Interferon-γ Limits Diabetogenic CD8+ T-Cell Effector Responses in Type 1 Diabetes

Abstract

Type 1 diabetes development in the NOD mouse model is widely reported to be dependent on high-level production by autoreactive CD4⁺ and CD8⁺ T cells of interferon-γ (IFN-γ), generally considered a proinflammatory cytokine. However, IFN-γ can also participate in tolerance-induction pathways, indicating it is not solely proinflammatory. This study addresses how IFN-γ can suppress activation of diabetogenic CD8⁺ T cells. CD8⁺ T cells transgenically expressing the diabetogenic AI4 T-cell receptor adoptively transferred disease to otherwise unmanipulated NOD.IFN-γ^null , but not standard NOD, mice. AI4 T cells only underwent vigorous intrasplenic proliferation in NOD.IFN-γ^null recipients. Disease-protective IFN-γ could be derived from any lymphocyte source and suppressed diabetogenic CD8⁺ T-cell responses both directly and through an intermediary nonlymphoid cell population. Suppression was not dependent on regulatory T cells, but was associated with increased inhibitory STAT1 to STAT4 expression levels in pathogenic AI4 T cells. Importantly, IFN-γ exposure during activation reduced the cytotoxicity of human-origin type 1 diabetes-relevant autoreactive CD8⁺ T cells. Collectively, these results indicate that rather than marking the most proinflammatory lymphocytes in diabetes development, IFN-γ production could represent an attempted limitation of pathogenic CD8⁺ T-cell activation. Thus, great care should be taken when designing possible diabetic intervention approaches modulating IFN-γ production.

Figure 1

IFN-γ–deficient but not standard NOD mice develop AI4 T cell–induced T1D. A: Diabetes development in female NOD and NOD.IFN-γ^null mice injected i.v. at 6 weeks of age with 1 × 10⁷ NOD.Rag1^null.AI4 splenocytes. Survival curves compared by log-rank test. B and C: In vivo proliferation and activation of CFSE-labeled NOD.Rag1^null.AI4 T cells in PLNs of NOD and NOD.IFN-γ^null mice. B: CFSE dilution of AI4 T cells in PLNs of NOD and NOD.IFN-γ^null mice at 3 days posttransfer. Representative histograms are shown in the left panel, and mean fluorescence intensity (MFI) of CFSE staining of AI4 T cells is shown in the right panel. C: The frequency of AI4 CD8⁺ T cells among live PLN cells at 3 days posttransfer. D: CFSE dilution and activation of AI4 T cells in spleens of NOD and NOD.IFN-γ^null mice at 8 days posttransfer. Results for each quadrant represent the mean ± SE of three mice per treatment. B–D represent results from a single experiment. *P < 0.05 determined by one-way ANOVA.

Figure 2

IFN-γ–producing CD4⁺ T cells suppress diabetogenic CD8⁺ T cells through mechanisms that do not involve quantitative or functional variations in Tregs. A: Quantitative PCR analysis of IFN-γ mRNA expression by host-type CD4⁺ and CD8⁺ (Vα8⁻) T cells and B cells purified from spleens of NOD mice 3 days postadoptive transfer with 2 × 10⁷ NOD.Rag1^null.AI4 splenocytes (post-AT) or untreated NOD mice (untreated). Results represent the mean ± SE of three samples per treatment. B: Diabetes incidence for female NOD.scid mice injected at 6–8 weeks of age with 1 × 10⁷ NOD.Rag1^null.AI4 splenocytes in the presence or absence of 3 × 10⁶ CD4⁺ T cells purified from NOD or NOD.IFN-γ^null donors. C: Frequencies, numbers, and mean fluorescence intensity (MFI) of FoxP3 antibody staining of splenic CD4⁺CD25⁺FoxP3⁺ Tregs in NOD and NOD.IFN-γ^null mice. Results represent the mean ± SE of five mice per treatment. D: Crisscross cultures were established to assess the ability of CD4⁺CD25⁺ Tregs from NOD and NOD.IFN-γ^null mice to suppress the anti-CD3–stimulated proliferation of CD4⁺CD25⁻ effectors from both strains (assessed by flow cytometic detection of CFSE dilution). E: Beginning at 6 weeks of age, NOD female mice received three biweekly i.p. injections with the Treg-depleting CD25-specific PC61 antibody or a rat IgG1 isotype control. One week after the first treatment, mice in both groups were injected i.v. with 1 × 10⁷ NOD.Rag1^null.AI4 splenocytes and subsequently monitored for diabetes. Survival curves compared by log-rank test. *P < 0.05 determined by one-way ANOVA, **P < 0.01 determined by unpaired t test. Teff, effector T cell; WT, wild type.

