An altered CD8+ T cell epitope of insulin prevents type 1 diabetes in humanized NOD mice

Abstract

Autoreactive CD8⁺ T cells, which play an indispensable role in β cell destruction, represent an emerging target for the prevention of type 1 diabetes (T1D). Altered peptide ligands (APLs) can efficiently induce antigen-specific T cells anergy, apoptosis or shifts in the immune response. Here, we found that HLA-A*0201-restricted CD8⁺ T cell responses against a primary β-cell autoantigen insulin epitope InsB1_5-14 were present in both NOD.β2m^null.HHD NOD mice and T1D patients. We generated several APL candidates for InsB1_5-14 by residue substitution at the p6 position. Only H6F exhibited an inhibitory effect on mInsB1_5-14-specific CD8⁺ T cell responses in vitro. H6F treatment significantly reduced the T1D incidence, which was accompanied by diminished autoreactive CD8⁺ T cell responses to mInsB1_5-14, inhibited infiltration of CD8⁺ and CD4⁺ T cells in the pancreas and reduced pro-inflammatory cytokine production in pancreatic and splenic T cells in NOD.β2m^null.HHD mice. Mechanistically, H6F treatment significantly augmented a tiny portion of CD8⁺CD25⁺Foxp3⁺ T cells in the spleen and especially in the pancreas. This subset exhibited typical Treg phenotypes and required peptide-specific restimulation to exert immunosuppressive activity. Therefore, this APL H6F may be a promising candidate with potential clinical application value for antigen-specific prevention of T1D.

Keywords: Altered peptide ligand; CD8+CD25+Foxp3+ regulatory T cells; InsB15–14; NOD.β2m null .HHD mice; Type 1 diabetes.

Fig. 1

Design and selection of a potential antagonist peptide for mIns1B_5–14. a The modeled structure of the mIns1B_5–14-HLA-A*0201 complex showing that the p5 and p6 positions of mIns1B_5–14 bulged out of the binding groove, which were more accessible for TCR inspection. b T2 cell-based peptide binding assays for HLA-A*0201 were performed by FACS analysis. T2 cells were incubated with or without the indicated peptides (10 μg/ml) and the level of surface HLA-A2 molecules was detected by flow cytometry. The H-2K^b-binding peptide OVA_257-264 was used as a negative control. Filled histograms, no peptide; open histograms, plus peptide. c The fluorescence index (FI) was calculated as follows: FI = (mean fluorescence intensity with the given peptide―mean fluorescence intensity without peptide)/(mean fluorescence intensity without peptide). Bars represent the mean ± SEM of three independent experiments. d, e The proliferation of splenocytes stimulated with mIns1B_5–14 (10 μg/ml) plus itself (10 μg/ml) or the indicated APLs (10 μg/ml) (d) and H6F at four different concentrations (e) was measured by [³H] thymidine incorporation. Bars represent the mean ± SEM of seven independent experiments. Significance was determined by an unpaired t-test

Fig. 2

Intraperitoneal immunization with the APL H6F prevented the onset of T1D in NOD.β2m^null.HDD mice. Female NOD.β2m^null.HHD mice received weekly intraperitoneal injections of 100 μg of peptide H6F, mIns1B_5-14, or OVA_257-264 from 4 to 9 weeks of age (n = 12 or 13). a The mice were monitored for diabetes development. Data were obtained from three independent experiments. *P < 0.05 and ** P < 0.01. b, c Histopathological evaluation of pancreatic sections from indicated peptide-treated NOD.β2m^null.HHD mice at the age of 12 weeks. Pancreatic sections were stained with H&E and scored for insulitis. Representative micrographs (200× magnification) (b) and histologic scores (c) from each group (n = 10) are shown. Insulitis scoring was performed according to the following criteria: 0, no infiltration; 1, peri-insulitis; 2, insulitis with <50% islet area infiltration; and 3, insulitis with >50% islet area infiltration. d Frequencies and absolute numbers of CD4⁺ and CD8⁺ T cells (pre-gated on a live CD3⁺ population) infiltrated in pancreata from the indicated peptide-treated NOD.β2m^null.HHD mice at the age of 12 weeks. The results for absolute numbers of these CD4⁺ and CD8⁺ T cells are expressed as the mean ± SD (each symbol represents a sample of pooled pancreatic infiltrating cells from four mice). e The mRNA expression levels of indicated cytokines in the pancreas in each peptide-treated group of nondiabetic mice (n = 6) were quantified by real-time RT-PCR. The data are presented as fold-change compared to the mRNA levels expressed in pancreata from control peptide-treated mice. Significance was determined by an unpaired t-test

Fig. 3

Treatment with H6F notably reduced IFN-γ and IL-17 secretion by splenic T cells in the NOD.β2m^null.HDD mice. The percentages of CD4⁺IFN-γ⁺ (a), CD8⁺IFN-γ⁺ (b), CD4⁺IL-17A⁺ (c), and CD8⁺IL-17A⁺ (d) T cells in spleens from the indicated peptide-treated nondiabetic NOD.β2m^null.HHD mice at 12 weeks of age (n = 8) were determined by flow cytometry. Representative FACS density plots and statistical analyses are shown. The data are expressed as the mean ± SD. Significance was determined by an unpaired t-test

Fig. 4

Treatment with H6F resulted in loss of mIns1B_5–14 autoreactive CD8⁺ T cell responses. a Representative statistical analysis of the average number of IFN-γ-positive spots derived from splenic CD8⁺ T cell responses to control peptide or mIns1B_5-14 within a treatment group (n = 5) are shown. The data are expressed as the mean ± SEM. Significance was determined by a paired t-test. b A representative image of IFN-γ ELISPOT assay for monitoring mIns1B_5-14-specific splenic CD8⁺ T cell responses in each peptide-treated group of nondiabetic NOD.β2m^null.HHD mice at the age of 12 weeks. The average number of IFN-γ-positive spots per 2 × 10⁵ splenic CD8⁺ T cells in triplicate cultures was calculated. T2 cells pulsed with mIns1B_5-14 or control peptide (50 µg/ml) were used as stimulators

