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The Journal of Immunology, 2004, 172: 915-922.
Copyright © 2004 by The American Association of Immunologists

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

Agnès Hartemann-Heurtier*,{dagger}, Lennart T. Mars*,{ddagger}, Nadège Bercovici2,*, Sabine Desbois*,{ddagger}, Christophe Cambouris2,*, Eliane Piaggio*,{ddagger}, Jacques Zappulla*,{ddagger}, Abdelhadi Saoudi{ddagger} and Roland S. Liblau3,*,{ddagger}

* Institut National de la Scientifique et de la Santé Recherche Médicale Unité 546, Faculté de Médecine Pitié-Salpêtrière, Paris, France; {dagger} Department of Diabetology and Metabolism, Pitié-Salpêtrière Hospital, Paris, France; and {ddagger} Institut National de la Scientifique et de la Santé Recherche Médicale Unité 563, Purpan Hospital, Toulouse, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 superagonist 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 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Type 1 diabetes is the end result of an autoimmune disorder in which the pancreatic {beta} islet cells are selectively destroyed by the immune system (1, 2, 3). Among the therapeutic strategies aimed at specifically inhibiting the pathogenic autoimmune response without affecting the protective function of the immune system, Ag-specific immunotherapies have shown promise in animal models of type 1 diabetes. These approaches rely on the administration of self-Ags or self-peptides binding to MHC molecules to silence potentially pathogenic T cells via apoptosis, anergy, immune deviation, and/or dominant mechanisms of immunoregulation. As a result, systemic or mucosal administration of soluble self-Ags has been shown to prevent diabetes in experimental models (4, 5, 6, 7, 8, 9). A related strategy is based on the administration of antigenic self-peptides with a single amino acid substitution at important TCR contact residues (referred to as altered peptide ligands or APLs4). This approach has been successfully applied in several rodent models of autoimmune diseases (10, 11, 12, 13). APLs bound to MHC molecules can induce both quantitatively and qualitatively different signals in the responding T cells as compared with the native peptide and are categorized according to their biological activity (14). Agonist peptides induce proliferation, cytokine release, and/or cytotoxicity by the activated T cells. Weak agonists similarly elicit the full spectrum of T cell functions but at a higher concentration than the agonist peptide. Partial agonists promote some but not all T cell functions and, therefore, may alter the pattern of cytokine production (10) or lead to anergy (15). Some APLs, which fail to induce any obvious T cell activation, can behave as TCR antagonists as they inhibit T cell responses to agonist peptides when both ligands are recognized simultaneously by the T cells (16). Finally, superagonist or heteroclitic peptides stimulate T cell responses at lower concentrations than the native peptide (17, 18).

Because of their ability to modulate T cell responses, APLs have 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 rodents to control pathogenic autoreactive CD4+ T cells and thereby prevent or treat experimental autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE) or diabetes in nonobese diabetic (NOD) mice (10, 11, 12, 13).

In type 1 diabetes, both the CD4+ and CD8+ T cell subsets are involved in the destructive autoimmune process, with the autoreactive CD8+ T cells playing a pivotal role during all phases of disease development (21, 22, 23). Therapeutic strategies aimed at blocking these CD8+ T cell-dependent events could, therefore, prevent or inhibit disease progression. On this basis, MHC class I-binding self-peptides have been injected systemically to target pathogenic autoreactive CD8+ T cells either before or during an ongoing autoimmune process in both NODs (23) and transgenic mouse models of autoimmune diabetes (24, 25). Despite this successful application of self-peptides in models of type 1 diabetes, strategies involving MHC class I-binding APLs in vivo have not received much attention (23). Moreover, little is known about the effect of APLs on already differentiated CD8+ T cells, an important parameter if the APL treatment is intended to control an ongoing disease in predisposed individuals or even in recently diagnosed patients. To specifically address this question, we generated APLs from a Kd-restricted influenza virus hemagglutinin (HA) peptide (HA512–520), which were functionally characterized in vitro by their ability to trigger CD8+ T cells expressing a transgenic TCR specific for HA512–520. Then, we set out to test the efficacy of a superagonist APL in controlling autoimmune diabetes induced by the adoptive transfer of activated HA-specific CD8+ T cells into rat insulin promoter-HA-transgenic recipient mice (26).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

