Abstract A006: Tissue resident memory (TRM) T cells and dendritic cells form an in situ archetype for improved response to immune checkpoint therapy in metastatic melanoma
Authors: Di Pietro Andrea , Au Lewis , Crock Patrick , Thio Niko , Pizzolla Angela , Bacac Marina , Schlenker Ramona , Rao Aparna , Spain L , Gyorki David , Sandhu Shahneen , McArthur Grant , Neeson Paul J 
Published: October 2024

INTRODUCTION: Immune checkpoint inhibitors (ICI) in metastatic melanoma results in durable responses in ∼50% of patients. Our understanding of the tumour immune microenvironment (TME) features underpinning ICI response or resistance is currently incomplete. METHODS: To address this issue, we explored the TME of melanoma lymph node metastases (MLN mets) at a spatial (n = 32) and a single cell level (n=24). MLN mets were either from ICI-responder and ICI-resistant patients or untreated controls. We used multiplex IHC and our SPIAT algorithm to examine the spatial immune context of the TME, and also analyzed gene expression by RNAseq. Cells were also isolated from fresh tissue before or after ICI and analyzed by single cell RNAseq and TCRseq to derive high dimension data on T cells and their clonality, all other immune and melanoma cells. Finally, we performed cell-cell interactome and neighborhood analyses to reveal which cell and molecular interactions were associated with response. RESULTS: In patients who responded to ICI, both CD4+ and CD8+ tissue resident memory (TRM) T cell subset density were significantly increased within the tumor, and this correlated with closer distance to melanoma cells. (p&lt;0.05). Single cell data revealed multiple clusters of immune cells (T, B, NK, Mø, DC) and melanoma cells. CD8+ T cell clustering revealed heterogeneous differentiation states. In ICI responders, we observed enrichment for TRM with an earlier transcriptional profile (TCF1+ Tpex.TRM and early TEM.TRM) and lower expression of the exhausted T cell (Texh) gene signature (p&lt;0.05). Hyperexpanded CD8+ T cell clones were present in the TRM cell cluster and were enriched in the responder patients but not in those with resistant disease. CD4+ T cells showed both TRM and TFH clusters with shared expression of multiple genes, with CD4+ TRM also expressed a cytotoxicity gene module. Additionally, Mø and melanoma cells in resistant disease had significantly higher IFNg and IFN-I response gene expression levels compared to tumors from responders (p&lt;0.05), suggesting that chronic activation of T cells in LN mets could lead to the observed Texh phenotype. Interactome analysis displayed putative interactions between CD4+ and CD8+ TRM via the CCL4-CCR5 axis in the ICI responders. This was confirmed by spatial analysis of neighborhood archetypes, which showed significantly increased cell-cell contact between both TRM subsets within the tumor of responders (p&lt;0.05). Finally, conventional dendritic cells interact with both CD8+ and CD4+ TRM subsets in the tumor of responders, providing key signals for maintenance of the TRM pool within the tumor. CONCLUSIONS: MLN mets that respond to ICI are characterized by increased TRM comprising hyperexpanded TCR clones and complex TRM-containing neighborhood archetypes. Whilst these tumors show increased global inflammation, at a single cell level the resistant tumors have increased Texh and greater expression of IFN-regulated genes in melanoma cells and macrophages suggesting ineffective response to chronic antigen stimulation.

Citation Format: Andrea Di Pietro, Lewis Au, Patrick Crock, Niko Thio, Angela Pizzolla, Marina Bacac, Ramona Schlenker, Aparna Rao, L Spain, David Gyorki, Shahneen Sandhu, Grant McArthur, Paul J Neeson. Tissue resident memory (TRM) T cells and dendritic cells form an in situ archetype for improved response to immune checkpoint therapy in metastatic melanoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr A006.