3-loaded target population was not affected analyzed by an investigator blinded to the treatment group. (quantitatively comparable to that of mice not injected with TC1), 920 A SUPERAGONIST SELF-PEPTIDE BLOCKS CD8-MEDIATED DIABETES Downloaded from FIGURE 5. In vivo cytotoxic properties of HA-specific CD8ϩ T cells following peptide treatment. A, Ins-HA mice were transferred with 2 ϫ 106 Thy1.1 ␮ TC1 and treated on day 1 with 0.5 g of either Cw3, HA512–520, or A517G peptide. On day 4 posttransfer, pancreatic lymph node cells were recovered and labeled with fluorescent mAbs. The histograms represent the percentage of Thy1.1ϩ cells among CD8ϩ T cells in pancreatic lymph nodes. The data ϫ 6 ␮ are representative of two independent experiments. B, Ins-HA mice were injected with 2 10 TC1, treated on day 1 with 0.5 g of either Cw3, HA512–520, or A517G peptide, and on day 4 received a mixture of HA-loaded spleen cells labeled with high fluorescence intensity (CFSEhigh) and Cw3-loaded spleen cells labeled with moderate fluorescence intensity (CFSEdull). The Ins-HA recipient mice were sacrificed 18 h later, their spleens were harvested, and the two differentially labeled target cell populations were quantified by flow cytometry. Control Ins-HA mice, which were neither injected with TC1 nor treated http://www.jimmunol.org/ with peptide (upper panel), are included. For each animal, the number of HA-loaded target cells was normalized to the number of control Cw3-loaded target cells. The data shown are from one of four independent experiments. C, Same experiment as B but Ins-HA mice were treated on day 1 after TC1 transfer with 25 ␮g of peptide. The data are representative of three independent experiments.

revealing a potent in vivo HA-specific cytotoxicity by transferred A single amino acid substitution, at an important peptide/TCR TC1. Remarkably, following injection of 0.5 ␮g of peptide (Fig. contact position, is sufficient to dramatically alter T cell responses 5B), the killing of HA-loaded cells was of comparable magnitude induced by TCR engagement. Some APLs act as TCR antagonists, by guest on September 29, 2021 ϭ in Cw3- and HA512–520-treated mice ( p 0.6), whereas in inhibiting antigenic peptide-induced T cell functions. In human A517G-treated mice, it was partially yet significantly reduced diabetes, two in vitro studies have attempted to take advantage of (␹2 ϭ 58; p Ͻ 0.0001; n ϭ 5 mice in each group). However, using antagonistic APLs to modulate potentially pathogenic autoreactive the high 25-␮g dose of peptide (Fig. 5C), the killing of HA-loaded CD4ϩ T cells (32, 33). The in vivo therapeutic potential of antag- target cells was greatly reduced and of comparable magnitude in onistic self-peptide analogues has been successfully demonstrated

HA512–520- or A517G-treated mice (Fig. 5C). Interestingly, at the in EAE using disease prevention protocols (34). For potential hu- 2.5-␮g peptide dose, which provides protection from diabetes us- man applications, stopping an already ongoing autoimmune pro-

ing either the native HA512–520 or the A517G peptides, a signifi- cess is obviously more relevant. Self-peptide APLs with partial cantly stronger inhibition of in vivo cytotoxicity was afforded by agonist activity, which favor in vivo differentiation of autoreactive the superagonist peptide as compared with the native HA peptide CD4 T cells into a nonpathogenic Th2 subtype, have been shown (␹2 ϭ 33.9; p Ͻ 0.0001; n ϭ 3 mice in each group). to exert such a therapeutic effect. Indeed, in a murine model of Taken together, these data indicate that, unlike mice treated with CD4ϩ T cell-mediated EAE, both clinical and pathological man- ␮ 0.5 gHA512–520, animals injected with the superagonist A517G ifestations regressed in an IL-4-dependent manner following i.v. (0.5 ␮g) are protected from diabetes and display a markedly re- injection of a myelin basic protein APL (11). These results led to duced insulitis, contrasting with preserved (IFN-␥ secretion) or phase II clinical trials in multiple sclerosis in which repeated s.c. moderately blunted (cytotoxicity) HA-specific CD8 T cell injections of an APL derived from myelin basic protein (83–99) functions. favored Th2 development and, in some patients, clinical stabiliza- tion (35). Similarly, in the NOD model, an Ins B chain peptide,

Discussion B9–23, with alanine substitutions at positions 16 and 19, elicited

