Re-utilization of germinal centers in multiple Peyer's patches results in highly synchronized,oligoclonal, and affinity-matured gut IgA responses

Whereas gut IgA responses to the microbiota may be multi-centered and diverse, little is known about IgA responses to T-cell-dependent antigens following oral immunizations. Using a novel approach, gut IgA responses to oral hapten (4-hydroxy-3-nitrophenyl)acetyl-cholera toxin (NP-CT) conjugates were followed at the cellular and molecular level. Surprisingly, these responses were highly synchronized, strongly oligoclonal, and dominated by affinity matured cells. Extensive lineage trees revealed clonal relationships between NP-specific IgA cells in gut inductive and effector sites, suggesting expansion of the same B-cell clone in multiple Peyer's patches (PPs). Adoptive transfer experiments showed that this was achieved through re-utilization of already existing germinal centers (GCs) in multiple PPs by previously activated GC GL7⁺ B cells, provided oral NP-CT was given before cell transfer. Taken together, these results explain why repeated oral immunizations are mandatory for an effective oral vaccine.     

B-cell activation following murine cytomegalovirus infection: implications for autoimmunity.

Infection of susceptible mice with murine cytomegalovirus (MCMV) induces persistent inflammation, and the production of autoantibodies reactive with large numbers of proteins from all major organs. However the roles of polyclonal B-cell activation, autoreactive T-helper cells and host-virus cross-reactions in these phenomena have not been evaluated. The present study reveals six- to 20-fold increases in serum immunoglobulin levels in MCMV-infected BALB/c and CBA mice, with IgG3 and IgG2b most affected. Titres of antibodies reactive with autologous tissues and ovalbumin (OVA) also increased following MCMV infection, whilst responses to a synthetic antigen [polyvinyl pyrrolidone (PVP)] were unaffected or depressed. IgG2a was the isotype most affected in responses to OVA, MCMV antigens and autologous tissues, suggesting interferon-gamma (IFN-gamma) may contribute to responses induced in the presence of the relevant antigen. Increases in total and antigen-specific immunoglobulin levels were CD4 dependent, as they were reduced in infected mice depleted of these cells with anti-CD4 antibodies. Serological changes were preceded by B-cell expansion and activation evident from increased cell yields, frequencies of cells releasing immunoglobulin and proliferation of T-depleted spleen and lymph node preparations. Numbers of mature B cells and macrophages increased in the lymph nodes, but B-1a (CD5+ Ig+) cell counts remained low. Alterations in the B-cell phenotypic profiles were more complex in the spleen, but correction for increased cell yields revealed increases in some subpopulations.     

滤泡树突状细胞与生发中心反应研究的新进展

生发中心是在T细胞依赖性抗体应答过程中于外周淋巴组织内形成的一个特殊的结构。在GC内,受抗原刺激而活化的B细胞进行克隆扩增、IgV区基因的体细胞高度突变、亲和力成熟以及同类型转换,最终形成记忆性B细胞或是产生Ig的浆细胞。在GC内B细胞增殖的同时,也启动了凋亡机制,以确保最终形成的记忆B细胞或浆细胞对抗原的高度特异性。FDCs是参与再次免疫应答的重要细胞,它主要是通过表面的FcR和CR将免疫复合物结合在细胞膜上,并选择性的将抗原递呈给表达高亲和力BCR的B细胞,使之激活并产生抗体或形成记忆B细胞。因此,FDCs在生发中心反应、免疫记忆的维持、B细胞的分化、成熟以及记忆B细胞的形成具有极其重要的作用。但最近的研究对FDCs及其结合的免疫复合物的重要性提出了质疑,认为FDCs在生发中心反应、B细胞的分化、成熟以及记忆B细胞的形成中的作用很可能是非特异性的,并对驻留在FDCs表面的免疫复合物的其它潜在功能进行了讨论。     

Lymphoid tissue-resident Alcaligenes LPS induces IgA production without excessive inflammatory responses via weak TLR4 agonist activity

Alcaligenes are opportunistic commensal bacteria that reside in gut-associated lymphoid tissues such as Peyer's patches (PPs); however, how they create and maintain their homeostatic environment, without inducing an excessive inflammatory response remained unclear. We show here that Alcaligenes-derived lipopolysaccharide (Alcaligenes LPS) acts as a weak agonist of toll-like receptor 4 and promotes IL-6 production from dendritic cells, which consequently enhances IgA production. The inflammatory activity of Alcaligenes LPS was weaker than that of Escherichia coli-derived LPS and therefore no excessive inflammation was induced by Alcaligenes LPS in vitro or in vivo. Alcaligenes LPS also showed adjuvanticity, inducing antigen-specific immune responses without excessive inflammation. These findings reveal the presence of commensal bacteria-mediated homeostatic inflammatory conditions within PPs that produce optimal IgA induction without causing pathogenic inflammation and suggest that Alcaligenes LPS could be a safe and potent adjuvant.     

