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.

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.

Influence of deoxynivalenol-contaminated feed on the immune response of pigs after PRRSV vaccination and infection.

The impact of the Fusarium mycotoxin deoxynivalenol (DON) on the immune response against porcine reproductive and respiratory syndrome virus (PRRSV) vaccination and infection was investigated. Forty-two weaned piglets were separated into seven groups and received three different diets: Low DON (1.09 ppm), High DON (2.81 ppm) or No DON. These three treatments were split further into either vaccinated (Ingelvac PRRSFLEX EU) and challenged with PRRSV 28 days post-vaccination, or only infected at day 28. A seventh group received no DON, no vaccination, and no infection. Two weeks after challenge infection, when pigs were euthanized, the number of IFN-γ producing lymphocytes in the blood of vaccinated animals was lower in pigs on High DON compared to animals on Low DON or No DON. Intracellular cytokine staining showed that vaccinated animals fed with the Low DON diet had higher frequencies of TNF-α/IFN-γ co-producing CD4+ T cells than the other two vaccinated groups, particularly in lung tissue. Vaccinated animals on High DON had similar viral loads in the lung as the non-vaccinated groups, but several animals of the Low DON or No DON group receiving vaccination had reduced titers. In these two groups, there was a negative correlation between lung virus titers and vaccine-specific TNF-α/IFN-γ co-producing CD4+ T cells located either in lung tissue or blood. These results indicate that after PRRSV vaccination and infection, high levels of DON negatively influence immune parameters and clearance of the virus, whereas low DON concentrations have immunomodulatory effects.

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-αß+ 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.

Influence of PRRSV-1 vaccination and infection on mononuclear immune cells at the maternal-fetal interface.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most devastating viruses for the global swine industry. Infection during late gestation causes reproductive failure but the local immune response in utero remains poorly understood. In this study, an experimental PRRSV-infection model with two different PRRSV-1 field isolates was used to investigate the immune cell phenotypes at the maternal-fetal interface during late gestation. In addition, phenotypic changes induced by a modified live virus (MLV, ReproCyc® PRRS EU) vaccine were studied. Vaccinated (n = 12) and non-vaccinated pregnant gilts (n = 12) were challenged with either one of the PRRSV-1 field isolates (low vs. high virulent, LV or HV) or sham-inoculated at day 84 of gestation. Twenty-one days post infection all gilts were euthanized and the fetal preservation status for all fetuses per litter was assessed. Leukocytes from the maternal-fetal interface were isolated and PRRSV-induced changes were investigated using ex vivo phenotyping by flow cytometry. PRRSV load in tissue from the maternal endometrium (ME) and fetal placenta (FP) was determined by RT-qPCR. In the ME, a vast increase in CD8ß T cells with CD8αposCD27dim early effector phenotype was found for fetuses from the non-vaccinated LV and HV-challenged gilts, compared to non-treated and vaccinated-only controls. HV-challenged fetuses also showed significant increases of lymphocytes with effector phenotypes in the FP, including NKp46pos NK cells, CD8αhigh γδ T cells, as well as CD8αposCD27pos/dim CD4 and CD8 T cells. In vaccinated animals, this common activation of effector phenotypes was more confined and the fetal preservation status significantly improved. Furthermore, a negative correlation between the viral load and CD163highCD169pos mononuclear phagocytic cells was observed in the FP of HV-infected animals. These results suggest that the strong expansion of effector lymphocytes in gilts that were only infected causes immune-pathogenesis rather than protection. In contrast, the attenuated MLV seems to dampen this effect, yet presumably induces memory cells that limit reproductive failure. This work provides valuable insights into changes of local immune cell phenotypes following PRRSV vaccination and infection.

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-αß T cells were classified by their CD8ß+ or CD4 expression. For monitoring their differentiation, expression of CD27 and perforin was investigated for CD8ß++ 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.

