Immunology

It has been known for the past 85 years that mucosal responses can be stimulated by local presentation of antigen and that the gut immune system is capable of mounting both primary and secondary responses to potentially harmful antigens while avoiding the expression of damaging responses to harmless dietary proteins. How these types of responses are induced and regulated remains unclear. In the gut attention has for some time been focused on Peyer's patches (PP) due to evidence that they play a vital role in the induction of humoral and cellular responses. Moreover, it has been established that MHC class II molecules are found in the gut mucosa on a variety of cell types outside PP, namely the lamina propria (LP). Fed antigens have also been detected in the LP and studies have shown that LP cells can stimulate allogeneic mixed lymphocyte responses and are capable of presenting soluble protein antigen to naïve T cells. This article reviews the present understanding of the possible roles of PP and LP in intestinal immunity in terms of induction, regulation, surveillance of immune responses and the antigen presenting cell types involved.     

Mucosal delivery of native and recombinant protein vaccines against Trichostrongylus colubriformis

This paper describes a series of five pilot trials to test the feasibility of inducing a protective mucosal immune response against a non-blood-feeding intestinal nematode by delivery of antigens across the mucosal epithelium. A number of antigen preparations from Trichostrongylus colubriformis (viable larvae, larval homogenate and recombinant 17 kDa excretory-secretory protein) were delivered to the luminal surface of the mucosal epithelium overlying jejunal or rectal lymphoid tissue in cellulose or chitosan formulations. Significant protection was induced following delivery of viable larvae, larval homogenate or recombinant protein to the epithelium overlying rectal Peyer’s patches, and recombinant protein to the epithelium overlying jejunal Peyer’s patches. Viable larvae were associated with a jejunal IgE/IgG1 response, while the 17 kDa antigen was associated with a jejunal IgA response. The results demonstrate that delivery of Trichostrongylus native and recombinant antigens across the epithelium overlying rectal lymphoid patches can result in significant protective immunity even in the absence of adjuvant. They warrant the further investigation of appropriate mucosal delivery methods and adjuvants for induction of protective mucosal responses to stages and species of gastrointestinal helminths which do not ingest serum antibodies.     

Specific immunosuppression by passive antibody. V. Participation of macrophages in reversal of suppression by peritoneal exudate cells from immune animals

Thioglycollate-stimulated peritoneal exudate cells (PEC), harvested from mice immunized against sheep erythrocytes (SRBC) and transferred to normal syngeneic recipients, reverse the immunosuppression caused by passively administered anti-SRBC antibody. Macrophages purified from PEC on BSA gradients did not reverse immunosuppression; neither did suspensions of cells from mesenteric lymph nodes of immune mice. Mixtures of the purified macrophages and lymph node cells were fully capable of reversing immunosuppression. Thus, two types of cell, one a macrophage and one a lymphocyte, are required. Both must be compatible with the recipient mice at the H-2 complex. However, only the macrophages must necessarily be obtained from an immune donor. When “immune” macrophages were preincubated in vitro with “normal” lymph node cells before transfer to antibody-treated syngeneic recipients, a significant reversal of the immunosuppressive effect occurred. The ability of whole PEC or spleen cells to reverse the immunosuppressive effect of passive antibody is acquired rapidly after injection of a single low dose of antigen. Development of this ability precedes the appearance, in the circulation, of immunosuppressive antibody.     

Simultaneous induction of antigen-specific IgA helper T cells and IgG suppressor T cells in the murine Peyer's patch after protein feeding