Could the airway epithelium play an important role in mucosal immunoglobulin A production?

Immunoglobulin (Ig) A is the major immunoglobulin of the healthy respiratory tract and is thought to be the most important immunoglobulin for lung defence. The basis for the preferential generation of IgA-secreting cells in the airway mucosa remains unclear. Given the half-life of 5 days for the majority of IgA plasma cells, many IgA plasma cells must develop daily from B cells to guarantee a continuous supply of IgA antibodies in the airway mucosa. For this, the surrounding cells must provide a constant supply of cytokines necessary for B-cell isotype switch, growth and differentiation into IgA-secreting plasma cells. Studies with CD4+ T-cell knockout mice, T-cell receptor knockout mice and mice made transgenic for CTLA4-Ig demonstrate normal mucosal IgA isotype switch, differentiation and IgA production, thereby suggesting that T cells are not critical for mucosal IgA production, and that other cell sources may be more important. Also, the bronchus-associated lymphoid tissue (BALT), which is believed to be the major site where IgA isotype switch and differentiation of B cells into plasma cells occur with the help of cytokines released by T cells, is not a constitutive feature of the normal human lung. This indicates that other parts of the respiratory tract must carry out the BALT function. We have recently demonstrated that healthy human airway epithelial cells constitutively produce IL-5, a major cytokine implicated in the growth and differentiation of post-switch mIgA+ B cells to IgA-producing plasma cells. Several studies have recently reported that the human airway epithelium also constitutively produces IL-2, TGFbeta, IL-6 and IL-10, factors which are essential for B-cell clonal proliferation, IgA isotype switch and differentiation into IgA-producing plasma cells. The close proximity of B cells to the airway epithelium probably ensures a constant supply of growth and differentiation factors necessary for mucosal IgA production. In addition, the epithelial cells produce a glycoprotein, called the secretory component, which not only confers increased stability to S-IgA, but is also quantitatively the most important receptor of the mucosal immune system, since it is responsible for the external transport of locally produced polymeric IgA and IgM. Recent studies also suggest a possible role for epithelial cells in antigen presentation. Dendritic cells situated within the airway epithelium could directly present antigens to B cells and direct their isotype switch towards IgA1 and IgA2 with the help of cytokines produced by epithelial cells. Airway epithelial cells could therefore play a major role in the production of mucosal IgA antibodies which are essential for airway mucosal defence.     

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.     

Microfold cell-dependent antigen transport alleviates infectious colitis by inducing antigen-specific cellular immunity

Infectious colitis is one of the most common health issues worldwide. Microfold (M) cells actively transport luminal antigens to gut-associated lymphoid tissue to induce IgA responses; however, it remains unknown whether M cells contribute to the induction of cellular immune responses. Here we report that M cell-dependent antigen transport plays a critical role in the induction of Th1, Th17, and Th22 responses against gut commensals in the steady state. The establishment of commensal-specific cellular immunity was a prerequisite for preventing bacterial dissemination during enteropathogenic Citrobacter rodentium infection. Therefore, M cell-null mice developed severe colitis with increased bacterial dissemination. This abnormality was associated with mucosal barrier dysfunction. These observations suggest that antigen transport by M cells may help maintain gut immune homeostasis by eliciting antigen-specific cellular immune responses.     

Molecular insights into the mechanisms of M-cell differentiation and transcytosis in the mucosa-associated lymphoid tissues

Microfold cells (M cells), which are located in the follicle-associated epithelium (FAE) covering mucosal lymphoid follicles, are specialized epithelial cells that initiate mucosal immune responses. These cells take luminal antigens and transport them via transcytosis across the FAE to the antigen-presenting cells underneath. Several intestinal pathogens exploit M cells as their portal for entry to invade the host and cause disease conditions. Recent studies have revealed that the uptake of antigens by M cells is essential for efficient antigen-specific IgA production and that this process likely maintains the homeostasis of mucosal tissues. The present article reviews recent advances in understanding the molecular mechanism of M-cell differentiation and describes the molecules expressed by M cells that are associated with antigen uptake and/or the transcytosis process. Current efforts to augment M-cell-mediated uptake for use in the development of effective mucosal vaccines are also discussed.     

