IgA Class Switching Research Articles

Eosinophils Promote Generation and Maintenance of Immunoglobulin-A-Expressing Plasma Cells and Contribute to Gut Immune Homeostasis

Although in normal lamina propria (LP) large numbers of eosinophils are present, little is known about their role in mucosal immunity at steady state. Here we show that eosinophils are needed to maintain immune homeostasis in gut-associated tissues. By using eosinophil-deficient ΔdblGATA-1 and PHIL mice or an eosinophil-specific depletion model, we found a reduction in immunoglobulin A(+) (IgA(+)) plasma cell numbers and in secreted IgA. Eosinophil-deficient mice also showed defects in the intestinal mucous shield and alterations in microbiota composition in the gut lumen. In addition, TGF-β-dependent events including class switching to IgA in Peyer's patches (PP), the formation of CD103(+) T cells including Foxp3(+) regulatory (Treg), and also CD103(+) dendritic cells were disturbed. In vitro cultures showed that eosinophils produce factors that promote T-independent IgA class switching. Our findings show that eosinophils are important players for immune homeostasis in gut-associated tissues and add to data suggesting that eosinophils can promote tissue integrity.

Recent progress in mucosal immunology and vaccine development

Recent progress in mucosal immunology and vaccine development

SUMO Proteins are not Involved in TGF-β1-induced, Smad3/4-mediated Germline α Transcription, but PIASy Suppresses it in CH12F3-2A B Cells.

TGF-β induces IgA class switching by B cells. We previously reported that Smad3 and Smad4, pivotal TGF-β signal-transducing transcription factors, mediate germline (GL) α transcription induced by TGF-β1, resulting in IgA switching by mouse B cells. Post-translational sumoylation of Smad3 and Smad4 regulates TGF-β-induced transcriptional activation in certain cell types. In the present study, we investigated the effect of sumoylation on TGF-β1-induced, Smad3/4-mediated GLα transcription and IgA switching by mouse B cell line, CH12F3-2A. Overexpression of small ubiquitin-like modifier (SUMO)-1, SUMO-2 or SUMO-3 did not affect TGF-β1-induced, Smad3/4-mediated GLα promoter activity, expression of endogenous GLα transcripts, surface IgA expression, and IgA production. Next, we tested the effect of the E3 ligase PIASy on TGF-β1-induced, Smad3/4-mediated GLα promoter activity. We found that PIASy overexpression suppresses the GLα promoter activity in cooperation with histone deacetylase 1. Taken together, these results suggest that SUMO itself does not affect regulation of GLα transcription and IgA switching induced by TGF-β1/Smad3/4, while PIASy acts as a repressor.

Detection and Characterization of R‐loops at the Murine Immunoglobulin S α Region

IgA is the most abundant antibody in mammals. However, the mechanism of its class switching is still not clear. The formation of the R‐loops has been proposed to play a crucial role during mammalian class switch recombination. Hence, we investigate if R‐loops exist at Sα. In addition, the correlation between R‐loops and IgA class switching is also been investigated. Here, we are the first to provide a systematic evaluation of R‐loops at Sα (IgA) in vitro and in situ in CH12F3–2A cells, which is a unique cell model system for class switch recombination for its consistent switching to IgA upon stimulation. R‐loops form at Sα, despite variable G density and relatively few GGGG clusters in Sα. The short distance from the promoter to Sα may compensate for the less robust R‐loop‐forming factors at Sα relative to other switch regions. R‐loops have distinct features specific to Sα. Therefore, R‐loops at the Sα region further support R‐loop formation as a general feature of all stimulated switch regions. Furthermore, the IgA class switching is altered in the deficiency of R‐loops‐related enzymes in CH12F3–2A cells.

