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.