Organized Gut-associated Lymphoid Tissue Research Articles

Crosstalk between CD8+ T cells and mesenchymal stromal cells in intestine homeostasis and immunity.

The intestine represents the most complex cellular network in the whole body. It is constantly faced with multiple types of immunostimulatory agents encompassing from food antigen, gut microbiome, metabolic waste products, and dead cell debris. Within the intestine, most T cells are found in three primary compartments: the organized gut-associated lymphoid tissue, the lamina propria, and the epithelium. The well-orchestrated epithelial-immune-microbial interaction is critically important for the precise immune response. The main role of intestinal mesenchymal stromal cells is to support a structural framework within the gut wall. However, recent evidence from stromal cell studies indicates that they also possess significant immunomodulatory functions, such as maintaining intestinal tolerance via the expression of PDL1/2 and MHC-II molecules, and promoting the development of CD103+ dendritic cells, and IgA+ plasma cells, thereby enhancing intestinal homeostasis. In this review, we will summarize the current understanding of CD8+ T cells and stromal cells alongside the intestinal tract and discuss the reciprocal interactions between T subsets and mesenchymal stromal cell populations. We will focus on how the tissue residency, migration, and function of CD8+ T cells could be potentially regulated by mesenchymal stromal cell populations and explore the molecular mediators, such as TGF-β, IL-33, and MHC-II molecules that might influence these processes. Finally, we discuss the potential pathophysiological impact of such interaction in intestine hemostasis as well as diseases of inflammation, infection, and malignancies.

Intestinal macrophages in Peyer's patches, sacculus rotundus and appendix of Angora rabbit.

The largest pool of macrophages in the body is harboured by the intestinal mucosa. As the principal phagocytic component of the immune system, macrophages are essential for maintaining mucosal homeostasis as they prevent commensal bacteria from adhering to mucosal epithelial cells. This study provides a RAM11 immunohistochemical and electron microscopic investigation of the existence, localization and distribution of intestinal macrophages in organized gut-associated lymphoid tissue (GALT), including Peyer's patches (PPs), the sacculus rotundus (SR) and the appendix, in the Angora rabbit. Although rabbit intestinal macrophages did not express the tissue macrophage marker macrosialin (CD68), they expressed RAM11. RAM11-positive intestinal macrophages were mostly localized to the subepithelial dome region, interfollicular area and germinal centres (GCs) of the GALT and the lamina propria or submucosa of the ileum and jejunum devoid of PPs and were also observed in the follicle-associated epithelium of PPs, but not in that of the SR and appendix. RAM11-positive macrophages containing engulfed apoptotic bodies were present in the GCs of the lymphoid follicles in the GALT. Electron microscopy further revealed multiple macrophages containing apoptotic bodies within the GCs of the follicles in the GALT. Some macrophage aggregations were observed in the GC and between the GC and the corona region of the follicles in the SR and appendix. Rabbit intestinal macrophages thus undertake both potent phagocytic activity and the efficient scavenging of apoptotic cells. Immunohistochemical data suggest that RAM11 can be reliably used for the determination of intestinal macrophages in the GALT of rabbits.

The human intestinal B-cell response.

The intestinal immune system is chronically challenged by a huge plethora of antigens derived from the lumen. B-cell responses in organized gut-associated lymphoid tissues and regional lymph nodes that are driven chronically by gut antigens generate the largest population of antibody-producing cells in the body: the gut lamina propria plasma cells. Although animal studies have provided insights into mechanisms that underpin this dynamic process, some very fundamental differences in this system appear to exist between species. Importantly, this prevents extrapolation from mice to humans to inform translational research questions. Therefore, in this review we will describe the structures and mechanisms involved in the propagation, dissemination, and regulation of this immense plasma cell population in man. Uniquely, we will seek our evidence exclusively from studies of human cells and tissues.

IgA subclass switch recombination in human mucosal and systemic immune compartments.

