T-bet+ B cells are induced by human viral infections and dominate the HIV gp140 response

Abstract

Humoral immunity is critical for viral control, but the identity and mechanisms regulating human antiviral B cells are unclear. Here, we characterized human B cells expressing T-bet and analyzed their dynamics during viral infections. T-bet+ B cells demonstrated an activated phenotype, a distinct transcriptional profile, and were enriched for expression of the antiviral immunoglobulin isotypes IgG1 and IgG3. T-bet+ B cells expanded following yellow fever virus and vaccinia virus vaccinations and also during early acute HIV infection. Viremic HIV-infected individuals maintained a large T-bet+ B cell population during chronic infection that was associated with increased serum and cell-associated IgG1 and IgG3 expression. The HIV gp140-specific B cell response was dominated by T-bet-expressing memory B cells, and we observed a concomitant biasing of gp140-specific serum immunoglobulin to the IgG1 isotype. These findings suggest that T-bet induction promotes antiviral immunoglobulin isotype switching and development of a distinct T-bet+ B cell subset that is maintained by viremia and coordinates the HIV Env-specific humoral response.

Keywords: AIDS/HIV; Immunology.

Figure 1. T-bet expression in memory B cell subsets from healthy human peripheral blood.

(A) Representative flow cytometry plots of T-bet expression in peripheral blood B cell subsets from a single donor. (B) T-bet expression frequency per subset in a 10-donor cohort. Bars on this and all following plots represent mean ± SEM. Trans., transitional B cell.(C) T-bet expression frequency of memory B cells expressing different Ig isotypes. (D) CD21/CD27–based gating scheme of total CD38^loCD10^– B cells from a representative donor. AM, activating memory, CD27⁺CD21^–; RM, resting memory, CD27⁺CD21⁺; TLM, tissue-like memory, CD27^–CD21^–. (E) T-bet expression frequency of CD21/CD27–derived B cell subsets. (F) T-bet median fluorescence intensity (MFI) of T-bet⁺ cells from each B cell subset. (G) Gating of CD21^–CD85j^hi and CD21^–CD85j^lo B cell subsets from a representative donor’s total CD38^loCD10^– B cells. (H) T-bet expression histogram of CD85j-gated B cell subsets from donor depicted in G. (I) T-bet expression frequency of CD85j-gated B cell subsets in 10-donor cohort. (J) Expression frequency of homing receptors (CD11c, CXCR3), activation markers (CD69, CD71, CD86, CD95), and inhibitory receptors (PD-1, FcRL4/5) by CD38^loCD10^– B cell subsets. Statistical comparisons in B, C, E, F, and J calculated using repeated-measures 1-way ANOVA with Tukey’s multiple comparisons test. Statistical comparison in I calculated using paired t test. *P ≥ 0.01 to < 0.05; **P ≥ 0.001 to < 0.01; ***P < 0.001.

Figure 2. Longitudinal T-bet⁺ B cell dynamics in yellow fever virus–vaccinated, vaccinia virus–vaccinated, or acutely HIV-infected individuals.

Longitudinal T-bet expression in memory B cells of (A) a yellow fever vaccinee and (B) a vaccinia vaccinee. Time of blood sampling after vaccination is depicted. (C) T-bet expression frequency in memory B cells of yellow fever vaccinees (n = 5 for all plots except E). Donor samples were binned to organize approximate weekly time points. (D) T-bet^hiCD85j^hi cell frequency of total memory B cells in yellow fever vaccinees. (E) Ki67 expression frequency of T-bet^hiCD85j^hi cells in yellow fever vaccines (n = 4). (F) T-bet median fluorescence intensity (MFI) of T-bet^hiCD85j^hi cells (blue) and resting memory (RM) cells (black) in yellow fever vaccinees. No statistical differences were observed between RM time points. (G) T-bet expression in memory B cells of an acutely HIV-infected individual before infection and shortly after peak of viremia. (H) Memory B cell T-bet expression frequency from 7-donor cohort of acutely HIV-infected individuals at preinfection, acute, and chronic infection time points. Note that intracellular T-bet staining of early acute HIV samples in G and H was performed using a BD Cytofix/Cytoperm Kit. Statistical comparisons in C, D, E, F, and H calculated using repeated-measures 1-way ANOVA with Tukey’s multiple comparisons test. *P ≥ 0.01 to < 0.05; **P ≥ 0.001 to < 0.01.

Figure 3. T-bet expression in B cells and antibody isotype repertoires during HIV infection.

