IgA Class Switching Research Articles

POS-387 ABNORMAL B CELL RESPONSE IN IGA NEPHROPATHY

Progression of IgA nephropathy (IgAN) has been shown to be associated with the increased serum level of galactose-deficient IgA1 and autoantibodies against the abnormally glycosylated this IgA1. Since abnormal antibody production seems to be the key feature in the pathogenesis of IgAN, we investigated characteristics of B cells by using IgAN prone mice with (O-ddY) or without (NO-ddY) full onset of this disease. Moreover, we evaluated class switch (CS) to IgA from B cells in O-ddY mice using our novel culture system mimicking germinal center in mucosa, by which nearly 50 % of B cells undergo class switch (CS) to IgA. To examine CS to IgA, naïve splenic B cells from O-ddY or NO-ddY mice were cultured for seven days under the newly developed culture system. The frequency of IgA CS was evaluated by flow cytometry. Sorted naïve B cells from spleen of O-ddY or NO-ddY mice were stimulated with various reagents (agonist of TLR1,2,4,7 or 9, TGF-β, CD40 or membrane-bound IgM) for 2 days and the proliferation of splenic B cells was evaluated by Thymidine-uptake analysis. We revealed that the frequency of CS to IgA from naïve B cells were comparable between O-ddY and NO-ddY mice. We found that B cells of O-ddY mice exhibited elevated proliferation rate than those of NO-ddY mice in response to stimuli through CD40 and membrane-bound IgM. These data indicate that B cells in O-ddY mice are hyper-sensitive to stimuli by antigen and T-cell help without increasing the frequency of IgA class switch and suggest that such abnormal B cell response might be involved in the pathogenesis of IgAN.

Enhanced BAFF expression is negatively correlated with a suppressed isotype-switched memory B cell pool in CVID patients with complications

Common variable immunodeficiency (CVID) is a primary immunodeficiency due to selected genetic defects. This syndrome is frequently associated with non-infectious complications such as interstitial lung disease, enteropathy, autoimmunity and malignancies, with lack of isotype-switched B cells. B cell–activating factor (BAFF) is one of the TNF family members important for IgA class switching and plasma cell survival. Excessive levels of BAFF are found in CVID patients. In this study, we investigated the correlation of BAFF levels with isotype-switched memory B cells. Serum BAFF levels in 70 CVID patients and 15 healthy controls were measured by ELISA. The percentages of isotype switched-memory B cells were measured by flow cytometry. The correlation between BAFF and isotype-switched memory B cells in CVID patients with and without complications were analyzed by Pearson’s correlation. The serum BAFF levels in all CVID patients were significantly higher than healthy controls (p<0.0001), whereas BAFF levels are higher in the group with non-infectious complications than the group without complications (p <0.005). In contrast, isotype-switched memory B cells were significantly lower in CVID patients than healthy controls (p <0.001). Furthermore, BAFF levels in all CVID patients and the group with complications were negatively correlated to isotype-switched memory B cells (p=0.012). Our data suggests that excessive BAFF expression in CVID patients with non-infectious complications may cause suppression of memory B cell switching. Alternatively exhaustive switching memory B cell pools in CVID patient may drive upregulation of BAFF, promoting the non-infectious complications. Future study to reveal the underlying mechanism is needed.

High levels of gut-homing immunoglobulin A+ B lymphocytes support the pathogenic role of intestinal mucosal hyperresponsiveness in immunoglobulin A nephropathy patients.

BackgroundImmunoglobulin A nephropathy (IgAN) is the most frequent primary glomerulonephritis. The role of the microbiota and mucosal immunity in the pathogenesis of IgAN remains a key element. To date, the hypothetical relationship between commensal bacteria, elevated tumour necrosis factor (TNF) superfamily member 13 [also known as B-cell activating factor (BAFF)] levels, perturbed homoeostasis of intestinal-activated B cells and intestinal IgA class switch has not been clearly shown in IgAN patients.MethodsWe studied the intestinal–renal axis connections, analysing levels of BAFF, TNF ligand superfamily member 13 (APRIL) and intestinal-activated B cells in IgAN patients, healthy subjects (HSs) and patients with non-IgA glomerulonephritides.ResultsIgAN patients had increased serum levels of BAFF cytokine, correlating with higher amounts of five specific microbiota metabolites, and high APRIL cytokine serum levels. We also found that subjects with IgAN have a higher level of circulating gut-homing (CCR9+ β7 integrin+) regultory B cells, memory B cells and IgA+ memory B cells compared with HSs. Finally, we found that IgAN patients had high levels of both total plasmablasts (PBs) and intestinal-homing PBs. Interestingly, PBs significantly increased in IgAN but not in patients with other glomerulonephritides.ConclusionsOur results demonstrate a significant difference in the amount of intestinal-activated B lymphocytes between IgAN patients and HSs, confirming the hypothesis of the pathogenic role of intestinal mucosal hyperresponsiveness in IgAN. The intestinal–renal axis plays a crucial role in IgAN and several factors may contribute to its complex pathogenesis and provide an important area of research for novel targeted therapies to modulate progression of the disease.

