FIG. 3.

Assessment of IFN-γ-producing HSV-1 gB-specific CD8⁺ T cells during acute infection with HSV-1 in CD4-depleted, B6-MHC-II^−/−, and WT mice. Mice were infected with HSV-1 in the hind FP, and the draining PLN were harvested on day 5 p.i. The lymph node cells were stimulated in vitro with gB peptide or control (VSV) 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 IFN-γ. (A) Representative dot plots show the frequency of CD8⁺ IFN-γ⁺ T cells in the draining lymph nodes. Numbers in the gated regions are the percentages of CD8⁺ IFN-γ⁺ T cells. Numbers beside the boxed area indicate the MFI for IFN-γ staining within the gated region. (B) (Top panels) Absolute numbers of IFN-γ producing CD8⁺ T cells (four mice/group; *, P = 0.01 [left]; three mice/group; *, P = 0.002 [right]). (Bottom panels) MFI of IFN-γ staining by gB-specific CD8⁺ T cells in the gated region (four mice/group; *, P = 0.004 [left]; three mice/group; *, P = 0.01 [right]). (C) Representative histograms showing expression of IFN-γ and CD25 on gated CD8⁺ T cells. Numbers in the quadrants are percentages of CD8⁺ cells which are IFN-γ⁺ CD25⁻ (quadrant 1) or IFN-γ⁺ CD25⁺ (quadrant 2). Numbers in parentheses are the MFI for IFN-γ staining. Data are representative of two independent experiments (means ± SEM). P values of <0.05 were considered significant.