TGF-β and BAFF derived from CD4+CD25+Foxp3+ T cells mediate mouse IgA isotype switching

TGF-β1 is generally accepted as the physiological IgA isotype switch factor. Nevertheless, it is unclear as to which cells in mucus-associated lymphoid tissue provide this cytokine to B cells. Regulatory T cells (Tregs) play immune-suppressive roles by secreting inhibitory cytokines such as TGF-β and IL-10. Thus, it is plausible that Tregs are involved in IgA class switch recombination (CSR) in MALT. We explored, in the present study, the possibility that CD4+CD25+ T cells facilitate IgA CSR in murine B cells. In cocultures, CD4+CD25+Foxp3+ T cells stimulated IgA production by splenic B cells to a greater extent than did CD4+CD25−Foxp3− T cells. This effect was markedly abrogated by the addition of anti-TGF-β1 Ab. Additionally, IgA production was paralleled by an increase in germ line transcript α (GLTα), an indicator of IgA CSR. In contrast, CD4+CD25−Foxp3− T cells were more potent at inducing GLTγ1 and GLTɛ production by cocultured splenic B cells than were CD4+CD25+Foxp3+ T cells. Consistent with these results, phenotypic analyses revealed that TGF-β1 and IL-4 were predominantly expressed by CD4+CD25+Foxp3+ T cells and CD4+CD25−Foxp3− T cells, respectively. Furthermore, CD4+CD25+ T cells strongly expressed BAFF, which led to activation-induced deaminase (AID) expression in B cells. Taken together, our results suggest that CD4+CD25+ Tregs have an important effect on IgA isotype commitment by expressing TGF-β1 and BAFF in MALT.

IgA Production and Tonsillar Focal Infection in IgA Nephropathy

IgA nephropathy (IgAN), the common primary glomerulonephritis, is a tonsillar focal infection characterized by the qualitative abnormality of IgA in circulation and IgA deposition in the renal mesangium. Mesangial deposition of IgA, which is composed predominantly of poorly galactosylated polymeric IgA1 (pIgA1), seems to be the initiating event in the pathogenesis of IgAN. The origin of poorly galactosylated IgA, however, remains unclear. Recent studies suggest that the mesangial polymeric IgA1 deposition could be derived from mucosally primed plasma cells. B cells may undergo IgA class switching to acquire the expression of IgA via T-cell-dependent or T-cell-independent pathways in mucosa-associated lymphoid tissue and then differentiate to IgA plasma cells or home in on systemic sites. Dendritic cells, including plasmacytoid dendritic cells and another type of antigen-retaining cell, follicular dendritic cells, have an irreplaceable role in IgA class-switch mechanisms by producing IgA-inducing signals. Furthermore, an increased number of pIgA1-secreting plasma cells in the bone marrow and tonsil, as well as increased IgA class switching, have been found in IgAN, providing a link between the mucosal immunity and IgAN. The favorable effect of tonsillectomy on patients with IgAN showed that tonsillar focal infection may be closely related to pIgA1 deposition in glomerular mesangium of patients with IgAN and at least a part of pIgA1 may originate from affected tonsils. Therefore, the indication for tonsillectomy should be considered in patients with IgA nephropathy, especially at a mild or early stage, to prevent future renal deterioration. In this paper, we focus on IgA class switching and the role of tonsils with focal infection in IgAN.

Oral-Nasopharyngeal Dendritic Cells Mediate T Cell-Independent IgA Class Switching on B-1 B Cells

Native cholera toxin (nCT) as a nasal adjuvant was shown to elicit increased levels of T-independent S-IgA antibody (Ab) responses through IL-5- IL-5 receptor interactions between CD4+ T cells and IgA+ B-1 B cells in murine submandibular glands (SMGs) and nasal passages (NPs). Here, we further investigate whether oral-nasopharyngeal dendritic cells (DCs) play a central role in the induction of B-1 B cell IgA class switch recombination (CSR) for the enhancement of T cell-independent (TI) mucosal S-IgA Ab responses. High expression levels of activation-induced cytidine deaminase, Iα-Cμ circulation transcripts and Iμ-Cα transcripts were seen on B-1 B cells purified from SMGs and NPs of both TCRβ−/− mice and wild-type mice given nasal trinitrophenyl (TNP)-LPS plus nCT, than in the same tissues of mice given nCT or TNP-LPS alone. Further, DCs from SMGs, NPs and NALT of mice given nasal TNP-LPS plus nCT expressed significantly higher levels of a proliferation-inducing ligand (APRIL) than those in mice given TNP-LPS or nCT alone, whereas the B-1 B cells in SMGs and NPs showed elevated levels of transmembrane activator and calcium modulator cyclophilin ligand interactor (TACI) expression. Interestingly, high frequencies of IgA+ B-1 B cells were induced when peritoneal IgA− IgM+ B cells were stimulated with mucosal DCs from mice given nasal TNP-LPS plus nCT. Taken together, these findings show that nasal nCT plays a key role in the enhancement of mucosal DC-mediated TI IgA CSR by B-1 B cells through their interactions with APRIL and TACI.

