Extended Data Figure 10. CMTM6 regulates tumour-specific T cell activity by regulating PD-L1 levels.
a. Schematic overview of T-cell and tumour co-culture assays to evaluate PD-L1-dependent inhibition of T-cell activity. b. Increased PD-1 expression on NY-ESO-192-100 HLA-CW3 restricted T-lymphocytes following 24 h co-culture with LM-MEL-53 melanoma. Staining with APC-conjugated anti-PD-1 antibody is completely blocked following addition of anti-PD-1 antibody nivolumab (BMS) at a saturating concentration (10μg/ml). c-fg CMTM6 regulates the anti-tumour activity of NY-ESO-1 antigen-specific T lymphocytes c-e. NY-ESO-192-100 HLA-CW3 restricted or NY-ESO-1157-165 HLA-A2 restricted CTLs were incubated with the indicated melanoma cells at an effector:target ratio of 1:1 (c&e) or 1:2 (d) for 24-48 h at 37 °C, then washed off. CTL mediated tumour lysis was determined by MTS assay and normalized to control wells with no CTLs.. Error bars represent SEM (n = 3). * p < 0.05. f&g. Enhanced T-cell activation following co-culture with a CMTM6-depleted cancer cells. f. NY-ESO-192-100 HLA-CW3 restricted T-lymphocytes were cultured with CMTM6 sgRNA, PD-L1 sgRNA or control vector transduced LM-MEL-53 melanoma cells stably expressing Cas9 for 24h prior to incubation with Brefeldin A for 6 h and intracellular staining for TNF-α. Where indicated, 10μg/ml nivolumab (anti-PD-1 antibody) was added to the co-cultures. c-f. Graphs show triplicates (mean + s.e.m.) *P < 0.05, unpaired two-tailed t-test. g. Supernatants from 3 day co-cultures of NY-ESO-192-100 CTLs and control vector or CMTM6 sgRNA transduced LM-MEL-53 cells were analysed by multiplex bead immunoassay (Luminex). Chemokines detected at increased levels following inclusion of 10μg/ml nivolumab (anti-PD-1 antibody) in the co-cultures are shown. Graphs show triplicates (mean + s.e.m). * p < 0.05, unpaired two-tailed t-test with Holm-Sidak correction for multiple testing. h. Efficiency of CMTM6 depletion was assessed by qRT-PCR in B16-OVA cells transduced with shRNA targeting CMTM6 or a non-targeting control shRNA. i&j. Growth of CMTM6-deficient and control shRNA expressing B16-OVA tumours injected subcutaneously in C57BL/6 mice. Data are from two independent experiments. Where indicated, mice were treated with anti-CTLA-4 or isotype control antibody from day 4 after tumour cell injection.