Sex-Specific Immune Responses to Seasonal Influenza Vaccination in Diabetic Individuals: Implications for Vaccine Efficacy

Figure 3

Evaluating the proportional and activity alterations in CD8T and CD4T cells and their subsets Treg and Th17 cell population. (a) The CD3+ cells are gated out from the live single splenocytes and the CD4T and CD8T populations are analyzed taking the unstained live single cells as reference gating. (b) The population percentage of cytotoxic CD8T cells and (c) the population percentage of helper CD4T cells were determined. The percentage exhibited in the Y axis is the total percentage within the 10,000 splenocytes analyzed. Hence, for absolute number conversion of CD8T or CD4T percentage: 1% in the Y axis = 100 cells. (d) The CD3+CD4+ T cell population gated out on the splenocytes (as in (a)) is analyzed for the expression of regulatory T cell (Treg) markers FOXP3 and Th17 cell marker RORgT. (e) Treg population (CD3+CD4+ FOXP3+) and (f) Th17 cell population (CD3+CD4+RORgT+) percentage of total helper CD4T cells were determined. The comparison between the absolute number of Treg and Th17 cells is provided in Figures S3(a) and S3(b). (g) The bar diagram of costimulatory marker CD28 expression on CD8T cells is determined by analyzing the MFI. The MFI and histogram plots are provided in Figure S3(c). (h and i) The MFI values were analyzed to determine the level of intracellular expression of proinflammatory cytokine of IFNγ as observed by flow cytometric staining of the permeabilized CD8T (h) and CD4T (i) cells, respectively. D−, nondiabetic groups; D+, diabetic groups. Data in graphs are the representative images derived from at least four independent experiments (, , and ). For each group n = 5, the error bar indicates the standard deviation.

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