Batada, N. | bioRxiv Science

bioRxiv2025

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

Gamma delta ({gamma}{delta}) T cells are a subset of T cells that express MHC-independent {gamma}{delta} 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 {gamma}{delta} 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 {gamma}{delta} T cells and clinical research concerning the applications of {gamma}{delta} T cell immunotherapy require successful {gamma}{delta} 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 V{delta}1+ 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 V{delta}1+ 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 V{delta}1+ T cells in both stimulation conditions. These results provide optimized conditions for scalable in vitro expansion of peripheral blood V{delta}1+ T cells from nonhuman primate models, supporting downstream applications in immunophenotyping, functional assays, and preclinical modeling of {gamma}{delta} T cell-based immunotherapies.

cell biology↗

bioRxiv2021

Single-cell analysis uncovers differential regulation of lung γδ T cell subsets by the co-inhibitory molecules, PD-1 and TIM-3

IL-17A-producing {gamma}{delta} T cells within the lung consist of both V{gamma}6+ tissue-resident cells and V{gamma}4+ circulating cells that play important roles in homeostasis, inflammation, infection, tumor progression and metastasis. How these {gamma}{delta} T cell subsets are regulated in the lung environment during homeostasis and cancer remains poorly understood. Using single-cell RNA sequencing and flow cytometry, we show that lung V{gamma}6+ cells express a repertoire of cell surface molecules distinctive from V{gamma}4+ cells, including PD-1 and ICOS. We found that PD-1 functions as a co-inhibitory molecule on V{gamma}6+ cells to reduce IL-17A production, whereas manipulation of ICOS signaling fails to affect IL-17A in V{gamma}6+ cells. In a mammary tumor model, ICOS and PD-1 expression on lung V{gamma}6+ cells remained stable. However, V{gamma}6+ and V{gamma}4+ cells within the lung pre-metastatic niche increased expression of IL-17A, IL-17F, amphiregulin (AREG) and TIM-3 in response to tumor-derived IL-1{beta} and IL-23, where the upregulation of TIM-3 was specific to V{gamma}4+ cells. Inhibition of either PD-1 or TIM-3 in mammary tumor-bearing mice further increased IL-17A by V{gamma}6+ and V{gamma}4+ cells, indicating that both PD-1 and TIM-3 function as negative regulators of IL-17A-producing {gamma}{delta} T cell subsets. Together, these data demonstrate how lung {gamma}{delta} T cell subsets are differentially controlled by co-inhibitory molecules in steady-state and cancer.

immunology↗