(A) T-bet expression in memory B cells of a representative HIV-negative donor and an HIV⁺ chronic progressor (progressor). (B) T-bet expression frequency of memory B cells in HIV-negative, progressor, HIV⁺ viremic controller (VC), HIV⁺ elite controller (EC), and HIV⁺ antiretroviral therapy–treated donors (ART); n = 10 donors per group. Statistical comparison calculated using 1-way ANOVA with Tukey’s multiple comparisons test. (C) Phenotype of T-bet⁺ B cell population from each cohort in B. Bars represent SEM. (D) T-bet median fluorescence intensity (MFI) of total T-bet⁺ cells from each cohort. Statistical comparison calculated using 1-way ANOVA with Tukey’s multiple comparisons test. (E) Frequency of IgG isotype–expressing B cells within total memory B cell compartment of HIV-negative individuals and progressors (HIV⁺) (n = 10 donors per group). Horizontal bars in B, D, and E represent the mean ± SEM. Statistics in E and F calculated using unpaired t test. (F) Absolute concentration of total serum antibodies by isotype from progressor (HIV⁺) and age/ethnicity–matched HIV-negative cohorts (n = 10 donors per group). (G) Correlations between T-bet expression frequency in memory B cells and serum antibody titers (ng/ml). Statistics calculated using Spearman correlation. *P ≥ 0.01 to < 0.05; **P ≥ 0.001 to < 0.01; ***P < 0.001.

Figure 4. Transcriptional analyses of T-bet^hiCD85j^hi cells and other B cell subsets in HIV-negative donors and during HIV infection.

(A) Heatmap depicting relative RNA transcript expression levels for 91 targets (1 per row) in HIV-negative donors (n = 4). Each column represents a specific B cell subset (colored bars above and below heatmap) sorted from 1 donor. (B) t-Distributed stochastic neighbor embedding (tSNE) analysis of transcriptional relationships between sorted B cell subsets from 4 healthy donors. Each color represents a sorted B cell subset and clusters are highlighted with the corresponding color. (C) AICDA transcript expression (log₂ units) of HIV-negative donors (gray; n = 4) and progressors (black; n = 5) per B cell subset. Trans., transitional; RM, resting memory; PB, plasmablast. Each data point represents transcript expression from 1 individual. Horizontal bars represent the mean ± SEM. Statistical comparison calculated using repeated-measures ANOVA with Tukey’s multiple comparisons. (D) Quantitative reverse transcription PCR of AICDA transcript in CD21^–CD27^– B cells of progressors transfected with control or T-bet siRNA. Values represent AICDA transcript level as a fraction of no-siRNA treatment condition. Statistical comparison calculated using paired t test. (E) Comparison of transcript expression levels for 91 gene targets between healthy donors (n = 4) and progressors (n = 5). Each data point represents the mean expression value calculated for HIV-negative donors (x value) and progressors (y value). Lines represent 90% prediction bands of calculated linear regression. Activation-induced cytidine deaminase (AICDA) transcript is depicted in red. *P ≥ 0.01 to < 0.05; **P ≥ 0.001 to < 0.01 (C and D).

Figure 5. gp140-specific memory B cell phenotypes and serum Ig isotypes.

(A) gp140-specific serum Ig titers by isotype from progressors (n = 10). Titers were normalized using age/ethnicity–matched controls. Statistical comparison calculated using repeated-measures 1-way ANOVA with Tukey’s multiple comparisons test. (B) Representative staining of gp140-specific class-switched (IgD^–IgM^–) memory B cells from a progressor. (C) Frequency of gp140-specific cells within total class-switched memory B cells by cohort. n = 10 for all cohorts except progressors (n = 11). (D) T-bet expression of gp140-specific B cells (red) and total B cells (black) from a representative progressor. (E) Frequency of T-bet expression within gp140-specific cells from HIV⁺ cohorts (n = 10 progressors, n = 10 viremic controllers [VC], n = 6 elite controllers [EC]). All donors with less than 40 gp140-specific cells (including all antiretroviral therapy [ART] individuals) were excluded from analyses in E and F. No statistical differences were observed between donor groups. (F) Frequency of gp140-specific cells with T-bet^hiCD85j^hi phenotype by cohort. For C, E, and F, statistical comparisons were calculated using 1-way ANOVA with Tukey’s multiple comparisons test. *P ≥ 0.01 to < 0.05; **P ≥ 0.001 to < 0.01; ***P < 0.001.