Abstract
Pigs are important livestock and comprehensive understanding of their immune responses in infections is critical to improve vaccines and therapies. Moreover, similarities between human and swine physiology suggest that pigs are a superior animal model for immunological studies. However, paucity of experimental tools for a systematic analysis of the immune responses in pigs represent a major disadvantage. To evaluate the pig as a biomedical model and additionally expand the knowledge of rare immune cell populations in swine, we established a multicolor flow cytometry analysis platform of surface marker expression and cellular responses for porcine invariant Natural Killer T cells (iNKT). In humans, iNKT cells are among the first line defenders in various tissues, respond to CD1d-restricted antigens and become rapidly activated. Naïve porcine iNKT cells were CD3+/CD4−/CD8+ or CD3+/CD4−/CD8− and displayed an effector- or memory-like phenotype (CD25+/ICOS+/CD5hi/CD45RA−/CCR7 ± /CD27+). Based on their expression of the transcription factors T bet and the iNKT cell-specific promyelocytic leukemia zinc finger protein (PLZF), porcine iNKT cells were differentiated into functional subsets. Analogous to human iNKT cells, in vitro stimulation of porcine leukocytes with the CD1d ligand α-galactosylceramide resulted in rapid iNKT cell proliferation, evidenced by an increase in frequency and Ki-67 expression. Moreover, this approach revealed CD25, CD5, ICOS, and the major histocompatibility complex class II (MHC II) as activation markers on porcine iNKT cells. Activated iNKT cells also expressed interferon-γ, upregulated perforin expression, and displayed degranulation. In steady state, iNKT cell frequency was highest in newborn piglets and decreased with age. Upon infection with two viruses of high relevance to swine and humans, iNKT cells expanded. Animals infected with African swine fever virus displayed an increase of iNKT cell frequency in peripheral blood, regional lymph nodes, and lungs. During Influenza A virus infection, iNKT cell percentage increased in blood, lung lymph nodes, and broncho-alveolar lavage. Our in-depth characterization of porcine iNKT cells contributes to a better understanding of porcine immune responses, thereby facilitating the design of innovative interventions against infectious diseases. Moreover, we provide new evidence that endorses the suitability of the pig as a biomedical model for iNKT cell research.
Highlights
Biomedical research is in need of large animal models that reflect human infectious diseases better than current rodent models [1, 2]
In contrast to the vast heterogeneity of T cell receptors (TCR) among conventional CD3+ T cells, invariant Natural Killer T (iNKT) cells possess a semi-invariant TCR. This TCR is restricted to the non-classical major histocompatibility complex (MHC) class I-related CD1d, presenting lipid or glycolipid antigens. iNKT cells can be activated antigen-dependently with glycolipids derived from microbes or the host by TCR-CD1d interactions or antigen-independently via cytokines, mainly interleukin-(IL-)12 and IL-18 or type I interferons (IFN)
After exclusion of doublet cells, live CD3+ T cells (cTC) were gated as CD3+/PBS57 Tet− cells and iNKT cells were defined as CD3+/PBS57 Tet+ among the lymphocyte gate [Figure 1A [48]]
Summary
Biomedical research is in need of large animal models that reflect human infectious diseases better than current rodent models [1, 2]. Leukocytes at systemic and peripheral sites are eminently important for control of microbial colonization and defense against infections by induction of protective immunity [11] One of those leukocyte subsets are invariant Natural Killer T (iNKT) cells. In contrast to the vast heterogeneity of T cell receptors (TCR) among conventional CD3+ T cells (cTC), iNKT cells possess a semi-invariant TCR This TCR is restricted to the non-classical major histocompatibility complex (MHC) class I-related CD1d, presenting lipid or glycolipid antigens. INKT cells rapidly proliferate and secrete effector molecules like IFNγ, IL-17 or granulocyte-macrophage colonystimulating factor after activation [14]. They are able to lyse infected cells by perforin and Fas/FasL interaction [15,16,17,18]. Differentiated subsets of human iNKT cells are not as well-defined as in mice [27]
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