The effects of feeding the dietary protein antigen, ovalbumin (OVA), on OVA-specific IgG and IgA immune responses involving Peyer's patches (PP) and mesenteric lymph nodes (MLN) were examined. Mice were administered soluble OVA by gastric intubation. One to 3 days later, PP, MLN, or spleen cells from these donor mice were adoptively transferred into normal syngeneic recipients. After two subsequent immunizations, spleens from the recipient mice were assayed for IgA and IgG anti-OVA plaque-forming cell (PFC) responses. None of the tissues from normal (unfed) mice had the inherent ability to alter recipients' IgG or IgA PFC responses. Within 1 day of OVA feeding, however, cells were generated in the PP that could augment recipients' IgA anti-OVA PFC responses and suppress IgG PFC responses. Three days after OVA feeding, these cells were present in MLN as well, and whereas the IgG suppressor cell also appeared to migrate to spleen, the IgA helper cell did not. The cells mediating antigen-specific IgG suppression and IgA help were both T cells but could be distinguished by surface phenotype. We therefore conclude that protein feeding induces differential, isotype-specific immunoregulation in gut-associated lymphoid tissues, part of which is mediated by an antigen-specific IgA helper T cell.     

Localization of fatty acid binding protein of epidermal type common to dendritic cells and presumptive macrophages in Peyer’s patches and epithelial M cells of mouse intestine

Fatty acid binding protein of epidermal type (E-FABP) was expressed/localized in most, if not all, populations of the dendritic cells in the subepithelial domes, follicles and interfollicular regions of Peyer’s patches and presumptive macrophages in their germinal centers, and all M cells in the follicle-associated epithelium of mouse intestine. The immunoreactivity in both of the cell populations makes it easy to recognize the accumulation of DCs in the subepithelial domes in close proximity to the base of M cells, which is essential for luminal antigens to be transported to Peyer’s patches. E-FABP may play some important roles in the mucosal immune reaction through Peyer’s patches and associated structures.     

Oral scrapie infection modifies the homeostasis of Peyer’s patches’ dendritic cells

In transmitted prion diseases the immune system supports the replication and the propagation of the pathogenic agent (PrPSc). DCs, which are mobile cells present in large numbers within lymph organs, are suspected to carry prions through the lymphoid system and to transfer them towards the peripheral nervous system. In this study, C57Bl/6 mice were orally inoculated with PrPSc (scrapie strain 139A) and sacrificed at the preclinical stages of the disease. Immunolabelled cryosections of Peyer’s patches were analysed by confocal microscopy. Membrane prion protein expression was studied by flow cytometry. In Peyer’s patches (PP), dissected at day one and day 105 after oral exposure to scrapie, we observed an increased population of DCs localised in the follicular-associated epithelium. On day 105, PrPSc was found in the follicles inside the PP of prion-infected mice. A subset of Peyer’s patches DCs, which did not express cellular prion protein on their surface in non-infected mice conditions, was prion-positive in scrapie conditions. Within Peyer’s patches oral scrapie exposure thus induced modifications of the homeostasis of DCs at the preclinical stages of the disease. These results give new arguments in favour of the implication of DCs in prion diseases.     

Successful priming and tolerization of T cells to orally administered antigens in B-cell-deficient mice

B cells have been shown to function as APCs capable of inducing both T cell priming and tolerization. Recently, B cells were also revealed to be essential in the organogenesis of Payer's patches (PPs), which have been supposed to play an important role in the initiation of mucosal immune responses. In this study, we examined the roles of B cells in T cell response to orally administrated antigen using B-cell-deficient mice. It was revealed that (1) both a single high dose and repeated low doses of orally administered OVA successfully induced tolerance of T cells in B-cell-deficient mice and (2) oral administration of OVA with cholera toxin successfully primed T cells in B-cell-deficient mice. Thus, it was revealed that B cells are not required for both priming and tolerization of T cells to orally administered antigens. These results also contradict the supposed roles of PPs in mucosal immune responses.     

