FIG. 5.

DC depletion impedes HSV-1-specific CD8⁺ T-cell activation and function. For tetramer analysis, PLN and splenocyte cells were harvested from day 4-infected animals and surface stained for expression of CD8a. The stained lymphocytes were incubated with the allophycocyanin-conjugated MHC class I tetramer containing the gB peptide for 1 h at 4°C and washed twice prior to flow cytometric analysis. Representative histograms from the PLNs and spleen of nontreated, DT-treated, and PBS-treated mice are depicted in panels A and B, respectively. Numbers in the upper right quadrant represent the frequency of gB⁺ CD8⁺ T cells. (C and D) The bars represent the total number of CD8a⁺ gB⁺ double-positive cells in the PLNs and spleen on day 4. Each bar represents the mean value obtained from a minimum of two experiments using a minimum of three mice per treatment. The error bars represent the standard deviation from the mean. Statistical analysis using the Wilcoxon sum of ranks test showed a significant difference on day 4 p.i. between PBS plus HSV-1 versus DT plus HSV-1 (P ≤ 0.05) mice and HSV-1 versus DT plus HSV-1 (P ≤ 0.05) DTR-Tg mice. (E) Intracellular staining was used to gauge the total number of HSV-1-specific CD8 T cells producing IFN-γ in the PLNs of DT-treated and PBS-treated DTR-Tg mice on day 4 post-HSV-1 infection. On day 4 p.i., PLNs were harvested and dissociated into a single cell suspension by mechanical dissociation. PLN cells (10⁶) were placed in each well of a 96-well U-bottomed plate and cultured for 6 h in the presence of 5 × 10⁴ gB peptide-pulsed spleen cells used to restimulate HSV-1-specific IFN-γ production. VSV peptide-pulsed spleen cells served as a negative stimulator control. After 6 h, the restimulated PLN cells were surface stained for expression of CD8, fixed, and permeabilized and stained for expression of intracellular IFN-γ. The bars represent the mean number of total CD8 T cells producing IFN-γ per popliteal lymph node. Each bar represents the mean value obtained from a minimum of two experiments using a minimum of three mice per treatment. The error bars represent the standard deviation from the mean.