Fig 2: Delineation and quantification of different types of CD4+ T cells based upon their cytokines production by FACS after ex-vivo stimulation of splenic leukocytes by PMA. Intracellular cytokines transport blockers IMB were included during stimulation. (A) Various cytokines, IL-10, TNF-a, IFN-?, IL-4, and IL-21 producing CD4+ T cells increased significantly in young mice after PMA stimulation while high levels of IFN-? and IL-4 cytokine producing CD4+ T cells are observed in old mice even without PMA stimulation. High numbers of IL-10 cytokine producing CD4+ cells were observed in all young and old mice irrespective of infection. (B) Th1 response was stimulated in all infected mice with most pronounced change in T cells of B. microti infected mice after PMA stimulation. Th2 response was most pronounced in all infected old C3H mice. High Th17 cells stimulation in young infected mice could indicate inflammatory response; however, a much higher Tregs response in these mice appears to maintain splenic immune cells homeostasis preventing fatal disease. Lowest ratio of Tregs/Th17 cells was observed in old B. microti infected mice but mice did not appear sick or lethargic at this stage of infection. Each bar represents the mean ± s.d. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig 3: Scheme of Flow-based quantification of various intracellular cytokines production, and of different types of CD4+ cell, and different cytokine producing CD8+ cells in infected mice after in vitro stimulation with PMA+IMB. After cell sorting for CD45+ cells (top left), cells were stimulated and then marked with antibodies against mouse CD4 (FITC), and CD8 (AF 647) cells (top center). Cells were then fixed, permeabilized, and stained for intracellular cytokines: IFN-?-PB, TNF-a-APC.Cy7, IL-21-e-Fluor 711, IL-10-PerCP/Cy5.5, and IL-4-BV605 (2nd row). Then CD4+ cells producing different cytokines were quantified (results presented in Figure 5A). In CD4+ subpopulation, cells that were positive for IFN-?, but negative for other four cytokines were identified as Th1 cells (bottom left). CD4+ cells that had IL-10 but no other cytokines were defined as Tregs, whereas cells producing both IL-4 and IL-10 cytokines, but not IL-21, IFN-?, and TNF-a were identified as Th2 (bottom center two boxes). CD4+ cells that produced only IL-21 were identified as Th17 (bottom right) and results are presented in Figure 5B). CD8 cells producing different cytokines are presented in Figure 6.