Development of B lymphocytes
Based on studies of sheep, the ileal Peyer’s patches (IPP) have been regarded as a type of primary lymphoid tissue similar to the bursa of Fabricius in chicken. It was assumed that there is massive antigen-independent B cell repertoire development in the IPP and that diversification occurs by somatic hypermutation and/or gene conversion. This concept has permeated other scientific reports, reviews and immunology textbooks for more than 20 years and due to the similarity in the organization of IPP, was broadened also to other species that include members of artiodactyls (including pig) and probably some cetaceans and carnivores. However, our previous data were in conflict with this presumption (Sinkora and Butler, 2009). For this reason we have designed a series of experiments using germ-free and gnotobiotic piglets to demonstrate dispensability of ileal Peyer's patches for B cell development. The results disprove the concept that porcine ileal Peyer's patches are a significant source of B cells, are required for systemic B cell generation and maintenance and are a site of B cell lymphogenesis in swine (Butler and Sinkora, 2013). We have also clearly shown that maturation of B cells in ileal Peyer's patches is antigen-dependent and it is heavily dependent on colonization (Potockova et al., 2015; Butler et al., 2017), which indicates that ileal Peyer's patches are a secondary lymphoid tissue that appear important in immune responses to colonizing bacteria. On the other hand, using flow cytometry sorting and semi-quantitative PCR detection of rearrangement-specific transcripts and DNA products we show that the bone marrow is a major B lymphopoietic organ of pigs (Sinkora and Sinkorova, 2014). Our most recent work resolved the course and differentiation pathways of B cell maturation in the bone marrow and showed that immunoglobulin light chain precedes heavy chain gene rearrangement during the development of B cells in swine (Sinkora et al., 2017).
Peripheral B lymphocytes
Our studies of B lymphocytes in pigs also involve characterization of B cell maturation in the periphery. Ontogenetic and in vitro culture studies, analysis of cell size, expression of CD11b and class-switched phenotype together with proliferation and cell death measurement, revealed four subpopulations of functionally different B cells in the periphery according to expression of CD2 and CD21 (Sinkora et al., 2013). Moreover, we have shown that CD21 molecules are always present on all mature B cells but can be expressed in at least two different forms CD21a and CD21b. We have also described that end-stage B cells can express these different form of CD21, which can be significant for their function, and that expression of these forms can be used for monitoring of B cell maturation during infection (Sinkora et al., 2014, Butler et al., 2014) and colonization (Potockova et al., 2015).
Development of αβ T lymphocytes
In comparison to other species, swine express some differences in the αβ T cell pool that may reflect modifications in T lymphocyte ontogeny and differentiation (Sinkora et al., 2005a) . For this reason, we have characterized development of αβ T cell in thymus, which is the primary lymphocyte tissue for these cells. Despite unique features of peripheral αβ T cells, developing thymocytes in pigs mostly resemble those in other species (Sinkora et al., 2000) . However, long gestation in swine allows us to describe successive colonization waves of thymus by hemopoietic progenitors during embryogenesis. We have also shown that the influx of thymocyte progenitors is discontinuous. This is not possible in mice because waves of thymic colonization overlap. The data suggest that porcine αβ thymocytes require about 15 days to fully differentiate, while γδ thymocytes do so in less than 3 days. We also observed that the porcine IL-2Rα (CD25), a typical differentiation marker of pre-T cells in mice and humans, was not expressed on thymocyte precursors in pigs and could only be found on mature thymocytes. Finally, surface immunophenotyping and cell cycle analysis of thymocyte subsets allowed us to propose a model for αβ T cell lymphopoiesis in swine.
Development of γδ T lymphocytes
Based on the expression of cell surface markers we have characterized the ontogeny of γδ thymocytes. Using the precise definition of developmental pathways for conventional γδ thymocytes (Sinkora et al., 2005b) , the populations of γδ thymocytes was found to have no counterpart in the periphery (Sinkora et al., 2007) . In total, 12 subpopulations of porcine γδ thymocytes based on their expression of CD1, CD2, CD4, CD8 and CD45RC were characterized. Our observation also indicates that two subsets of peripheral γδ T lymphocytes differing in MHC-II expression exist, depend of their origin during ontogeny. We have proposed that one subset acquires CD8αα in the thymus while the second acquires CD8αα as a result of stimulation in the periphery.
