926 Comprehensive single-cell immune profiling of tumor-infiltrating lymphocytes in acral melanoma

Background Acral melanoma is the most prevalent melanoma subtype among non-Caucasians and its distinct characteristics make it less responsive than cutaneous melanoma to immunotherapy. Whereas patients with cutaneous melanoma often respond to immune checkpoint inhibitors (ICI), current ICIs have shown limited efficacy against acral melanoma. To enhance immunotherapy effectiveness, numerous new therapeutic agents, often in combination with existing drugs, are being explored. However, selecting appropriate agents requires a deeper understanding of the tumor immune microenvironment. Yet, the characteristics of tumor-infiltrating T lymphocytes (TILs) in acral melanoma remain poorly elucidated, impeding the development of effective therapeutic strategies. Methods We isolated CD3+CD45+ TILs from five treatment-naïve primary acral melanoma patients and five treatment-naïve primary cutaneous melanoma patients and subjected them to single-cell RNA sequencing coupled with single-cell TCR sequencing analysis. Additionally, we evaluated the tumor reactivity of expanded CD8 clonotypes in vitro (figure 1). Results We obtained transcriptomes from 38,333 T cells, clustering CD4 and CD8 TILs into 15 and 18 subsets, respectively. Testing 23 clonally expanded CD8 TCRs against autologous and acral melanoma cell lines identified 13 tumor-reactive CD8 TCRs. Furthermore, assessment of eight relatively expanded CD4 Treg TCRs revealed four with tumor reactivity. Compared to cutaneous melanoma, acral melanoma exhibited lower frequencies of tumor-reactive CD8 clusters and an enrichment of regulatory T cells. Tumor-reactive CD8 TILs displayed heterogeneous expression of co-inhibitory molecules. Notably, resident memory phenotype cells selectively expressed KLRC1 (NKG2A), which negatively regulates CD8 T cell activation. NKG2Ahigh PD-1high TIL clone cells demonstrated increased degranulation and IFNγ secretion upon NKG2A or PD-1 blockade, with a more pronounced effect observed with combined NKG2A and PD-1 blockade. Survival analysis of 38 melanoma patients revealed NKG2A expression correlated with improved overall survival, indicative of tumor-reactive TIL presence. However, this survival benefit was absent in patients with high tumor HLA-E expression (the sole NKG2A ligand). Conclusions Our results reveal differences in immune cell composition between acral melanoma and cutaneous melanoma, with acral melanoma containing lower frequencies of tumor-reactive CD8 TIL and an enrichment of regulatory T cells. Further, expression of NKG2A in acral melanoma TILs was indicative of tumor-reactivity and correlated with improved overall survival, except in patients whose tumors displayed high HLA-E expression, suggesting potential therapeutic implications. These distinctions in the tumor immune microenvironment between acral and cutaneous melanoma likely impact immunotherapy efficacy and overall patient prognosis. Therefore, our insights will open avenues for the development of more refined immunotherapy approaches in acral melanoma. Acknowledgements I would like to express my sincere gratitude to Matthew M Gubin for his invaluable feedback, which greatly contributed to the refinement of this poster presentation. Ethics Approval This study was performed with the approval of the Institutional Review Board (282–224) of Sapporo Medical University (Sapporo, Japan) and in accordance with the Declaration of Helsinki. All patients provided written informed consent for the collection of tissue and blood samples for research.