Ha peptide mab

PBMCs were labeled with mAbs against CD20, CD10, CD27, and IgG, and sorted for naïve, transitional, CD27⁻, or unswitched (IgG⁻) memory B cells using the FACSAria III (Becton Dickinson), ensuring >98% purity of the recovered populations. Transitional, naïve, or unswitched memory B cells were cultured in 96-well round-bottom plates (30–40 × 10³ cells per well for CFSE analysis or 5 × 10³ cells per well to determine Ig secretion). B cells were stimulated with 200 ng/ml CD40L cross-linked to 50 ng/ml HA Peptide mAb (R&D Systems) alone or together with 50 ng/ml IL-21 (PeproTech), 1 μg/ml CpG 2006 (Sigma-Aldrich), 0.05% heat-killed, or formalin-fixed Staphylococcus aureus Cowan I strain (SAC; Sigma-Aldrich). Varying doses of the PI3K p110δ-specific inhibitor leniolisib (Hoegenauer et al., 2017 (link); Rao et al., 2017 (link)) and equivalent doses of DMSO were also added.
B cell viability was determined using the Zombie Aqua Viability dye (BioLegend) and proliferation was measured by CFSE (eBioscience) as described previously (Avery et al., 2010 (link); Hodgkin et al., 1996 (link)). Secretion of IgM, IgG, and IgA by in vitro cultured human transitional, naïve, and memory B cells was determined using Ig heavy-chain specific ELISAs, as described previously (Avery et al., 2005 (link)).

PBMCs were labeled with mAbs against CD20, CD27, and CD10 and transitional (CD20⁺CD10⁺CD27⁻) or naive (CD20⁺CD10⁻CD27⁻) B cells were then sorted using a FACSAria III (Becton Dickinson; Cuss et al., 2006 (link); Suryani et al., 2010 (link)). Purity of the recovered populations was >98%. Transitional and naive B cells were then cultured in 96-well U-bottom plates (Falcon; 20–40 × 10³/200 µl well) for 4–5 d to determine proliferation and gene expression by qPCR and microarray analysis, or 5 × 10³/200 µl well for 5–7 d to determine Ig secretion. B cells were stimulated with 200 ng/ml CD40L cross-linked to 50 ng/ml HA Peptide mAb (R&D Systems) alone or together with 100 U/ml IL-4, 100 U/ml IL-10 (provided by R. de Waal Malefyt, DNAX Research Institute, Palo Alto, CA), 50 ng/ml IL-21 (PeproTech), 2.5 µg/ml F(ab’)₂ fragment of goat anti-IgA/IgG/IgM (H+L; Jackson ImmunoResearch), 1 µg/ml CpG 2006 (Sigma-Aldrich), and varying doses of the PI3K p110δ specific inhibitor leniolisib (Hoegenauer et al., 2017 (link); Rao et al., 2017 (link); Novartis Pharma), and equivalent doses of DMSO.