Characterization of conventional CD4+ T cells as well as Treg cells during pulmonary PCM.

(A) Representative FACS plots demonstrating the gating strategy for CD4+ T cells and T reg cells. (B) Expansion of CD4+ T lymphocytes during infection with P. brasiliensis. Percent (left) and total number (right) of CD4+Foxp3- T cells and CD4+Foxp3+ T cells were analyzed by flow cytometry in the lungs of infected and uninfected C57BL/6 Foxp3GFP mice. Numbers indicate mean ± SD for at least five mice per group and are representative of three independent experiments (* p < 0.05; ** p < 0.005; *** p < 0.001). (C, D) Flow cytometric characterization of CD4+Foxp3- T cells (C) and CD4+Foxp3+ T cells (D) indicate an activated status at the site of infection of both cell subsets, as shown by CTLA-4, GITR, ICOS, and PD-1 expression. Solid lines represent infected mice, whereas dashed lines indicate uninfected mice. Median fluorescence intensity (MFI) values were calculated from all CD4+Foxp3- T cells or CD4+Foxp3GFP+ T cells. Bars reflect mean ± SD of three independent experiments with five mice per group (* p < 0.05; ** p < 0.005; *** p < 0.001).