Frequency of infection.

†<p>Method 1: Assuming no multiple infection.</p>‡<p>Method 2: Assuming maximal multiple infection.</p>*<p>Poisson predicted values in parentheses.</p>**<p>Geometric mean of infection values.</p

Number of identical and distinct HIV-1 sequences.

<p>Number of identical and distinct HIV-1 sequences.</p

Evaluation of viral compartmentalization.

*<p>P values<0.05 (for Fst and SM) and bootstrap values>0.95 were considered statistically significant evidence of compartmentalization. P-values<0.05 and bootstrap values >0.95 are shown in bold and underlined.</p>**<p>Statistically significant evidence for compartmentalization after Bonferroni correction for multiple comparisons.</p>°<p>The number of migration events between the different populations in each phylogenetic tree.</p>†<p>AI: Association index, where 0 indicates maximum phylogenetic structure and 1 indicates panmixia. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003432#s2" target="_blank">Results</a> in italic type are results that changed (either to no significance of compartmentalization or significance of compartmentalization) when running the same analyses with collapsed identical sequences or the same number of sequences from each compartment. N/A Not applicable.</p

Frequency of HIV-1 infection of memory and naïve cells in blood and lymph nodes.

<p>(a) Frequency of infection of memory CD4⁺ T-cells isolated from lymph node tissue vs memory CD4⁺ T-cells isolated from peripheral blood. (b) Frequency of infection of memory CD4⁺ T-cells vs naïve CD4⁺ T-cells isolated from lymph node tissue. Frequency of infection for each patient and cell type was calculated using two methods. Method 1 assumes that each observed amplicon is assumed to derive from a unique infected cell (i.e., no multiple infection); Method 2 assumes that each row with two or more amplicons represents a multiply infected cell (assuming a maximal rate of multiple infection). Frequency of infection is shown as total number of cells per HIV-1 DNA molecule. Differences in frequency of infection between the different cellular compartments and cell types (here represented as p-values in the figure) are based on paired <i>t</i> tests of log-transformed frequencies of infection.</p

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 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

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

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

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