Fig 1: TSPAN4 impacts PD‐1 binding and T‐cell activation. (A, B) Cells were incubated with recombinant PD‐1 at different concentrations, and PD‐1 binding was measured by flow cytometry. Percentage PD‐1+ cells after recombinant PD‐1 treatment in BLM (A) or Mel624 (B) TSPAN4 KD or NT cells. Data are from n = 3 biological replicates shown as mean ± SEM. Significance was determined by ordinary two‐way ANOVA with Šidák's multiple comparison's test (panel A: ns, P > 0.9999, ns, P = 0.4303, *P = 0.0355, ***P = 0.0002, ***P = 0.0001, panel B: ns, P > 0.9999, *P = 0.030, ****P < 0.0001). (C) Schematic of Jurkat coculture assay. Melanoma cell lines are gp100 peptide‐pulsed and treated with or without durvalumab, and co‐cultured with Jurkat T cells. After overnight incubation, supernatant is taken for ELISA. Created in BioRender. Franken, G. (2025) https://BioRender.com/wi89t1a. (D, G) IL‐2 ELISA experiment results from Jurkat T‐cell coculture assay with BLM (D) or Mel624 (G) TSPAN4 KD and NT cells, representative of n = 4 biological replicates. Dots are from n = 3 technical replicates and are shown as mean ± SD. Significance was determined by ordinary one‐way ANOVA with Tukey's multiple comparisons test (panel D: *P = 0.0476, **P = 0.0034, panel G: ns, P = 0.1828, *P = 0.0328). (E, H) IL‐2 fold change of durvalumab‐treated over isotype‐treated samples in TSPAN4 KD and NT BLM (E) or Mel624 (H) cells. Dots are from n = 4 biological replicates and conditions belonging to the same experiment are connected with a line. Significance was determined by paired one‐tailed Student's t‐test (panel E: *P = 0.0385, panel H: *P = 0.0275). (F, I) IL‐2 fold change of durvalumab‐treated over isotype‐treated samples in TSPAN4 KD and NT BLM (F) or Mel624 (I) cells, normalized to the NT condition. Dots are from n = 4 biological replicates, and data is shown as mean ± SEM. Significance was determined by one sample t‐test (panel F: *P = 0.0313, panel I: *P = 0.0335). (J, K) Overall survival in patients with skin cutaneous melanoma with high or low mRNA expression levels of PD‐L1 (J) and TSPAN4 (K).