Gene expression of peripheral blood mononuclear cells and CD8+ T cells from gilts after PRRSV infection

Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive-stranded RNA virus, which emerged in Europe and U.S.A. in the late 1980s and has since caused huge economic losses. Infection with PRRSV causes mild to severe respiratory and reproductive clinical symptoms in pigs. Alteration of the host immune response by PRRSV is associated with the increased susceptibility to secondary viral and bacterial infections resulting in more serious and chronic disease. However, the expression profiles underlying innate and adaptive immune responses to PRRSV infection are yet to be further elucidated. In this study, we investigated gene expression profiles of PBMCs and CD8+ T cells after PRRSV AUT15-33 infection. We identified the highest number of differentially expressed genes in PBMCs and CD8+ T cells at 7 dpi and 21 dpi, respectively. The gene expression profile of PBMCs from infected animals was dominated by a strong innate immune response at 7 dpi which persisted through 14 dpi and 21 dpi and was accompanied by involvement of adaptive immunity. The gene expression pattern of CD8+ T cells showed a strong adaptive immune response to PRRSV, leading to the formation of highly differentiated CD8+ T cells starting from 14 dpi. The hallmark of the CD8+ T-cell response was the increased expression of effector and cytolytic genes (PRF1, GZMA, GZMB, GZMK, KLRK1, KLRD1, FASL, NKG7), with the highest levels observed at 21 dpi. Temporal clustering analysis of DEGs of PBMCs and CD8+ T cells from PRRSV-infected animals revealed three and four clusters, respectively, suggesting tight transcriptional regulation of both the innate and the adaptive immune response to PRRSV. The main cluster of PBMCs was related to the innate immune response to PRRSV, while the main clusters of CD8+ T cells represented the initial transformation and differentiation of these cells in response to the PRRSV infection. Together, we provided extensive transcriptomics data explaining gene signatures of the immune response of PBMCs and CD8+ T cells after PRRSV infection. Additionally, our study provides potential biomarker targets useful for vaccine and therapeutics development

Species comparison: human and minipig PBMC reactivity under the influence of immunomodulating compounds in vitro

Considering the similarities between swine and humans, it is a logical consequence to use swine as a translational model in research and drug development, including non-clinical safety. Here, we compared the reactivity of peripheral blood mononuclear cells (PBMCs) from humans and minipigs under the influence of different compounds in vitro. We conducted a flow cytometry-based proliferation assay that focused on the T-cell response to three different stimuli: concanavalin A (ConA), phytohemagglutinin-L (PHA-L), and staphylococcal Enterotoxin B (SEB). Furthermore, four approved immunosuppressive drugs-abatacept, belatacept, rapamycin, and tofacitinib-which are used for the treatment of rheumatoid arthritis or rejection in transplant recipients, were combined with the different stimuli. This allowed us to study the effect of suppressive drugs in comparison with the different stimuli in both species. We examined proliferating T cells (CD3+) and investigated the presence of TCR-αβ+ and TCR-γδ+ T cells. Differences in the response of T cells of the two species under these various conditions were evident. CD4+ T cells were more activated within humans, whereas CD8+ T cells were generally more abundant in swine. The effectiveness of the used humanized antibodies is most likely related to the conserved structure of CTLA-4 as abatacept induced a much stronger reduction in swine compared with belatacept. The reduction of proliferation of rapamycin and tofacitinib was highly dependent on the used stimuli. We further investigated the effect of the immunosuppressive compounds on antigen-specific restimulation of pigs immunized against porcine circovirus 2 (PCV2). Treatment with all four compounds resulted in a clear reduction of the proliferative response, with rapamycin showing the strongest effect. In conclusion, our findings indicate that the effectiveness of suppressive compounds is highly dependent on the stimuli used and must be carefully selected to ensure accurate results. The results highlight the importance of considering the response of T cells in different species when evaluating the potential of an immunomodulatory drug

Characterization of the immune system of Ellegaard Göttingen Minipigs - An important large animal model in experimental medicine