Expression of IgA class switching gene in tonsillar mononuclear cells in patients with IgA nephropathy

There are many reports suggesting a relationship between the tonsillar autoimmune response and the pathogenesis of IgA nephropathy (IgAN). Hyperproduction of IgA and IgA1 in tonsils could be caused by activation of the Ig class switching recombination (CSR). αGLT (germline transcripts) plays a critical role in the initiation of switching from Cμ to Cα, resulting in production of IgA. Activation-induced cytidine deaminase (AID) is a molecule essential for CSR and Ig gene conversion. The aim of this study was to investigate IgA and IgA1 levels in the supernatant of tonsillar mononuclear cells (TMCs) and the expression of Iα-Cα germline transcript and AID in TMCs stimulated with lipopolysaccharide or hemolytic streptococcus in IgAN patients and chronic tonsillitis patients. 27 IgAN patients were admitted into our hospital from Jan. 2009 to Feb. 2010. Another 27 patients with chronic tonsillitis but without renal disease were selected as the control group. Tonsillar lymphocytes were isolated by density gradient centrifugation using Lymphocyte Separation Medium. The amount of IgA or IgA1 secreted in the culture supernatants was determined by specific enzyme-linked immunosorbent assay. Expressions of Iα-Cα germline transcript and AID mRNA were examined by reverse transcription real-time PCR. The AID protein was determined by Western blotting. The production of IgA and IgA1 protein, especially the ratio of IgA1/IgA in TMCs stimulated with or without 10μg/ml of lipopolysaccharide or 1×10(8)cfu/ml of hemolytic streptococcus, were significantly increased in the IgAN group compared with that in the non-IgAN group (P<0.05), and the IgA and IgA1 levels in TMCs stimulated with 10μg/ml of lipopolysaccharide or 1×10(8)cfu/ml of hemolytic streptococcus were markedly increased in patients with IgAN compared with the control group (P<0.05).The expressions of Iα-Cα and AID mRNA were significantly upregulated in TMCs stimulated with 10μg/ml of lipopolysaccharide or 1×10(8)cfu/ml of hemolytic streptococcus in patients with IgAN compared with control group (P<0.05). The expression of AID protein in TMCs stimulated with or without 10μg/ml of lipopolysaccharide or 1×10(8)cfu/ml of hemolytic streptococcus was significantly increased in the IgAN group compared with that in the non-IgAN group (P<0.05). The expression of AID protein in TMCs stimulated with 10μg/ml of lipopolysaccharide or 1×10(8)cfu/ml of hemolytic streptococcus was significantly increased in patients with IgAN compared with the control group (P<0.05, P<0.01). Lipopolysaccharide or hemolytic streptococcus can induce the production of IgA and IgA1 and the expression of AID and Iα-Cα in TMCs from patients with IgAN. Our results indicate that the TMCs from patients with IgAN are capable of producing high levels of IgA and IgA1 when stimulated with lipopolysaccharide or hemolytic streptococcus, which may be due to the increased expression of AID and Iα-Cα.

Prominent Role for Plasmacytoid Dendritic Cells in Mucosal T Cell-Independent IgA Induction

Although both conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs) are present in the gut-associated lymphoid tissues (GALT), the roles of pDCs in the gut remain largely unknown. Here we show a critical role for pDCs in Tcell-independent (TI) IgA production by B cells in the GALT. When pDCs of the mesenteric lymph nodes (MLNs) and Peyer's patches (PPs) (which are representative GALT) were cultured with naive B cells to induce TI IgA class switch recombination (CSR), IgA production was substantially higher than in cocultures of these cells with cDCs. IgA production was dependent on APRIL and BAFF production by pDCs. Importantly, pDC expression of APRIL and BAFF was dependent on stromal cell-derived type I IFN signaling under steady-state conditions. Our findings provide insight into the molecular basis of pDC conditioning to induce mucosal TI IgA production, which may lead to improvements in vaccination strategies and treatment for mucosal-related disorders.