Human immunoglobulin A (IgA) comprises two IgA subclasses, IgA1 and IgA2, whose distribution has been shown by immunohistochemistry to be different in various body compartments. In comparison with systemic immune compartments, we investigated the IgA switch profiles at the molecular level in salivary and lacrimal glands, nasal mucosa, and proximal and distal gut mucosa. Direct switching from IgM to IgA1 or IgA2 predominated in all immune compartments analyzed. Similar composition of the Sμ-Sα1 and Sμ-Sα2 junctions was observed, including microhomology usage, which suggested that there is no major difference in the actual recombination mechanism utilized during IgA subclass switching. The proportion of IgA1/IgA2 switch recombination events largely paralleled the previously published immunohistochemical representation of IgA1(+) and IgA2(+) plasma cells, implying that the local subclass distribution generally reflects precommitted memory/effector B cells that have undergone IgA subclass switching before extravasation at the effector site. The extremely low or undetectable levels of activation-induced cytidine deaminase (AID) and Iα-Cμ circle transcripts in intestinal lamina propria samples as compared with Peyer's patches suggest that the cellular IgA subclass distribution outside of organized gut-associated lymphoid tissue is only to a minor extent, if at all, influenced by in situ switching.

Development of CD27+ marginal zone B cells requires GALT

In species other than mouse, little is known about the origin and development of marginal zone (MZ) B cells. Using cross-reactive antibodies, we identified and characterized splenic MZ B cells in rabbits as CD27(+) CD23(-). In rabbits in which organized gut-associated lymphoid tissue (GALT) was surgically removed at birth, we found only CD23(+) follicular (FO) B cells and almost no CD27(+) MZ B cells in the spleen, indicating that GALT is required for the development of splenic MZ B cells. These findings lead us to suggest that commensal microbiota contribute to the development of MZ B cells.

A rat model of mild intestinal inflammation induced byStaphylococcus aureusenterotoxin B

The epithelial barrier of the intestine and the gut-associated lymphoid tissue (GALT) protects the host against luminal pathogenic micro-organisms. This is important at weaning, when animals are exposed to infectious agents and stresses. We have developed a rat model of intestinal inflammation post weaning, based on the systemic administration of Staphylococcus aureus enterotoxin B (SEB). Since the inflammatory response obtained is mild, the food intake pattern is not affected, which makes this model useful for studies of nutritional therapies for intestinal inflammatory disease. SEB increased T-lymphocytes in Peyer's patches and the number of activated T-lymphocytes in mesenteric lymph nodes (organized GALT). In the lamina propria, SEB increased activated T-lymphocytes as well as cytotoxic and natural killer-cell populations of the diffuse GALT. It also increased pro-inflammatory cytokines and inflammatory mediators in both Peyer's patches and mucosa. Rats given SEB had higher paracellular permeability to macromolecules, which was associated with a reduction in epithelial tightness. This model was used to examine whether dietary supplementation with spray-dried animal plasma proteins affects intestinal inflammation. Results showed that dietary plasma proteins can attenuate the mucosal immune response in both organized and diffuse GALT and that these effects are mediated by a reduction in the production of pro-inflammatory cytokines.

Dietary plasma proteins, the intestinal immune system, and the barrier functions of the intestinal mucosa1

ABSTRACTThe intestinal mucosa contributes to homeostasis by preventing the entrance of biological and chemical agents across the epithelium that could alter the stability of the system. This protective function is especially important at the time of weaning, when animals are exposed to infectious agents and to numerous stresses such as the change of environment and diet. Diets supplemented with spray-dried plasma or plasma protein fractions have been shown to improve growth performance of farm animals and have been proposed as an alternative to antibiotics. In this review, we summarize our findings on the mechanism of action of dietary plasma proteins using a rat model of intestinal inflammation, based on the administration of Staphylococcus aureus enterotoxin B (SEB). Staphylococcal enterotoxin B activates the gut-associated lymphoid tissue (GALT), increasing T-lymphocytes in Peyer's patches and the number of activated T lymphocytes in mesenteric lymph nodes (organized GALT). In the lamina propria SEB increased cytotoxic T δγ and natural killer cell populations of the diffuse GALT. Staphyloccocal enterotoxin B significantly increased proinflammatory cytokines in Peyer's patches and mucosa. Plasma protein supplements modulated the mucosal immune response in organized and diffuse GALT, protecting GALT from possible excessive activation by the SEB challenge. These effects are accompanied by a reduction of proinflammatory cytokine production, supporting the view that changes in cytokine production mediate the effects of dietary plasma proteins during intestinal inflammation. The increase in mucosal permeability and intestinal secretion induced by SEB was associated with decreased expression of mucosal tight-junction and adherent-junction proteins. Both plasma and plasma protein fractions prevented the effects of SEB on intestinal permeability, thus reducing the exposure of the host to microbial and food antigens across the interstitial space. These findings indicate that dietary plasma proteins modulate functional and structural properties of the intestinal mucosa.