Alternate mucosal immune system: organized Peyer's patches are not required for IgA responses in the gastrointestinal tract

The progeny of mice treated with lymphotoxin (LT)-beta receptor (LTbetaR) and Ig (LTbetaR-Ig) lack Peyer's patches but not mesenteric lymph nodes (MLN). In this study, we used this approach to determine the importance of Peyer's patches for induction of mucosal IgA Ab responses in the murine gastrointestinal tract. Immunohistochemical analysis revealed that LTbetaR-Ig-treated, Peyer's patch null (PP null) mice possessed significant numbers of IgA-positive (IgA+) plasma cells in the intestinal lamina propria. Further, oral immunization of PP null mice with OVA plus cholera toxin as mucosal adjuvant resulted in Ag-specific mucosal IgA and serum IgG Ab responses. OVA-specific CD4+ T cells of the Th2 type were induced in MLN and spleen of PP null mice. In contrast, when TNF and LT-alpha double knockout (TNF/LT-alpha-/-) mice, which lack both Peyer's patches and MLN, were orally immunized with OVA plus cholera toxin, neither mucosal IgA nor serum IgG anti-OVA Abs were induced. On the other hand, LTbetaR-Ig- and TNF receptor 55-Ig-treated normal adult mice elicited OVA- and cholera toxin B subunit-specific mucosal IgA responses, indicating that both LT-alphabeta and TNF/LT-alpha pathways do not contribute for class switching for IgA Ab responses. These results show that the MLN plays a more important role than had been appreciated until now for the induction of both mucosal and systemic Ab responses after oral immunization. Further, organized Peyer's patches are not a strict requirement for induction of mucosal IgA Ab responses in the gastrointestinal tract.     

In vitro studies of the rabbit immune system: I. Hemolytic plaque-forming response of dissociated spleen cells from normal and primed rabbits

The conditions for the in vitro generation of primary and secondary immune responses by rabbit spleen cells to sheep red blood cell (SRBC) antigen have been examined. Spleen cells from many normal and all previously immunized rabbits are capable of producing in vitro plaque-forming cell (PFC) responses when cultured as dissociated cell suspensions in the presence of antigen. Primed spleen cells generate approximately 100 times the number of PFCs obtained in normal cultures with a shorter lag period. Both types of cultures demonstrate a period of exponential increase in PFCs during which the doubling time is 12–14 hr. This increase occurs after 1 day of culture of spleen cells from primed rabbits and after 4 days of culture of spleen cells from unprimed rabbits. The PFCs which arise in cultures of primed cells appear not to be the progeny of those generated in vivo but to be derived from an increased number of PFC precursors. Repeated immunization of the spleen cell donor is required to produce significant numbers of indirect (IgG) PFC or indirect precursors; most of the PFC found after a single immunization in vivo or in vitro are direct (IgM). There is no evidence for conversion of IgM to IgG PFC in vitro. This system should provide a means for further identification of the cellular interactions involved in the immune response of the rabbit.     

Phenotypic Analysis of a Population of IgA+ Cells in the Follicle-Associated Epithelium of Mouse Peyer's Patches

The follicle-associated epithelium (FAE) selectively transports prions, viruses, pathogenic bacteria, commensal microflora, and even secretory IgA (SIgA)-immune complexes from the intestinal lumen to underlying gut-associated lymphoid tissues like Peyer’s patches. The FAE consists of a single layer of columnar epithelial cells that includes enterocytes and M (microfold) cells, intermingled with dendritic cells (DCs), macrophages, and naïve and memory B and T lymphocytes. In this report we describe a population of IgA⁺ cells that reside within and immediately below the FAE in mouse Peyer’s patches. Immunofluorescence microscopy analysis indicated that the FAE-associated IgA⁺ cells were negative for surface antigen markers specific for B cells (B220), T cells (CD3), DCs (CD11c), and plasma cells (CD138). The IgA⁺ cells were also negative Ki-67 and IRF4, indicating that they are not mature B cells or plasma cells. The IgA⁺ cells were, however, often found in close proximity to DCs, leading us to speculate that the population of IgA⁺ cells in the FAE constitutes an atypical subset of B cells involved in mucosal antigen surveillance and/or immune recall.     

