Anti tcrvα7.2 pe

For QRT-PCR experiments and functional studies, PBMC were stained with anti-TCRVα7.2-PE (Miltenyi Biotec), washed, and incubated with anti-PE microbeads (Miltenyi Biotec). After washing, cells were processed for positive selection with the autoMacs Pro Separator (Miltenyi Biotec) and the positive TCRVα7.2 fraction was collected. These cells were further stained with anti-CD5, anti-CD8, anti-CD45RA, and anti-CD161, and the CD5⁺CD8⁺TCRVα7.2⁺CD45RA^–CD161⁺ MAIT cells and the CD5⁺CD8⁺TCRVα7.2⁺CD45RA⁻CD161⁻ conventional memory CD8 were Facs-Sorted. For microarray analysis, untouched CD8 T cells from 4 healthy donors were purified from fresh PBMC with the CD8 T cell Isolation Kit (Miltenyi Biotec), according to the manufacturer’s instructions. The purified fractions were stained with anti-TCR Vα7.2, CD45RA and CD161, and the TCRVα7.2⁺CD45RA⁻CD161⁺ MAIT cells and the TCRVα7.2⁻CD45RA⁻CD161⁻ memory CD8 T cell fractions were electronically sorted to a purity >98%. Cell sorting was performed with a FacsAria SORP equipped with four lasers (488 nm, 633 nm, 405 nm, and 375 nm) (Becton Dickinson).

For immunophenotyping, cells were stained in PBS supplemented with 0.5% BSA and 2mM EDTA for 15min at 4°C with following fluorochrome-conjugated antibodies: anti-CD69-FITC or anti-CD3-FITC, anti-TCR Vα7.2-PE, anti-CD161-APC (all from Miltenyi Biotec, Germany), anti-CD223(LAG3)-FITC, anti-CD154(CD40L)-FITC (Biolegend, USA). Dead cells were excluded using propidium iodide (Miltenyi Biotec, Germany). MAIT cells were identified based on their TCR Vα7.2 and CD161 expression. Cell surface expression was analyzed on a FACS Calibur (BD Bioscience). Cell sorting was carried out using FACS ARIA II (BD Bioscience). Data were analyzed using FlowJo™ Software Version 10.5.3 (Becton, Dickinson and Company).