CL4-TCR (V{beta}8.2, V{alpha}10.3) mice are transgenic for an HA512–520 (IYSTVASSL)-specific, H-2Kd-restricted TCR (27). Ins-HA mice express HA specifically in the pancreatic {beta} islet cells (28). Hemizygous CL4-TCR and Ins-HA mice were backcrossed at least eight times onto the BALB/c genetic background. In addition, CL4-TCR mice were crossed at least twice with congenic Thy1.1 BALB/c mice. Mice were bred and manipulated in a specific pathogen-free animal facility (Salpêtrière Medical School, Paris, France) in keeping with the European Union legislation on animal care.

Peptides

All APLs (>70% pure) and Cw3 (RYLKNGKETL), a control Kd-binding peptide, were purchased from Chiron (Melbourne, Australia). For further in vitro and in vivo analyses, the HA512–520, S514I, A517G, and Cw3 peptides (>95% pure) were synthesized by the Neosystem Laboratory (Strasbourg, France).

Measurement of peptide binding to Kd

Binding of peptides to Kd was measured in a competition assay with the radiolabeled S9I peptide (SYIPSAEKI) with known affinity for the Kd molecule (29). The concentration of competitor HA peptide resulting in 50% inhibition of the binding of the probe peptide (IC50) was determined. The relative affinity of a given APL for Kd is the APL IC50:wild-type HA peptide IC50 ratio.

T cell proliferation

Spleen single-cell suspensions from CL4-TCR-transgenic mice were stimulated with increasing concentrations (0–3 x.105 nM) of peptide in complete RPMI 1640 supplemented with 10% FCS (Life Technologies, Paisley, U.K.) for 42 h in 96-well flat-bottom microtiter plates (106 cells/well in 200 µl). Proliferation was measured by [3H]thymidine (Amersham, Arlington Heights, IL) incorporation for the last 18 h of culture. SEMs were consistently <10% of the mean.

Cytokine measurement and flow cytometry

Cytokine production was assessed following stimulation of 105 naive CD8+ T cells (isolated using MACS beads to typically >98% purity) with 106 irradiated BALB/c spleen cells with 3 x 10-1–3 x 103 nM peptide in 200 µl of complete RPMI 1640 medium. In ex vivo experiments, splenocytes from Ins-HA recipient mice (106 cells/ml) were cultured with or without 1 µM peptide. Supernatants were collected after a 24-, 48-, or 72-h incubation, aliquoted, and stored at -20°C until use. Cytokine determination was assessed by sandwich ELISA as previously described (26). The detection limit of the ELISA was 40 pg/ml for IFN-{gamma}, 25 pg/ml for IL-4, and 30 pg/ml for IL-10.

Expression of T cell activation markers was assessed after a 24-h in vitro stimulation of 5 x 106/ml spleen cells from CL4-TCR mice with increasing concentrations of peptide. Triple staining was performed using PE-conjugated anti-CD4 mAb, FITC-conjugated anti-CD8 mAb (Caltag Laboratories, Burlingame, CA), and biotinylated mAbs (BD PharMingen, San Diego, CA) specific for mouse CD25 (clone 7D4), CD69 (clone HI.2F3), CD62-L (clone Mel-14), or CD90.1 (Thy1.1) followed by streptavidin-TC (Caltag Laboratories). Cells were acquired on a FACScan flow cytometer (BD Biosciences, Mountain View, CA) and analyzed using CellQuest (BD Biosciences).

