Peyer's Patches Germinal Centers Research Articles

Evaluation of B Cell Proliferation in vivo by EdU Incorporation Assay.

Generation of antibodies is crucial for establishing enduring protection from invading pathogens, as well as for maintaining homeostasis with commensal bacteria at mucosal surfaces. Chronic exposure to microbiota- and dietary- derived antigens results in continuous production of antibody producing cells within the Peyer's patch germinal center structures. Recently, we have shown that B cells responding to gut-derived antigens colonize the subepithelial dome (SED) in Peyer's patches and rapidly proliferate independently of their relative BCR affinity. To evaluate B cell proliferation within different niches in Peyer's patches, we applied in vivo EdU incorporation assay as described in this protocol.

Antigen sampling by intestinal M cells is the principal pathway initiating mucosal IgA production to commensal enteric bacteria.

Secretory IgA (SIgA) directed against gut resident bacteria enables the mammalian mucosal immune system to establish homeostasis with the commensal gut microbiota after weaning. Germinal centers (GCs) in Peyer's patches (PPs) are the principal inductive sites where naive B cells specific for bacterial antigens encounter their cognate antigens and receive T-cell help driving their differentiation into IgA-producing plasma cells. We investigated the role of antigen sampling by intestinal M cells in initiating the SIgA response to gut bacteria by developing mice in which receptor activator of nuclear factor-κB ligand (RANKL)-dependent M-cell differentiation was abrogated by conditional deletion of Tnfrsf11a in the intestinal epithelium. Mice without intestinal M cells had profound delays in PP GC maturation and emergence of lamina propria IgA plasma cells, resulting in diminished levels of fecal SIgA that persisted into adulthood. We conclude that M-cell-mediated sampling of commensal bacteria is a required initial step for the efficient induction of intestinal SIgA.

Segmented Filamentous Bacterium Uses Secondary and Tertiary Lymphoid Tissues to Induce Gut IgA and Specific T Helper 17 Cell Responses

Segmented filamentous bacterium (SFB) is a symbiont that drives postnatal maturation of gut adaptive immune responses. In contrast to nonpathogenic E. coli, SFB stimulated vigorous development of Peyer's patches germinal centers but paradoxically induced only a low frequency of specific immunoglobulin A (IgA)-secreting cells with delayed accumulation of somatic mutations. Moreover, blocking Peyer's patch development abolished IgA responses to E. coli, but not to SFB. Indeed, SFB stimulated the postnatal development of isolated lymphoid follicles and tertiary lymphoid tissue, which substituted for Peyer's patches as inductive sites for intestinal IgA and SFB-specific T helper 17 (Th17) cell responses. Strikingly, in mice depleted of gut organized lymphoid tissue, SFB still induced a substantial but nonspecific intestinal Th17 cell response. These results demonstrate that SFB has the remarkable capacity to induce and stimulate multiple types of intestinal lymphoid tissues that cooperate to generate potent IgA and Th17 cell responses displaying only limited target specificity.

DNA polymerases β and λ do not directly affect Ig variable region somatic hypermutation although their absence reduces the frequency of mutations

During somatic hypermutation (SHM) of antibody variable (V) region genes, activation-induced cytidine deaminase (AID) converts dC to dU, and dUs can either be excised by uracil DNA glycosylase (UNG), by mismatch repair, or replicated over. If UNG excises the dU, the abasic site could be cleaved by AP-endonuclease (APE), introducing the single-strand DNA breaks (SSBs) required for generating mutations at A:T bp, which are known to depend upon mismatch repair and DNA Pol η. DNA Pol β or λ could instead repair the lesion correctly. To assess the involvement of Pols β and λ in SHM of antibody genes, we analyzed mutations in the VDJh4 3' flanking region in Peyer's patch germinal center (GC) B cells from polβ(-/-)polλ(-/-), polλ(-/-), and polβ(-/-) mice. We find that deficiency of either or both polymerases results in a modest but significant decrease in V region SHM, with Pol β having a greater effect, but there is no effect on mutation specificity, suggesting they have no direct role in SHM. Instead, the effect on SHM appears to be due to a role for these enzymes in GC B cell proliferation or viability. The results suggest that the BER pathway is not important during V region SHM for generating mutations at A:T bp. Furthermore, this implies that most of the SSBs required for Pol η to enter and create A:T mutations are likely generated during replication instead. These results contrast with the inhibitory effect of Pol β on mutations at the Ig Sμ locus, Sμ DSBs and class switch recombination (CSR) reported previously. We show here that B cells deficient in Pol λ or both Pol β and λ proliferate normally in culture and undergo slightly elevated CSR, as shown previously for Pol β-deficient B cells.

