Beyond FoxP3-Identification of a Chicken Regulatory T Cell Signature.

Area of Science:

• Immunology
• Avian Biology
• Cellular Biology

Background:

• Regulatory T cells (Tregs) are vital for immune homeostasis but are poorly understood in birds.
• Existing mammalian Treg markers may not fully apply to avian species, necessitating species-specific research.

Purpose of the Study:

• To identify reliable phenotypic markers for characterizing chicken regulatory T cells (Tregs).
• To investigate the evolutionary conservation and species-specific differences in Treg markers.

Main Methods:

• RNA sequencing of chicken CD4+ splenocyte subpopulations (CD25 negative, low, high).
• Evaluation of additional markers including CTLA-4 and GITR.
• Development and application of a novel chicken-specific CTLA-4 antibody.
• Single-cell RNA sequencing for high-resolution analysis.

Main Results:

• CD25 expression alone is insufficient to identify chicken Tregs, as FOXP3 is present in both CD25low and CD25high populations.
• A novel chicken-specific CTLA-4 antibody revealed intracellular staining, differing from mammalian expression.
• Distinct FOXP3+ clusters expressing CTLA-4 and GITR were identified via single-cell RNA sequencing.
• The combination of CD4+/CD25+/CTLA-4+/GITR+ provides the most accurate characterization of chicken Tregs to date.

Conclusions:

• Chicken Tregs exhibit both conserved and unique features compared to mammalian Tregs.
• The identified marker combination (CD4+/CD25+/CTLA-4+/GITR+) advances the characterization of avian Tregs.
• This research provides a foundation for future functional studies of chicken Tregs and avian immunology.