The frequency of less differentiated PD-1^high CD127^high CD4 T cells is reduced compared with more differentiated subsets in advanced HIV infection.

<p><b>(A)</b> Gating strategy to define differentiation status of CD127, PD-1 and CTLA-4 expression by CD4 T cells. Differentiation was defined by gating on CD27 and CD45RA with CD27^highCD45RA^high (referred to as <b>Naïve</b>), CD27^highCD45RA^low (<b>Early/Intermediate</b>), and CD27^lowCD45RA^low (<b>Late</b>). <b>(B)</b> Distribution plots from HIV- infected subjects compared to HIV-uninfected (open circles, n = 15) from two cohorts with HIV infection: Cohort 1 (median CD4 count 525 cells/μl, filled circles, n = 31); and Cohort 2 with more advanced infection (median CD4 count 148 cells/μl, filled squares, n = 14) of PD-1 and PD-1/CTLA-4 expression by differentiation status and CD127 (IL-7Ra) staining demonstrating an altered/reduced frequency of PD-1^high CTLA-4^high/low CD127^high CD4 T cells of early/intermediate differentiation compared to more differentiated subsets which show increased PD-1 expression with more advanced HIV infection. Plots include median and interquartile range, *p< 0.05, **p< 0.001, ***p< 0.0001 by Kruskal-Wallis or Mann-Whitney test.</p

PD-1^highCD127^high Early/Intermediate CD4 T cells express the HIV coreceptor CCR5, activation markers HLA-DR and CD38, and demonstrate evidence of TCR stimulation.

<p><b>(A)</b> Bar graphs showing the relative frequency of CD4 T cell populations expressing several chemokine receptors (CCR4, CCR5, CCR6, and CCR7) and <b>(B)</b> markers of activation/differentiation (BTLA, HLA-DR, and CD38) (n = 7 HIV-infected donors). All populations are CD127^high. MFI, mean fluorescence intensity; bars represent mean and SEM, *p< 0.05, after correction by Dunn’s multiple comparisons test. (<b>C</b>) Evidence of recent TCR stimulation was assessed based on telomerase expression by qRT-PCR assay of sorted populations (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144767#pone.0144767.s003" target="_blank">S3 Fig</a> for gating strategy). Individual differences between differentiation subsets (shown for each individual by a connecting line) were statistically significant (p = 0.02, Friedman test).</p

Seletctive loss of PD-1^highCTLA-4^low/highCD127^high Early/Intermediate CD4 T cells occurs with higher plasma HIV-1 viral RNA levels and higher cell-associated viral DNA.

<p><b>(A)</b> Scatter plots of HIV-1 viral RNA and fitted regression lines for total (naïve and memory) CD8 and CD4 T cells demonstrating increased PD-1 expression with higher viral replication. However, for CD4 T cells of Early/Intermediate differentiation expressing CD127 and PD-1 or PD-1/CTLA-4 there is a negative association compared with more differentated (CD127^low) CD4 T cells. Spearman rank correlation coefficients and associated p-values are shown. <b>(B)</b> Donors (n = 14, five from Cohort 1 and nine from Cohort 3) with HIV Gag-specific CD4 T-cell responses are more differentiated (CD127^low) and co-express both PD-1 and CTLA-4. <b>(C)</b> Cell-associated HIV-1 <i>gag</i> DNA (no. copies/cell) for sorted T cell populations (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144767#pone.0144767.s003" target="_blank">S3 Fig</a> for gating strategy). Individual differences between differentiation subsets (shown for each individual by a connecting line) are statistically significant (p = 0.031 by Friedman test). <b>(D)</b> PD-1^highCTLA-4^lowCD127^high Early/Intermediate CD4 T cells are increased after antiretroviral therapy. Relative frequencies of bulk CD4 populations before and after initiation of combination antiretroviral therapy (cART). Connected symbols represent pre-cART and 48 weeks post-cART (Cohort 2, n = 14, Wilcoxon matched-pairs test, p-values shown in figure). The PD-1^highCTLA-4^low CD127^high group is analyzed separately for subjects who started cART with a CD4 count less than 200.</p

PD-1^highCD127^high Early/Intermediate CD4T cells maintain broad cytokine production.

<p><b>(A)</b> Cytokine production after polyclonal stimulation (anti-CD3 with anti-CD28 and anti-CD49d co-stimulation) measured by bead-based Luminex technology of fresh, sorted CD4 T cells from HIV-uninfected donors (n = 5, *p< 0.05 by Friedman test for each cytokine across cell populations). <b>(B)</b> Percent Ki67⁺ staining cells for CD127^high and CD127^low early/intermediate CD4 T cells from HIV-infected Cohort 1 (n = 11). <b>(C)</b> Differentiation phenotype of IFN-g or IL-17a positive cells detected after (6h) <i>ex vivo</i> SEB stimulation for HIV-infected donors (n = 5). No differences were statistically significant (Mann-Whitney test) <b>(D)</b> The relative frequency of the CCR6^highCXCR5^high population within the CCR7^highPD-1^highCD127^high Intermediate CD4 T cell population is decreased in HIV-infected (n = 15) compared to uninfected (n = 8) individuals (p = 0.0004, Mann-Whitney test).</p