Secretome analysis (Olink® Target 48 Cytokine panel) of MAIT cell-derived media after the exposure to IL-33, IL-12p70 or their combination

Project description This study intends to explore, by using the Olink® Target 48 Cytokine panel, the secretome derived from human circulating MAIT cells that were exposed to a combination of different stimuli, including IL-33 and IL-12p70. Sample procession protocol MAIT cells were isolated from PBMCs using a 5-OP-RU-loaded PE-conjugated MR1 tetramer (kindly provided by the National Institutes of Health (NIH) Tetramer Facility) combined with αPE microbeads/ QuadroMACS™ Separator magnets (Miltenyi Biotec), as previously reported (26). 2x105 MAITs were then cultured in a 96 well round (U) bottom plate for 48 hours in the presence of IL-2 (10 ng/mL), IL-7 (10 ng/mL) and combinations of the following reagents: IL-12p70 (50 ng/mL), IL-33 (50 ng/mL). Proteomic characterization of the secretome was performed using media derived from culturing these tetramer-enriched MAIT cells (conditions: un-stimulated, IL-33, IL-12p70 and IL-33+IL-12p70) by Proximity Extension Assay (PEA; Olink®, Sweden). The samples were analyzed with the Olink® Target 48 Cytokine panel including 40 proteins. All samples were revised for quality control, and protein levels are reported as absolute concentration values (pg/mL). Data processing protocol Here we provided 3 different files (3 different running plates) containing raw data of the analysed samples by Olink® Target 48 Cytokine panel. In order to discard potential Hooke effects, each sample was analysed using different dilutions of the original media (non-diluted, 1:20, 1:50 and 1:100). Final concentration values (in pg/mL) was obtained from the mean of the different concentrations applying their respective dilution factor.