Hla dr clone l243

To validate the changes in class II HLA observed at transcriptomic level with IFN-γ and LPS treatment, we assessed class II expression on primary monocytes by multiparametric flow cytometry. Monocytes were stimulated, as described, before being washed and stained with violet viability dye (Invitrogen) as per the manufacturer’s instructions. To reduce nonspecific staining, a three-step procedure adapted from a protocol shared by R. Apps (National Cancer Institute, National Institutes of Health) was used. Human immunoglobulin G (IgG) was added to each tube, followed by mouse anti-human antibodies directed against HLA-DP (clone B7/21, Abcam), HLA-DQ (clone SPV-L3, Abcam), HLA-DR (clone L243, BD Biosciences), or all class II alleles (clone Tu39 and SK10/SPV-L3 combined) or an isotype control. After 20 min, cells were washed and stained with sheep anti-mouse PE-labeled antibody (Sigma) and sheep IgG.
After a further 20 min, cells were washed and stained with mouse anti-human CD14–FITC (fluorescein isothiocyanate) (clone 61D3, eBioscience) and mouse IgG (Sigma). Cells were fixed with the paraformaldehyde containing Reagent A (Life Technologies), and data were acquired immediately on a FACSCanto (BD Biosciences) machine. A minimum of 10,000 gated monocytes were acquired for each sample. Fluorescence-minus-one controls were used to set gate thresholds.

Thawed PBMCs were surface stained with Aqua amine reactive viability dye (Invitrogen) and mAbs to CD45 (clone D058–1283, BD Biosciences), CD3 (clone SP34–2, BD Biosciences), CD4 (clone L200, BD Biosciences), CD8 (clone 3B5, Invitrogen), CD95 (clone DX2, BioLegend), CD28 (clone CD28.2, Beckman), CD127 (clone hIL-7R-M21, BD Biosciences), PD-1 (clone EH12.2H7, BioLegend), and HLA-DR (clone L243, BD Biosciences) for 20 min at room temperature, fixed, and then permeabilized with FoxP3 Fix/Perm Buffers (eBioscience) per the manufacturer’s instructions. Permeabilized cells were then stained intracellularly for Ki67 (clone B56, BD Biosciences) and FoxP3 (clone PCH101, eBioscience) for 30 min at 4°C, washed in FoxP3 Perm/Wash buffer, fixed in 0.5% paraformaldehyde and analyzed by flow cytometry on a BD LSRII flow cytometer (BD Biosciences) and analyzed using Flowjo Software (Tree Star Inc).

Healthy, HLA typed B cells were isolated with EasySep™ B cell enrichment kit as per protocol (StemCell Technologies, Vancouver, BC, Canada) and expanded as previously published³⁷ . Concisely, B cells were co-incubated with irradiated (43Grays) NIH-3T3-CD154 fibroblasts (generously donated by Professor P.S. Heeger, Mount Sinai, New York, NY, USA), in the described media. Prior to cryopreservation the B-cells were analysed for MHC-I (HLA-ABC, clone: B9.12.1, BD Pharmingen, San Jose, CA) and MHC-II (HLA-DR, clone: L243, BD Bioscience, San Jose, CA) expression by flow cytometry and supernatants examined for Epstein-Barr Virus RNA by Polymerase Chain Reaction.