Interest in Ellegaard Göttingen Minipigs (EGMs) as a model in experimental medicine is continuously growing. The aim of this project is to increase the knowledge of the immune system of EGMs as information is still scarce. Therefore, we studied the postnatal maturation of their immune system from birth until 126 weeks of age. For the first 26 weeks of the study, animals were kept under pathogen-reduced conditions (SPF) and afterwards under conventional housing conditions. The development of the immune system was analyzed by monitoring changes in total numbers of leukocytes and lymphocytes of ten individuals and the composition of leukocyte populations by multi-color flow cytometry (FCM). We followed the presence of monocytes using monoclonal antibodies (mAbs) against CD172a+ and CD163+ and B cells based on the expression of CD79a. NK cells were distinguished as CD3-CD16+CD8α+/dim cells and further subdivided using NKp46 (CD335) expression into NKp46-, NKp46+, and NKp46high NK cells. T-cell receptor (TCR) γδ T cells were defined by the expression of TCR-γδ and different subsets were determined by their CD2 and perforin expression. TCR-αβ T cells were classified by their CD8β+ or CD4 expression. For monitoring their differentiation, expression of CD27 and perforin was investigated for CD8β++ T cells and CD8α together with CD27 for CD4+ T cells. We clearly detected a postnatal development of immune cell composition and identified phenotypes indicative of differentiation within the respective leukocyte subsets. Examination of the development of the antigen-specific immune system after transfer to different distinct housing conditions and after vaccination against common porcine pathogens such as porcine circovirus 2 (PCV2) revealed a markedly increased presence of more differentiated CD8+ and CD4+ T cells with central and effector memory T-cell phenotypes. To complement the findings, a PCV2 vaccine-specific antigen was used for in vitro restimulation experiments. We demonstrated antigen-specific proliferation of CD4+CD8α+CD27+ central and CD4+CD8α+CD27- effector memory T cells as well as antigen-specific production of TNF-α and IFN-γ. This study of postnatal immune development defines basic cellular immune parameters of EGMs and represents an important milestone for the use of EGMs for immunological questions in experimental medicine

Comparative analysis of swine leukocyte antigen gene diversity in Göttingen Minipigs

Worldwide, pigs represent economically important farm animals, also representing a preferred preclinical large animal model for biomedical studies. The need for swine leukocyte antigen (SLA) typing is increasing with the expanded use of pigs in translational research, infection studies, and for veterinary vaccine design. Göttingen Minipigs (GMP) attract increasing attention as valuable model for pharmacological studies and transplantation research. This study represents a first-time assessment of the SLA gene diversity in Göttingen Minipigs in combination with a comparative metadata analysis with commercial pig lines. As Göttingen Minipigs could harbor private as well as potential novel SLA allele combinations, future research projects would benefit from the characterization of their SLA background. In 209 Göttingen Minipigs, SLA class I (SLA-1, SLA-2, SLA-3) and class II (DRB1, DQB1, DQA) genes were characterized by PCR-based low-resolution (Lr) haplotyping. Criteria and nomenclature used for SLA haplotyping were proposed by the ISAG/IUIS-VIC SLA Nomenclature Committee. Haplotypes were assigned based on the comparison with already known breed or farm-specific allele group combinations. In total, 14 SLA class I and five SLA class II haplotypes were identified in the studied cohort, to manifest in 26 SLA class I but only seven SLA class II genotypes. The most common SLA class I haplotypes Lr-24.0 (SLA-1*15XX or Blank-SLA-3*04:04-SLA-2*06:01~02) and Lr-GMP-3.0 (SLA-1*16:02-SLA-3*03:04-SLA-2*17:01) occurred at frequencies of 23.44 and 18.66%, respectively. For SLA class II, the most prevalent haplotypes Lr-0.21 (DRB1*01XX-DQB1*05XX-DQA*04XX) and Lr-0.03 (DRB1*03:02-DQB1*03:01-DQA*01XX) occurred at frequencies of 38.28 and 30.38%. The comparative metadata analysis revealed that Göttingen Minipigs only share six SLA class I and two SLA class II haplotypes with commercial pig lines. More importantly, despite the limited number of SLA class I haplotypes, the high genotype diversity being observed necessitates pre-experimental SLA background assessment of Göttingen Minipigs in regenerative medicine, allo-transplantation, and xenograft research