Extended Data Figure 10: CMTM6 regulates tumour-specific T cell activity by regulating PD-L1 levels.
From: CMTM6 maintains the expression of PD-L1 and regulates anti-tumour immunity
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 (10 μg ml−1). c–g, 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. 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 24 h before incubation with brefeldin A for 6 h and intracellular staining for TNFα. Where indicated, 10 μg ml–1 nivolumab (anti-PD-1 antibody) was added to the co-cultures. Graphs show triplicates (mean + s.e.m.). *P < 0.05, unpaired two-tailed t-test (c–f). 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 10 μg ml−1 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.
