Cytolytic Function for Pseudorabies Virus-Stimulated Porcine CD4 + CD8 dull+ Lymphocytes

We previously observed that pseudorabies (PRV) virus-specific killing in vitro was mediated by CD6⁺CD8⁺ lymphocytes. Also a high percentage of CD4⁺ lymphocytes, among these CD6⁺CD8⁺ lymphocytes, was observed. The purpose of this study was, therefore, to further characterize the killing ability of PRV-stimulated CD4⁺CD8⁺ lymphocytes. Peripheral blood mononuclear cells (PBMC) were isolated from blood of PRV-immune pigs and were stimulated in vitro with PRV. After 6 days, the frequency of CD4⁺CD8⁺ lymphocytes in peripheral blood was determined by flow cytometry analyses. Lymphocytes were separated using a magnet-activated cell sorter or a FACSVantage SE, and the cytolytic activity of the isolated populations was determined. Flow cytometry analyses demonstrated that PRV stimulation of immune PBMC resulted in the occurrence of 26% ± 4% CD4⁺CD8^dull+ lymphocytes. We further demonstrated that killing by PRV-stimulated PBMC was mediated by CD4⁺CD8^dull+ T lymphocytes and CD4⁻CD8⁺ T lymphocytes (classic cytolytic T lymphocytes and natural killer cells). The CD4⁺CD^8dull+ T lymphocytes showed major histocompatibility complex (MHC) II-restricted PRV-specific killing. The CD4⁻CD8⁺ T lymphocytes showed both PRV-specific and natural killing. The CD4⁺CD8^dull+ lymphocytes, which are unique in the pig, seemed to have a more heterogeneous function than was earlier demonstrated. In conclusion, we demonstrated that PRV-specific CD4⁺CD8^dull+ lymphocytes are able to kill PRV-infected target cells in a MHC II-restricted manner.