B-cell memory is usually defined by the persistence of elevated levels of specific antibodies, the presence of memory B cells of the IgG or IgA isotype, and faster secondary antibody responses with higher antibody affinities than primary responses after reexposure to antigens. 1 In vivo antigen-specific activation and differentiation of B cells occur in germinal centers, where naive B cells (CD19+, CD27−, and IgD+) undergo activation, proliferation, somatic hypermutation of rearranged V region genes, immunoglobulin isotype switching, and subsequent selection by antigens, mature into antibody-producing plasma cells, or alternatively become memory B cells (CD19+, CD27+, and IgD−). 2–4 Memory B cells are long-living cells that recirculate throughout the lymphatic tissue and after encountering antigens migrate via the blood to the bone marrow, a homing site of immunoglobulin secreting cells (SCs). 5 Usually, memory B cells are defined by their phenotype and represent ∼15% to 40% of the circulating B cells. 6
HIV-1 infection leads to a progressive loss of immune functions. This process starts during the early stages of infection and involves both T and B lymphocytes. 7 There is evidence of in vivo B-lymphocyte hyperreactivity as shown by polyclonal hypergammaglobulinemia 8 and the expression of activation markers of B cells (CD38+ and CD71+) that spontaneously secrete antibodies specific to HIV-1 in vitro. 9,10 In contrast, in vivo humoral immune response after immunization and B-lymphocyte responses to in vitro antigen or mitogen stimulation are impaired, whereas anti-HIV-1 antibodies directed to gag-encoded proteins decrease in advanced stages of AIDS. In addition, HIV-1 infection induces a clonal deletion of B-cell subsets bearing the VH3 family surface immunoglobulin in patients with AIDS-defining clinical conditions. 11 In spite of all these B-cell abnormalities, only minor modifications of the percentage and number of circulating memory B cells are observed. 6
Because activation of specific memory B cells through interaction between the B-cell receptor and antigen ligation has, to our knowledge, never been reported, we hypothesized that in vitro polyclonal activation of memory B cells by CD40L ligation and IL-2 and IL-10, which induces these cells to differentiate into immunoglobulin SCs, 12,13 could also lead to differentiation of HIV-1–specific memory B cells into anti-HIV-1 antibody SCs. To test this hypothesis, circulating B cells from 10 HIV-1–infected patients were highly purified and then stimulated using the CD40–CD40L system. Anti-HIV-1 antibody SCs were enumerated using an HIV-1–specific ELISPOT assay, and anti-HIV-1–specific antibodies were detected in B-cell culture supernatants.
PATIENTS AND METHODS
B cells from HIV-1–infected patients were purified and cultured with or without CD40L-expressing mouse fibroblasts and adequate cytokines. After 5 days, polyclonal κ or λ L-chain SCs in B-cell cultures were counted using a standard immunoglobulin ELISPOT assay as previously described. 14 Anti-HIV-1–specific antibody SCs were enumerated using an anti-HIV-1–specific antibody ELISPOT assay, and anti-HIV-1–specific antibodies were detected in supernatants of B-cell cultures using standard ELISA and immunoblotting assays (Fig. 1).
Patients and Healthy Controls
Ten HIV-1–infected patients with detectable plasma viral loads (between 111 and 12,566 copies/mL) were included in the study after providing written informed consent. The University Hospital Committee (Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale de l'Hôpital de Montpellier, France [no. 98-7564]) approved this study. Three healthy controls were also included in the study.
The numbers of memory and naive B cells in peripheral blood lymphocyte samples as well as in B-cell–enriched preparations were determined by flow cytometry (XL-apparatus; Beckman–Coulter, Villepinte, France). One hundred microliters of centrifuged and washed EDTA-treated blood was incubated for 30 minutes with RPMI 1640. Then, the cells were incubated with a mixture of phycoerythrin cyanin 5.1–conjugated anti-CD19 (Beckman–Coulter), phycoerythrin-conjugated anti-CD27 (Beckman–Coulter), and fluorescein isothiocyanate–conjugated anti-IgD (Tebu, Le Perray en Yvelines, France) antibodies during 10 minutes. Red blood cells were lysed by using the IMMUNOPREP system (Beckman–Coulter), and the remaining cells were fixed with 1% formaldehyde. We analyzed the lymphocytes on the basis of size/structure and CD19 expression. Memory B cells were defined as the CD27+ and IgD− population, and naive B cells were defined as the IgD+ and CD27− population (Fig. 2). CD4+ T cells were counted using the tetraONE SYSTEM (Beckman–Coulter) after staining the cells with CYTO-STAT tetraCHROME (CD45/CD4/CD8/CD3; Beckman–Coulter). In addition, the absolute number of B-cell and CD4+ T-cell populations was determined using calibrated Flow Count fluorospheres (Beckman–Coulter) according to the manufacturer's instructions. Adjustments for compensations and positivity thresholds of each sample were carried out using isotopic controls.
