RPS6 acts as a translational checkpoint to limit effector skewing and sustain CD8 T cell memory Free

Background: Durable CD8 T cell memory underpins protective immunity and effective immunotherapies. The mammalian target of rapamycin complex 1 (mTORC1) orchestrates the balance between effector and memory CD8 T cell fates, promoting effector differentiation but, when overly or chronically active, driving increased effector memory (Tem) and terminal differentiation at the expense of central memory T cell (Tcm) formation. Ribosomal protein S6 (RPS6) phosphorylation (pRPS6) is a canonical downstream event in the mTORC1–S6K pathway and is typically considered a passive readout of pathway activity. Prevailing models, largely based on mTORC1 inhibition by agents like rapamycin, infer that reduced pRPS6 leads to diminished glycolysis, constraining effector differentiation and enhancing memory. However, these models have not directly tested whether pRPS6 itself actively regulates CD8 T cell fate. Since effector differentiation is tightly controlled by T-bet expression and glycolytic reprogramming downstream of mTORC1, a buffering mechanism is likely required to prevent unchecked effector skewing. Here, we investigate whether pRPS6 acts as a regulatory node that shapes the metabolic and translational programming of CD8 T cells.

Central Hypothesis: We propose that pRPS6 serves as a downstream translational and metabolic buffer in the mTORC1 pathway, tempering both T-bet function and glycolytic activity to sustain central memory differentiation and prevent effector over-skewing.

Methods: Using Rps6^5A/5A knock-in mice with non-phosphorylatable mutations, we examined CD8 T cell differentiation, functions, and metabolism. Phenotypic analysis by flow cytometry included markers of effector and memory differentiation (CD44, CD62L) to define Tem and Tcm subsets, as well as transcription factors associated with effector/memory programming (T-bet) and exhaustion/dysfunction (TOX). Metabolic profiling was conducted using extracellular flux Seahorse assays to assess glycolysis (extracellular acidification rate [ECAR]) and mitochondrial respiration (oxygen consumption rate [OCR]). Signaling activation states were determined by phospho-flow cytometry and Western blot. Functional assays assessed proliferation and cytokine production (granzyme B, IFN-γ, TNF).

Results: Rps6^5A/5A CD8 T cells displayed heightened glycolytic activity and mitochondrial functional alterations, contrasting with the canonical rapamycin-based model in which reduced pRPS6 is interpreted as a passive marker of low glycolysis and enhanced memory formation. These cells strongly skewed toward an effector phenotype with increased granzyme B and TNF production but displayed a loss of Tcm subsets. Extracellular flux analysis revealed significantly elevated glycolytic flux (increased ECAR) and reduced mitochondrial respiration (lower OCR) in Rps6^5A/5A cells compared with wild-type controls, reflecting a metabolic shift favoring glycolysis over oxidative metabolism. These metabolic changes were associated with increased T-bet protein levels, likely reflecting enhanced translational efficiency, supporting a role for pRPS6 as a critical buffer that restrains mTORC1-driven T-bet accumulation and excessive glycolytic programming.

Significance: Our data challenge the prevailing paradigm that reduction of pRPS6 universally promotes memory differentiation through metabolic downregulation. Instead, loss of RPS6 phosphorylation removes a key checkpoint on glycolysis and T-bet translation, driving excessive effector differentiation and impairing central memory formation. Thus, pRPS6 plays an essential active role in maintaining metabolic balance and limiting excessive effector fate, which is necessary for the development of durable CD8 T cell memory.

Conclusions: This study establishes pRPS6 as a key checkpoint that fine-tunes mTORC1–T-bet signaling, redefining its role from a passive output indicator to an active modulator of T cell fate. Therapeutic modulation of this axis may offer novel strategies to optimize immune memory and enhance immunotherapy efficacy. Future investigations will elucidate the molecular mechanisms underlying pRPS6-mediated translational control and metabolic regulation, with the goal of translating these findings to clinical applications.