Acute infection of mice with lactic dehydrogenase virus (LDV) impairs the antigen-presenting capacity of their macrophages

The infection of mice with lactic dehydrogenase virus (LDV) is characterized by elevated levels of various plasma enzymes such as lactic dehydrogenase, malic dehydrogenase, and others. This elevation is probably the consequence of a defect in the clearance capacity of the virus-affected reticuloendothelial cells, which were found to serve as the targets for LDV infection. Since macrophages play a pivotal role in the induction and regulation of cellular immune responses, we tested the antigen-presenting capacity of macrophages from LDV-infected mice, using a system in which in vitro reactivation of memory T cells depends on specific antigen presentation by macrophages. Our experiments revealed that the antigen-presenting capacity of spleen, lymph node, and peritoneal antigen-presenting macrophages from LDV-infected mice was impaired. This impairment, however, was not due to a defective cellular concentration capacity of antigen, since no difference in the uptake of radiolabeled antigen by uninfected and acutely LDV-infected macrophages was observed. Similarly one cannot attribute the impaired presentation capacity to suppressor cells, since we found that LDV-infected macrophages are not differentially immunosuppressive in the specific in vitro assays used. The analysis of peritoneal macrophages for their expression of Ia antigens revealed that the proportion of Ia-positive macrophages among the LDV-infected peritoneal cells is reduced in comparison to their proportion in noninfected mice. Our results suggest, therefore, that infection of macrophages by LDV is followed by an impairment of their antigen-presenting capacity, probably due to a reduction in the relative proportion of Ia-positive macrophages. These results indicate that the virus either impairs the expression of membrane-associated antigen-presenting components (such as the Ia determinants), thus damaging antigen presentation, or is responsible for the elimination of Ia-positive cells from the peritoneum.     

Orally administered <Emphasis Type="Italic">Bifidobacterium</Emphasis> triggers immune responses following capture by CD11c<Superscript>+</Superscript> cells in Peyer’s patches and cecal patches

We have investigated the immunomodulatory mechanisms of Bifidobacterium pseudocatenulatum JCM7041 (Bp) as model of probiotics following oral administration to mice. This study was conducted with the aim of clarifying the mechanism of immunomodulation induced by oral administration of probiotic bacteria through elucidation of the detailed mechanism of transfer of orally administered bacterial cells within the body and the interaction between bacterial cells and cells of the immune tissues. We observed the localization of Bp in mice following oral administration, showing that Bp was surrounded by CD11c⁺ cells in Peyer’s patches (PP) and cecal patches (CP). These results indicated that Bp might induce CD11c⁺ cell-mediated immune responses directly. Furthermore, IL-10 and IL-12p40 production by Thy1.2⁻ cells, including CD11c⁺ cells, increased significantly. Production of IL-10 and IL-12p40 by bone marrow-derived dendritic cells (BMDC) was significantly increased by Bp stimulation. These results suggest that oral administration of Bp induces immune responses directly following capture by CD11c⁺ dendritic cells (DCs). Subsequently, we observed oral administration of Bp for 1 week induced IgA and IgA-associated cytokine production by CP and PP cells, suggesting that Bp induced DC-mediated immune responses on CP as well as PP.     

Regulation of the In Vitro Secondary Cell-Mediated Cytotoxic Response against Syngeneic FBL-3 Leukemia by Macrophages

Depletion of macrophages from immune spleen cells by treatment with carbonyl iron and magnet or by in vivo treatment with carrageenan enhanced the in vitro secondary cell-mediated cytotoxic response against a syngeneic Friend virus-induced leukemia, FBL-3 cells of C57BL/6 mice. However, further depletion of macrophages by passing the carbonyl iron-treated immune spleen cells through a nylon wool column abrogated the cytotoxic response. The addition of splenic macrophage-enriched preparations from either FBL-3-immune or normal mice suppressed the cytotoxic response of immune spleen cells treated with carbonyl iron and magnet. This suppressive effect of splenic macrophages presented a marked contrast with the enhancing effect of normal peritoneal macrophages on the same cell-mediated cytotoxic response, indicating regulation of the generation of killer T cells against a syngeneic tumor by functionally distinct macrophages. The suppressed cell-mediated cytotoxic response against FBL-3 cells by immune spleen cells was augmented by the addition of indomethacin to the culture medium, and this augmentation with indomethacin was greatly decreased by depletion of phagocytic cells from the immune spleen by treatment with carbonyl iron and magnet. The mechanisms of regulation of the cell-mediated cytotoxic response with soluble factors released from macrophages are discussed.     