Identification and characterization of novel gut-associated lymphoid tissues in rat small intestine

The crypt lamina propria of the mouse small intestine has been shown to harbor multiple tiny clusters filled with c-kit- and interleukin 7 receptor (IL-7R)-positive lympho-hemopoietic cells (cryptopatches; CPs). However, it has remained an open question whether similar lymphoid tissue are present in the gastrointesitinal tract in other animals. In the present study, we investigated whether the small intestine of rats harbored lymphoid tissues similar to mouse CPs. Immunohistochemical and flow cytometric analyses were carried out using various antibodies, including those to c-kit and IL-7R molecules. Lymphocyte-filled villi (LFVs), populated predominantly with c-kit- and IL-7 receptor (IL-7R)-positive cells and less with T cell receptor (TCR)-alphabeta T cells were found throughout the small intestine of young adult rats. Although LFVs were absent from fetal rat intestine, they were first detected at around 2 weeks after birth. Notably, in most LFVs that settled in the antimesenteric wall of the small intestine in young adult rats, immunoglobulin M-positive B cells were also detectable at the bottom of the LFVs. In aged rats, lymphocytes in some LFVs displayed a different phenotype, comprising a large B-cell area that included a germinal center. Thus, these clusters represent the first description of isolated lymphoid follicles (ILFs) in the rat small intestine. The present study provides the first evidence for c-kit- and IL-7R-positive lymphocyte clusters in the rat small intestine. Our data also indicating that LFVs and ILFs may constitute novel organized gut-associated lymphoid tissues in lamina propria of the rat small intestine.

Human Intestinal IgA Response Is Generated in the Organized Gut-Associated Lymphoid Tissue but Not in the Lamina Propria

The intestinal lamina propria has traditionally been viewed as the effector site of mucosal immune responses. However, this view has been challenged with the identification, in the murine lamina propria, of an in situ class switch DNA recombination pathway to IgA. In this study, we tested the hypothesis that in situ class switching occurs in the human lamina propria. Immunohistochemistry was used to analyze tissue microenvironments and RT-PCR to look for molecular evidence of Ig class switching and to track clonally related cells of B lineage. We found no evidence of proliferation of either lamina propria CD20+ or CD19+ cells or evidence of activation-induced cytidine deaminase mRNA expression outside the organized gut-associated lymphoid tissue, although I alpha-C alpha immunoglobulin germ-line gene transcript expression could be identified in the lamina propria. We identified clonally related cells, including IgA and IgM isotype-switched variants, in multiple samples known to be free of activation-induced cytidine deaminase, organized lymphoid tissue, or cellular proliferation. For 4 groups of cells, the patterns of somatic mutations on the rearranged IgV(H)5 gene segment were more similar between cells from distant sites than from their immediate neighbors, implying dissemination of cells from a common set of precursors. Our data are inconsistent with a model in which precursors of human IgA-secreting plasma cells are induced or expanded in the lamina propria. The human lamina propria is therefore likely to solely be an effector site of intestinal secretory IgA responses that originate from the organized gut-associated lymphoid tissues.