Functions of Runx in IgA class switch recombination

Runt-related (Runx) transcriptional regulators play essential roles in various cell fate determination processes, and dysfunction of these regulators causes many human diseases. Considerable insight into the functions of Runx proteins was provided mainly by studies of hematopoietic and skeletal disorders. Recently, extensive investigations have revealed new functions of these transcription factors in immune cell differentiation and functioning. In the present review, we discuss the mechanisms of selective IgA production in the intestine and report the involvement of Runx proteins in this process.

Increases in IgA+ B cells in Peyer's patches during milk-borne mouse mammary tumor virus infection are influenced by Toll-like receptor 4 and are completely dependent on the superantigen response

Mouse mammary tumor virus (MMTV) is a milk-borne betaretrovirus that has developed strategies to exploit and subvert the host immune system. Although mammary glands are the final target of infection, Peyer's patches (PP) are the entry site of the virus. Herein, we show that the infection induces increases in the number of PP IgA(+) B cells and higher expression of the α circular transcript, which is a specific marker of the switch to IgA. In addition, IgA(+) B-cell increases correlated with higher levels of cytokines related to IgA class switching, such as interleukin (IL)-5 and IL-6. Of interest, the increases in IgA(+) B cells were lower in Toll-like receptor 4-deficient mice and were completely dependent on the presence of superantigen-reactive T cells. Our results point to a novel mechanism involved in MMTV infection and suggest that IgA(+) B cells may play an important role in carrying the virus to the mammary glands.

Enemy at the gates: dendritic cells and immunity to mucosal pathogens

Dendritic cells (DC) are diverse and specialized hematopoietic cells serving as an essential bridge between innate and adaptive immunity. DC exist in all lymphoid and nonlymphoid organs and are amongst the first responders to infection at epithelial surfaces including mucosal tissues. DC of the lung, gut, and vaginal mucosa display different phenotypes and functions reflecting each unique tissue and, in contrast to their counterparts in spleen and lymph nodes, are constantly exposed to both harmful and benign factors of their environments. Mucosal DC recognize and respond to pathogens through engagement of pattern recognition receptors, and activated DC migrate to draining lymph nodes to induce adaptive immune responses. The specialized function of DC aids in the induction of immunity and pathogen control at the mucosa. Such specialization includes the potent antiviral interferon response of plasmacytoid DC to viral nucleic acids, the ability of mucosal DC to capture organisms in the gut lumen, the capacity of DC to cross-present antigen from other infected cells, and the ability of mucosal DC to initiate IgA class switching in B cells. DC plasticity is also critical in the immune response to mucosal pathogens, as DC can respond to the microenvironment and sense pathogen-induced stress in neighboring epithelial cells. Finally, DC interact with each other through crosstalk to promote antigen presentation and T-cell immunity. Together, these processes condition mucosal DC for the induction of a tailored immune response to pathogens.

T Cell-Independent IgA Class Switch Recombination Is Restricted to the GALT and Occurs Prior to Manifest Germinal Center Formation

