Mitochondrial One-Carbon Metabolism Drives CD34-Lineage Cells to Differentiate Into T Follicular Helper Cells to Form Tertiary Lymphoid Organs in Transplant Arteriosclerosis

BACKGROUND: Allograft arteriosclerosis, a significant cause of graft failure, is linked to the formation of tertiary lymphoid organs. T follicular helper (Tfh) cells are a vital subset of helper T cells that control the formation of the germinal center in tertiary lymphoid organs. Thus, understanding the origins and regulatory mechanisms of Tfh cells in allograft arteriosclerosis is essential for developing targeted therapies. METHODS: We used a lineage-tracing strategy to track Tfh cell fate in mouse models. Single-cell RNA sequencing, flow cytometry, and immunofluorescence staining were employed to analyze cell populations in remodeled arteries 2 and 4 weeks after transplantation. Additionally, we used VEGFR-3 inhibitors and lymph node dissection to suppress lymphatic vessel formation. Metabolic signatures and flux in different cell types were investigated using ultrahigh-performance liquid chromatography and high-resolution mass spectrometry–based metabolomics. CD4 + T cell–specific MTHFD2 knockout mice were used to corroborate our hypothesis about the role of mitochondrial one-carbon metabolism in Tfh cell differentiation. Mechanisms discovered in vivo were also tested ex vivo. RESULTS: CD34-lineage cells were found to be the major source of cells differentiating into T cell populations in allograft arteries. CD34-lineage cells mainly originated from the thymus, with drainage through lymphatic vessels, and differentiated into effective T cells around grafting arteries. Using CD34 lineage-tracing mice and single-cell RNA sequencing, we identified a Tfh cell population derived from CD34-lineage CD4 + T cells. Untargeted and targeted metabolomics revealed distinct upregulation of one-carbon metabolism during CD4 + T-to-Tfh cell differentiation. Supplementation of amino acids essential for one-carbon metabolism, such as serine, methionine or glycine, facilitated differentiation from CD4 + T to Tfh cells. Using deuterium-labeled serine, we found that the mitochondrial one-carbon pathway is predominant. Inhibition of the mitochondrial one-carbon metabolic enzyme MTHFD2 by administration of DS18561882 or generating CD4 + T cell–specific MTHFD2 knockout mice, significantly inhibited the numbers of Tfh cells and tertiary lymphoid organ formation as well as vascular remodeling. CONCLUSIONS: This study provides insights into the critical role of mitochondrial one-carbon metabolism and MTHFD2 in governing the differentiation of CD34-lineage cells into Tfh cells, which contributes to tertiary lymphoid organ formation in transplant vasculopathy, offering potential therapeutic targets to enhance transplant outcomes.