Circulating memory B-cell subpopulations are affected differently by HIV infection and antiretroviral therapy
D'Orsogna, Lloyd Ja; Krueger, Rom Gb; McKinnon, Elizabeth Jc; French, Martyn Aa,d
From the aDepartment of Clinical Immunology and Immunogenetics, Royal Perth Hospital, Perth, Australia
bFlow Cytometry Unit, Core Services Laboratory, Royal Perth Hospital, Perth, Australia
cCentre for Clinical Immunology and Biomedical Statistics, Murdoch University, Perth, Australia
dSchool of Surgery and Pathology, University of Western Australia, Perth, Australia.
Received 16 November, 2006
Revised 16 May, 2007
Accepted 22 May, 2007
Correspondence to Martyn French, Department of Clinical Immunology and Immunogenetics, Royal Perth Hospital, GPO Box X2213, Perth, WA 6847, Australia. E-mail: firstname.lastname@example.org
Objective: To determine if the depletion of IgM memory B cells might contribute to the increased susceptibility of HIV patients to pneumococcal infection, memory B-cell subpopulations were investigated in HIV patients, including patients receiving antiretroviral therapy (ART).
Methods: Blood B cells with the phenotype of IgM memory B cells (CD27+, IgM+) and switched memory B cells (CD27+, IgM−) were measured in antiretroviral-treated (n = 32) and untreated (n = 24) HIV patients and non-HIV controls (n = 35). Serum levels of IgG and IgG2 antibodies to pneumococcal polysaccharides, IgG, IgG subclasses, IgM and IgA were also assayed in HIV patients.
Results: Switched memory B-cell counts were lower than controls in HIV patients (P < 0.01) irrespective of antiretroviral status and correlated with CD4 T-cell counts (r = 0.56, P = 0.001) in treated patients. In untreated patients, IgM memory B-cell counts correlated with CD4 T-cell counts (r = 0.73, P < 0.0001) reflecting higher values than controls in patients with CD4 T-cell counts greater than 300 cells/μl (P = 0.004) and lower values than controls in patients with CD4 T-cell counts below 300 cells/μl (P = 0.0001). There was no relationship between serum levels of pneumococcal antibodies and IgM or switched memory B cells.
Conclusion: The depletion of IgM memory B cells in untreated HIV patients with a CD4 T-cell count below 300 cells/μl might be a risk factor for pneumococcal infection. The depletion of switched memory B cells is a complication of HIV infection irrespective of ART and might contribute to impaired IgG antibody responses. Memory B-cell subpopulations might predict the risk of pneumococcal sepsis more accurately than the CD4 T-cell count or pneumococcal antibody levels.
Pneumococcal disease occurs more often than normal in HIV patients, with an incidence reported to be up to one hundred times greater than in the general population [1,2], and it may also be recurrent. The effect of combination antiretroviral therapy (ART) on the susceptibility of HIV patients to invasive pneumococcal disease is unclear, with some studies showing a decline , whereas others have not . Risk factors for invasive pneumococcal disease include intravenous drug use, smoking cigarettes, excessive alcohol intake, other co-morbidities and CD4 T-cell deficiency [3,4]. The immunological risk factors for pneumococcal disease in HIV patients are not well delineated.
It is well recognized that HIV-induced immune dysfunction includes B-cell activation and the impaired production of antibodies that is partly related to a deficiency of memory B cells [5–8]. The CD27 molecule is a cell-surface marker that identifies somatically mutated peripheral memory B cells [9–11]. The human B-cell compartment contains memory B cells (CD19+, CD27+) that express IgM (IgM memory B cells) and a further subset of cells in which immunoglobulin production has switched to isotypes other than IgM (switched memory B cells) [12,13]. IgM memory B cells possess a prediversified IgM antigen receptor and are capable of responding immediately to the antigens of encapsulated bacteria in a T-cell-independent fashion [10,14]. In contrast, switched memory B cells require T-cell co-stimulation to produce IgG and other isotypes of antibody. Blood IgM memory B cells appear to be the circulating counterpart of marginal zone B cells that are present in the spleen [12,15]. A deficiency of IgM memory B cells in asplenic individuals or patients with common variable immunodeficiency syndrome has been associated with an increased susceptibility to invasive pneumococcal disease [10,16,17].