We generated a panel of APLs by introducing single amino acid Th2 responses that were cross-reactive with the native B9–23 pep- substitutions in a MHC class I-binding ␤ islet cell neoself-peptide. tide and could inhibit diabetes progression even when given late in These APLs elicited CD8ϩ T cell functions ranging from antago- the disease process (13). nist to superagonist activity when TCR contact residues were mu- Since the phenomenon of TCR superagonism has been de- tated. The relevant findings reported in this study are that i.v. in- scribed for both CD4ϩ (17, 18, 36) and CD8ϩ T cells (37), we jection of the superagonist APL was more effective than injection wondered whether the use of a superagonist APL derived from a ␤ of the wild-type self-peptide in protection from autoimmune dia- cell autoantigen would prove of therapeutic value. We tested this betes and that the APL not only promoted deletion of pathogenic hypothesis in a transgenic model of CD8-mediated autoimmune CD8ϩ T cells, but also decreased the cytotoxicity and the migra- diabetes in which the pathogenic immune response targets a neo-

tion/accumulation of persistent islet cell-specific T cells in the self-Ag (HA512–520). One HA peptide analogue, A517G, exhibited pancreas. heteroclitic properties in vitro and was, therefore, considered a The Journal of Immunology 921 good candidate to modulate the fate of autoreactive CD8ϩ T cells produce chemokines, such as CXCL9 and CXCL10, recruiting lo- in vivo. Indeed, we found that, compared with the wild-type pep- cally additional T cells and initiating a pathogenic cascade (41). tide, the superagonist APL was more efficient at low doses in con- This chain reaction may be blocked after the superagonist peptide trolling the development of diabetes. This protection is strikingly treatment, which inhibits accumulation of T cells in the pancreas. illustrated by the major reduction of intraislet infiltration, whereas In our particular model, the superiority of the APL is revealed at the native peptide treatment failed to prevent severe insulitis. a narrow concentration range, but the qualitative differences ob- Several mechanisms may concur to induce tolerance following served after triggering activated CD8ϩ T cells with a superagonist systemic Ag exposure (7). Repeated triggering of mature activated rather than with the native peptide could be exploited in other T cells is known to promote activation-induced cell death (AICD) models. Most therapeutic strategies involving APLs rely on the (38). In our model, both the native and the superagonist peptides, expansion of regulatory populations and are most efficient early in at the 0.5-␮g dose, significantly reduced the number of autoanti- the autoimmune process. In view of the pathogenic contribution of gen-specific CD8ϩ T cells in secondary lymphoid tissue, as com- autoreactive CD8ϩ T cells, targeting already activated autoreactive pared with control peptide-injected mice. This decrease is likely to CD8ϩ T cells is a relevant therapeutic approach during ongoing be a result of AICD rather than the inhibition of cell division as disease. As suggested by data in the NOD system, the pathogenic CSFE dilution in the autoreactive CD8ϩ T cells was considerable CD8ϩ T cell response in diabetes might not be prone to determi- in all groups. However, a similar and sizeable proportion of Thy1.1 nant spreading as commonly seen for CD4ϩ T cells and appears to CFSEneg HA-specific CD8ϩ T cells escaped AICD in both HA- be rather focused in terms of self-peptide recognition pattern (23, and A517G-treated mice. The increased efficacy of the superago- 42, 43). In human autoimmune diabetes, identification and char- ϩ nist peptide at a low dose therefore likely results from additional acterization of ␤ islet cell-specific CD8 T cells using MHC:pep- Downloaded from functional alterations imprinted on the autoreactive T cells by the tide tetramer should provide a rapid and individualized means of superagonist, but not by the native peptide. identifying those patients harboring an expanded autoreactive When assessing the cytokine secretion profile of the persistent CD8ϩ T cell population (44, 45). On this basis, immunotherapeu- autoreactive HA-specific CD8ϩ T cells in the superagonist- vs tic strategies such as the one described in the present study might wild-type peptide-treated mice, no blatant differences were ob- be considered.

