Triple costimulation via CD80, 4-1BB, and CD83 ligand elicits the long-term growth of Vγ9Vδ2 T cells in low levels of IL-2

Human gamma delta T cells play important roles in the regulation of infection and cancer. To understand the roles of costimulatory signals in activation and expansion ex vivo, V gamma 9V delta 2 T cells were grown with artificial APCs that express CD83, 4-1BB ligand, and/or CD32, which allowed a loading of alpha CD3 and alpha CD28 antibodies. The costimulatory signals through CD80, 4-1BB, and CD83 ligand in low levels of IL-2 triggered an explosive ex vivo proliferation of V gamma 9V delta 2 T cells capable of secreting high levels of IL-2, IFN-gamma, and TNF-alpha. Moreover, the triple-costimulatory signals cause augmented cell viabilities for long-term growth of V gamma 9V delta 2 T cells, resulting in phenotypic changes to CD272(-)CD45RA(+) effector memory-like cells. Notably, we observed that CD83 ligand signaling is crucial to promote ex vivo expansion, survival, and cytolytic effector functions of V gamma 9V delta 2 T cells. In contrast, 4-1BB signaling is moderately important in up-regulating surface molecules on V gamma 9V delta 2 T cells. Consequently, gd T cells stimulated in the presence of triple-costimulatory signals have diverse cytolytic effector molecules, including perforin, granzyme A, granzyme B, and Fas ligand, eliciting potent cytolytic activities against tumor cells. Overall, our results provide insights into the roles of costimulatory signals in manufacturing long-lived and fully functional V gamma 9V delta 2 T cells that could be useful against cancers.