p97-mediated proteostasis is a checkpoint for late-stage thymocyte positive selection.

AAA+ ATPase p97 is a central regulator of protein homeostasis, yet its role in late-stage thymocyte development remains undefined. Here, we demonstrate that T-cell-specific ablation of p97 in mice severely blocks the double-positive (DP) to single-positive (SP) transition, with a pronounced defect in CD8+ lineage commitment. Using both genetic deletion and acute pharmacological inhibition, we revealed a stage- and lineage-specific requirement for p97, with DP thymocytes being most sensitive to p97 loss. This failure in late-stage positive selection leads to intrathymic developmental arrest of immature DP cells and profound peripheral T-cell lymphopenia. Mechanistically, p97 deficiency results in the accumulation of ubiquitinated proteins, triggering the unfolded protein response and apoptosis in thymocytes. Furthermore, we identified a critical requirement for p97 in sustaining IL-7 receptor (IL-7R) expression and JAK signaling. Strikingly, pharmacological activation of JAK partially rescued SP thymocyte development in p97-deficient mice. Our findings establish p97-mediated protein homeostasis as a previously uncharacterized, cell-intrinsic checkpoint that is indispensable for late-stage positive selection by preventing proteostatic collapse and ensuring the fidelity of IL-7R signaling.