Background: Tumour-resident (TR) T cells play a critical role in melanoma immunosurveillance, and increased intratumoral TR cell abundance is associated with improved clinical outcomes following immune checkpoint inhibitor (ICI) therapy. Despite this association, the functional contribution of TR cells to ICI responsiveness and the mechanisms governing their spatial organisation within tumours remain incompletely understood. In parallel, chronic inflammatory signalling drives melanoma dedifferentiation, promoting immune evasion and resistance to ICI therapy. How tumour-intrinsic plasticity intersects with immune cells and organisation within the tumour microenvironment (TME) to determine ICI response or resistance remains undefined.
Methods: Lymph node (LN) melanoma metastases from untreated and ICI-treated patients (n = 42) were profiled using single-cell multi-omic sequencing, multiplex immunohistochemistry (mIHC), bulk RNA-sequencing, and spatial transcriptomics (VisiumHD). Resulting high-dimensional datasets were integrated and analysed using R-based computational pipelines.
Results: ICI-responsive tumours exhibited a pro-inflammatory transcriptional landscape with enrichment of TR cell signatures. mIHC confirmed accumulation of both precursor and terminally differentiated CD8⁺ and CD4⁺ TR cells in the TME of responsive tumours, whereas depleted in resistant cases. Single-cell analyses revealed TR differentiation states, with effector and cytotoxic phenotypes enriched in responders and terminally exhausted CD8⁺ TR cells predominating in resistant tumours. In responders, CD8⁺ and CD4⁺ TR cells displayed cytotoxic programs characterised by IFNG, GZMB, and NKG7. CD8⁺ TR cells also showed clonal expansion compared to resistant patients. Functional cytotoxicity of both CD4⁺ and CD8⁺ TR cells was confirmed by co-culture with autologous melanoma cells.
Spatial and interactome analyses identified a responder-specific immune triad comprising DC3 dendritic cells, CD8⁺ and CD4⁺ TR cells, spatially co-localised with melanoma cells. In contrast, ICB-resistant lymph node metastases exhibited loss of these immune triads. Resistant melanoma cells also displayed increased type I interferon and IFNG responses, activation of IL6/JAK/STAT3 signalling pathway, and a de-differentiated neural crest-like transcriptional state, with spatial transcriptomics confirming enrichment of IFNG response and neural crest gene signatures.
Gene regulatory network analysis integrating single-cell transcriptomic and chromatin accessibility data revealed extensive rewiring in resistant melanoma cells, including downregulation of differentiation-repressing transcription factors such as KLF4. In silico perturbation of these networks predicted phenotypic reversion of de-differentiated melanoma states.
Conclusion: ICI response in melanoma LN metastases is associated with TR cell-centred immune triads and preserved tumour differentiation, whereas resistance is marked by immune exclusion and melanoma de-differentiation driven by rewired regulatory networks. These findings identify actionable transcriptional targets to restore tumour immunogenicity in ICI-resistant melanoma.