Figure 7. LN Trm cells are found in melanoma-involved lymph nodes in humans, where their transcriptional signature portends improved survival.

(A) Immunohistochemistry on patient melanoma-involved sentinel lymph nodes, stained with anti-CD8 (green) and anti-CD69 (red), with co-stained cells (black; arrows) detected throughout regions. Image of LN is from a single patient, but with similar cells identified in a total of n=4 patients. (B) Immunofluorescence of metastatic melanoma patient tumor-involved LN, with CD8⁺CD103⁺CD69⁺ triple-stained cells appearing white. Image is representative of specimens from n=2 patients analyzed. (C-F) ScRNAseq and paired TCR seq of 501 CD8⁺ T cells FACS sorted from a tumor-invaded LN of a melanoma patient with oligometastatic disease. (C) tSNE clustering, revealing four distinct transcriptional clusters. (D) Representative expression of T_RM-associated genes, red circle highlights the Hu_LN1 cluster; each dot corresponds to a single cell. (E) Average Z-transformed normalized expression of representative Trm and Tcirm genes across the clusters. (F) tSNE projection of clusters indicating (in color) individual cells comprising the five most highly expanded TCR clonotypes. (G) Schematic diagram depicting workflow for generating memory T cell subset transcriptional signatures from mouse scRNAseq dataset, and applying signatures to melanoma patient specimen data from The Cancer Genome Atlas. (C) Kaplan-Meier plots indicating the prognostic value of enrichment of the indicated single cell-derived binary memory T cell signatures (skin Trm, lung Tcirm, and LN Trm) in all TCGA melanoma specimens (top, n=431), or regional lymph node metastases only (bottom; n=213); patients were stratified into high and low groups based on the mean. (I) Forest plot showing the hazard ratios estimated based on multivariate Cox proportional hazards regression analysis of TCGA metastatic melanoma specimens (n=345).