Intestinal location- and age-specific variation of intraepithelial T lymphocytes and mucosal microbiota in pigs.

Intraepithelial T lymphocytes (T-IELs) are T cells located within the epithelium that provide a critical line of immune defense in the intestinal tract. In pigs, T-IEL abundances and phenotypes are used to infer putative T-IEL functions and vary by intestinal location and age, though investigations regarding porcine T-IELs are relatively limited. In this study, we expand on analyses of porcine intestinal T-IELs to include additional phenotypic designations not previously recognized in pigs. We describe non-conventional CD8α+CD8ß- αß T-IELs that were most prevalent in the distal intestinal tract and primarily CD16+CD27-, a phenotype suggestive of innate-like activation and an activated cell state. Additional T-IEL populations included CD8α+CD8ß+ αß, CD2+CD8α+ γδ, and CD2+CD8α- γδ T-IELs, with increasing proportions of CD16+CD27- phenotype in the distal intestine. Thus, putative non-conventional, activated T-IELs were most abundant in the distal intestine within multiple γδ and αß T-IEL populations. A comparison of T-IEL and respective mucosal microbial community structures across jejunum, ileum, and cecum of 5- and 7-week-old pigs revealed largest community differences were tissue-dependent for both T-IELs and the microbiota. Between 5 and 7 weeks of age, the largest shifts in microbial community compositions occurred in the large intestine, while the largest shifts in T-IEL communities were in the small intestine. Therefore, results indicate different rates of community maturation and stabilization for porcine T-IELs and the mucosal microbiota for proximal versus distal intestinal locations between 5 and 7 weeks of age. Collectively, data emphasize the intestinal tract as a site of location- and age-specific T-IEL and microbial communities that have important implications for understanding intestinal health in pigs.

Regional epithelial cell diversity in the small intestine of pigs.

Understanding regional distribution and specialization of small intestinal epithelial cells is crucial for developing methods to control appetite, stress, and nutrient uptake in swine. To establish a better understanding of specific epithelial cells found across different regions of the small intestine in pigs, we utilized single-cell RNA sequencing (scRNA-seq) to recover and analyze epithelial cells from duodenum, jejunum, and ileum. Cells identified included crypt cells, enterocytes, BEST4 enterocytes, goblet cells, and enteroendocrine (EE) cells. EE cells were divided into two subsets based on the level of expression of the EE lineage commitment gene, NEUROD1. NEUROD1hi EE cells had minimal expression of hormone-encoding genes and were dissimilar to EE cells in humans and mice, indicating a subset of EE cells unique to pigs. Recently discovered BEST4 enterocytes were detected in both crypts and villi throughout the small intestine via in situ staining, unlike in humans, where BEST4 enterocytes are found only in small intestinal villi. Proximal-to-distal gradients of expression were noted for hormone-encoding genes in EE cells and nutrient transport genes in enterocytes via scRNA-seq, demonstrating regional specialization. Regional gene expression in EE cells and enterocytes was validated via quantitative PCR (qPCR) analysis of RNA isolated from epithelial cells of different small intestinal locations. Though many genes had similar patterns of regional expression when assessed by qPCR of total epithelial cells, some regional expression was only detected via scRNA-seq, highlighting advantages of scRNA-seq to deconvolute cell type-specific regional gene expression when compared to analysis of bulk samples. Overall, results provide new information on regional localization and transcriptional profiles of epithelial cells in the pig small intestine.

Cells lining the intestinal tract (i.e., epithelial cells) provide a barrier to the outside environment but also play important specialized roles in nutrient absorption and secretion of mucus or hormones involved in controlling appetite and digestion. While similar cell types can be found throughout the small intestine, they have even more specialized function depending on region of the small intestine. Identification and characterization of intestinal epithelial cells are foundational to promoting pig intestinal health for optimal growth. Our research identified six types of epithelial cells across the small intestine of pigs. Enterocytes, an absorptive cell type, shared commonalities with human enterocytes, but a population of enteroendocrine cells, which secrete hormones, was unique to pigs. The location of certain epithelial cells in the intestine was identified and informed the relationship between various epithelial cell types. Overall, a clearer understanding of specific epithelial cells in the porcine intestine is provided, proving a critical foundation to further research aimed at maximizing pig intestinal health.

Intraepithelial lymphocytes in the pig intestine: T cell and innate lymphoid cell contributions to intestinal barrier immunity.

Intraepithelial lymphocytes (IELs) include T cells and innate lymphoid cells that are important mediators of intestinal immunity and barrier defense, yet most knowledge of IELs is derived from the study of humans and rodent models. Pigs are an important global food source and promising biomedical model, yet relatively little is known about IELs in the porcine intestine, especially during formative ages of intestinal development. Due to the biological significance of IELs, global importance of pig health, and potential of early life events to influence IELs, we collate current knowledge of porcine IEL functional and phenotypic maturation in the context of the developing intestinal tract and outline areas where further research is needed. Based on available findings, we formulate probable implications of IELs on intestinal and overall health outcomes and highlight key findings in relation to human IELs to emphasize potential applicability of pigs as a biomedical model for intestinal IEL research. Review of current literature suggests the study of porcine intestinal IELs as an exciting research frontier with dual application for betterment of animal and human health.