Identification of MHC-I-Presented Porcine Respiratory and Reproductive Syndrome Virus (PRRSV) Peptides Reveals Immunogenic Epitopes within Several Non-Structural Proteins Recognized by CD8+ T Cells.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most relevant porcine pathogens worldwide. Active control of the disease relies on modified live virus vaccines (MLVs), as most inactivated vaccines provide very limited protection. Neutralizing antibodies occur late in infection; therefore, CD8+ T cells are considered important correlates of protection and are a frequent focus of investigation. Our aim was to identify viral peptides naturally bound by the class I major histocompatibility complex (MHC-I) and to confirm their ability to stimulate CD8+ T cells. For this purpose, we immunoprecipitated MHC-I/peptide complexes of PRRSV (strain AUT15-33) -infected cells (SLA-I Lr-Hp 35.0/24 mod) to isolate the viral epitopes and analyzed them with liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Furthermore, we employed these identified peptides to stimulate peripheral blood mononuclear cells (PBMCs) of previously PRRSV-infected pigs and measured the PRRSV-specific CD8+ T-cell response with an intracellular cytokine staining (ICS). Our data revealed that PRRSV non-structural proteins (NSPs), encoded in open reading frame 1a and 1b (ORF1), present the major source of MHC-I-presented peptides. Additionally, we show that our identified epitopes are able to trigger IFNγ responses in vitro. These findings are a basis for understanding the proteasomal degradation of PRRSV proteins, the cellular ability to display them via MHC-I, and their potential to restimulate CD8+ T cells.

An Equine Model for Vaccination against a Hepacivirus: Insights into Host Responses to E2 Recombinant Protein Vaccination and Subsequent Equine Hepacivirus Inoculation.

Equine hepacivirus (EqHV) is the closest known genetic homologue of hepatitis C virus. An effective prophylactic vaccine is currently not available for either of these hepaciviruses. The equine as potential surrogate model for hepacivirus vaccine studies was investigated, while equine host responses following vaccination with EqHV E2 recombinant protein and subsequent EqHV inoculation were elucidated. Four ponies received prime and booster vaccinations (recombinant protein, adjuvant) four weeks apart (day -55 and -27). Two control ponies received adjuvant only. Ponies were inoculated with EqHV RNA-positive plasma on day 0. Blood samples and liver biopsies were collected over 26 weeks (day -70 to +112). Serum analyses included detection of EqHV RNA, isotypes of E2-specific immunoglobulin G (IgG), nonstructural protein 3-specific IgG, haematology, serum biochemistry, and metabolomics. Liver tissue analyses included EqHV RNA detection, RNA sequencing, histopathology, immunohistochemistry, and fluorescent in situ hybridization. Al-though vaccination did not result in complete protective immunity against experimental EqHV inoculation, the majority of vaccinated ponies cleared the serum EqHV RNA earlier than the control ponies. The majority of vaccinated ponies appeared to recover from the EqHV-associated liver insult earlier than the control ponies. The equine model shows promise as a surrogate model for future hepacivirus vaccine research.

Co-Expression of the B-Cell Key Transcription Factors Blimp-1 and IRF4 Identifies Plasma Cells in the Pig.

Antibody-secreting plasma cells (PCs) have remained largely uncharacterized for years in the field of porcine immunology. For an in-depth study of porcine PCs, we identified cross-reactive antibodies against three key transcription factors: PR domain zinc finger protein-1 (Blimp-1), interferon regulatory factor 4 (IRF4), and paired box 5 (Pax5). A distinct Blimp-1+IRF4+ cell population was found in cells isolated from blood, spleen, lymph nodes, bone marrow, and lung of healthy pigs. These cells showed a downregulation of Pax5 compared to other B cells. Within Blimp-1+IRF4+ B cells, IgM-, IgG-, and IgA-expressing cells were identified and immunoglobulin-class distribution was clearly different between the anatomical locations, with IgA+ PCs dominating in lung tissue and IgM+ PCs dominating in the spleen. Expression patterns of Ki-67, MHC-II, CD9, and CD28 were investigated in the different organs. A high expression of Ki-67 was observed in blood, suggesting a plasmablast stage. Blimp-1+IRF4+ cells showed an overall lower expression of MHC-II compared to regular B cells, confirming a progressive loss in B-cell differentiation toward the PC stage. CD28 showed slightly elevated expression levels in Blimp-1+IRF4+ cells in most organs, a phenotype that is also described for PCs in mice and humans. This was not seen for CD9. We further developed a FACS-sorting strategy for live porcine PCs for functional assays. CD3-CD16-CD172a- sorted cells with a CD49dhighFSC-Ahigh phenotype contained Blimp-1+IRF4+ cells and were capable of spontaneous IgG production, thus confirming PC identity. These results reveal fundamental phenotypes of porcine PCs and will facilitate the study of this specific B-cell subset in the future.

