Blockade of IFN-γ or TNF-α prevents abrogation of diabetes by secondary viral infection. (a) Cumulative incidence of diabetes in RIP-LCMV-NP mice infected i.p. with 10⁵ PFUs of LCMV-Arm on day 0 and 10⁵ PFUs of LCMV-Past 1 month after LCMV-Arm infection. Blood glucose was measured in weekly intervals, and values greater than 300 mg/dl were considered diabetic. The number of diabetic mice and the total number of animals used per group are indicated in brackets. Human TNFR55-IgG1 fusion protein (38, 39) or neutralizing rat anti–mouse IFN-γ antibody (Pharmingen, San Diego, California, USA) were given at 100 μg (i.v.) or 50 μg (i.v.), respectively, at days 1, 3, 6, and 9 after secondary (Past) infection. (b) In vitro hyperstimulation with D^b(NP₃₉₆)-tetramers (16 hours at 37°C) of lymphocytes isolated from spleen and PDLN of C57BL/6 mice at 1 week after LCMV-Arm infection. Apoptosis of D^b(NP₃₉₆)-specific CD8 T cells was determined by staining with annexin V–phycoerythrin conjugate in parallel to staining with D^b(NP₃₉₆)-tetramers. Cells were incubated in medium alone (control) or in the presence of human TNFR55-IgG1 fusion protein (100 μg/well) or neutralizing rat anti–mouse IFN-γ antibody (100 μg/well). *Among splenocytes, significant differences in the number of D^b(NP₃₉₆)-specific CD8 T cells that undergo apoptosis (annexin V^hi cells) were observed after blockade of TNF-α compared with the control stimulation, but not after IFN-γ blockade (Student’s t test, P < 0.05). **PDLNs were collected from three mice, and cells were pooled in order to increase the number of D^b(NP₃₉₆)-specific CD8 T cells within the experimental setup.