Article Extended Data Fig. 5 | Phenotyping of CD8 + T cells in TDLNs. ( a ) Volcano plot of DEGs (P < 0.05 or P < 0.1; detected by whole-transcriptome GeoMx DSP) in CD8 + T cells from the TDLN paracortex comparing disease-free (DF) and recurrence (R) patients with stage III melanoma (n = 5 per group). ( b ) Enrichment of biological pathways among all DEGs ( P < 0.05) upregulated in R versus DF CD8 + T cells. ( c ) Scaled average gene expression levels of selected DEGs (from a ) based on associated with the indicated protein groups or functions. ( d ) Heat map with scaled (Z-score) RNA expression levels for DEGs ( P < 0.05) between TDLN paracortex CD8 + T cells from DF and R patients with stage III melanoma. Columns represent individual patients; associations with published tumour-associated phenotypic signatures are indicated. ( e ) Representative immunofluorescence image of a representative paracortex ROI displaying CD8, CD68 and CD11c-positive cells with LAMP3 and CXCL13 expression marking mRegDCs and activated CD8 + T cells, respectively. GC: germinal centre, F: follicle, IF: interfollicular area, C: capsule. ( f , g ) GSEA testing the enrichment of indicated transcriptional signatures (as detected by whole- transcriptome GeoMx DSP) for CD8 + T cells, comparing paracortex stage III cells from R versus DF patients with melanoma. Dot size indicates the adjusted P value; dot colour indicates normalized enrichment scores (NES). In f , pathways passing statistical significance (i.e. pan-TEX genes UP ) are indicated by a black line; all pathways shown in e are statistically significant. ( h ) Box plots displaying marker expression for different cell types, visualized per ROI (R = 24 & DF = 30 ROIs from 5 or 6 patients respectively; 4–5 ROIs per patient), within the paracortex. Inverse hyperbolic sine-transformed IMC protein levels for TCF1 and TOX are shown in CD8 + T cells, divided by Ki-67 status. Values are displayed for the paracortex (PC) and intratumoral (IT) zone of the TDLN, on a log 2 - transformed y axis (points represent individual ROIs). ( i ) Box plots: normalized PD-1 (left) and TOX (right) protein levels from IMC analysis of intratumoral (IT) CD8 + T cells (DF = 45,859 cells; R = 41,100 cells), comparing DF (n = 6) and R (n = 5) patients with stage III melanoma. Correlation plots (Pearson) denote associations between CXCL13 RNA levels (GeoMx) in CD8 + T cells from the paracortex (PC) and the IT PD-1 (left) or TOX (right) levels (IMC) on CD8 + T cells from the same patients across both outcome groups. P values were calculated using a two-sided Wilcoxon rank-sum test ( h , i ), a LMM ( a , d ), hypergeometric test and the Benjamini–Hochberg (BH) procedure as implemented by Metascape ( b ), or weighted Kolmogorov–Smirnov–like running-sum statistic as implemented by GSEA ( f , g ). ** P < 0.01, **** P < 0.0001 (Exact P values in Supplementary Data).