A viral epitope that mimics a self antigen can accelerate but not initiate autoimmune diabetes

Abstract

We document here that infection of prediabetic mice with a virus expressing an H-2Kb-restricted mimic ligand to a self epitope present on beta cells accelerates the development of autoimmune diabetes. Immunization with the mimic ligand expanded autoreactive T cell populations, which was followed by their trafficking to the islets, as visualized in situ by tetramer staining. In contrast, the mimic ligand did not generate sufficient autoreactive T cells in naive mice to initiate disease. Diabetes acceleration did not occur in H-2Kb-deficient mice or in mice tolerized to the mimic ligand. Thus, arenavirus-expressed mimics of self antigens accelerate a previously established autoimmune process. Sequential heterologous viral infections might therefore act in concert to precipitate clinical autoimmune disease, even if single exposure to a viral mimic does not always cause sufficient tissue destruction.

Figure 1

Molecular mimicry can accelerate but not easily initiate autoimmune diabetes. (A) Molecular mimicry is insufficient to prime naive autoreactive CD8 T cells and cause autoimmune diabetes. RIP-LCMV-NP mice were infected with 10⁵ PFU LCMV-Arm (open circles), LCMV-Arm-Var (filled triangles), or PV alone (filled circles). (B) Primed autoreactive cells can become activated via molecular mimicry and accelerate disease. RIP-LCMV-NP mice were infected with either 10⁵ PFU LCMV-Arm (open circles, filled circles) or 10⁵ PFU PV (open triangles, filled triangles) on day 0 and, as indicated, received a secondary inoculation (2^nd inf.) with PV (open triangles, filled circles) 28 days after the priming LCMV infection. As a comparison, the incidence data to RIP-LCMV-NP mice infected with LCMV alone are displayed (open circles). For both studies, blood glucose values were determined at weekly intervals. Mice with blood glucose levels above 300 mg/dl were considered diabetic. It is evident from these studies that secondary infection but not primary infection with PV can accelerate T1D development. Statistical analysis was done using the log rank test. Note that the diabetes onset curves for the groups LCMV alone versus LCMV-PV are significantly different (P = 0.0066).

Figure 2

CD8 T cell populations specific for the mimicking epitope PV-NP₂₀₅ are significantly expanded after sequential infection with PV. (A and B) RIP-LCMV-NP and RIP-LCMV-NP × K^b(–) mice were infected with 10⁵ PFU LCMV or PV. After 4 weeks, the mice received a secondary infection of either LCMV or PV. (A) Intracellular cytokine staining (ICCS) after stimulation with PV-NP₂₀₅ is displayed for 1 representative mouse infected first with LCMV (LCMV alone) and then with PV (LCMV-PV) (mean frequencies are indicated in boxed areas). (B) The frequency of epitope-specific CD8 T cells in the blood was determined by ICCS for IFN-γ after stimulation with the indicated peptides (key) immediately before (upper panel) and 7 days after (lower panel) secondary infection. (C) Numbers of H-2K^b–restricted PV-NP₂₀₅–specific lymphocytes after LCMV or PV infection, assessed by ICCS for IFN-γ. Splenocytes were harvested on day 35 from mice that received LCMV at day 0 (d0) and, for the PV group, PV at day 28 (d28). Means (± SEM) are displayed. (D) Lytic precursors after LCMV-NP₃₉₆ versus PV-NP₂₀₅ antigenic stimulation for 10 days. In addition to lytic activity, IFN-γ production was assessed in the supernatant of each well; wells with IFN-γ levels of more than 0.05 ng/ml by ELISA were counted as positive. IFN-γ production was on average 13 (± 3.5) ng/ml in LCMV-NP₃₉₆–stimulated cultures and 7.1 (± 3.1) ng/ml in PV-NP₂₀₅–stimulated cultures. This experiment was repeated three times and mean values (± SEM) are displayed. *P < 0.05.

