Altered Memory Circulating T Follicular Helper-B Cell Interaction in Early Acute HIV Infection

Abstract

The RV254 cohort of HIV-infected very early acute (4thG stage 1 and 2) (stage 1/2) and late acute (4thG stage 3) (stage 3) individuals was used to study T helper- B cell responses in acute HIV infection and the impact of early antiretroviral treatment (ART) on T and B cell function. To investigate this, the function of circulating T follicular helper cells (cTfh) from this cohort was examined, and cTfh and memory B cell populations were phenotyped. Impaired cTfh cell function was observed in individuals treated in stage 3 when compared to stage 1/2. The cTfh/B cell cocultures showed lower B cell survival and IgG secretion at stage 3 compared to stage 1/2. This coincided with lower IL-10 and increased RANTES and TNF-α suggesting a role for inflammation in altering cTfh and B cell responses. Elevated plasma viral load in stage 3 was found to correlate with decreased cTfh-mediated B cell IgG production indicating a role for increased viremia in cTfh impairment and dysfunctional humoral response. Phenotypic perturbations were also evident in the mature B cell compartment, most notably a decrease in resting memory B cells in stage 3 compared to stage 1/2, coinciding with higher viremia. Our coculture assay also suggested that intrinsic memory B cell defects could contribute to the impaired response despite at a lower level. Overall, cTfh-mediated B cell responses are significantly altered in stage 3 compared to stage 1/2, coinciding with increased inflammation and a reduction in memory B cells. These data suggest that early ART for acutely HIV infected individuals could prevent immune dysregulation while preserving cTfh function and B cell memory.

Fig 1. cTfh-mediated memory B cell help is impaired in late stage acute HIV-infected individuals.

PBMCs from week 0 stage 1/2 (n = 9) and stage 3 (n = 4–7) individuals were sorted. cTfh (CD3⁺CD4⁺CD45RA^-CXCR5⁺CXCR3^-) or the less efficient T-helper cells (CD3⁺CD4⁺CD45RA^-CXCR5⁺CXCR3⁺) denoted CXCR5⁺CXCR3⁺ were placed in culture with autologous resting memory B cells (CD19⁺CD10^-CD21⁺CD27⁺) in the presence of SEB. Quantification of cTfh-mediated B cell help was carried out by measuring (A) total and (B) HIV-specific IgG ELISA in 7 day culture supernatant. Absolute numbers of live (C) B and (D) T cells were quantified. (E) Correlation analysis was carried out between the absolute number of B cells and HIV-specific IgG. (F) HIV-specific IgG ELISA in 7 day culture supernatant from cultures of CXCR5⁺CXCR3⁺ T cells and memory B cells was also quantified. For (A)-(D) and (F) bars represent mean ±SD. Symbols on the graphs represent stage 1/2 individuals (black circles) and stage 3 individuals (black squares) and statistics were carried out using the Mann-Whitney non-parametric test. For (E) symbols represent stage 1/2 individuals (black circles) and stage 3 individuals (black squares) and correlation data was analyzed using Spearman r test. * P< 0.05. Results were significant if P < 0.05.

Fig 2. IL-10 and TNF-α levels in cocultures of cTfh and B cells from late stage acute individuals influence HIV-specific IgG production.

Cocultures of cTfh and autologous resting memory B cells from week 0 stage 1/2 (n = 9) and stage 3 (n = 7) individuals were analyzed for the presence of cytokines including (A) RANTES, (B) TNF-α and (C) IL-10. Correlation analysis was carried out between (D) TNF-α; (E) IL-10 and HIV-specific IgG. For (A)-(C) bars represent mean ±SD. Symbols on the graphs represent stage 1/2 individuals (black circles) and Stage 3 individuals (black squares) and statistics were carried out using the Mann-Whitney non-parametric test. For (D) and (E) symbols represent stage 1/2 individuals (black circles) and stage 3 individuals (black squares) and correlation data was analyzed using Spearman r test. * P< 0.05.