Porcine Plasmacytoid Dendritic Cells Are Unique in Their Expression of a Functional NKp46 Receptor.

The activating receptor NKp46 shows a unique expression pattern on porcine leukocytes. We showed already that in swine not all NK cells express NKp46 and that CD3+NKp46+ lymphocytes form a T-cell subset with unique functional properties. Here we demonstrate the expression of NKp46 on CD4highCD14-CD172a+ porcine plasmacytoid dendritic cells (pDCs). Multicolor flow cytometry analyses revealed that the vast majority of porcine pDCs (94.2% ± 4) express NKp46 ex vivo and have an increased expression on the single-cell level compared to NK cells. FSC/SSChighCD4highNKp46+ cells produced high levels of IFN-α after CpG ODN 2216 stimulation, a hallmark of pDC function. Following receptor triggering with plate-bound monoclonal antibodies against NKp46, phosphorylation of signaling molecules downstream of NKp46 was analyzed in pDCs and NK cells. Comparable to NK cells, NKp46 triggering led to an upregulation of the phosphorylated ribosomal protein S6 (pS6) in pDCs, indicating an active signaling pathway of NKp46 in porcine pDCs. Nevertheless, a defined effector function of the NK-associated receptor on porcine pDCs could not be demonstrated yet. NKp46-mediated cytotoxicity, as shown for NK cells, does not seem to occur, as NKp46+ pDCs did not express perforin. Yet, NKp46 triggering seems to contribute to cytokine production in porcine pDCs, as induction of TNF-α was observed in a small pDC subset after NKp46 cross-linking. To our knowledge, this is the first report on NKp46 expression on pDCs in a mammalian species, showing that this receptor contributes to pDC activation and function.

Transcriptome Profiling of Porcine Naïve, Intermediate and Terminally Differentiated CD8+ T Cells.

The pig has the potential to become a leading research model for human diseases, pharmacological and transplantation studies. Since there are many similarities between humans and pigs, especially concerning anatomy, physiology and metabolism, there is necessity for a better understanding of the porcine immune system. In adaptive immunity, cytotoxic T lymphocytes (CTLs) are essential for host defense. However, most data on CTLs come from studies in mice, non-human primates and humans, while detailed information about porcine CD8+ CTLs is still sparse. Aim of this study was to analyze transcriptomes of three subsets of porcine CD8ß+ T-cell subsets by using next-generation sequencing technology. Specifically, we described transcriptional profiles of subsets defined by their CD11a/CD27 expression pattern, postulated as naïve (CD8ß+CD27+CD11alow), intermediate differentiated (CD8ß+CD27dimCD11a+), and terminally differentiated cells (CD8ß+CD27-CD11ahigh). Cells were analyzed in ex vivo condition as well as upon in vitro stimulation with concanavalin A (ConA) and PMA/ionomycin. Our analyses show that the highest number of differentially expressed genes was identified between naïve and terminally differentiated CD8+ T-cell subsets, underlining their difference in gene expression signature and respective differentiation stages. Moreover, genes related to early (IL7-R, CCR7, SELL, TCF7, LEF1, BACH2, SATB1, ZEB1 and BCL2) and late (KLRG1, TBX21, PRDM1, CX3CR1, ZEB2, ZNF683, BATF, EZH2 and ID2) stages of CD8+ T-cell differentiation were highly expressed in the naïve and terminally differentiated CD8+ T-cell subsets, respectively. Intermediate differentiated CD8+ T-cell subsets shared a more comparable gene expression profile associated with later stages of T-cell differentiation. Genes associated with cytolytic activity (GNLY, PRF1, GZMB, FASL, IFNG and TNF) were highly expressed in terminally and intermediate differentiated CD8+ T-cell subsets, while naïve CD8+ T cells lacked expression even after in vitro stimulation. Overall, PMA/ionomycin stimulation induced much stronger upregulation of genes compared to stimulation with ConA. Taken together, we provided comprehensive results showing transcriptional profiles of three differentiation stages of porcine CD8+ T-cell subsets. In addition, our study provides a powerful toolbox for the identification of candidate markers to characterize porcine immune cell subsets in more detail.

