RPS6 Phosphorylation Shapes Effector/Memory Balance and Recall Function of CD8 T Cells 2305349

Abstract

Abstract Introduction Durable CD8 T cell memory is critical for long‑term protection and immunotherapy. mTORC1 promotes effector differentiation but, when overactive, limits T cell memory formation. Phosphorylation of ribosomal protein S6 (pRPS6), a classic mTORC1-S6K output, is usually viewed as a passive readout. Whether pRPS6 itself shapes CD8 T cell fate, metabolism, and recall capacity remains unclear. Methods Rps6 5A/5A knock-in mice with non-phosphorylatable RPS6 were crossed with pmel transgenic mice. Effector and memory subsets were defined by CD44/CD62L. Metabolism was evaluated by Seahorse assays (ECAR, OCR). T-bet protein was quantified by intracellular staining. For chronic in vitro stimulation, CD8 T cells were restimulated with irradiated B16 melanoma every 3—4 days, and cytotoxicity was measured by RTCA and cytokine ELISA. For in vivo memory and tumor control, 5-day effector CD8 T cells from Rps6 wt/wt or Rps6 5A/5A pmel mice were rested 30 days in tumor-free hosts, then challenged with B16. Results Rps6 5A/5A CD8 T cells displayed increased glycolytic flux, higher T‑bet, and reduced Tcm frequencies, indicating a glycolysis‑biased, effector‑skewed phenotype. Under repeated restimulation with B16 cells, Rps6 5A/5A pmel CD8 T cells outperformed wild‑type cells in short‑term target killing. In contrast, when 5-day effector CD8 T cells were rested in vivo for 30 days, and then tumor-challenged, Rps6 5A/5A pmel cells controlled tumors less effectively than WT, revealing impaired memory phase protection despite robust early effector activity. Conclusion These data identify pRPS6 as a regulatory checkpoint rather than a passive mTORC1 marker. By limiting T‑bet expression and restraining glycolytic overactivation, pRPS6 helps maintain CD8 T cell memory potential. Loss of pRPS6 enhances effector function yet compromises long-term tumor control, revealing a trade-off between immediate cytotoxicity and durable CD8 T cell memory. Funding Source NIH (1R01CA250514-01) Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)