Figure 2

Multiplex IHC reveals the broader immune context of metastatic melanoma. FFPE sections of metastatic melanoma from MelTIL024, s.c. arm metastasis (A) and MelTIL026, pelvic lymph node metastasis (B) were stained by OPAL for the “T-cell Panel” including SOX10 (melanoma marker), PDL1, CD3, CD8, CD4 and FOXP3 and in the “Pan-Immune Panel” SOX10, PDL1, CD3, CD20, CD68 and CD11c. Stained sections were imaged on the Vectra Automated Imaging System and composite HPF images displayed. The T cell Panel showed the distribution of tumour cells (purple), PDL1 (membranous yellow) on either melanoma or infiltrating APCs, CD8+ T cells (green), CD4+ T cells (pink) and T regulatory T cells (orange nucleus). The pan-immune panel showed distribution of tumour cells (purple), PDL1 (membranous yellow) on either melanoma or infiltrating APCs, CD3+ T cells (red), CD20+ B cells (orange), CD68+ macrophages (white cytoplasmic), and CD11c+ likely dendritic cells (green membranous). High-powered fields were selected based on highest TIL density, and cell populations measured using inform v2.2 software. Immune context quantitation from the T cell panel and pan immune panel was recorded and plotted in Graph Pad PRISM as counts per HPF and % of total cells for each resected metastasis. Collated data for all Mel-TIL samples in the study cohort is depicted in (C) including the % of T cells (CD3), B cells (CD20), macrophages (CD68) and CD11c⁺ (likely dendritic cells) in three tumour regions (IT, TM and S) as well as the average of these regions. Statistical analysis (Mann Whitney) was performed across the cohort, significantly different data is represented by *(p < 0.05), **(p < 0.01) and ****(p < 0.0001).