The Sensing of Environmental Stimuli by Follicular Dendritic Cells Promotes Immunoglobulin A Generation in the Gut

In the Peyer's patches (PPs), germinal centers (GCs) are chronically induced by bacteria and are the major sites for generation of gut immunoglobulin A (IgA) immune responses. Whether follicular dendritic cells (FDCs) within the GCs directly contribute to the IgA production in PPs is unknown. We showed here that direct stimulation of FDCs by bacterial products and retinoic acid synergistically enhanced the expression of the chemokine CXCL13, the survival factor BAFF, and molecules that facilitate the secretion and activation of the cytokine TGF-beta1. A reduced production of these molecules by PP FDCs associated with deficiencies in the Toll-like receptor pathway or vitamin A resulted in decreased numbers of GC B cells and defective generation of IgA(+) B cells within PP GCs. Our data indicate that PP FDCs are conditioned by environmental stimuli to express key factors for B cell migration, survival, and preferential generation of IgA in gut.

Differences in transcriptomic profile and IgA repertoire between jejunal and ileal Peyer's patches

In many species such as sheep and pig, there are two types of Peyer's patches (PP): several discrete patches in the jejunum and a long and continuous patch in the ileum. Most of the immunoglobulin A in the gut is generated by B-cells in the PP germinal centers. Moreover, swine like ovine ileal PP might be important for antigen independent B-cell repertoire diversification. We examined, by quantitative real-time PCR, the expression of 36 transcripts of antimicrobial peptides, chemokines, interleukines, Toll-like receptors and transcription factors from both PP and we highlighted the differences by a principal component analysis. Ileal PP was characterized by a higher mRNA expression of CCL28, IL5, IL10, TLR2 and TLR4 while jejunal PP showed higher mRNA expression of antimicrobial peptides, CCL25, FOXP3, IL4, T-Bet, TSLP and SOCS2. Then, we analyzed some VDJ rearrangements to assess immunoglobulin repertoire diversity in jejunal and ileal PP from weaned piglets. The IgA and IgM repertoires were more diverse in ileal than in jejunal piglet PP. All these results could be related to the rarefaction of interfollicular T-cell zone and the presence in ileal versus jejunal lumen of a more diversified microflora. These findings shed a light on the functional differences between both PP.

The downstream enhancer, Ed, positively regulates Ig kappa gene expression and somatic hypermutation

The mouse Ig kappa locus has three known enhancers: the MAR/intronic enhancer (MEi), the 3′enhancer (E3′) and the further downstream enhancer (Ed). Previous studies have shown that both MEi and E3′ enhancers are required for maximal gene rearrangement of the locus, and that E3′ is also required for maximal expression and somatic hypermutation (SHM). To functionally elucidate Ed in vivo, we generated knockout mice with a targeted germ line deletion of Ed. Ed deleted homozygous mice (Ed−/−) have moderately reduced numbers of kappa expressing B cells and correspondingly increased numbers of lambda expressing B cells in spleen. Ed−/− mice also have decreased kappa mRNA expression in resting and T‐cell dependent activated splenic B cells and reduced kappa chains in sera. However, our analysis indicates that kappa gene rearrangement is normal in Ed−/− mice. In addition, our results show that Ed−/− mice exhibit reduced SHM in the kappa gene J‐C intronic region in Peyer's patch germinal center B cells. We conclude that Ed positively regulates Ig kappa gene expression and SHM, but not gene rearrangement.Research supported by grants GM29935 and AI067906 from NIH and I‐0823 from the Robert A. Welch Foundation.

