Harnessing arginase 2-specific CD8+ T cells to target immunosuppressive cutaneous T-cell lymphoma.

Abstract

Arginase 2 (ARG2) is a metabolic enzyme that reduces local L-arginine levels in the tumour microenvironment, impairing T-cell function and suppressing antitumour 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 healthy donors and individuals with cancer, 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. To determine whether malignant CTCL cells express high levels of ARG2, similarly to Tregs, and whether they can be targeted by ARG2-specific CD8+ T cells as a novel immunotherapeutic strategy. ARG2 expression was analysed in eight CTCL cell lines by Western blotting and in lesions from patients with CTCL using publicly available single-cell RNA sequencing datasets. Immunosuppressive features of the cell lines were evaluated by measuring interleukin (IL)-10 and transforming growth factor (TGF)-β secretion, assessing the expression of immunoregulatory surface proteins and testing their ability to suppress interferon (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 enzyme-linked immunosorbent spot assays. To confirm ARG2-dependent T-cell recognition, we modulated ARG2 expression through overexpression and CRISPR-Cas9-mediated knockdown. ARG2 expression was highly heterogeneous in CTCL cell lines and in malignant T cells from 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. 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.