Epithelium-Intrinsic MicroRNAs Contribute to Mucosal Immune Homeostasis by Promoting M-Cell Maturation

M cells in the follicle-associated epithelium (FAE) of Peyer’s patches (PPs) serve as a main portal for external antigens and function as a sentinel in mucosal immune responses. The scarcity of these cells has hampered identification of M cell-specific molecules. Recent efforts have begun to provide insight into antigen transcytosis and differentiation of M cells; however, the molecular mechanisms underlying these processes are not fully elucidated. Small non-coding RNAs including microRNA (miRNA) have been reported to regulate gene expression and control various biological processes such as cellular differentiation and function. To evaluate the expression of miRNAs in FAE, including M cells, we previously performed microarray analysis comparing intestinal villous epithelium (VE) and PP FAE. Here we confirmed FAE specific miRNA expression levels by quantitative PCR. To gain insight into miRNA function, we generated mice with intestinal epithelial cell-specific deletion of Dicer1 (Dicer^ΔIEC) and analyzed intestinal phenotypes, including M-cell differentiation, morphology and function. Dicer^ΔIEC mice had a marked decrease in M cells compared to control floxed Dicer mice, suggesting an essential role of miRNAs in maturation of these cells. Furthermore, transmission electron microscopic analysis revealed that depletion of miRNA caused the loss of endosomal structures in M cells. In addition, antigen uptake by M cells was impaired in Dicer^ΔIEC mice. These results suggest that miRNAs play a significant role in M cell differentiation and help secure mucosal immune homeostasis.     

PP4 Is Essential for Germinal Center Formation and Class Switch Recombination in Mice

PP4 is a serine/threonine phosphatase required for immunoglobulin (Ig) VDJ recombination and pro-B/pre-B cell development in mice. To elucidate the role of PP4 in mature B cells, we ablated the catalytic subunit of murine PP4 in vivo utilizing the CD23 promoter and cre-loxP recombination and generated CD23^crePP4^F/F mice. The development of follicular and marginal zone B cells was unaffected in these mutants, but the proliferation of mature PP4-deficient B cells stimulated by in vitro treatment with either anti-IgM antibody (Ab) or LPS was partially impaired. Interestingly, the induction of CD80 and CD86 expression on these stimulated B cells was normal. Basal levels of serum Igs of all isotypes were strongly reduced in CD23^crePP4^F/F mice, and their B cells showed a reduced efficiency of class switch recombination (CSR) in vitro upon stimulation by LPS or LPS plus IL-4. When CD23^crePP4^F/F mice were challenged with either the T cell-dependent antigen TNP-KLH or the T cell-independent antigen TNP-Ficoll, or by H1N1 virus infection, the mutant animals failed to form germinal centers (GCs) in the spleen and the draining mediastinal lymph nodes, and did not efficiently mount antigen-specific humoral responses. In the resting state, PP4-deficient B cells exhibited pre-existing DNA fragmentation. Upon stimulation by DNA-damaging drug etoposide in vitro, mutant B cells showed increased cleavage of caspase 3. In addition, the mutant B cells displayed impaired CD40-mediated MAPK activation, abnormal IgM-mediated NF-κB activation, and reduced S phase entry upon IgM/CD40-stimulation. Taken together, our results establish a novel role for PP4 in CSR, and reveal crucial functions for PP4 in the maintenance of genomic stability, GC formation, and B cell-mediated immune responses.     

