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

Abstract

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.

Keywords: T lymphocyte; biomedical model; epithelial barrier; innate lymphoid cell 1; intestinal epithelium; intestinal lymphocyte; porcine; swine.

Figure 1

Identification of IELs from H&E staining in pig intestine. H&E-stained tissue section of jejunum from a ~9-week-old pig. IELs are identified as dark purple, round nuclei within the epithelial layer. Two each of IELs in apical epithelium (red arrows), nuclear-level epithelium (gold arrows), and basement membrane (light blue arrows) are indicated in the panel on the right. H&E (hematoxylin and eosin); IEL (intraepithelial lymphocyte).

Figure 2

RNA-ISH staining of potential IEL markers in porcine ileum. Sections of ileum from a ~7.5-week-old pig stained for mRNA transcripts (red staining): ITGAE (A), CD69 (B), NCR1 (C), CD161 (D), CCR9 (E), IFNG (F), IL10 (G), TGFB1 (H). Probes were custom-designed from Advanced Cell Diagnostics with the following catalog numbers: ITGAE (#590611); CD69 (#590601); NCR1 (#553131); KLRB1 (#490871, product labeled CD161); CCR9 (#1062631); IFNG (#490821); IL10 (#442791); TGFB1 (#444951). A protocol for RNA-ISH staining is described previously (57). RNA-ISH (RNA in situ hybridization).

Figure 3

Dual in situ detection of αβ and γδ T cells in pig intestine. Section of ileum from a ~6-week-old pig stained for CD3ε protein (red) and TRDC transcript (dark green). TRDC ⁺ cells (dark green) are identified as γδ T cells, while TRDC ^-CD3e⁺ cells (red) are identified as αβ T cells. The staining protocol was further optimized from previous work (57, 66) and is available at dx.doi.org/10.17504/protocols.io.q26g7yro1gwz/v1.

Figure 4

EDTA incubation time influences epithelial cell removal in porcine intestinal tissues. H&E sections of porcine small intestinal tissue subjected to incubation in EDTA solution for various amounts of time. Tissue was derived from terminal ileum of a ~3-month-old pig. After dissociating mucus from tissue [described elsewhere (66)], ~2 grams of tissue was placed into 200 mL of epithelial removal solution (5mM EDTA and 2% FCS in HBSS) and incubated at 37°C, 200 rpm for amounts of time specified above each image panel. Tissues were not transferred into fresh EDTA solution throughout the duration of specified incubation time. Cell recovery did not substantially increase from 2 hr 35 min to either 4 hr 20 min or 6 hr 30 min incubations. This figure is not intended as a reference for an EDTA incubation time for optimal epithelial removal but to emphasize that individual optimization of methods for epithelial cell removal will be necessary based on technical and biological contexts of an experiment. EDTA (ethylenediamine tetraacetic acid); FCS (fetal calf serum); H&E (hematoxylin and eosin); HBSS (Hank’s balanced salt solution); hr (hour); min (minute); mL (milliliters); mM (millimolar); rpm (rotations per minute).

Figure 5

Co-expression of CD16 and MHC-II on γδ T-IELs in porcine jejunum. (A) Representative flow cytometry gating showing the expression of CD16 and MHC-II on gated γδ T-IELs in epithelial-enriched cell fractions from jejunum of a 6-week-old pig. (B) Stacked bar plot of the percentage of CD16⁺ γδ T-IELs that are MHC-II⁺ (turquoise) versus MHC-II^- (salmon). Data are derived from jejunum of eight 6-week-old pigs. Epithelial-enriched cell fractions were isolated from jejunum and stained for flow cytometry as previously described (67). Detection reagents used for flow cytometry staining are as follows: Fixable Viability Dye eFluor780 (ThermoFisher Scientific 65-0865-14); anti-CD3ε-PE-Cy7 (clone BB23-8E6-8C8; BD 561477); anti-γδTCR (clone PGBL22A; Washington State University PG2032; conjugated to mFluor510 fluorophore by Caprico Biotechnologies); anti-CD16 (clone G7; BioRad MCA1971GA) detected with anti-mouse IgG1 (clone A85-1; BD 740234); MHC-II (clone 2E9/13; BioRad MCA2314GA) detected with anti-mouse IgG2b-BUV496 (clone R12-3; BD 750517). FSC-A (forward-scatter area); FSC-H (forward-scatter height); Ig (immunoglobulin); MHC (major histocompatibility complex); SSC-A (side-scatter area); T-IEL (intraepithelial T lymphocyte); TCR (T cell receptor).