The Natural Cytotoxicity Receptor NKp44 (NCR2, CD336) Is Expressed on the Majority of Porcine NK Cells Ex Vivo Without Stimulation.

Natural killer (NK) cells have been studied extensively in humans and mice for their vital role in the vertebrate innate immune system. They are known to rapidly eliminate tumors or virus infected cells in an immune response utilizing their lytic properties. The natural cytotoxicity receptors (NCRs) NKp30 (NCR3), NKp44 (NCR2), and NKp46 (NCR1) are important mediators of NK-cell cytotoxicity. NKp44 expression was reported for NK cells in humans as well as in some non-human primates and found exclusively on activated NK cells. Previously, no information was available on NKp44 protein expression and its role in porcine lymphocytes due to the lack of species-specific monoclonal antibodies (mAbs). For this study, porcine-specific anti-NKp44 mAbs were generated and their reactivity was tested on blood and tissue derived NK cells in pigs of different age classes. Interestingly, NKp44 expression was detected ex vivo already on resting NK cells; moreover, the frequency of NKp44+ NK cells was higher than that of NKp46+ NK cells in most animals analyzed. Upon in vitro stimulation with IL-2 or IL-15, the frequency of NKp44+ NK cells, as well as the intensity of NKp44 expression at the single cell level, were increased. Since little is known about swine NK cells, the generation of a mAb (clone 54-1) against NKp44 will greatly aid in elucidating the mechanisms underlying the differentiation, functionality, and activation of porcine NK cells.

Composition of lymphocyte subpopulations in normal and mildly reactive peripheral lymph nodes in cats.

OBJECTIVES: Flow cytometric (FCM) immunophenotyping of lymphoid tissue aspirates is an available adjunct for feline lymphoma diagnostics. Reference data have only been established for feline peripheral blood. Studies investigating the composition of normal and mildly reactive feline lymph nodes (LNs) are lacking. The aim of this prospective study was to establish reference data for lymphocyte subpopulations in normal and mildly reactive feline peripheral LNs using a standardised multicolour panel of antibodies. METHODS: Macroscopically inconspicuous mandibular and/or popliteal LNs from 31 adult cats, which were euthanased for reasons other than haematological diseases, were excised and processed within 5 h after death. Multicolour flow cytometry using eight different feline-specific, anti-canine and human cross-reactive monoclonal antibodies used in current diagnostic marker panels was performed after cytological exclusion of pathological states and complemented by lymphocyte clonality testing, histopathology and immunohistochemistry (IHC) to ensure the absence of lymphoid disease. RESULTS: Of 31 cats, the immunophenotyping data of 24 individuals could be included as histopathology and clonality testing excluded a pathological condition. Lymphocyte populations showed the following positive antibody reactions: CD18+ 86.3% ± 13.86%, CD3+ 54.81% ± 11.10%, CD5+ 57.39% ± 12.66%, CD21+ 40.42% ± 12.40%, CD79alphacy+ (CD79αcy) 30.41% ± 13.49% and CD14+ 0.75% ± 1.35%. There were 30.88% ± 13.48% CD4+ and 12.91% ± 6.68% CD8+ cells. Cytology revealed a mixed population of mostly lymphoid cells in all samples. The absence of a monoclonal/oligoclonal neoplastic population was confirmed by lymphocyte clonality testing. Histopathology and IHC showed a normal or mildly reactive pattern in all cases. CONCLUSIONS AND RELEVANCE: This study establishes FCM immunophenotyping data of lymphocyte populations of normal and mildly reactive feline peripheral LNs. For the first time, anti-CD5, CD4, CD8 and CD21 reference data in normal and mildly reactive feline peripheral LNs are presented. CD18, CD3, CD14 and CD79αcy have been used to establish reference data for the first time in any feline material.

