Function of Mucosa-Associated Lymphoid Tissue in Antibody Formation

Abundant evidence supports the notion that human intestinal plasma cells are largely derived from B cells initially activated in gut-associated lymphoid tissue (GALT). Nevertheless, insufficient knowledge exists about the uptake, processing, and presentation of luminal antigens occurring in GALT to accomplish priming and sustained expansion of mucosal B cells. Also, it is unclear how the germinal center reaction so strikingly promotes class switch to IgA and expression of J chain, although the commensal microbiota appears to contribute to both diversification and memory. B-cell migration from GALT to the intestinal lamina propria is guided by rather well-defined adhesion molecules and chemokines/chemokine receptors, but the cues directing homing to secretory effector sites beyond the gut require better definition. In this respect, the role of human Waldeyer's ring (including adenoids and the palatine tonsils) as a regional mucosa-associated lymphoid tissue must be better defined, although the balance of evidence suggests that it functions as nasopharynx-associated lymphoid tissue (NALT) like the characteristic NALT structures in rodents. Altogether, data suggest a remarkable compartmentalization of the mucosal immune system that must be taken into account in the development of effective local vaccines to protect specifically the airways, small and large intestines, and the female genital tract.     

The B-cell system of human mucosae and exocrine glands

Summary: The mucosae and exocrine glands harbour the largest activated B-cell system of the body, amounting to some 80–90% of all immunoglobulins (Ig)-producing cells. The major product of these immunocytes is polymeric (p)IgA (mainly dimers) with associated J chain. Both pIgA and pentameric IgM contain a binding site for the polymeric Ig receptor (pIgR), or secretory component (SC), which is a requirement for their active external transport through secretory epithelia. The pIgR/SC binding site depends on covalent incorporation of the J chain into the quaternary structure of the polymers when they are produced by the local immunocytes. This important differentiation characteristic appears to be sufficient functional justification for the J chain to be expressed also by most B cells terminating at secretory effector sites with IgD or IgG production; they probably represent a ‘spin-off’ from sequential downstream C_H switching on its way to pIgA expression, thus apparently reflecting a maturational stage of effector B-cell clones compatible with homing to these sites. Observations in IgA-deficient individuals suggest that the magnitude of this homing is fairly well maintained even when the differentiation pathway to IgA is blocked. Certain microenvironmental elements such as specific cytokines and dendritic cells appear to be required for induction of IgA synthesis, but it remains virtually unknown why this isotype normally is such a dominating product of local immunocytes and why they have such a high level of J chain expression. Also, despite the recent identification of some important requirements in terms of adhesion molecules (e.g. integrin α4β7 and MAdCAM-1) that explain the “gut-seeking” properties of enterically induced B cells, the origin of regionalized homing of B cells to secretory effector sites outside the gut remains elusive. Moreover, little is known about immune regulation underlying the striking disparity of both the class (IgD, IgM) and subclass (IgA1, IgA2, IgGI, IgG2) production patterns shown by local iinmttnocytes in various regions of the body, although the topical microbiota and other environmental stimuli might be important. Rational design of local vaccines will depend on better knowledge of both inductive and migratory properties of human mucosal B cells.     

Mucosal Immunity: Induction, Dissemination, and Effector Functions

Prevention of infections by vaccination remains a compelling goal to improve public health. Most infections involve the mucosae, but the development of vaccines against many of these pathogens has yet to be successful. Mucosal vaccines would make immunization procedures easier, be better suited for mass administration, and most efficiently induce immune exclusion – a term coined for non-inflammatory antibody shielding of internal body surfaces – mediated principally by secretory immunoglobulin A (SIgA). The exported antibodies are polymeric, mainly IgA dimers (pIgA) – produced by local plasma cells stimulated by antigens that target the mucosae. SIgA was early shown to be complexed with an epithelial glycoprotein – the secretory component (SC). In 1974, a common SC-dependent transport of pIgA and pentameric IgM was proposed. From the basolateral surface, pIg-SC complexes are taken up by endocytosis and finally extruded into the lumen. Membrane SC is now referred to as polymeric Ig receptor (pIgR). In 1980, it was shown to be synthesized as a larger transmembrane protein – first cloned from rabbit and then from human. Mice deficient for pIgR showed that this is the only receptor responsible for epithelial transport of IgA and IgM. In the gut, induction of B cells occurs in gut-associated lymphoid tissue, particularly the Peyer's patches, but also in mesenteric lymph nodes. Plasma cell differentiation is accomplished in the lamina propria to which the memory/effector cells home. The airways also receive such cells from nasopharynx-associated lymphoid tissue – but by different homing receptors. Such compartmentalization is a challenge for development of mucosal vaccines.     

