GSK-3 regulates CD4-CD8 cooperation for super-armed CD8+ cytolytic T cells in immunotherapy against tumors

Although the efficacy of immune checkpoint blockade (ICB) depends on coordinated activity across progenitor and terminal effector CD4⁺ and CD8⁺ T-cell compartments, the cell-intrinsic pathways that enable this cooperation remain incompletely defined. Here, we identify glycogen synthase kinase-3 (GSK-3) as a central regulator of TCF-1⁺ progenitor and memory CD8⁺ T-cell differentiation, where reduced GSK-3 expression enhances antiviral and anti-tumor immunity. In GSK-3 knockdown (GSK-3 KD) mice, diminished GSK-3 reshaped basal T-cell homeostasis, skewing differentiation toward memory-phenotype subsets even in the absence of antigenic stimulation. During chronic LCMV Cl13 infection, GSK-3 KD promoted the expansion and proliferation of GP33-specific memory-precursor effector cells (MPECs). Metabolic profiling by Seahorse and SCENITH revealed increased glycolysis and oxidative phosphorylation in GSK-3 KD TCF-1⁺ stem-like CD8⁺ T cells, with greater GLUT1 expression and mitochondrial mass, indicative of enhanced metabolic adaptability. GSK-3 was also required for regulatory T-cell (Treg) suppressive function, while GSK-3 inhibition and reduced expression synergized with PD-1 blockade to "super-arm" cytolytic CD8⁺ T cells, characterized by upregulation of perforin and seven distinct granzymes. Notably, whereas B16-F10 tumor rejection in wild-type mice relied on CD4⁺ CTLA-4⁺ Tregs, tumor control in GSK-3 KD mice required CD4⁺ T-cell help for optimal granzyme induction and tumor rejection. Together, these findings define a GSK-3-PD-1 signaling axis that links metabolic and differentiation programs to govern T-cell fate and cytotoxicity, providing a mechanistic framework for overcoming ICB resistance.