Human-like Response of Pig T Cells to Superagonistic Anti-CD28 Monoclonal Antibodies.

Because of its size, anatomical similarities, and now also accessibility to genetic manipulations, pigs are used as animal models for human diseases and immune system development. However, expression and function of CD28, the most important costimulatory receptor expressed by T cells, so far is poorly understood in this species. Using a newly generated mAb (mAb 3D11) with specificity for pig CD28, we detected CD28 on CD8+ and CD4+ αß T cells. Among γδ T cells, CD28 expression was restricted to a small CD2+ subpopulation of phenotypically naive cells. Functionally, CD28 ligation with mAb 3D11-costimulated porcine T cells, enhanced proliferation and cytokine secretion in vitro. We used a second, likewise newly generated but superagonistic, anti-CD28 mAb (CD28-SA; mAb 4D12) to test the function of CD28 on porcine T cells in a pilot study in vivo. Injection of the CD28-SA into pigs in vivo showed a very similar dose-response relationship as in humans (i.e., 100 µg/kg body weight [BW]) of CD28-SA induced a cytokine release syndrome that was avoided at a dose of 10 µg/kg BW and below. The data further suggest that low-dose (10 µg/kg BW) CD28-SA infusion was sufficient to increase the proportion of Foxp3+ regulatory T cells among CD4+ T cells in vivo. The pig is thus a suitable animal model for testing novel immunotherapeutics. Moreover, data from our pilot study in pigs further suggest that low-dose CD28-SA infusion might allow for selective expansion of CD4+ regulatory T cells in humans.

T-Cell Cytokine Response in Salmonella Typhimurium-Vaccinated versus Infected Pigs.

Vaccination with the live attenuated vaccine Salmoporc is an effective measure to control Salmonella Typhimurium (STM) in affected swine populations. However, the cellular immune response evoked by the Salmoporc vaccine including differences in vaccinated pigs versus non-vaccinated pigs upon STM infection have not been characterized yet. To investigate this, tissue-derived porcine lymphocytes from different treatment groups (vaccination-only, vaccination and infection, infection-only, untreated controls) were stimulated in vitro with heat-inactivated STM and abundances of IFN-γ, TNF-α and/or IL-17A-producing T-cell subsets were compared across organs and treatment groups. Overall, our results show the induction of a strong CD4+ T-cell response after STM infection, both locally and systemically. Low-level induction of STM-specific cytokine-producing CD4+ T cells, notably for the IFN-γ/TNF-α co-producing phenotype, was detected after vaccination-only. Numerous significant contrasts in cytokine-producing T-cell phenotypes were observed after infection in vaccinated and infected versus infected-only animals. These results suggest that vaccine-induced STM-specific cytokine-producing CD4+ T cells contribute to local immunity in the gut and may limit the spread of STM to lymph nodes and systemic organs. Hence, our study provides insights into the underlying immune mechanisms that account for the efficacy of the Salmoporc vaccine.

Flow Cytometric Assessment of Ki-67 Expression in Lymphocytes From Physiologic Lymph Nodes, Lymphoma Cell Populations and Remnant Normal Cell Populations From Lymphomatous Lymph Nodes.

