Figure 1.
Time course of T cell cytokine production after infection with LCMV clone 13: the role of IL-10 in promoting viral persistence. (A) Spleen cells were isolated from BALB/c mice on days 0, 5, 7, 14, 20, 40, and 94 after infection with 105 PFU LCMV Armstrong or 2 × 106 PFU LCMV clone 13 and cultured for 48 h. The concentration of IL-10 present in the culture supernatants was measured by ELISA. Data are plotted as IL-10 (pg/ml) produced per 106 cells and are means of three mice per time point. The experiment is representative of three similar experiments. (B) To reveal the origin of IL-10–producing cell subsets, BALB/c splenocytes were isolated and infected in vitro for 48 h with LCMV Armstrong or LCMV clone 13 without or with anti–IL-10R antibody treatment (multiplicity of infection = 3). Next, CD4+, CD8+, and CD11c+ cells were enriched using MACS beads, and the supernatant of the purified populations was analyzed for IL-10 production by ELISA. The graph indicates the levels of IL-10 (pg/ml) in the supernatant of CD4+, CD8+, and CD11c+ cells. Data are representative of one experiment with five mice per group. (C) Sensitive direct in vivo intracellular cytokine assay was performed to detect in vivo cytokine expression in LCMV-specific T cells, as described by Liu et al. (reference 61). In brief, mice were injected i.v. with 0.5 mg BFA on days 5 and 7 after infection with 105 PFU LCMV Armstrong or 2 × 106 PFU LCMV clone 13. Spleens were harvested 6 h after BFA injection, and direct in vivo intracellular cytokine detection was performed after staining with fluorescent antibodies to CD4, CD8, IFN-γ, and TNF-α. Mean values per spleen of three to five individual mice are shown. The experiment is representative of two similar experiments. (D) PD-1 expression was detected on CD4+ and CD8+ T cells from naive or day 7 LCMV Armstrong– or LCMV clone 13–infected mice by labeling splenocytes with fluorescent antibodies to CD4, CD8, and PD-1. Percentages of PD-1–expressing T cells are shown. Data show one representative mouse per group (three mice total per group). (E) IL-10−/− and wild-type mice were infected with 2 × 106 PFU LCMV clone 13, and viral titers were detected in kidney and liver from three mice per group by RT-PCR 3 wk after infection. Data are shown as mean LCMV genome copies per mg organ ± SEM. (F) Cytokine expression was monitored in wild-type and IL-10−/− mice 7 d after LCMV clone 13 infection by intracellular cytokine staining. Splenocytes were stimulated with BFA and GP33-41 or GP61-80 peptide for 6 h. Intracellular cytokine detection was performed by staining with fluorescent antibodies to CD4, CD8, IFN-γ, and TNF-α. The IL-10/IFN-γ ratio of CD4+ and CD8+ T cells is shown as mean values of three individual mice per group. Statistical analysis was performed using the Student's t test. *, P < 0.01; **, P < 0.001; and ***, P < 0.0001.