General method for the detection and in vitro expansion of equine cytolytic T lymphocytes

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.