Immunoregulatory mechanisms triggered by viral infections protect from type 1 diabetes in mice (original) (raw)
Acute infection of prediabetic mice with CVB3 or LCMV delays T1D onset and diminishes disease incidence. In the NOD model of spontaneous autoimmune diabetes, we used 2 different RNA viruses that, upon infection, prevent rather than induce the disease: CVB3, an enterovirus that causes a systemic, acute infection that is nonpersisting but leads to immune-mediated myocarditis; and LCMV Armstrong, an arenavirus that causes a systemic, acute infection that is asymptomatic and also nonpersisting. The majority of mice infected with CVB3 or LCMV did not develop T1D (Figure 1) (although mice challenged with CVB3 developed signs of virus-related illness). This effect of these viruses on T1D development was observed in earlier studies by us and others (7, 11, 12), but the precise underlying mechanisms were not elucidated. Interestingly, we observed that CVB3 and LCMV infections had 2 distinct effects on T1D — a delay in the onset of overt diabetes and a decrease in disease incidence, which we sought to explain mechanistically.
Acute infection of prediabetic NOD mice with CVB3 or LCMV delays T1D onset and diminishes disease incidence. Cumulative diabetes incidence over time in NOD mice left untreated (Naive) or infected at 9 weeks of age with CVB3 (orange triangles) or LCMV (green inverted triangles). ***P < 0.001.
Viral infection of prediabetic mice causes PD-L1 upregulation on lymphoid cells and prevents the expansion of diabetogenic CD8+ T cells expressing PD-1. We first assessed the early changes that occurred in the lymphoid organs of NOD mice infected with CVB3 or LCMV. In particular, we assessed accessory, inhibitory molecules often upregulated during viral infection. Expression of PD-L1 was variable in CVB3-infected mice, but both CVB3 and LCMV strongly increased PD-L1 levels transiently on lymphoid cells in the pancreatic LNs and spleen of NOD mice (Figure 2A and Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI38503DS1). At the time at which infection was performed, the majority of autoreactive, diabetogenic CD8+ T cells specific for the β cell antigen islet-specific glucose-6-phosphate catalytic-related protein (IGRP) (35, 36) showed an activated phenotype and expressed the PD-L1 receptor PD-1 (Figure 2B). The PD-L1/PD-1 interaction has been shown to control not only T cell immunity in viral infection (14, 15), but also autoimmune T cell responses, notably in T1D (16, 18). Our observations thus suggested that viral infection of NOD mice rendered diabetogenic CD8+ T cells highly susceptible to PD-1–mediated impairment. Accordingly, whereas, as previously described (35), IGRP-specific CD8+ T cells expanded significantly in the pancreatic LN and spleen of untreated NOD mice, the frequency of these cells remained low in mice infected with CVB3 or LCMV (Figure 2C).
CVB3 or LCMV infection of prediabetic NOD mice causes PD-L1 upregulation on lymphoid cells and prevents the expansion of IGRP-specific CD8+ T cells expressing PD-1. (A) Percentage of PD-L1hi cells over time in the pancreatic LN and spleen of individual NOD mice left untreated or infected at 9 weeks of age with CVB3 (orange triangles) or LCMV (green inverted triangles), as measured by flow cytometry. (B) PD-1 and CD44 expression by IGRP-specific or -nonspecific (Other) CD8+ T cells in the pancreatic LN and spleen of 9-week-old naive NOD mice, as measured by flow cytometry after staining with NRP-V7 tetramer. Shown are representative flow cytometry contour plots. Numbers indicate the percentage of PD-1+CD44hi cells ± SD for 3 individual mice per group. (C) Percentage of IGRP-specific CD8+ T cells over time in the pancreatic LN and spleen of individual NOD mice left untreated or infected at 9 weeks of age with CVB3 or LCMV, as measured by flow cytometry after staining with NRP-V7 tetramer. In A and C, symbols represent individual values, and horizontal lines denote mean. *P < 0.05, **P < 0.005, ***P < 0.001.