Regional specialization in the mucosal immune system: primed cells do not always home along the same track

According to the current paradigm of lymphocyte trafficking, primed B and T cells extravasate in the intestinal lamina propria chiefly by means of the mucosal homing receptor alpha4beta7, which interacts with the vascular addressin MAdCAM-1. However, as discussed here, this mechanism cannot explain the preferential homing of B cells with a high level of J-chain expression to mucosal effector sites outside the gut.     

Immunoglobulin- and J chain-producing cells associated with lymphoid follicles in the human appendix, colon and ileum, including Peyer''s patches.

Immunohistochemistry of B cells associated with normal human Peyer's patches and solitary lymphoid follicles of the ileum, colon and appendix mucosa showed that local accumulation of IgG-producing cells is a common feature of gut-associated lymphoid tissue (GALT). These immunocytes have strikingly down-regulated J-chain expression, indicating that they belong to mature memory clones. They are located mainly in the dome areas, alongside the follicles, and to a lesser extent in the germinal centres, and are accompanied by a much smaller number of J-chain negative IgA- and IgM-producing cells. It is concluded that B cells of mature memory clones are retained in GALT, whereas relatively early counterparts with a high J chain-expressing potential probably emigrate rapidly after stimulation and seed distant secretory sites where they undergo terminal differentiation to produce mainly J chain-containing dimeric IgA.     

Gut lymphocyte migration: we are halfway 'home'

The gastrointestinal immune system consists of immune cells in organized gut-associated lymphoreticular tissues (GALT) and diffuse lamina propria, which give rise to mucosal secretory IgA antibody responses. A recent study showed that the retinoic acid produced by GALT dendritic cells (DCs) imprints B cells for gut homing. Surprisingly, GALT DCs, together with interleukin-5 (IL-5) and IL-6, also provided a milieu for both B cell switching to IgA and IgA synthesis.     

Human Intestinal B-Cell Blasts and Plasma Cells Express the Mucosal Homing Receptor Integrin α4β7

Interactions between homing receptors on circulating leucocytes and endothelial addressins regulate tissue-specific cellular extravasation. Although integrin á4β7 appears to be the main receptor for guthoming T lymphocytes, less is known about molecules mediating mucosal B cell homing. Expression of integrin α4β7 on B lymphocytes, B cell blasts, and plasma cells in human gut-associated lymphoid tissue (GALT; the Peyer's patches and appendix) and lamina propria was studied by multi-colour immunofluorescence applied on cryosections. Isolated mononuclear cells from the same tissue compartments were examined by flow cytometry and compared with peripheral blood B cells. Integrin α4β7 was expressed by IgA⁺ B cell blasts and plasma cells (CD38^high) in the lamina propria, B cell blasts in GALT, and sIgD⁺ B lymphocytes in peripheral blood. In contrast, GALT sIgD⁺ B lymphocytes were negative or only weakly positive for α4β7. These results suggested that B lymphocytes down-regulate αAβ7 upon extravasation in GALT but up-regulate this integrin after antigen-priming. Thus, α4β7 may be a homing receptor also for B cell blasts extravasating in the gut lamina propria, where this integrin is maintained on plasma cells, perhaps as a local retention factor.     