served. However, injection of the superagonist peptide, but not the http://www.jimmunol.org/ native peptide, resulted in reduced cytotoxicity against HA-loaded Acknowledgments targets in vivo. This phenotype is reminiscent of HIV-specific We thank K. Bailly, Dr. C. Vizler, and Dr. N. Pardigon for help with the d CD8ϩ T cells from infected patients with uncontrolled viral load, peptide K binding experiments and early aspects of the work and Dr. B. which retain their capacity to produce IFN-␥ but exhibit reduced Salomon for helpful suggestions on this manuscript. perforin expression and cytotoxic properties as compared with ϩ References CMV-specific CD8 T cells from the same donor (39). However, 1. Tisch, R., and H. McDevitt. 1996. Insulin-dependent diabetes mellitus. Cell inhibition of in vivo cytotoxicity might only be one of the mech- 85:291. anisms by which the peptides protect from diabetes. Indeed, at the 2. Delovitch, T. L., and B. Singh. 1997. The nonobese diabetic mouse as a model ␮ of autoimmune diabetes: immune dysregulation gets the NOD. Immunity 7:727. by guest on September 29, 2021 2.5- g dose of peptide, although the superagonist peptide is sig- 3. Mathis D., L. Vence, and C. Benoist. 2001. ␤-cell death during progression to nificantly more efficient in blocking cytotoxicity, both peptides diabetes. Nature 414:792. prevent diabetes. 4. Weiner, H. L. 1997. Oral tolerance: immune mechanisms and treatment of au- toimmune diseases. Immunol. Today 18:335. An alternative interpretation of the data stems from recent ad- 5. Maron R., M. Guerau-de-Arellano, X. Zhang, and H. L. Weiner. 2001. Oral vances in the understanding of the selection and shaping of the administration of insulin to neonates suppresses spontaneous and cyclophosph- amide induced diabetes in the NOD mouse. J. Autoimmun. 16:21. autoimmune T cell repertoire. For instance, in the NOD mouse 6. von Herrath M. G., T. Dyrberg, and M. B. Oldstone. 1996. Oral insulin treatment model, injection of a superagonist self-peptide analogue was able suppresses virus-induced antigen-specific destruction of beta cells and prevents to prevent diabetes by efficiently eliminating the high avidity-spe- autoimmune diabetes in transgenic mice. J. Clin. Invest. 98:1324. ϩ 7. Liblau R., R. Tisch, N. Bercovici, and H. O. McDevitt. 1997. Systemic antigen cific CD8 T cells while driving the expansion of low avidity, in the treatment of T-cell-mediated autoimmune diseases. Immunol. Today 18: nonpathogenic CD8 T cells (23). These observations reflect the 599. natural ability of the autoreactive T cell repertoire to be tuned in 8. Daniel, D., and D. R. Wegmann. 1996. Protection of nonobese diabetic mice from diabetes by intranasal or subcutaneous administration of insulin peptide B9–23. vivo based on the strength of the antigenic stimulus, so that strong Proc. Natl. Acad. Sci. USA 93:956. stimuli select a weakly reactive repertoire through deletion of high 9. Ploix, C., I. Bergerot, A. Durand, C. Czerkinsky, J. Holmgren, and C. Thivolet. 1999. Oral administration of cholera toxin B-insulin conjugates protects NOD avidity-specific T cells (23, 36). Interestingly, the tuning phenom- mice from autoimmune diabetes by inducing CD4ϩ regulatory T-cells. Diabetes enon is not restricted to a polyclonal T cell population but also 48:2150. applies to mature T cells expressing a given TCR (36, 40). In light 10. Nicholson, L. B., J. M. Greer, R. A. Sobel, M. B. Lees, and V. K. Kuchroo. 