Virally induced upregulation of PD-L1 in prediabetic mice prevents the expansion of diabetogenic CD8+ T cells and is responsible for the delayed onset but not decreased incidence of T1D. We assessed whether impaired expansion of IGRP-specific, diabetogenic CD8+ T cells was caused by the increase in PD-L1 expression triggered by viral infection. We used LCMV for this purpose, as PD-L1 was systematically upregulated in the lymphoid organs of LCMV-infected but not all CVB3-infected mice (Figure 2A). As PD-L1 blockade in NOD mice accelerated diabetes in a previous study (18), we avoided using neutralizing antibodies against PD-L1 to address this issue. Instead, we used siRNA technology with the goal of suppressing de novo expression of PD-L1 transiently in LCMV-infected mice. The efficacy of different siRNAs specific for PD-L1 was first assessed in vitro (Figure 3A and Supplemental Figure 2, A and B) to identify a candidate that could significantly inhibit upregulation of PD-L1 in the pancreatic LN and spleen of LCMV-infected NOD mice (Figure 3B). Of note, 21 days after infection, the level of expression of surface PD-L1 was reduced to normal levels in both siRNA-treated and nontreated mice (data not shown), suggesting that PD-L1 expression was indeed inhibited and not delayed by siRNA treatment. NOD mice were then challenged with LCMV and simultaneously treated with the selected PD-L1–specific siRNA or empty vehicle. We observed that LCMV infection failed to prevent the expansion of IGRP-specific CD8+ T cells in the pancreatic LN and spleen of mice treated with PD-L1 siRNA (Figure 3C). On the other hand, PD-L1 siRNA treatment did not appear to affect IGRP-specific cells in terms of PD-1 and CD44 expression (Supplemental Figure 2C). Furthermore, the delay in onset of overt diabetes mediated by LCMV infection was abolished by PD-L1 siRNA treatment, while the reduction in disease incidence was not reversed (Figure 3D). These results indicated that virally induced upregulation of PD-L1 on lymphoid cells prevented the expansion of diabetogenic CD8+ T cells and was the cause for the delayed onset but not the decreased incidence of T1D observed after viral infection.
LCMV-induced upregulation of PD-L1 in prediabetic NOD mice prevents the expansion of IGRP-specific CD8+ T cells and delays the onset of T1D. (A) Different siRNAs specific for PD-L1 were assessed for efficacy by infection of NOD splenocytes for 24 hours with LCMV in vitro (MOI, 1) after transfection with different siRNAs (in duplicate). Shown is inhibition of PD-L1 upregulation, calculated as the percentage of PD-L1hi cells in populations infected with LCMV and transfected with siRNA relative to the percentage of PD-L1hi cells in the population infected with LCMV and transfected with scrambled-sequence siRNA, ± SD for duplicate samples. (B) Percentage of PD-L1hi cells over time in the pancreatic LN and spleen of individual NOD mice infected at 9 weeks of age with LCMV and simultaneously injected with cationic vehicle alone or containing 150 μg PD-L1 siRNA 76238, as measured by flow cytometry. (C) Percentage of IGRP-specific CD8+ T cells in the pancreatic LN and spleen of individual 12-week-old NOD mice injected 21 days previously with cationic vehicle alone and left untreated or simultaneously infected with LCMV (green inverted triangles) or injected 21 days previously with cationic vehicle containing PD-L1 siRNA 76238 and simultaneously infected with LCMV (white inverted triangles), as measured by flow cytometry after staining with NRP-V7 tetramer. In B and C, symbols represent individual values, and horizontal lines denote mean. (D) Cumulative diabetes incidence over time in NOD mice injected at 9 weeks of age with cationic vehicle alone and left untreated or simultaneously infected with LCMV or injected 21 days previously with cationic vehicle containing PD-L1 siRNA 76238 and simultaneously infected with LCMV. *P < 0.05, **P < 0.005, ***P < 0.001.
The frequency of CD4+CD25+ Tregs and their capacity to produce TGF-β are increased in virally immune NOD mice. The upregulation of PD-L1 and the failure of diabetogenic CD8+ T cells to accumulate in virally infected mice caused only a delay in the onset of overt diabetes, indicating that additional factors contributed to full, long-term protection from T1D upon viral infection. It has been suggested that so-called natural CD4+CD25+ Tregs, which are crucial players in the maintenance of peripheral tolerance, progressively lose their capacity to control autoreactive T cells in the NOD model (37–39), thereby permitting T1D. We therefore assessed the number and function of these cells in virally challenged NOD mice. After viral clearance, the pancreatic LN and spleen of CVB3- and LCMV-immune NOD mice showed a significant increase in CD4+CD25+ T cells (Figure 4A). The vast majority of these cells (more than 95%) expressed Foxp3 along with low levels of CD127 (Figure 4B), indicating that they were indeed Tregs (20, 40). Furthermore, these cells expressed CTLA-4 along with high levels of glucocorticoid-induced tumor necrosis factor receptor (GITR; Figure 4B), suggesting that they might be natural Tregs (41, 42). Interestingly, while the percentage of TGF-β–producing cells was somewhat variable in CVB3-challenged mice, it was significantly higher in lymphoid organs compared with naive mice, a scenario that was also found in NOD mice following LCMV infection (Figure 4D). CD4+CD25+ T cells from both naive and virally immune mice were capable of producing IFN-γ but not IL-10 (Figure 4C). In sum, viral infection caused an increase in the frequency of CD4+CD25+ Tregs and their capacity to produce TGF-β.