Characterization of HIV-1-specific cytotoxic T lymphocytes expressing the mucosal lymphocyte integrin CD103 in rectal and duodenal lymphoid tissue of HIV-1-infected subjects

Acute HIV-1 infection depletes CD4(+) T cells in gut-associated lymphoid tissue (GALT). The failure of containment of local viral replication, and consequent CD4(+) T cell depletion, might be due to delayed mobilization of effector CD8(+) T cells or absence of functioning HIV-1-specific CD8(+) T cell effectors within GALT. No studies have addressed human intestinal HIV-1-specific CD8(+) T cell functions. We sought to determine whether functional HIV-1-specific CTL were present in GALT and whether the repertoire differed from HIV-1-specific CTL isolated from peripheral blood mononuclear cells. From three HIV-1-infected subjects, we isolated HIV-1-specific CD8(+) T cells expressing the mucosal lymphocyte integrin CD103 from GALT. These antigen-specific effector cells could be expanded in vitro and lysed target cells in an MHC class I-restricted manner. HIV-1-specific CTL could be isolated from both duodenal and rectal GALT sites, indicating that CD8(+) effectors were widespread through GALT tissue. The breadth and antigenic specificities of GALT CTL appeared to differ from those in peripheral blood in some cases. In summary, we found HIV-1-specific CD8(+) effector T cells in GALT, despite HIV-1-induced CD4(+) T cell lymphopenia. This suggests that HIV-1-specific CTL in gut tissue can be maintained with limited CD4(+) T cell help.     

Gut- and bronchus-associated lymphoid tissue

Bronchus-associated and gut-associated lymphoid tissues (BALT and GALT) have both functional and morphologic similarities and are involved in seeding lung, gut, and other mucosal sites with predominantly IgA-containing B cells. Both types of lymphoid tissue are engaged in the regulation and the controlled amplification of immune responses, which vary from positive mucosal responses in both mucosae and peripheral tissues to local mucosal responses and systemic tolerance. Their further involvement in provision of cells destined to reside in the epithelial compartment of the body appears likely but requires further investigation. Their role in the provision of precursors of mucosal mast cells must also be explored further, but some participation in this event appears likely. The mucosa-associated lymphoid tissue (MALT) system appears to be integrated with the systemic immune system but may be considered as separate from it in several functional ways.     

Distribution of beta 7 integrins in human intestinal mucosa and organized gut-associated lymphoid tissue.

Two alternative integrins involved in mucosal homing (alpha 4 beta 7) or epithelial retention (alpha E beta 7) of lymphocytes were examined in the human gut. The distribution of the beta 7 subunit [monoclonal antibody (mAb) M301] was bimodal in that it was strongly expressed by alpha E beta 7 + cells but weakly by alpha 4 beta 7 + cells. More than 90% of intraepithelial lymphocytes (IEL), including the minor subsets of CD4+, T-cell receptor (TCR) gamma/delta +, and CD3- cells, expressed alpha E beta 7 as did most lamina propria CD8+ (88%) and a fraction (36%) of CD4+ lymphocytes. Conversely, B-lineage cells (CD19+) and macrophages (CD68+) were negative. In gut-associated lymphoid tissue (GALT: Peyer's patches and appendix) only a few (< 5%) cells were positive for alpha E beta 7 (confined to CD8+ lymphocytes and CD11c+ putative dendritic cells). A relatively small fraction of IEL (30-50%) expressed alpha 4 beta 7 (mAb Act-1), while most (70%) lamina propria T and B lymphocytes, blasts, plasma cells and macrophages were positive. In GALT, T lymphocytes expressed similar levels of alpha 4 beta 7 as in the lamina propria whereas relatively few B lymphocytes (< 50%) were positive. Isolated lamina propria CD8+, CD4+, CD19+, and CD38+ cells contained mRNA for alpha 4 and the former three subsets as well as appendix CD8+ cells also for beta 7 while only lamina propria CD8+ cells had mRNA for alpha E. Together, the results suggested that alpha E beta 7 and alpha 4 beta 7 are differentially regulated in inductive sites and effector sites of the human gut. Because lymphoid cells at both sites expressed mainly alpha 4 beta 7, this integrin may be a homing receptor on memory and effector cells bound for lamina propria as well as on naive lymphocytes extravasating in GALT. Conversely, because alpha E beta 7 was mainly expressed by CD8+ cells in epithelium and lamina propria, it was probably induced after extravasation, in agreement with the observation that IEL and a fraction of lamina propria T lymphocytes (mainly CD8+ cells) generally expressed higher levels of beta 7 than most CD4+ and B cells. Also a subset of putative dendritic cells located near the follicle-associated epithelium of GALT expressed alpha E beta 7, perhaps reflecting epithelial interaction during primary immune responses.     

