Discrimination of different subsets of cytolytic cells in pseudorabies virus-immune and naive pigs.

We previously observed that pseudorabies virus (PRV)-induced, cell-mediated cytolysis in pigs includes killing by natural killer (NK) cells. We also observed that IL-2 stimulation in vitro of naive PBMC expands porcine NK cells. The purpose of this study was to compare the phenotypes of the cytolytic subsets stimulated in vitro by PRV and by IL-2. PBMC were isolated from blood of PRV-immune and naive pigs and stimulated in vitro with PRV or IL-2. After 6 days, the frequency of various lymphocyte subsets in these cultured PBMC was determined by flow cytometry: the cells were separated with a magnet-activated cell sorter and the cytolytic activity of the separated populations was determined. When lymphocytes were separated and analysed with FACScan, the following lymphocyte subsets were discriminated: CD6(+) CD8(bright+) CD4(-) (CTL phenotype), CD6(+) CD8(dull+) CD4(+) (the fraction containing memory T helper cells), CD6(+) CD8(-) CD4(+) (T helper cell phenotype), CD6(-) CD8(dull+) CD4(-) gammadelta-T(+) ( gammadelta-T cell phenotype), CD6(-) CD8(dull+) CD4(-) gammadelta-T(-) (NK phenotype) and CD6(-) CD8(-) CD4(-) gammadelta-T(-) or gammadelta-T(+). Flow cytometry analysis demonstrated that PRV stimulation of immune PBMC resulted in the occurrence of more CD6(+) CD8(+) and CD4(+) CD8(+) and fewer CD6(-) CD8(+) and gammadelta-T(+) CD8(+) lymphocytes than IL-2 stimulation of naive PBMC (P<0.05). It was demonstrated further that killing by PRV-stimulated PBMC was mediated mainly by CD6(+) CD8(+) T lymphocytes. Killing by IL-2-stimulated PBMC was mediated mainly by CD6(-) CD8(+) T lymphocytes. These results demonstrate that both natural killing and killing by classical PRV-specific CTL were detected in PRV-immune pigs, whereas IL-2 stimulation of PBMC isolated from naive pigs mainly induced natural killing.

Changes of leukocyte phenotype and function in the broncho-alveolar lavage fluid of pigs infected with porcine reproductive and respiratory syndrome virus: a role for CD8(+) cells.

Porcine reproductive and respiratory virus (PRRSV) primarily infects and destroys alveolar macrophages of the pig. The aim of the present study was to characterize the changes of leukocyte populations in the broncho-alveolar lavage fluid (BALF) of PRRSV-infected pigs. Piglets were inoculated intranasally with PRRSV strain LV ter Huurne. On various days post-infection the piglets were sacrificed and the lungs removed, washed semi-quantitatively and analysed by flow cytometry. The total number of recovered BALF cells increased approximately 10 times between day 10 and day 21 of infection and decreased thereafter. The number of small low-autofluorescent cells (SLAC), i.e. lymphocytic and monocytic cells, increased very strongly from day 2 until day 21 of infection; in contrast, the number of large highly autofluorescent cells (LHAC), i.e. mostly macrophages, remained constant until day 14 of infection, increased slightly on day 21 and then decreased. On day 21 of infection in specific-pathogen-free piglets approximately 60% of the SLAC consisted of CD2(+)CD8(+)CD4(-)gammadeltaTCR(-) cells, which were partly CD8(+)CD6(+) and partly CD8(+)CD6(-). These phenotypes correspond to that of cytotoxic T-cells and natural killer cells respectively. From these results we can conclude that during a PRRSV infection the total number of BALF cells increases mainly due to an influx of lymphocytic cells with a cytolytic phenotype.

Cytolytic function for pseudorabies virus-stimulated porcine CD4+ CD8dull+ 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+ CD8dull+ lymphocytes. We further demonstrated that killing by PRV-stimulated PBMC was mediated by CD4+ CD8dull+ T lymphocytes and CD4- CD8+ T lymphocytes (classic cytolytic T lymphocytes and natural killer cells). The CD4+ CD8dull+ 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+ CD8dull+ 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+ CD8dull+ lymphocytes are able to kill PRV-infected target cells in a MHC II-restricted manner.

Interaction of pseudorabies virus with immortalized porcine B cells: influence on surface class I and II major histocompatibility complex and immunoglobulin M expression.

We examined whether the L14 cell line, an immortalized B cell line originating from inbred miniature pigs of the MHC haplotype d/d, could be useful to study T cell responses of pigs to pseudorabies virus (PRV). Compared with porcine kidney cells, the replication of PRV in L14 cells was slower and yielded lower quantities of infectious virus, which agrees with the reported poor replication of PRV in peripheral blood lymphocytes of swine. The virus yield and the number of L14 cells expressing the viral glycoprotein gE were both maximal at 48 h postinfection, when approximately 90% of all viable L14 cells expressed gE. Morphologically detectable effects of PRV replication in L14 cells were not obvious, but the number of viable cells at 72 h postinfection was lower in infected cultures than in uninfected cultures. Major histocompatibility complex (MHC) class I and II antigen expression was significantly higher at different time points postinfection on infected than on uninfected L14 cells. In contrast, expression of IgM appeared very slightly reduced on infected L14 cells, indicating a selective influence of PRV on cellular protein expression. PRV-infected L14 cells were lysed by lymphocytes from PRV-immune minipigs of MHC haplotype d/d, indicating their usefulness in in vitro cytolytic assays.

Cellular immune response to hog cholera virus (HCV): T cells of immune pigs proliferate in vitro upon stimulation with live HCV, but the E1 envelope glycoprotein is not a major T-cell antigen.

T-cell responses of pigs to hog cholera virus (HCV) have reportedly been absent or difficult to detect. Therefore, little is known about cellular immunity to HCV. In this study, we used an attenuated strain of pseudorabies virus expressing the envelope glycoprotein E1 of HCV and purified recombinant E1 to examine whether the E1 protein is a target antigen recognized by the T cells of HCV-immune pigs. We were unable to identify the E1 protein as a major target antigen recognized by the T cells of HCV-immune animals. However, such cells proliferated in vitro upon stimulation with viable HCV antigen. The lymphoproliferative response to HCV was strictly time and dose dependent and could be induced upon stimulation by live but not by UV light-inactivated HCV. Depletion studies demonstrated that lymphoproliferation depended on the presence of CD2+CD8bright+ lymphocytes, but CD2+CD4+ cells also contributed to the lymphoproliferative response. The primary lymphoproliferative response in animals inoculated with 10(7) 50% tissue culture infective doses of strain Brescia 2.1.1 was stronger than that observed in animals inoculated with 10(3) 50% tissue culture infective doses of the Cedipest strain. A remarkable finding was the increase in non-antigen-specific lymphoproliferation upon inoculation of the animals with HCV strains. This immunological phenomenon may mask a specific T-cell response to the virus.