General method for the detection and in vitro expansion of equine cytolytic T lymphocytes.
Abstract: Equine immunological research is hindered by the lack of a simple yet reliable general protocol by which to assay CTL activity specific for viral or parasitic antigens. We present here the first comprehensive analysis of the parameters necessary to reliably culture equine T cells and to analyze the antigen specific cytolytic activity of T lymphocytes utilizing the equine infectious anemia virus (EIAV) infection of outbred ponies as a source for in vivo primed T lymphocytes. Effective long-term in vitro culture of equine T cells was determined to require minimally 200 U/ml of recombinant human IL-2. We demonstrated that pokeweed mitogen (PWM) stimulated PBMC generated large quantities of MHC class I and MHC class II expressing autologous lymphoblasts that were used initially to activate and expand antigen specific T lymphocytes and later to serve as a source of target cells in standard chromium release assays. The source of antigen expressed by the PWM lymphoblasts was a recombinant vaccinia virus vector which carried sequences encoding various antigens of interest, but most specifically, the envelope glycoprotein of EIAV. Secondary in vitro stimulation of the T lymphocytes by autologous PWM lymphoblasts expressing EIAV envelope glycoprotein was maximal using a ratio of 10 T cells to one stimulator cell. After antigen stimulation, responding T lymphocytes had antigen specific cytolytic activity and were of both the CD4 and CD8 lineage. The methodology presented here should provide an effective and reliable means by which to analyze the cytolytic activity of equine T lymphocytes to other foreign antigens. Furthermore, we suggest that this method derived for the equine animal model should be applicable to other mammalian and avian model systems that currently lack an effective means by which to analyze antigen specific CTL activity.
Publication Date: 1998-07-22 PubMed ID: 9671126DOI: 10.1016/s0022-1759(98)00024-6Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
- Research Support
- U.S. Gov't
- P.H.S.
Summary
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This study presents a new method for cultivating and analysing the virus or parasite-targeting activity of cytolytic T lymphocytes in horses, a key aspect of immunity that has been difficult to investigate in this species due to lack of a reliable technique.
Research Aim and Methodology
- The goal of the study was to find a reliable way to grow and investigate the pathogen-specific activity of cytolytic T lymphocytes (CTLs), a type of immune cell, in horses. These cells’ activity was previously hard to identify in horses due to the lack of an efficient method.
- Researchers used equine infectious anemia virus (EIAV) infection in outbred ponies to source the T lymphocytes which had interacted with a virus in the body (“in vivo”). The extracted lymphocytes were then cultured in a lab (“in vitro”).
- To cultivate the T cells effectively, a minimum of 200 U/ml of recombinant human Interleukin-2 (IL-2), an immune system modulator, was found necessary.
- Pokeweed mitogen (PWM) was used to stimulate the generation of a large quantity of Major Histocompatibility Complex (MHC) class I and II, important molecules in immune system response, in autologous lymphoblasts (type of white blood cell). These lymphoblasts served twofold: they initiated activation and multiplication of T lymphocytes, and were later used as target cells for the T cells’ cytolytic activity in lab tests.
- The antigen (virus or parasite specific markers) that the lymphoblasts expressed came from a recombinant vaccinia virus vector. This virus carried the sequences encoding the antigens of interest, especially the envelope glycoprotein of the EIAV, which was used as the antigen of interest for this study.
Findings and Implications
- The researchers found that using a ratio of 10 T lymphocytes to one stimulator cell produced maximum stimulation in vitro.
- After antigen stimulation, responding T lymphocytes showed pathogen-specific cytolytic activity, demonstrating their role in immune response. Both CD4 and CD8 T lymphocytes were stimulated, highlighting the versatility of this method.
- The method developed here provides a solution to the existing scientific problem of analysing the cytolytic activity of equine T lymphocytes against foreign antigens.
- The research team suggests that this procedure developed for horses could also be used for other mammalian and avian species which currently lack an efficient method for investigating antigen-specific CTL activity.
Cite This Article
APA
Hammond SA, Issel CJ, Montelaro RC.
(1998).
General method for the detection and in vitro expansion of equine cytolytic T lymphocytes.
J Immunol Methods, 213(1), 73-85.
https://doi.org/10.1016/s0022-1759(98)00024-6 Publication
Researcher Affiliations
- Department of Molecular Genetics and Biochemistry, School of Medicine, University of Pittsburgh, PA 15261, USA.
MeSH Terms
- Animals
- Antigen-Presenting Cells
- Cell Division
- Cells, Cultured
- Horses
- Humans
- Immunologic Memory
- Immunophenotyping
- Interleukin-2 / pharmacology
- Lymphocyte Activation
- Pokeweed Mitogens / pharmacology
- T-Lymphocytes, Cytotoxic / cytology
- T-Lymphocytes, Cytotoxic / immunology
Grant Funding
- 5RO1 AI25850 / NIAID NIH HHS
- 5T32 AI07487 / NIAID NIH HHS
Citations
This article has been cited 1 times.- Mealey RH, Sharif A, Ellis SA, Littke MH, Leib SR, McGuire TC. Early detection of dominant Env-specific and subdominant Gag-specific CD8+ lymphocytes in equine infectious anemia virus-infected horses using major histocompatibility complex class I/peptide tetrameric complexes. Virology 2005 Aug 15;339(1):110-26.
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