Der Gastrointestinaltrakt als Immunorgan: Das darmassoziierte Immunsystem

Summary The gastrointestinal mucosa separates the intraluminal gastrointestinal fluid, which contains a high number of antigens from different sources, and prevents free access of antigens to the body. Simultaneously, it allows some vital host-environment interactions. A number of unspecific factors are important in preventing antigen invasion. The specific mucosal immunity is related to secretory IgA. IgA is derived from mucosal plasma cells after antigen-induced proliferation of its precursors in Peyer's patches. These IgA-positive B-lymphoblasts migrate through the systemic circulation and then home to the mucosa. IgA is translocated as a dimer to the gut lumen after attachment to the secretory component (SC). Part of it is excreted into the bile via small bile ducts after portal and possibly systemic circulation and binding to SC. T cells and mast cells are also considered to show migration and homing phenomena. In addition to the gut, some other mucosa-associated lymphoid tissues, (e.g. bronchus, mammary, salivary and lacrimal glands as well as the female genital tract), can participate in homing. Little is known about the local regulatory mechanisms, which allow an immunoglobulin class specifity of immune responses. Induction of local immunity and specific systemic tolerance seems to be a characteristic immune response of the gut-associated lymphoid tissue (GALT). The knowledge of the local immune system allows a better understanding of many aspects of gastrointestinal pathology, especially in immuno-inflammatory and immunoproliferative diseases as well as in gastrointestinal immunodeficiency syndromes.

     

Generation of Immunoglobulin diversity in human gut-associated lymphoid tissue

The organised gut associated lymphoid tissue (GALT) exists adjacent to an extensive and diverse luminal flora. The follicle associated epithelium and associated dendritic cells and lymphocytes form a tightly fortified gateway between the flora and the host that permits connectivity between them and chronic activation of the lymphoid compartment. As a consequence, plasma cell precursors are generated continuously, and in abundance, in GALT by clonal proliferation. Clonal proliferation alone on this scale would reduce the spectrum of B cell specificity. To compensate, GALT also houses molecular machinery that diversifies the receptor repertoire by somatic hypermutation, class switch recombination and receptor revision. These three processes of enhancing the diversity of mature B cells ensure that although clonally related plasma cells may secrete immunoglobulin side by side in the mucosa they rarely have identical antigen binding sites.     

The gut microbiota and its role in the development of allergic disease: a wider perspective

