CD8A and melanoma: To further evaluate the T cell composition and spatial location in the TME, we performed multiplex immunofluorescence (mIF) staining (Fig. 2d, e; Supplementary Fig. 2b) on the same baseline tumor resections that were used for sequencing (Fig. 1a, cohort 1 and 2), and found a significant enrichment of FOXP3+ Tregs in AYA compared to adult melanomas (Fig. 2f; median = 110 vs 12.3 cells/mm2, P = 0.023), and a trend of lower CD8+:FOXP3+ T cell ratio in AYA melanomas (Fig. 2g).