IL-10-producing B cells are induced early in HIV-1 infection and suppress HIV-1-specific T cell responses

Abstract

A rare subset of IL-10-producing B cells, named regulatory B cells (Bregs), suppresses adaptive immune responses and inflammation in mice. In this study, we examined the role of IL-10-producing B cells in HIV-1 infection. Compared to uninfected controls, IL-10-producing B cell frequencies were elevated in both blood and sigmoid colon during the early and chronic phase of untreated HIV-1 infection. Ex vivo IL-10-producing B cell frequency in early HIV-1 infection directly correlated with viral load. IL-10-producing B cells from HIV-1 infected individuals were enriched in CD19(+)TIM-1(+) B cells and were enriched for specificity to trimeric HIV-1 envelope protein. Anti-retroviral therapy was associated with reduced IL-10-producing B cell frequencies. Treatment of B cells from healthy donors with microbial metabolites and Toll-like receptor (TLR) agonists could induce an IL-10 producing phenotype, suggesting that the elevated bacterial translocation characteristic of HIV-1 infection may promote IL-10-producing B cell development. Similar to regulatory B cells found in mice, IL-10-producing B cells from HIV-1-infected individuals suppressed HIV-1-specific T cell responses in vitro, and this suppression is IL-10-dependent. Also, ex vivo IL-10-producing B cell frequency inversely correlated with contemporaneous ex vivo HIV-1-specific T cell responses. Our findings show that IL-10-producing B cells are induced early in HIV-1 infection, can be HIV-1 specific, and are able to inhibit effective anti-HIV-1 T cell responses. HIV-1 may dysregulate B cells toward Bregs as an immune evasion strategy.

Figure 1. IL-10-producing B cell frequency is elevated in PBMC of untreated HIV-1 infection.

(A) Gating strategy for identification of ex vivo IL-10-producing B cells in PBMC. (B) Flow cytometry dot plots of IL-10-producing B cells in unstimulated PBMC and PMA/ionomycin-stimulated PBMC from representative HIV-1 infected individuals and a healthy donor. (C) Summary data of IL-10-producing B cell frequencies in unstimulated PBMC from a cohort of HIV-1 infected individuals at different stages, healthy donors, and HCV mono-infected individuals. Each circle represents one individual. (D) IL-10 production from purified B cells of healthy donors and untreated HIV-1 infected individuals. B cells were purified from PBMC and cultured in unstimulated medium for 12 h and then IL-10 in the supernatant was measured by a Luminex assay. (E) Summary data showing IL-10-producing B cell frequency in PBMC stimulated 5 h with PMA/ionomycin from healthy donors and HIV-1 infected individuals. HIV-1 neg: healthy donors. CI: untreated chronic HIV-1 infection. TCI: treated chronic HIV-1 infection. EI: untreated early HIV-1 infection. LTNP: long-term non-progressors. HCV: HCV mono-infection. Isotype: representative IL-10 antibody isotype control from the same EI patient shown in Figure 1B. *: P<0.05. **: P<0.01. ***: P<0.001. N.S.: non-significant (Kruskal-Wallis one-way ANOVA and Dunn’s test). For flow cytometry analysis, >1.5 million events in the lymphocyte gate were collected.

Figure 2. Correlation of IL-10-producing B cell frequency with HIV-1 plasma viral load and CD4⁺ T cell count in untreated early HIV-1 infection.

Correlation of IL-10-producing B cell frequency in (A) ex vivo unstimulated PBMC and (B) PBMC stimulated with PMA/ionomycin for 5 h with viral load. Correlation of IL-10-producing B cell frequency in (C) unstimulated PBMC and (D) PBMC stimulated with PMA/ionomycin for 5 h with CD4⁺ T cell count. Ten HIV-1 EI subjects were tested. Pearson’s correlation was used for correlation analysis.

Figure 3. Phenotype of IL-10-producing B cells in HIV-1 infection.

(A) Representative phenotype of IL-10-producing B cells in the unstimulated PBMC of HIV-1 viremic individuals. (B) TIM-1 expression on IL-10-producing B cells and non-IL-10-producing B cells in unstimulated PBMC (Medium) or PBMC stimulated 5 h with PMA/ionomycin (PMA/Ionomycin) of eight individuals (4 healthy donors, 2 HIV-1 infected EI subjects, and 2 HIV-1 infected CI subjects). Filled shapes represent IL-10-producing B cells and open shapes represent non-IL-10-producing B cells. *: P<0.05 (Wilcoxon rank-sum test). (C) Representative activation/maturation marker expression of IL-10-producing B cells (black dots) in the unstimulated PBMC of HIV-1 viremic individuals overlaying on total B cells (grey dots). Numbers in each quadrant indicate the percentage of IL-10-producing B cells bearing the quadrant’s phenotype. (D) Percentage of IL-10-producing B cells exhibiting the CD10⁻ phenotype or the CD27⁺ phenotype. Shown is the summary from 5 HIV-1 viremic individuals.