Positive selection of the peripheral B cell repertoire in gut-associated lymphoid tissues

Gut-associated lymphoid tissues (GALTs) interact with intestinal microflora to drive GALT development and diversify the primary antibody repertoire; however, the molecular mechanisms that link these events remain elusive. Alicia rabbits provide an excellent model to investigate the relationship between GALT, intestinal microflora, and modulation of the antibody repertoire. Most B cells in neonatal Alicia rabbits express VHn allotype immunoglobulin (Ig)M. Within weeks, the number of VHn B cells decreases, whereas VHa allotype B cells increase in number and become predominant. We hypothesized that the repertoire shift from VHn to VHa B cells results from interactions between GALT and intestinal microflora. To test this hypothesis, we surgically removed organized GALT from newborn Alicia pups and ligated the appendix to sequester it from intestinal microflora. Flow cytometry and nucleotide sequence analyses revealed that the VHn to VHa repertoire shift did not occur, demonstrating the requirement for interactions between GALT and intestinal microflora in the selective expansion of VHa B cells. By comparing amino acid sequences of VHn and VHa Ig, we identified a putative VH ligand binding site for a bacterial or endogenous B cell superantigen. We propose that interaction of such a superantigen with VHa B cells results in their selective expansion.

Identification of multiple isolated lymphoid follicles on the antimesenteric wall of the mouse small intestine.

We have revealed that 100-200 clusters, filled with closely packed lymphocytes, can be found throughout the length of the antimesenteric wall of the mouse small intestine. They are composed of a large B cell area, including a germinal center, and epithelia overlying the clusters contain M cells. A large fraction of B cells displays B220+ CD19+ CD23+ IgM(low)IgD(high)CD5(-)Mac-1(-) phenotype, and the composition of IgA+ B cells is smaller but substantial. To our knowledge, these clusters are the first identification of isolated lymphoid follicles (ILF) in mouse small intestine. ILF can be first detected at 7 (BALB/c mice) and 25 (C57BL/6 mice) days after birth, and lymphoid clusters equivalent in terms of cellular mass to ILF are present in germfree, athymic nude, RAG-2(-/-), TCR-beta(-/-), and Ig mu-chain mutant (mu(-/-)) mice, although c-kit+ cells outnumber B220+ cells in germfree and athymic nude mice, and most lymphoid residents are c-kit+ B220(-) in RAG-2(-/-), TCR-beta(-/-), and mu(-/-) mice. ILF develop normally in the progeny of transplacentally manipulated Peyer's patch (PP)-deficient mice, and decreased numbers of conspicuously atrophied ILF are present in IL-7Ralpha(-/-) PP(null) mice. Neither ILF nor PP are detectable in lymphotoxin alpha(-/-) and aly/aly mice that retain well-developed cryptopatches (CP) and thymus-independent subsets of intraepithelial T cells, whereas ILF, PP, CP, and thymus-independent subsets of intraepithelial T cells disappear from common cytokine receptor gamma-chain mutant mice. These findings indicate that ILF, PP, and CP constitute three distinct organized gut-associated lymphoid tissues that reside in the lamina propria of the mouse small intestine.

Intestinal microflora and diversification of the rabbit antibody repertoire.

The rabbit establishes its primary Ab repertoire by somatically diversifying an initial repertoire that is limited by restricted VH gene segment usage during VDJ gene rearrangement. Somatic diversification occurs in gut-associated lymphoid tissue (GALT), and by about 1-2 mo of age nearly all Ig VDJ genes are somatically diversified. In other species that are known to establish their primary Ab repertoire by somatic diversification, such as chicken, sheep, and cattle, diversification appears to be developmentally regulated: it begins before birth and occurs independent of exogenous factors. Because somatic diversification in rabbit occurs well after birth in GALT, the diversification process may not be developmentally regulated, but may require interaction with exogenous factors derived from the gut. To test this hypothesis, we examined Ab repertoire diversification in rabbits in which the appendix was ligated shortly after birth to prevent microbial colonization and all other organized GALT was surgically removed. We found that by 12 wk of age nearly 90% of the Ig VDJ genes in PBL were undiversified, indicating that intestinal microflora are required for somatically diversifying the Ab repertoire. We also examined repertoire diversification in sterilely derived remote colony rabbits that were hand raised away from contact with conventional rabbits and thereby acquired a different gut microflora. In these remote colony rabbits, GALT was underdeveloped, and 70% of the Ig VDJ genes in PBL were undiversified. We conclude that specific, currently unidentified intestinal microflora are required for Ab repertoire diversification.

