Thioreductase-Containing Epitopes Inhibit the Development of Type 1 Diabetes in the NOD Mouse Model

Abstract

Autoreactive CD4(+) T cells recognizing islet-derived antigens play a primary role in type 1 diabetes. Specific suppression of such cells therefore represents a strategic target for the cure of the disease. We have developed a methodology by which CD4(+) T cells acquire apoptosis-inducing properties on antigen-presenting cells after cognate recognition of natural sequence epitopes. We describe here that inclusion of a thiol-disulfide oxidoreductase (thioreductase) motif within the flanking residues of a single MHC class II-restricted GAD65 epitope induces GAD65-specific cytolytic CD4(+) T cells (cCD4(+) T). The latter, obtained either in vitro or by active immunization, acquire an effector memory phenotype and lyse APCs by a Fas-FasL interaction. Furthermore, cCD4(+) T cells eliminate by apoptosis activated bystander CD4(+) T cells recognizing alternative epitopes processed by the same APC. Active immunization with a GAD65 class II-restricted thioreductase-containing T cell epitope protects mice from diabetes and abrogates insulitis. Passive transfer of in vitro-elicited cCD4(+) T cells establishes that such cells are efficient in suppressing autoimmunity. These findings provide strong evidence for a new vaccination strategy to prevent type 1 diabetes.

Keywords: MHC class II epitopes; NOD mouse; antigen-specific; cytolytic CD4+ T cells; type 1 diabetes.

Figure 1

NOD mice immunized with CCGAD65 peptide in SPF animal facilities. (A) Four-week-old female NOD mice received four subcutaneous injections of 50 μg of peptide in alum with a 7-day interval. Mice were randomly divided in five cohorts of five groups each treated with the same immunization protocol. The first group was immunized with the CCGAD65 peptide in alum, the second with the loss-of-function AAGAD65 peptide in alum, the third group with the non-relevant CCHEL peptide in alum, the fourth group received alum alone, and the fifth group remained untreated. Results represent diabetes-free survival rates at, from top to bottom at 40 weeks, 43% for CCGAD65-immunized mice (n = 47), 38% for AAGAD65-immunized mice (n = 50), 35% for CCHEL-immunized mice (n = 23), 21% for alum-treated mice (n = 24), 17% for untreated control group (n = 23), *P < 0.05 and P > 0.1 reported as NS (Mantel–Cox log-rank test). (B,C) Pancreatic sections of mice sacrificed after two consecutive weekly glycemia above 300 mg/dl or after remaining normoglycemic during 40 weeks were stained with H&E and insulitis was scored by examining a minimum of 40 islets per mouse. (B) Individual II are represented for each group with line representing mean index; from left to right, CCGAD65 peptide in alum (n = 46, mean II = 0.51), AAGAD65 peptide in alum (n = 47, mean II = 0.63), CCHEL peptide in alum (n = 21, mean II = 0.63), alum alone (n = 23, mean II = 0.67), and untreated (n = 19, mean II = 0.70). Statistical significance was calculated with Kruskal–Wallis ANOVA test, P < 0.0001 followed by Dunn’s multiple comparison test as noted, *P < 0.05. (C) Grading of insulitis of the various groups as indicated. Statistical significance was calculated based on insulitis-free islets with Kruskal–Wallis ANOVA test, P < 0.0001 and Dunn’s multiple comparison test as indicated, *P < 0.05.

Figure 2

Characterization of CCGAD65-induced CD4⁺ T cells. (A) The proliferative response of CCGAD65-induced CD4⁺ T cells (pooled from three previously CCGAD65-immunized mice) to either CCGAD65 peptide (continuous line) or the natural counterpart (WTGAD65, dashed line) was assayed after 72 h in cocultures with Mitomycine C^®-treated T-cell-depleted splenocytes loaded with the indicated peptide concentrations. ³H-thymidine was added for the last 12 h of culture. Error bars represent 1 SD. Data from one experiment representative of three experiments performed in triplicate wells. (B) CD4⁺ T cells induced either with WTGAD65 peptide (left panels) or CCGAD65 peptide (right panels) were analyzed by FACS at day 10 of stimulation. Results are from one experiment representative of three independent experiments. Additional information can be found as Figures S3 and S4 in Supplementary Material.