Recently, we reported that CD40(-/-) mice, exhibiting exclusively T cell-independent IgA class switch recombination (CSR), demonstrated near normal levels of IgA plasma cells in the gut lamina propria (LP), despite the complete lack of germinal centers (GCs). In this study, we have extended our analysis focusing on how to reconcile these findings using flow cytometry and molecular markers for IgA CSR. In agreement with our previous results with small intestinal LP, the colon LP was found to host IgA CSR only when lymphoid follicles were present. Thus, no IgA CSR was observed in the nonorganized colon LP. By contrast, the Peyer's patch (PP) was the dominant IgA CSR site in both CD40(-/-) and wild type (WT) mice, and they both hosted similar levels of mRNA expression for B cell activating factor of the TNF family, a proliferation inducing ligand, and inducible NO synthase, potential switch-factors for IgA. Unexpectedly, we found that PP B cells undergoing IgA CSR were GL7-intermediate. These cells had not undergone somatic hypermutations (SHMs), whereas GL7-high cells in WT PP, which exhibited GCs, were heavily mutated. Moreover, IgA plasma cells in the LP of CD40(-/-) mice demonstrated few mutations in their Ig V regions, whereas WT LP B cells from different sites showed extensive SHMs, which were also clonally related. Therefore, IgA CSR can occur in PP at a stage preceding manifest GC (GL7-intermediate), whereas SHM require GC formations (GL7-high). These findings reconcile that IgA CSR can occur in PP in the absence of GC with the fact that CD40(-/-) mice host near normal levels of IgA plasma cells in the LP.

Requirement for Runx Proteins in IgA Class Switching Acting Downstream of TGF-β1 and Retinoic Acid Signaling

IgA is a specific isotype required for mucosal immunity and is the most abundant Ab produced in vivo. Recently, several inductive signals for IgA class switch recombination have been identified; however, the molecular details of the action of these signals and the specific factors acting in B cells remain elusive. In this study, we show that combination of retinoic acid (RA) and TGF-beta1 with other factors induced a much higher frequency of IgA-switched cells than reported previously. In addition, IgA production is severely impaired in Runx2-Runx3 double-deficient mice. In Runx2-Runx3-deficient B cells, both RA- and TGF-beta1-dependent inductions of alpha germline transcription are completely blocked. These data suggest that Runx proteins play an essential role in IgA class switching acting downstream of RA and TGF-beta1 signaling.

Does Arkadia contribute to TGF-β1-induced IgA expression through up-regulation of Smad signaling in IgA nephropathy?

Immunoglobulin A nephropathy (IgAN) is an immune-complex-mediated glomerulonephritis characterized by the presence of IgA deposits in mesangial and paramesangial regions. However, the exact mechanism involved in IgA deposition is still unknown. TGF-β(1) that mediates the progression of IgAN is well established as a critical IgA class (isotype) switching factor, and Smad proteins are critical intracellular mediators in the expression of TGF-β(1)-targeted genes, which suggest that TGF-β signaling has been implicated in the primary pathogenesis of IgAN. Arkadia, an E3 ubiquitin ligase, can amplify TGF-β signaling through regulating Smads degradation. When these findings are considered together, it is of interest to explore how Arkadia and Smad signaling affect TGF-β(1)-induced IgA expression in IgAN. Therefore, we propose that Arkadia could positively contribute to TGF-β(1)-induced IgA secretion through up-regulation of Smad signaling in the pathogenesis of IgAN.

25-Hydroxycholesterol secreted by macrophages in response to Toll-like receptor activation suppresses immunoglobulin A production

25-Hydroxycholesterol is produced in mammalian tissues. The function of this oxysterol is unknown. Here we describe a central role for 25-hydroxycholesterol in regulating the immune system. In initial experiments, we found that stimulation of macrophage Toll-like receptors (TLR) induced expression of cholesterol 25-hydroxylase and the synthesis of 25-hydroxycholesterol. Treatment of naïve B cells with nanomolar concentrations of 25-hydroxycholesterol suppressed IL-2-mediated stimulation of B cell proliferation, repressed activation-induced cytidine deaminase (AID) expression, and blocked class switch recombination, leading to markedly decreased IgA production. Consistent with these findings, deletion of the mouse cholesterol 25-hydroxylase gene caused an increase in serum IgA. Conversely, inactivation of the CYP7B1 oxysterol 7alpha-hydroxylase, which degrades 25-hydroxycholesterol, decreased serum IgA. The suppression of IgA class switching in B cells by a macrophage-derived sterol in response to TLR activation provides a mechanism for local and systemic negative regulation of the adaptive immune response by the innate immune system.

