Skeletal muscle as an active immune niche orchestrating adaptive responses to replicon RNA vaccination 2232181

Introduction Self-amplifying replicon RNA (repRNA) vaccines formulated with the cationic nanocarrier LION™ induce strong adaptive immunity with low systemic reactogenicity. However, the mechanisms linking localized intramuscular delivery to systemic T- and B-cell responses remain poorly understood. Methods We combined in vivo murine immunization models, engineered repRNA variants, flow cytometry, in situ hybridization, and in vitro muscle—APC—T cell co-culture systems to dissect interactions between transfected muscle cells, antigen-presenting cells (APCs), and lymphocytes. Results Intramuscular administration of repRNA/LION generated a muscle-resident immune niche. Transfected myocytes transferred RNA and protein antigens to infiltrating monocyte-derived dendritic cells, which cross-primed CD8+ T cells. Antibody induction required CD4+ T cells, which expanded locally and exhibited follicular helper—like features. CD138+ antibody-secreting cells accumulated in muscle, and their frequencies correlated with serum IgG titers. Conclusion Our findings reveal skeletal muscle as an active immunological hub rather than a passive site of antigen expression. Local antigen transfer and immune cell interactions orchestrate systemic T- and B-cell responses to repRNA/LION vaccination, providing mechanistic insights to guide safer and more durable RNA vaccine design. Funding Source NIH/NIAID grant R01AI180195, a Career Development Award from the American Society of Gene & Cell Therapy Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)