nTregs and DP cells exhibit superior permissiveness to infection. Matched total memory (CD45RA−) and the four subsets of CD45RA+CCR7+ CD4+ T-cells (nTregs, nT, DP, and DN cells) were isolated by flow cytometry from the PBMCs of CI on ART subjects as in Additional file 1 and 2: Figure S1-S2, respectively. Levels of integrated (A) and Gag
(C) HIV-DNA were quantified by real-time PCR (equivalent of 105 cells/test in triplicates) in matched samples from five CI on ART subjects (Table 3, CI #4, 6, 8, 12, 16). HIV-DNA copies were normalized to CD3 levels and expressed as HIV-DNA copies/106 cells. (A, C) Shown are results from individuals donors. (B, D) Shown are statistical analysis of relative integrated and Gag HIV-DNA levels in naive T-cell subsets (mean ± SD, n = 4); HIV-DNA levels in conventional naive cells (nT) were considered 100%. (E-F) PBMCs from HIV-uninfected subjects (n = 17) were stained on the surface with CD3, CD4, CD45RA, CCR7, CD25, CD127, and CCR5 or CXCR4 Abs. The viability dye Vivid was used to exclude dead cells. The nTregs, nT, and DP cells (identified as in Figure 2A), together with total memory CD4+ T-cells (identified as in Figure 4E), were analyzed for the expression of CCR5 and CXCR4. Shown are the frequencies of CCR5+
(E) and CXCR4+
(F) T-cells within each subset of HIV-uninfected subjects (n = 17; Table 1, HIV- #1,2,5,10,14,15,18,20,21,23-30). The Friedman and Dunns’ post-test p-values are indicated on the graphs (*, p < 0.05; **, p < 0.01; ***, p < 0.001).