Healthy controls (HC) were recruited among members of the McGill University community. Well-characterized patients with confirmed MS (Additional file 1: Table S1) were recruited at the MS Clinic of the Montreal Neurological Institute and Hospital (MNI/H). All participants provided informed consent as part of a protocol approved by the university’s internal review board (IRB). All MS patients were untreated at the time of blood draw, had not received any immune-modulating treatments within at least 6 months prior to sampling, and had not been treated with steroids within at least 30 days prior to sampling.
B cell and B cell subset isolation
B cells were isolated from fresh antecubital venous blood, as previously described . Briefly, peripheral blood mononuclear cells (PBMC) were isolated from 100 to 120 ml venous blood of untreated MS patients and healthy volunteers using standard density-gradient centrifugation on Ficoll-Paque (Pharmacia Biotech). Magnetic beads (MACS, Miltenyi Biotec) were used according to the manufacturer’s instructions to isolate CD19+ B cells by positive selection, and their purity was confirmed by flow cytometry (routinely > 98% pure). B cells were then washed and resuspended in serum-free X-Vivo 10 medium (Lonza, Walkersville, MD). For experiments with B cell subsets, total B cells were initially sorted from PBMC by CD19+ MACS separation and then stained for CD20+ (2H7), CD27+ (M-T271), IgD (IA6-2), CD24 (ML5), and CD38 (HIT2), all from BD Bioscience. The total B cells were subsequently sorted (using a BD LSRFortessa, BD Bioscience) into transitional (CD20+CD24+CD38+), naive (CD20+CD27−IgD+), or memory (CD20+CD27+IgD−/+) B cell subsets with routine purity confirmation (typically > 93%).
Astrocyte isolation and culture
Human fetal astrocytes (HFA) were isolated as previously described . In brief, fetal CNS tissue age 17–22 weeks of gestation, obtained from the Albert Einstein College of Medicine Human Fetal Tissue Repository (Bronx, NY), was first dissociated using trypsin-EDTA (Invitrogen Life Technologies) and DNase I (Roche, Laval, QC), followed by mechanical dissociation. The cell suspension was then washed and seeded in complete Dulbecco’s modified Eagle’s medium (DMEM; containing 10% fetal calf serum (FCS), penicillin, streptomycin, l-glutamine, and glucose) into poly-l-lysine-coated flasks, at a concentration of 3–5 × 106 cells/mL. To obtain near-pure astrocyte cultures, the HFA were further enriched in DMEM by at least three passages, starting 2 weeks post isolation. Astrocytes for our experiments, isolated from over 40 independent preparations, were used between passages 3 and 5 and > 90% pure, as determined by glial fibrillary acidic protein (GFAP, clone EPG724) immunostaining as previously described . Purified astrocytes were then washed twice in phosphate-buffered saline (PBS) and plated in complete DMEM at a density of 0.1 × 106 cells/well in 300 μL (48-well plates) or at 2.4 × 105 cells/well in 500 μL (24-well plates). Upon reaching confluency (usually following 24 h in culture), the astrocytes were again washed in PBS, and 300 or 500 μL serum-free X-Vivo 10 medium supplemented with penicillin, streptomycin, and l-glutamine was added to each well. Astrocytes were either left unstimulated or stimulated (Additional file 2: Figure S1) with a combination of IFNγ (10 ng/mL) and IL-1β (10 ng/mL) as previously reported . After 24 h of incubation, the astrocytes were again washed thoroughly in PBS to minimize possible carry-over effects of activating cytokines, and fresh medium was added. Where indicated, astrocytes were either used in co-culture experiments with B cells (described below) or maintained in culture for an additional 24 h, with astrocyte-conditioned medium (ACM) then collected and stored at − 80 °C until use.
