Anti cd38 hit2

Peripheral blood mononuclear cells (PBMCs) were isolated by standard density-gradient centrifugation (Lymphoprep Fresenus Kabi, Norway). B cells (CD19 + ) and T cells (CD3 + ) were positively selected using magnetic beads (Miltenyi Biotec, Germany) according to the manufacturer's instructions. For isolation of marginal zone-like B cells, B cells (CD19 + ) were stained with anti-CD27 brilliant Violet 605 (O323) (Biolegend, USA) and anti-IgD VioBlue (REA740, Miltenyi Biotec) mAb. CD19 + CD27 + IgD + B cells were sorted with a BD Influx cell sorter (BD Bioscience, USA). FACS sorted naïve and memory B cells (as above) were activated with CpG (2.5 µg/ml) for 24 hours and then stained with anti-CD86-PE (IT2.2, Biolegend), anti-CD69-FITC (FN50, Immunotools), anti-HLADR-APC (G46-6, BD Biosciences), and anti-CD71-PE (CY1G4, Biolegend). For carboxyfluorescein succinimidyl ester (CFSE) dilution assays, PBMCs were stained with CFSE and then activated with anti-CD3 and anti-CD28 mAb (for T cells), or with CpG or IL-21/CD40L (for B cells). Cells were stimulated for 5 days and then assessed by flow cytometry. For CMV ELISPOT assays patient and HC PBMCs were stimulated CMV antigens using the T-track CMV kit (Lophius). For detailed B cell phenotyping the following additional antibodies were used: anti-CD24 (IML5, Biolegend), anti-CD38 (HIT2, BD Biosciences), anti-TACI (1A1, Biolegend).