Myocarditis is a predominant cause of congestive heart failure and sudden death in children and young adolescents that can lead to dilated cardiomyopathy. Lymphocytic myocarditis mediated by T cells can result from the recognition of cardiac antigens that may involve CD4 or CD8 T cells or both. In this report, we describe the generation of T cell receptor (TCR) transgenic mice on a C57BL/6 genetic background specific to cardiac myosin heavy chain (Myhc)-α 334–352 and make the following observations: First, we verified that Myhc-α 334–352 was immunogenic in wild-type C57BL/6 mice and induced antigen-specific CD4 T cell responses despite being a poor binder of IA^b; however, the immunized animals developed only mild myocarditis. Second, TCRs specific to Myhc-α 334–352 in transgenic mice were expressed in both CD4 and CD8 T cells, suggesting that the expression of epitope-specific TCR is common to both cell types. Third, although T cells from naïve transgenic mice did not respond to Myhc-α 334–352, both CD4 and CD8 T cells from animals immunized with Myhc-α 334–352 responded to the peptide, indicating that antigen priming is necessary to break tolerance. Fourth, although the transgenic T cells could produce significant amounts of interferon-γ and interleukin-17, the immunized animals developed only mild disease, indicating that other soluble factors might be necessary for developing severe myocarditis. Alternatively, the C57BL/6 genetic background might be a major contributing factor for resistance to the development of myocarditis. Taken together, our model permits the determination of the roles of both CD4 and CD8 T cells to understand the disease-resistance mechanisms of myocarditis in a single transgenic system antigen-specifically.

Background: We recently generated T cell receptor (TCR) transgenic (Tg) mice specific to cardiac myosin heavy chain-α (Myhc-α 334–352) on both myocarditis-resistant (C57BL/6) and susceptible (A/J) genetic backgrounds. We noted that the antigen-specific TCRs were expressed in CD4⁺ and CD8⁺ T cells in both strains, but their responses differed. While the T cells from naïve Tg C57BL/6 mice do not respond to Myhc-α 334–352, whereas those from A/J mice spontaneously respond to the antigen, suggesting their underlying molecular mechanisms might differ. Methods: To investigate the mechanisms of differences in the antigen-responsiveness between the Tg C57BL/6 and A/J mice, we performed bulk RNA sequencing on CD4⁺ and CD8⁺ T cells sorted by flow cytometry. Differentially expressed genes, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, gene set enrichment analysis (GSEA) of GO and KEGG, and transcription factor (TF) network analyses were performed to identify pathways and regulators of immune responses. Results: First, the principal component analysis of the transcriptomic profiles distinguished CD4⁺ from CD8⁺ T cells, which also differed between the two strains. Second, the differentially expressed cytokine and cytotoxicity genes revealed similar patterns between CD4⁺ and CD8⁺ T cells. Importantly, KEGG enrichment analysis revealed downregulated pathways in both CD4⁺ and CD8⁺ T cells that are associated with viral myocarditis, and various autoimmune conditions in C57BL/6 as compared to A/J mice. Similarly, the GSEA of GO revealed negative regulation of heart contraction and positive regulation of cardiac muscle hypertrophy processes were negatively enriched in CD4⁺ T cells of C57BL/6 mice. Finally, by generating the transcription factor (TF) networks, 22 TFs were found common to both CD4⁺ and CD8⁺ T cells, whereas eight TFs were unique to CD4⁺ or CD8⁺ T cells that have a role in T cell activation, tolerance, and T regulatory cells. Conclusions: Our data provide new insights into the transcriptomic profiles that may contribute to the genetic resistance mechanisms for developing cardiac autoimmunity.