Development of M cells within the follicle‐associated epithelium of Peyer's Patches depends on expression of TRANCE/RANKL by stromal cells

M cells are specialized epithelial cells found in the follicle‐associated epithelium (FAE) covering Peyer's patches (PP). M cells sample small particulate antigens from the intestinal lumen and transport these antigens to intraepithelial pockets found on the basal aspect of M cells. PP from mice lacking B cells (μMT knockout mice) have a marked reduction (over 150‐fold) in the overall number of intestinal M cells, but the specific receptor‐ligand interaction(s) central to promoting normal M cell differentiation within the FAE have not yet been identified. We now report that PP from mice deficient in the TNF superfamily member TRANCE/RANKL (TNFSF11) display a defect in M cell development that is even more pervasive than that of μMT mice. M cells identified by whole mount immunofluorescence microscopy using rhodamine‐conjugated Ulex europaeus lectin (UEA‐1) are essentially absent from PP in the proximal and central portions of the small intestine. Rare UEA‐1+ M cells are present in a few follicles from the most distal PP of TRANCE‐deficient mice, but these M cells don't have the normal radial symmetry of FAE M cells and are instead abnormally clustered near the apex of the dome. The M cell deficiency in the PP of TRANCE‐deficient mice is associated with impaired formation of PP germinal centers and decreased fecal IgA. TRANCE is selectively expressed by subepithelial stromal cells located in the dome area of PP. These findings indicate that local production of TRANCE by PP stromal cells is required for the normal induction and differentiation of M cells within the FAE.Supported by the NIH (DK‐64730, DK‐64399).

Rapidity and multiplicity of synthesis and expression of immunoglobulin isotypes by B lymphocytes in the small intestine.

An immunofluorescence double labeling assay was used to examine the kinetics of intestinal B lymphocytes with concurrent expression of multiple antibody isotypes in the mucosal tissues of rats infected with Trichinella spiralis (TS) muscle larvae for 1 to 15 days. As compared to the uninfected controls (day 0), the non-Peyer's patch tissues of the small intestine contained a significantly increased number of dual antibody-expressing B cells as early as 3 days after infection with a maximum proliferation of these B cells on days 7 and 10. These results indicate the rapidity of B cell response in the small intestine. Similar results were observed in the germinal centers of the Peyer's patches. The non-germinal centers of the Peyer's patch tissues showed delayed kinetics in B cell activation which occurred 10 days after infection. Quantification of the total number of B cells in these tissues was also carried out by staining the CD45RA marker on B cells with the OX33 monoclonal antibody. When comparing the total numbers of B cells with the numbers of B cells expressing dual isotypes of antibodies, our results showed the numbers of dual-expressing B cells (IgA:IgE, IgM:IgE, IgG1:IgE, IgG2a:IgE, IgG2b:IgE, and IgG2c:IgE), when combined, were over 7 times that of the total number of OX33-labeled B cells on day 7 in the small intestine. The dual-expressing B cells in the Peyer's patch-germinal center were more than 5 times that of the OX33-labeled B cells on day 15. These results therefore suggest that the dual-expressing B cells most likely synthesized and expressed more than two isotypes of antibodies during the peak days of the humoral response. Such phenomenon was not observed in non-germinal centers of the Peyer's patch tissues.

Germinal center B cells in Peyer's patches of aged mice exhibit a normal activation phenotype and highly mutated IgM genes

Systemic and mucosal humoral immune responses have been reported to exhibit an age related decline. However, differences in primary and memory responses at mucosal sites in aged mice have been noted. In an effort to begin characterizing deficiencies in the mucosal system of aged mice, we examined the B cell compartment of gut associated Peyer's patch lymphoid tissue. To our surprise, we found that germinal center (GC) B cells from aged B6D2F1 mice (24–26 months) were present at similar frequencies and exhibited a normal activation phenotype such as upregulation of B7.1, B7.2 and CD44, and downregulation of CD23, CD62L and CD38 as that observed in younger mice (2.5–4 months). As expected, Peyer's patch GC B cells from aged mice expressing V HX24 genes displayed higher somatic mutation frequencies compared with younger mice. However, this was particularly striking in IgM sequences where high mutational loads suggested we were sampling memory cells. It is conceivable that B-cells expressing these genes reflect the presence of a mucosal memory compartment in aged mice that either retains flexibility in effector function or is committed to the secretion of IgM antibody.

Decreased apoptosis in the ileum and ileal Peyer's patches: a feature after infection with rabbit enteropathogenic Escherichia coli O103.