Adoptive transfer of diabetes and peptide treatment

Diabetogenic TC1 cells were generated from CL4-TCR mice as previously described (26). Briefly, purified naive CD8 T cells were stimulated in vitro with HA512–520 peptide-pulsed irradiated splenocytes in the presence of IL-2 (1 ng/ml) and IL-12 (20 ng/ml; a kind gift from Genetics Institute, Cambridge, MA). After 6 days of culture, the live cells were isolated using a Ficoll gradient and directly used for adoptive transfer experiments. Nonirradiated 6- to 8-wk-old Ins-HA recipients received 2 x 106 HA-specific TC1 i.v. in 0.2 ml of PBS. Adoptively transferred mice were treated with 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 (Glukotest; Roche Diagnostic, Mannheim, Germany). Diabetes was confirmed by measurement of blood glucose using a One Touch Ultra glucometer (Lifescan, Milpitas, CA). Mice were considered diabetic if blood glucose levels were above 2 g/L on two consecutive tests. For histological analyses, pancreata were fixed in formol solution and processed for paraffin embedding. Sections were stained with H&E and the degree of insulitis was evaluated microscopically.

In vivo CD8+ T cell tracking

Activated Thy1.1 diabetogenic CD8+ T cells were labeled for 10 min with 5 µM CFSE (Molecular Probes, Eugene, OR), washed, and transferred i.v. into Ins-HA mice (2 x 106 cells/mouse). On day 1 posttransfer, Ins-HA mice were injected with 0.5 µg peptide (Cw3, HA512–520, or APL). Four days posttransfer, transferred CD8+ T cells were detected and enumerated in lymphoid tissue by virtue of their Thy1.1 expression.

In vivo cytotoxicity assay

To prepare target cells, BALB/c spleen cells were pulsed with either 1 µM HA512–520 or Cw3 peptide (2 h at 37°C), washed, and labeled with CFSE at a final concentration of 5 µM for HA-loaded cells or 1 µM for Cw3-loaded cells. A combination of 20 x 106 HA-pulsed and 10 x 106 Cw3-pulsed target cells were injected i.v. into Ins-HA mice that had received unlabeled 2 x 106 TC1 96 h earlier and had been treated with peptide (Cw3, HA512–520, or APL) 72 h earlier. Control Ins-HA mice received CFSE-labeled splenocytes but did not receive TC1 cells nor peptide. Spleens from Ins-HA recipient mice were removed 18 h after the injection of target cells and a single-cell suspension was prepared. The number of cells with high fluorescence intensity (HA loaded) or moderate fluorescence intensity (Cw3 loaded) was determined by FACS analysis (30).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of altered peptide ligands

To generate APLs able to modulate a diabetogenic CD8+ T cell response, we first identified the residues of the HA512–520 peptide that are in contact with the CL4-TCR when presented in the context of Kd. A panel of peptide analogues was generated by single amino acid substitutions at all positions except for the canonical Kd anchor residues Y513 and L520. Each residue was substituted with either the small alanine amino acid (except for alanine at position 517, which was changed for glycine) or the bulky isoleucine or leucine amino acid.

We then measured the ability of these peptide analogues to bind the Kd molecule. All peptides have a Kd-binding affinity similar to that of the wild-type peptide, with a relative affinity ranging from 0.4 to 2 (Table I and Fig. 1A). This allowed us to compare the capacity of the wild-type and the mutated peptides to elicit the proliferation of naive CL4-TCR CD8+ T cells. As shown in Table I, an alanine or an isoleucine substitution at positions 516, 518, and 519 moderately reduced the ability of the mutated peptide to trigger T cell proliferation, indicating that these positions are not essential TCR-contact residues (Table I and data not shown). In contrast, substituting the threonine at position 515 for either an alanine (T515A) or an isoleucine (T515I) resulted in a drastic (>100,000-fold) decrease in agonist activity. Further mutated peptides revealed that among the few tolerated substitutions at position 515 was the most conservative T515S change (Table I). In contrast, T515F and T515D induced little or no proliferative response (Fig. 1B). Albeit to a lesser extent, similar observations were made for position 514, at which a variable agonist activity was obtained depending on the nature of the substituting amino acid: S514A is a weak agonist whereas S514I is unable to induce proliferation (Table I). Fig. 1B shows representative T cell proliferative responses with 514 or 515 mutated peptides. Taken together, these results suggest that these residues are in direct contact with the CL4-TCR; position 515 fulfils the criteria for a primary TCR contact residue (14), while position 514 is likely an important secondary TCR contact residue. We therefore investigated whether the nonagonist peptide mutants at position 514 or 515 exhibited antagonist properties. A reproducible antagonist activity was only observed for peptide S514I (data not shown).