Peripheral γδ T lymphocytes in swine
In order to characterize peripheral γδ T cells in swine we investigated expressions of CD25, CD11b, CD52, SWC7, MHC-II and the family of CD45 molecules on these cells. We have found that they differ depending on bacterial colonization, age of animals and/or status of cell activation (Stepanova and Sinkora, 2012) . These studies imply that CD2/CD8 subsets of porcine γδ T cells may represent functionally different cells. For this reason, these subpopulations of γδ T lymphocytes were studied in more detail. Sorting and following in vitro cultivation showed that CD2 expression can be used for definition of two lineages of γδ cells (Stepanova and Sinkora, 2013; Potockova et al., 2015) . Because CD2— γδ T cells are missing in the blood of humans and mice, but are obvious in other members of γδ high species such as ruminants and birds, our findings support the idea that circulating CD2— γδ T cells are a specific lineage (Sinkora and Butler, 2016).
In addition to the work concerning porcine γδ T cells we have also investigated murine γδ T cells expressing canonical Vδ1-Dδ2-Jδ2 chain, which completely lacks junctional diversity (Holtmeier et al., 2010). We showed that γδ T cells expressing this canonical TCRδ chain are not unique to the skin and reproductive sites as expected from other mouse studies. We also discovered other γδ T cells expressing fetal type Vδ1 chains, which were shared among different organs and animals. These findings indicate that γδ T cells expressing conserved Vδ1 chains continuously recirculate throughout the organism and rapidly respond to stress-induced self antigens.
Involvement of porcine lymphocytes in viral diseases
Our laboratory also participated in studies of the porcine immune system during experimental infection by swine influenza virus (SIV), porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2). Germ-free piglets were used since their response could be directly correlated to the viral infection. Using the phenotypic profile of T, B and NK cells and their subsets and utilizing our knowledge about these lymphoid cells in swine we have shown that PRRSV and PCV2 negatively modulate the host immune system by different mechanisms which may explain their persistence (Sinkora et al., 2014; Butler et al., 2014). The most expressive differences were observed as regards the B cells and their subsets: while massive overproduction of immunoglobulins was observed in the PRRSV infection (up to 20mg/ml), the PCV2 infection was characteristic of their decrease. Molecular analysis showed an expansion of dominant B cell clones in PRRSV infection as a result of T-independent proliferation of naive B cells without any repertoire diversification and affinity maturation. The PCV2 infection was characteristic of αβ T cell dysregulation expressed by increased numbers of cytotoxic T cells at the expenses of helper T cells. Our results explain overall lymphoid hyperplasia, hypergammaglobulinemia and autoimmunity in the case of PRRSV infection and also immunosuppressive effect of PCV2 infection. New findings in a field of PRRSV lead us to recent hypothesis that immune dysregulation is caused by altered thymocyte development when PRRSV-specific T cells are selectively eliminated, which cause their absence in the periphery. The "hole" in T cell repertoire allows acceptation of PRRSV epitopes as self-antigens so that specific immune response against PRRSV is missing. Without adequate PRRSV-specific T cells, selection of B cells capable of producing high affinity neutralizing antibodies is impaired as is generation of cytotoxic T cells (Butler et al., 2019). This hypothesis is going to be investigated in our further studies.
Involvement of porcine lymphocytes in tumors
Using a porcine model, we also describe Melanoma-Associated T-lymphocytes (MATL) in peripheral blood that increase during melanoma regression. These MATL possess the CD4+CD8hi phenotype and they have their direct counterparts in Tumor Infiltrating Lymphocytes (TIL) isolated from melanoma loci. Both MATL and TIL have a similar expression of selected markers indicating that they represent effector/memory αβ T-cell subset. Importantly, TIL isolated from different pigs and different melanoma loci among the same pig have similar phenotype and distribution, indicating that the composition of the MATL and TIL compartment is identical. Analysis of sorted cells from regressing pigs revealed a unique MATL subpopulation with mono-specific T-cell receptor that was further analyzed by sequencing. These results indicate that pigs regressing melanomas possess a characteristic population of recirculating T-cells playing a role in tumor control and regression (Cizkova et al., 2019).
Involvement of porcine lymphocytes in cystic fibrosis
Our laboratory also participated in the investigation of inflammatory cells that participate in the destruction of the pancreas during the course of cystic fibrosis (CF). In CF, pancreatic disease begins in utero and progresses over time to the complete destruction of the beta cells. This is an unusual type of autoimmune inflammation that develops in fetal life. The study was performed on the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene knock-out pig model. Unlike mice, these CFTR–/– pigs develop pathological changes similar to humans. We discovered the activation of both the innate and adaptive immune systems during destruction of pancreas indicating that immune reaction may be initiated during fetal life, in a period of time when only tolerance should be expected (Abu-El-Haija et al., 2011).