1995. An altered peptide ligand mediates immune deviation and prevents autoimmune of these observations, it is conceivable that, in the present study, encephalomyelitis. Immunity 3:397. the persisting ␤ cell-specific T cells in superagonist-treated mice 11. Brocke, S., K. Gijbels, M. Allegretta, I. Ferber, C. Piercy, T. Blankenstein, were desensitized. This shift in avidity might therefore have pre- R. Martin, U. Utz, N. Karin, D. Mitchell, et al. 1996. Treatment of experimental encephalomyelitis with a peptide analogue of myelin basic protein. Nature vented T cell responses to the native HA expressed on the pan- 379:343. creatic ␤ cells. This is supported by our data, which show a re- 12. Gaur, A., S. A. Boehme, D. Chalmers, P. D. Crowe, A. Pahuja, N. Ling, S. Brocke, L. Steinman, and P. J. Conlon. 1997. Amelioration of relapsing ex- duced, but by no means eliminated, cytotoxicity toward wild-type perimental autoimmune encephalomyelitis with altered myelin basic protein pep- HA peptide-spiked target cells. tides involves different cellular mechanisms. J. Neuroimmunol. 74:149. Whether or not a result of tuning, the change in the functional 13. Alleva, D. G., A. Gaur, L. Jin, D. Wegmann, P. A. Gottlieb, A. Pahuja, ϩ E. B. Johnson, T. Motheral, A. Putnam, P. D. Crowe, et al. 2002. Immunological properties of HA-specific CD8 T cells in superagonist-treated characterization and therapeutic activity of an altered-peptide ligand, NBI-6024, Ins-HA mice was associated with a marked defect in their migra- based on the immunodominant type 1 diabetes autoantigen insulin B-chain (9– tion from pancreatic lymph nodes to the islets and/or in their local 23) peptide. Diabetes 51:2126. 14. Sloan-Lancaster, J., and P. M. Allen. 1996. Altered peptide ligand-induced partial accumulation. Together with the difference in the diabetes protec- T cell activation: molecular mechanisms and role in T . Annu. Rev. tion curves, this was the most striking difference observed when Immunol. 14:1. 15. Sloan-Lancaster, J., B. D. Evavold, and P. M. Allen. 1993. Induction of T-cell we compared the two peptide treatments. The release of proin- anergy by altered T-cell- ligand on live antigen-presenting cells. Nature flammatory cytokines by islet-specific T cells induces the ␤ cells to 363:156. 922 A SUPERAGONIST SELF-PEPTIDE BLOCKS CD8-MEDIATED DIABETES

16. De Magistris, M. T., J. Alexander, M. Coggeshall, A. Altman, F. C. Gaeta, autoimmune diabetes: most ␤-cells die through inflammatory cytokines and not H. M. Grey, and A. Sette. 1992. Antigen analog-major histocompatibility com- perforin from autoreactive (anti-viral) cytotoxic T-lymphocytes. Diabetes plexes act as antagonists of the T cell receptor. Cell 68:625. 49:1801. 17. Nicholson, L. B., H. Waldner, A. M. Carrizosa, A. Sette, M. Collins, and 32. Geluk, A., K. E. van Meijgaarden, B. O. Roep, and T. H. Ottenhoff. 1998. Altered V. K. Kuchroo. 1998. Heteroclitic proliferative responses and changes in cyto- peptide ligands of islet autoantigen Imogen 38 inhibit antigen specific T cell kine profile induced by altered peptides: implications for autoimmunity. Proc. reactivity in human type-1 diabetes. J. Autoimmun. 4:353. Natl. Acad. Sci. USA 95:264. 33. Nepom, G. T., J. D. Lippolis, F. M. White, S. Masewicz, J. A. Marto, A. Herman, 18. Vergelli, M., B. Hemmer, M. Kalbus, A. B. Vogt, N. Ling, P. Conlon, C. J. Luckey, B. Falk, J. Shabanowitz, D. F. Hunt, V. H. Engelhard, and J. E. Coligan, H. McFarland, and R. Martin. 