The common mucosal immune system and current strategies for induction of immune responses in external secretions

The selective induction of antibodies in external secretions is desirable for the prevention of various systemic as well as predominantly mucosa-restricted infections. An enormous surface area of mucosal membranes is protected primarily by antibodies that belong, in many species, to the IgA isotype. Such antibodies are produced locally by large numbers of IgA-containing plasma cells distributed in subepithelial spaces of mucosal membranes and in the stroma of secretory glands. In humans and in some animal species, plasma-derived IgA antibodies do not enter external secretions in significant quantities and systemically administered preformed IgA antibodies would be of little use for passive immunization. Systemic administration of microbial antigens may boost an effective S-IgA immune response only in a situation whereby an immunized individual had previously encountered the same antigen by the mucosal route. Local injection of antigen in the vicinity of secretory glands is usually accompanied by an undesirable concomitant systemic response and frequently requires the addition of adjuvants that are unacceptable for administration in humans. Immunization routes that involve ingestion or possibly inhalation of antigens lead to the induction of not only local but also generalized immune responses manifested by the parallel appearance of S-Iga antibodies to ingested or inhaled antigens in secretions of glands distant from the site of immunization. Based on extensive studies in animal models as well as in humans, convincing evidence is available that antigen-sensitized and IgA-committed precursors of plasma cells from GALT are disseminated to the gut, other mucosa-associated tissues, and exocrine glands. However, due to the limited absorption of desired antigens from the gut lumen of orally immunized individuals, repeated large doses of antigens are required for an effective S-IgA response. Novel antigen delivery systems for the stimulation of such responses are currently being examined in several laboratories. Live attenuated or genetically manipulated bacteria expressing other microbial antigens have also been used for selective colonization of gut-associated lymphoid tissues. Unique antigen packaging and the use of adjuvants suitable for oral administration hold promise for an efficient antigen delivery to critical tissues in the intestine and deserve extensive exploration. The oral immunization route appears to have many advantages over systemic immunization.(ABSTRACT TRUNCATED AT 400 WORDS)     

Unique properties of memory B cells of different isotypes

Summary: Memory antibody responses are typically seen to T-cell-dependent antigens and are characterized by the rapid production of high titers of high-affinity antigen-specific antibody. The hallmark of T-cell-dependent memory B cells is their expression of a somatically mutated, isotype-switched B-cell antigen receptor, features that are mainly generated in germinal centers. Classical studies have focused on isotype-switched memory B cells (mainly IgG isotype) and demonstrated their unique intrinsic properties in terms of localization and responsiveness to antigen re-exposure. However, recent advances in monitoring antigen-experienced B cells have revealed the considerable heterogeneity of memory B cells, which include unswitched IgM⁺ and/or unmutated memory B cells. The IgM and IgG type memory B cells reside in distinct locations and appear to possess distinct origins and effector functions, together orchestrating humoral memory responses.     

Mucosal homeostasis: role of interleukins, isotype-specific factors and contrasuppression in the IgA response

Oral ingestion of antigen elicits immune responses at mucosal sites where humoral immunity is largely due to antibodies of the IgA isotype. This is often accompanied by suppression of systemic responses to the same antigen, a state termed oral tolerance. This IgA response is regulated by interactions between T cell subsets found at IgA inductive tissues, i.e., the gut-associated lymphoreticular tissue (GALT) or Peyer's patches (PP). PP T helper (Th) cells support IgA responses, and interleukins 5 (IL-5) and IL-6 can augment secretion of this isotype. Subsets of Th cells may also express Fc receptors for IgA (Fc alpha R) and secrete Fc alpha R as an IgA-binding factor (IBF alpha). Membrane-derived Fc alpha R is a glycoprotein of 38,000 M.W. and this molecule induces selective increases in IgA secreting cells (as determined by the ELISPOT assay) in PP B cell cultures. Fc alpha R+ T cell lines have been shown to secrete IBF alpha as well as IL-5 both of which promote IgA synthesis. Recombinant IL-5 (rIL-5) and rIL-6 induce IgA synthesis mainly by PP B cell blasts, and principally act on surface IgA-positive (sIgA+) B cells for these responses. Another form of mucosal regulation is provided by T contrasuppressor (Tcs) cells, which abrogate oral tolerance when adoptively transferred to mice and restore systemic responsiveness to the antigen sheep erythrocyte (SRBC). Tcs cells from mice systemically primed with SRBC support IgM and IgG subclass responses, while Tcs cells from orally primed mice support IgM, IgG subclass and IgA anti-SRBC responses. These Tcs cells are CD3+, CD4-, 8- and are antigen-specific. These regulatory cells may use the gamma-delta (gamma-delta) form of T cell receptor for antigen recognition.     

