Abnormal B cell memory subsets dominate HIV-specific responses in infected individuals

Abstract

Recently, several neutralizing anti-HIV antibodies have been isolated from memory B cells of HIV-infected individuals. Despite extensive evidence of B cell dysfunction in HIV disease, little is known about the cells from which these rare HIV-specific antibodies originate. Accordingly, we used HIV envelope gp140 and CD4 or coreceptor (CoR) binding site (bs) mutant probes to evaluate HIV-specific responses in peripheral blood B cells of HIV-infected individuals at various stages of infection. In contrast to non-HIV responses, HIV-specific responses against gp140 were enriched within abnormal B cells, namely activated and exhausted memory subsets, which are largely absent in the blood of uninfected individuals. Responses against the CoRbs, which is a poorly neutralizing epitope, arose early, whereas those against the well-characterized neutralizing epitope CD4bs were delayed and infrequent. Enrichment of the HIV-specific response within resting memory B cells, the predominant subset in uninfected individuals, did occur in certain infected individuals who maintained low levels of plasma viremia and immune activation with or without antiretroviral therapy. The distribution of HIV-specific responses among memory B cell subsets was corroborated by transcriptional analyses. Taken together, our findings provide valuable insight into virus-specific B cell responses in HIV infection and demonstrate that memory B cell abnormalities may contribute to the ineffectiveness of the antibody response in infected individuals.

Figure 1. Identification of HIV-specific B cells by FACS analysis.

Mature (CD10^–) B cells isolated from the peripheral blood of representative HIV-infected untreated and uninfected individuals and stained for CD20, IgG, and 3 gp140 probes. (A) Binding of gp140-WT probe to B cells shown by CD20 and IgG. (B) Binding of gp140-WT probe to B cells shown by gp140 probes with point mutations at CD4 (gp140ΔCD4bs) and CoR (gp140ΔCoRbs) bs. The numbers refer to the percentage of cells in each quadrant or the percentage of cells within the gated population relative to the total number of cells shown in the dot plot.

Figure 2. Frequencies of HIV-specific B cells in early and chronic HIV infection.

(A) Cross-sectional FACS analysis of B cells from individuals at early and chronic phases of infection stained with gp140 probes. Top panels include untreated viremic individuals, and bottom panels include individuals who began ART during their early or chronic phase of infection and were aviremic at the time of analysis. Horizontal bars show median values, and P values below graphs represent comparisons between viremic and aviremic individuals for a corresponding early or chronic stage of infection. Longitudinal FACS analysis of gp140-WT binding to B cells of (B) HIV-infected individuals before and after reduction of viremia by ART and (C) HIV-viremic untreated individuals at early and later chronic phases of infection. Median time after initiation of ART was 18 months.

Figure 3. Frequencies of HIV-specific B cells by subset.

(A) Mature (CD10^–) B cells isolated from the peripheral blood of a representative HIV-infected untreated individual and stained for CD20, IgG, CD21, CD27, and 3 gp140 probes. Gates were set on IgG⁺ B cells, followed by gating on B cells that were positive for gp140, gp140-CD4bs, and gp140-CoRbs and displaying the expression of the probe-gated B cells by the subset-identifying markers CD21 and CD27. (B) Pie chart analysis of gp140-binding B cells by subset for 42 HIV-viremic individuals and comparison of gp140-binding frequencies between subsets. (C) Similar analyses as in B, but for epitope specificities within gp140 (CD4bs and CoRbs); “other” refers to responses to gp140 after subtraction of those specific to CD4bs and CoRbs. Horizontal bars show mean values. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. The numbers in A refer to the percentage of cells in each quadrant or the percentage of cells within the gated population relative to the total number of cells shown in the dot plot. AM, activated memory; IM, intermediate memory; RM, resting memory; TLM, tissue-like memory.

Figure 4. Immunologic and virologic associations with HIV-specific responses among resting memory B cells.

(A) Correlations between the proportion of gp140-specific responses within resting memory B cells, and expression of CD38 on CD8⁺ T cells, HIV plasma viremia, and CD4⁺ T cell count or percentage in peripheral blood. Effects of reduction of HIV plasma viremia by ART on the (B) frequency and (C) distribution of the gp140-specific response among B cell subsets. The P values in B represent comparisons of frequencies before and after ART for each subset.

Figure 5. Comparisons between gp140-, influenza- and tetanus-specific B cells.

Mature (CD10^–) B cells isolated from the peripheral blood of representative untreated HIV-infected individuals vaccinated with influenza or tetanus and stained for CD20, IgG, and 3 probes. (A) Binding of gp140, influenza (H1-CA09), and tetanus (TTCF) probes to B cells gated on CD20 and IgG. (B) Probe-binding frequencies shown by antigen for all IgG⁺ B cells and (C) shown as median among each B cell subset. Horizontal bars show median values, and significant differences are identified in the subset with the higher value. *P < 0.05; **P < 0.01; ***P < 0.001. The numbers in A refer to the percentage of cells within the gated population relative to the total number of cells shown in the dot plot. I, influenza; T, tetanus; H, HIV gp140.

Figure 6. Transcriptional analysis by low-density array.

Heatmap and hierarchical clustering of 29 genes and their levels of expression are shown for 3 sorted IgG⁺ memory B cell subsets from 6 HIV-viremic individuals (S1–S6). The subsets were resting, activated, and tissue-like memory B cells. Genes that were significantly different in expression between resting memory and the other 2 subsets are highlighted in bold. The asterisk identifies genes that were significantly different in expression between gp140-sorted IgG⁺ B cells of individuals with strong versus weak pro–resting memory profiles. An FDR of less than 10% was considered significant. Bootstrap and k-means clustering analyses were used to confirm that the separation of tissue-like memory samples from the rest (activated memory and resting memory) was highly robust.