Background/Purpose: mRNA vaccines are safe and remarkably effective, but rare cases of vaccine-induced autoimmunity have been reported, particularly in patients with underlying inflammatory disease. Mechanistic insight into such cases is limited by the fact that little is known of the proximal events leading to CD4⁺ T cell priming after mRNA vaccination. Vaccine antigen derived from cells at the immunization site, such as muscle cells, is presumed to activate CD4⁺ T cells through uptake by antigen presenting cells (APCs) and processing via the classical exogenous pathway. However, APCs themselves efficiently produce mRNA-encoded antigen, and in analogous viral infection models, virus-specific CD4⁺ T cell responses are driven primarily by endogenous antigens presented via non-classical pathways. We investigated whether a similar phenomenon underlies CD4⁺ T cell responses to mRNA vaccines.

Methods: We developed a series of mRNA vaccines that encode a model viral protein (influenza nucleoprotein, NP), followed by specific microRNA (miR) targeting sequences that allow for differential expression of vaccine antigen in different cell types. Three different NP-miR mRNA constructs were generated. “NP-miR142t” contains the targeting sequence for hematopoietic-specific miR-142, leading to inhibition of NP expression within hematopoietic cells – and thus reliance on exogenous antigen for presentation to CD4⁺ T cells. “NP-miR206t” contains the targeting sequence for muscle cell-specific miR-206, thus excluding NP expression from muscle cells and forcing APCs to rely primarily on endogenous antigen. A control “NP-scrambled” construct contains scrambled miR binding sites that are not targeted by any miRs. Cell-type specificity of miR targeting was validated in vitro by flow cytometry and Western blot. Mice were immunized intramuscularly with mRNA encapsulated in lipid nanoparticles to evaluate NP-specific CD4⁺ T cell responses in vivo.

Results: Mice immunized with NP-miR142t demonstrated significantly lower frequencies of NP-specific CD4⁺ T cells by both IFNg ELISpot and tetramer staining, compared to mice immunized with NP-scrambled, suggesting that antigen expressed within hematopoietic cells is critical to the CD4⁺ T cell response. Immunization with NP-miR206t resulted in only a modest reduction in NP-specific CD4⁺ T cells, despite significantly decreased NP protein levels within muscle.

Conclusion: Optimal CD4⁺ T cell activation by mRNA vaccines requires antigen expression in APCs and other hematopoietic cells, suggesting that endogenous antigen presentation is a major driver of the CD4⁺ T cell response. Contrary to the prevailing paradigm, exogenous vaccine antigen derived from muscle and other cells may not be the primary source of antigen activating vaccine-specific CD4⁺ T cells. Future studies will expand upon these findings to determine how restriction of antigen source – and therefore processing via exogenous or endogenous pathways – impacts adaptive immune responses to mRNA vaccines more broadly.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/expression-of-mrna-vaccine-antigen-in-hematopoietic-cells-is-necessary-for-induction-of-optimal-vaccine-specific-cd4-t-cell-responses/