MAIT cell gating strategy validation, risk factor study and MR1 tetramer staining
We validated the gating strategy of CD3+CD8+CD161+CCR6+live lymphocytes used in Cohort 2 as a comparable measurement of MAIT cells to the “gold-standard” measurement of CD3+CD8+CD161+TCR Vα7.2+ live lymphocytes, in Cohort 3 patients with melanoma at baseline and 35 healthy blood donor controls.
Activation of gut-isolated MAIT cells was confirmed using human MR1/5-OP-RU tetramer provided by the USA National Institute of Health Tetramer Facility (Emory University, USA). Tetramers were generated using BV421-streptavidin (Biolegend) according to the NIH Tetramer Facility instructions. Tetramer staining was performed using a 1/200 dilution in cellular suspension at room temperature for 40min prior to washing with PBS+0.05%BSA+1%EDTA and proceeding with extracellular antibody staining.