6 research outputs found
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<sub>FH</sub>) 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<sub>FH</sub> cells. Here, we characterize the phenotype and in vitro B cell helper activity of peripheral T<sub>FH</sub> 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<sup>high</sup>CXCR5<sup>high</sup>CCR6<sup>high</sup>PD-1<sup>high</sup> 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<sub>FH</sub> cells and memory B cells, or with neutralization activity in untreated HIV infection in our cohort. Furthermore, we found that within the peripheral T<sub>FH</sub> population, the expression level of T<sub>FH</sub>-associated genes more closely resembles a memory, non-T<sub>FH</sub> population, as opposed to a T<sub>FH</sub> 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<sub>FH</sub> cells.</p></div
Characterization of peripheral T<sub>FH</sub> 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<sup>low</sup> (pink), CM CCR7<sup>high</sup>CXCR5<sup>low</sup> (orange), CM CCR7<sup>high</sup>CXCR5<sup>high</sup>CCR6<sup>low</sup>PD-1<sup>high</sup> (green), CM CCR7<sup>high</sup>CXCR5<sup>high</sup>CCR6<sup>high</sup>PD-1<sup>low</sup> (blue) and CCR7<sup>high</sup>CXCR5<sup>high</sup>CCR6<sup>high</sup>PD-1<sup>high</sup> (red). Before gating on CCR6 and PD-1, cells were first gated on CD150<sup>high</sup>. 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<sup>high</sup>CD27<sup>low</sup>IgD<sup>−</sup>) 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
Relationship between pT<sub>FH</sub> cells and T<sub>FH</sub> 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<sup>high</sup> cells in T<sub>FH</sub> (CXCR5<sup>high</sup>PD-1<sup>high</sup>) and non-T<sub>FH</sub> populations. (<b>B</b>) Bar graphs showing the frequency of CCR6<sup>high</sup> cells in T<sub>FH</sub> and non-T<sub>FH</sub> populations in human tonsils (n = 5). (<b>C</b>) Heatmap analysis of selected genes from RNA-seq data comparing pT<sub>FH</sub> cells (CXCR5<sup>high</sup>CCR6<sup>high</sup>PD-1<sup>high</sup>) from HIV-uninfected donors, pT<sub>FH</sub> cells from HIV-infected donors, non-T<sub>FH</sub> CD4 memory tonsil cells (CM CD57<sup>low</sup>PD-1<sup>dim</sup>CCR7<sup>high</sup>CCR5<sup>low</sup>CXCR4<sup>low</sup>), non-germinal center T<sub>FH</sub> tonsil cells (CM CD57<sup>low</sup>PD-1<sup>high</sup>CCR7<sup>low</sup>CXCR5<sup>high</sup>) and germinal center T<sub>FH</sub> tonsil cells (CM PD-1<sup>high</sup>CD57<sup>high</sup>) 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
Progressive loss of pT<sub>FH</sub> cells in HIV infection.
<p>(<b>A</b>) Pooled data showing the frequency (%) of CXCR5<sup>high</sup>, CXCR5<sup>high</sup>CCR6<sup>high</sup> and CXCR5<sup>high</sup>CCR6<sup>high</sup>PD-1<sup>high</sup> 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<sup>high</sup>, CXCR5<sup>high</sup>CCR6<sup>high</sup> and CXCR5<sup>high</sup>CCR6<sup>high</sup>PD-1<sup>high</sup> 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<sup>high</sup>, CXCR5<sup>high</sup>CCR6<sup>high</sup> and CXCR5<sup>high</sup>CCR6<sup>high</sup>PD-1<sup>high</sup> 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
Functional characteristics of pT<sub>FH</sub> 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
Impaired B cell help by pT<sub>FH</sub> cells in HIV infection.
<p>(<b>A</b>) CCR7<sup>high</sup>CXCR5<sup>low</sup> and CCR7<sup>high</sup>CXCR5<sup>high</sup>CCR6<sup>high</sup> CM CD4 T cells isolated from PBMCs were cultured with autologous naïve B cells (CD19<sup>high</sup>CD27<sup>low</sup>IgD<sup>−</sup>) 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