Inducing CD8⁺ Tregs Limits Atherosclerosis via Notch-Mediated VSMC Quiescence

CD8⁺ (cluster of differentiation 8) T cells contribute to atherosclerosis, but how they affect VSMCs (vascular smooth muscle cells) is unclear. The protective role of CD8⁺ regulatory T cells (Tregs) and in vivo induction strategies also remain undefined.

We analyzed human atherosclerosis single-cell RNA sequencing to map immune-VSMC communication. Atherosclerosis was modeled in adeno-associated virus-Pcsk9 or Apoe^-/- mice. CD8⁺ T-cell activity was modulated by adoptive transfer of CD45.1⁺CD8⁺ T cells into syngeneic CD45.2⁺ Apoe^-/- recipients (gain-of-function) or nonlytic anti-CD8α antibodies (loss-of-function). VSMC-specific Notch loss was tested in Myh11^CreER;Rbpj^fl/fl;Apoe^-/- mice. Ligand-receptor interactions were perturbed with neutralizing antibodies. Lineage tracing was performed using Myh11^CreER;Rosa^YFP;Apoe^-/- mice. CD8⁺CD44⁺CD122⁺ Tregs were adoptively transferred to assess therapeutic potential. Transcription factor chromatin immunoprecipitation sequencing (ChIP-seq) was used to define VSMC gene regulation.

CD8⁺ T cells are the dominant immune partners communicating with VSMCs via CD8α in human plaques. Transferred CD8⁺ T cells localize to the media and inhibit VSMC Notch signaling. VSMC Notch ablation accelerates atherosclerosis, enhancing VSMC proliferation, inflammation, and phenotypic switching. CD8α blockade induces CD8⁺CD44⁺CD122⁺ Tregs, which, partly via IL-10 (interleukin-10), activate VSMC Notch, suppress proliferation, and limit VSMC-to-macrophage transdifferentiation. These Tregs express higher Notch ligands than conventional CD8⁺ T cells and decline with a high-cholesterol diet. Their adoptive transfer slows atherosclerosis progression. Blocking Notch1-Jag2 (Jagged 2), or synergistically blocking DLL1/4 (delta-like ligand 1/4) and Jag1/2, negates Treg protection. Mechanistically, Notch-RBPJ (recombination signal binding protein for immunoglobulin kappa J region) activates Hes1, which suppresses Klf4 and Rel via Hes1/EZH2-mediated trimethylation of histone H3 at lysine 27.

CD8⁺ T cells promote atherosclerosis by inhibiting VSMC Notch; CD8⁺CD44⁺CD122⁺ Tregs restore Notch, preserve VSMC quiescence, and stabilize plaques. The CD8⁺ Treg-Notch axis is a novel and promising therapeutic target against atherosclerosis.