Significant changes occur in intestinal epithelial cells after infection with enteropathogenic Escherichia coli (EPEC). However, it is unclear whether this pathogen alters rates of apoptosis. By using a naturally occurring weaned rabbit infection model, we determined physiological levels of apoptosis in rabbit ileum and ileal Peyer's patches (PP) and compared them to those found after infection with adherent rabbit EPEC (REPEC O103). Various REPEC O103 strains were first tested in vitro for characteristic virulence features. Rabbits were then inoculated with the REPEC O103 strains that infected cultured cells the most efficiently. After experimental infection, intestinal samples were examined by light and electron microscopy. Simultaneously, ileal apoptosis was assessed by using terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) and caspase 3 assays and by apoptotic cell counts based on morphology (hematoxylin-and-eosin staining). The highest physiological apoptotic indices were measured in PP germinal centers (median = 14.7%), followed by PP domed villi (8.1%), tips of absorptive villi (3.8%), and ileal crypt regions (0.5%). Severe infection with REPEC O103 resulted in a significant decrease in apoptosis in PP germinal centers (determined by TUNEL assay; P = 0.01), in the tips of ileal absorptive villi (determined by H&E staining; P = 0.04), and in whole ileal cell lysates (determined by caspase 3 assay; P = 0.001). We concluded that REPEC O103 does not promote apoptosis. Furthermore, we cannot rule out the possibility that REPEC O103, in fact, decreases apoptotic levels in the rabbit ileum.

Sequence analysis of human IgVH genes indicates that ileal lamina propria plasma cells are derived from Peyer's patches.

The origin of human ileal lamina propria (LP) plasma cells has been investigated using a technique based on microdissection of cells from immunohistochemically stained tissue sections. We have sequenced rearranged IgVH4.2 1, IgVH 5 family and IgVH6 genes from Peyer's patch germinal center (GC) cells and plasma cells from the ileal LP. Clonally related cells were identified by comparison of their complementarity determining region (CDR) 3 sequences and patterns of somatic mutation. We observed Ig genes from B cells in the GC of Peyer's patches which were related to Ig genes from plasma cells in the ileal LP, demonstrating that clonally related cells span these sites. In addition, groups of clonally related. but diversified plasma cells were common in the lamina propria. All Ig genes isolated from LP plasma cells were heavily mutated. The distribution of mutations in the CDR of the Ig heavy chain variable region (VH) genes which were considered to be the expressed alleles in LP plasma cells was consistent with an affinity-matured response. These observations provide compelling evidence for the origin of human ileal plasma cells from the Peyer's patches.

The preference for switching to IgA expression by Peyer's patch germinal center B cells is likely due to the intrinsic influence of their microenvironment.

This study was aimed at determining whether the chronically activated physiologic state of Peyer's patch (PP) tissue is primarily responsible for the IgA isotype preference expressed by PP germinal centers (GC) and memory B cells. We have used reovirus type 1/Lang to stimulate acute, de novo GC reactions in lymph nodes (LN) or PP to test the possibility that the surface (s)IgA component of enteric responses is peculiar to the local gut microenvironment whether or not PP are in a state of chronic activation. GC were raised in PP of germ-free mice by oral administration of virus and in lymph nodes (LN) of conventionally reared mice by local parenteral infection. Transient GC reactions were found to develop with similar time courses in both PP and LN after both primary and secondary local infections with reovirus. sIgA+ B cells, which were the major non-sIgM+/sIgD+ population found to arise in GC of PP, were not found in the LN. In LN, sIgG1+ B cells comprised the predominant non-IgM/IgD bearing population that arise after local infection. Lymphoid fragment cultures of PP initiated in vitro as early as 5 days after in vivo infection contained detectable secreted reovirus-specific IgA, whereas IgG1 but no IgA was found in LN cultures. Northern blot analysis to detect C alpha and C gamma 1 germline transcripts further substantiated a site-related bias in the expression of non-IgM/IgD isotypes that was manifest within a few days after infection. In summation, these observations taken together suggest that the preference for generating sIgA+ B cells in PP may be the result of intrinsic features of their gut microenvironment.

