Regulatory T cells (Tregs), defined by the lineage-specific transcription factor FoxP3, are crucial for immune regulation and have been studied extensively in mammals. However, avian Tregs remain poorly characterized, leaving gaps in our understanding of their evolutionary conservation and unique features. In this study, we investigated the phenotype of chicken Tregs to define reliable markers for their identification and characterization. We analyzed CD4+ splenocytes sorted into CD25negative, CD25low, and CD25high subpopulations using RNA sequencing. FOXP3 and other Treg-associated genes were expressed in both CD25low and CD25high populations, showing that CD25 expression alone is insufficient to distinguish chicken Tregs. To refine the marker profile, we evaluated additional markers, including CTLA-4 and GITR. Notably, we describe for the first time a chicken-specific CTLA-4 antibody, which uniquely stains CTLA-4 exclusively in intracellular (ic) compartments, distinguishing it from mammalian counterparts. Single-cell RNA sequencing further confirmed distinct FOXP3+ clusters enriched for expression of CTLA4 and TNFRSF18 (encoding GITR). While CTLA-4's ic expression limits usability in functional assays, the combination of CD4+/CD25+/CTLA-4+/GITR+ represents the most accurate characterization of putative chicken Tregs to date. These findings highlight evolutionary conservation and species-specific differences in Treg markers, providing the foundation for future studies on chicken Treg functionality.
