Fig. 5

The CD3⁺PRF1⁺ clusters contain various cell types with different gene expression patterns characteristic of cytotoxic lymphocyte function. Clusters within the CD3⁺PRF1⁺ lymphocyte major cell group was further analyzed and annotated by differential gene expression. a UMAP subset of CD3⁺PRF1⁺ lymphocyte clusters (left) and of re-clustering analysis with putative cluster annotations (right). Selected axis ranges excluded < 5 cells in CD3⁺PRF1⁺ group from UMAP subset plot. b Frequency of each cell cluster within the CD3⁺PRF1⁺ lymphocyte group per horse. c Hierarchical clustering (integrated PCA dimensions) of CD3⁺PRF1⁺ lymphocyte major cell group. d Heatmap of differentially expressed genes (adjusted p value < 0.05, log₂ fold-change > 0.58 for each cluster versus all other clusters, expressed > 25% of cluster). e Dot plot of select genes associated with cytotoxic lymphocyte populations differentially expressed across CD3⁺PRF1⁺ lymphocyte clusters. Dot size is proportional to number of cells with detectable expression of indicated gene. Dot color intensity indicates average gene expression values scaled across plotted clusters. *Gene ID ENSECAG00000006663 is labeled FCGR3A/B and Gene ID ENSECAG00000031528 is labeled KLRD1 (CD94) based on Ensembl/NCBI annotations. f Hierarchical clustering of equine PBMC scRNA-Seq data (CD3⁺PRF1⁺ lymphocyte clusters) and human PBMC scRNA-Seq data (non-naïve CD8⁺ T cells, NK cells, NKT cells). Median-normalized average expression values for highly variable human/horse one-to-one orthologs were calculated for each cluster, and clustering was performed on Pearson distances by Ward’s method