Anti tim 3

For the analysis of MHC molecules expression by SARC-L1, cells were stained with the
following fluorescent conjugated monoclonal antibodies: Anti-2K^b, (clone
AF6-88.5.5.3,); anti-H-2K^dand anti-I^A/I^E(Ebiosciences, France); anti-HLA-A2 (BD Biosciences, France); anti-HLA-DR (Diaclone,
France) or with their respectively isotype control. For tumor infiltrating immune
cells analysis, tumor cells were mechanically dissociated (GentleMax, Miltenyi,
France) tumor infiltrating lymphocytes were separated on a Percoll (Sigma-Aldrich,
France) density gradient. Lymphocytes layer was collected and stained with the
following fluorescent conjugated monoclonal antibodies: anti-CD3, anti-CD4, anti-
CD8, anti-Gr1, anti-PD-L1 (Biolegend, France), anti-CD11b, anti- FoxP₃,
anti-CD25 (eBiosciences, France), anti-PD-1, and anti-TIM-3 (Miltenyi, France),
fixable viability stain (BD, France). The tumor cells fraction was stained
withanti-CD45, anti-PD-L1, anti-HLA-A2 and anti-HLA-DR (BD Biosciences, France) and
fixable viability stain (Biolegend, France). Samples were acquired on a FACS Canto II
(BD Biosciences, France) and analyzed with the BDFacsDIVA or FlowJo software.

For flow cytometry (fluorescence-associated cell sorting, FACS) analysis, PBMCs were washed in staining buffer (SB) (2% fetal bovine serum, FBS, 0,1% sodium azide in phosphate-buffered saline) and, after blocking for 10 min with SB plus Ab serum 20%, were stained for 30 min with the following monoclonal antibodies: anti-CD107a, anti-TIM-3 and anti-PD-L1 (Miltenyi Biotec). Stained cells were washed two times, resuspended in SB, acquired on FACS ACCURI C6 and analyzed using ACCURI C6 software (BD Biosciences).