Characterization of monoclonal antibodies to equine interleukin-10 and detection of T regulatory 1 cells in horses.
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 200pg/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-gamma (IFN-gamma) indicated that PMA and ionomycin stimulation induced 80% IL-10(+)/IFN-gamma(+) lymphocytes, while only 5% IL-10(+)/IL-4(+) cells were observed. By calculation, at least 60% of the IL-10(+)/IFN-gamma(+) cells were CD4(+) lymphocytes. This expression profile corresponds to the recently described T regulatory 1 (T(R)1) 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.
Publication Date: 2007-10-24 PubMed ID: 18061278DOI: 10.1016/j.vetimm.2007.10.012Google Scholar: Lookup
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- Journal Article
- Research Support
- N.I.H.
- Extramural
- Research Support
- Non-U.S. Gov't
- Research Support
- U.S. Gov't
- Non-P.H.S.
Summary
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This research paper details the creation and analysis of three antibodies that target Equine Interleukin-10 (IL-10), an important regulator in horse immune responses. The results confirmed the specificity of the antibodies for equine IL-10 and their potential for detecting IL-10 in controlled laboratory conditions.
Creation and Analysis of Monoclonal Antibodies
- The study produced three monoclonal antibodies (mAbs), specifically against equine IL-10. IL-10 is a critical immune system protein that suppresses inflammatory responses and impedes the activation of certain immune cells.
- The specificity of these antibodies was then analyzed using diverse recombinant equine cytokine/IgG fusion proteins, confirming that the antibodies were unique to equine IL-10.
Antibody Use in ELISA
- Two of the three mAbs were selected for use in an Enzyme-Linked Immunosorbent Assay (ELISA). This illustrated their capacity to detect IL-10 secretions in horse peripheral blood mononuclear cells (PBMC) in certain conditions.
- The ELISA demonstrated a sensitive detection level for secreted IL-10 at around 200pg/ml.
Flow Cytometry Analysis
- The research also evaluated the intracellular IL-10 production in horse PBMCs using flow cytometry, a technique used to analyze characteristics of cells in a sample.
- For stimulation, PBMCs were exposed to a variety of compounds including Phorbol 12-myristate 13-acetate (PMA) and ionomycin, which were both combined with the secretion blocker Brefeldin A.
- PMA-stimulated lymphocytes had a distinguishable presence of IL-10, while it was absent in the control samples.
Identification of Immune Cell Types
- Approximately 80% of detected IL-10 cells were CD4(+) lymphocytes while around 15% were CD8(+) cells.
- With the addition of other proteins for double staining, 80% of IL-10 positive cells were also shown to be interferon-gamma positive. Around 5% of the cells were found to be positive for both IL-10 and IL-4.
- The experiment figured at least 60% of IL-10 and interferon-gamma positive cells were CD4(+) lymphocytes. This profile aligns with the regulatory T1 cell phenotype.
Summary of Findings
- In conclusion, the newly produced antibodies could effectively denote native equine IL-10 using ELISA and flow cytometry. This finding paved the way for the further exploration of IL-10’s significant regulatory function in horse immune systems.
Cite This Article
APA
Wagner B, Hillegas JM, Brinker DR, Horohov DW, Antczak DF.
(2007).
Characterization of monoclonal antibodies to equine interleukin-10 and detection of T regulatory 1 cells in horses.
Vet Immunol Immunopathol, 122(1-2), 57-64.
https://doi.org/10.1016/j.vetimm.2007.10.012 Publication
Researcher Affiliations
- Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA. bw73@cornell.edu
MeSH Terms
- Animals
- Antibodies, Monoclonal / immunology
- Enzyme-Linked Immunosorbent Assay
- Horses / immunology
- Interferon-gamma / biosynthesis
- Interleukin-10 / blood
- Interleukin-10 / immunology
- Interleukin-4 / biosynthesis
- T-Lymphocytes, Regulatory / classification
- T-Lymphocytes, Regulatory / immunology
- Tetradecanoylphorbol Acetate / pharmacology
Grant Funding
- 1 R01 HD049545 / NICHD NIH HHS
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