Easysep human cd8 positive selection kit

Peripheral blood mononuclear cells (PBMCs) were isolated from up to 300 ml whole blood donations using Lymphoprep (Stemcell Technologies) and density gradient centrifugation. CD8 T cells were depleted by positive selection using the EasySep™ Human CD8 Positive Selection Kit (Stemcell Technologies) and EasySep™ magnet (Stemcell Technologies). Depletion of CD8 T cells was confirmed by staining an aliquot of 50,000 CD8 depleted PBMC with anti-CD3-FITC (clone: OKT3), anti-CD4-PE (clone: OKT4) and anti-CD8-APC (clone: HIT8a) fluorescent antibodies and analysis with an Accuri C6 flow cytometer and software (BD Biosciences).
Antigen reactivity of CD8 T cell depleted PBMC to the peptide pools was determined where indicated by IFN-γ ELISpot assay (see below), and cells were subsequently stimulated with the various antigen peptide pools (see below) at 1 µg/ml each peptide. After 48 hours of antigen stimulation, an aliquot of ~ 50,000 CD8 depleted PBMC of each condition was stained for anti-CD4-APC (clone: OKT4) and anti-CD69-PE (clone: FN50) and analyzed using the Accuri C6 flow cytometer and software (BD Biosciences). All antibodies for flow cytometry were obtained from BioLegend.

CD8⁺ T cells were first isolated from peripheral blood of healthy controls or Sarcoidosis patients using EasySep Human CD8 Positive Selection Kit (StemCell Technologies, Canada). The cells were then cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1X penicillin-streptomycin antibiotics (Invitrogen, CA), and stimulated by plate-bound anti-CD3 (5 ng/ml) and anti-CD28 (2 ng/ml) antibodies (both from eBioscience, CA), with or without IL-17A (100 ng/ml) for 3 days.

CD8⁺ T cells were isolated with EasySep Human CD8 Positive Selection Kit (Stem Cell Technologies) from bcTumor or EasySep Human Memory CD8⁺ Enrichment Kit from bcPBMCs. T cells were pre-stained with fluorescent anti-PD-1 antibody immediately before culture setup. The CD19⁺ B cell line C1R:A2 (kindly donated by the laboratory of Jill Slansky) was used at a 1:1 effector/target ratio. In order to target CD8⁺ T cells to this cell line, we added bi-specific CD19-directed CD3 T-cell engager Blincyto (Blinatumomab, Amgen, Thousand Oaks, CA) to cultures at a final concentration of 125 ng/ml. Targets and effectors were cultured at 37 °C for 4 h to allow for cytotoxic T lymphocyte (CTL) effector function. Detection of CD107 mobilization was done as previously described^{61 (link)}. Anti-CD107 antibodies were included at the start of the CTL assay with 7.2 µM of monensin. Change (Δ) in frequency of CD107 mobilizing T cells from co-cultures with and without bi-specific antibodies are reported. For cytotoxicity assays, similar co-culture conditions were utilized but carried out overnight instead. The following day, CD19⁺ target cells were quantified by flow cytometry using Precision Count Beads (Biolegend). Percent cytotoxicity was calculated as (no. of control well target cells − no. of target cells in co-culture)/(no. of control well target cells).