Optimized In Vitro Expansion of Vδ1+ γδT Cells from Nonhuman Primate Peripheral Blood

Gamma delta (γδ) T cells are a subset of T cells that express MHC-independent γδ T cell receptors (TCRs) and can perform the same T-helper functions as CD4+ cells as well as cytotoxic functions like CD8+ T cells. The MHC-independent nature of γδ TCRs allows them to recognize a larger diversity of antigens, including self and non-self-antigens, making them ideal candidates for immunotherapeutic interventions. Both translational science efforts to better understand the physiology of γδ T cells and clinical research concerning the applications of γδ T cell immunotherapy require successful γδ T cell expansion techniques. Building on prior methods and recent innovations, in this study we optimized in vitro methods for rapid and efficient expansion of Vδ1 + T cells from stimulated PBMCs. CD3-stimulated PBMCs from rhesus macaques in the presence of phospho-vitamin C (pVC) and IL-15 achieved up to a 6561-fold expansion (average 2559-fold) increase of Vδ1 + T cells after 9-day culture. Comparable expansion was obtained with phytohemagglutinin (PHA)-stimulated PBMCs, achieving up to 7574-fold expansion (average 2040-fold) increase when IL-7 and IL-18 were added alongside IL-15 and pVC. Notably, inclusion of pVC significantly enhanced the expansion of Vδ1 + T cells in both stimulation conditions. These results provide optimized conditions for scalable in vitro expansion of peripheral blood Vδ1 + T cells from nonhuman primate models, supporting downstream applications in immunophenotyping, functional assays, and preclinical modeling of γδ T cell–based immunotherapies.