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

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

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

MOESM1 of Pre-clinical evaluation of CYP 2D6 dependent drug–drug interactions between primaquine and SSRI/SNRI antidepressants

Additional file 1. Reference compounds utilized in Primaquine pharmacokinetic study. (A) The structures of Primaquine (PQ) and the marker for 5-hydroxylation (5,6-ortho-quinone) are shown. The liquid chromatography and mass spectrometry parameters utilized for quantitation of PQ and the phenolic metabolite. Shown are the observed m/z ions, product ms/ms ions, cone voltages, collision energies, and electrospray polarities utilized for quantitation. Additionally, the retention times for each molecule are also indicated. Separate standard curves and analyses of PK samples were conducted for each analyte. (B) Liver pharmacokinetics of a single 20 mg/kg dose of primaquine alone (blue line), or co-administered with fluoxetine (red line). (C) Liver pharmacokinetics of the 5,6-orthoquinone metabolite from a single 20 mg/kg dose of primaquine alone (blue line), or upon co-administration with fluoxetine (red line)

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

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

Baseline characteristics of acute HIV-infected subjects.

*<p>MHAbce: A multi-region hybridization assay distinguishes subtypes B, C and CRF01_AE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033948#pone.0033948-Kijak1" target="_blank">[12]</a>. One sample was not done due to low plasma HIV RNA. MSM: Men who have sex with men, NRTI: Nucleoside reverse transcriptase inhibitor, NNRTI: Non-nucleoside reverse transcriptase inhibitor, PI: Protease inhibitor.</p

Decline in plasma cytokines following 24 weeks of antiretroviral treatment.

<p>Interferon (IFN)-α (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033948#pone-0033948-g006" target="_blank"><b>Figure 6A</b></a>); interleukin (IL)-17 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033948#pone-0033948-g006" target="_blank"><b>Figure 6B</b></a>); interferon gamma-induced protein (IP)-10 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033948#pone-0033948-g006" target="_blank"><b>Figure 6C</b></a>). All cytokine levels were significantly reduced following treatment.</p

HIV reservoir size by total HIV DNA quantification in peripheral blood mononuclear cells and CD4+ T cells of acute-HIV infected subjects.

<p>Footnote: At baseline, the HIV reservoir size, determined by the frequencies of HIV-infected cells expressed as HIV DNA copies per million peripheral blood mononuclear cells (PBMCs) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033948#pone-0033948-g005" target="_blank"><b>Figure 5A</b></a>) or calculated per million CD4+ T cells (based on CD4 frequency among PBMCs measured by flow cytometry) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033948#pone-0033948-g005" target="_blank"><b>Figure 5B</b></a>), increased with progression of Fiebig stage. The amount of total HIV DNA in PBMCs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033948#pone-0033948-g005" target="_blank"><b>Figure 5C</b></a>) and in CD4+ T cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033948#pone-0033948-g005" target="_blank"><b>Figure 5D</b></a>) at baseline predicted the HIV reservoir size after 24 weeks of antiretroviral treatment. The median total HIV DNA in PBMCs of subjects treated during acute HIV infection in this study was lower than that of subjects treated during chronic HIV infection. In addition, some acutely treated subjects achieved HIV DNA in PBMCs as low as that in elite controllers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033948#pone-0033948-g005" target="_blank"><b>Figure 5E</b></a>). For all figures, each data point represents an individual subject. For <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033948#pone-0033948-g005" target="_blank">figures 5A to 5D</a>, the horizontal bars represent the median values. For <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033948#pone-0033948-g005" target="_blank">figure 5E</a>, megaHAART (highly active antiretroviral therapy) represents subjects in this study who initiated 5-drug antiretroviral regimen during acute HIV infection (n = 15). Data from two control groups are included here as HAART chronic and ECs (elite controllers). HAART chronic represents subjects who initiated standard antiretroviral therapy during the chronic HIV infection stage and had received treatment for a mean duration of 56 months (n = 14). Elite controllers represent subjects whom HIV RNA are undetectable in the absence of antiretroviral therapy (n = 13).</p