Monoclonal antibodies for equine CD25 improve detection of regulatory T cells in horses.

Overview

• This study developed new monoclonal antibodies (mAbs) specific to equine CD25 to improve the detection and characterization of regulatory T cells (Tregs) in horses.
• The research compared the performance of these newly developed equine-specific CD25 mAbs with an existing anti-human CD25 antibody commonly used in equine studies, demonstrating enhanced staining quality and more accurate identification of Tregs using the equine-specific antibodies.

Background

• CD25 is the interleukin-2 receptor α-chain present on the surface of various immune cells and is a key marker for identifying regulatory T cells (Tregs).
• Tregs play a crucial role in maintaining immune tolerance and hemostasis, influencing the development and progression of diseases such as allergies, autoimmunity, cancer, and chronic inflammation across different species.
• In horses, characterization of Tregs has previously relied on cross-reactive antibodies developed against human CD25, which may have limitations in specificity and sensitivity for equine cells.

Objectives

• To develop monoclonal antibodies specifically targeting equine CD25 for better accuracy in detecting Tregs and other CD25-expressing cells.
• To compare these equine-specific mAbs with an existing anti-human CD25 antibody with respect to staining patterns and detection efficiency in flow cytometry analyses.

Methods

• Generation of monoclonal antibodies directed against equine CD25.
• Flow cytometry was used to analyze staining patterns of equine peripheral blood lymphocytes using the new equine CD25 mAbs and the anti-human CD25 antibody.
• Two independent laboratories conducted separate comparative analyses to validate the findings.
• Peripheral blood mononuclear cells (PBMCs) were stimulated with pokeweed mitogen and also examined ex vivo and after culture without stimulation to assess differences in Treg detection.

Results

• Both the equine CD25 monoclonal antibody (notably clone 15-1) and the anti-human CD25 antibody showed similar staining patterns for equine peripheral blood lymphocytes.
• Comparable percentages of CD4+CD25+ and CD4+CD25+FOXP3+ Tregs were detected after pokeweed mitogen stimulation using both antibodies.
• The equine CD25 mAb 15-1 exhibited stronger staining intensity compared to the anti-human CD25 antibody, enhancing the resolution of CD25-positive cells.
• Higher percentages of Tregs and other CD25-expressing cells were identified using the equine-specific mAb both in freshly isolated cells (ex vivo) and in cultured PBMCs without stimulation.

Conclusions and Implications

• Monoclonal antibodies specifically developed for equine CD25 provide an improved tool for detecting and phenotyping regulatory T cells in horses.
• The increased sensitivity and staining intensity with these equine CD25 mAbs allow for more accurate identification of Tregs, which is important for veterinary immunology research.
• This advancement may facilitate better understanding of Treg roles in equine health and diseases, potentially contributing to improved diagnostics and therapies for immune-mediated disorders in horses.