Evidence for HIV-associated B cell exhaustion in a dysfunctional memory B cell compartment in HIV-infected viremic individuals

Abstract

Human immunodeficiency virus (HIV) disease leads to impaired B cell and antibody responses through mechanisms that remain poorly defined. A unique memory B cell subpopulation (CD20(hi)/CD27(lo)/CD21(lo)) in human tonsillar tissues was recently defined by the expression of the inhibitory receptor Fc-receptor-like-4 (FCRL4). In this study, we describe a similar B cell subpopulation in the blood of HIV-viremic individuals. FCRL4 expression was increased on B cells of HIV-viremic compared with HIV-aviremic and HIV-negative individuals. It was enriched on B cells with a tissuelike memory phenotype (CD20(hi)/CD27(-)/CD21(lo)) when compared with B cells with a classical memory (CD27(+)) or naive (CD27(-)/CD21(hi)) B cell phenotype. Tissuelike memory B cells expressed patterns of homing and inhibitory receptors similar to those described for antigen-specific T cell exhaustion. The tissuelike memory B cells proliferated poorly in response to B cell stimuli, which is consistent with high-level expression of multiple inhibitory receptors. Immunoglobulin diversities and replication histories were lower in tissuelike, compared with classical, memory B cells, which is consistent with premature exhaustion. Strikingly, HIV-specific responses were enriched in these exhausted tissuelike memory B cells, whereas total immunoglobulin and influenza-specific responses were enriched in classical memory B cells. These data suggest that HIV-associated premature exhaustion of B cells may contribute to poor antibody responses against HIV in infected individuals.

Figure 1.

Phenotypic characterization of tissuelike memory B cells in the peripheral blood of HIV-viremic individuals. (A) Color-coded gating on CD20/CD21-stained B cells of a representative HIV-viremic individual was used to identify CD20^hi/CD21^lo activated (CD27^int) and tissuelike (CD27⁻) memory B cells (green); CD20^int/CD21^hi resting memory (CD27^int) and naive (CD27⁻) B cells (red); and CD20⁻/CD27^hi/CD21^lo plasmablasts (blue). (B) Percentage of expression of FCRL4 on peripheral blood B cells of HIV-viremic (n = 16), HIV-aviremic (n = 12), and HIV-negative individuals (n = 12). Horizontal bars indicate medians. (C–E) Color-coded gating on CD27/CD21-stained B cells (C and E) of a representative HIV-viremic individual was used to establish expression of FCRL4 (C), frequency of FCRL4 on B cell subpopulations of 12 HIV-viremic individuals (D), and other inhibitory/homing receptors and adhesion molecules (E) on CD20^hi/CD27⁻/CD21^lo tissuelike memory (black), CD27⁺ classical memory (blue), and CD20^int/CD27⁻/CD21^hi naive (red) B cells. MFI, mean fluorescence intensity. All stains included CD19, which was used to establish the B cell gate. Immature/transitional B cells were excluded by performing stains on CD10-depleted B cells or on PBMCs gated on CD19⁺/CD10⁻ cells.

Figure 2.

Distinct properties of tissuelike memory B cells isolated from the peripheral blood of HIV-viremic individuals. Evaluation of the number of cell divisions undergone in vivo by KREC analysis on mature (CD10⁻) B cells of a representative (A) and a group of HIV-viremic individuals (B; n = 8) after fractionation into classical memory (CD27⁺), tissuelike memory (CD27⁻/CD21^lo), and naive (CD27⁻/CD21^hi) B cells. Evaluation of Ig VH3 diversity by restriction enzyme-based hotspot analysis on mature B cells of a representative (C) and a group of HIV-viremic individuals (D; n = 8) after fractionation into classical memory, tissuelike memory, and naive B cells. Each individual is identified by a different color in B and D.

Figure 3.

Low proliferative capacity of tissuelike memory B cells isolated from the peripheral blood of HIV-viremic individuals. (A and B) Proliferation was measured in response to various B cell stimuli in the absence (A) or presence (B) of cytokines on mature B cells that had been fractionated into classical memory, tissuelike memory, and naive B cells (n = 8). Anti-Ig refers to goat anti–human IgG/A/M antibodies, and CpG refers to CpG type B, which is described in the Materials and methods. Horizontal bars indicate medians, and tissue memory in the graphs refers to tissuelike memory.

Figure 4.

Enrichment of HIV-specific ASCs in tissuelike memory B cells contrasted by enrichment of total and influenza-specific ASCs in classical memory B cells. Total Ig ASCs(A) and Ig isotypes of total ASCs (B) in classical memory, tissuelike memory, and naive B cells after in vitro polyclonal stimulation (n = 13). Total influenza-specific ASCs (C) and Ig isotypes of influenza-specific ASCs (D) in classical memory, tissuelike memory, and naive B cells after in vitro polyclonal stimulation (n = 12). Total HIV-specific ASCs (E) and Ig isotypes of HIV-specific ASCs (F) in classical memory, tissuelike memory, and naive B cells after in vitro polyclonal stimulation (n = 13). V01-V15 along the x axis of B, D, and F identifies each individual tested, and the asterisk indicates that only IgG was tested on this individual. Horizontal bars indicate medians, and tissue memory in the graphs refers to tissuelike memory.