1997. Modifications of peptide B. S. Nepom. 2001. Identification and modulation of a naturally processed T cell ligands enhancing T cell responsiveness imply large numbers of stimulatory li- epitope from the diabetes-associated autoantigen human glutamic acid decarbox- gands for autoreactive T cells. J. Immunol. 158:3746. ylase 65 (hGAD65). Proc. Natl. Acad. Sci. USA 4:1763. 19. Gottlieb, P. A., and G. S. Eisenbarth. 2002. Insulin-specific tolerance in diabetes. 34. Wauben, M. H., C. J. Boog, R. van der Zee, I. Joosten, A. Schlief, and Clin. Immunol. 102:2. W. van Eden. 1992. Disease inhibition by major histocompatibility complex 20. Bielekova, B., and R. Martin. 2001. Antigen-specific immunomodulation via al- binding peptide analogues of disease-associated epitopes: more than blocking tered peptide ligands. J. Mol. Med. 79:552. alone. J. Exp. Med. 176:667. 21. DiLorenzo, T. P., R. T. Graser, T. Ono, G. J. Christianson, H. D. Chapman, 35. Kappos, L., G. Comi, H. Panitch, J. Oger, J. Antel, P. Conlon, and L. Steinman. D. C. Roopenian, S. G. Nathenson, and D. V. Serreze. 1998. Major histocom- 2000. Induction of a non-encephalitogenic type 2 T helper-cell autoimmune re- patibility complex class I-restricted T cells are required for all but the end stages sponse in multiple sclerosis after administration of an altered peptide ligand in a of diabetes development in nonobese diabetic mice and use a prevalent T cell placebo-controlled, randomized phase II trial: The Altered Peptide Ligand in receptor ␣ chain gene rearrangement. Proc. Natl. Acad. Sci. USA 95:12538. Relapsing MS Study Group. Nat. Med. 6:1176. 22. Liblau, R. S., F. S. Wong, L. T. Mars, and P. Santamaria. 2002. Autoreactive 36. Anderton, S. M., C. G. Radu, P. A. Lowrey, E. S. Ward, and D. C. Wraith. 2001. CD8 T cells in organ-specific autoimmunity: emerging targets for therapeutic Negative selection during the peripheral immune response to antigen. J. Exp. intervention. Immunity 17:1. Med. 193:1. 23. Amrani, A., J. Verdaguer, P. Serra, S. Tafuro, R. Tan, and P. Santamaria. 2000. 37. Dressel, A., J. L. Chin, A. Sette, R. Gausling, P. Hollsberg, and D. A. Hafler. Progression of autoimmune diabetes driven by avidity maturation of a T-cell 1997. Autoantigen recognition by human CD8 T cell clones: enhanced agonist population. Nature 406:739. response induced by altered peptide ligands. J. Immunol. 159:4943. Downloaded from 24. Aichele, P., D. Kyburz, P. S. Ohashi, B. Odermatt, R. M. Zinkernagel, 38. Lenardo, M., K. M. Chan, F. Hornung, H. McFarland, R. Siegel, J. Wang, and H. Hengartner, and H. Pircher. 1994. Peptide-induced T-cell tolerance to prevent L. Zheng. 1999. Mature T lymphocyte apoptosis–immune regulation in a dy- autoimmune diabetes in a transgenic mouse model. Proc. Natl. Acad. Sci. USA namic and unpredictable antigenic environment. Annu. Rev. Immunol. 17:221. 91:444. 39. Appay, V., D. F. Nixon, S. M. Donahoe, G. M. Gillespie, T. Dong, A. King, 25. Bercovici, N., A. Heurtier, C. Vizler, N. Pardigon, C. Cambouris, P. Desreumaux, G. S. Ogg, H. M. Spiegel, C. Conlon, C. A. Spina, et al. 2000. HIV-specific and R. Liblau. 2000. Systemic administration of agonist peptide blocks the pro- CD8ϩ T cells produce antiviral cytokines but are impaired in cytolytic function. gression of spontaneous CD8-mediated autoimmune diabetes in transgenic mice J. Exp. Med. 192:63.