Novel vaccination strategies for the control of mucosal infection.

Enteric disease remains one of the greatest causes of mortality and morbidity in both human and veterinary species. There has been a remarkable lack of success in vaccination to control mucosal disease and it is therefore apparent that novel strategies are required to achieve effective mucosal immunity. Basic studies described in this paper have addressed problems associated with antigen handling and the induction of an immune response in the intestine, and the subsequent dissemination of effector cells and molecules to intestinal and extra-intestinal submucosal regions. Effective induction of IgA responses is dependent on T-cell help and requires cognate interactions between T cells and B cells within organized gut-associated lymphoid tissue (GALT). The delivery of an IgA antibody response to mucosal sites is also a T cell dependent but antigen driven process. The normal route by which antigen is taken up by GALT is via the epithelial surface but antigen presented in this way via villus epithelial cells generates predominantly a suppressor response. Strategies designed to overcome this effect include the use of powerful adjuvants (such as cholera toxin, muramyldipeptide and phorbol esters), the use of immunogenic carriers, or delivery via liposomes, microspheres or genetically engineered viral or bacterial vectors. Alternatively, the feasibility of accessing GALT via the serosal surface by administration of intraperitoneal antigen in oil emulsion has been explored and a vaccine formulation (Auspharm (patent pending)) has been developed which is suitable for IP delivery in commercial applications.

Cell-mediated cytotoxicity of mononuclear cells isolated from cancer and normal mucosa of the stomach

Mononuclear cells from the organized gut-associated lymphoid tissues of gastric cancer patients or cancer-free individuals were examined for the number of K cells in antibody-dependent cellular cytotoxicity and target cell reduction in microcytotoxicity assays. Mononuclear cells were isolated from cancer tissue, gastric mucosa, intestinal mucosa, peripheral blood, spleens, tonsils, thymus, and the mesenteric lymph nodes of gastric cancer patients or the non-gastric cancer group. K cells against sheep erythrocytes were found in peripheral blood mononuclear cells of both gastric cancer patients and cancer-free individuals, and to a lesser extent in mucosal mononuclear cells from gastric cancerous and noncancerous areas and spleens of both groups. No significant differences in the number of K cells were observed between mononuclear cells from gastric cancerous areas and noncancerous areas. On the other hand, microcytotoxicity assay effector cell activities against allogeneic gastric cancer cell lines were significantly higher in mucosal mononuclear cells from gastric cancerous areas than those from noncancerous areas. Peripheral blood mononuclear cells of gastric cancer patients showed relatively low microcytotoxicity assay activity at an effector to target cell ratio of 10:1, but not 30:1 as compared with that in mucosal mononuclear cells from gastric cancerous areas, spleens, and peripheral blood mononuclear cells of the non-gastric cancer group. Cells from lymph nodes, tonsils, and thymus did not have K cells and showed low microcytotoxicity assay activities. These results demonstrate that mucosal mononuclear cells from gastric cancerous areas may possibly show different behavior from those from noncancerous areas with regard to cell-mediated cytotoxicity.

Cytotoxic effector cell function in organized gut-associated lymphoid tissue (GALT)

Lymphocytes from the organized gut-associated lymphoid tissues (GALT) of adult guinea pigs were examined for surface markers characteristic of T and B lymphocytes and for their capacity to function as effector cells in mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC) reactions. GALT lymphocytes formed rosettes with rabbit erythrocytes, a T-cell marker, and underwent proliferative responses in vitro in the presence of phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM). GALT lymphocytes were cytotoxic in vitro for erythrocyte and DBA mastocytoma targets in the presence of PHA. A population of GALT lymphocytes bound aggregated γ-globulin; however, they functioned poorly in ADCC reactions. Thus, organized GALT in the guinea pig contains lymphocytes capable of functioning in T-cell-dependent MICC reactions but either lacks the effector cell population which mediates ADCC or contains an effector cell which functions poorly in ADCC.