Decreased survival of B cells of HIV-viremic patients mediated by altered expression of receptors of the TNF superfamily

Abstract

Human immunodeficiency virus (HIV) infection leads to numerous perturbations of B cells through mechanisms that remain elusive. We performed DNA microarray, phenotypic, and functional analyses in an effort to elucidate mechanisms of B cell perturbation associated with ongoing HIV replication. 42 genes were up-regulated in B cells of HIV-viremic patients when compared with HIV-aviremic and HIV-negative patients, the majority of which were interferon (IFN)-stimulated or associated with terminal differentiation. Flow cytometry confirmed these increases and indicated that CD21(low) B cells, enhanced in HIV-viremic patients, were largely responsible for the changes. Increased expression of the tumor necrosis factor (TNF) superfamily (TNFSF) receptor CD95 correlated with increased susceptibility to CD95-mediated apoptosis of CD21(low) B cells, which, in turn, correlated with HIV plasma viremia. Increased expression of BCMA, a weak TNFSF receptor for B lymphocyte stimulator (BLyS), on CD21(low) B cells was associated with a concomitant reduction in the expression of the more potent BLyS receptor, BAFF-R, that resulted in reduced BLyS binding and BLyS-mediated survival. These findings demonstrate that altered expression of genes associated with IFN stimulation and terminal differentiation in B cells of HIV-viremic patients lead to an increased propensity to cell death, which may have substantial deleterious effects on B cell responsiveness to antigenic stimulation.

Figure 1.

Genes up-regulated greater than twofold in B cells of 10 HIV-viremic patients compared with 10 HIV-aviremic patients and 10 HIV-negative donors. The results depict all genes whose profiles were significantly different between HIV-viremic patients and the other groups of individuals (grouped together), as evaluated by T-statistics. The genes are ordered by the ratio of mean values between these two groups and represent one out of two independent hybridizations. A cut-off of twofold was used to produce this gene set. Relative levels of gene expression (median-centered normalized expression values) are shown in color according to the scale at the bottom, where red indicates up-regulation and green indicates down-regulation. Data points with CV ≥0.8 are depicted in gray. The categories of B and B-TD refer to genes associated with B cell activities and B cell terminal differentiation, respectively. The values in the column on the left represent median signal intensities obtained after linear normalization and averaging of duplicate spot signals for each clone.

Figure 2.

Phenotyping of B cell markers found to be up-regulated in HIV-viremic patients by microarray analyses. A gate was set on CD19-positive B cells, and cells were analyzed for expression of BCMA, CD95, and CD38 relative to CD21. Representative profiles from an HIV-viremic patient, an HIV-aviremic patient, and an HIV-negative individual are shown. Inset values shown for CD95 and CD38 represent percentage of B cells expressing high-intensity receptors delineated by the top horizontal bar.

Figure 3.

Flow cytometric analysis of BCMA (A), CD38 (B), and CD95 (C) expression relative to CD21 on B cells. Levels of expression measured by gating on CD19-positive B cells are shown for nine HIV-viremic patients, eight HIV-aviremic patients, and nine HIV-negative individuals.

Figure 3.

Flow cytometric analysis of BCMA (A), CD38 (B), and CD95 (C) expression relative to CD21 on B cells. Levels of expression measured by gating on CD19-positive B cells are shown for nine HIV-viremic patients, eight HIV-aviremic patients, and nine HIV-negative individuals.

Figure 3.

Flow cytometric analysis of BCMA (A), CD38 (B), and CD95 (C) expression relative to CD21 on B cells. Levels of expression measured by gating on CD19-positive B cells are shown for nine HIV-viremic patients, eight HIV-aviremic patients, and nine HIV-negative individuals.

Figure 4.

B cell responses to FasL. B cells were incubated at 4°C or at 37°C with and without FasL for 2 h and stained for CD21 and annexin V. (A) FACS^® plots on live-gated cells are shown for a representative HIV-viremic patient, HIV-aviremic patient, and HIV-negative individual (see Table S1 for complete dataset). Correlation between percentage of annexin V staining in B cells where levels of staining without FasL were subtracted from levels with FasL and (B) percentage of CD95 expression on B cells or (C) levels of HIV plasma viremia in the HIV-infected patients (limit of detection was 50 copies/ml of plasma).

Figure 4.

B cell responses to FasL. B cells were incubated at 4°C or at 37°C with and without FasL for 2 h and stained for CD21 and annexin V. (A) FACS^® plots on live-gated cells are shown for a representative HIV-viremic patient, HIV-aviremic patient, and HIV-negative individual (see Table S1 for complete dataset). Correlation between percentage of annexin V staining in B cells where levels of staining without FasL were subtracted from levels with FasL and (B) percentage of CD95 expression on B cells or (C) levels of HIV plasma viremia in the HIV-infected patients (limit of detection was 50 copies/ml of plasma).

Figure 5.

Representative expression of BCMA, TACI, and BAFF-R, as well as BLyS-binding, on B cells of HIV-viremic, HIV-aviremic, and HIV-negative individuals. A gate was set on CD19-positive B cells, and cells were analyzed for expression of BCMA, TACI, and BAFF-R relative to CD21. Representative profiles from an HIV-viremic patient, an HIV-aviremic patient, and an HIV-negative donor are shown (see Table S1 for complete dataset).

Figure 6.

Levels of (A) BAFF-R expression and (B) BLyS-binding on CD21^low versus CD21^high B cells of HIV-viremic patients. Levels of BAFF-R expression and BLyS-binding were measured on CD19-gated B cells of 16 HIV-viremic patients and are expressed as the percentage of BAFF-R expression on the CD21^low and CD21^high populations for each patient. (C) Survival effect of BLyS measured on B cells of HIV-viremic patients fractionated into CD21^low and CD21^high subsets. The percent survival after 16 h incubation was calculated as the fraction of annexin-positive cells measured in the presence of BLyS over annexin-positive cells measured in the absence of BLyS multiplied by 100.