Intestinal single-cell atlas reveals novel lymphocytes in pigs with similarities to human cells.

Lymphocytes can heavily influence intestinal health, but resolving intestinal lymphocyte function is challenging as the intestine contains a vastly heterogeneous mixture of cells. Pigs are an advantageous biomedical model, but deeper understanding of intestinal lymphocytes is warranted to improve model utility. Twenty-six cell types were identified in the porcine ileum by single-cell RNA sequencing and further compared with cells in human and murine ileum. Though general consensus of cell subsets across species was revealed, some porcine-specific lymphocyte subsets were identified. Differential tissue dissection and in situ analyses conferred spatial context, revealing similar locations of lymphocyte subsets in Peyer's patches and epithelium in pig-to-human comparisons. Like humans, activated and effector lymphocytes were abundant in the ileum but not periphery of pigs, suggesting tissue-specific and/or activation-associated gene expression. Gene signatures for peripheral and ileal innate lymphoid cells newly discovered in pigs were defined and highlighted similarities to human innate lymphoid cells. Overall, we reveal novel lymphocyte subsets in pigs and highlight utility of pigs for intestinal research applications.

Reference Transcriptomes of Porcine Peripheral Immune Cells Created Through Bulk and Single-Cell RNA Sequencing.

Pigs are a valuable human biomedical model and an important protein source supporting global food security. The transcriptomes of peripheral blood immune cells in pigs were defined at the bulk cell-type and single cell levels. First, eight cell types were isolated in bulk from peripheral blood mononuclear cells (PBMCs) by cell sorting, representing Myeloid, NK cells and specific populations of T and B-cells. Transcriptomes for each bulk population of cells were generated by RNA-seq with 10,974 expressed genes detected. Pairwise comparisons between cell types revealed specific expression, while enrichment analysis identified 1,885 to 3,591 significantly enriched genes across all 8 cell types. Gene Ontology analysis for the top 25% of significantly enriched genes (SEG) showed high enrichment of biological processes related to the nature of each cell type. Comparison of gene expression indicated highly significant correlations between pig cells and corresponding human PBMC bulk RNA-seq data available in Haemopedia. Second, higher resolution of distinct cell populations was obtained by single-cell RNA-sequencing (scRNA-seq) of PBMC. Seven PBMC samples were partitioned and sequenced that produced 28,810 single cell transcriptomes distributed across 36 clusters and classified into 13 general cell types including plasmacytoid dendritic cells (DC), conventional DCs, monocytes, B-cell, conventional CD4 and CD8 αß T-cells, NK cells, and γδ T-cells. Signature gene sets from the human Haemopedia data were assessed for relative enrichment in genes expressed in pig cells and integration of pig scRNA-seq with a public human scRNA-seq dataset provided further validation for similarity between human and pig data. The sorted porcine bulk RNAseq dataset informed classification of scRNA-seq PBMC populations; specifically, an integration of the datasets showed that the pig bulk RNAseq data helped define the CD4CD8 double-positive T-cell populations in the scRNA-seq data. Overall, the data provides deep and well-validated transcriptomic data from sorted PBMC populations and the first single-cell transcriptomic data for porcine PBMCs. This resource will be invaluable for annotation of pig genes controlling immunogenetic traits as part of the porcine Functional Annotation of Animal Genomes (FAANG) project, as well as further study of, and development of new reagents for, porcine immunology.

Severity of bovine tuberculosis is associated with innate immune-biased transcriptional signatures of whole blood in early weeks after experimental Mycobacterium bovis infection.

Mycobacterium bovis, the causative agent of bovine tuberculosis, is a pathogen that impacts both animal and human health. Consequently, there is a need to improve understanding of disease dynamics, identification of infected animals, and characterization of the basis of immune protection. This study assessed the transcriptional changes occurring in cattle during the early weeks following a M. bovis infection. RNA-seq analysis of whole blood-cell transcriptomes revealed two distinct transcriptional clusters of infected cattle at both 4- and 10-weeks post-infection that correlated with disease severity. Cattle exhibiting more severe disease were transcriptionally divergent from uninfected animals. At 4-weeks post-infection, 25 genes had commonly increased expression in infected cattle compared to uninfected cattle regardless of disease severity. Ten weeks post-infection, differential gene expression was only observed when severely-affected cattle were compared to uninfected cattle. This indicates a transcriptional divergence based on clinical status following infection. In cattle with more severe disease, biological processes and cell type enrichment analyses revealed overrepresentation of innate immune-related processes and cell types in infected animals. Collectively, our findings demonstrate two distinct transcriptional profiles occur in cattle following M. bovis infection, which correlate to clinical status.

Intraepithelial T Cells Diverge by Intestinal Location as Pigs Age.

