PD-1 and CTLA-4 expression on PPD-specific CD4 T-cells in response to TB treatment.

<p><b>A.</b> Representative plot showing the gating scheme used to identify PD-1, CTLA-4, and 2B4 expression on PPD-specific CD4 T-cells. Single, live, CD3⁺, CD4⁺ cells were gated for CD27 and CD45RO. The naïve CD4 T cell population was identified (CD27⁺CD45RO^-) and selected to set gates for PD-1, CTLA-4 and 2B4 as this population typically does not express inhibitory molecules. These gates were then applied to PPD-specific and total CD4 T-cell populations. <b>B.</b> Expression of PD-1, CTLA-4, and 2B4 on PPD-specific CD4 T-cells in untreated and treated TB disease. <b>C.</b> Correlation between baseline CD4 T-cell count and frequency of PD-1 and CTLA-4 expression on PPD-specific CD4 T-cells in untreated and treated TB disease. Lines of best fit, along with Spearman’s rank correlation coefficient and corresponding p-values are shown <b>D.</b> Expression of PD-1, CTLA-4, and 2B4 on CMV-specific CD4 T-cells in untreated and treated TB disease in our HIV-TB and TB cohorts. <b>E</b>. Expression of PD-1, CTLA-4, and 2B4 on total CD4 T-cells in untreated and treated TB disease in our HIV-TB and TB cohorts. <b>F</b>. Bar graph depicts the co-expression patterns of PD-1, CTLA-4, and 2B4 on PPD-specific CD4 T-cells in untreated and treated TB disease in our HIV-TB and TB cohorts. To assess expression of inhibitory molecules on PPD and CMV-specific CD4 T-cells, only samples with at least 50 cytokine positive cells and 2-fold higher responses than negative control samples were included to allow for a statistically valid analysis. * denotes p<0.05, ** p<0.01, *** p<0.001 by Wilcoxon matched-pairs signed rank test. $denotes p<0.05,$ $p<0.01,$ $$ p<0.001 by Mann-Whitney test.</p

TB therapy alters maturation phenotype of PPD-specific CD4 T-cells.

<p><b>A.</b> Representative example of differentiation marker expression on PPD-specific CD4 T-cells. PPD-specific CD4 T-cells (red dots) are overlaid onto density plots of CD27 and CD45RO and CD27 and CD57, gated on total CD4 T-cells. <b>B.</b> Frequency of PPD-specific CD4 T-cells expressing CD27⁺CD45RO⁺ (CM), CD27^-CD45RO⁺ (EM), and CD57⁺ (TD) phenotypes. <b>C.</b> Correlation between baseline CD4 T-cell count and frequency of PPD-specific CD4 T-cells expressing CM and TD phenotypes in untreated and treated TB disease. Lines of best fit, along with Spearman’s rank correlation coefficient and corresponding p-values are shown <b>D.</b> Frequency of CMV-specific CD4 T-cells expressing CM, EM, and TD phenotypes in our HIV-TB and TB cohorts. <b>E.</b> Frequency of naïve, CM, EM, and TD subsets on total CD4 T-cells in untreated and treated TB disease in our HIV-TB and TB cohorts. To assess maturation phenotype on PPD and CMV-specific CD4 T-cells, only samples with at least 50 cytokine positive cells and 2-fold higher responses than negative control samples were included to allow for a statistically valid analysis. The Wilcoxon matched-pairs signed rank test was used for paired comparisons (HIV-TB cohort) while the Mann-Whitney test was used to analyze unpaired data (TB cohort and comparisons between cohorts). * denotes p<0.05, ** p<0.01, *** p<0.001 by Wilcoxon matched-pairs signed rank test. $denotes p<0.05,$ $p<0.01,$ $$ p<0.001 by Mann-Whitney test.</p

TB therapy alters the functional profile of PPD-specific CD4 T-cells.

<p><b>A.</b> Representative plot showing the gating scheme used to identify cytokine/chemokine positive cells. Single, live, CD3⁺, CD4⁺ T cells were gated for CD27 and CD45RO to identify the total CD4 memory population. Cytokine/chemokine gates were then applied to the total CD4 memory population to identify cytokine/chemokine positive cells. <b>B.</b> Total frequency of IFN-γ, TNF-α, IL-2, and MIP-1β produced by memory CD4 T-cells in untreated and treated TB disease within our HIV-TB and TB cohorts. Background cytokine/chemokine production from the negative control sample was subtracted. <b>C.</b> Pie graph displaying the proportion of cytokine/chemokine⁺ CD4 T-cells producing all 4 cytokines/chemokines (light grey wedge) or any combination of 3 cytokines/chemokines (medium gray), 2 cytokines/chemokines (dark grey), or a single cytokine/chemokine (black wedge) in untreated and treated TB disease. The bar graph depicts the relative contribution of each cytokine/chemokine producing subset to the overall PPD-specific CD4 T-cell response. “G” denotes IFN-γ, “2” denotes IL-2, “T” denotes TNF-α, and “M” denotes MIP-1β. For all bar graphs bars represent the interquartile range (IQR), horizontal lines denote the median, and whiskers the 10^th and 90^th percentiles. Solid bars represent our HIV-TB cohort, patterned bars represent our TB cohort. Light gray represents untreated TB disease while dark gray represents treated TB disease. Statistical analysis was performed using the Wilcoxon matched-pairs signed rank test for paired data (HIV-TB cohort) and the Mann-Whitney test for unpaired data (TB cohort or comparisons between cohorts).* denotes p<0.05, ** p<0.01, *** p<0.001 by Wilcoxon matched-pairs signed rank test. $denotes p<0.05,$ $p<0.01,$ $$ p<0.001 by Mann-Whitney test.</p

Characteristics of ACTG 5221 (A5221) Study Population.

<p>Characteristics of ACTG 5221 (A5221) Study Population.</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

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

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

Treatment with native heterodimeric IL-15 increases cytotoxic lymphocytes and reduces SHIV RNA in lymph nodes

<div><p>B cell follicles in secondary lymphoid tissues represent an immune privileged sanctuary for AIDS viruses, in part because cytotoxic CD8⁺ T cells are mostly excluded from entering the follicles that harbor infected T follicular helper (T_FH) cells. We studied the effects of native heterodimeric IL-15 (hetIL-15) treatment on uninfected rhesus macaques and on macaques that had spontaneously controlled SHIV infection to low levels of chronic viremia. hetIL-15 increased effector CD8⁺ T lymphocytes with high granzyme B content in blood, mucosal sites and lymph nodes, including virus-specific MHC-peptide tetramer+ CD8⁺ cells in LN. Following hetIL-15 treatment, multiplexed quantitative image analysis (histo-cytometry) of LN revealed increased numbers of granzyme B⁺ T cells in B cell follicles and SHIV RNA was decreased in plasma and in LN. Based on these properties, hetIL-15 shows promise as a potential component in combination immunotherapy regimens to target AIDS virus sanctuaries and reduce long-term viral reservoirs in HIV-1 infected individuals.</p><p><b>Trial registration:</b> ClinicalTrials.gov <a href="https://clinicaltrials.gov/ct2/show/NCT02452268" target="_blank">NCT02452268</a></p></div

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