In treated HIV patients who had achieved undetectable plasma HIV-RNA levels, switched memory B-cell counts (median 14.6, IQR 8.7–28.0 cells/μl) were lower than in controls (P = 0.01; Fig. 1). Similar results were obtained for proportions of switched memory B cells. This decrease was not associated with the duration of ART, and was still observed when the analysis was restricted to those who had been receiving ART for more than 4 years (n = 19, P = 0.01). In contrast, IgM memory B-cell counts (median 14.8, IQR 8.4–23.8 cells/ml) were not significantly different from controls, and there were no significant correlations between IgM memory B-cell counts and counts of total, naive and memory CD4 T cells (P > 0.2) or CD8 T cells (P > 0.9).
Neither total, IgM nor switched memory B-cell counts or proportions correlated with serum levels of IgG or IgG2 antibodies to pneumococcal polysaccharides in untreated or treated patients (Table 2). In treated patients, total and switched memory B-cell counts correlated with serum levels of IgG1 (P = 0.02) and marginally with IgG3 (P < 0.07). Otherwise, there were no correlations between serum levels of IgG subclasses, IgA or IgM and memory B cells in untreated or treated patients.
We have shown for the first time that HIV-associated depletion of memory B cells affects components of the circulating memory B-cell compartment (IgM or switched memory B cells) differently, and that these abnormalities are not fully corrected by ART. IgM memory B cells were increased in untreated HIV patients with a CD4 T-cell count above 300 cells/μl but became depleted as the acquired immunodeficiency progressed. Low IgM memory B-cell counts were particularly associated with a CD4 T-cell count below 300 cells/μl, suggesting that the previously reported association of pneumococcal disease with low CD4 T-cell counts in HIV patients  might, at least partly, be a consequence of IgM memory B-cell depletion. Switched memory B cells were lower than non-HIV controls in both untreated and treated HIV patients.
Our observation that IgM memory B cells were higher in untreated HIV patients with a CD4 T-cell count above 300 cells/μl when compared with both untreated patients with a CD4 T-cell count below 300 cells/μl and non-HIV controls was unexpected. Possible explanations include a T-cell-independent marginal zone B-cell response to HIV, as demonstrated in mice for other viral antigens , or B-cell activation induced by polysaccharide antigens of microbial products, such as lipopolysaccharide, which is reported to be increased in the plasma of patients with HIV infection as a consequence of microbial translocation across the mucosa of the intestine .
The abnormalities of IgM memory B-cell counts demonstrated in untreated HIV patients were not demonstrated in treated patients (see Table 2 and Fig. 2), suggesting that ART corrects the underlying immune defect. This might partly explain the lower rates of pneumococcal disease in patients receiving ART reported in some studies . In contrast, switched memory B cells remained lower than controls in treated patients, in whom there was a correlation with CD4 T-cell counts (see Table 2).
Defects in memory B-cell switching may contribute to the impairment of antibody production and B-cell memory that is characteristic of HIV infection [5,8,18] and affects IgG antibody responses to vaccine antigens such as pneumococcal polysaccharides  and influenza virus . Nagase et al.  found that hypergammaglobulinaemia in HIV patients was associated with a deficiency of total memory B cells and with bone marrow plasmacytosis, and suggested that the depletion of circulating memory B cells was a consequence of HIV-associated immune activation that resulted in the differentiation of memory B cells into plasma cells. Antibody responses to unconjugated pneumococcal polysaccharides are improved by ART [23–25], possibly reflecting the correction of HIV-induced abnormalities of IgM memory B cells demonstrated in this study. Antibody responses may, however, not be optimal in patients with CD4 T-cell counts below 200 cells/μl .
Serum levels of IgG and IgG2 antibodies to pneumococcal polysaccharides did not correlate with IgM or switched memory B-cell counts or proportions in either untreated or treated HIV patients. These findings suggest that the ‘steady state’ serum level of IgG antibodies to pneumococcal polysaccharides is not influenced by the number or proportion of circulating IgM or switched memory B cells in HIV patients. Barry et al.  reported that serum pneumococcal antibody levels showed no correlation with rates of invasive pneumococcal disease in HIV patients. Therefore, IgM memory B-cell counts, and possibly switched memory B-cell counts, might be better indicators of susceptibility to pneumococcal disease than serum levels of pneumococcal polysaccharide-specific IgG antibodies.
The findings of our study have important implications for clinical practice. First, susceptibility to pneumococcal disease in HIV patients might be predicted more effectively by monitoring subpopulations of memory B cells, particularly IgM memory B cells, as well as CD4 T-cell counts. The method of assessing memory B-cell subpopulations described here is readily applicable to routine diagnostic laboratories.
Second, our finding that HIV patients receiving effective ART have lower than normal switched memory B cells might have implications for clinical trials of vaccines to increase pathogen-specific IgG antibody levels in HIV patients receiving ART. For example, trials of unconjugated pneumococcal polysaccharide vaccines (T-cell independent) or protein-conjugated pneumococcal polysaccharide vaccines (T-cell dependent) might include the stratification of patients according to the count or proportion of IgM memory B cells (T-cell independent) and switched memory B cells (T-cell dependent). Finally, our study may also have important ramifications for the design of a therapeutic vaccine to increase HIV-specific antibody levels. The depletion of switched memory B cells that exists in HIV patients receiving effective ART may result in impaired IgG antibody responses to HIV antigens.
In conclusion, we have shown in untreated HIV patients that IgM memory B cells are increased in patients with a CD4 T-cell count above 300 cells/μl but decreased in patients with a CD4 T-cell count below 300 cells/μl, and that switched memory B cells are low in both untreated and treated HIV patients. We suggest that the measurement of memory B-cell subpopulations might have clinical utility in predicting the susceptibility of HIV patients to pneumococcal disease. This should be tested in clinical studies.
The authors are grateful to the Australian Red Cross Blood Service for providing blood samples from non-HIV controls. They also thank Nick Acquarola for helpful assistance with the flow cytometry.
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