Expansion of HIV-specific T follicular helper cells in chronic HIV infection

Abstract

HIV targets CD4 T cells, which are required for the induction of high-affinity antibody responses and the formation of long-lived B cell memory. The depletion of antigen-specific CD4 T cells during HIV infection is therefore believed to impede the development of protective B cell immunity. Although several different HIV-related B cell dysfunctions have been described, the role of CD4 T follicular helper (TFH) cells in HIV infection remains unknown. Here, we assessed HIV-specific TFH responses in the lymph nodes of treatment-naive and antiretroviral-treated HIV-infected individuals. Strikingly, both the bulk TFH and HIV-specific TFH cell populations were significantly expanded in chronic HIV infection and were highly associated with viremia. In particular, GAG-specific TFH cells were detected at significantly higher levels in the lymph nodes compared with those of GP120-specific TFH cells and showed preferential secretion of the helper cytokine IL-21. In addition, TFH cell expansion was associated with an increase of germinal center B cells and plasma cells as well as IgG1 hypersecretion. Thus, our study suggests that high levels of HIV viremia drive the expansion of TFH cells, which in turn leads to perturbations of B cell differentiation, resulting in dysregulated antibody production.

Figure 1. High frequency of CXCR5⁺PD-1^hi CD4 T cells with BCL6 expression in human lymph nodes.

(A) Representative plots of CXCR5 and PD-1 expression on CD4 T cells in lymph nodes and corresponding PBMCs of HIV-infected individuals. Numbers represent fraction (%) of total CD4 T cells. (B) The frequency of CXCR5⁺PD-1^hi cells of CD4 T cells is significantly higher in lymph nodes (average, 4.3% ± 7%) compared with periphery (average, 0.04% ± 0.04%) (P < 0.0001). Symbols represent individual samples; horizontal bars represent mean; and error bars show SEM. (C) Median fluorescence intensity of BCL6 expression based on CXCR5 or PD-1 expression in different CD4 T cell subsets in the lymph node. The highest BCL6 expression was detected within the CXCR5⁺PD-1^hi CD4 T cell subset (n = 31). In box-and-whisker plots, horizontal bars indicate the medians, boxes indicate 25th to 75th percentiles, and whiskers represent 1.5 times the interquartile distance (Tukey). B and C include all HIV-infected and uninfected individuals (n = 31).

Figure 2. TFH cells in the lymph node are predominantly effector memory cells and secrete high levels of IL-21.

(A) Representative flow plots of IFN-γ and IL-21 production of TFH cells in the lymph node. Numbers represent fraction (%) of total CD4 T cells. (B) Frequency of cytokine production dependent on CXCR5 and PD-1 expression in CD4 T cell subsets in the lymph node and periphery. Symbols represent individual samples; horizontal bars represent mean; and error bars show SEM. (C) Definition of memory phenotype of CD4 T cell subsets in the lymph node demonstrated the majority of CXCR5⁺PD-1^hi cells to be effector memory cells. Naive CCR7⁺CD45RA⁺, central memory (CM) CCR7⁺CD45RA^–, TEMRA CCR7^–CD45RA⁺, and effector memory (EM) CCR7^–CD45RA^– (n = 19) cells are shown. In box-and-whisker plots, horizontal bars indicate the medians, boxes indicate 25th to 75th percentiles, and whiskers represent 1.5 times the interquartile distance (Tukey).

Figure 3. Expansion of HIV-specific TFH cells in chronic infection.

Mononuclear cells from lymph nodes were stimulated with overlapping peptide pools for HIV-1 GAG and GP120 for 6 hours and subsequently examined by intracellular multiparameter flow cytometry. (A) Significantly higher frequencies of CXCR5⁺PD-1^hi CD4 T cells can be detected in chronic HIV-positive (treatment-naive [nTx]) individuals (6.1% ± 8.7%) compared with those in ART-treated (Tx) (3.3% ± 3.5%) and HIV-negative (0.5% ± 0.6%) individuals. (B and C) Significantly higher BCL6 expression was detected in CXCR5⁺PD-1^hi CD4 T cells in chronic HIV-positive individuals compared with that in uninfected individuals, as shown by representative histogram plot and median fluorescence intensity level, respectively (P < 0.01). In box-and-whisker plots, horizontal bars indicate the medians, boxes indicate 25th to 75th percentiles, and whiskers represent 1.5 times the interquartile distance (Tukey). (D and E) Higher frequency of GAG- and GP120-specific cytokine responses, respectively, in CXCR5⁺PD-1^hi CD4 T cells in lymph nodes of treatment-naive individuals compared with that in individuals on ART, and the overall CD4 T cell responses in periphery. (A, D, and E) Symbols represent individual samples; horizontal bars represent mean; and error bars show SEM.

Figure 4. Skewing within the B cell compartment in HIV-infected individuals is correlated with TFH cell frequency.

Viable B cells were defined as CD3^–CD14^–CD16^–CD19⁺ and further characterized based on the B mature (Bm) classification: Bm1 (naive) CD38^–IgD⁺, Bm2 (activated naive) CD38⁺IgD⁺, Bm2′ (pregerminal center) CD38⁺⁺IgD⁺, Bm3/4 (germinal center) CD38⁺⁺IgD^–, Bm5 early (early memory) CD38⁺IgD^–, Bm5 late (late memory) CD38^–IgD^–, and plasma cells (PCs) CD38⁺⁺⁺IgD^–. (A) Gating strategy and representative flow plots. Numbers represent fraction (%) of CD3^–CD14^–CD16^–CD19⁺ cells. (B) Skewing in frequency of B cell subset in chronically infected, treatment-naive (nTx) individuals compared with that in treated (Tx) and uninfected individuals. In box-and-whisker plots, horizontal bars indicate the medians, boxes indicate 25th to 75th percentiles, and whiskers represent 1.5 times the interquartile distance (Tukey). (C) Significant correlations between B cell subsets and TFH cell frequency in the lymph nodes of uninfected (white squares) and HIV-infected, untreated (black triangles) and treated (black circles) individuals.

Figure 5. Hypergammaglobulinemia is associated with BCL6 expression in TFH cells.

Significant correlation between the expression of BCL6 in TFH cells and levels of total serum IgG and IgG1 antibody levels in HIV-infected untreated (black triangles) and treated (black circles) individuals.