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Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus
Published on: May 7, 2012
General method for the detection and in vitro expansion of equine cytolytic T lymphocytes
S A Hammond1, C J Issel, R C Montelaro
1Department of Molecular Genetics and Biochemistry, School of Medicine, University of Pittsburgh, PA 15261, USA.
Insights
This study establishes a reliable method for culturing equine T cells and analyzing their antigen-specific cytotoxic T lymphocyte (CTL) activity. The protocol enables effective assessment of T cell responses against viral and parasitic antigens in horses.
Area of Science:
- Veterinary Immunology
- Cellular Immunology
- Equine Infectious Diseases
Background:
- Equine immunological research is limited by the absence of a standardized protocol for assessing T-cell activity against specific antigens.
- Developing such a method is crucial for understanding and combating viral and parasitic infections in horses.
Purpose of the Study:
- To develop and validate a reliable protocol for culturing equine T cells.
- To analyze antigen-specific cytotoxic T lymphocyte (CTL) activity in equine T cells.
- To establish a method applicable to various foreign antigens and potentially other animal models.
Main Methods:
- Equine T cells were cultured long-term using recombinant human IL-2 (200 U/ml).
- Pokeweed mitogen (PWM) stimulated peripheral blood mononuclear cells (PBMCs) generated autologous lymphoblasts expressing MHC class I and II.
- Antigen specificity was introduced using a recombinant vaccinia virus vector encoding the equine infectious anemia virus (EIAV) envelope glycoprotein.
- Secondary in vitro stimulation involved a 10:1 ratio of T cells to stimulator cells for optimal expansion of antigen-specific T lymphocytes.
- Cytolytic activity was measured using standard chromium release assays with antigen-expressing lymphoblasts as target cells.
Main Results:
- Effective long-term in vitro culture of equine T cells requires a minimum of 200 U/ml of recombinant human IL-2.
- PWM-stimulated PBMCs successfully generated antigen-presenting lymphoblasts.
- Optimal secondary stimulation of T lymphocytes occurred at a 10:1 T cell to stimulator cell ratio.
- Antigen-stimulated T lymphocytes exhibited specific cytolytic activity and included both CD4+ and CD8+ T cell populations.
- The developed methodology proved effective in analyzing antigen-specific CTL activity.
Conclusions:
- A reliable protocol for culturing equine T cells and assaying antigen-specific CTL activity has been established.
- This method utilizes EIAV-infected ponies as a model and is adaptable for other antigens.
- The protocol is expected to significantly advance equine immunological research and may be applicable to other species.
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.
