Heterogeneity of T-cells in periapical lesions and in vitro validation of the proangiogenic effect of GZMA on HUVECs.

Abstract

T-cells are key immunomodulatory cells in periapical lesions. This study aimed to explore the roles of T-cells in chronic apical periodontitis (CAP) using single-cell RNA sequencing and to further investigate Granzyme A (GZMA) in angiogenesis regulation. A total of five CAP samples were collected for single-cell RNA sequencing. We performed sub-cluster and lineage-tracing analyses for T-cells. According to differential gene expression, distinct biological functions enriched in T-cells of CAP were presented by Gene Set Enrichment Analysis (GSEA) and compared with healthy gingiva (data obtained from the GEO database). CellChat was used to explore potential ligand-receptor interactions between T-cells and endothelial cells in CAP. The coculture of primary human umbilical vein endothelial cells (HUVECs) and Jurkat T-cells, as well as the addition of GZMA recombinant protein, was used to validate the predicted pair of GZMA and_Coagulation Factor II Thrombin Receptor (F2R) by RT-PCR, angiogenesis, and migration assays. A transcriptomic atlas of 44,746 individual cells was constructed from the periapical lesions of five patients with CAP by single-cell RNA-seq, and eight cell types were identified. We identified nine subsets of T cells and deciphered the cellular heterogeneity of T-cells in CAP at the functional level by sub-clustering and GSEA. Lineage-tracing revealed a distinct lineage of T-cells in CAP and predicted the transition of the T cellular state upon CAP. GSEA revealed multiple biological processes and relevant angiogenesis genes upregulated in CAP T-cells. GZMA-F2R pairs were predicted by cell-cell interactions in CAP. High expression of GZMA and F2R was observed in the coculture of HUVECs and Jurkat T-cells, and the proangiogenic capacity of the GZMA recombinant protein was emphasized by in vitro experiments. Our study provides novel insights into the heterogeneity of T-cells in periapical lesions and reveals the potential role of GZMA in T-cells in regulating angiogenesis in HUVECs.