Purification and Stimulation of Peripheral Blood B Lymphocytes
B cells were isolated from EDTA-treated blood samples using a Rosette sep B-cell enrichment cocktail (Stemcell Technologies, Meylan, France) according to the manufacturer's instructions. The enriched B-cell population, containing >98% of CD19+ cells, was frozen in liquid nitrogen until use. In some experiments, memory B cells were negatively selected. Briefly, enriched B cells were incubated with anti-IgD human monoclonal antibody (Sigma Chemical Company, St. Louis, MO) coupled via a short DNA fragment to magnetic beads in the CELLection Kit (Dynal Biotech, Compiégne, France) in cold phosphate-buffered saline (Eurobio, Les Ullis, France) supplemented with 2% fetal calf serum at 4°C under gentle shaking. After 30 minutes of incubation, the cells were washed and passed through a magnetic field, and naive B cells were detached from the bead surface by DNase. The purity and the viability of the naive and memory B-cell pools were both >95%. One hundred thousand purified B cells per well were cultured in Microtest 96-well tissue culture plates (35172; Becton Dickinson, Myelan, France) with or without 104 CD40L-expressing mouse fibroblasts treated with 0.1 μg mitomycin C (Sigma Chemical Company) in RPMI 1640 supplemented with 10% fetal calf serum, 2 mm l-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 0.5 ng IL-2 (R & D, Oxon, United Kingdom), and 1 ng IL-10 (R & D).
Enumeration of Immunoglobulin SCs
κ or λ L-chain SCs were enumerated using an immunoglobulin-specific 2-color ELISPOT assay as previously described. 14 Spots were counted using the automate reader KS ELISPOT (Carl Zeiss Vision GmbH, Germany). Results were expressed as the number of immunospots per 106 B lymphocytes tested.
Enumeration of Anti-HIV-1–Specific Antibody SCs
Anti-HIV-1–specific antibody SCs were assessed using the previously described ELISPOT assay 14 with modifications. Briefly, 96-well microtiter plates (Nunc, Roskilde, Denmark) using Immobilon-P membrane as solid phase (Millipore Corporation, Bedford, MA) were coated overnight at 4°C with mouse monoclonal anti-human IgG (γ), IgA (α), or IgM (μ) (Tebu). Cells (5 × 104 per well) were incubated for 18 hours; after extensive washings with phosphate-buffered saline, purified horseradish peroxidase–labeled HIV-1 peptides mimicking the immunodominant epitopes of the HIV-1 envelope glycoproteins and a horseradish peroxidase–labeled nucleocapsid recombinant protein (Bio-Rad, Marnes-la-Coquette, France) were added for 1 hour. After phosphate-buffered saline washings, 3-amino-9-ethyl-carbazol (Sigma Chemical Company) was added to each well, insoluble red-colored precipitates were obtained within 5 to 10 minutes, and the immunoenzymatic reaction was stopped with distilled water.
Detection of Anti-HIV-1 Antibodies in Cell Culture Supernatants
Antibodies specific to HIV-1 were detected in supernatants of 10-day cultures by ELISA as previously described. 15,16 Briefly, anti-HIV-1 antibodies were detected using a commercial ELISA kit (Genscreen, Bio-Rad) according to the manufacturer's instructions with some modifications. Culture supernatants were incubated for 90 minutes at 37°C, and results were expressed as the ratio of the absorbance at 492 nm of the patient's culture supernatant to the absorbance (mean ± 2 SDs) of B-cell culture supernatants from HIV-1–seronegative controls. Anti-HIV-1 antibody specificities were determined using an immunoblotting method according to the manufacturer's instructions (Bio-Rad).
Circulating Memory B Cells From HIV-1–Infected Patients Differentiated Into Immunoglobulin SCs
Purified B cells from 10 HIV-1–infected patients were cultured with or without mouse fibroblast cells expressing CD40L in medium containing IL-2 and IL-10. At day 5 of the cell culture, κ or λ L-chain SCs were enumerated by ELISPOT assay. Polyclonal stimulation of B cells induced more κ and λ L-chain SCs (30,871 ± 22,305 and 49,290 ± 53,296, respectively) than unstimulated B cells (101.6 ± 70.8 and 78.3 ± 44.9, respectively). Negatively purified memory B cells from controls also induced more κ or λ L-chain SCs (1999,303 ± 96,588 and 239,166 ± 31,879, respectively), whereas only few κ and λ L-chain SCs were enumerated in CD40L-activated naive B cells (57.5 ± 95 and 77.5 ± 45, respectively). Similar results were obtained when immunoglobulin SCs were detected using an IgG/IgA or IgG/IgM 2-color ELISPOT assay (authors' unpublished data). These findings clearly indicate that only memory B cells differentiated into immunoglobulin SCs.