The gut microbiota are critical in the homoeostasis of multiple interconnected host metabolic and immune networks. If early microbial colonization is delayed, the gut‐associated lymphoid tissues (GALT) fail to develop, leading to persistent immune dysregulation in mice. Microbial colonization has also been proposed as a major driver for the normal age‐related maturation of both Th1 and T regulatory (Treg) pathways that appear important in suppressing early propensity for Th2 allergic responses. There is emerging evidence that resident symbionts induce tolerogenic gut‐associated Treg cells and dendritic cells that ensure the preferential growth of symbionts; keeping pathogenic strains in check and constraining proinflammatory Th1, Th2, and Th17 clones. Some effects of symbionts are mediated by short‐chain fatty acids, which play a critical role in mucosal integrity and local and systemic metabolic function and stimulate the regulatory immune responses. The homoeostatic IL‐10/TGF‐β dominated tolerogenic response within the GALT also signals the production of secretory IgA, which have a regulating role in mucosal integrity. Contrary to the ‘sterile womb’ paradigm, recent studies suggest that maternal microbial transfer to the offspring begins during pregnancy, providing a pioneer microbiome. It is likely that appropriate microbial stimulation both pre‐ and postnatally is required for optimal Th1 and Treg development to avoid the pathophysiological processes leading to allergy. Disturbed gut colonization patterns have been associated with allergic disease, but whether microbial variation is the cause or effect of these diseases is still under investigation. We are far from understanding what constitutes a ‘healthy gut microbiome’ that promotes tolerance. This remains a major limitation and might explain some of the inconsistency in human intervention studies with prebiotics and probiotics. Multidisciplinary integrative approaches with researchers working in networks, using harmonized outcomes and methodologies, are needed to advance our understanding in this field.     

Mucosal immunoglobulins and B cells of teleost fish

As physical barriers that separate teleost fish from the external environment, mucosae are also active immunological sites that protect them against exposure to microbes and stressors. In mammals, the sites where antigens are sampled from mucosal surfaces and where stimulation of naïve T and B lymphocytes occurs are known as inductive sites and are constituted by mucosa-associated lymphoid tissue (MALT). According to anatomical location, the MALT in teleost fish is subdivided into gut-associated lymphoid tissue (GALT), skin-associated lymphoid tissue (SALT), and gill-associated lymphoid tissue (GIALT). All MALT contain a variety of leukocytes, including, but not limited to, T cells, B cells, plasma cells, macrophages and granulocytes. Secretory immunoglobulins are produced mainly by plasmablasts and plasma cells, and play key roles in the maintenance of mucosal homeostasis. Until recently, teleost fish B cells were thought to express only two classes of immunoglobulins, IgM and IgD, in which IgM was thought to be the only one responding to pathogens both in systemic and mucosal compartments. However, a third teleost immunoglobulin class, IgT/IgZ, was discovered in 2005, and it has recently been shown to behave as the prevalent immunoglobulin in gut mucosal immune responses. The purpose of this review is to summarise the current knowledge of mucosal immunoglobulins and B cells of fish MALT. Moreover, we attempt to integrate the existing knowledge on both basic and applied research findings on fish mucosal immune responses, with the goal to provide new directions that may facilitate the development of novel vaccination strategies that stimulate not only systemic, but also mucosal immunity.     

Mucosal immunoglobulins and B cells of teleost fish

As physical barriers that separate teleost fish from the external environment, mucosae are also active immunological sites that protect them against exposure to microbes and stressors. In mammals, the sites where antigens are sampled from mucosal surfaces and where stimulation of naïve T and B lymphocytes occurs are known as inductive sites and are constituted by mucosa-associated lymphoid tissue (MALT). According to anatomical location, the MALT in teleost fish is subdivided into gut-associated lymphoid tissue (GALT), skin-associated lymphoid tissue (SALT), and gill-associated lymphoid tissue (GIALT). All MALT contain a variety of leukocytes, including, but not limited to, T cells, B cells, plasma cells, macrophages and granulocytes. Secretory immunoglobulins are produced mainly by plasmablasts and plasma cells, and play key roles in the maintenance of mucosal homeostasis. Until recently, teleost fish B cells were thought to express only two classes of immunoglobulins, IgM and IgD, in which IgM was thought to be the only one responding to pathogens both in systemic and mucosal compartments. However, a third teleost immunoglobulin class, IgT/IgZ, was discovered in 2005, and it has recently been shown to behave as the prevalent immunoglobulin in gut mucosal immune responses. The purpose of this review is to summarise the current knowledge of mucosal immunoglobulins and B cells of fish MALT. Moreover, we attempt to integrate the existing knowledge on both basic and applied research findings on fish mucosal immune responses, with the goal to provide new directions that may facilitate the development of novel vaccination strategies that stimulate not only systemic, but also mucosal immunity.     