Fig 3. Elevated plasma viral load is associated with reduction in the cTfh-dependent humoral response.

(A) Viral load was quantified in the plasma from stage 1/2 (n = 24) and from stage 3 (n = 30) HIV-infected individuals at day 0 prior to ART initiation. Correlation analysis was carried out between plasma viral load at day 0 and cTfh and memory 7 day culture supernatant (B) -Total IgG, (C) -HIV-specific IgG, (D) absolute number of B cells, (E) IL-10, (F) RANTES and (G) TNF-α from stage 1/2 (n = 8) and stage 3 (n = 5–7). For (A) bars represent mean SD, symbols on the graphs represent stage 1/2 individuals (black circles) and stage 3 individuals (black squares) and statistics were carried out using the Mann-Whitney non-parametric test. For (B)-(G) symbols represent stage 1/2 individuals (black circles) and stage 3 individuals (black squares) and correlation data was analyzed using Spearman r test.* P < 0.05.

Fig 4. Elevated plasma viral load is also associated with a T cell-independent hyperactivated B cell response.

Plasma at day 0 and days 5, 14, 56, 84, 168, 252, 336, 504 and 672 of ART, were analyzed by ELISA for (A) Total IgG and (B) HIV-specific IgG. PBMCs from week 0 Stage 1 and 2 (n = 8) and Stage 3 (n = 7) patients were analyzed by flow cytometry for terminally differentiated plasmablasts identified as CD19⁺CD10^-CD20^-CD21^-CD38^hiCD27^hi cells. (C) Plasmablasts from stage 1/2 and stage 3 were compared to each other and (D) together with plasma viral load. For graphs (A) and (B) bars represent mean SEM and statistics were carried out using multiple T tests comparing stage 1/2 to stage 3 at the different time points. Symbols on the graphs represent Stage 1/2 individuals (black circles) and Stage 3 individuals (black squares). For (C) bars represent mean ±SD, symbols on the graphs represent stage 1/2 individuals (black circles) and stage 3 individuals (black squares) and statistics carried out using the Mann-Whitney non-parametric test. For (D) symbols represent stage 1/2 individuals (black circles) and stage 3 individuals (black squares) and correlation data was analyzed using Spearman r test. * P < 0.05.

Fig 5. Impact of early acute HIV infection on blood memory B cell subsets.

PBMCs from week 0 stage 1/2 (n = 17) and stage 3 (n = 16) patients were analyzed by flow cytometry for resting memory (RM) B cells CD19⁺CD10^-CD21⁺CD27⁺ and (A) compared with each other and (B) with plasma viral load. (C) Comparisons were also done with PBMCs from week 72 of ART (W72) (n = 19 and n = 18 respectively), chronic HIV infected patients (n = 14) and HIV negative patients (n = 14). PBMCs from week 0 stage 1/2 (n = 17) and stage 3 (n = 16) patients were analyzed by flow cytometry for (D) Total B cells which were identified as CD19⁺; (E) resting naïve (RN) B cells CD19⁺CD10^-CD21⁺CD27^-IgD⁺ (F) activated memory (AM) B cells CD19⁺CD10^-CD21^-CD27⁺ cells, (G) Tissue-like (TL) B cells CD19⁺CD10^-CD21^-CD27^-. For (A) and (E)-(H), bars represent mean ±SD. Symbols on the graphs represent stage 1/2 individuals (black circles) and stage 3 individuals (black squares). Statistics were carried out using the Mann-Whitney non-parametric test. For (B) symbols represent stage 1/2 individuals (black circles) and stage 3 individuals (black squares) and correlation data was analyzed using Spearman r test. * P < 0.05. For (C), symbols represent individuals from week 0 stage 1/2 (black circles), week 0 stage 3 (black squares), week 72 ART stage 1/2 (upward pointing black triangles), week 72 ART stage 3 (downward pointing black triangles), chronic individuals (black diamonds) and HIV negative (open circles). Statistics were carried out using Kruskal-Wallis multiple comparison test. * P< 0.05.