Figure 3

Sequential infection with LCMV and PV results in accumulation of PV-NP₂₀₅–specific CD8 T cells in the islets of Langerhans. (A) RIP-NP mice were infected with 10⁵ PFU LCMV. After 4 weeks, one group of mice received a secondary infection of PV (10⁵ PFU, i.p.). Left panels, pancreata were harvested at week 3 after secondary infection and 6-μm tissue sections were stained for cellular infiltration with a monoclonal antibody against CD8. Sections were counterstained with hematoxylin. Right panels, pancreata were harvested at day 5 after secondary infection and 6-μm tissue sections were cut and were stained for CD8 T cells with rhodamine X–conjugated anti-CD8 (red) and for PV-NP₂₀₅–specific CD8 T cells with allophycocyanin-conjugated H-2K^b–PV-NP₂₀₅ tetramers (green). Note that only after sequential infection with LCMV followed by PV are PV-NP₂₀₅–specific CD8 T lymphocytes (yellow) found in the islets of Langerhans. Original magnification, ×20. (B) Expansion of PV-NP₂₀₅–specific CD8 T cell populations in blood and pancreatic lymph nodes after secondary PV infection. Upper panels, flow cytometry of PV-NP₂₀₅–specific CD8 T cells in the blood of LCMV-immune mice that did or did not receive secondary infection with PV, as detected by H-2K^b–PV-NP₂₀₅ tetramers; mean frequencies are indicated in boxed areas. Lower panel, frequencies of PV-NP₂₀₅–specific CD8 T cells were determined by flow cytometry using H-2K^b–PV-NP₂₀₅ tetramer staining and by ICCS for IFN-γ expression after 5 hours of in vitro stimulation with PV-NP₂₀₅ peptide.

Figure 4

H-2K^b–restricted, autoreactive, LCMV/PV-NP₂₀₅–specific cross-reactive CD8 T cells mediate the acceleration of diabetes. (A and B) RIP-LCMV-NP or RIP-LCMV-NP × K^b(–) mice were infected with LCMV or PV. After 4 weeks, the mice received a secondary infection of PV. (A) Blood glucose of RIP-LCMV-NP, RIP-LCMV-NP × K^b(–), and RIP-LCMV-NP × K^b(+) littermates was measured in weekly intervals. The diabetes onset curves (blood glucose values > 300 mg/dl) for the groups [RIP-LCMV-NP × K^b(–) vs. RIP-LCMV-NP × K^b(+)] are significantly different (log rank test; P = 0.0167). (B) Pancreas sections from 3–4 mice per group at week 3 after secondary infection with PV were stained for cellular infiltration of CD8 T cells. Sections of 1 representative RIP-LCMV-NP × K^b(–) and RIP-LCMV-NP × K^b(+) mouse are shown. Original magnification, ×20. (C) Mice were tolerized to PV-NP₂₀₅ by injection of 2 × 10⁷ ECDI–PV-NP₂₀₅–coupled autologous splenocytes (ECDI + NP₂₀₅) or with 2 × 10⁷ splenocytes treated with EDCI alone, 5 days before infection with 10⁵ PFU LCMV. After 4 weeks, mice were infected with PV. Diabetes incidence (blood glucose values > 300 mg/dl) at week 4 after PV infection is displayed; numbers of mice analyzed per group are indicated in parentheses. (D and E) Groups of 3–4 mice were infected with LCMV. After 4 weeks, the mice received 100 μg of PV-NP₂₀₅ peptide or an H-2K^b–restricted control peptide (OVA; SIINFEKL). In addition, mice received three injections of poly(I:C) (7.5 μg/g body mass) at the time of peptide injection and then at days 2 and 4 thereafter. Controls received PV-NP₂₀₅ only or PV infection. (D) The frequency of blood LCMV/PV-NP₂₀₅–specific cross-reactive CD8 T cells was assessed by flow cytometry using H-2K^b–PV-NP₂₀₅ tetramers (day 7 after peptide injection). (E) Mean blood glucose values (± SEM) measured at week 2 after peptide and/or poly(I:C) injection is displayed.