without bystander damage. J. Immunol. 165:202. 40. Munder, M., E. Bettelli, L. Monney, J. M. Slavik, L. B. Nicholson, and http://www.jimmunol.org/ 26. Vizler, C., N. Bercovici, A. Heurtier, N. Pardigon, K. Goude, K. Bailly, V. K. Kuchroo. 2002. Reduced self reactivity of an autoreactive T cell after C. Combadiere, and R. S. Liblau. 2000. Relative diabetogenic properties of islet- activation with cross-reactive non-self-ligand. J. Exp. Med. 196:1151. specific Tc1 and Tc2 cells in immunocompetent hosts. J. Immunol. 165:6314. 41. Frigerio, S., T. Junt, B. Lu, C. Gerard, U. Zumsteg, G. A. Hollander, and L. Piali. 27. Morgan, D. J., R. Liblau, B. Scott, S. Fleck, H. O. McDevitt, N. Sarvetnick, 2002. ␤ cells are responsible for CXCR3-mediated T-cell infiltration in insulitis. D. Lo, and L. A. Sherman. 1996. CD8 ϩ T cell-mediated spontaneous diabetes in Nat. Med. 8:1414. neonatal mice. J. Immunol. 157:978. 42. Wong F. S., J. Karttunen, C. Dumont, L. Wen, I. Visintin, I. M. Pilip, N. Shastri, 28. Lo, D., J. Freedman, S. Hesse, R. D. Palmiter, R. L. Brinster, and L. A. Sherman. E. G. Pamer, and C. A. Janeway, Jr. 1999. Identification of an MHC class I-re- 1992. Peripheral tolerance to an islet cell-specific hemagglutinin transgene affects stricted autoantigen in type 1 diabetes by screening an organ-specific cDNA both CD4ϩ and CD8ϩ T cells. Eur. J. Immunol. 22:1013. library. Nat. Med. 5:1026. 29. Quesnel, A., S. C. Hsu, A. Delmas, M. W. Steward, Y. Trudelle, and 43. Trudeau, J. D., C. Kelly-Smith, C. B. Verchere, J. F. Elliott, J. P. Dutz, J. P. Abastado. 1996. Efficient binding to the MHC class I Kd molecule of syn- D. T. Finegood, P. Santamaria, and R. Tan. 2003. Prediction of spontaneous

thetic peptides in which the anchoring position 2 does not fit the consensus motif. autoimmune diabetes in NOD mice by quantification of autoreactive T cells in by guest on September 29, 2021 FEBS Lett. 387:42. peripheral blood. J. Clin. Invest. 111:217. 30. Aichele, P., K. Brduscha-Riem, S. Oehen, B. Odermatt, R. M. Zinkernagel, 44. Reijonen, H., E. J. Novak, S. Kochik, A. Heninger, A. W. Liu, W. W. Kwok, and H. Hengartner, and H. Pircher. 1997. Peptide antigen treatment of naive and G. T. Nepom. 2002. Detection of GAD65-specific T-cells by major histocom- virus-immune mice: antigen-specific tolerance versus immunopathology. Immu- patibility complex class II tetramers in type 1 diabetic patients and at-risk sub- nity 6:519. jects. Diabetes 5:1375. 31. Seewaldt, S., H. E. Thomas, M. Ejrnaes, U. Christen, T. Wolfe, E. Rodrigo, 45. Eisenbarth, G. S., and B. L. Kotzin. 2003. Enumerating autoreactive T cells in B. Coon, B. Michelsen, T. W. Kay, and M. G. von Herrath. 2000. Virus-induced peripheral blood: a big step in diabetes prediction. J. Clin. Invest. 111:179.