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  		Table I. Altered peptide ligands for the CL4-TCRa

 


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  		FIGURE 1. Properties of selected APLs with substitutions at position 514 or 515. A, The affinity of individual APLs for the Kd molecule was determined as described in Materials and Methods. Two independent experiments were performed with similar results. B, Proliferation of splenic T cells from CL4-TCR mice that were stimulated with increasing concentrations of HA512–520 or APLs. Results are expressed as the mean of [3H]thymidine uptake obtained in triplicate. Two independent experiments were performed with similar results.

 
Interestingly, substituting the alanine at position 517 with a bulky residue such as valine (A517V) or serine (A517S) reduced the agonist activity, while shortening its side chain by introducing a glycine (A517G) significantly enhanced the agonist capacity (Table I). This would suggest that, in a similar manner to positions 514 and 515, the side chain of residue 517 is available to contact the TCR. However, this contact appears to inhibit, rather than support, the productive interaction of the Kd-peptide complex with the CL4-TCR, giving rise to enhanced proliferation when the 517 side chain is trimmed.

A517G is a superagonist peptide

The A517G peptide has a higher agonistic activity than the wild-type HA peptide on the CL4-TCR CD8+ T cells, shifting the proliferation dose-response curve to the left by a factor of 10–30, but without altering the maximum proliferative response (Fig. 2A). Since the A517G peptide has a nearly identical affinity for the Kd molecule to that of the wild-type peptide (relative affinity: 0.9) the superagonist activity is attributable to improved interactions of the Kd-peptide complex with the CL4-TCR following removal of a methyl group.



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  		FIGURE 2. Functional characteristics of A517G, a superagonist HA peptide analogue. A, Proliferation of splenic T cells from CL4-TCR mice that were stimulated with increasing concentrations of HA512–520 or A517G peptide. Results are expressed as the mean of [3H]thymidine uptake obtained in triplicate and are representative of four independent experiments. B, Cell surface phenotype of CD8+ 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 CD8+ T cell population, are representative of three independent experiments. C, IFN-{gamma} production of CD8+ T cells from CL4-TCR mice stimulated with HA512–520 or A517G peptide. CD8+ 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-{gamma} 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 <10%. 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 µg/ml anti-CD3 mAb.

 
To further investigate the superagonist properties of the A517G peptide, the expression of activation markers on CD8+ T cells was assessed after a 24-h in vitro stimulation with peptide. As shown in Fig. 2B, 0.3 nM A517G induced the expression of CD69 and CD25 on most CD8+ T cells, whereas <50% of the CD8+ T cells expressed these molecules when stimulated with a 10-fold higher concentration of the native HA peptide. The CD62-L down-regulation induced by either peptide was, however, similar (data not shown).

To test whether cytokine production would be influenced by the HA peptide analogue, we measured the release of IFN-{gamma}, IL-4, and IL-10 by CD8+ T cells activated by HA peptide- or A517G-pulsed APCs. As shown in Fig. 2C, a 10-fold higher concentration of A517G (30 nM) compared with HA (3 nM) was required to induce an optimal production of IFN-{gamma}, suggesting that the superagonist A517G elicits a modified signal. Nevertheless, the cytokine profile of the CD8+ T cells was not shifted toward a TC2 type since neither IL-10 nor IL-4 production was detected (data not shown).

The in vitro response of preactivated HA-specific TC1 cells to the A517G superagonist and to the wild-type HA peptide was also 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 activation-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. However, 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 prevents or blocks diabetes in (Ins-HA x 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 x 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 i.v. dose of either Cw3, HA512–520, or A517G peptide. High HA512–520 peptide doses (25 µg, n = 7 mice, data not shown) or 2.5 µg (Fig. 3A) completely blocked diabetes (lethal diabetes in all 9 Cw3-treated mice vs 0 of 10 HA512–520-treated mice; p = 10-4, Fisher’s exact test). A similar protective effect was observed with a single i.v. dose of 25 µg (n = 7 mice, data not shown) or 2.5 µg (Fig. 3A) of A517G, demonstrating that high doses of both peptides are similarly potent in preventing diabetes.



