Figure 5. Intracellular cytokine staining detection of VV-specific CD8⁺ and CD4⁺ T-cell responses.

Splenocyte single-cell suspensions described in Figure 4 were stimulated in vitro with mock- or VV-WR-infected syngeneic A20 cells in the presence of IL-2 and Brefeldin A. After 16-18 hours of co-culture, the cells were surface stained with anti-CD4 and anti-CD8 antibodies, fixed, permeabilized, intracellularly stained with anti-IL-2 and -IFN-γ antibodies, and then analyzed by flow cytometry. Live lymphocytes were first identified and gated by FSC and SSC parameters and then subgated based on CD8 or CD4 expression. Panels depict the background (mock stimulated)-subtracted frequency of VV-specific CD8⁺ (A,B,E,F) or CD4⁺ (C,D,G,H) T cells expressing the indicated cytokines, as the arithmetic mean ± standard deviation of individually analyzed spleens (n = 4-5). These frequencies were <0.02% for both T-cell subsets in the spleens of naïve BALB/c and JH-KO mice (not shown). Although splenocytes from all of the immunodeficient groups were analyzed similarly, data corresponding to the depleted T-cell subset(s) is not shown due to >99% depletion efficiency. *P-value < 0.05.