Discovery of Seven ROS-Sensitive Immune Checkpoints and 46 Ligands Mediating Immune Suppression Through T cell-APC Networks

<b>Rationale:</b> The functional landscape of immune checkpoints (ICs) operating on CD4⁺FoxP3⁺ regulatory T cells (Tregs) remain incompletely defined. Although canonical IC pathways are well characterized, the full spectrum of IC molecules governing Treg-mediated immune regulation across physiological and pathological contexts has not been fully explored. <b>Methods:</b> We performed a comprehensive, multi-dataset transcriptomic screening of Treg membrane proteins to identify candidate immune checkpoints. This approach yielded 151 putative novel ICs, including 45 Treg-specific molecules and 106 FoxP3⁺-upregulated candidates. Cross-referencing these candidates with ten well-established IC-deficient models refined the list to 85 high-confidence ICs. A subsequent high-stringency, integrating expression specificity, functional relevance, and cross-dataset consistency, was applied to identify seven the most robust candidates. Ligand-receptor interaction mapping was then performed to define associated IC ligands and characterize their cellular expression patterns. <b>Results:</b> This integrative analysis identified seven previously unrecognized immune checkpoints: CEP55, CD38, EHD4, CD200R1, PRC1, RAPH1, and CD86 expressed across Tregs and multiple T cell subsets. Ligand interaction mapping further revealed 46 corresponding IC ligands, predominantly expressed on antigen-presenting cells and tumor cells. Together, these IC-ligand interactions form extensive regulatory networks that modulate immune signaling and inflammatory responses. <b>Conclusion:</b> Our study delineates a comprehensive immune checkpoint-ligand network encompassing seven novel ICs and 46 associated ligands, providing mechanistic insight into Treg- and T cell-mediated immune regulation. This expanded IC landscape broadens the current repertoire of immune modulatory pathways and highlights new therapeutic opportunities across cancer, autoimmune disorders, infectious diseases, transplantation immunology, inflammatory conditions, and cardiovascular diseases.