Titanji and colleagues [8,18] recently demonstrated that the loss of total memory B cells in chronic HIV infection impairs the maintenance of long term ‘serological’ memory as evidenced by reduced in-vitro production of IgM and IgG antibodies against measles virus and Streptococcus pneumoniae antigens after activation of peripheral blood mononuclear cells. The relationship between impaired antibody responses to S. pneumoniae antigens and memory B-cell and CD4 T-cell deficiency was not, however, reported.
The aim of this study was to clarify memory B-cell deficiency further in HIV patients, in particular to determine if there is a deficiency of IgM memory B cells that might increase susceptibility to invasive pneumococcal disease, and also to determine what effect ART has on this. We also aimed to determine if ‘steady state’ pneumococcal antibody levels in serum are related to memory B-cell counts or proportions.
Patients and controls
We undertook a cross-sectional study of 24 HIV patients not receiving ART (untreated), 32 HIV patients receiving ART (treated), and 35 non-HIV controls. HIV patients receiving ART had been treated for at least 6 months with at least three drugs from two separate classes, and had undetectable (< 50 log10 copies/ml) plasma HIV RNA at the time of assessment. Nineteen of the untreated patients were ART naive and five had ceased ART at least 10 months before study participation. All subjects were older than 18 years of age. The demographic characteristics of patients and controls are given in Table 1. Laboratory assessments were performed on a single occasion between March 2005 and June 2006. Written informed consent was obtained from all patients and controls.
Analysis of memory B cells by flow cytometry
Leukocytes were enriched from a 10 ml peripheral blood sample anticoagulated with ethylenediaminetetraacetic acid by collecting a phosphate-buffered saline (PBS)-washed buffy coat and resuspending it in approximately 1 ml of PBS flow buffer containing 2% fetal calf serum. The following antibodies coupled with fluoroscein isothiocyanate (FITC), R-phycoerythrin (PE), or the tandem dye R-phycoeythrin cyanin 5.1 (PC5) were used for flow cytometry; anti-CD19-PC5 (clone J4.119; Immunotech, Beckman Coulter, Inc., Fullerton, California, USA), anti-CD27-PE (clone IA4-CD27; Immunotech), anti-IgM-FITC (polyclonal fab’2; Dako, Glostrup, Denmark), isotype control-PE (clone 679.1Mc7; Immunotech), isotype control-FITC (fab’2; Dako). An aliquot of 100 μl of diluted buffy coat was stained for 10 min at RT with appropriate amounts (5 or 10 μl) of the following antibodies: CD19-PC5, CD27-PE, IgM-FITC in one tube and CD19-PC5, isotype control-PE, isotype control-FITC in a second tube. After incubation, the cell/antibody mixture was processed in a Beckman Coulter Multi-Q Prep instrument to lyse, stabilize and fix the cells before acquisition and analysis on the flow cytometer. Three-colour data acquisition was performed using a Beckman Coulter XL-MCL flow cytometer with system II analysis software or a Beckman Coulter Elite-ESP flow cytometer with Elite analysis software. Validation data were obtained on replicate samples from both instruments to ensure the reliability and comparability of data acquired from either cytometer.
Assays of plasma HIV RNA, T-cell subpopulations and serum levels of immunoglobulins and pneumococcal polysaccharide antibodies
HIV-1 RNA in plasma was quantified using the Amplicor polymerase chain reaction HIV-1 monitor assay (Roche, Branchburg, New Jersey, USA) version 1.5. Flow cytometry was used to quantify T-cell subpopulations, including naive (CD45RA+) and memory (CD45RO+) CD4 T cells. Serum levels of total IgG, IgM, IgA and IgG subclasses were assayed by nephelometry. Serum levels of total IgG and IgG2 antibodies to pneumococcal polysaccharides were assayed by enzyme-linked immunosorbent assay (Evolis; BioRad, Hercules, California, USA) using monoclonal antibodies from Binding Site (Birmingham, UK).
