The infection with HIV-1 leads to the progressive loss of immune functions, a process starting during the early stages of infection and involving both T and B lymphocytes [1,2]. Polyclonal B-cell activation is a major hallmark of B-cell function dysregulation in HIV-1-infected patients. In fact, hypergammaglobulinaemia is often observed in HIV-1-infected individuals and also their peripheral lymphocytes spontaneously produce high levels of immunoglobulins (Ig) in vitro[3,4]. Despite the hyperactivation status of B cells, a severe impairment in B-cell response has been shown following in vivo immunization of infected patients with recall antigens and in vitro stimulation with mitogens or antigens [5–7]. The mechanism underlying HIV-1-induced polyclonal B-cell activation is poorly understood but it has been suggested that both viral proteins (Tat, Nef, gp120) and cytokines [TNF-a, interleukin (IL)-6, IL-10)] are involved in promoting this dysfunction [8–12].
Abnormal expression of the CD40 ligand (CD40L) on CD4 T cells has been proposed to play a role in the impaired B-cell response to T cell-dependent antigens [8,13]. In line with this, the ability of B cells from HIV-1-infected individuals to respond to triggering through CD40 and the B-cell receptor was shown to be inversely correlated with the clinical stage of disease . Recently, CD40-activated B lymphocytes have been described as a potential viral reservoir , though the low level of HIV-1 infection in B cells excluded a major direct role of the virus in B-cell functional defects. The role of apoptosis through Fas/Fas ligand (FasL) pathway in B-cell impairment during HIV-1 infection remains to be clarified . Our group has provided evidence that loss of humoral immunity in HIV-1-infected individuals correlates with B-cell apoptosis and increased levels of FasL expression on B cells [17,18].
Like Fas, the CD40 and CD27 molecules belong to the tumour necrosis factor receptor family. The interaction between these receptors and their respective ligands plays a crucial role in the homeostatic regulation of immune responses. The process of B-cell terminal differentiation is regulated by the interaction between CD40L and CD70 expressed on activated T cells, and their respective receptors CD40 and CD27 expressed on B cells . Activation of the CD40/CD40L pathway mediates Ig class switching and memory B-cell commitment whereas the ligation of CD27 by CD70 is believed to occur in vivo after CD40 activation of memory B cells by T lymphocytes, leading to plasma cell differentiation [19–24]. Indeed, CD27 has recently been recognized as a marker for memory B cells, previously identified as the IgD-fraction of B cells [25–27].
In the present study we analysed the phenotype of peripheral B lymphocytes and their functional response to T cell-dependent stimuli in vitro. We also evaluated the putative role of the CD27–CD70 pathway in virus-induced polyclonal B cell activation.
Materials and methods
Patients and blood donors
A total of 36 HIV-1-infected subjects and 34 age-matched blood donors were included in this study after informed consent was obtained. Among the infected patients, 15 were drug naive and 21 were undergoing highly active antiretroviral therapy (HAART) including at least one protease inhibitor. According to the Centers for Disease Control and Prevention classification, 11 out of 36 patients were classified with AIDS (CDC A3, B3, and C), five patients were in stage B infection and 20 in stage A infection. The immunological characteristics of the study participants are given in Table 1.
Peripheral blood monononuclear cells (PBMC) from HIV-1-infected patients and healthy controls were purified from 20 ml EDTA-treated whole blood by centrifugation over a Ficoll-Hypaque (Amersham Pharmacia Biotech, Uppsala, Sweden) density gradient. For the in vitro study on purified B cells 30 ml whole blood was used. Plasma samples were collected and stored at −70°C until analysis. B lymphocytes were obtained by positive selection using anti-CD19 monoclonal antibody (MAb)-coated magnetic beads (Dynal, Oslo, Norway) following removal of the beads with Detachabead (Dynal) according to the manufacturer's recommendations. The selected cell population was shown to contain 96–98% CD20 B lymphocytes as assessed by FACS analysis with anti-CD20 RPECy5-conjugated MAb (DAKO, Copenhagen, Denmark).
