Fig. 5: IFNG-induced acquisition of the NC-like phenotype enhances secretion of T cell-suppressive sEVs by melanoma DCCs. | Nature Cancer

Fig. 5: IFNG-induced acquisition of the NC-like phenotype enhances secretion of T cell-suppressive sEVs by melanoma DCCs.

From: MCSP+ metastasis founder cells activate immunosuppression early in human melanoma metastatic colonization

Fig. 5

a, Flow cytometric analysis of MelDCC 5a, 6, 10a and 11 for Melan A and NGFR expression before (day 0) and after 4 weeks of IFNG treatment (day 28) or after 4 weeks of IFNG treatment followed by 48 h in IFNG-free EV production medium (day 30). b, Fold change in the number of sEVs secreted per million MelDCCs over 48 h, either untreated or treated with IFNG for 28 days, followed by 48 h in IFNG-free EV production medium (n = 6 technical replicates each). c, WB analysis of EV pellets (2K, 10K and 100K) isolated from MelDCC 10a, using antibodies for small (CD81 and TSG101) and large (GRP94) EV markers or the pan-EV marker HSP70. L, whole-cell lysate. d, TEM of 100K preparations. Left, negative staining; right, freeze-etching. e,f, Flow cytometric analysis of proliferation and effector cytokine production (IFNG and GZMB) in polyclonal (e) or MART127L26-35-specific (f) CD8 T cells exposed to sEV from MelDCC 10a or PBS for 18 h before (−18 h) or after anti-CD3/CD28 stimulation (+18 h) (n = 6 technical replicates each). g, Cytotoxic activity of MART127L26-35-specific CD8 T cells exposed or not to sEVs from MelDCC 10a 18 h before anti-CD3/CD28 stimulation. CD8 T cells were harvested on day 4 and added at an effector-to-target ratio of 1:1 to MART127L26-35-loaded CFSE-labeled T2 cells. Nonloaded, CellTrace violet-labeled T2 cells served as nontarget controls (n = 6 technical replicates each). h, Flow cytometric analysis of Ki67+ CD8 T cells in human PCLSs (n = 4 patients) 5 days after anti-CD3/CD28 stimulation and exposure to high or low doses of sEVs (sEVs produced by 6.25 × 106 or 1.25 × 106 MelDCC 10a cells) or 5 µM pimecrolimus. Nonstimulated PCLSs served as the control. sEVs or pimecrolimus was added 18 h after stimulation. Shown is the median (line) with range (whiskers) delineating the minimum and maximum values. P values in h were determined using a one-way ANOVA with Dunnett’s post hoc multiple-comparison test (two-sided). Boxes represent the median, lower quartile and upper quartile, with individual data points illustrating the data distribution.

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