Proliferation, degranulation and cytokine production by FLUAVsw-specific CD8β
T cells. Violet-labeled PBMCs isolated at 2, 6 and 9 weeks post primary infection were restimulated twice with FLUAVsw (infection strain, MOI = 0.1; day 0 and day 5 of culture) and analyzed by FCM on day 6 of culture. (A) CD8β+ T cells were gated (not shown) and analyzed for violet proliferation, surface CD107a expression and intracellular accumulation of IFN-γ and TNF-α. Contour plots show all possible marker combinations. Exemplary raw data of one non-infected control animal (#3) and one infected animal (#8) is shown for week 6 post primary infection. (B) Boolean gating was applied in order to identify multifunctional CD8β+ T cells, and resulting CD8β+ T-cell subsets were grouped by number of functions. Displayed is the frequency of CD8β+ T cells carrying out two or more functions within total CD8β+ T cells. Data of six FLUAVsw-infected animals (#4-9) and two non-infected control animals (#1, #3) is shown.