Bulk RNAseq Analysis of Cardiac Myosin-Specific CD4+ and CD8+ T Cells Reveals Distinct Transcriptomic Profiles Between Myocarditis-Resistant and Susceptible Mice

Figures

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  Figure 1. Bulk RNAseq analysis of CD4+ and CD8+ T cells from Tg C57BL/6 and A/J mice. (a) Flow charts for bulk RNAseq. Schematic representation of the methodology employed for bulk RNAseq. The CD3+ T cells enriched by negative selection from C57BL/6 and A/J mice (n = 3) were flow cytometrically sorted to purify CD4+ and CD8+ T cells. The RNA samples corresponding to each subset were subjected to bulk RNAseq. (b) Comparison of transcriptomic profiles. Distribution of CD4+ and CD8+ Tg T cells from C57BL/6 and A/J mice (n = 3). The PCA plot was generated using the gene expression value quantified as FPKM from individual samples to visualize the variance and clustering patterns between the two mouse strains and T cell subtypes.

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  Figure 2. RNAseq analysis in the Tg C57BL/6 and A/J mice reveals distinct gene expression patterns in CD4+ and CD8+ T cells. (a) Comparison of RNA transcripts. Distribution of uniquely expressed and overlapped genes in CD4+ and CD8+ T cell populations sorted from C57BL/6 and A/J mice (n = 3) using a cutoff of 10 for expressed genes. (b) DEGs. Volcano plots showing differentially expressed genes in CD4+ and CD8+ T cells from C57BL/6 and A/J mice (n = 3). The plots display genes that are significantly differentially expressed based on the Padj threshold of < 0.05 and a log2 fold change > 1. The upregulated genes are indicated in red dots, while downregulated genes are shown in green dots. (c) Selected DEGs in C57BL/6 in relation to A/J. The DEGs associated with immune response were identified in CD4+ and CD8+ T cells based on the Padj threshold of 0.05 and a log2 fold change >2. Upregulated genes are shown in red; in contrast, downregulated genes are shown in blue.

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  Figure 3. Validation of selected DEGs by qPCR in CD4+ (a) and CD8+ T (b) cells from Tg mice. CD4+ and CD8+ T cells were sorted flow cytometrically from Tg mice. Total RNA was extracted, and qPCR was performed to assess the expression of selected genes in each T cell subset (panel a, CD4+ T cells; panel b, CD8+ T cells). Gene expression levels were normalized to GAPDH, and relative expressions were calculated using the 2−(∆∆Ct) method. Data are presented as mean ± SEM, with n = 3 mice per group. Statistical significance was determined using a Student’s t-test; * p < 0.05 was considered significant.

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  Figure 4. GO enrichment analysis revealed varied biological processes in CD4+ and CD8+ T cells. (a) CD4⁺ T cells. The left panel shows terms enriched among upregulated genes, and the right panel shows terms enriched among downregulated genes in C57BL/6 relation to A/J mice (n = 3). (b) CD8⁺ T cells. The left and right panels show terms enriched in upregulated and downregulated genes. The size of a point represents the number of genes annotated to a specific GO term, and the color represents the significance level of the enrichment.

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  Figure 5. KEGG enrichment analysis revealed varied pathways associated with various disease conditions and metabolic pathways in CD4+ and CD8+ T cells. (a) CD4+ T cells. Bulk RNA-seq analysis of KEGG enrichment of DEGs in CD4+ T cells from C57BL/6 mice compared to A/J mice (n = 3). The top panel displays enriched KEGG pathways associated with downregulated genes. (b) CD8+ T cells. KEGG enrichment of DEGs in CD8+ T cells from C57BL/6 mice compared to A/J mice (n = 3). The left and right panels show pathways enriched in upregulated and downregulated genes, respectively. The size of a point represents the number of genes annotated to a specific KEGG pathway. The color represents the significance level of enrichment.

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  Figure 6. GSEA reveals differentially enriched GO and KEGG pathways in CD4⁺ and CD8⁺ T cells. (a) GSEA_GO analysis. GSEA of GO term enrichment revealed both positively and negatively enriched pathways in CD4+ and CD8+ T cells in C57BL/6 compared to A/J mice (n = 3). (b) GSEA_KEGG analysis. GSEA of KEGG revealed positively enriched pathways in CD4+ T cells in C57BL/6 compared to A/J mice (n = 3). In each GSEA plot, the top portion shows the enrichment score, representing the degree of overrepresentation of the gene set across the ranked list. Red indicates upregulated genes and blue indicates downregulated genes. The vertical black lines indicate the positions of genes from the selected gene set within this ranked list. The bottom portion of the plot illustrates the rank metric used to order genes. Enrichment plots represent NES, with significance determined using an FDR q-value < 0.25 and a NES > 1.5.

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  Figure 7. Transcription factor network analysis revealed TFs common to both CD4+ and CD8+ T cells in addition to a set of TFs unique to each subset. Transcription factor network analysis of DEGs in CD4+ (left panel) and CD8+ T cells (right panel) from C57BL/6 in comparison to A/J Tg mice (n = 3) using a Padj value cut off <0.05 and a log2 fold change ≥2. Shown are the 30 transcription factors with the highest downstream regulation effects. The resulting master regulator network is visualized as a circular layout, and the TF-target subnetwork is visualized in a tree layout. Orange nodes represent transcription factors, and blue nodes represent regulated genes that are not transcription factors. Directed edges indicate interactions or regulatory relationships. Red node borders link upregulated genes to their corresponding master regulators, while blue edges connect downregulated genes to their predicted regulators. Black edges indicate a mixed regulation, meaning that one node is upregulated and the other is downregulated.