To assess whether loss of memory B cells in HIV-1-infected patients could affect capability of producing IgG in response to in vitro activation, we purified B cells from 10 HIV-1-infected patients and six normal controls. The B cells were activated with agonistic anti-CD40 MAb or with CD70-transfected cells in the presence of IL-2 and IL-10, or left unstimulated. Unstimulated B lymphocytes from HIV-1-infected subjects produced significantly higher levels of IgG than those from uninfected subjects (P < 0.05;Table 2). IgG secretion upon stimulation through CD40 was higher in HIV-1-infected patients than in controls, although the difference was not significant statistically (P = 0.1). Surprisingly, CD70-dependent IgG secretion by B cells from HIV-1-positive subjects was increased twofold compared with that of HIV-1-negative controls (P < 0.05), despite the lower proportion of memory B cells present in the culture. However, the ratios between activation-induced and spontaneous IgG were similar in HIV-1-positive and -negative subjects (data not shown).
HIV-1 causes severe damage to the immune system as early as the asymptomatic phase of infection. The mechanisms leading to immunological dysfunction detected in the T- and B-cell compartments are both directly mediated by the virus and driven by virus-induced chronic immune activation. In parallel with cell-mediated immunity, a functional humoral immunity appears to be important in reducing the spread of HIV-1  and might play a fundamental role in the control of the opportunistic and secondary infections commonly observed in HIV-1-infected subjects.
The mechanisms underlying B-lymphocyte dysfunction are not well characterized. In the present study we have investigated the phenotype and functionality of peripheral B cells. We observed that the B-cell population expressing the CD27 antigen and representing the memory subset is reduced significantly during HIV-1 infection. Because CD27 can be cleaved from the cell surface, we considered the possibility that memory B cells could not be detected because of the release of soluble CD27. Parallel analysis of IgD and CD27 expression showed that the reduced numbers of CD27-positive B cells detected in HIV-1-positive subjects actually results from a selective loss of the memory B-cell subset. The numbers of memory B cells and the CD4 cell counts were not correlated, and similar numbers of memory B cells were detected in subjects with CD4 cell count below and above 390 × 106 cells/l. In addition, it was found that subjects undergoing HAART and drug-naive subjects had similar levels of peripheral memory B lymphocytes. These findings suggest that the loss of memory B cells might occur early after infection and may not be corrected by therapy.
The finding that memory B cells are reduced in HIV-1-infected subjects in parallel with the observed polyclonal B cell activation prompted us to analyse the relationship between memory B cells and activated T cells. Activated CD70 T lymphocytes promote differentiation of memory B cells into plasma cells via CD27 ligation [22,34]. Similarly to Wolthers et al., we observed that the frequency of CD70 T cells ex vivo was higher in HIV-1-positive subjects than in HIV-1-negative controls. We report here that the numbers of CD70-expressing T cells is inversely correlated with the percentage of memory B cells. This finding suggests that the activation status of T cells, driven by circulating HIV-1, may trigger constitutive differentiation of B cells into antibody-secreting cells, resulting in hypergammaglobulinaemia and exhaustion of the memory B-cell pool. This hypothesis is supported further by our finding that among patients undergoing HAART, patients with undetectable viral load had a higher percentage of memory B cells than those with detectable virus.
To understand if the altered phenotype of peripheral B cells was accompanied by a functional defect in Ig production, we stimulated peripheral B cells via CD40 or CD27 ligation. We found that B cells from HIV-1-infected subjects produced an increased amount of IgG upon CD40 activation compared with B cells from healthy donors. Surprisingly, despite the reduced number of memory (CD27) B cells present in culture, we found that CD70-induced IgG production was higher in HIV-1-infected subjects than in the control group. Although a normal or higher response to CD40 ligation has already been described [8,13] this is the first report that the CD27–CD70 interaction is not impaired during HIV-1 infection and that the pool of memory B cells, although reduced, is still functional. The hyperactivation status of B cells, shown by the high spontaneous IgG secretion in vitro, may explain why the CD70-dependent IgG production is elevated in HIV-1-infected subjects in spite of the reduced number of memory B cells.
Dysregulation of the Fas/FasL pathway is involved in the increased lymphocyte apoptosis observed during HIV-1 infection . Previous studies from our group have shown that B cells from HIV-1-infected subjects are primed for apoptosis in vivo and overexpress FasL . We hereby report that memory B cells from HIV-1-infected patients express high levels of Fas ex vivo and strongly upregulate Fas and, to a lesser extent, FasL expression upon culture in vitro. As plasma cells express high levels of FasL , the higher expression of FasL detected on memory B cells may represent the first step towards terminal differentiation into antibody-secreting cells. These findings further confirm that memory B lymphocytes from HIV-1-infected subjects are activated in vivo. In this context, it is tempting to speculate that the upregulated expression of both Fas and FasL on memory B cells may trigger an autocrine apoptotic circuit or alternatively render these cells targets for apoptosis induced by FasL-activated T cells or macrophages . Investigations on the susceptibility of such B cells to undergo Fas-mediated apoptosis are currently ongoing in our laboratory.
On the basis of our observations we suggest two potential mechanisms underlying the loss of memory B lymphocytes during HIV-1 infection. As a result of the constitutive T-cell activation occurring during the infection, memory B cells acquire an activated phenotype. This process induces activated memory B cells to undergo two different, although not mutually exclusive pathways: terminal differentiation into plasma cells and Fas-mediated apoptosis. Both of these processes may account for the loss of memory B cells during infection.
Recently, polyclonal B-cell activation was shown to be reduced in response to combination antiretroviral therapy . On the other hand, the loss of B-cell function seems to be rapid and persistent after HIV-1 infection . Indeed, we observed that the numbers of memory B cells were not related to CD4 cell counts, disease stage or therapy status, suggesting that loss of memory B cells may occur early during the infection and may not be corrected by antiviral therapy. Plasma cells are located mainly in the bone marrow and signs of plasmacytosis have already been reported in HIV-1 infection . The parallel analysis of peripheral blood and bone marrow samples from HIV-1-infected subjects would be important to verify that HIV-1-induced polyclonal B-cell activation results in loss of memory B cells in the periphery and accumulation of plasma cells in the bone marrow. Recently, the engagement of CD134L on B cells was shown to increase the rate of IgG production without affecting the process of plasma cell generation . As CD134 represents a T-cell activation antigen that is upregulated during HIV-1 infection , it would be interesting to study if this receptor and its ligand play a role in the HIV-1-induced polyclonal B-cell activation.
Together with severe T-cell dysfunction, loss of B cell memory may represent an important pathogenic mechanism underlying the lack of response after immunization with recall antigens and the susceptibility to opportunistic infections frequently observed in HIV-1-infected subjects. The pathogenic mechanism underlying loss of memory B cells needs to be investigated further.
The authors thank M. Halvarsson for providing clinical data and the patients who contributed to the study. We thank M. Zazzi for critical reading of the manuscript.
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