Transient hepatic reconstitution of thymic factors using mRNA rejuvenates T cell immunity, and improves vaccination and cancer immunotherapy responses in aged hosts

Abstract Introduction: Aging impairs immune function and leads to increased vulnerability to infection, malignancy, and vaccine failure.Immunosenescence, marked by reduced thymic output, naive T cell attrition, and progenitor exhaustion, undermines vaccine responses and anti-tumor immunity in aged individuals. While cytokine-based and stem cell-modifying therapies have attempted to reverse this decline, they remain limited by toxicity, durability, or translational feasibility. Here, we present an mRNA approach that transiently reconstitutes thymic signaling pathways in an unrelated organ. By expressing Delta-like ligand 1 (DLL1), Fms-like tyrosine kinase 3 ligand (FLT3-L), and interleukin-7 (IL-7) - collectively termed DFI - in the liver, we observed rejuvenated aged immunity via coordinated modulation of thymopoiesis, lymphoid progenitors, and immune function. Methods: We identified age-related decline of Notch, IL-7, and FLT3-L signaling in thymic epithelial cells and peripheral T cells using spatial transcriptomics (Slide-seq v2) and single-cell RNA-seq across the human and murine lifespan. Modified mRNAs encoding DFI were encapsulated in SM-102 lipid nanoparticles (LNPs) and administered to aged mice (72 weeks). Hepatic expression, pharmacokinetics, and tissue specificity were assessed via RIBOmap and immunofluorescence. Functional impact on thymopoiesis, hematopoiesis, and immune function was assessed via flow cytometry, V(D)J sequencing, TREC analysis, peptide vaccination, tumor challenge (B16-OVA and MC38-OVA), and immune checkpoint inhibitor (ICI) therapy. Autoimmune safety was evaluated in NOD, Act-mOVA, and EAE models. Results: DFI mRNA was selectively translated in hepatocytes, yielding membrane-bound DLL1 and secreted IL-7 and FLT3-L without off-target organ expression. This induced expansion of naïve CD4⁺ and CD8⁺ T cells, increased TRECs, and restored thymic cellularity in aged mice. Mechanistically, DFI did not reprogram hematopoietic stem cell (HSC) fate or reverse myeloid bias but significantly expanded common lymphoid progenitors (CLPs) in the bone marrow. These CLPs exhibited increased CCR9 expression and thymus-homing potential, consistent with peripheral DLL1-mediated Notch signaling and improved thymic seeding. Early thymocyte subsets (DN1-DN3) were selectively enriched, accompanied by rapid induction of Rag2 and Nur77 in thymocytes, supporting enhanced de novo thymopoiesis. Despite HSC aging signatures remaining intact, DFI circumvented upstream deficits by expanding committed lymphoid progenitors and reinvigorating thymic output. Beyond the T cell compartment, DFI restored conventional dendritic cell type 1 (cDC1) populations and boosted co-stimulatory molecule expression (CD40, CD83, CD86). In parallel, it decreased dysfunctional age-associated B cells and increased follicular B cells, resulting in more robust antigen-specific IgG responses and IgM-to-IgG class-switching after peptide vaccination. Functionally, DFI pre-treatment doubled vaccine-specific CD8⁺ T cells in aged mice and improved antigen recall cytokine production (IFN-γ, IL-2). In tumor models, DFI conditioning enhanced spontaneous rejection of MC38-OVA and B16-OVA tumors and improved survival. It restored intratumoral CD8⁺ T cell infiltration and diversity and reversed age-associated T cell exhaustion signatures. When combined with ICI, DFI synergistically enhanced tumor control and survival in aged mice with established tumors. Importantly, DFI did not trigger autoimmune responses. In NOD mice, DFI did not accelerate type 1 diabetes or increase autoreactive TCRs. In Act-mOVA mice, tolerance to endogenous OVA was preserved. In the EAE model, DFI increased peripheral MOG-specific T cells but did not worsen CNS inflammation or clinical scores. DFI's immunostimulatory effects were strictly transient, dissipating 4 weeks post-treatment without long-term adverse effects. Conclusions: Transient hepatic expression of DLL1, IL-7, and FLT3-L safely and effectively reactivates thymopoiesis, expands functional T cell pools, and enhances antigen-specific immune responses in aged hosts. By bypassing stem cell-intrinsic defects and rejuvenating the thymic niche via liver-derived trophic cues, DFI overcomes major roadblocks of immune aging. This mRNA-based, non-integrating platform supports scalable, cyclic immunostimulation without autoimmunity, suggesting a viable strategy to enhance vaccine efficacy and cancer immunotherapy in elderly patients.