Characterization of plasmablasts in the blood of HIV-infected viremic individuals: evidence for nonspecific immune activation

Abstract

Terminal differentiation of B cells and hypergammaglobulinemia are hallmarks of B-cell hyperactivity in HIV disease. Plasmablasts are terminally differentiating B cells that circulate transiently in the blood following infection or vaccination; however, in HIV infection, they arise early and are maintained at abnormally high levels in viremic individuals. Here we show that only a small fraction of plasmablasts in the blood of viremic individuals is HIV specific. Assessment of plasmablast immunoglobulin isotype distribution revealed increased IgG(+) plasmablasts in early and most prominently during chronic HIV viremia, contrasting with a predominantly IgA(+) plasmablast profile in HIV-negative individuals or in aviremic HIV-infected individuals on treatment. Of note, IgG is the predominant immunoglobulin isotype of plasmablasts that arise transiently in the blood following parenteral immunization. Serum immunoglobulin levels were also elevated in HIV-infected viremic individuals, especially IgG, and correlated with levels of IgG(+) plasmablasts. Several soluble factors associated with immune activation were also increased in the sera of HIV-infected individuals, especially in viremic individuals, and correlated with serum immunoglobulin levels, particularly IgG. Thus, our data suggest that while plasmablasts in the blood may contribute to the HIV-specific immune response, the majority of these cells are not HIV specific and arise early, likely from indirect immune-activating effects of HIV replication, and reflect over time the effects of chronic antigenic stimulation. Such B-cell dysregulation may help explain why the antibody response is inadequate in HIV-infected individuals, even during early infection.

Fig 1

Frequencies of total Ig and gp140-specific ASCs in the blood of HIV-infected individuals. Frequencies of ASCs were measured by ELISPOT assay on B cells isolated from the blood of early and chronically infected HIV-viremic individuals. (A) Percent gp140 ASC response, defined as percent gp140-specific of total Ig (IgG, IgA, and IgM) ASC frequency, was measured on peripheral blood B cells isolated from 6 early and 9 chronically infected HIV-viremic individuals. Horizontal bars represent medians. (B) Frequencies of total Ig and gp140-specific ASCs, as well as percent gp140-specific ASCs, were measured before and after ART for 6 HIV-viremic individuals each in early and chronic HIV infection. Duration on ART was 12 months, with a viral load below the limit of detection at the time of sample collection. (C) Flow cytometric profiles of HIV gp140 binding to B cells (CD20⁺ CD3⁻) (left graph) of a representative HIV-viremic individual are shown. The numbers in each box represent percent expression of IgG among gated B cells (middle graph) and percent gp140 binding among IgG⁺ B cells (right graph). (D and E) Correlation (D) and comparison (E) between percent gp140-specific ASCs and percent gp140 binding of IgG⁺ B cells measured by flow cytometry on B cells isolated from individuals in panel A.

Fig 2

Frequencies of IgG⁺, IgA⁺, and IgM/D⁺ PBs in the blood of early infected HIV-viremic (EV; n = 24), chronically infected HIV-viremic (CV; n = 36), chronically infected HIV-aviremic (CAV; n = 30), and HIV-negative (HN; n = 25) individuals. (A) The percentage or fraction of PBs, identified as CD19⁺ CD20⁻ CD27⁺⁺ CD3⁻, expressing each Ig isotype was determined by flow cytometry. The profiles shown are of a representative chronically infected HIV-viremic individual. The percentages in the pie charts represent means for IgG, IgA, and IgM/D (B); the horizontal bars in graphs for IgG (C), IgA (D), and IgM/D (E) represent median values for each group.

Fig 3

Correlations between peripheral blood PB counts and CD4⁺ T-cell count (left graph) and HIV plasma viremia (right graph). Data include samples from the four groups described in Table 1, with the exception that the HN group was not included in the right graph.

Fig 4

Longitudinal analysis of Ig isotype among PBs in the blood of HIV-infected individuals. PBs of eight individuals each from early (A) and chronically (B) HIV-infected groups were analyzed before and after initiation of ART for Ig isotype distribution. Individuals in the early group were receiving ART for an average of 12 months, and individuals in the chronic group were receiving ART for an average of 6 months.

Fig 5

Ig levels in the sera of early infected HIV-viremic (EV; n = 33), chronically infected HIV-viremic (CV; n = 33), chronically infected HIV-aviremic (CAV; n = 34), and HIV-negative (HN; n = 30) individuals. Horizontal bars represent median values for each group. Ig classes and subclasses reported include IgG (A), IgA (B), IgM (C), IgG1 (D), and IgG3 (E).

Fig 6

Levels of soluble factors TNFRII, MIG, ICAM, IL-10, and IP-10 in the sera of early infected HIV-viremic (EV; n = 33), chronically infected HIV-viremic (CV; n = 32), chronically infected HIV-aviremic (CAV; n = 34), and HIV-negative (HN; n = 29) individuals. Horizontal bars represent median values for each group.

Fig 7

Correlations between serum Ig levels and peripheral blood PB counts for corresponding Ig isotype (A), levels of serum IgG and soluble factors (B), and HIV plasma viremia and soluble factors (C). Data in panels A and B include samples from the four groups described in Table 1. Data in panel C include samples from the three HIV groups described in Table 1.