Presented antigen from damaged pancreatic beta cells activates autoreactive T cells in virus-mediated autoimmune diabetes

Abstract

The induction of autoimmunity by viruses has been attributed to numerous mechanisms. In mice, coxsackievirus B4 (CB4) induces insulin-dependent diabetes mellitus (IDDM) resembling the final step of disease progression in humans. The immune response following the viral insult clearly precipitates IDDM. However, the molecular pathway between viral infection and the subsequent activation of T cells specific for islet antigen has not been elucidated. These T cells could become activated through exposure to sequestered antigens released by damaged beta cells, or they could have responded to factors secreted by the inflammatory response itself. To distinguish between these possibilities, we treated mice harboring a diabetogenic T cell repertoire with either the islet-damaging agent streptozotocin (STZ) or poly I:C, which nonspecifically activates T cells. Significantly, only treatment of mice with STZ resulted in IDDM and mimicked the effects observed following CB4 infection. Furthermore, antigen-presenting cells from STZ-treated mice were shown to directly activate autoreactive T cells and induce diabetes. Therefore, the primary role of CB4 in the precipitation of IDDM is to damage tissue, causing release and presentation of sequestered islet antigen. These events stimulate autoreactive T cells and thereby initiate disease.

Figure 1

(a) Incidence of diabetes in BDC2.5 mice following poly I:C treatment. BDC Tg mice and nontransgenic littermate controls were treated with 100 μg of poly I:C. Alternatively, BDC Tg mice were infected with 100 pfu of CB4. Mice were monitored twice weekly for blood glucose, and mice with two consecutive values over 300 mg/dl were considered diabetic. Both BDC2.5 mice (n = 12) and their nontransgenic littermates (n = 12) were analyzed and compared with untreated BDC Tg mice (n = 20). (b) Incidence of diabetes in BDC2.5 mice following STZ treatment. BDC Tg mice and nontransgenic littermates were treated with single doses of 0, 40, 80, or 160 mg/kg of STZ. Mice were monitored twice weekly for blood glucose, and mice with two consecutive values over 300 mg/dl were considered diabetic. (c) Incidence of diabetes in NOD/Shi mice following STZ treatment. Twelve-week-old NOD/Shi mice (n = 29) were treated with 80 mg/kg of STZ. Mice were monitored twice weekly for blood glucose, and mice with two consecutive values over 300 mg/dl were considered diabetic. Treated mice were analyzed and compared with untreated NOD/Shi mice (n = 10). Mice from all groups were age matched. Groups from b and c were compared for statistical significance by standard χ-square tests. Of the relevant comparisons, significant differences were observed between STZ-treated (80 mg/kg) BDC mice and STZ-treated (80 mg/kg) NOD mice at both 2 (P < 0.001) and 4 (P < 0.001) weeks after treatment.

Figure 2

(a) Histological analysis of pancreata of treated BDC Tg and NOD mice. Representative sections of the pancreas from STZ-treated BDC Tg mice and poly I:C–treated BDC Tg mice reveal islet cell integrity and function. The function of β cells was confirmed by immunohistochemistry using antibody to insulin (INS) (×400), present as brown staining. Mice were sacrificed at 2 weeks after treatment. (b) Immunohistological analysis of the islets from treated BDC Tg mice for activated T cells. Representative immunostained sections of islets from the pancreas of either STZ- or poly I:C–treated BDC Tg mice. Immunohistochemical staining for either CD4 or CD25 was performed and revealed CD4⁺ lymphocytes in both mice, but only STZ-treated mice have activated CD4⁺ cells as demonstrated by CD25 staining. Immunostained cells are brown (×400). The mice were sacrificed at 14 days after treatment. (c) Immunohistological analysis of pancreata from treated BDC Tg and NOD mice for macrophages. Representative immunostained sections of the pancreas from CB4-infected and STZ- and poly I:C–treated BDC Tg and NOD mice reveal differences in the number and activation state of resident macrophages. Antibody to the macrophage activation marker F4/80 was used to identify activated macrophages, which, following CB4 infection and STZ treatment of BDC Tg mice, appear to become activated in the peri-insular inflammation and to mobilize into the islets of Langerhans during insulitis (×400). Brown anti-macrophage staining is present over macrophages. Mice were sacrificed 2 weeks after infection or treatment.

Figure 3

Analysis of the activation state of lymphocytes from BDC2.5 mice following treatment with either poly I:C (a) or STZ (b). Cells were stained with antibody to V_β4 TCR, CD4, and antibody to either CD25, CD44, or CD69. Cells were analyzed by FACS and compared to isotype controls. Shown are histograms of mean fluorescent intensities generated by gating on 10,000–20,000 CD4⁺, V_β4⁺ cells, or V_β4⁺, CD4^– cells. (a). For both a and b, the plots are representative of one of six mice analyzed, and all six had similar results. (a) Analysis of peripheral blood lymphocytes before (narrow) and three days following poly I:C treatment (wide) from identical mouse are displayed. No shift of activation is observed in the V_β4⁺, CD4⁺ T cell population while activation is observed in the V_β4⁺, CD4^– (CD8⁺) T cell population posttreatment (PT). Changes in cell populations and gated markers CD4⁺, V_β4⁺: CD25, 5.7% to 7.2%; CD44-high, 28.6% to 25.2%; CD69, 6.7% to 6.9% and CD4^–, V_β4⁺: CD25, 2.9% to 27.8%; CD 44-high, 22.9% to 65.0%; CD69, 10.7% to 41.8%. (b) Analysis of lymphocytes from the spleen and pancreatic lymph node before (narrow) and seven days following STZ treatment (wide) are displayed. Mice were analyzed at three, five, and seven days PT. Similar results were observed at five days, but not at three days. A dramatic shift in all three activation markers was observed in the spleen PT. A less dramatic shift was observed in the pancreatic lymph node.Changes in cell populations and gated markers in spleen CD4⁺ V_β4⁺ : CD25, 10.4% to 32.9%; CD44-high, 19.8% to 56.5%; CD69, 9.0% to 32.3%; and in pancreatic lymph nodes, CD4⁺ V_β4⁺ : CD25, 5.4% to 25.3%; CD44-high, 7.2% to 18.9%; CD69, 8.7% to 27.0%.

Figure 4

Presentation of endogenous antigen by APCs from STZ-treated mice activates β cell–specific BDC T cells. Adherent mononuclear cells from NOD mice treated with STZ or PBS were prepared, irradiated, and cultured with BDC2.5 Tg splenocytes (2 × 10⁵) at various ratios. Shown is a summary of results from the 4:1 ratio, T cell (2 × 10⁵): APC (5 × 10⁴). Also shown are the results of culturing these cells at 4:1 in the presence of antigenic peptide (7 μM) (20) specific for the BDC T cell. Cultures were pulsed with 1 μCi of ³H thymidine at 48 hours and harvested 18 hours thereafter. The data presented are from four separate experiments involving a total of eight mice in each group representing the APC populations. Values are presented as stimulation index + SEM as compared with BDC splenocytes incubated without additional APCs or antigen. Background in all experiments was between 500 and 2500 cpm.