A 16-colour spectral flow cytometry panel to characterise T cell immunophenotypes in canine oral melanoma

Advances in immunophenotyping of tumour-infiltrating lymphocytes (TILs) have improved our understanding of prognostic biomarkers and immune targets in human melanoma. However, whether the tumour-immune landscape in canine oral malignant melanoma (COMM) is concordant with that of human melanoma has not been properly defined. To address this gap, we developed a 16-colour spectral flow cytometry panel to characterise TIL phenotypic and functional profiles in COMM. Validation using mitogen-stimulated peripheral blood mononuclear cells from healthy dogs ( n = 5) demonstrated robust identification of major T cell lineages, including regulatory T cells (T _regs ) and memory subsets, and reliable evaluation of their activation and exhaustion status. COMM patients ( n = 8) displayed a distinct protumour microenvironment, characterised by an increased proportion of T _regs , enrichment of tumour-specific exhausted-like T cells co-expressing programmed cell death protein 1 (PD-1) and tumour necrosis factor receptor 2 (TNFR2), and a reduction in cytotoxic CD8 _⁺ and natural killer T (NKT) cell populations compared with tissue-resident ( n = 4) and circulating ( n = 8) lymphocytes. Analysis of additional solid tumours, including a mast cell tumour, nerve sheath tumour and adrenal cortical carcinoma, further supported the capability of the panel to identify similar patterns of immune dysregulation across diverse canine tumour landscapes. Collectively, this work describes the first detailed evaluation of canine TIL immunophenotyping using spectral flow cytometry and provides insights into the immunosuppressive mechanisms shaping the tumour microenvironment in COMM. These findings not only increase our understanding of canine tumour immunology but also identify potential immune targets and support ongoing comparative immuno-oncology efforts.