TLR3 stimulation rejuvenates aged mesenchymal stem cells and restores immunosuppressive function in graft-versus-host...

Graft-versus-host disease (GVHD) remains a major complication of allogeneic hematopoietic stem cell transplantation, particularly in patients who are refractory to corticosteroids. Mesenchymal stem cells (MSCs) possess immunosuppressive properties and are being explored as a treatment for GVHD. However, their therapeutic efficacy declines with extensive in vitro expansion due to cellular aging. Therefore, this study aimed to investigate the effects of MSC aging on GVHD outcomes and explore strategies to rejuvenate aged MSCs. Specifically, we compared the therapeutic efficacies of early (P5) and late passage (P15) human MSCs in a murine model of GVHD. Single-cell RNA sequencing was performed to identify molecular alterations associated with MSC aging. Polyinosinic:polycytidylic acid [poly(I:C)], a Toll-like receptor 3 (TLR3) agonist, was used to stimulate aged MSCs. Early passage MSCs significantly alleviated the severity of GVHD, improved survival, and reduced systemic inflammation (p < 0.05). In contrast, late-passage MSCs showed minimal therapeutic effect. Single-cell transcriptomics revealed that MSC aging is associated with the loss of immunoregulatory subpopulations and downregulation of TLR3 signaling. Notably, poly(I:C) priming partially reversed the senescence phenotypes and restored the immunosuppressive capacity of aged MSCs, resulting in enhanced suppression of T cell proliferation, increased T cell apoptosis and G1 accumulation, and reduced IFN-related readouts (p < 0.05).Mechanistically, replicative senescence impairs the immunoregulatory potency of MSCs by disrupting TLR3-mediated signaling pathways. Overall, TLR3 activation by poly(I:C) rejuvenates aged MSCs and restores their therapeutic function, providing a clinically translatable strategy for enhancing MSC-based immunotherapies for GVHD.