Impaired naive and memory B-cell responsiveness to TLR9 stimulation in human immunodeficiency virus infection

Abstract

Toll-like receptor 9 (TLR9) agonists such as unmethylated bacterial CpG DNAs activate B lymphocytes directly, potentially influencing their function and homeostasis. To assess B-cell responsiveness to TLR9 agonists in human immunodeficiency virus (HIV) disease, we examined the ability of naive and memory B cells to proliferation and to increase surface expression of CD80 in response to CpG oligonucleotides (ODN). CpG ODN induced expression of CD80 similarly in B cells from HIV-infected persons and from healthy controls. In contrast, proliferation responses to CpG ODN were markedly impaired in both naive and memory B-cell subsets from HIV-infected persons. Naive B-cell proliferation defects were related to plasma HIV RNA and, among memory B cells, to the frequencies of CD21-negative cells. Importantly, TLR9 mRNA levels were significantly diminished in freshly prepared naive B cells and especially so in memory B cells from HIV-positive viremic donors, suggesting a possible underlying mechanism for the observed functional impairments. Dose-response studies indicated that optimal induction of CD80 expression was achieved with much lower concentrations of CpG ODN than optimal induction of proliferation. We propose that the relatively low threshold of activation that is required for CD80 induction by CpG ODN might explain the preservation of this response in B cells from HIV-infected persons despite diminished TLR9 expression. Impaired responsiveness to TLR9 agonists may contribute to defects in humoral immunity in HIV infection.

FIG. 1.

Impaired expansion of naive and memory B cells in response to CpG ODN in viremic HIV-1 infection. PBMCs were labeled with CFSE and cultured with or without CpG ODN2006 at 6 μg/ml for 7 days. (A) Naive B-cell (CD19⁺ CD27⁻) and memory B-cell (CD19⁺ CD27⁺) expansion was evaluated by flow cytometric measurement of dye dilution. (B) Summary data (medians and interquartile ranges) of B-cell proliferation by CpG ODN for HIV-seronegative controls and for HIV-infected patients with undetectable (≤400 copies/ml) and detectable (>400 copies/ml) plasma HIV viremia. (C) Medians of B-cell proliferation for HIV-seronegative controls, HIV⁺ aviremic patients receiving HAART, HIV⁺ viremic patients receiving HAART, and untreated HIV⁺ viremic patients. P values were calculated by one-way ANOVA tests.

FIG. 2.

Increased CD80 expression on memory B cells in HIV infection. PBMCs were analyzed by flow cytometry following staining with antibodies to CD19, CD27, and CD80. (A) Percentage of CD80 expression from one representative analysis among freshly isolated cells. (B) Percentages of CD80⁺ naive and memory B cells among freshly isolated cells. (C) Percentages of CD80⁺ cells among naive and memory B cells within unseparated PBMCs cultured overnight with or without CpG ODN. (D) Median percentage of cells that were induced to express CD80 by CpG ODN 2006 after 20 h of cultivation. The comparison among three groups was performed as noted above, and the comparison in the last panel was among HIV⁻ donors, HIV⁺ patients with CD4 levels of >500/μl, HIV⁺ patients with CD4 levels of 200 to 500/μl, and HIV⁺ patients with CD4 levels of <200/μl. One-way ANOVA and the Tukey test were used. (E) CD4 cell counts in relation to CpG-induced CD80 expression on memory B cells among HIV⁺ viremic donors. Data were analyzed using Spearman's correlation.

FIG. 3.

CD21-positive B-cell frequencies in HIV infection are inversely related to plasma HIV RNA level and CD80 expression. (A) B-cell CD21 expression in one representative analysis among gated naive (CD27⁻) and memory (CD27⁺) B (CD19⁺) B cells. (B) Median percentages of CD21⁺ naive and memory B cells in freshly isolated cells; comparisons were performed using one-way ANOVA and the Tukey test. (C) Among patients, plasma HIV RNA levels are inversely correlated with CD21 frequencies among freshly isolated naive and memory B cells (P < 0.001, Spearman's correlation). (D) In B-cell subsets from HIV-infected patients, the expression of CD80 was inversely related to the frequency of CD21⁺ cells in both freshly isolated naive (P = 0.001) and memory (P < 0.001) B cells (Spearman's correlation).

FIG. 4.

CD21 expression predicts memory B-cell proliferation failure, and plasma HIV RNA level predicts naive B-cell proliferation failure, in response to CpG ODN in HIV infection. (A) Age, CD4⁺ T-cell number, plasma HIV RNA level, CD21 and CD80 expression among freshly isolated naive and memory B cells, and induction of CD80 by CpG ODN in overnight cultures were analyzed for relationships to CpG-induced B-cell proliferation by Spearman's correlation. (B) Multiple regression analysis indicates that plasma HIV RNA level was the only independent predictor of naive B-cell proliferation responses and that CD21 level was the only independent predictor of memory B-cell proliferation in response to CpG ODN in HIV infection.

FIG. 5.

Diminished TLR9 mRNA levels in both memory and naive B cells from viremic HIV⁺ patients. TLR9 mRNA levels were assessed by real-time PCR in purified memory and naive B cells obtained ex vivo (A) and in purified memory and naive B cells cultured with or without CpG ODN for 20 h (B) from five HIV⁻ donors and six HIV⁺ viremic donors (plasma HIV RNA levels of >400 copies/ml). Medians ± standard errors of the means of TLR9 mRNA copies/1,000 GADPH are compared between HIV⁻ and HIV⁺ viremic donors by the Wilcoxon rank test (*, P < 0.05).

FIG. 6.

Different thresholds for optimal CD80 induction and proliferation among B cells in response to CpG ODN. PBMCs from two healthy donors and two HIV⁺ donors were cultured with increasing concentrations of CpG ODN 2006 (0.01 to 24 μg/ml). The percentage of B cells (CD19⁺) induced to express CD80 was evaluated after 20 h of cultivation (A), and proliferation responses were measured by dilution of CFSE dye after 7 days (B).