Analyses presented here consider total, IgM or switched memory B cells as counts derived from the total lymphocyte count or as proportions of total B cells. The median and interquartile range (IQR) are used as summary statistics. Comparative analyses are non-parametric and include the Mann–Whitney U test and Spearman's rank correlation test. P < 0.05 is considered to be significant. Analyses were carried out using S-PLUS 7.0 for Windows (Insightful Corp., Seattle, Washington, USA).
Switched memory B cells correlated with CD4 T-cell counts in non-HIV controls
There was no effect of sex or age on either the counts or proportions of memory B-cell subpopulations in non-HIV controls. Both the counts and proportions of total memory B cells correlated with CD4 T-cell counts (r = 0.43, P = 0.009 and r = 0.36, P = 0.03, respectively). Switched memory B-cell counts correlated strongly with CD4 T-cell counts (r = 0.48, P = 0.003) and memory CD4 T-cell counts (r = 0.46, P = 0.005), whereas IgM memory B-cell counts correlated with naive CD4 T cells (r = 0.38, P = 0.03) but not memory CD4 T cells. Associations between memory B-cell counts and CD8 T cells were similar to those observed for CD4 T cells (Table 2).
Memory B-cell subpopulations are altered in untreated HIV patients
In untreated HIV patients, switched memory B-cell counts (median 14.0, IQR 3.5–26.6 cells/μl) were lower than in non-HIV controls (median 23.6, IQR 16.4–40.7 cells/μl; P = 0.004; Fig. 1) but showed only a marginal correlation with CD4 T-cell counts (Table 2). Similar results were obtained for proportions of switched memory B cells. In contrast, neither total memory B-cell counts (median 42.1, IQR 20.9–57.4 cells/μl) nor IgM memory B-cell counts (median 20.9, IQR 4.3–40.7 cells/μl) were significantly different from non-HIV controls (Fig. 1). Strong observed associations of both total and IgM memory B-cell counts with CD4 T-cell counts (r > 0.73, P < 0.0001; Table 2), however, reflected increased IgM memory B-cell counts and proportions in patients with CD4 T-cell counts above 300 cells/μl (P = 0.004 and P = 0.0001, respectively) and decreased IgM memory B-cell counts and proportions in patients with CD4 T-cell counts below 300 cells/μl (P = 0.0001 and P = 0.05, respectively), compared with non-HIV controls (Fig. 2). This pattern of co-segregation was also seen for both naive and memory CD4 T cells (Table 2). IgM memory B-cell counts and proportions did not correlate with CD8 T-cell counts (Table 2), but correlated inversely with the plasma HIV-RNA level (r < −0.45, P < 0.05).
Switched memory B cells remained low in HIV patients receiving antiretroviral therapy
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).
Serum levels of pneumococcal antibodies did not correlate with memory B cells
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.
In contrast with the findings of our study, De Milito et al.  showed that HIV patients with low memory B cells had lower serum levels of antibody to several antigens (tetanus toxoid, measles virus and HIV-1). Their analysis was undertaken with data on total memory B cells (not IgM or switched memory B cells), however, and they did not examine antibodies to pneumococcal polysaccharides. Furthermore, patients were not subdivided according to treatment status. Therefore, the relationship between memory B-cell subpopulations and serum pneumococcal antibody levels in HIV patients is not clearly defined. Lanzavecchia et al.  found that tetanus-specific memory B cells, but not total memory B cells, correlated with tetanus-specific antibody production in healthy controls. Therefore, future studies in HIV patients should examine the relationship between pneumococcal-specific memory B cells and serum pneumococcal antibody levels.
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.
Conflicts of interest: None.
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HIV; IgM memory B cells; pneumococcal antibody; switched memory B cells
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