HIV-1 evades virus-specific IgG2 and IgA responses by targeting systemic and intestinal B cells via long-range intercellular conduits

Contact-dependent communication between immune cells generates protection, but also facilitates viral spread. We found that macrophages formed long-range actin-propelled conduits in response to negative factor (Nef), a human immunodeficiency virus type-1 (HIV-1) protein with immunosuppressive functions. Conduits attenuated immunoglobulin G2 (IgG2) and IgA class switching in systemic and intestinal lymphoid follicles by shuttling Nef from infected macrophages to B cells through a guanine exchange factor-dependent pathway involving the amino-terminal anchor, central core and carboxy-terminal flexible loop of Nef. By showing stronger virus-specific IgG2 and IgA responses in patients harboring Nef-deficient virions, our data suggest that HIV-1 exploits intercellular highways as a “Trojan horse” to deliver Nef to B cells and evade humoral immunity systemically and at mucosal sites of entry.

Expression and purification of APRIL by auto-induction

APRIL (A proliferation-inducing ligand) is a newly-identified member of the tumor necrosis factor family that induces pleiotropic biological responses, including immunological responses, IgA class switch and cell growth. It is associated with multiple diseases such as cancer and autoimmune diseases. High levels of APRIL mRNA can be detected in transformed cell lines and several malignant tumors; heparin sulfate proteoglycans (HSPG) are also involved in the APRIL tumor cell proliferation induction response. The interaction of APRIL and HSPG occurs through an N-terminal basic region on APRIL. We successfully expressed recombinant APRIL using an auto-induction system in Escherichia coli. By using in situ cleavage, we generated the mature form of APRIL, comprising its N-terminal basic region, which has full biological activity: receptor binding capability and proliferation induction activity.

High circulating immunoglobulin A levels in infants are associated with intestinal toxigenic Staphylococcus aureus and a lower frequency of eczema

Intestinal bacteria trigger IgA production and delayed maturation of mucosal IgA response is linked to allergy development. Our aim was to investigate if plasma levels of IgA or APRIL (a proliferation inducing ligand), an important factor for IgA class switch recombination, in infancy correlates with intestinal colonization by any specific bacteria or yeast. We also examined if plasma IgA or APRIL levels are related to sensitization and the development of eczema. IgA was quantified in plasma obtained from infants at birth and at 4 and 18 months of age and APRIL was measured at 4 months of age. Colonization by major bacterial groups and yeast was followed in the first 8 weeks of life by quantitative culture of stool samples. A clinical evaluation regarding the presence of allergen-specific IgE or eczema and eosinophil counts in blood was performed at 18 months of age. In multiple linear regression analysis, only colonization by Staphylococcus aureus strains producing toxins with superantigen function (SEA-D or TSST-1) made an independent contribution to plasma IgA levels at 4 months of age. Further, increased levels of APRIL in plasma at 4 months were negatively associated with sensitization while IgA plasma levels were inversely correlated to eczema development and blood eosinophil counts at 18 months of age. Early intestinal colonization by toxigenic S. aureus strains seems to promote systemic IgA responses. Furthermore, high levels of APRIL and IgA in the circulation at 4 months of age seem to correlate negatively with allergy development.

A T cell-dependent mechanism for the induction of human mucosal homing IgA-secreting plasmablasts (39.5)

Abstract Mucosal IgA secreted by local plasma cells (PCs) is a critical component of mucosal immunity. Although IgA class switching can occur at mucosal sites in a T cell-independent manner, high-affinity PCs are optimally generated in germinal centers (GCs) in a T cell-dependent fashion. However, the mechanism of how CD4+ helper T cells induce mucosal-homing IgA-PCs remains unclear. We show here that TGFb1 and IL-21, produced by follicular helper T cells (TFH), synergize to generate abundant IgA-plasmablasts (PBs) that express CCR10. In the presence of IL-21, TGFb1 promotes naive B cell proliferation and differentiation. In addition it overrides IL-21-induced IgG class switching in favor of IgA. Furthermore, in combination with IL-21, TGFb1 downregulates CXCR5, while upregulating CCR10 on PBs, enabling their exit from GCs and migration towards local mucosa. This is supported by the presence of CCR10+IgA+ cells in tonsil GCs. In conclusion, TFH, through IL-21 and TGFb1 contribute to the generation of human mucosal-homing IgA+ PCs. Thus, mucosal vaccines should aim to induce robust TFH responses.