International Immunopharmacology · Nov 14, 2026
RHOA–ROCK hyperactivation is associated with impaired migratory persistence and an effector-associated phenotype in aged CD8+ T cells
By Kan S, Jiang W, Hu Z et al.
Background Immune aging is characterized by impaired immune memory, manifested as increased susceptibility to previously encountered pathogens and reduced vaccine efficacy, yet the underlying mechanisms remain incompletely understood. Here, we investigated whether age-related RHOA hyperactivation, together with reduced migratory persistence, skews the fate decision of CD8⁺ T cells away from memory commitment toward terminal effector differentiation. Methods Naïve CD8⁺ T cells were isolated from peripheral blood of young and older healthy donors. Clustering capacity and migratory persistence were assessed by Incucyte live-cell imaging and single-cell trajectory tracking. Differentiation phenotypes were analyzed by flow cytometry. Single-cell RNA sequencing and module scoring were performed to identify key molecular pathways, with validation by Western blotting. Pharmacological inhibition of RHOA–ROCK pathway and mechanical cluster disruption were used to establish causal relationships. Results In qualitative assays, Aged CD8⁺ T cells formed fewer activation-induced clusters in vitro and displayed reduced migratory persistence relative to young cells. Flow cytometry revealed accelerated effector differentiation in aged CD8⁺ T cells, accompanied by diminished memory commitment. Single-cell RNA sequencing uncovered a transcriptional signature consistent with RHOA hyperactivation, together with elevated senescence-associated secretory phenotype (SASP) and exhaustion signatures and dysregulated cytoskeletal dynamics. Biochemical assays confirmed elevated RHOA activity (GTP-bound RHOA) and sustained cofilin phosphorylation, the latter indicating cofilin inactivation and impaired actin depolymerization. GSEA showed enrichment of mTORC1 signaling and concomitant suppression of WNT/β-catenin signaling in aged CD8⁺ T cells, consistent with a cell-fate bias toward terminal effector differentiation at the expense of memory commitment. Importantly, pharmacological inhibition of RHOA–ROCK pathway restored CD62L and CD127 expression in aged CD8⁺ T cells, consistent with at least partial restoration of an effector-associated phenotype. Conclusion In aged CD8⁺ T cells, RHOA hyperactivation impairs cofilin-mediated actin depolymerization, compromising activation-induced clustering and sustaining mTORC1 signaling while repressing WNT/β-catenin-dependent memory programs, thereby skewing differentiation toward a terminal effector fate. Pharmacological inhibition of RHOA–ROCK pathway partially reverses these alterations, suggesting that this pathway may represent a potential therapeutic target for counteracting CD8⁺ T cell immune aging.