Anti cd69 percp cy5.5 clone fn50

The cells obtained from the digestion of the biopsies were washed and resuspended in PBS, then collected and transferred into 5 mL tubes for flow cytometry, and then incubated for 15 min in the dark with the Zombie Violet™ fixable viability stain (Biolegend, San Diego, CA, USA). The cells were then washed and resuspended in FACS buffer (PBS, 2% FBS, 2 mM FBS EDTA) to proceed with the labeling with antibodies (and their isotopic controls) for the recognition of surface markers. For immunophenotyping of T lymphocytes and TRM, the cells were labeled with anti-CD3 FITC clone UCHT1, anti-CD103 PE clone Ber-ACT8, anti-CD69 PerCP-Cy5.5 clone FN50 (Biolegend, San Diego, CA, USA), anti-CD8 PE-Cy7 clone HIT8a, anti-CD45 APC-Cy7 clone 2D1. In addition, the following antibodies were used for immunophenotyping NK and γδ T cells: anti-CD56 PE clone B159, anti-CD16 PECy7 clone 3G8, anti-Vδ2 APC clone B6 (Biolegend, San Diego, CA, USA). All the antibodies used, except for anti-CD69 and anti-Vδ2, were obtained from the BD Biosciences Company, (Franklin Lakes, NJ, USA). The samples were acquired using a BD FACSAria flow cytometer at least 50,000 viable cells were acquired for each sample. The gating strategy for the detection of T lymphocytes and TRM T cells is shown in supplemental Figure S1. The data obtained were analyzed using the FlowJo software (BD Biosciences).

Cells were harvested and washed twice with PBS containing 1% BSA (flow cytometry buffer). The following antibody cocktail was used to phenotype moDCs: anti-CCR7-APC (clone G043H7, BioLegend), anti-CD40-BV605 (clone 5C3, BD), anti-CD86-FITC (clone FUN-1, BD Pharmingen), anti-HLA-DR-PE-CY7 (clone G46-6, BD Pharmingen), and anti-HLA-A2-BV421 (clone BB7.2, BioLegend). Cells were also stained with 7-AAD (BioLegend) to discriminate live from dead cells. T cell phenotyping was performed using the following antibodies: anti-CD8-BV421 (clone RPA-T8, BioLegend), anti-CD45RA-PE (clone HI100, BD), anti-CD45RO-APC (clone UCHL1, BD) and MHC dextramer Mel-A-PE or -APC (HLA-A∗0201/ELAGIGILTV, WB2162, Immudex) for CD8⁺ T cells and anti-CD4-FITC (clone OKT4, BioLegend), anti-CD69-PerCP-CY5.5 (clone FN50, BioLegend) and anti-OX40-APC (clone ACT35, BioLegend) for CD4⁺ T cells. T cells were also stained with 7-AAD (BioLegend) to discriminate live from dead cells. Cells were acquired on the LSR Fortessa flow cytometer and analyzed with FlowJo software, version 10.0.