T lymphocytes that express CD4 and the alpha beta-T cell receptor but lack Thy-1. Preferential localization in Peyer's patches.

Thy-1- T cells expressing CD4 and the alpha beta-TCR have been identified in murine lymphoid tissues. These cells are particularly prevalent in Peyer's patches (PP), representing 17 +/- 3% of PP CD4 T cells, whereas they are much less prevalent in spleen, lymph nodes, lamina propria, or peritoneum. Phenotypic studies of fresh-isolated PP T cells demonstrate that all PP CD4 T cells (both Thy-1- and Thy-1+) express CD3, alpha beta-TCR, and CD5 (Lyt-1), whereas none coexpress CD8 (Lyt-2). Thy-1- and Thy-1+ CD4 T cell lines generated from PP also coexpress CD3 and alpha beta-TCR, but are heterogeneous in expression of CD5 and again do not coexpress CD8. Further studies revealed that Thy-1- CD4+ T cells were not present in nude mice. Short term stimulation of Thy-1+ CD4+ PP T cells with anti-CD3 resulted in loss of Thy-1 in a substantial fraction of these cells. Functional studies of Thy-1- and Thy-1+ CD4+ PP T cells indicate that fresh-isolated Thy-1- CD4+ cells do not proliferate in response to insoluble anti-CD3 but do proliferate when stimulated with soluble anti-CD3 in the presence of feeder cells. In contrast, Thy-1+ CD4+ cells proliferate well to both stimuli. However, Thy-1- CD4+ PP T cells adapted to in vitro culture exhibit vigorous proliferative responses when stimulated with either form of anti-CD3. Evaluation of lymphokine secretion by fresh-isolated Thy-1- and Thy-1+ CD4+ PP T cells revealed that both make substantial amounts of IL-2; however, Thy-1- T cells made less IL-4 than their Thy-1+ counterparts. Neither population made IL-5 or IFN-gamma. Similarly, Thy-1- and Thy-1+ CD4 T cell lines made similar amounts of IL-2; again Thy-1- T cells made less IL-4; and in this case Thy-1- T cells made IL-5 albeit significantly less than the Thy-1+ cells. Finally, immunohistochemical studies suggested that many of the CD4+ T cells in PP germinal centers were Thy-1-, indicating that Thy-1- and Thy-1+ CD4 T cells differ in their distribution within the PP. These studies thus define a phenotypically and functionally distinct T cell population which is most prevalent in murine Peyer's patches.

Surface phenotype of Peyer's patch germinal center cells: implications for the role of germinal centers in B cell differentiation.

The surface phenotype of Peyer's patch germinal center lymphoid cells in the mouse is described. It is confirmed that most germinal center lymphocytes bind high levels of peanut agglutination (PNA), a lectin with specificity for terminal galactosyl residues. It is shown that germinal center lymphocytes can be identified in cell suspensions as a discrete PNAhi population distinct from other B cells, plasma cells, and most T cells, which bind only low levels of PNA. Using fluorescence-labeled PNA as a marker in dual fluorescence studies, we found that the majority of Peyer's patch germinal center cells are B lymphocytes: PNAhi Peyer's patch cells express B220, the B lineage-specific form of the T200 family of molecules, as well as low levels of surface Ig. They do not express the T cell-lineage antigens Thy-1, Lyt-1, or Lyt-2 (only 1 to 3% positive). They bear lower levels of H2-K than PNAlo B cells, but two to three times the level of surface I-A-encoded determinants. A discrete but variable subpopulation of PNAhi Peyer's patch cells bear ThB in AKR/c mice, but BALB/c PNAhi lymphocytes are ThB-. About 10 to 30% bear surface IgM or IgG, but in contrast to essentially all PNAlo B lymphocytes in this site, they express no detectable surface IgD. The majority of Peyer's patch germinal center cells bear surface IgA, and this IgA is allelically excluded in F1 mice, indicating it is synthesized by the germinal center cells themselves. In fact, germinal centers contain most of the IgA-bearing cells in Peyer's patches (70 to 85%). These findings lend considerable support to the concept that germinal centers in Peyer's patches are the site of generation of precursors of the IgA-secreting plasma cells that characterize mucosal immune responses, and also suggest that germinal centers may play an important role in the process of heavy chain class switching.