Date Full Report Received01/05/2015
Date Abstract Report Received01/05/2015
InvestigationInstitution: Kansas State University
Primary Investigator: Melinda Wilkerson
Co-Investigators: Raymond R. Rowland, Catherine Ewen
Funded ByNational Pork Board
Objectives: The purpose of our studies was to understand how adaptive immunity contributes to the susceptibility of nursery-age piglets to porcine reproductive and respiratory syndrome virus (PRRSV). Typically, adaptive immunity, comprised of T and B cells, is central to the host’s ability to control and clear viral infections, through the generation of antibodies and cell-mediated immunity. However, a previous study conducted by our research group observed that pigs that were deficient in T and B cells, due to a genetic defect that led to severe combined immunodeficiency (SCID), developed less severe infections when exposed to PRRSV than normal littermates. This led us to speculate that some component of adaptive immunity was actually enhancing the ability of PRRSV to infect normal pigs. Therefore, we sought to utilize pigs with SCID in order to understand viral infection in the absence of adaptive immunity. Additionally, we proposed to selectively add back a population of T cells from normal donors in order to understand how these cells may be contributing to the susceptibility of young animals to PRRSV.
Method: We received pregnant sows from Iowa State University and, upon farrowing, screened the litters for the genetic defect and identified normal and SCID pigs. For our study that looked at acute PRRSV infection in SCID pigs, one litter was exposed to virus shortly after weaning. Serum was collected a numerous timepoints to assess levels of viral infection, and to monitor anti-viral responses. At the peak of acute infection, animals were sacrificed, and bronchoalveolar lavage fluid (BALF) and lung tissue were collected for analysis. Lung samples were subjected to immunopathological assessment to score tissue damage, while serum and BALF samples were monitored for anti-viral cytokine production.
In studies where we sought to repopulate SCID piglets with T cells, we adoptively transferred either mature T cells, or T cell precursors from normal pigs into SCID animals by intravenous injection. We collected blood samples at various timepoints to monitor whether T cells had become engrafted in the recipient pigs. At the end of the experiments, we collected blood, BALF, lymph nodes, spleen, gut, and thymus tissue to evaluate the level of T cell reconstitution. Tissue samples were subjected to immunopathological and immunohistochemical analysis for the detection of T cells. In addition, lymphocytes were isolated from these tissues, stained with antibodies, and analyzed by flow cytometry in order to determine what subsets of T cells were present in these animals.
Research Findings: As we had observed in a prior study, animals with SCID once again developed lower levels of circulating virus than their normal littermates. Pathology was also lower in many of the SCID pigs, which was surprising given their lack of adaptive immunity. Furthermore, these animals also produced lower levels of anti-viral cytokines, suggesting that the lower viremia loads were not a result of a more vigorous immune response. We also did not detect T cell-derived cytokines in serum of BALF, suggesting these mediators were not yet contributing to viral clearance, or suppressing the acute anti-viral immune response.
In reconstitution experiments, we detected the successful engraftment of T cells in SCID piglets that received T cell precursor cells, but not in those that received purified, mature T cells. These recipients developed a full repertoire of the T cell population, including a subset known to dampen immune responses, and one that may play a role in regulating macrophage permissiveness to PRRSV. Further studies may now be performed using our T cell reconstitution procedure to evaluate what role of T cells may play during acute PRRSV infection.
These results demonstrate that the pig SCID model has tremendous potential and utility for understanding how the different facets of the immune system contribute to PRRSV infection. We have developed a critical component of the model, as T cell reconstitution studies may lead to the identification of new host targets, and the generation of more successful vaccines in the future.
Melinda J. Wilkerson, DVM, MS, PhD
Director of Clinical Immunology/Flow Cytometry
Dept. Diagnostic Medicine/Pathobiology
Kansas State University
Raymond R. R. Rowland, Ph.D.
Department of Diagnostic Medicine and Pathobiology
Kansas State University
Catherine Ewen, PhD
Research Assistant Professor, Immunology
Associate Director Flow Cytometry Core Facility
Diagnostic Medicine/ Pathobiology
Kansas State University