Within the spectrum of T-cell populations, tissue-resident memory T-cells (TRM) have emerged as key players in the immune control of melanoma, and their abundance is associated with a more favourable prognosis in response to immune checkpoint inhibitor (ICI) treatment. However, approximately half of the patients develop resistance to the treatment and, therefore, it is imperative to assess the role of TRM in this subgroup to refine therapeutic strategies. In this context, we hypothesised that TRM at the tumour site show features of exhaustion, resulting in the loss of a functional response and contributing to melanoma immune escape.
To characterise features associated with resistance, we conducted a comprehensive immune-profiling of T-cells in fresh surgical or formalin-fixed paraffin-embedded samples of lymph node metastases obtained from untreated, ICI-resistant, and ICI-responder patients with stage III/IV melanoma. Responder patients exhibited a higher expression of genes associated with a functional immune response. A curated gene module to analyse the TRM signature was applied among the patient groups, revealing a significantly higher z-score in ICI-responders. This was validated by multiplex immunofluorescence analysis which demonstrated an enrichment of both precursors (CD103+CD69+TCF1+) and terminally differentiated (CD103+CD69+TCF1-) CD8+ and CD4+ TRM within the tumour areas in the responder group, however, their proportion was significantly decreased in the resistant group.
While we validated the correlation between TRM abundance and the response to ICI, we further conducted single-cell RNA-Seq within the same patient cohort to assess whether CD8+ TRM exist in various differentiation states and functional polarisations based on the treatment status. Importantly, we observed that TRM were exhausted in the ICI-resistant tumours while effector/Tpex TRM were increased in ICI-responsive metastatic melanoma, displaying features of those seen in long term non-progressors on anti-PD1 treatment including expression of IFN-g and TNF. In addition, hyper-expanded TCR clones were enriched in CD8+ TRM clusters and were associated with ICI-responder and untreated groups, inferring an efficient response towards cancer cells. Interactome analyses were then performed to understand which cells could potentially support CD8+ TRM functions. We observed that CD8+ TRM interact with CD4+ TRM in the responder group, with the latter also showing cytotoxic features.
In conclusion, our study suggests that a pro-inflammatory microenvironment and specific cell-to-cell interactions play a crucial role in establishing functional TRM responses, contributing to a positive clinical outcome following ICI treatment. Conversely, the absence or diminished tumour responses by exhausted TRM were associated with ICI resistance or immune escape.