Characterization of conventional CD4<sup>+</sup> T cells as well as Treg cells during pulmonary PCM.

<p>(A) Representative FACS plots demonstrating the gating strategy for CD4<sup>+</sup> T cells and T reg cells. (B) Expansion of CD4<sup>+</sup> T lymphocytes during infection with <i>P</i>. <i>brasiliensis</i>. Percent (left) and total number (right) of CD4<sup>+</sup>Foxp3<sup>-</sup> T cells and CD4<sup>+</sup>Foxp3<sup>+</sup> T cells were analyzed by flow cytometry in the lungs of infected and uninfected C57BL/6 Foxp3<sup>GFP</sup> mice. Numbers indicate mean ± SD for at least five mice per group and are representative of three independent experiments (* <i>p</i> < 0.05; ** <i>p</i> < 0.005; *** <i>p</i> < 0.001). (C, D) Flow cytometric characterization of CD4<sup>+</sup>Foxp3<sup>-</sup> T cells (C) and CD4<sup>+</sup>Foxp3<sup>+</sup> 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<sup>+</sup>Foxp3<sup>-</sup> T cells or CD4<sup>+</sup>Foxp3<sup>GFP+</sup> T cells. Bars reflect mean ± SD of three independent experiments with five mice per group (* <i>p</i> < 0.05; ** <i>p</i> < 0.005; *** <i>p</i> < 0.001).</p>