Primary antibody responses to thymus-independent antigens in the lungs and hilar lymph nodes of mice.

B lymphocytes from the pulmonary lymphoid tissues were stimulated with a variety of thymus-independent (TI) antigens by intratracheal (i.t.) immunization. Immune responses in the lungs and hilar lymph nodes (HLN), which are part of the localized lymphoid tissue, as well as in the spleen, the systemic lymphoid organ, were studied. Thus, primary i.t. immunization of mice with the TI-1 antigen trinitrophenyl-lipopolysaccharide (TNP-LPS) elicited both antigen-specific and polyclonal plaque-forming cell responses from HLN, lung, and splenic B lymphocytes. These responses appeared as early as 3 days after immunization and declined by day 7. Similar immunization with another TI-1 antigen, TNP-Brucella abortus, resulted in anti-TNP responses in both pulmonary and systemic lymphoid tissues, although the kinetics of the antibody response were different than those to TNP-LPS. Interestingly an i.t. immunization with a TI-2 antigen, TNP-Ficoll, failed to induce an anti-TNP PFC response from HLN and lung B cells, although there was good antibody formation from splenic B cells. Antibody response to TNP-Ficoll was restored in pulmonary tissues when mice were immunized with TNP-Ficoll mixed with unconjugated B. abortus. In conclusion, our results indicate that TI-1 and TI-2 antigens differ in their ability to induce antibody responses in the pulmonary lymphoid tissues. The inability of TNP-Ficoll to elicit an antibody response in pulmonary lymphoid tissues has significance in the development of vaccines containing bacterial polysaccharides.     

Memory B-cell diversity: From early generation to tissue residency and reactivation

Memory B cells (MBCs) have a crucial function in providing an enhanced response to repeated infections. Upon antigen encounter, MBC can either rapidly differentiate to antibody secreting cells or enter germinal centers (GC) to further diversify and affinity mature. Understanding how and when MBC are formed, where they reside and how they select their fate upon reactivation has profound implications for designing strategies to improve targeted, next-generation vaccines. Recent studies have crystallized much of our knowledge on MBC but also reported several surprising discoveries and gaps in our current understanding. Here, we review the latest advancements in the field and highlight current unknowns. In particular, we focus on timing and cues leading to MBC generation before and during the GC reaction, discuss how MBC become resident in mucosal tissues, and finally, provide an overview of factors shaping MBC fate-decision upon reactivation in mucosal and lymphoid tissues.     

Immunohistochemical characterization of cellular immunoglobulins and epithelial marker antigens in Warthin's tumor

Lymphoid and epithelial cell marker studies based on paired immunofluorescence staining were performed on ethanol-fixed specimens from six Warthin's tumors of the parotid gland. A polyclonal pattern of isotype and light-chain expression was demonstrated for immunoglobulin-producing cells and afforded definitive evidence for the reactive nature of B-cell proliferation. The average percentages of IgG, IgA, IgM, IgD, and IgE immunocytes were 48.6, 38.5, 8.9, 3.3, and 0.7, respectively. The percentages of J-chain-positive cells within the first four isotypes were 11.3, 47.0, 67.2, and 64.4. Both features were more typical of immune responses in lymphoid tissues than in exocrine glands. In five of the six specimens, IgE was present in a prominent lacy pattern in some follicular centers, often extending to lymphocyte membranes of the mantle zone. Mast cells positive for IgE were seen in all cases. The two latter features indicate that type 1 hypersensitivity might contribute to the lesion. Parts of the tumor epithelium stained selectively for dimeric IgA and secretory component (SC), signifying secretory capacity. In addition, lactoferrin and carcinoembryonic antigen (CEA) occasionally were present in a narrow cytoplasmic luminal rim. Carcinoembryonic antigen was also seen in papillary epithelial projections. Lysozyme was found in isolated epithelial cells, whereas amylase was completely lacking. Except for the presence of CEA, this pattern of epithelial markers resembled that seen in striated ducts of normal salivary glands.     

