The class III PI3-kinase VPS34 is a mediator of endocytosis, trafficking of intracellular membranes and vesicles, and autophagy in various cell types. Given its central role in key cellbiological processes, we explored whether VPS34 is critical for immune cells by generating conditional knockout mice where exon 21 of Pik3c3 (the VPS34 gene) is deleted specifically in regulatory T (Treg) cells or activated CD8+ T cells, respectively. We found that mice with VPS34-deficient Treg cells died within 6 weeks of birth from an autoimmune lymphoproliferative disease, similar to mice lacking Treg cells. However, VPS34- deficient Treg cells developed normally and populated the peripheral lymphoid organs, demonstrating a critical role for VPS34 in Treg cell suppressive functions rather than survival. However, none of the known Treg cell suppressive mechanisms were impaired by the loss of VPS34. Nonetheless, VPS34-deficient Treg cells had a competitive disadvantage and impaired activation compared to VPS34-sufficient Treg cells, suggesting that VPS34 is required for Treg cells maturation or the survival of mature Treg cells. In an attempt to determine which cellular processes are affected by the loss of VPS34, we performed proteomic profiling, and while the results could not provide a definite clue about the mechanism leading to the observed phenotype in mice with VPS34-deficient Treg cells, they suggested that loss of VPS34 induces a state of heightened metabolic activity in Treg cells.

Autophagy is critical for the formation of memory CD8+ T cells, while it is dispensable during the effector phase. Since VPS34 is required for the induction of autophagy, it was striking to observe that deletion of VPS34 in activated CD8+ T cells did not result in a phenotype similar to mice with autophagy (ATG7)-deficient CD8+ T cells. Rather, mice with VPS34-deficient, activated CD8+ T cells displayed reduced proportions of antigen-specific CD8+ T cells and reduced effector functions at the peak of expansion after infection with Listeria monocytogenes, while memory formation seemed intact. Results from in vitro data suggested that while proliferation was intact, the transition through the cell cycle was impaired in VPS34-deficient, activated CD8+ T cells.