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  		FIGURE 3. Blockade of adoptive transfer of diabetes in superagonist or agonist peptide-treated mice. Nonirradiated adult Ins-HA recipient mice were injected i.v. with 2 x 106 activated HA-specific CD8+ T cells and 24 h later received the indicated quantity of peptide. Glycosuria was assessed three times a week for 30 days and diabetes was confirmed by blood glucose measurements. A, The animals received 2.5 µg of either Cw3 (n = 9), HA512–520 (n = 10), or A517G peptide (n = 10). The comparison of the protective effect of HA512–520 and A517G was not significant, p = 0.80, Fisher’s exact test. B, The animals received a single injection of 0.5 µg (n = 12 mice per group; p < 10-4) or 0.1 µg of either HA512–520 or A517G peptide (n = 8 mice/group; p = 0.46).

 
Strikingly, with a dose of 0.5 µg the effect was quite different between HA512–520 and A517G peptides. Indeed, 11 of 12 mice injected with 0.5 µg HA512–520 peptide developed persistent diabetes, 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 = 10-4). Moreover, pancreata from additional groups of mice, treated with 0.5 µg of either Cw3, HA512–520, or A517G peptide, were analyzed histologically on day 4 after TC1 transfer. A significantly greater number of islets remained free of infiltration in mice treated with A517G compared with HA512–520, whereas a lower number of islets exhibited an invading insulitis (Fig. 4, p < 10-4). Thirty days posttransfer, 85% of islets were still free of insulitis in A517G-treated animals (data not shown). When the dose of peptide was reduced even further, to 0.1 µg, the superagonist peptide showed partial therapeutic properties; however, these proved not to be statistically significant (Fig. 3B). Taken together, these data show that both the native and the superagonist peptide are able to protect against CD8+ T cell-mediated diabetes. However, the superagonist is significantly more effective at low doses.



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  		FIGURE 4. Inhibition of insulitis in superagonist peptide-treated Ins-HA mice. Ins-HA mice were transferred with 2 x 106 TC1 and treated on day 1 with 0.5 µg Cw3 (n = 4; 532 islets), HA512–520 (n = 4; 445 islets), or A517G peptide (n = 4; 522 islets). Pancreata were removed on day 4, and histological analysis was performed after H&E staining. Islets were analyzed by an investigator blinded to the treatment group.

 
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 {beta} cell-specific CD8+ 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 x 106 TC1/mouse). Mice were treated 24 h later with a single dose of 0.5 µg of Cw3, HA512–520, or A517G peptide. Four days posttransfer, the proportion of CD8+ T cells from donor origin (Thy1.1+) in the pancreatic lymph nodes of HA-treated (n = 4) or A517G-treated mice (n = 5) was reduced to 42.4 ± 12.0% and 36.7 ± 5.4% of the value of Cw3-treated mice, respectively (Fig. 5A). This reduction was not different between the HA- and A517G peptide-treated groups (p = 0.65, unpaired Student’s t test). The reduction was even more pronounced in the spleen of HA-treated (32.2 ± 4.7%) and A517G-treated (22.5 ± 2.7%) animals (p = 0.12, comparing HA-treated and A517G-treated groups). As expected, the preactivated TC1 cells proliferated vigorously following transfer into Ins-HA mice. Before transfer, the activated TC1 cells were brightly labeled with CSFE, whereas 4 days after transfer the vast majority of Thy1.1 donor cells had completely diluted their CFSE fluorescence in all groups (mean percentage of splenic Thy1.1 TC1 cells negative for CFSE fluorescence: 99.3% in two Cw3-, 98.4% in two HA-, and 98.1% in two A517G-treated mice), indicating a high rate of cell division. Collectively, these results suggest that both the wild-type and the superagonist peptide deleted most transferred CD8+ T cells; however, a substantial population of HA-specific Thy1.1 CD8+ T cells persisted in both groups.



