Optimized Multiple Amplification Protocol for the Production of Allogeneic Human Vγ9Vδ2 T Lymphocytes for Adoptive Cell Transfer Immunotherapy

Cancer remains a major therapeutic challenge despite substantial advances in diagnosis and treatment, including immune checkpoint blockade. Among emerging immunotherapeutic approaches, adoptive cell transfer (ACT) has attracted growing interest. Human peripheral Vγ9Vδ2 T cells are promising candidates for ACT because they combine rapid and potent antitumor functions with major histocompatibility complex (MHC)-independent tumor recognition, enabling allogeneic use with limited risk of graft-versus-host disease. This raises the possibility of generating standardized Vγ9Vδ2 T-cell banks from healthy donors for off-the-shelf immunotherapy. Here, we provide preclinical evidence supporting the suitability of allogeneic human Vγ9Vδ2 T cells for ACT. We characterized peripheral blood Vγ9Vδ2 T cells from healthy donors after successive antigen-specific and non-specific amplification steps, assessing their phenotype, effector functions, and metabolic state. Amplified cells maintained a strong pro-inflammatory Th1-like profile, preserved cytotoxic activity, and did not produce immunoregulatory cytokines. They also displayed high purity, a predominant effector memory phenotype, reduced expression of several inhibitory immune checkpoints, and sustained antitumor reactivity. Altogether, these findings support the development of allogeneic Vγ9Vδ2 T-cell products as a scalable platform for next-generation cancer immunotherapies.