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Major histocompatibility complex class I-restricted cytotoxic T-lymphocyte responses in horses infected with equine
G Allen1, M Yeargan, L R Costa
1Department of Veterinary Science, University of Kentucky, Lexington, Kentucky 40546.
Insights
A new assay reliably detects equine herpesvirus 1 (EHV-1)-specific cytotoxic T-lymphocyte (CTL) activity in horses. This EHV-1 CTL response develops quickly after infection and is mediated by CD8+ T cells.
Area of Science:
- Veterinary Immunology
- Virology
- Cellular Immunology
Background:
- Equine herpesvirus 1 (EHV-1) is a significant pathogen causing respiratory disease, abortion, and neurological signs in horses.
- Understanding the equine immune response, particularly cell-mediated immunity, is crucial for developing effective vaccines and treatments.
- Cytotoxic T-lymphocyte (CTL) responses play a key role in controlling viral infections by eliminating infected cells.
Purpose of the Study:
- To develop and optimize a sensitive and reproducible experimental system for detecting EHV-1-specific CTL activity in horses.
- To characterize the kinetics and phenotype of the equine CTL response following experimental EHV-1 infection.
- To establish a reliable assay for future studies on EHV-1 immunopathogenesis.
Main Methods:
- Peripheral blood mononuclear cells (PBMC) from EHV-1-immune horses were restimulated in vitro with live EHV-1.
- Cytotoxic activity was assessed using a 51Cr release assay with EHV-1-infected lymphoblast targets.
- Optimal conditions for CTL generation and target cell preparation were systematically identified.
- Effector cell phenotype was determined using flow cytometry (CD8+ T cells) and MHC restriction was assessed.
Main Results:
- An optimized system for generating and assaying EHV-1-specific equine CTLs was established.
- EHV-1-specific CTLs were detected in horses as early as 1 week post-infection, peaking at 2-3 weeks and persisting for up to a year.
- The primary effector cells were CD8+ T lymphocytes, mediating virus-specific and MHC class I-restricted cytotoxicity.
Conclusions:
- A reliable and convenient experimental system for EHV-1 CTL detection in horses has been developed.
- The study defines the kinetics and cellular basis of the equine CTL response to EHV-1 infection.
- This system provides a valuable tool for further research into EHV-1 immunity and pathogenesis.
Abstract:
An experimental system that permits sensitive and reproducible detection of equine herpesvirus 1 (EHV-1)-specific cytotoxic T-lymphocyte (CTL) activity in the horse was developed. Peripheral blood mononuclear cells (PBMC) collected from immune horses were restimulated in vitro by culture with live EHV-1. Cytotoxic activity against virus-infected, pokeweed mitogen-stimulated lymphoblast targets was assessed in a 4-h 51Cr release assay. The optimal conditions for in vitro stimulation of equine memory CTLs and for preparation of EHV-1-infected target cells expressing viral antigens were systematically identified by individually testing the effects of variations in responder cell concentration, culture medium composition, serum type, incubation time, antigen form, and exogenous mediator content. By using this optimized system for generation and assay of equine CTLs, the development of EHV-1-specific cytotoxic responses in 12 horses was evaluated after experimental viral infection. CTLs with the capacity for killing EHV-1-infected target cells were detected in equine PBMC as early as 1 week postinfection, reached maximal levels by 2 to 3 weeks, and remained detectable for a year after infection. Equine effector cells mediating lysis of EHV-1-infected targets were predominantly CD8+ T lymphocytes, and the cytotoxicity was specific for virus and restricted by major histocompatibility complex class I molecules. The results define a reliable and convenient experimental system for generation and assay of EHV-1 CTLs which can now be used for more-detailed characterization of the equine CTL response to infection by this herpesvirus pathogen.