Flow cytometric analysis of CD4⁺ and CD8⁺ IFN-γ producing lymphocytes. PBMC from foals of different age groups and from adult horses were stimulated with PMA and ionomycin. The cells were stained for intracellular IFN-γ and cell surface CD4 or CD8 expression. (A) Flow cytometric analysis of one representative horse per age group. (B) Relative percentages of CD4⁺/IFN-γ⁺ and (C) CD8⁺/IFN-γ⁺ T cells in foals and adult horses within the IFN-γ producing cells (= 100%). (D) Percentages of IFN-γ⁺/IL-10⁺ lymphocytes in foals and adult horses. (E) A tri-color staining was performed on stimulated PBMC to confirm that the majority of the IFN-γ⁺/IL-10⁺ cells are CD4⁺ T cells. Gates were set on lymphocytes (see Fig. 1A) and on IFN-γ⁺ cells (left panel). The IFN-γ⁺ lymphocytes were then analyzed for IL-10 and CD4 staining (right panel). The horizontal lines within the data sets B, C and D represent medians. *** p < 0.001; ** p = 0.001 to < 0.01; ns = not significant.