Evaluation of equine xenogeneic mixed lymphocyte reactions using 5-ethynyl-2’-deoxyuridine (EdU)

Abstract

Allogeneic solid organ transplantation is currently the only treatment option for end stage organ disease. The shortage of available donor organs has driven efforts to utilize xenogeneic organs for transplantation. In vitro methods for evaluating immune-compatibility are a quick and low cost means of screening novel tissue products prior to more involved, expensive, and invasive live animal studies. Recently, a new analog of the DNA base thymidine, 5-ethynyl-2’-deoxyuridine (EdU), was developed. It may be used in a fast, efficient and specific means of evaluating cell proliferation via flow cytometry. This study was designed to test and optimize this platform for assessing equine xenogeneic one-way mixed lymphocyte reaction (MLR) to porcine stimulator cells. Furthermore, it was hypothesized that an enriched T-lymphocyte (T-cell) population would generate a stronger proliferative response to stimulation, and higher levels of cytokine production when compared to unfractionated peripheral blood mononuclear cells (PBMCs). PBMCs and T-cells were isolated from 3 horses and 4 pigs. Equine xenogeneic MLRs were set up using porcine allogeneic MLRs as a reference for clinically acceptable levels of cell proliferation. Equine T-cells showed significantly greater EdU incorporation in one-way xenogeneic MLRs than equine PBMCs. However, there was no significant difference in cell proliferation between porcine T-cell and PBMC as responders in allogenic one-way MLRs. Given the results of this study, we consider that enriched equine T-cells should be used in preference to unfractionated PBMCs when attempting to evaluate the equine xenogeneic response using the EdU assay as an indicator of suitability for transplant in vivo.