Fig. 6

TIGIT-28 can help mitigating T cell hypofunction. a Schematic representation of the hypofunction induction assay following repetitive antigen exposure developed herein. Initially, 10⁵ cancer cells were seeded in a 6-well plate and 4 h later 10⁶ transduced T cells were added (day = 0). 2 days afterwards, previously co-cultured T-cells were transferred to a new plate, previously seeded with 10⁵ new cancer cells. This process was repeated 4 times, totaling 8 days of co-culture. On day 8, these T-cells were used in an additional co-culture for cytokine secretion assessment. b-c TIGIT-28/F4 or ctrl/F4 transduced T cells were conditioned in co-culture with 888A2 (antigen positive) or HeLa (antigen negative - control) for 8 days as described above. On day 9, these T cells were co-cultured with either 888A2/155 or SK-MEL23/155. b IFNγ or c TNF-α secreted in the co-culture supernatant was measured by ELISA. Cytokine concentrations were normalized to the secretion observed in the positive control Ctrl/F4 group exposed to antigen negative tumor cells (HeLa) for 8 days (with an average secretion of IFNγ of 2530 pg/ml for 888A2, 1903 pg/ml for SK-MEL23 and with an average secretion of TNF-α of 2475 pg/ml for 888A2, 1170 pg/ml for SK-MEL23). These results are representative of three independent experiments, performed with at least two different donors (*: p < 0.05, calculated using a Student’s paired t-test). d The surface expression of CD226 (DNAM), an antagonist to TIGIT, was analyzed on TIGIT-28/F4 or Ctrl/F4 transduced T cells that were co-cultured with 888A2/155 (Ag⁺ target) or HeLa (Ag⁻ target) cells for several days. On day 2 and 5 (after the beginning of the conditioning co-culture), these cells were stained with anti-CD226 and analyzed by flow cytometry. These results are presented as the MFI average of three independent experiments, performed with at least two different donors (*: p < 0.05, calculated using a Student’s paired t-test)