Figure 3

Antigen-presenting cells are induced into apoptosis by cCD4⁺ T cells. Splenic B cells isolated by magnetic beads from naive female NOD mice and activated overnight with LPS were cocultured for 24 h with CD4⁺ T cells generated either with the natural GAD65 peptide (white histograms) or with the CCGAD65 peptide (all other columns) in the presence of the indicated peptide (2 μM). Vertical axis represents B cell apoptosis measured by Annexin V binding. Addition of antibody toward FasL is shown in hatched column 7, and inhibitor toward GZB is shown in cross-hatched column 8. Statistical significance was calculated with one-way ANOVA test, P < 0.0001 and Dunnett’s multiple comparison test as indicated, *P < 0.05. Error bars represent 1 SD. Data are representative of three independent experiments.

Figure 4

Induction of apoptosis of bystander CD4⁺ T cells. CD4⁺ T cells isolated from female NOD mice previously immunized with WTGAD65 peptide and expanded in vitro served as bystander CD4⁺ T cells. (A) These cells were CFSE stained and cocultured with APCs loaded with WTGAD65 peptide (left column) showing basal mortality rate measured by Annexin V binding (vertical axis). cCD4⁺ T cells generated with CCGAD65 peptide were added to this coculture (right column). A control well was added in which the cCD4⁺ T cells were replaced with the same number of unlabeled CD4⁺ T cells generated with WTGAD65 (middle column). Error bars represent 1 SD. Statistical significance was calculated with one-way ANOVA test, P < 0.0001 followed by Dunnett’s multiple comparison test as indicated, *P < 0.05. (B) Same CFSE-stained cell line was used in this setting in coculture with APCs and cCD4⁺ T cells generated with CCGAD65 in the presence of the indicated peptide and mortality was measured by Annexin V expression (vertical axis). Hatched column 4 shows the effect of FasL antibody addition to the coculture, cross-hatched column 5 shows addition of GZB inhibitor, and black column 6 shows combination of both blockers. Error bars represent 1 SD. ****P < 0.0001 (One-way ANOVA test and Dunnett’s multiple comparison test). Data are representative of three independent experiments.

Figure 5

NOD mice transferred with cCD4⁺ T cells induced by CCGAD65. Naive mice were passively transferred at 6 weeks of age with cCD4⁺ T cells (2 × 10⁵) obtained from mice preimmunized with CCGAD65 peptide (from 8 to 12 weeks and sacrificed at 15 weeks) and expanded in vitro with CCGAD65. Mice were followed up with weekly blood glucose measurements until 40 weeks of age. (A) Diabetes-free survival curves are shown for mice transferred with CCGAD65-generated CD4⁺ T cells (n = 11), compared to the group transferred with AAGAD65-generated CD4⁺ T cells (n = 12) as indicated, *P < 0.05 (Mantel–Cox log-rank test). (B) Pancreatic sections of normoglycemic mice at 40 weeks of age transferred either with CCGAD65-induced CD4⁺ T cells or AAGAD65-induced CD4⁺ T cells were stained with H&E and insulitis was scored for at least 40 islets per mouse. Left panel: vertical axis represents insulitis index for each mouse, line indicates mean per group, treated group (n = 8) was compared to control group (n = 4). Statistical significance was calculated using two-tailed Mann–Whitney, **P < 0.01. Right panel: vertical axis represents percentage of insulitis-free islets per mouse, line indicates mean per group. Treated group was compared to control, **P < 0.01 (two-tailed Mann–Whitney test).

Figure 6

Diabetes-free survival in female Ins2^−/− NOD mice immunized with CCGAD65 peptide. In conventional animal facilities, 4-week-old female Ins2^−/− NOD mice received four weekly subcutaneous injections of 50 μg of CCGAD65 (n = 11) or AAGAD65 (n = 10) or CCHEL (n = 8) peptide in alum as indicated. Control group (n = 11) remained untreated. Blood glucose levels were monitored weekly up to 20 weeks of age. Vertical axis represents percentage of diabetes-free survival. Results from two independent experiments, *P < 0.05, **P < 0.005 (Mantel–Cox log-rank test).