B cell:astrocyte co-cultures
For direct B cell:astrocyte co-cultures, astrocytes were isolated as described above and plated at a density of 2.25 × 105 cells in 300 μL per well in 48-well plates in DMEM supplemented with 10% FCS until they reached 80% confluence (2–3 days). DMEM was then removed and B cells, purified from the periphery of healthy donors, were directly co-cultured at a density of 3 × 105 cells in 300 μL of X-Vivo 10. Survival responses were measured after 5 days of co-culture, while activation changes were assessed after 48 h of co-culture. For B cell:astrocyte transwell co-cultures, astrocytes were isolated and plated at a density of 2.4 × 105 cells/well in 500 μL in 24-well plates and were either left unstimulated, or stimulated, as described above. BD Falcon cell-culture inserts (0.4 μm pore diameter) were then placed into the wells, and freshly isolated B cells were added to the upper compartments at a density of 2 × 105 B cells in 200 μL serum-free X-Vivo 10 medium for the indicated duration, at which time the B cells were collected from the upper well and survival and activation were measured. For experiments assessing the effects of astrocytes on B cell subsets, the sorted B cell subsets were exposed in transwell to astrocytes, as described above, for 40 h. For experiments utilizing ACM, B cells were plated at a density of 1.5 × 105 cells in 150-μL serum-free X-Vivo 10 medium per well in U-bottom 96-well plates, and 50 μL of ACM (or control medium, as indicated) was added to each well (representing 25% of the final volume). For functional blocking experiments, neutralizing antibodies to human IL-6 (clone 6708) human IL-15 (clone 34559) and BAFF (clone 148725), as well as appropriate isotype controls (all R&D Systems), were incubated at a final concentration of 1 μg/ml with ACM for at least 20 min prior to addition to the B cell cultures. At the end of all cultures, B cell survival and expression of the T cell co-stimulatory molecule CD86 were assessed by flow cytometry, as described below.
T cell isolation and allogeneic stimulation
To determine whether astrocyte exposure can modulate the capacity of B cells to induce T cell responses, B cells that were pre-exposed to astrocyte-soluble factors in the transwell system described above were washed and co-cultured with T cells isolated from different healthy donors. Following PBMC separation, CD4+ T cells were isolated by positive selection using standard MACS separation (Miltenyi Biotec). T cell purity was confirmed by flow cytometry (routinely > 98%), and the T cells were then washed, resuspended in serum-free X-Vivo 15 medium, and stained with CFSE (20 μL CFSE 1% per mL). Allogeneic B cells that were previously cultured for 48 h under the different conditions described above were added to the freshly isolated CD4+ T cells in 300 μL serum-free X-Vivo 15 medium in 48-well plates at a B cell:T cell ratio of 1:4 (0.75 × 105 B cells per 0.3 × 106 T cells). T cells were also cultured alone, as a negative control, or with 2 μg/mL phytohemagglutinin (PHA, Sigma), as a positive control for T cell proliferation. Following 6 days in culture, the T cells were harvested and analyzed by flow cytometry as described below.
Flow cytometry for T cell and B cell responses
Depending on the assay, B cell survival was assessed following 2, 3, or 5 days in culture by co-staining for CD20 (anti-CD20, 2H7, BD Bioscience), 7AAD (BD Bioscience), and Annexin V (BD Bioscience), with forward- (FSC) and side-scatter (SSC) properties also considered. Surviving B cells we considered to be Annexin V−/7AAD− CD20+. B cell activation was assessed by quantifying upregulation of surface expression of the co-stimulatory molecule CD86 (anti-CD86, FUN-1, BD Bioscience). Following 2 days in culture, B cells were collected and washed using PBS containing 5% FCS, incubated with CD20 and CD86 antibodies for 20 min at 4°C, washed again and stained with Annexin V and 7AAD for 10 min at room temperature. T cell proliferation was quantified based on CFSE dilution following 6 days of culture as described above. All FACS acquisition was done using either FACSCalibur or LSRFortessa (BD Biosciences), and data were analyzed using FlowJo flow-cytometry10 analysis software (TreeStar, OR, USA).
GraphPad Prism (versions 7 and 8) was used for all statistical analyses. Student’s unpaired t tests were used for statistical comparisons between two groups when the assumption of normal distribution was deemed appropriate. One-way ANOVA was used to compare across groups or conditions, and two-way ANOVA was used to compare several groups across different conditions.