Altered frequency of memory Th17 during HIV infection in relationship with the paucity of naive-like nTreg and DP cells. (A-D) Memory CD4+ T-cells isolated by negative selection using magnetic beads (Miltenyi) were stimulated with PMA/Ionomycin and brefeldin A for 5 hours. Shown are: (A) representative flow cytometry dot plots illustrating IL-17A and IFN-γ expression on CD3+CD8− T-cells from one HIV- and one CI on ART; (B) statistical analysis of the frequency of IL-17A+IFN-γ− and IL-17A+IFN-γ+ cells in HIV- controls (n = 5; Table 1, HIV- #2, 10, 14, 31, 32) versus CI on ART subject (n = 8; Table 3, CI #3, 7–11, 14, 18); (C) the co-expression of CCR6, CD26 and CD161 with IL-17A in one representative HIV- control; and (D) statistical analysis for the frequency of IL-17A-expressing cells within CD3+CD8− T-cells co-expressing (+++) or lacking (---) CCR6, CD26, and CD161. (E) Shown is the gating strategy for memory CD4+ T-cells (CD45RA−) with a mTh17 (CCR6+CD26+CD161+) phenotype in one representative donor. (F) The frequency and counts of mTh17 cells were analyzed in HIV- (n = 18; Table 1, HIV- #1-3, 5, 9–18, 20–23), RI (n = 15, Table 2, RI 11–15), and CI on ART (n = 17; Table 3, CI #1-12, 14–18) subjects. Each symbol represents a different subject. The (B) Mann–Whitney, (D) Wilcoxon, and (F) Kruskal-Wallis and Dunns post test p-values are indicated on the graphs (*, p < 0.05; **, p < 0.01; ***, p < 0.001). (G) Linear regression (LR) and Spearman correlation (SC) models were applied to determine the relationship between mTh17 cell counts and the counts of nTregs (left panel), DP (middle panel) and total naive-like CD25+ T-cells (right panel) in CI on ART. LR p and r2 and SC p and r values are indicated on the graphs. For studies in Figure 4G, subjects were identical to those included in Figure 4 F for which matched samples were available.