Abstract 2283: An immunosuppressive ectoenzyme-expressing T cell population is associated with non-responsiveness to immune checkpoint blockade in metastatic melanoma patients

Abstract The development of antibodies to block immune checkpoints like PD-1, CTLA-4, PD-L1 or LAG-3 has been a major advance in cancer therapy. This has led to the approval of multiple antibodies for metastatic melanoma and other malignancies. A large proportion of the patients do not benefit from CTLA-4 or PD-1 antagonistic antibodies. Understanding the mechanisms of resistance to immunotherapy is thus critical to improve its effectiveness. Previously we identified and validated a novel population of cells characterized by phenotype CD3+CD4+CD127-GARP-CD38+CD39+ that was associated with non-responsiveness to immune checkpoint blockade (ICB) in metastatic melanoma patients (Woods et al, 2020). CD38 and CD39 are ectonucleotidases, that convert intracellular ATP or NADPH to extracellular adenosine which has suppressive effect on T cell functions. We used high-dimension flow cytometry and bulk RNA seq to characterize the phenotype of these cells and evaluated the functions by T cell suppression assays. All characterizations and functional experiments were performed on peripheral blood samples from metastatic melanoma patients undergoing anti-PD1 therapy. The characterization of this population by flow cytomtery shows an enhanced expression of activation markers (4-1BB, HLA-DR, OX-40, GITR), inhibitory receptors (Tim3, CTLA-4, PD-1) and ligands (PD-L1, CD244) and an exhausted phenotype (TOXhiTCF1low). Further functional studies performed on these cells showed a suppressive phenotype, as observed by a significant decrease in the proliferation of target autologous CD4 (p=0.001) and CD8 (p=0.0012) T cells when in co-culture with these cells, compared to the controls. The transcriptomic features of these cells showed an upregulation of genes associated with proliferation (e.g. CDC20, TK1, DHFR, MYL6B), as well as exhaustion (e.g. upregulated TOX), consistent with the flow cytometry data. We also evaluated the presence of these cells and their correlation with the outcome in other melanoma datasets. By performing gene set variation analysis (GSVA) on existing single-cell RNA seq datasets of tumor derived immune cells in melanoma, we have found that an intra-tumoral CD3+CD4+CD127-GARP-CD38+CD39+ signature is associated with resistance to immunotherapy (p= 0.000003). We identified a novel population of ectonucleotidase expressing T cells in peripheral blood and tumor to be associated with immunotherapy resistance in metastatic melanoma patients. This population showed an exhausted phenotype and suppressed autologous T-cells in vitro. Our data identifies CD3+CD4+CD127-GARP-CD38+CD39+ cells as a potential target to enhance the efficacy of immunotherapy in advanced melanoma patients. Citation Format: Ankita Mitra, Brian Thompson, David M. Woods, Jeffrey S. Weber. An immunosuppressive ectoenzyme-expressing T cell population is associated with non-responsiveness to immune checkpoint blockade in metastatic melanoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2283.