Anti-tumour Activity of Canine Activated T-Cells and Cytokine-Induced Killer Cells against Canine Malignant Melanoma In Vitro

: Adoptive cell transfer (ACT) immunotherapy represents a promising therapeutic approach for cancer treatment, utilising ex vivo-expanded immune effector cells such as T-lymphocytes, natural killer (NK) cells, and cytokine-induced killer (CIK) cells. CIK cells are characterised by dual T-cell and NK cell-like properties, exhibiting high proliferative capacity, cytolytic activity, and non-MHC-restricted tumour recognition. Canine malignant melanoma (CMM) is an aggressive neoplasm with significant translational relevance to human melanoma, yet the cytotoxic potential of CIK cells against CMM remains unexplored. This study aimed to expand and functionally characterise canine CIK and T-cells from healthy donors, comparing their cytotoxic activity against CMM cell lines in allogeneic and autologous settings. Peripheral blood mononuclear cells were isolated and expanded using standardised protocols with IFN-γ, anti-CD3/CD28 activation, and IL-2 supplementation. Flow cytometry confirmed successful expansion with CIK cells showing 85% NKp46 expression and T-cells reaching 91.96% CD8 expression, with comparable expansion folds (7.94 vs. 7.88). Cytotoxicity assays against seven CMM cell lines demonstrated robust anti-tumour activity for both effector populations, with CIK cells achieving 61.5% mean killing and T-cells 59.8% in allogeneic assays at 10:1 effector-to-target ratios. Pearson's correlation analysis revealed strong negative correlations between effector cell numbers and target viability (r = -0.960 for CIK cells; r = -0.790 for T cells). Cytokine profiling showed distinct secretion patterns, with T-cells producing a broader cytokine repertoire. These findings establish the feasibility and comparable efficacy of both CIK cells and T-cells against CMM, supporting their therapeutic development for canine melanoma immunotherapy.