Fig. 2

VEGF-A promotes the cytotoxic function of CD4 CTLs. A The graph-based clustering and t-SNE algorithm were applied in CD4⁺ T cells from GSE106543 (pink), GSE149652 (green), GSE179292 (purple) and SRP226183 (blue). Clusters denoted by the same color scheme were labeled with inferred cell types (bottom). B Heatmap from scRNA-seq analysis via expression data of the top 20 genes differentially expressed for each cluster (denoted by colored bars at the top). Key genes for each cell type were shown on the left margin. Genes in red rectangle represented cytotoxicity related. C The stack plots showed the ratio of each cluster of CD4 CTLs. Each color represented different cell cluster. D Pseudo-time trajectory analysis showed the differentiation stage of each CD4 CTLs subset according to the cytotoxicity. Pink represented cluster 2, green was cluster 6, blue was cluster 7 and purple was cluster 10. E Dot plots showed the expression of GZMB, PRF1, NKG7, GZMK and VEGFR1 in each CD4 CTLs subset respectively. Color scale represented z-score and dot size represented percentages of cells. F GSEA showed (upper) cytotoxic related processes and (bottom) VEGF and cytotoxicity pathways were obviously enriched in CD4 CTLs with higher expression of VEGFR1 compared with lower VEGFR1 expression. G Representative histogram plots showed the expression of GrmB and Prf in CD4 CTLs in the CON and VEGF-A group. The quantification of the proportions of GrmB⁺ and Prf⁺ in CD4 CTLs was displayed on the right (N = 3). Blue was CON and red for VEGF-A treated. Error bars showed SEM. The data were representative of at least three biological replicates. CON: control; Grm: granzyme; Prf: perforin. *P < 0.05, ****P < 0.0001