IgA synthesis: a form of functional immune adaptation extending beyond gut

Immunoglobulin A (IgA) is the most abundantly produced antibody isotype in mammals. The primary function of IgA is to maintain homeostasis at mucosal surfaces. IgA is generated in specialized gut associated lymphoid tissues (GALT) by T cell-dependent and T cell-independent mechanisms. Studies in mice have demonstrated that IgA diversification has an essential role in the regulation of gut microbiota. Aberrant bacterial growth, by activating innate and adaptive immune cells, has emerged as a risk factor for inflammatory diseases such as metabolic disorders and autoimmune diseases. Dynamic diversification of IgA shields bacterial antigens preventing inflammatory responses, but when IgA regulation is suboptimal aberrant bacterial growth and inflammation can ensue.     

Mucosal Homeostasis: Role of Interleukins,Isotype-specific factors and Contrasuppression in the IgA response

Oral ingestion of antigen elicits immune responses at mucosal sites where humoral immunity is largely due to antibodies of the IgA isotype. This is often accompanied by suppression of systemic responses to the same antigen, a state termed oral tolerance. This IgA response is regulated by interactions between T cell subsets found at IgA inductive tissues, i.e., the gut-associated lymphoreticular tissue (GALT) or Peyer's patches (PP). PP T helper (Th) cells support IgA responses, and interleukins 5 (IL-5) and IL-6 can augment secretion of this isotype. Subsets of Th cells may also express Fc receptors for IgA (FcαR) and secrete FcαR as an IgA-binding factor (IBFα). Membrane-derived FcαR is a glycoprotein of 38,000 M.W. and this molecule induces selective increases in IgA secreting cells (as determined by the ELISPOT assay) in PP B cell cultures. FcαR⁺ T cell lines have been shown to secrete IBFα as well as IL-5 both of which promote IgA synthesis. Recombinant IL-5 (rIL-5) and rIL-6 induce IgA synthesis mainly by PP B cell blasts, and principally act on surface IgA-positive (sIgA⁺) B cells for these responses. Another form of mucosal regulation is provided by T contrasuppressor (Tcs) cells, which abrogate oral tolerance when adoptively transferred to mice and restore systemic responsiveness to the antigen sheep erythrocyte (SRBC). Tcs cells from mice systemically primed with SRBC support IgM and IgG subclass responses, while Tcs cells from orally primed mice support IgM, IgG subclass and IgA anti-SRBC responses. These Tcs cells are CD3⁺, CD4^?, 8^? and are antigen-specific. These regulatory cells may use the gamma-delta (γ-δ) form of T cell receptor for antigen recognition.     

Intra-intestinal priming leads to antigen-specific IgA memory cells in peripheral lymphoid organs.

The aim of this study was to gain more insight into the mechanism of IgA memory formation by testing the effects of intra-intestinal antigen priming on various booster routes. To obtain a primary immune response trinitrophenyl conjugated keyhole limpet haemocyanin (KLH-TNP) was injected into the lumen of the small intestines of mice. For secondary immune responses mice were boosted intra-intestinally, intravenously or subcutaneously. The distribution of antigen specific cells in situ was demonstrated by enzyme histochemistry whereas quantification of TNP-specific cells was performed with a plaque-forming cell assay. After single or repeated intra-intestinal antigen administrations both primary and secondary immune responses in terms of specific antibody containing cells were mainly located in the spleen. The anti-TNP antibody-containing cells produced predominantly IgM during the primary and IgM, IgG and IgA during the secondary response. In mesenteric lymph nodes and villi antigen-specific cells were detected sporadically. When intra-intestinal priming was followed by intravenous or subcutaneous booster injections most anti-TNP antibody-producing cells were demonstrated in the spleen and in the draining popliteal lymph nodes. In contrast to repeated intravenous or subcutaneous immunizations alone, these organs contained, besides specific IgM and IgG cells, many TNP-specific cells producing IgA antibodies. This result demonstrates that the production of IgA antibodies is not restricted to mucosa-associated lymphoid tissues. IgA memory cells are induced in mucosa associated lymphoid tissues, probably in Peyer's patches, will consecutively migrate throughout the whole lymphoid system and can be triggered by renewed antigen contact to become IgA plasma cells.