Figure 6: CMTM6 is necessary for and enables an increase of PD-L1 in cells with CD58 loss.
(A) Flow cytometry analysis of surface expression of PD-L1 in 2686 control, CD58 KO, CD58/CMTM6 DKO, and CD58 KO/CMTM6 OE cells after 72 h with 10 ng/mL IFN-ɣ .
(B) Co-IP of PD-L1 and CD58 with CMTM6 pulldown in 2686 WT, CMTM6 KO, and CMTM6 OE cells after 72 h with 10 ng/mL IFN-ɣ.
(C) Co-IP of CMTM6 and PD-L1 with CD58 pulldown in 2686 WT, CD58 KO, CD58-TM, and CD58GPI OE cells after 72 h with 10 ng/mL IFN-ɣ.
(D) Relative gene expression of CD274 in 2686 WT and CD58 KO cells after 72 h with or without 10 ng/ml IFN-ɣ.
(E) Co-IP of CD58 and PD-L1 with CMTM6 pulldown in 2686 WT, CD58 KO, and CD58-TM OE cells after 72 h with 10 ng/mL IFN-ɣ.
(F) Remaining PD-L1 and HLA-A,B,C in 2686 WT, CD58 KO, and CD58-TM OE cells (pre-stimulated with 10 ng/mL IFN-ɣ for 72 h) after 3 and 6 hours of incubation at 37 °C post-staining with fluorophore-conjugated antibodies, in the presence or absence of chloroquine or concanamycin A, as assessed by flow cytometry.
(G) Co-IP of PD-L1–6His and CD58–6His protein with GST-CMTM6 protein from a mixture of 4 μg PD-L1–6His, 1 μg GST-CMTM6, and increasing amounts of CD58–6His purified recombinant proteins.
(H) Surface expression of PD-L1 in 2686 control, CD274 KO, CD274 OE, and CD274H1Amut OE cells after 72 h with 10 ng/mL IFN-ɣ. Counts normalized to mode.
(I) Co-IP of CMTM6 with PD-L1 pulldown in 2686 control, CD274 KO, CD274 OE, and CD274H1Amut OE cells after 72 h 10 ng/mL with IFN-ɣ.
Representative blot shown from two independent experiments (B, C, E, G, I). Experiments performed in duplicate (A, F) or with four technical replicates (D), with independent experiments (A, D) or representative experiment (F) shown. Statistical analysis performed using two-sided T-test (D, F) and one-way ANOVA with Tukey’s multiple comparisons testing (A). Data represent mean ± SD.See also Figure S7.