T cells resident within the intestinal epithelium play a central role in barrier integrity and provide a first line of immune defense. Intraepithelial T cells (IETs) are among the earliest immune cells to populate and protect intestinal tissues, thereby giving them an important role in shaping gut health early in life. In pigs, IETs are poorly defined, and their maturation in young pigs has not been well-studied. Given the importance of IETs in contributing to early life and long-term intestinal health through interactions with epithelial cells, the microbiota, and additional environmental factors, a deeper characterization of IETs in pigs is warranted. The objective of this study was to analyze age- and intestinal location-dependent changes in IETs across multiple sites of the small and large intestine in pigs between 4- and 8-weeks of age. IETs increased in abundance over time and belonged to both γδ and αß T cell lineages. Similar compositions of IETs were identified across intestinal sites in 4-week-old pigs, but compositions diverged between intestinal sites as pigs aged. CD2+CD8α+ γδ T cells and CD4-CD8α+ αß T cells comprised >78% of total IETs at all intestinal locations and ages examined. Greater percentages of γδ IETs were present in large intestine compared to small intestine in older pigs. Small intestinal tissues had greater percentages of CD2+CD8α- γδ IETs, while CD2+CD8α+ γδ IET percentages were greater in the large intestine. Percentages of CD4-CD8α+ αß IETs increased over time across all intestinal sites. Moreover, percentages of CD27+ cells decreased in ileum and large intestine over time, indicating increased IET activation as pigs aged. Percentages of CD27+ cells were also higher in small intestine compared to large intestine at later timepoints. Results herein emphasize 4- to 8-weeks of age as a critical window of IET maturation and suggest strong associations between intestinal location and age with IET heterogeneity in pigs.

CD3ε+ Cells in Pigs With Severe Combined Immunodeficiency Due to Defects in ARTEMIS.

Severe combined immunodeficiency (SCID) is described as the lack of functional T and B cells. In some cases, mutant genes encoding proteins involved in the process of VDJ recombination retain partial activity and are classified as hypomorphs. Hypomorphic activity in the products from these genes can function in the development of T and B cells and is referred to as a leaky phenotype in patients and animals diagnosed with SCID. We previously described two natural, single nucleotide variants in ARTEMIS (DCLR1EC) in a line of Yorkshire pigs that resulted in SCID. One allele contains a splice site mutation within intron 8 of the ARTEMIS gene (ART16), while the other mutation is within exon 10 that results in a premature stop codon (ART12). While initially characterized as SCID and lacking normal levels of circulating lymphoid cells, low levels of CD3ε+ cells can be detected in most SCID animals. Upon further assessment, we found that ART16/16, and ART12/12 SCID pigs had abnormally small populations of CD3ε+ cells, but not CD79α+ cells, in circulation and lymph nodes. Newborn pigs (0 days of age) had CD3ε+ cells within lymph nodes prior to any environmental exposure. CD3ε+ cells in SCID pigs appeared to have a skewed CD4α+CD8α+CD8ß- T helper memory phenotype. Additionally, in some pigs, rearranged VDJ joints were detected in lymph node cells as probed by PCR amplification of TCRδ V5 and J1 genomic loci, as well as TCRß V20 and J1.1, providing molecular evidence of residual Artemis activity. We additionally confirmed that TCRα and TCRδ constant region transcripts were expressed in the thymic and lymph node tissues of SCID pigs; although the expression pattern was abnormal compared to carrier animals. The leaky phenotype is important to characterize, as SCID pigs are an important tool for biomedical research and this additional phenotype may need to be considered. The pig model also provides a relevant model for hypomorphic human SCID patients.

Novel Engraftment and T Cell Differentiation of Human Hematopoietic Cells in ART-/-IL2RG-/Y SCID Pigs.

Pigs with severe combined immunodeficiency (SCID) are an emerging biomedical animal model. Swine are anatomically and physiologically more similar to humans than mice, making them an invaluable tool for preclinical regenerative medicine and cancer research. One essential step in further developing this model is the immunological humanization of SCID pigs. In this work we have generated T- B- NK- SCID pigs through site directed CRISPR/Cas9 mutagenesis of IL2RG within a naturally occurring DCLRE1C (ARTEMIS)-/- genetic background. We confirmed ART-/-IL2RG-/Y pigs lacked T, B, and NK cells in both peripheral blood and lymphoid tissues. Additionally, we successfully performed a bone marrow transplant on one ART-/-IL2RG-/Y male SCID pig with bone marrow from a complete swine leukocyte antigen (SLA) matched donor without conditioning to reconstitute porcine T and NK cells. Next, we performed in utero injections of cultured human CD34+ selected cord blood cells into the fetal ART-/-IL2RG-/Y SCID pigs. At birth, human CD45+ CD3ε+ cells were detected in cord and peripheral blood of in utero injected SCID piglets. Human leukocytes were also detected within the bone marrow, spleen, liver, thymus, and mesenteric lymph nodes of these animals. Taken together, we describe critical steps forwards the development of an immunologically humanized SCID pig model.