Pe conjugated anti ki67

Tumor cells were resuspended in 100 μl staining buffer after blocking Fc receptors, then incubated with APC-conjugated anti-B7-H4 (cloneMIH43, #358,108) or isotype control (BioLegend, CA, USA) for 30 min. After washing and centrifuging in staining buffer, cells were fixed and permeabilized, and then performed intracellular staining of PE-conjugated anti-Ki67 (BioLegend, CA, USA). After staining, cells were evaluated using BD FACS Verse and analyzed using FlowJo 10 (BD Biosciences, CA, USA).

BrdU (Roche, France) was added at the bottom of the well (transwell experiments), or to the co-culture medium for 18 h. In co-culture experiments, MAIT cells were then carefully aspirated, and adherent HMF were washed once with 1× PBS and their DNA synthesis was estimated by a colorimetric BrdU ELISA test (Roche, France) as per the manufacturer’s instruction. For the experiments with MR1-blocking antibodies, HMFs were incubated with purified anti-MR1 antibody at 20 μg/ml (26.5, BioLegend, France) or with isotype control antibody for 2 h at 37 °C. Pre-activated or non-activated MAIT cells were washed and co-cultured with anti-MR1 (26.5, BioLegend)-exposed HMF. Cells were then processed for DNA synthesis as described above. In separate experiments, FACS analysis of Ki-67 was performed in HMF upon co-culture with MAIT cells, using phycoerythrin (PE)-conjugated anti-Ki-67 (BioLegend, France) by intracellular staining as per the manufacturer’s instructions.