Immunophenotyping Pig Immune Cells by Flow Cytometry
Why pigs? 2019 is the year of the Pig on the Chinese calendar, and pigs have been part of human history for a very long time. Pigs were domesticated independently in Europe and Asia ~9000 years ago from wild boar (Guiffra, 2000), and have been part of human agricultural practices since then. Because they share many physiological and anatomical similarities to humans, the use of the pig in biomedical research has steadily increased (Figure 1). Although dogs and non-human primates (NHP) have traditionally been used for this purpose, ethical concerns have increased the demand for alternatives (Swindle, 2012). Examples of the use of the pig in biomedical research include wound healing studies (skin), renal studies, GI disease, and genetically modified pig models of Cystic Fibrosis, heart disease/arrhythmias, and transplant.
Pork is the most widely eaten meat in the world, accounting for over 36% of the world meat intake. Thus raising healthy, pathogen resistant pigs is a major focus of research. Vaccines are the primary method to prevent infectious disease in pigs from zoonotic pathogens (Influenza, Salmonella, Erypsipelas, and Campylobacter), and animal-specific pathogens (Porcine reproductive and respiratory syndrome virus (PRRSV), Mycoplasma hyopneumoniae and Foot-and Mouth disease (Muztagh, 2014)). Pigs are the model of choice for the assessment of human vaccines for cancer and influenza (Mair, 2014; Overgaard, 2015). Because the focus on vaccine research in the pig, the basic knowledge of the porcine immune system is more extensive than that of the dog or old world monkeys (Bode, 2010). Pig-specific reagents for pig immune cells are available, and studies involving immune research in the pig have steadily increased (Figure 2).
The immune system of the pig is divided into the lymphoid (T, B and NK Cells), and myeloid (monocytes, dendritic cells, and all granulocytes, including neutrophils, eosinophils and basophils) lineages (Figure 3). Pigs have some unique aspects to their immunology (Rubic-Schneider, 2016).
• In general pigs have a higher number circulating leukocytes than other large animals
• The normal range of white blood cells (WBC) is 11,000-24,000 k/μL for adult pigs
• Like canines and humans, pigs have higher Neutrophils (50-70%) than NHP and rodents
• Pigs have a lower ratio of CD4:CD8 T cells (generally less than 1.0 in adult pigs)
• Pigs express more DP (double positive) CD4+/CD8+ cells than other species, and DP cells increase with age
• DP T cells are thought to have a memory phenotype
• DP T cells Increase after infection or vaccination
• Pigs have a higher percentage of γδ T cells than other species
• γδ T cells numbers are highest in younger animals (35-40% of total lymphocytes, 20% in adults)
• γδ T cells express SLA-DR (Swine Leukocyte Antigen-DR) and CD8 and may have cytolytic activity and memory
• Thought to result from “inverted” lymph node structure in pig
• CD8 is expressed as a homo-or heterodimer of α and β isoforms:
• CD8β is only found on CD8high cells
• Unlike other species, SLA-DR is preferentially expressed on CD8+ T cells and can be expressed on resting or activated cells
• Monocyte subtypes are defined by CD14 and CD163 (and not CD14/CD16)
At FCSL, we recognize the increasing use of the pig in biomedical research and have developed and currently offer Immunophenotyping panels to identify pig immune cellsOur pig Immunophenotyping panels identify the major cells of the lymphoid lineage: T cells, (helper CD4+CD8- T cells, Cytoxic CD4-CD8+ T cells, DP CD4+/CD8+ cells and γδ T cells), B cells and NK cells, and the myeloid lineage (monocytes). In addition we can monitor the activation status of Cytotoxic CD4-CD8+ and DP CD4+CD8+ cells using SLA-DR, and by using CD163 to identify activated monocytes. We are currently optimizing our methods to identify Regulatory T cells (Tregs) by intracellular FoxP3, and intracellular Interferon Gamma (INFγ) in CD8+ populations.
We recently presented a poster entitled “Immunophenotyping Pig Immune Cells: a Powerful Tool for Pharmacology and Toxicology Studies”, at the Society of Toxicology (SOT) meeting in Baltimore, MD, March 10-14, 2019. We are pleased to add the Pig to our existing immunophenotyping panels for humans, Non-Human Primates, canines, rat and mouse.
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