Fig. 3: Single cell transcriptomic analysis of endogenous and T_TCR-C4 CD8⁺ T-cell states and their differentiation dynamics.

A Heatmap showing the top 10 differentially expressed genes per cluster. The “top 10” refers to the 10 genes with the most significant differential expression across the identified clusters. The dendrogram on the left displays the similarity between the 13 clusters, which were determined through unsupervised clustering based on gene co-expression patterns. The top dendrogram shows the relationships between the genes based on their expression patterns. K-means algorithm grouped the 13 clusters into 5 main categories, labeled 1 to 5 on the left side of the heatmap. Blue indicates low expression; red, high. B UMAP plot of the CD8⁺ T cells (endogenous and T_TCR-C4⁺) on a two-dimensional space. T_TCR-C4⁺ cells were identified using scGate, an R package which scores cells based on TCR_C4 expression and defines thresholds to classify cells as positive or negative for the population of interest. TCR_C4⁺ cells are colored in dark red, while TCR_C4^- cells (endogenous) are represented in light gray. C UMAP plot displaying the two-dimensional distribution of annotated CD8⁺ T-cell transcriptional states, colored by subset. D Violin plot illustrating the distribution of CD8⁺ T-cell subsets identified through gene expression (Fig. 3A) and TCR_C4 score (Fig. 3B) along the principal component 1 (PC_1) axis. Each violin represents a different CD8⁺ T-cell state. The proximity of each subset along PC_1 (y-axis) indicates transcriptional similarity, with subsets closer together showing more similar gene expression profiles. E Heatmap showing the differential expression of manually curated genes across the five annotated CD8⁺ T-cell subsets. Blue and red indicate the relative expression levels of each marker within each subset, with blue representing lower expression and red indicating higher expression. F Bar plot showing differential gene expression analysis comparing T_TCR-C4 and NKL/Temra (T_TCR-C4^-) CD8⁺ T cells. Differential expression was performed using a two-sided Wilcoxon rank-sum test implemented in the Seurat package. P-values were adjusted for multiple testing using the Bonferroni correction. Genes with an adjusted p-value (padj) <0.01 and log2 fold change (logFC) > 0.5 or < −0.5 were included. Bars represent log2 fold change values, with red indicating genes upregulated in T_TCR-C4 and green representing genes upregulated in NKL/Temra.