VPS34-Mediated Autophagic Flux Supports γδ T Cell Survival and Anti-Tumor Immunity by Preserving Metabolic and Iron Homeostasis

Abstract γδ T cells serve as key effectors of immune surveillance, and their survival and effector functions are tightly governed by precise metabolic regulation. However, the molecular mechanisms orchestrating metabolic homeostasis in γδ T cells remain incompletely defined. Here, we demonstrate that VPS34 is an indispensable regulator of γδ T cell development, peripheral homeostasis, and anti‑tumor immunity by maintaining intact autophagic flux. Using LC3 dual‑fluorescence reporter mice, we observed peak autophagic activity at the thymic DN stage, with intrinsically higher basal autophagy in thymic and peripheral γδ T cells compared with αβ T cells. T‑cell‑specific VPS34 deletion disrupts autophagic turnover and causes accumulation of LC3B, p62 and autophagosomes. While VPS34 ablation perturbs the shared DN3-to-DN4 thymic checkpoint for αβ and γδ T progenitors, peripheral CD44 hi effector γδ T cells are selectively vulnerable to VPS34 loss. VPS34-deficient γδ T cells show impaired terminal maturation, skewed TCRγ usage, and reduced numbers across lymphoid and barrier tissues. Mechanistically, VPS34 deletion induces mitochondrial dysfunction and metabolic reprogramming in γδ T cells, accompanied by cellular iron overload, elevated CD71 and CD36 expression, and excessive mitochondrial and lipid‑derived ROS. The resultant oxidative stress drives concurrent ferroptosis and apoptotic cell death. Both genetic and pharmacological VPS34 inhibition reprograms γδ T cell cytokine responses, dampening IFN-γ while boosting IL-17A production, and compromises γδ T-cell-mediated anti-tumor protection in melanoma adoptive‑transfer models. Supporting these observations, metformin‑driven autophagy augmentation elevates IFN-γ production and boosts anti‑tumor activity in WT γδ T cells. Our findings illustrate how VPS34‑dependent autophagy maintains γδ T cell fitness and anti-tumor immunity via coordinated control of metabolism and iron homeostasis, providing mechanistic insights for future γδ T‑cell‑based immunotherapeutic development.