Figure 4. TIM-1⁺ B cells were enriched with HIV-1 trimeric Env-specific B cells.

PBMCs were incubated with a FLAG-tagged HIV-1 YU2 gp140 trimer (see Materials and Methods) and then stained with anti-FLAG mAb, CD3, CD19, IgD, and TIM-1 mAbs. (A) Representative flow cytometry dot plot showing gp140 and TIM-1 co-staining of IgD⁻ B cells from an HIV-1 viremic individual. Staining of the same HIV-1 viremic individual without gp140 trimer (No gp140) and staining of an HIV-1 uninfected subject (HIV-1 uninfected) were used as gating controls for gp140 gating. (B) Summary of gp140 staining on TIM-1⁺ IgD⁻ and TIM-1⁻ IgD⁻ B cells from five HIV-1 viremic individuals. **: P<0.01. (Wilcoxon rank-sum test).

Figure 5. IL-10-producing B cells can suppress HIV-1-specific T cell responses.

Purified T cells from seven HIV-1 EI individuals were cocultured at 1∶1 ratio with autologous purified TIM-1⁺ B cells or TIM-1⁻ B cells for 3 d and stimulated with HIV-1 Gag antigen (HIV-1 Gag Antigen) or without (Medium) during the last 6 h in the presence of GolgiStop and GolgiPlug. HIV-1 Gag-specific T cell cytokine production/degranulation was calculated as percentage of cytokine/degranulation positive T cells in T cells stimulated with HIV-1 Gag subtracting background from T cells cultured in medium. % change of HIV-1 Gag-specific T effector cells was calculated as 100% × (percentage of HIV-1 Gag-specific T effector cells from T cells co-cultured with TIM-1⁺ B cells ÷ percentage of HIV-1 Gag-specific T effector cells from T cells co-cultured with TIM-1⁻ B cells) (A) Flow cytometry dot plots of CD8⁺ T cell Gag responses from one representative HIV-1 EI individual. (B) Summary of CD8⁺ T cell responses from the seven HIV-1 EI subjects. (C) Flow cytometry dot plots of CD4⁺ T cell responses from one representative HIV-1 EI individual. (D) Summary of CD4⁺ T cell responses from the seven HIV-1 EI subjects. (E) IL-10-producing B cells suppressed HIV-1-specific T cell responses through IL-10. Purified T cells from HIV-1 EI individuals were cocultured with autologous purified TIM-1⁺ B cells with or without soluble IL-10 receptor (sIL-10R) or TIM-1⁻ B cells for 3 d. HIV-1 Gag antigen was added during the last 6 h in the presence of GolgiStop and GolgiPlug. Shown is a representative sample taken from three HIV-1 EI individuals. *: P<0.05. N.S.: non-significant (Wilcoxon matched-pairs test).

Figure 6. Correlation of IL-10-producing B cell frequency with HIV-1-specific T cell proliferation in early HIV-1 infection.

CFSE-labeled PBMCs from ten HIV-1 EI subjects were stimulated with HIV-1 Gag pool for 6 d and proliferation of CD4⁺ and CD8⁺ T cells were analyzed with flow cytometry. These data were then correlated with contemporaneous IL-10-producing B cell frequencies in ex vivo unstimulated B cells or PMA/ionomycin stimulated B cells. (A) and (B) show proliferation of CD4⁺ and CD8⁺ T cells for the HIV-1 EI subjects grouped according to their IL-10-producing B cell frequency in unstimulated PBMC (IL-10-producing B cell frequency ≥0.05% or <0.05%). **: P<0.01. (Wilcoxon rank-sum test). (C) and (D) show correlation of frequency of IL-10-producing B cell in PBMC stimulated with PMA/ionomycin with proliferation of CD4⁺ and CD8⁺ T cells. Pearson’s correlation was used for correlation analysis.

Figure 7. IL-10-producing B cell frequency is elevated by increased microbial translocation.

(A) B cells were purified from PBMC of healthy subjects and then cultured in medium and treated with agonists for TLR-2 (Pam3CSK4), TLR-4 (LPS), TLR-7 (Imiquimod) and TLR-9 (CpG) for 12 h. IL-10 in the supernatant was measured. *: P<0.05. **: P<0.01 (Wilcoxon rank-sum test). (B) Single cells were prepared from sigmoid colons of healthy subjects, HIV-1 CI and EI individuals (four subjects for each group were tested) as described in Materials and Methods and cultured in medium in the presence of GolgiStop and GolgiPlug for 6 h. Cells were then stained with IL-10, CD3, and CD19 mAbs and analyzed with flow-cytometry. *: P<0.05. N.S.: non-significant (Kruskal-Wallis one-way ANOVA and Dunn’s test).