To determine the efficiency of both isolation and activation of memory B cells, the number of κ and λ L-chain SCs obtained after 5 days of culture was compared with that of memory B cells as determined in the blood by their phenotype marker. As shown in Table 1, the number of memory B cells, which had differentiated into immunoglobulin SCs, represented 55% to 98% of circulating memory B cells. These findings suggest that under our experimental conditions, B-cell isolation, B-cell cryopreservation, and in vitro polyclonal B-cell stimulation were optimized to detect rare circulating HIV-1 antigen–specific B lymphocytes dispersed among the other B lymphocytes.
Detection and Enumeration of Circulating HIV-1–Specific Memory B Cells
Stimulated purified B cells from 10 HIV-1–infected patients were tested using a modified form of our ELISPOT assay 14 to enumerate anti-HIV-1 isotype–specific antibody SCs. Between 10 and 190 B cells per 1,000,000 B cells tested were able to differentiate into cells secreting anti-HIV-1–specific-IgG, IgA, or IgM (Table 1). These cells represented about 1 × 10−4 to 1 × 10−5 of the total circulating B cells and 1 × 10−2 to 1 × 10−3 of the total SCs. HIV-1–specific memory B cells were detected for all tested patients independently of the plasma viral load and the immune status.
In addition, anti-HIV-1 antibodies were detected by ELISA in culture supernatants of B cells stimulated by the CD40L system during 10 days. Results clearly indicated that the absorbance measured for cell culture supernatants from activated B cells was higher than the cutoff of the ELISA that was established using unstimulated B cells from HIV-1–infected patients or B cells from healthy controls (Table 1). As shown in Figure 3, culture supernatants from activated B cells from HIV-1–infected patients contained antibodies directed to HIV-1 gp160, 120, and 41 as determined using an HIV-1–specific immunoblotting assay; the level of antibodies specific to HIV-1 gag- and pol-encoded proteins in these culture supernatants was low or in some even cases undetectable.
In this study, circulating B lymphocytes from HIV-1–infected patients and healthy controls were highly purified and stimulated via the CD40–CD40L system. Cell purification, cryopreservation, and culture conditions were optimized to recover the greatest possible number of functionally circulating B cells. Under our laboratory conditions, enumeration of immunoglobulin SCs at the end of the B-cell culture represented more than one half of the circulating memory B cells. Consequently, a sufficient number of B cells was usually recovered in 20 mL of blood, allowing us to detect HIV-1–specific memory B cells by their synthesis of anti-HIV-1–specific antibodies. For all the patients tested, most circulating memory B cells differentiated into immunoglobulin SCs, and 1 × 10−2 to 1 × 10−3 of them secreted anti-HIV-1–specific antibodies of the IgG, IgA, or IgM isotype. The enumeration of anti-HIV-1–specific IgM SCs by ELISPOT assay also confirmed the fact that anti-HIV-1–specific IgM memory B cells can be identified in HIV-1–infected patients. Antibodies specific to HIV-1 proteins and glycoproteins were detected in culture supernatants of stimulated B cells, but antibodies to the viral proteins p24 and p34 were less frequently found. This discrepancy can be explained by the fact that anti-p24 antibody production is T helper cell dependent, whereas anti-gp120 antibodies can also be produced via a T helper cell–independent pathway. 17
Our results raise the question as to whether several HIV-1–specific B-cell subpopulations circulate in HIV-1–infected patients. Because freshly isolated nonactivated B cells did not spontaneously secrete anti-HIV-1–specific antibodies, two distinct sets of circulating functional HIV-1–specific B cells could be identified in HIV-1–infected patients with detectable plasma viral loads: B lymphocytes responsible for the spontaneous ex vivo anti-HIV-1–specific antibody secretion 18–20 and HIV-1–specific resting memory B cells. These findings suggest that the CD40L–B cell activation method could explore a quiescent pool of HIV-1–specific memory B cells that differentiate into plasma cells in vitro and produce specific anti-HIV-1 antibodies.
HIV-1–specific memory B cells were found to circulate in blood from HIV-1–infected patients. Our findings add to the evidence explaining the mechanisms of generation of HIV-1–specific memory B cells by HIV-1 antigens and the possible modifications of the number of these cells in patients receiving highly active antiretroviral therapy over a long time. A clinical study investigating the population of circulating HIV-1–specific memory B cells in patients with long-term nonprogressing HIV-1 infection could be helpful to determine the possibility of these cells in the production of neutralizing anti-HIV-1 antibodies. Finally, we suggest that this new functional B-cell concept could be applied for a better understanding of the mucosal B-cell repertory during HIV-1 infection and in vaccination models.
The authors thank Beckman–Coulter for their help with this study. They are indebted to S.L. Salhi for carefully reading the manuscript.
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