Recent literature suggests conventional flow cytometric (FCM) immunophenotyping complemented by Ki-67 FCM assessment as a reliable tool to classify canine lymphomas. Ki-67 expression assessed by FCM is rarely reported in canine lymphoma cases and reference data for normal canine lymph nodes are missing. Moreover, nothing is known about the Ki-67 expression within the occasionally observed remnant cell population within the gates of normal lymphocytes in lymphoma cases. Aim of this study was to compare flow cytometric Ki-67 expression of lymphocyte populations from normal canine lymph nodes, lymphoma cells from World-Health-Organisation (WHO) classified lymphoma patient samples and their neighboring normal remnant cell population. Cryopreserved lymphocyte cell suspensions from normal lymph nodes from eight dogs free of lymphoma served as reference material. Fourteen cases diagnosed by cytology, FCM, clonality testing, histopathology including immunohistochemistry consisting of 10 DLBCL, 1 MZL, 1 PTCL and 2 TZL showed a residual small lymphocyte population and were investigated. The Ki-67 expression in normal canine lymphoid tissue was 3.19 ± 2.17%. Mean Ki-67 expression in the malignant cell populations was 41 ± 24.36%. Ki-67 positivity was 12.34 ± 10.68% in the residual physiologic lymphocyte population, which otherwise exhibited a physiologic immunophenotype pattern. This ratio was equivalent (n = 3) or lower (n = 11) than the Ki-67 expression of the malignant cell population within the sample. This is the first report of FCM derived Ki-67 expression combined with immunophenotype patterns in normal canine lymph nodes, compared with lymphoma cell populations and residual normal cell populations of lymphoma cases diagnosed by state of the art technology.

Expression of CD9 on porcine lymphocytes and its relation to T cell differentiation and cytokine production.

In this work, we report on two novel monoclonal antibodies, specific for porcine CD9. CD9 is a tetraspanin that is expressed on a wide variety of cells. We phenotyped porcine immune cell subsets and found that CD9 was expressed on all monocytes as well as a subset of B cells. CD9 was variably expressed on T cells, with CD4 T cells containing the highest frequency of CD9+ cells. CD9 expression positively correlated with the frequency of central memory CD4 T cells in ex vivo PBMC. Therefore, we proceeded to explore CD9 as a marker of T cell function. Here we observed that CD9 was expressed on the vast majority of long-lived influenza A virus-specific effector cells that retained the capacity for cytokine production in response to in vitro recall antigen. Therefore, the new antibodies enable the detection of a cell surface molecule with functional relevance to T cells. Considering the importance of CD9 in membrane remodelling across many cell types, they will also benefit the wider field of swine biomedical research.

NK and T Cell Differentiation at the Maternal-Fetal Interface in Sows During Late Gestation.

The phenotype and function of immune cells that reside at the maternal-fetal interface in humans and mice have been, and still are, extensively studied with the aim to fully comprehend the complex immunology of pregnancy. In pigs, information regarding immune cell phenotypes is limited and mainly focused on early gestation whereas late gestation has not yet been investigated. We designed a unique methodology tailored to the porcine epitheliochorial placenta, which allowed us to address immune phenotypes separately in the maternal endometrium (ME) and fetal placenta (FP) by flow cytometry. In-depth phenotyping of NK cells, non-conventional and conventional T cells within maternal blood (mBld), ME, FP, and fetal spleen (fSpln) revealed major differences between these anatomic sites. In both maternal compartments, all NK cells were perforin+ and had NKp46-defined phenotypes indicative of late-stage differentiation. Likewise, T cells with a highly differentiated phenotype including CD2+CD8α+CD27dim/-perforin+ γδ T cells, CD27-perforin+ cytolytic T cells (CTLs), and T-bet+ CD4+CD8α+CD27- effector memory T (Tem) cells prevailed within these compartments. The presence of highly differentiated T cells was also reflected in the number of cells that had the capacity to produce IFN-γ. In the FP, we found NK cells and T cell populations with a naive phenotype including CD2+CD8α-CD27+perforin- γδ T cells, T-bet-CD4+CD8α-CD27+ T cells, and CD27+perforin- CTLs. However, also non-naive T cell phenotypes including CD2+CD8α+CD27+perforin- γδ T cells, T-bet+CD4+CD8α+CD27- Tem cells, and a substantial proportion of CD27-perforin+ CTLs resided within this anatomic site. Currently, the origin or the cues that steer the differentiation of these putative effector cells are unclear. In the fSpln, NKp46high NK cells and T cells with a naive phenotype prevailed. This study demonstrated that antigen-experienced immune cell phenotypes reside at the maternal-fetal interface, including the FP. Our methodology and our findings open avenues to study NK and T cell function over the course of gestation. In addition, this study lays a foundation to explore the interplay between immune cells and pathogens affecting swine reproduction.

