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

Bettina Wagner, Julia M. Hillegas, Danielle R. Brinker, David W. Horohov, Douglas F. Antczak

Research output: Contribution to journalArticlepeer-review

50 Scopus citations

Abstract

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 (TR1) 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.

Original languageEnglish
Pages (from-to)57-64
Number of pages8
JournalVeterinary Immunology and Immunopathology
Volume122
Issue number1-2
DOIs
StatePublished - Mar 15 2008

Bibliographical note

Funding Information:
This work was supported by NIH grant # 1 R01 HD049545 and the Zweig Memorial Fund for Equine Research. The characterization of the equine anti-IL10 mAs was also supported by USDA Grant #2005-01812 (The US Veterinary Immune Reagent Network).

Keywords

  • ELISA
  • Equine cytokines
  • Flow cytometry
  • Interleukin-10
  • T1 cells

ASJC Scopus subject areas

  • Immunology
  • General Veterinary

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