Fig. 5

Treatment with H6F notably increased the frequency of CD8⁺CD25⁺Foxp3⁺ T cells in NOD.β2m^null.HHD mice. Spleen-, pancreatic lymph node (PLN)-, inguinal lymph node (ILN)- and pancreas-infiltrating (PIL) cells were freshly isolated from the indicated peptide-treated nondiabetic NOD.β2m^null.HHD mice at the age of 12 weeks (n = 8–16). a The percentages of CD8⁺CD25⁺ T cells and CD8⁺CD25⁺Foxp3⁺ T cells in the spleen and PLNs were determined by flow cytometry. The data are expressed as the mean ± SD. b Comparison of the frequencies of CD8⁺CD25⁺ and CD8⁺CD25⁺Foxp3⁺ T cells in ILNs, PLNs, and spleens from H6F-treated nondiabetic NOD.β2m^null.HHD mice (n = 8). The data are expressed as the mean ± SD. c Representative FACS plots showing the frequencies of CD8⁺CD25⁺ and CD8⁺CD25⁺Foxp3⁺ T cells among PIL cells pooled from four nondiabetic NOD.β2m^null.HHD mice with the indicated peptide treatments. d Comparison of the frequencies of CD8⁺CD25⁺Foxp3⁺ T cells among PIL cells and PLNs from the same group of NOD.β2m^null.HHD mice treated with mIns1B_5-14 or H6F (n = 16). The results of the PIL cells are expressed as the mean ± SD (n = 4, each sample derived from pooled pancreatic infiltrating cells from four mice), and the PLN results are expressed as the mean ± SD (n = 16). Significance was determined by the Mann–Whitney test

Fig. 6

Phenotypic characterization of CD8⁺CD25⁺ T cells in the NOD.β2m^null.HHD mice. CD8⁺CD25⁺ and CD8⁺CD25⁻ T cells were freshly isolated from the spleens of H6F-treated nondiabetic NOD.β2m^null.HHD mice at the age of 12 weeks (n = 5) and these cells were phenotyped by flow cytometry analysis. a Representative flow cytometry phenotypic profiles of CD8⁺CD25⁺ and CD8⁺CD25^- T cells in H6F-treated NOD.β2m^null.HHD mice. The numbers above the line indicate the event numbers of cells with a positive phenotype in the indicated cell subsets, and the numbers below the line indicate the total numbers of the indicated CD8⁺CD25⁺ T cells or CD8⁺CD25^- T cells. b The mean percentages of Foxp3⁺, CTLA-4⁺, CD62L⁺, CD103⁺, IFN-γ⁺ and IL-17A⁺ in CD8⁺CD25⁺ (open bars) or CD8⁺CD25^- (gray bars) T cells from H6F-treated NOD.β2m^null.HHD mice (n = 5). ***Compared to the same phenotype or cytokine production of CD8⁺CD25⁺ T cells, P < 0.001. The data are expressed as the mean ± SD. c The mean percentages of Foxp3⁺, CTLA-4⁺, CD103⁺, and CD62L⁺ in splenic CD8⁺CD25⁺ T cells or d the mean percentages of IFN-γ⁺ and IL-17A⁺ in splenic CD8⁺CD25^- T cells isolated from different peptide-treated NOD.β2m^null.HHD mice (n = 5). The data are expressed as the mean ± SD. Significance was determined by an unpaired t-test

Fig. 7

Splenic CD8⁺CD25⁺ T cells in the H6F-treated NOD.β2m^null.HHD mice exerted ligand-specific suppressive activity. a Suppression of CD4⁺CD25^- T cell proliferation by splenic CD8⁺CD25⁺ T cells or CD4⁺CD25⁺ T cells. CD4⁺CD25^- T cells (100,000 cells/well) were co-cultured in triplicate with varied numbers of CD8⁺CD25⁺ (open bars) or CD4⁺CD25⁺ (gray bars) T cells isolated from the same H6F-treated mice at 1∶0, 10∶1, 5∶1, 2∶1, 1∶1 and 0∶1 ratios in the presence of anti-CD3/CD28-coupled beads for 3 days. ^*P < 0.05 and ^**P < 0.01 compared with the [³H] thymidine uptake of CD4⁺CD25^- T cells alone. b The proliferation of pre-activated CD4⁺CD25^- T cells (50,000 cells/well) cocultured with mitomycin C-treated syngeneic splenocytes (250,000 cells/well) loaded with 10 μg/ml OVA_257-264 (gray bars), 10 μg /ml H6F (open bars), or no peptide (black bar) in the presence of an equal number of CD8⁺CD25⁺ T cells purified from the spleens of H6F-treated NOD.β2m^null.HHD mice. **P < 0.01 compared with the [³H] thymidine uptake of CD4⁺CD25^- T cells cocultured with mitomycin C-treated syngeneic splenocytes alone. c IFN-γ and IL-17A in the cell culture supernatants from (b) were assayed by ELISA. ^***P < 0.001 and ^**P < 0.01 compared with IFN-γ and IL-17A in the cell culture supernatants of CD4⁺CD25^- T cells cocultured with mitomycin C-treated syngeneic splenocytes alone. The data are presented as the mean of triplicate cultures and are representative of three independent experiments. Significance was determined by an unpaired t-test