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

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

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

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

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

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

Safety and pharmacokinetics of the Fc-modified HIV-1 human monoclonal antibody VRC01LS: A Phase 1 open-label clinical trial in healthy adults

<div><p>Background</p><p>VRC01 is a human broadly neutralizing monoclonal antibody (bnMAb) against the CD4-binding site of the HIV-1 envelope glycoprotein (Env) that is currently being evaluated in a Phase IIb adult HIV-1 prevention efficacy trial. VRC01LS is a modified version of VRC01, designed for extended serum half-life by increased binding affinity to the neonatal Fc receptor.</p><p>Methods and findings</p><p>This Phase I dose-escalation study of VRC01LS in HIV-negative healthy adults was conducted by the Vaccine Research Center (VRC) at the National Institutes of Health (NIH) Clinical Center (Bethesda, MD). The age range of the study volunteers was 21–50 years; 51% of study volunteers were male and 49% were female. Primary objectives were safety and tolerability of VRC01LS intravenous (IV) infusions at 5, 20, and 40 mg/kg infused once, 20 mg/kg given three times at 12-week intervals, and subcutaneous (SC) delivery at 5 mg/kg delivered once, or three times at 12-week intervals. Secondary objectives were pharmacokinetics (PK), serum neutralization activity, and development of antidrug antibodies. Enrollment began on November 16, 2015, and concluded on August 23, 2017. This report describes the safety data for the first 37 volunteers who received administrations of VRC01LS. There were no serious adverse events (SAEs) or dose-limiting toxicities. Mild malaise and myalgia were the most common adverse events (AEs). There were six AEs assessed as possibly related to VRC01LS administration, and all were mild in severity and resolved during the study. PK data were modeled based on the first dose of VRC01LS in the first 25 volunteers to complete their schedule of evaluations. The mean (±SD) serum concentration 12 weeks after one IV administration of 20 mg/kg or 40 mg/kg were 180 ± 43 μg/mL (<i>n</i> = 7) and 326 ± 35 μg/mL (<i>n</i> = 5), respectively. The mean (±SD) serum concentration 12 weeks after one IV and SC administration of 5 mg/kg were 40 ± 3 μg/mL (<i>n</i> = 2) and 25 ± 5 μg/mL (<i>n</i> = 9), respectively. Over the 5–40 mg/kg IV dose range (<i>n</i> = 16), the clearance was 36 ± 8 mL/d with an elimination half-life of 71 ± 18 days. VRC01LS retained its expected neutralizing activity in serum, and anti-VRC01 antibody responses were not detected. Potential limitations of this study include the small sample size typical of Phase I trials and the need to further describe the PK properties of VRC01LS administered on multiple occasions.</p><p>Conclusions</p><p>The human bnMAb VRC01LS was safe and well tolerated when delivered intravenously or subcutaneously. The half-life was more than 4-fold greater when compared to wild-type VRC01 historical data. The reduced clearance and extended half-life may make it possible to achieve therapeutic levels with less frequent and lower-dose administrations. This would potentially lower the costs of manufacturing and improve the practicality of using passively administered monoclonal antibodies (mAbs) for the prevention of HIV-1 infection.</p><p>Trial registration</p><p>ClinicalTrials.gov <a target="_blank">NCT02599896</a></p></div

Measurement of antibody serum concentration (μg/mL).

<p>(A) Serum VRC01LS concentrations (colored plots) are shown from first measurement through week 24 after a single administration. The infusion dose and route are as specified in the legend. All values are the mean of duplicate samples run in different wells within the same plate. Previously published VRC01 concentrations based on historical data (black plots) after administration at weeks 0 and 4 are shown for comparison. (B) Geometric mean serum VRC01LS concentrations per group over time. The dotted line at 10 μg/mL on each graph is shown as a reference value. IV, intravenous; SC, subcutaneous.</p

CONSORT flow diagram of the VRC 606 trial.

<p>Thirty-seven volunteers were allocated to 6 groups. All volunteers who received at least one VRC01LS administration were analyzed for safety and reactogenicity. All volunteers who completed the per-protocol dosing schedule by the time of manuscript submission (<i>n</i> = 25) were additionally analyzed for pharmacokinetic parameters, serum neutralization activity, anti-drug antibodies, and IgG1 GM allotype. These 25 volunteers were all the volunteers in groups 1–4, the first six volunteers in group 5, and the first five volunteers in group 6. GM, genetic marker; IgG1, immunoglobulin G subclass 1; IV, intravenous; SC, subcutaneous; VRC, Vaccine Research Center.</p

Post infusion serum antibody concentrations and PK parameters by dose and route.

<p>(A) C_max (top panel) showing expected dose-dependent increases of VRC01LS, t_1/2β (middle panel), and antibody clearance (bottom panel), showing similar values for doses and routes. Bars show mean values, with error bars indicating s.d. Previously published VRC01 data following 40 mg/kg infusion are shown for comparison. (B) VRC01LS and VRC01 serum concentrations 4 weeks after a single administration, as indicated on the x-axis. Each value is the mean of duplicate wells in the same plate. VRC01 data (black dots) were derived from historical data. C_max, maximal concentration; IV, intravenous; PK, pharmacokinetic; SC, subcutaneous; s.d., standard deviation; t_1/2β, elimination half-life.</p