Purified B cells were cultured at 2.5 × 105 cells/ml in RPMI 1640 supplemented with 10% foetal calf serum, antibiotics, IL-2 (200 U/ml, Peprotech, London, UK) and IL-10 (50 ng/ml, R & D Systems, Oxon, UK). The CD70-transfected B-cell line 300-19 was kindly provided by K. Agematsu (Shinshu University, Japan). Purified B cells were activated in the presence of IL-2 and IL-10 by anti-CD40 MAb (clone B-B20, 2 μg/ml; DIACLONE Research, Besancon, France) or by adding the CD70-transfected cells (ratio B cells : CD70 cells, 5:2) pretreated as reported previously . Unstimulated B cells were cultured with medium only. Cells were cultured in triplicate wells, each well containing 5 × 104 purified B cells. Cell culture supernatants were collected after 8 days and kept at −20°C for analysis of IgG content. For the study on Fas and FasL expression, B cells were cultured overnight in medium only.
IgG quantification in cell culture supernatants was performed by enzyme-linked immunosorbent assay as described previously . The amount of plasma Ig was measured by nephelometry.
Using two-colour flow cytometry, freshly isolated PBMC were stained with the following MAbs conjugated with flourescein isothiocyanate (FITC), phycoerythrin (PE) or RPE-Cy5: CD19-RPECy5, CD3-FITC, Fas-FITC, IgD-FITC (DAKO), CD27-FITC, CD27-PE, mouse anti-CD70 (Pharmingen, San Diego, California, USA), and FasL-FITC (Alexis Corporation, San Diego, California, USA). Ig isotype-matched FITC-, PE- and RPECy5-conjugated mouse antibodies (DAKO) were used as negative controls for non-specific staining. Briefly, 0.5 × 106 cells were stained with the appropriate MAbs for 30 min on ice in phosphate-buffered saline (PBS) with 2% foetal calf serum. After the final staining, the cells were washed twice with PBS/2% foetal calf serum and fixed in PBS containing 2% paraformaldehyde. Flow cytometry was performed with a FACScan instrument (Becton Dickinson, Mountain View, California, USA) using the Cellquest software (Becton Dickinson). Forward/side scatter dot plot was used to gate the live lymphocyte population and 25 000 cells/sample were analysed.
The expression of Fas and FasL on specific B-cell subpopulations was analysed by a three-colour flow cytometric analysis on freshly isolated PBMC and on cultured purified B cells from five HIV-1-infected patients and four uninfected controls. Expression of Fas and FasL was measured as the mean fluorescence intensity on the gate of live CD19+CD27+ (memory) and CD19+CD27− (naive) B cells. The mean fluorescence intensities of the control FITC MAb for the ex vivo and the overnight staining were 3.3 ± 1.4 and 3.1 ± 1.2 respectively.
Differences between HIV-1-infected patients and uninfected controls were analysed by Mann–Whitney test or Student's t test as appropriate. Correlation analysis between variables was performed by Spearman rank test or regression analysis. Data in the text are expressed as median and range or mean ± SEM.
