Harnessing Arginase-2-specific CD8+ T cells to target immunosuppressive Cutaneous T cell Lymphoma

Background Arginase-2 (ARG2) is a metabolic enzyme that reduces local L-arginine levels in the tumor microenvironment, impairing T-cell function and suppressing antitumor immunity. We previously identified proinflammatory CD8+ T cells that recognize an ARG2-derived peptide presented by HLA-B8. These ARG2-specific CD8+ T cells, found in both healthy donors and cancer patients, selectively targeted autologous regulatory T cells (Tregs) and cancer cells with high ARG2 expression. In advanced cutaneous T-cell lymphoma (CTCL), malignant T cells have been reported to adopt immunosuppressive features resembling those of Tregs. Objectives To determine whether malignant CTCL cells express high levels of ARG2, similar to Tregs, and whether they can be targeted by ARG2-specific CD8⁺ T cells as a novel immunotherapeutic strategy. Methods ARG2 expression was analyzed in eight CTCL cell lines by western blotting and in CTCL patient lesions using publicly available single-cell RNA sequencing datasets. Immunosuppressive features of the cell lines were evaluated by measuring IL-10 and TGF-β secretion, assessing the expression of immunoregulatory surface proteins, and testing their ability to suppress IFN-γ production in activated T cells. The SeAx cell line was transfected with an HLA-B8-encoding plasmid and used as target cells for ARG2-specific CD8⁺ T cells in IFN-γ ELISPOT assays. To confirm ARG2-dependent T-cell recognition, we modulated ARG2 expression through overexpression and CRISPR-Cas9-mediated knockdown. Results ARG2 expression was highly heterogeneous across both CTCL cell lines and malignant T cells from CTCL patient lesions. The CTCL cell lines also exhibited diverse immunosuppressive features, including IL-10 and TGF-β secretion, and suppressed IFN-γ production by activated T cells. SeAx cells displayed moderate ARG2 levels and were selected as a model to assess targeted recognition of CTCL cells. Following HLA-B8 transfection, ARG2-specific CD8+ T cells from several donors recognized and responded to SeAx cells, as demonstrated by increased IFN-γ secretion. Recognition required both ARG2 and HLA-B8, as responses were enhanced by ARG2 overexpression, diminished by CRISPR-Cas9-mediated knockdown, and absent in mock-transfected controls. Conclusions This study provides the first evidence that malignant CTCL cells can be targeted by ARG2-specific CD8⁺ T cells, highlighting ARG2 as a promising immunotherapeutic target in CTCL.