Regulatory T cells turn into multiple pro-inflammatory effector T cells in Mice with Atherosclerosis

Introduction/Rationale: Atherosclerosis is an immune-mediated disease in which regulatory T cells (Tregs) can lose lineage stability and convert into pro-inflammatory effector states (“exTregs”) that may accelerate plaque progression. However, the diversity, lineage relationships, and pathogenic roles of exTreg subsets in cardiovascular disease remain undefined.

Methods: Using Foxp3 lineage-tracker Apoe-/- mice, we uncovered two distinct Treg subsets (GFP+tdTomatolow and GFP+tdTomatohi), with only the tdTomatohi population differentiating into exTregs (GFP-tdTomatohi). To comprehensively characterize these populations, we performed large-scale single-cell CITE-seq and TCR-seq across spleen, draining (axillar and cervical), and non-draining lymph nodes from Apoe-/- mice on a Western diet.

Results: Density-based clustering and trajectory inference revealed at least seven exTreg subsets, including Tfh-like (the largest population), Th1-like, Th17-like, cytotoxic and proliferating exTreg-like populations. Pseudotime analyses supported a branched rather than linear trajectory, with tdTomatohi Tregs giving rise to multiple effector-like programs. TCR analyses showed clonal enrichment predominantly within Tfh- and cytotoxic-like exTregs, suggesting antigen-driven expansion. CITE-seq identified surface markers (e.g., CXCR5/CD185 for Tfh-like and CXCR6/CD186 for cytotoxic Th1-like exTregs). Spatially, exTregs accumulated in draining lymph nodes and spleen, and were detectable in atherosclerotic plaques by intravital microscopy.

Conclusion: Collectively, these data define a branched Treg-to-exTreg transition trajectory. Clonally expanded, antigen-experienced exTreg subsets that are expected to influence plaque biology.