The frequency of natural CD4+CD25+ Tregs and their capacity to produce TGF-β are increased in the lymphoid organs of CVB3- and LCMV- immune NOD mice. (A) Percentage of CD4+CD25+ T cells in the pancreatic LN and spleen of individual 12-week-old NOD mice left untreated or infected 21 days previously with CVB3 (orange triangles) or LCMV (green inverted triangles), as measured by flow cytometry. (B) Representative flow cytometry contour plots of Foxp3, CD127, CTLA-4, and glucocorticoid-induced tumor necrosis factor receptor (GITR) expression by CD4+CD25+ T cells in the pancreatic LN of individual 12-week-old NOD mice left untreated or infected 21 days previously with CVB3 or LCMV. Quadrants were defined based on isotype control stainings showing less than 0.2% positive cells for each parameter analyzed. Numbers indicate the percentage of cells in the corresponding quadrants. Comparable results were obtained in the spleen. (C) Representative flow cytometry contour plots of TGF-β, IL-10, IFN-γ, and TNF-α expression by CD4+CD25+ T cells from the pancreatic LN of individual 12-week-old NOD mice left untreated or infected 21 days previously with CVB3 or LCMV, as measured after PMA plus ionomycin stimulation. Quadrants were defined based on isotype control stainings showing less than 0.2% positive cells for each parameter analyzed. Numbers indicate the percentage of cells in the corresponding quadrants. Comparable results were obtained in the spleen. (D) Percentage of TGF-β–producing CD4+CD25+ T cells in the pancreatic LN and spleen of individual 12-week-old NOD mice left untreated or infected 21 days previously with CVB3 or LCMV, as measured by flow cytometry after PMA plus ionomycin stimulation. In A and D, symbols represent individual values, and horizontal lines denote mean. *P < 0.05, **P < 0.005, ***P < 0.001.
CD4+CD25+ Tregs modulated by viral infection in vivo are capable of diminishing T1D incidence through TGF-β production. We assessed whether the changes we observed in the Treg compartment of virally immune mice played a role in virally mediated prevention of T1D. We purified CD4+CD25+ T cells from naive, CVB3-immune, or LCMV-immune mice and injected them into naive, age-matched NOD recipients. The transferred CD4+CD25+ T cells were more than 95% pure and isolated 21 days after viral challenge, i.e., after viral clearance, in order to avoid injection of virus in these transfer experiments. When injected into prediabetic NOD mice, CD4+CD25+ T cells from CVB3-immune or LCMV-immune but not naive donors mediated a significant reduction in T1D incidence (Figure 5A). Based on our observation that virally exposed Tregs differed from their naive counterpart regarding TGF-β production (Figure 4, C and D), we asked whether TGF-β expression by naive Tregs would provide them with the capacity to promote tolerance in vivo. To this end, we purified CD4+CD25+ T cells from naive mice and transfected them with a TGF-β1–encoding cDNA before transferring them into naive prediabetic recipients. Transfection conditions yielded 8%–9% efficacy and allowed TGF-β expression by 4%–5% of the cells (Figure 5B), which was roughly comparable to the TGF-β production by CVB3- or LCMV-enhanced CD4+CD25+ T cells (Figure 4D). We found that naive CD4+CD25+ T cells transgenically expressing TGF-β could diminish T1D incidence in vivo (Figure 5C), suggesting that the enhanced tolerogenic function of virally modulated CD4+CD25+ T cells in T1D was exerted via their production of TGF-β.
CD4+CD25+ Tregs modulated during the prediabetic phase by CVB3 or LCMV infection in vivo are capable of diminishing T1D incidence in NOD mice through TGF-β production. (A) Cumulative diabetes incidence over time in NOD mice left untreated (None) or injected at 12 weeks of age with 5 × 105 CD4+CD25+ T cells purified from 12-week-old NOD donors left untreated (Naive Tregs) or infected 21 days previously with CVB3 (CVB3 Tregs) or LCMV (LCMV Tregs). CD4+CD25– T cells from CVB3- or LCMV-immune mice had no effect on disease outcome (data not shown). (B) The efficacy of transfection in CD4+CD25+ T cells was assessed by measuring by flow cytometry GFP and TGF-β expression in naive Tregs (purified as described in A) 15 hours after transfection with an empty plasmid (Control plasmid, top) or plasmid containing a cDNA (Encoding plasmid, bottom) encoding GFP (left) or TGF-β1 (right, assessed after PMA plus ionomycin stimulation). Shown are flow cytometry contour plots representative of 2 samples per group. Numbers indicate the percentage of cells in the corresponding gate/quadrant. (C) Cumulative diabetes incidence over time in NOD mice left untreated (None) or injected at 12 weeks of age with 5 × 105 naive Tregs transfected with an empty plasmid (Naive ctrl Tregs) or plasmid containing a cDNA encoding human TGF-β1 (Naive TGF-β Tregs). *P < 0.05, **P < 0.005.