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  		FIGURE 5. In vivo cytotoxic properties of HA-specific CD8+ T cells following peptide treatment. A, Ins-HA mice were transferred with 2 x 106 Thy1.1 TC1 and treated on day 1 with 0.5 µg 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 Thy1.1+ cells among CD8+ T cells in pancreatic lymph nodes. The data are representative of two independent experiments. B, Ins-HA mice were injected with 2 x 106 TC1, treated on day 1 with 0.5 µg 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 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 µg of peptide. The data are representative of three independent experiments.

 
To evaluate the functional properties of these remaining T cells, splenocytes from peptide-treated Ins-HA mice were recovered on day 4 after TC1 cell transfer and cultured with or without the native peptide for 72 h. A comparable production of IFN-{gamma} was detected in splenocyte cultures from HA512–520- or A517G-treated mice, both spontaneously (0.36 ± 0.31 and 0.39 ± 0.37 ng/ml, respectively; mean ± SEM of three independent experiments) and upon 1 µM HA512–520 peptide restimulation (1.8 ± 1.4 and 1.7 ± 1.6 ng/ml, respectively), whereas higher, although not statistically significantly so, IFN-{gamma} production was detected in Cw3-treated animals (0.72 ± 0.88 spontaneously and 3.11 ± 1.68 ng/ml upon HA peptide restimulation). No IFN-{gamma} was detected in cultures 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-{gamma} production (31) or a switch in the cytokine profile (10, 11, 12, 13).

We next investigated the in vivo cytotoxic capacity of the transferred TC1 cells after peptide treatment. Ins-HA mice received 2 x 106 preactivated TC1 and were treated on day 1 with 0.5 (Fig. 5B) or 25 (Fig. 5C) µg 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 CFSEdull cells was determined in the spleen 18 h later. As shown in Fig. 5, B and C, the transferred HA-loaded target cells were largely eliminated in TC1 recipients treated with the control peptide, whereas the Cw3-loaded target population was not affected (quantitatively comparable to that of mice not injected with TC1), revealing a potent in vivo HA-specific cytotoxicity by transferred TC1. Remarkably, following injection of 0.5 µg of peptide (Fig. 5B), the killing of HA-loaded cells was of comparable magnitude in Cw3- and HA512–520-treated mice (p = 0.6), whereas in A517G-treated mice, it was partially yet significantly reduced ({chi}2 = 58; p < 0.0001; n = 5 mice in each group). However, using the high 25-µg dose of peptide (Fig. 5C), the killing of HA-loaded target cells was greatly reduced and of comparable magnitude in HA512–520- or A517G-treated mice (Fig. 5C). Interestingly, at the 2.5-µg peptide dose, which provides protection from diabetes using either the native HA512–520 or the A517G peptides, a significantly stronger inhibition of in vivo cytotoxicity was afforded by the superagonist peptide as compared with the native HA peptide ({chi}2 = 33.9; p < 0.0001; n = 3 mice in each group).

Taken together, these data indicate that, unlike mice treated with 0.5 µg HA512–520, animals injected with the superagonist A517G (0.5 µg) are protected from diabetes and display a markedly reduced insulitis, contrasting with preserved (IFN-{gamma} secretion) or moderately blunted (cytotoxicity) HA-specific CD8 T cell functions.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We generated a panel of APLs by introducing single amino acid substitutions in a MHC class I-binding {beta} islet cell neoself-peptide. These APLs elicited CD8+ T cell functions ranging from antagonist to superagonist activity when TCR contact residues were mutated. The relevant findings reported in this study are that i.v. injection of the superagonist APL was more effective than injection of the wild-type self-peptide in protection from autoimmune diabetes and that the APL not only promoted deletion of pathogenic CD8+ T cells, but also decreased the cytotoxicity and the migration/accumulation of persistent islet cell-specific T cells in the pancreas.

