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 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.

OBJECTIVES: 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.

METHODS: 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.

RESULTS: 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.

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.