Treatment of prediabetic mice with IFN-α induces PD-L1 upregulation on lymphoid cells and prevents the expansion of diabetogenic CD8+ T cells in vivo. We investigated the mechanism by which viral infection could have induced PD-L1 upregulation in vivo. Previous work by others had shown that PD-L1 expression by microvascular endothelial cells can be acquired as a direct consequence of exposure to type I and II interferon (43). As these cytokines are produced in vast amounts during viral infections, we asked whether they might be capable of directly inducing PD-L1 upregulation on NOD lymphoid cells. We found that PD-L1 expression was increased on these cells in response to IFN-α or IFN-γ in vitro (Figure 6A). Similarly, administration of IFN-α to prediabetic NOD mice rapidly induced upregulation of PD-L1 on lymphoid cells in the pancreatic LN and spleen in vivo (Figure 6B). In order to determine whether the effects of this cytokine-induced PD-L1 expression on T1D were comparable to those of the virally triggered increase, we measured the frequency of diabetogenic CD8+ T cells in lymphoid organs. We observed that expansion of IGRP-specific CD8+ T cells in the pancreatic LN and spleen was significantly hampered by IFN-α treatment, similar to our observation using CVB3 or LCMV (Figure 2C). These results suggested that during viral infection, upregulation of PD-L1 on lymphoid cells and subsequent control over expanding diabetogenic CD8+ T cells was caused, at least in part, by interferon production.
Treatment of prediabetic NOD mice with IFN-α induces PD-L1 upregulation on lymphoid cells and prevents the expansion of IGRP-specific CD8+ T cells in vivo. (A) PD-L1 expression on NOD splenocytes incubated for 24 hours with LCMV (MOI, 1), IFN-α (104 U/ml), or IFN-γ (10 μg/ml), as measured by flow cytometry. Numbers indicate the percentage of PD-L1hi cells ± SD for 2 individual mice per group. (B) Percentage of PD-L1hi cells in the pancreatic LN and spleen of individual 9-week-old NOD mice left untreated (None) or injected 24 hours previously with 105 U IFN-α (blue diamonds), as measured by flow cytometry. (C) Percentage of IGRP-specific CD8+ T cells in the pancreatic LN and spleen of individual 12-week-old NOD mice left untreated or injected 21 days prior with 105 U IFN-α, as measured by flow cytometry. In B and C, symbols represent individual values, and horizontal lines denote mean. *P < 0.05, **P < 0.005, ***P < 0.001.
PD-L1 upregulation in prediabetic mice synergistically increases the capacity of virally enhanced CD4+CD25+ Tregs to protect from T1D. Our results so far indicated that viral infection was capable of modulating 2 distinct regulatory components of the immune system, both acting to protect from T1D. First, PD-L1 upregulation prevented the expansion of diabetogenic, PD-1–expressing CD8+ T cells and delayed the onset of overt diabetes. Second, CD4+CD25+ Tregs were enhanced in number and TGF-β–producing function and diminished diabetes incidence. Our finding that these 2 regulatory components had distinct, partially protective effects in T1D (Figure 3D and Figure 5A) suggested that upon viral infection, full protection might be conferred by their combination. We thus addressed whether virally enhanced CD4+CD25+ T cells might confer enhanced protection from T1D when diabetogenic CD8+ T cells had been previously curbed by upregulation of PD-L1 in vivo. To this end, we treated NOD mice with IFN-α and LCMV-enhanced CD4+CD25+ T cells in combination. IFN-α was administered at 9 weeks of age to confer upregulation of PD-L1 and hinder the accumulation of IGRP-specific diabetogenic CD8+ T cells (Figure 6, B and C), and CD4+CD25+ T cells were purified from LCMV-immune mice and injected at 12 weeks of age to diminish T1D incidence (Figure 5A). We found that treatment with IFN-α alone delayed the onset of T1D (consistent with a PD-L1–mediated effect), while injection of LCMV-exposed CD4+CD25+ Tregs alone diminished disease incidence (Figure 7). Strikingly, coinjection of IFN-α and LCMV-enhanced CD4+CD25+ Tregs was highly effective in protecting from T1D (Figure 7), to a degree comparable to LCMV infection (Figure 1). This indicated that virally enhanced CD4+CD25+ Tregs synergized with PD-L1 upregulation and ensuing impairment of autoreactive T cells to efficiently protect from T1D.
Early PD-L1 upregulation in prediabetic NOD mice synergistically increases the capacity of LCMV-enhanced CD4+CD25+ Tregs to protect from T1D. Cumulative diabetes incidence over time in NOD mice left untreated or injected at 9 weeks of age with 105 U IFN-α or at 11 weeks of age with 5 × 105 LCMV Tregs (purified as described in Figure 5A), or receiving both treatments (red squares). ***P < 0.001.