Memory and naive B lymphocytes during HIV-1 infection
To study the phenotype of peripheral B lymphocytes in HIV-1-infected patients, PBMC were double stained for CD19 and CD27, a marker of memory B cells . The number of memory (CD27) B lymphocytes was significantly lower in HIV-1-infected patients than in uninfected controls, both as a percentage of the B lymphocytes and as absolute number (P < 0.001 and P < 0.01 respectively;Fig. 1a). The frequency of circulating total B cells was comparable between the two groups (Table 1). The percentage of memory B-cells was lower in patients with AIDS (median, 11.9%; range, 6.1–31.6%) than in patients without AIDS (median, 19%; range, 6–54.6%) although the difference was not significant (P = 0.13). In addition, patients were subdivided into those with a CD4 cell count above or below the median value of 390 × 106 cells/l. We found that patients with CD4 cell counts < 390 × 106/l (n = 19) and patients with CD4 cell counts > 390 × 106/l (n = 17) had similar percentages of memory B cells (15.8% and 15.65%, respectively;P = 0.68). Moreover, no difference in the percentage of memory B cells was observed between drug-naive patients (median, 15.2%; range, 6.1–24.7%) and patients undergoing HAART (median, 19%, range, 6.1–54.6%;P = 0.19). Eight out of the 21 patients treated with HAART had undetectable plasma HIV-1 RNA. These patients had a higher percentage of memory B cells than patients with detectable circulating virus (19.9 ± 3.7% versus 13.8 ± 2%;P = 0.14), although the difference was not statistically significant.
As HIV-1 infection is associated with the release of surface molecules involved in immune responses , we investigated the possibility that memory B cells were not really decreased but that CD27 has been shed from the cell surface. To address this possibility, we measured IgD and CD27 expression in parallel in 15 HIV-1-positive and 12 HIV-negative subjects. The median percentage of IgD-B lymphocytes in HIV-1-infected patients was 14.07% (range, 8.5–40.6%) compared with 27.05% (range, 16.21–38.28%) in uninfected subjects (P = 0.01). Moreover, the percentage of IgD-B cells and CD27 B cells were similar and significantly correlated in both HIV-1-infected patients (Fig. 2) and controls (data not shown).
CD70 expression on T cells
The expression of CD70 is upregulated on T cells from HIV-1-infected patients upon in vitro stimulation . As CD70 expressed on activated T lymphocytes promotes the differentiation of CD27 B cells into plasma cells [19,24], we asked whether the expression of CD70 on T cells was related to the expression of CD27 on B cells in HIV-1 infection. We analysed freshly isolated PBMC from 14 HIV-1-infected patients and 12 uninfected controls. The percentage of T cells expressing CD70 in the HIV-1-infected group was higher than that in the control group (Fig. 3) and was inversely correlated with the CD4 cell count (r2 = 0.32;P < 0.05). Interestingly, in the HIV-1-positive group (but not in HIV-1-negative controls) the percentage of CD27 memory B cells was found to be inversely correlated to the proportion of CD70-expressing T cells (Fig. 4).
CD40- and CD27-dependent antibody secretion in HIV-1-infected subjects
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).
In addition to in vitro IgG production, plasma levels of Ig were measured (Table 1). Plasma IgG and IgA concentrations of HIV-1-infected patients were significantly elevated compared with those of uninfected individuals. The amount of plasma IgG was inversely correlated to the CD4 cell count (r2 = 0.14;P = 0.02) and titers of plasma IgG were higher in patients with AIDS than in subjects without AIDS (P < 0.05).
Fas and FasL expression on memory B cells
The high IgG secretion in vitro and the hypergammaglobulinaemia observed in HIV-1-infected patients indicated that B cells are hyperactivated during HIV-1 infection. It has been shown that in vitro-activated B cells upregulate the expression of both Fas and FasL [31,32]. We measured the expression of Fas and FasL on naive and memory B cells both ex vivo and on B cells cultured overnight from five HIV-1-positive and four HIV-1-negative subjects. We observed that Fas expression was increased on freshly isolated B cells from HIV-1-infected patients, with a higher level of expression on memory B cells (P < 0.01;Fig. 5). Following overnight culture, the expression of Fas was upregulated further on memory B cells but not on naive B cells (Fig. 5). The expression of FasL on B cells from HIV-1-infected subjects was also slightly upregulated after overnight culture compared with that of uninfected subjects (P = 0.01;Fig. 5), as previously shown by us .
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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Keywords:© 2001 Lippincott Williams & Wilkins, Inc.
activation; antibodies; B cell; FACS; pathogenesis