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 γδ 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, Vγ9Vδ2 T cells were grown with artificial APCs that express CD83, 4-1BB ligand, and/or CD32, which allowed a loading of αCD3 and αCD28 antibodies. The costimulatory signals through CD80, 4-1BB, and CD83 ligand in low levels of IL-2 triggered an explosive ex vivo proliferation of Vγ9Vδ2 T cells capable of secreting high levels of IL-2, IFN-γ, and TNF-α. Moreover, the triple-costimulatory signals cause augmented cell viabilities for long-term growth of Vγ9Vδ2 T cells, resulting in phenotypic changes to CD27(-)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 Vγ9Vδ2 T cells. In contrast, 4-1BB signaling is moderately important in up-regulating surface molecules on Vγ9Vδ2 T cells. Consequently, γδ 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 Vγ9Vδ2 T cells that could be useful against cancers.