Fig. 6: Melanoma DCCs suppress T cells through CD155 and CD276.
a, WB for various ICLs in MelDCC lines and their respective 100K pellets. b, Expression of ICLs in DCCs separated according to their phenotype (scRNA-seq data of DCCs; n = 164 cells). c, Flow cytometric analysis of CD8 T cell proliferation on day 4 after anti-CD3/CD28 stimulation and addition of PBS (n = 5 technical replicates) or MelDCC 10a sEVs in the presence or absence of 10 µg of anti-human TIGIT antibody or human IgG1 isotype control (all n = 6 technical replicates). The bar indicates the median. d, WB for CD155 and CD276 expression in MelDCC 10a controls or CD155;CD276 single or double knockout and their respective 100K pellets. KO, knockout; gRNA, guide RNA. e, Flow cytometric analysis of CD8 T cell proliferation and percentage of IFNG+ and GZMB+ CD8 T cells on day 4 after anti-CD3/CD28 stimulation and addition of PBS or sEVs of MelDCC 10a (wild-type sEVs) and MelDCC 10a with CD155;CD276 single or double knockout 18 h before anti-CD3/CD28 stimulation. Experiments with CD155 or CD276 single-knockout or CD155;CD276 double-knockout sEVs were performed independently with n = 11–12 technical replicates per condition and results were normalized to their respective PBS controls to enable a pooled comparative analysis. f,g, Flow cytometric analysis of CD226:TIGIT (f) and TNF (g) expression in CD8 T cells from LNs of patients with melanoma (n = 69 LNs) and patients without melanoma (n = 6 LNs). Mean fluorescence intensity of marker expression by CD8 T cells as a function of patient DCCD. The red line provides the LOESS regression where log10(DCCD + 1) is entered as a continuous variable (gray area, 95% CI; values for patients with and without melanoma are presented as red and gray dots). h, Synopsis of DCC phenotype switching during metastatic colony formation in human melanoma. Acquisition of the NC-like phenotype enables DCCs to suppress early CD8 T cell attack by immunosuppressive sEVs. M, melanocytic; U, undifferentiated; ET and LT, early and late transitory phenotypes. P values in f,g were determined using Pearson’s correlation (two-sided). Boxes represent the median, lower quartile and upper quartile, with individual data points illustrating the data distribution.
