Background: Tumour-resident (TR) T cells are central to melanoma immunosurveillance, and their intratumoral abundance correlates with improved outcomes following immune checkpoint inhibitor (ICI) therapy. However, their functional contribution to ICI response, including the impact of cellular cross-talk and spatial-organisation within the tumour microenvironment (TME), is incompletely defined. Concurrently, chronic inflammatory signalling promotes melanoma dedifferentiation and immune evasion, yet how tumour-intrinsic plasticity interacts with immune architecture to drive response or resistance is unclear.
Methods: Lymph node metastases from untreated and ICI-treated melanoma patients (n = 42) were analysed using single-cell RNA and ATAC sequencing, multiplex immunohistochemistry, bulk RNA sequencing, and spatial transcriptomics (VisiumHD). Multi-modal datasets were integrated using R-based computational frameworks.
Results: ICI-responsive tumours exhibited a pro-inflammatory transcriptional landscape with enrichment of TR cell signatures. Multiplex imaging confirmed accumulation of both precursor and terminally differentiated CD8⁺ and CD4⁺TR cells within the TME of responders, whereas these populations were reduced in resistant cases. Single-cell analyses identified distinct TR differentiation states, with effector and cytotoxic phenotypes enriched in responders and terminally exhausted CD8⁺ TR cells were characteristic of ICI-resistance. TR cells in responders expressed cytotoxic programs (IFNG, GZMB, NKG7) and demonstrated clonal expansion, with functional activity validated in co-culture assays.
Spatial and interactome analyses revealed a responder-specific immune triad comprising DC3 dendritic cells and CD8⁺and CD4⁺ TR cells, co-localised with melanoma cells. This organised immune niche was absent in resistant tumours. Instead, resistant melanoma cells exhibited increased type I interferon and IFNG signalling, activation of IL6/JAK/STAT3 pathways, and a neural crest-like dedifferentiated state, confirmed by spatial transcriptomics.
Gene regulatory network analysis integrating transcriptomic and chromatin accessibility data showed reduced activity of multiple differentiation-associated regulators such as KLF4, suggesting extensive rewiring in resistant melanoma cells. CRISPR/Cas9 perturbation of KLF4-regulated target genes significantly restored melanocytic differentiation programs and increased MHC-I/MHC-II expression, suggesting enhanced tumour immunogenicity with potential re-sensitisation to ICI therapy.
Conclusion: ICI response is associated with TR cell-centred immune triads and preserved tumour differentiation, whereas resistance is defined by immune disorganisation and tumour plasticity driven by rewired regulatory networks. Targeting these transcriptional programs represents can restore tumour immunogenicity and potentially overcome ICI-resistance.