FIG. 4.

Analysis of TNF-α-producing HSV-1 gB-specific CD8⁺ T cells following acute infection with HSV-1. WT and CD4-depleted mice were infected with HSV-1 in the FP, and draining PLN cells were harvested on day 5 p.i. and stimulated in vitro with gB peptide in the presence of brefeldin A for 5 h. The stimulated lymphocytes were then surface stained for the expression of CD8 and CD25, fixed, permeabilized, and stained for intracellular expression of TNF-α and IFN-γ. (A) Representative dot plots show the frequency of CD8⁺ TNF-α⁺ T cells in the draining lymph nodes. Numbers are the percentages of CD8⁺ TNF-α⁺ T cells. Numbers in parentheses are the MFI for TNF-α staining. (B) (Left) Absolute number of TNF-α-producing CD8⁺ T cells (three mice/group; *, P = 0.02). (Right) MFI of TNF-α staining by CD8⁺ T cells (three mice/group; *, P < 0.0001). Data shown are representative of two independent experiments (means ± SEM). (C) Representative histograms show percentages of gated CD8⁺ T cells which express IFN-γ only (upper left quadrant), IFN-γ and TNF-α (upper right quadrant), or TNF-α only (lower right quadrant). P values of <0.05 were considered significant.