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

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 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

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

Loss of Circulating CD4 T Cells with B Cell Helper Function during Chronic HIV Infection

<div><p>The interaction between follicular T helper cells (T_FH) and B cells in the lymph nodes and spleen has a major impact on the development of antigen-specific B cell responses during infection or vaccination. Recent studies described a functional equivalent of these cells among circulating CD4 T cells, referred to as peripheral T_FH cells. Here, we characterize the phenotype and in vitro B cell helper activity of peripheral T_FH populations, as well as the effect of HIV infection on these populations. In co-culture experiments we confirmed CXCR5+ cells from HIV-uninfected donors provide help to B cells and more specifically, we identified a CCR7^highCXCR5^highCCR6^highPD-1^high CD4 T cell population that secretes IL-21 and enhances isotype-switched immunoglobulin production. This population is significantly decreased in treatment-naïve, HIV-infected individuals and can be recovered after anti-retroviral therapy. We found impaired immunoglobulin production in co-cultures from HIV-infected individuals and found no correlation between the frequency of peripheral T_FH cells and memory B cells, or with neutralization activity in untreated HIV infection in our cohort. Furthermore, we found that within the peripheral T_FH population, the expression level of T_FH-associated genes more closely resembles a memory, non-T_FH population, as opposed to a T_FH population. Overall, our data identify a heterogeneous population of circulating CD4 T cells that provides <i>in vitro</i> help to B cells, and challenges the origin of these cells as memory T_FH cells.</p></div

Characterization of peripheral T_FH cells.

<p>(<b>A</b>) Left: Representative flow cytometry plots from HIV-uninfected PBMC showing the gating scheme for isolating T cell subsets for the T cell/B cell coculture assay. Isolated populations include naïve cells (brown), CM CCR7^low (pink), CM CCR7^highCXCR5^low (orange), CM CCR7^highCXCR5^highCCR6^lowPD-1^high (green), CM CCR7^highCXCR5^highCCR6^highPD-1^low (blue) and CCR7^highCXCR5^highCCR6^highPD-1^high (red). Before gating on CCR6 and PD-1, cells were first gated on CD150^high. Right: Scatter plot indicating the frequency of each population in HIV-uninfected subjects (<i>n</i> = 13). Cells were not gated on CD150 for phenotypic analysis. (<b>B</b>) Indicated CD4 T cell populations were cultured with autologous naïve B cells (CD19^highCD27^lowIgD⁻) in the presence of SEB for 12 days and Ig concentrations were measured from supernatants (n = 6). (<b>C</b>) Indicated CD4 T cell populations were cultured with autologous naïve B cells in the presence of SEB for 2 days and cytokine concentrations were measured from supernatants (n = 6). Horizontal lines indicate limit of detection. Significant differences were determined using the Friedman test with Dunn's multiple comparison post-test. *p<0.05; **p<0.01.</p

Functional characteristics of pT_FH cells and the impact of HIV.

<p>(<b>A</b>) Representative flow cytometry plots showing CM, CD154-positive, cytokine-positive cells after SEB stimulation. CD154-positive, cytokine-positive CD4 T cells, shown by contour plots (blue: HIV-uninfected; red: HIV-infected), are overlaid onto 2 dimensional density plots for CM CD4 T cells plotted against CCR7 and CD3, and CXCR5 and CCR6. (<b>B</b>) Bar graphs showing the frequency of SEB-stimulated CD154-positive, cytokine-positive cells that express CCR7, CXCR5 and CCR6 (Blue: uninfected; n = 5; Red: HIV-infected; n = 24). (<b>C</b>) Left: Gag-specific CD4+ T cells (CD154-positive, cytokine-positive) shown as red contour plots are overlaid onto 2 dimensional density plots for CM cells CD4 T cells plotted against CCR7 and CD3, and CXCR5 and CCR6. Right: Bar graphs showing the frequency of Gag-specific CD154-positive, cytokine-positive cells that express CCR7, CXCR5 and CCR6 (n = 14). *p<0.05.</p

Progressive loss of pT_FH cells in HIV infection.