Human IgA‐secreting cells induced by intestinal,but not systemic,immunization respond to CCL25 (TECK) and CCL28 (MEC)

Organ‐specific homing of lymphoid cells depends on the expression of tissue‐specific adhesion molecules and production of specific chemokines. CCL25 (TECK) and CCL28 (MEC) have been reported to direct circulating memory/effector B cells to mucosal tissues. Here, we examined if differential responsiveness to mucosal and systemic chemokines could explain the differential migration pattern of circulating human antibody‐secreting cells (ASC), induced by mucosal and systemic immunization. There was a robust migration of specific IgA‐ and IgM‐ASC induced by Salmonella vaccination toward the mucosal chemokines CCL25 and CCL28. In contrast, tetanus‐specific ASC migrated to the systemic chemokine CXCL12 (SDF‐1α) and showed no response to CCL25 or CCL28, not even tetanus‐specific IgA‐ASC. Cell sorting experiments demonstrated that Salmonella‐specific ASC co‐expressed CCR9 and CCR10. Our results show that induction site, rather than isotype commitment, determines the chemokine responsiveness and migration pattern of human effector B cells.     

Lymphocyte homing to the gut: attraction, adhesion, and commitment

Summary: Lymphocytes continuously migrate from the blood into the intestine. Naive lymphocytes leave the blood through high endothelial venules in Peyer's patches. During the multistep extravasation cascade, they sequentially roll on, firmly adhere to, and transmigrate through the endothelial layer using multiple adhesion molecules and chemotactic signals. In the organized lymphoid tissues of the gut, lymphocytes can become activated, if they meet their cognate antigens transported to Peyer's patches through the gut epithelium. During activation and proliferation, the lymphocytes become imprinted by the local dendritic cells, so that after returning to systemic circulation via the efferent lymphatic vasculature, they preferentially home to lamina propria of the gut to execute their effector functions. In inflammation, the recirculation routes of lymphocytes are altered, and these may explain the pathogenesis of certain extra‐intestinal manifestations of gut infections and inflammatory bowel diseases. The increased knowledge on the mechanisms that regulate lymphocyte homing and imprinting has clear applicability in designing more effective vaccination regimens. A detailed understanding of the mucosal homing has recently led to the development of the first successful anti‐adhesive therapeutics in human.     

Mucosal immune network in the gut for the control of infectious diseases

The common mucosal immune system (CMIS) consists of an integrated cross-communication pathway of lymphoid tissues made up of inductive and effector sites for host protection against pathogenic microorganisms. Major effector molecules of the CMIS include IgA antibodies and cytokines, chemokines and their corresponding receptors. Secretory IgA (S-IgA), the major immunoglobulin, is induced by gut-associated lymphoreticular tissue (GALT)-derived B cells with the help of Th1- and Th2-type CD4(+) T lymphocytes. Cytotoxic T lymphocytes (CTLs) in the mucosal epithelium, a subpopulation of intraepithelial lymphocytes (IELs), also help maintain the mucosal barrier. The CMIS is unique in that it can provide both positive and negative signals for the induction and regulation of immune responses in both the mucosal and systemic compartments after oral or nasal antigen exposure. Prevention of infection through mucosal surfaces can be achieved by the CMIS through connections between inductive (e.g. GALT) and effector tissues. When vaccine antigens are enterically administered together with mucosal adjuvants [e.g. cholera toxin (CT), heat-labile toxin produced by Escherichia coli (LT) and IL-12], antigen-specific Th1/Th2 and IgA B cell responses are induced simultaneously in the mucosal effector compartment. Since these antigen-specific immune responses are not generated by oral vaccine without mucosal adjuvant, safe and effective adjuvants for the induction of antigen-specific S-IgA and CTL responses are essential for the development of mucosal vaccines for protection against infectious diseases. Finally, recent findings suggest the presence of a CMIS-independent IgA induction pathway, which also must be considered in the development of mucosal vaccines.