NK cells and regulatory T cells (Tregs) phenotype analysis
cancer
CANCER, COLD, APC
Analysis of the expression of cell-surface and intracellular proteins in peripheral blood mononuclear cells (PBMCs) from cancer patients was performed by flow cytometry to evaluate NK cells and Tregs phenotype. PBMCs from cancer patients were utilized under the appropriate NCI Institutional Review Board approval (protocol code NCT03476681, first approved 03/26/2018; latest update 01/08/2020). To isolate PBMCs, blood from cancer patients was collected in Heparin Green Top tubes.For NK phenotype analysis, blood samples were drawn prior to the first infusion dose on C1D1, 72 h post EOI, 14 days post EOI and prior to C3D1. For Tregs phenotype analysis blood samples were drawn prior to the first infusion dose on C1D1, 14 days post EOI and prior to C3D1. Isolated PBMCs were cryopreserved in cryovials containing 95% human AB serum + 10% DMSO in liquid nitrogen. The day of the flow cytometry PBMCs were thawed and stained with primary anti-human mAbs in 1X PBS + 1% BSA (Teknova, Hollister, CA, USA) for 30 min at 4 °C.To detect the NK surface markers PBMCs were labeled with following antibodies: CD56 PE (clone 5.1H11), CD16 PerCP-Cy5.5 (clone 3G8), NKG2D BV421 (clone 1D11), NKp46 FITC (clone 9E2) (BioLegend, San Diego, CA, USA), CD107a APC-H7 (clone H4A3) (BD Biosciences, San Jose, CA, USA), CEACAM-1 APC (clone 283340) (VWR, Radnor, PA, USA). To detect surface Tregs markers and to evaluate the reactivity of NEO-201 to human Tregs, PBMCs were labeled with the following anti-human mAbs: CD4 FITC (clone OKT4), CD127 APC (clone A019D5), CD15s PE (clone FH6), NEO-201 Pacific Blue (BioLegend, San Diego, CA, USA), CD25 APC-H7 (clone M-A251) (BD Biosciences, San Jose, CA, USA).For Tregs analysis, after staining surface markers, cells were permeabilized with Fix/Perm Solution (eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set, Thermo Fisher Scientific) for 1 h at 4 °C to allow detection of intracellular transcription factors, and then stained in 100 uL of 1X Permeabilization Buffer for 1 h at room temperature in the dark with 2-4µL/sample of the anti-human Foxp3 PerCP-Cy5.5 mAb (clone 236A/E7, BD Biosciences, San Jose, CA, USA).After staining, cells were washed twice with cold 1X PBS and examined using a FACSVerse flow cytometer (BD Biosciences, San Jose, CA, USA). Analysis of cellular fluorescence was performed using BD FACSuite software (BD Biosciences, San Jose, CA, USA) and FlowJo 10.8.1. Positivity was determined by using fluorescence-minus-one controls.Complete staining procedure is reported in Supplementary materials and methods.