Abstract: BACKGROUND: The T cell immunosuppressive activity of mesenchymal stromal cells offers new hope for the treatment of autoimmune diseases. Amimatoside injection, a drug of human umbilical cord mesenchymal stromal cells, has been approved for the treatment of acute graft-versus-host disease (GVHD) primarily affecting the digestive tract, after steroid therapy failure, in patients aged 14 years and older. Therefore, further exploration of the T cell immunosuppressive potential of mesenchymal stromal cells could lay the foundation for the treatment of autoimmune diseases. 
OBJECTIVE: To investigate the impact of programmed cell death ligand 1 gene overexpression on the inhibition of CD4+ T cell proliferation by human umbilical cord mesenchymal stromal cells. 
METHODS: (1) Human umbilical cord mesenchymal stromal cells were cultured in vitro to passages 0, 1, 2, and 3, and the percentage of programmed death ligand 1-positive cells was detected by flow cytometry. (2) Human umbilical cord mesenchymal stromal cells were divided into an experimental group and a negative control group. The experimental group was modified with lentivirus-mediated programmed death ligand 1 gene, while the negative control group was transfected with blank plasmid vector lentivirus. The transfection efficiency was detected by flow cytometry, real-time fluorescence quantitative PCR, and western blot assay. (3) CD4+ T cells were enriched with magnetic beads from the peripheral blood of healthy subjects. T cells were labeled with carboxyfluorescein diacetate succinimidyl ester and co-cultured with human umbilical cord mesenchymal stromal cells from the experimental and negative control groups at a ratio of 5:1. The proportion of CD4+ T cells attenuated by carboxyfluorescein diacetate succinimidyl ester was measured by flow cytometry. (4) RNA sequencing was performed on human umbilical cord mesenchymal stromal cells from the experimental and negative control groups. Single-cell RNA sequencing was also performed on human umbilical cord mesenchymal stromal cells from the experimental group. Subpopulations were then grouped according to function. Bioinformatics analysis methods were used to depict heat maps of marker genes, enriched signaling pathways, and gene regulatory networks for each subpopulation.
RESULTS AND CONCLUSION: (1) With the increase in passage number, the percentage of programmed death ligand 1-positive cells in human umbilical cord mesenchymal stromal cells gradually decreased. (2) Human umbilical cord mesenchymal stromal cells stably overexpressing programmed death ligand 1 were successfully constructed, and the expression of programmed death ligand 1 in the experimental group was significantly increased. (3) Transcriptome sequencing data suggested that human umbilical cord mesenchymal stromal cells overexpressing programmed death ligand 1 could promote the upregulation of genes related to immune effector regulatory pathways. (4) Co-culture of CD4+ T cells with human umbilical cord mesenchymal stromal cells showed a significant downregulation of the proportion of CD4+ T cells in the experimental group. (5) Based on single-cell RNA sequencing results, human umbilical cord mesenchymal stromal cells overexpressing programmed death ligand 1 could be divided into three functional subpopulations, which were heterogeneous. The expression level of programmed death ligand 1 gene was higher in subpopulation 1, and the significantly high expression of histone methyltransferase SETDB1 may be closely related to the enhanced immunosuppressive function of T cells. These results demonstrate that overexpression of the programmed death ligand 1 gene significantly enhances the T cell immunosuppressive capacity of human umbilical cord mesenchymal stromal cells. Single-cell RNA sequencing allows for the effective stratification of human umbilical cord mesenchymal stromal cells based on their functional properties, providing theoretical support for improving the clinical efficacy of human umbilical cord mesenchymal stromal cell therapy.

Key words: autoimmune diseases, human umbilical cord mesenchymal stromal cells, programmed death ligand 1, immunosuppression, immune rejection, gene overexpression, methyltransferase, single-cell sequencing