Clusterin in human gut-associated lymphoid tissue, tonsils, and adenoids: localization to M cells and follicular dendritic cells

The follicle-associated epithelium (FAE) overlying the follicles of mucosa-associated lymphoid tissue is a key player in the initiation of mucosal immune responses. We recently reported strong clusterin expression in the FAE of murine Peyer’s patches. In this study, we examined the expression of clusterin in the human gut-associated lymphoid tissue (GALT) and Waldeyer’s ring. Immunohistochemistry for clusterin in human Peyer’s patches, appendix and colon lymphoid follicles revealed expression in M cells and in follicular dendritic cells (FDCs). Using cryo-immunogold electron microscopy in Peyer’s patches, we observed cytosolic immunoreactivity in M cells and labeling in the ER/Golgi biosynthetic pathway in FDCs. In palatine tonsils and adenoids, we demonstrated clusterin expression in germinal centers and in the lymphoepithelium in the crypts where M cells are localized. In conclusion, clusterin is expressed in M cells and follicular dendritic cells at inductive sites of human mucosa-associated lymphoid tissue suggesting a role for this protein in innate immune responses. Moreover, the use of clusterin as a human M cell marker could prove to be a valuable tool in future M cell research.     

Studies of the mechanisms for the induction of in vivo tumor immunity. VI. Induction of specific and nonspecific cell-mediated immunity in tumor-bearing hosts and its correlation with transplantation tumor immunity

Studies were performed to determine the development of cell-mediated cytotoxic response at tumor site in C57BL/6 mice bearing progressively growing FBL-3 ascites leukemia. The effectors isolated from tumor ascites are found to be highly cytotoxic for leukemic target cells. The levels of cytotoxicity obtained with effectors isolated from tumor site are generally higher than those obtained with immune mice. This cytotoxicity is both specific and nonspecific. The specific cytotoxicity against tumor-associated antigen is mainly mediated by T cells and the nonspecific cytotoxicity against unrelated tumor cells is mediated largely by macrophages. The T-cell-enriched preparation did not give significant natural killer activity. When testing the ability of these effectors to produce in vivo immunity against the challenge of FBL-3, it was found that only T cells could confer the transplantation-type immunity, but the immunity was transient. The macrophage-enriched preparation isolated from tumor ascites failed to give in vivo protection. These findings indicate that in FBL-3 system, mice with progressively growing tumors are able to develop immune response against tumor cells. However, this immunity is probably interfered with by a suppressor factor(s) or suppressor cells which restrict their activity to eliminate the tumor cells effectively.     

The state of CD4+ T cell activation is a major factor for determining the kinetics and location of T cell responses to oral antigen

Current models suggest that inductive immune responses to enteric Ag are initiated in Peyer's patches (PP) and mesenteric lymph nodes (MLN) followed by migration of activated, memory-like CD4(+) T cells to extralymphoid sites in the intestinal lamina propria (LP). The resultant immune system contains both naive and activated T cells. To examine the differential responses of naive and memory-like T cells to oral Ag, bone marrow chimeras (BMC) were generated. Irradiated BALB/c hosts were reconstituted with a mix of DO11.10 x RAG-1(-/-) and BALB/c bone marrow. In unprimed DO11.10 and BMC models, LP and PP DO11.10 T cells responded to oral Ag with similar kinetics. Responses of activated, memory-like T cells to oral Ag were examined in thymectomized BMC 60 days after i.p. immunization with OVA peptide in Freund's adjuvant (OVA(323-339)/CFA). Results indicate that i.p. OVA(323-339)/CFA generated a high proportion of memory-like CD45RB(low) DO11.10 T cells in peripheral lymphoid (40%) and intestinal LP (70%) tissue. Previously activated DO11.10 T cells in the LP responded to oral Ag earlier and at 50% higher levels compared with memory CD4(+) T cells localized to PP tissue. These data indicate that responses to oral Ag in antigenically naive animals are initiated in PP whereas in Ag-experienced animals LP T cells respond earlier and more vigorously than cells in PP. Taken together, these data suggest that previous activation alters the hierarchy of T cell responses to oral Ag by enhancing the efficiency of LP T cell activation.