A single amino acid substitution, at an important peptide/TCR contact position, is sufficient to dramatically alter T cell responses induced by TCR engagement. Some APLs act as TCR antagonists, inhibiting antigenic peptide-induced T cell functions. In human diabetes, two in vitro studies have attempted to take advantage of antagonistic APLs to modulate potentially pathogenic autoreactive CD4+ T cells (32, 33). The in vivo therapeutic potential of antagonistic self-peptide analogues has been successfully demonstrated in EAE using disease prevention protocols (34). For potential human applications, stopping an already ongoing autoimmune process is obviously more relevant. Self-peptide APLs with partial agonist activity, which favor in vivo differentiation of autoreactive CD4 T cells into a nonpathogenic Th2 subtype, have been shown to exert such a therapeutic effect. Indeed, in a murine model of CD4+ T cell-mediated EAE, both clinical and pathological manifestations regressed in an IL-4-dependent manner following i.v. injection of a myelin basic protein APL (11). These results led to phase II clinical trials in multiple sclerosis in which repeated s.c. injections of an APL derived from myelin basic protein (83–99) favored Th2 development and, in some patients, clinical stabilization (35). Similarly, in the NOD model, an Ins B chain peptide, B9–23, with alanine substitutions at positions 16 and 19, elicited Th2 responses that were cross-reactive with the native B9–23 peptide and could inhibit diabetes progression even when given late in the disease process (13).

Since the phenomenon of TCR superagonism has been described for both CD4+ (17, 18, 36) and CD8+ T cells (37), we wondered whether the use of a superagonist APL derived from a {beta} cell autoantigen would prove of therapeutic value. We tested this hypothesis in a transgenic model of CD8-mediated autoimmune diabetes in which the pathogenic immune response targets a neoself-Ag (HA512–520). One HA peptide analogue, A517G, exhibited heteroclitic properties in vitro and was, therefore, considered a good candidate to modulate the fate of autoreactive CD8+ T cells in vivo. Indeed, we found that, compared with the wild-type peptide, the superagonist APL was more efficient at low doses in controlling the development of diabetes. This protection is strikingly illustrated by the major reduction of intraislet infiltration, whereas the native peptide treatment failed to prevent severe insulitis.

Several mechanisms may concur to induce tolerance following systemic Ag exposure (7). Repeated triggering of mature activated T cells is known to promote activation-induced cell death (AICD) (38). In our model, both the native and the superagonist peptides, at the 0.5-µg dose, significantly reduced the number of autoantigen-specific CD8+ T cells in secondary lymphoid tissue, as compared with control peptide-injected mice. This decrease is likely to be a result of AICD rather than the inhibition of cell division as CSFE dilution in the autoreactive CD8+ T cells was considerable in all groups. However, a similar and sizeable proportion of Thy1.1 CFSEneg HA-specific CD8+ T cells escaped AICD in both HA- and A517G-treated mice. The increased efficacy of the superagonist peptide at a low dose therefore likely results from additional functional alterations imprinted on the autoreactive T cells by the superagonist, but not by the native peptide.

When assessing the cytokine secretion profile of the persistent autoreactive HA-specific CD8+ T cells in the superagonist- vs wild-type peptide-treated mice, no blatant differences were observed. However, injection of the superagonist peptide, but not the native peptide, resulted in reduced cytotoxicity against HA-loaded targets in vivo. This phenotype is reminiscent of HIV-specific CD8+ T cells from infected patients with uncontrolled viral load, which retain their capacity to produce IFN-{gamma} but exhibit reduced perforin expression and cytotoxic properties as compared with CMV-specific CD8+ T cells from the same donor (39). However, inhibition of in vivo cytotoxicity might only be one of the mechanisms by which the peptides protect from diabetes. Indeed, at the 2.5-µg dose of peptide, although the superagonist peptide is significantly more efficient in blocking cytotoxicity, both peptides prevent diabetes.

