Characterization of monoclonal antibodies to equine interleukin-10 and detection of T regulatory 1 cells in horses

Interleukin-10 (IL-10) terminates inflammatory immune responses and inhibits activation and effector functions of T-cells, monocytes, macrophages and dendritic cells. IL-10 has also been found to be a key cytokine expressed by subpopulations of regulatory T-cells. In this report, we describe the generation and characterization of three monoclonal antibodies (mAbs) to equine IL-10. The antibodies were found to be specific for equine IL-10 using different recombinant equine cytokine/IgG fusion proteins. Two of the anti-equine IL-10 mAbs were selected for ELISA to detect secreted IL-10 in supernatants of mitogen stimulated equine peripheral blood mononuclear cells (PBMC). The sensitivity of the ELISA for detecting secreted IL-10 was found to be around 200 pg/ml. The production of intracellular IL-10 was measured in equine PBMC by flow cytometry. PBMC were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin in the presence of the secretion blocker Brefeldin A. All three anti-IL-10 mAbs detected a positive population in PMA stimulated lymphocytes which was absent in the medium controls. Around 80% of the IL-10⁺ cells were CD4⁺. Another 15% were CD8⁺ cells. Double staining with IL-4 or interferon-γ (IFN-γ) indicated that PMA and ionomycin stimulation induced 80% IL-10⁺/IFN-γ⁺ lymphocytes, while only 5% IL-10⁺/IL-4⁺ cells were observed. By calculation, at least 60% of the IL-10⁺/IFN-γ⁺ cells were CD4⁺ lymphocytes. This expression profile corresponds to the recently described T regulatory 1 (T_R1) cell phenotype. In summary, the new mAbs to equine IL-10 detected native equine IL-10 by ELISA and flow cytometry and can be used for further characterization of this important regulatory cytokine in horses.