<p>(<b>A</b>) Pooled data showing the frequency (%) of CXCR5^high, CXCR5^highCCR6^high and CXCR5^highCCR6^highPD-1^high populations in total CD4 cells from PBMC from HIV uninfected (open circles; n = 13), HIV-infected (treatment-naïve), CD4 count >200 (light gray circles; n = 44), and HIV-infected (treatment-naïve), CD4 count <200 (black circles; n = 22). Significant differences between HIV-uninfected and HIV-infected subjects were determined using the Mann-Whitney U test. ***p<0.001; **p<0.01; *p<0.05. (<b>B</b>) Longitudinal analysis showing the frequency (%) of CXCR5^high, CXCR5^highCCR6^high and CXCR5^highCCR6^highPD-1^high populations in total CD4 cells or indicated populations in CXCR5-expressing cells (bottom row) from HIV-infected (treatment naïve) subjects (n = 10) over 36–48 months. No significant correlations were found. (<b>C</b>) Pooled data showing the frequency (%) of CXCR5^high, CXCR5^highCCR6^high and CXCR5^highCCR6^highPD-1^high populations in total CD4 cells from PBMC from HIV-uninfected subjects (open circles; n = 13) and HIV-infected subjects before (n = 14, week 0; black circles) and after ART (week 24, dark gray circles; week 48, light gray circles). Significant differences between HIV-uninfected and HIV-infected subjects were determined using the Mann-Whitney U test. Significant differences between subjects before and after ART were determined using the Wilcoxon matched-pairs signed rank test. ***p<0.001; **p<0.01; *p<0.05.</p

Relationship between pT_FH cells and T_FH cells in human tonsil.

<p>(<b>A</b>) Representative flow cytometry plots from HIV-uninfected, pediatric tonsils showing the gating scheme for determining the frequency of CCR6^high cells in T_FH (CXCR5^highPD-1^high) and non-T_FH populations. (<b>B</b>) Bar graphs showing the frequency of CCR6^high cells in T_FH and non-T_FH populations in human tonsils (n = 5). (<b>C</b>) Heatmap analysis of selected genes from RNA-seq data comparing pT_FH cells (CXCR5^highCCR6^highPD-1^high) from HIV-uninfected donors, pT_FH cells from HIV-infected donors, non-T_FH CD4 memory tonsil cells (CM CD57^lowPD-1^dimCCR7^highCCR5^lowCXCR4^low), non-germinal center T_FH tonsil cells (CM CD57^lowPD-1^highCCR7^lowCXCR5^high) and germinal center T_FH tonsil cells (CM PD-1^highCD57^high) from HIV-uninfected donors. (<b>D</b>) Top: Comparison of MAF expression on CD4 T cells from blood or tonsil. Bottom: Geometric mean (MFI) of MAF expression in the indicated populations of central memory CD4 T cells normalized to MAF MFI in naïve CD4 T cells.</p

Impaired B cell help by pT_FH cells in HIV infection.

<p>(<b>A</b>) CCR7^highCXCR5^low and CCR7^highCXCR5^highCCR6^high CM CD4 T cells isolated from PBMCs were cultured with autologous naïve B cells (CD19^highCD27^lowIgD⁻) in the presence of SEB for 12 days and Ig concentrations were measured from supernatants (HIV-uninfected, n = 8; HIV-infected (non-viremic), n = 5–7, HIV-infected (viremic), n = 1–2). Significant differences were determined using the Wilcoxon paired t-test or the Mann-Whitney test. *p<0.05; **p<0.01. (<b>B</b>) Top: HIV-uninfected PBMCs were incubated with indicated concentrations of CXCL-13 for 1 hour at 37°C (red) or 4°C (black). Bottom: Healthy PBMCs were incubated with 1 µg/mL CXCL13 for 10, 30, 60 or 120 minutes at 37°C (red) or 4°C (black). The frequency of CXCR5-positve CD4 T cells was calculated and normalized to time 0. (n = 3). (<b>C</b>) Top: Correlative analysis showing the frequency of CM CXCR5-positive CD4 T cells versus viral load (n = 27; r = −0.4036, P = 0.0368). Bottom: Correlative analysis showing the concentration of CXCL-13 in plasma or sera versus viral load (n = 27; r = 0.4414, P = 0.0165). Correlations were analyzed using the nonparametric Spearman test.</p