Deoxynivalenol Has the Capacity to Increase Transcription Factor Expression and Cytokine Production in Porcine T Cells.

Deoxynivalenol (DON) is a Fusarium mycotoxin that frequently contaminates the feed of farm animals. Pigs with their monogastric digestive system are in particular sensitive to DON-contaminated feed. At high concentrations, DON causes acute toxic effects, whereas lower concentrations lead to more subtle changes in the metabolism. This applies in particular to the immune system, for which immunosuppressive but also immunostimulatory phenomena have been described. Research in human and rodent cell lines indicates that this may be partially explained by a binding of DON to the ribosome and subsequent influences on cell signaling molecules like mitogen-activated protein kinases. However, a detailed understanding of the influence of DON on functional traits of porcine immune cells is still lacking. In this study, we investigated the influence of DON on transcription factor expression and cytokine production within CD4+, CD8+, and γδ T cells in vitro. At a DON concentration, that already negatively affects proliferation after Concanavalin A stimulation (0.8 µM) an increase of T-bet expression in CD4+ and CD8+ T cells was observed. This increase in T-bet expression coincided with elevated levels of IFN-γ and TNF-α producing T-cell populations. Increases in T-bet expression and cytokine production were found in proliferating and non-proliferating T cells, although increases were more prominent in proliferating cell subsets. Differently, IL-17A production by CD4+ T cells was not influenced by DON. In addition, frequencies of regulatory T cells and their expression of Foxp3 were not affected. In γδ T cells, GATA-3 expression was slightly reduced by DON, whereas T-bet levels were only slightly modulated and hence IFN-γ, TNF-α, or IL-17A production were not affected. Our results show for the single-cell level that DON has the capacity to modulate the expression of transcription factors and related cytokines. In particular, they suggest that for CD4+ and CD8+ T cells, DON can drive T-cell differentiation into a pro-inflammatory type-1 direction, probably depending on the already prevailing cytokine milieu. This could have beneficial or detrimental effects in ongoing immune responses to infection or vaccination.

Vaccination and Infection of Swine With Salmonella Typhimurium Induces a Systemic and Local Multifunctional CD4+ T-Cell Response.

The gram-negative facultative intracellular bacteria Salmonella Typhimurium (STM) often leads to subclinical infections in pigs, but can also cause severe enterocolitis in this species. Due to its high zoonotic potential, the pathogen is likewise dangerous for humans. Vaccination with a live attenuated STM strain (Salmoporc) is regarded as an effective method to control STM infections in affected pig herds. However, information on the cellular immune response of swine against STM is still scarce. In this study, we investigated the T-cell immune response in pigs that were vaccinated twice with Salmoporc followed by a challenge infection with a virulent STM strain. Blood- and organ-derived lymphocytes (spleen, tonsils, jejunal and ileocolic lymph nodes, jejunum, ileum) were stimulated in vitro with heat-inactivated STM. Subsequently, CD4+ T cells present in these cell preparations were analyzed for the production of IFN-γ, TNF-α, and IL-17A by flow cytometry and Boolean gating. Highest frequencies of STM-specific cytokine-producing CD4+ T cells were found in lamina propria lymphocytes of jejunum and ileum. Significant differences of the relative abundance of cytokine-producing phenotypes between control group and vaccinated + infected animals were detected in most organs, but dominated in gut and lymph node-residing CD4+ T cells. IL-17A producing CD4+ T cells dominated in gut and gut-draining lymph nodes, whereas IFN-γ/TNF-α co-producing CD4+ T cells were present in all locations. Additionally, the majority of cytokine-producing CD4+ T cells had a CD8α+CD27- phenotype, indicative of a late effector or effector memory stage of differentiation. In summary, we show that Salmonella-specific multifunctional CD4+ T cells exist in vaccinated and infected pigs, dominate in the gut and most likely contribute to protective immunity against STM in the pig.