Figure 3

IFN-γ produced by T or B cells suppresses AI4 T cells through both direct and indirect mechanisms. A: Diabetes incidence in female NOD and NOD.IFN-γR^null recipient mice injected i.v. at 6 weeks of age with 1 × 10⁷ NOD.Rag1^null.AI4 splenocytes. B: Diabetes incidence in female NOD, NOD.IFN-γR^null, and NOD.IFN-γ^null recipient mice injected i.v. at 6 weeks of age with 1 × 10⁶ purified AI4 T cells. C: Diabetes incidence in female NOD.Rag1^null and NOD.Rag1^null.IFN-γR^null recipient mice injected i.v. with 1 × 10⁶ purified AI4 T cells and 2 × 10⁶ purified NOD splenic CD4⁺ T cells. D: Diabetes incidence in female NOD.Rag1^null recipient mice injected i.v. with 1 × 10⁷ NOD.Rag1^null.AI4 splenocytes and 2 × 10⁶ purified CD4⁺ splenic T cells from NOD or NOD.IFN-γR^null donors. Survival curves compared by log-rank test.

Figure 4

Higher STAT1 but not STAT4 expression by transferred AI4 T cells is associated with the lesser ability of such effectors to induce diabetes in NOD and NOD.IFN-γR^null than NOD.IFN-γ^null recipients. A–C: Otherwise unmanipulated NOD or NOD.IFN-γ^null recipients were injected with 1 × 10⁶ AI4 T cells and analyzed for total STAT1 or STAT4 levels 2–7 days after transfer. A: Representative pattern (from day 4) of transferred CD8⁺ tetramer⁺ AI4 T cells from the indicated recipients showing STAT1 (left) or STAT4 (right) compared with fluorescence minus one (FMO) control stains. B: Quantification of STAT1 (left) or STAT4 (right) staining of splenic AI4 CD8⁺ T cells after transfer into the indicated recipients. C: Endogenous CD8⁺ tetramer⁻ T cells from the indicated recipients were analyzed for mean fluorescence intensity (MFI) of STAT1 (left) or STAT4 (right) staining after transfer of AI4 CD8⁺ T cells. B and C display combined data for days 2–7 posttransfer of AI4 T cells. D: Comparison of STAT1 expression by tetramer⁺ AI4 donor T cells before and 2 days after transfer into otherwise unmanipulated NOD.IFN-γ^null recipients. E: Otherwise unmanipulated NOD or NOD.IFN-γR^null recipients were injected with 1 × 10⁶ AI4 T cells and analyzed for STAT1 or STAT4 levels 2 days after transfer. Quantification of STAT1 (left) or STAT4 (right) staining of splenic AI4 CD8⁺ T cells 2 days after transfer into the indicated recipient. F: Endogenous CD8⁺ tetramer⁻ T cells from the indicated recipients were analyzed for MFI of STAT1 (left) or STAT4 (right) staining 2 days after transfer of AI4 CD8⁺ T cells. D–F display data from a single experiment. G and H: NOD, NOD.IFN-γ^null, or NOD.IFN-γR^null mice were irradiated (600 cGy) and injected with 1 × 10⁶ AI4 T cells. Two days posttransfer, AI4 T cells were analyzed for STAT1 or STAT4 levels. Left panels: Histograms showing STAT1 (G) or STAT4 (H) expression of AI4 T cells from the indicated recipients compared with an FMO control. Right panels: Quantification of MFI of STAT1 (G) or STAT4 (H) staining from one of two experiments showing n ≥ 3 per group. P values calculated using Mann–Whitney analysis.

Figure 5

IFN-γ exposure during activation reduces the cytotoxicity of human β-cell–reactive CD8⁺ T cells. A: Specific lysis of BL5 human β-cell line target cells coincubated at different E:T ratios with HLA-A*02-01–restricted IGRP-specific CD8⁺ T cells transduced in the presence or absence of 1,000 U/mL IFN-γ. B: Specific lysis of BL5 target cells coincubated with nondiabetogenic MART-1–specific CD8⁺ T cells transduced in the presence or absence of IFN-γ. BL5 cells were pre-exposed to 1,000 U/mL IFN-γ and washed before they were used in the cell-mediated lympholysis assays. P values calculated using a paired t test.