Ccr6 pe

For Western blot and quantitative real-time PCR (qRT-PCR) analyses, untouched T cells were separated using the Pan T Cell isolation Kit II (Miltenyi Biotec, Bergisch-Gladbach, Germany). The purity of recovered cells, assessed by flow cytometer, was ≥97%.
For apoptosis and ERβ analyses of Th17 cells (i.e., CD4⁺CD45RA⁻CCR6⁺CXCR3⁻), CD4⁺ T cells were separated from PBMC by positive selection using CD4 MicroBeads (Miltenyi Biotec), with a purity ≥97%, as determined by flow cytometer. Then, CD4⁺CD45RA⁻CCR6⁺CXCR3⁻ T cell subset was sorted by FACS (BD FACSAria; BD Biosciences) upon staining with the following mixture of mAb: CD4 PE/Cy7 (BD Biosciences), CD45RA FITC (BD Biosciences), CCR6 PE (Miltenyi Biotec), and CXCR3 APC (BD Biosciences). Sorted T cell subset was on average >95% pure as determined by postsorting flow cytometry analysis.

MLs phenotypes from patients’ samples of the ELYP cohort at different timepoints were performed using CD103-FITC, CD73-PE, CTLA4-APC, CD39-APC-Vio770, CCR6-PE, PD1-PE-Vio770, NKG2D-APC, CD45RO-APC-Vio770 (Miltenyi), CD3-AF700, CD161-PE-Cy5, CD8-BV605 and CD4-BV711 (BD Biosciences), . MLs phenotypes from patients’ samples of the REMIND and IMCO cohort at different timepoints were performed using CD69-FITC, CD45RO-PerCP, CD5-AF700, CD8-BV605, CD4-BV711 (BD Biosciences), NKG2D-APC and CD103-APC-Vio770 (Miltenyi). All samples were co-stained with DAPI to assess cell viability and diluted in optimal concentration in FACS buffer (Miltenyi).
For the allogenic cocultures, cells were then stained using the following antibodies: Annexin V-FITC, E-Cadherin-PE-Vio770 HLA-E-APC, MICA/MICB-APC-Vio770 (Miltenyi), HLA-ABC-AF700 (BioLegend), HLA-DPDQDR-BV510, CD45-BV605 (BD Biosciences) and ULBP 2/5/6-PE (RD). All samples were co-stained with DAPI to assess cell viability and diluted in optimal concentration in Annexin V binding buffer (Miltenyi).