Cd27 o323

We isolated peripheral blood mononuclear cells (PBMCs) from the blood samples using density-gradient centrifugation on Sepmate and RosetteSep DM-L Density Medium (Stemcell Technologies, Grenoble, France). Isolated PBMCs were stained for flow cytometry with antibodies (Abs) against CD3 (SK7; BD Biosciences, San Diego, CA), CD4 (SK3; BD Biosciences), CD45RA (HI100; Biolegend, San Diego, CA), CXCR5 (J252D4; Biolegend), CD19 (HIB19; Biolegend), CD27 (O323; Biolegend), or CD38 (HIT2; Biolegend) for 30 min at 4 °C. Stained cells were analyzed by multiparameter flow cytometry (FACS Verse, BD Biosciences) using FlowJo ver. 10 software (BD Biosciences).

Excised tonsils from three de-identified immunocompetent patients were diced and strained through a 100-μm filter. A single cell suspension of tonsillar mononuclear cells (MNCs) was created with Ficoll density gradient separation. Once isolated, MNC were counted and resuspended in organoid media (RPMI with L-glutamine, 10% FBS, 2 mM glutamine, 1X penicillin-streptomycin, 1 mM sodium pyruvate, 1X MEM non-essential amino acids, 10 mM HEPES buffer and 1 μg/ml of recombinant human B cell activating factor [BioLegend]) at a concentration of 6 × 10⁷ cells per ml. As previously described by Wagar et al. [32 (link)], MNC’s were transferred to permeable transwells (0.4-μm pore, 12-mm diameter; Millipore). transwells were inserted into standard 12-well polystyrene plates containing 1 ml of additional organoid media and placed in an incubator at 37 °C and 5% CO2. Lometrexol in phosphate-buffered saline was added, or not, to organoids after 48 h in culture. Organoid media with or without lometrexol was replaced every 3 days. On culture day 7, organoid MNCs were resuspended and stained at 4 °C with the anti-human CD38 (HIT2; BioLegend), CD27 (O323; BioLegend), CD19 (HIB19; BioLegend), and L/D aqua (Invitrogen). Cells were analyzed with LSRFortessa (BD Bioscience) and visualized with FlowJo software (TreeStar).