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Abstract: Surface expression of IL-2R-alpha (CD25) is widely used to identify activated lymphocyte populations, while interferon-gamma (IFN-γ) levels have been shown to be a good indicator of cell-mediated immunity (CMI) in pigs. To investigate the relationship between these two parameters, we developed an intracellular cytokine-staining assay and studied the kinetics of cytokine (IFN-γ and interleukin-10, IL-10) production relative to CD25 expression in porcine lymphocyte subpopulations, following immunization with a classical swine fever (CSF) vaccine. The number of activated memory T cells (CD4+CD8+CD25+ cells) increased slightly in the peripheral blood mononuclear cell (PBMC) population soon after vaccination, then diminished within a few weeks. The number of activated cytotoxic T cells (CD4−CD8+CD25+ cells) peaked approximately 2 weeks after the memory population. Although the number of IFN-γ producing cells detected in this experiment was relatively low, the CD4+CD8+ T cells were major IFN-γ producers in the PBMCs throughout the experiment. In another experiment, CSF-vaccinated pigs were challenged with a virulent classical swine fever virus (CSFV), and the kinetics of CD25 expression and cytokine productions were monitored. Following exposure to the virus, the number of IFN-γ producing cells in the PBMCs increased markedly in both the vaccinated and unvaccinated groups. The CD4−CD8+ cells were major IFN-γ producing cells in vaccinated pigs, while both CD4+CD8+ and CD4−CD8+ populations contributed to the IFN-γ production in the control group. Interestingly, the enhanced IFN-γ production was not associated with the upregulation of CD25 expression following the CSFV challenge. In addition, exposure to the virulent CSFV significantly increased interleukin-10 production by the CD4−CD8+ populations in PBMCs of the unvaccinated pigs. Taken together, our results indicated that CD25 expression and IFN-γ production were not tightly associated in porcine lymphocytes. In addition, the CD4−CD8+ lymphocytes of the PBMCs played a major role in cytokine productions following the CSFV challenge. [Copyright &y& Elsevier]

Abstract: Increases in numbers or activities of regulatory T lymphocytes (Tregs) have been linked to the establishments of several persistent infections. It has been previously shown that porcine reproductive and respiratory syndrome virus (PRRSV) can negatively modulate the host immune responses, resulting in persistent infection and secondary immunodeficiency. Recently, the existence of porcine CD4+CD25+ Tregs has been demonstrated. We investigated the effect of PRRSV on the CD4+CD25+ Tregs. The CD4+CD25+Foxp3+ T lymphocytes in the peripheral blood mononuclear cells (PBMCs) were identified, using the anti-human anti-Foxp3 monoclonal antibody. In vitro culture of porcine PBMC in the presence of PRRSV, but not classical swine fever virus, significantly increased the numbers of Foxp3+ lymphocytes, particularly in the CD4+CD25high subpopulation. The time-course study revealed that PRRSV significantly increased the numbers of viral-specific CD4+CD25highFoxp3+ subpopulation in the culture starting from 12h through the end of the observation period. Consistent to the results obtained by flow cytometry, enhanced Foxp3 gene expression was observed in the PBMC cultured with PRRSV in a time-course manner. The presence of monocyte-derived DC in the co-culture significantly enhanced the induction of CD4+CD25+ Foxp3+ T lymphocytes. The PRRSV-induced CD4+CD25high T lymphocytes exhibited suppressive activity when co-cultured with PHA-activated, autologous peripheral blood leukocytes, indicating the suppressive activity of the PRRSV-specific Tregs. In addition, PRRSV exposure significantly increased the numbers of PRRSV-specific CD4+CD25+Foxp3+ subpopulation in the PBMC of infected pigs at 10 days post-infection. In summary, the results indicated that PRRSV could increase the numbers of viral-specific, inducible regulatory T lymphocytes in the porcine PBMC, both in vitro and in vivo. The findings suggested the novel immunomodulatory mechanism induced by PRRSV. [Copyright &y& Elsevier]