An alternative interpretation of the data stems from recent advances in the understanding of the selection and shaping of the autoimmune T cell repertoire. For instance, in the NOD mouse model, injection of a superagonist self-peptide analogue was able to prevent diabetes by efficiently eliminating the high avidity-specific CD8+ T cells while driving the expansion of low avidity, nonpathogenic CD8 T cells (23). These observations reflect the natural ability of the autoreactive T cell repertoire to be tuned in vivo based on the strength of the antigenic stimulus, so that strong stimuli select a weakly reactive repertoire through deletion of high avidity-specific T cells (23, 36). Interestingly, the tuning phenomenon is not restricted to a polyclonal T cell population but also applies to mature T cells expressing a given TCR (36, 40). In light of these observations, it is conceivable that, in the present study, the persisting {beta} cell-specific T cells in superagonist-treated mice were desensitized. This shift in avidity might therefore have prevented T cell responses to the native HA expressed on the pancreatic {beta} cells. This is supported by our data, which show a reduced, but by no means eliminated, cytotoxicity toward wild-type HA peptide-spiked target cells.

Whether or not a result of tuning, the change in the functional properties of HA-specific CD8+ T cells in superagonist-treated Ins-HA mice was associated with a marked defect in their migration from pancreatic lymph nodes to the islets and/or in their local accumulation. Together with the difference in the diabetes protection curves, this was the most striking difference observed when we compared the two peptide treatments. The release of proinflammatory cytokines by islet-specific T cells induces the {beta} cells to produce chemokines, such as CXCL9 and CXCL10, recruiting locally additional T cells and initiating a pathogenic cascade (41). This chain reaction may be blocked after the superagonist peptide treatment, which inhibits accumulation of T cells in the pancreas.

In our particular model, the superiority of the APL is revealed at a narrow concentration range, but the qualitative differences observed after triggering activated CD8+ T cells with a superagonist rather than with the native peptide could be exploited in other models. Most therapeutic strategies involving APLs rely on the expansion of regulatory populations and are most efficient early in the autoimmune process. In view of the pathogenic contribution of autoreactive CD8+ T cells, targeting already activated autoreactive CD8+ T cells is a relevant therapeutic approach during ongoing disease. As suggested by data in the NOD system, the pathogenic CD8+ T cell response in diabetes might not be prone to determinant spreading as commonly seen for CD4+ T cells and appears to be rather focused in terms of self-peptide recognition pattern (23, 42, 43). In human autoimmune diabetes, identification and characterization of {beta} islet cell-specific CD8+ T cells using MHC:peptide tetramer should provide a rapid and individualized means of identifying those patients harboring an expanded autoreactive CD8+ T cell population (44, 45). On this basis, immunotherapeutic strategies such as the one described in the present study might be considered.


   Acknowledgments
 
We thank K. Bailly, Dr. C. Vizler, and Dr. N. Pardigon for help with the peptide Kd binding experiments and early aspects of the work and Dr. B. Salomon for helpful suggestions on this manuscript.


   Footnotes
 
1 This work was supported by the French Biomedical Research Institute (Institut National de la Scientifique et de la Santé Recherche Médicale), the French AIDS Research Agency (Agence Nationale de Recherches sur le SIDA), and the Juvenile Diabetes Foundation International. L.T.M. and E.P. were supported by postdoctoral fellowships from the Association pour la Recherche sur la Sclérose en Plaques (The French Multiple Sclerosis Society) and French Biomedical Research Foundation (Fondation pour la Recherche Médicale), respectively. Back

2 Current address: Immuno-Designed Molecules, Paris, France. Back

3 Address correspondence and reprint requests to Prof. Roland S. Liblau, Institut National de la Scientific et de la Santé Recherche Médicale Unité 563, Purpan Hospital, Place Dr Baylac, Toulouse 31000, France. E-mail address: rolandliblau{at}hotmail.com Back

4 Abbreviations used in this paper: APL, altered peptide ligand; EAE, experimental autoimmune encephalomyelitis, HA, hemagglutinin; Ins, Insulin; NOD, nonobese diabetic; AICD, activation-induced cell death. Back

Received for publication March 1, 2003. Accepted for publication November 4, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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