Eosinophils are dispensable for the regulation of IgA and Th17 responses in Giardia muris infection.

IgA and Th17 responses are pivotal for the control of Giardia infections. Eosinophils support IgA class switching, the survival of intestinal IgA+ plasma cells at steady state and can control Th17 activity in the small intestine. To see whether eosinophils regulate adaptive immune responses during giardiasis, we investigated Giardia muris infections in wild-type BALB/c and eosinophil-deficient ∆dblGATA-1 mice. Infected ∆dblGATA-1 mice did not differ markedly in parasite control from wild-type mice. Confirming previous studies, naive ∆dblGATA-1 mice displayed diminished IgA+ B cell frequencies in Peyer's patches. However, IgA class switching and intestinal IgA secretion in response to G muris infection were comparable in wild-type BALB/c and ∆dblGATA-1 mice. Both strains displayed similarly low intestinal Th17 responses, accompanied by a mild expansion of type 3 innate lymphoid cells (ILC3). Contrasting previous reports on overt small intestinal Th17 activity in eosinophil-deficient mice, IL-17A production is kept in check in the absence of eosinophils during Giardia infection. Suboptimal homeostatic IgA responses in the absence of eosinophils are transiently fostered in infected mice and the maintenance of IgA+ plasma cells appears to be restored during persisting Giardia infection.

Interleukin (IL)-21 Promotes the Differentiation of IgA-Producing Plasma Cells in Porcine Peyer's Patches via the JAK-STAT Signaling Pathway.

Secretory IgA is critical to prevent the invasion of pathogens via mucosa. However, the key factors and the mechanisms of IgA generation in the porcine gut are not well-understood. In this study, a panel of factors, including BAFF, APRIL, CD40L, TGF-β1, IL-6, IL-10, IL-17A, and IL-21, were employed to stimulate IgM+ B lymphocytes from porcine ileum Peyer's patches. The results showed that IL-21 significantly upregulated IgA production of B cells and facilitated cell proliferation and differentiation of antibody-secreting cells. In addition, three transcripts in porcine IgA class switch recombination (CSR), germ-line transcript α, post-switch transcript α, and circle transcript α, were first amplified by (nest-)PCR and sequenced. All these key indicators of IgA CSR were upregulated by IL-21 treatment. Furthermore, we found that IL-21 predominantly activated JAK1, STAT1, and STAT3 proteins and confirmed that the JAK-STAT signaling pathway was involved in porcine IgA CSR. Thus, IL-21 plays an important role in the proliferation and differentiation of IgA-secreting cells in porcine Peyer's patches through the JAK-STAT signaling pathway. These findings provide insights into the mucosal vaccine design by regulation of IL-21 for the prevention and control of enteric pathogens in the pig industry.

P0379BION-1301, A FULLY BLOCKING ANTIBODY TARGETING APRIL FOR THE TREATMENT OF IGA NEPHROPATHY: ASSESSMENT OF SAFETY, PHARMACOKINETICS AND PHARMACODYNAMICS IN LONG-TERM NONCLINICAL STUDIES

Abstract Background and Aims IgA Nephropathy (IgAN) is a common form of glomerulonephritis and its pathogenesis is thought to consist of sequential pathogenic hits, including the production of auto-antigen galactose-deficient IgA1 (Gd-IgA1) and auto-antibody formation ultimately resulting in immune complex deposition, inflammation and functional deterioration of the kidney. Serum levels of APRIL (A PRoliferation Inducing Ligand, TNFSF13) and levels of Gd-IgA1 are correlated with severity of disease (Zhao et al, 2012). As a ligand for the receptors BCMA and TACI, APRIL is thought to regulate differentiation and proliferation of B cells and plasma cells and IgA class switching. BION-1301 is a humanized antibody neutralizing APRIL. In nonhuman primates, BION-1301 reduced serum levels of IgA, IgM, and IgG with no drug-related toxicity (Dulos J, et al. ASN Annual Meeting. 2018). Here we describe the nonclinical safety assessment of BION-1301, to support its continued clinical development for the treatment of IgA Nephropathy. Method A 14-week repeat-dose toxicity and toxicokinetic/pharmacodynamic (TK/PD) study in non-human primates (NHP) was performed by biweekly intravenous bolus (IV) administration of BION-1301 at three different dose levels. To support a potential change in route of administration, a 4-week repeat-dose NHP bridging study was conducted with weekly subcutaneous (SC) administration of BION-1301 at three different dose levels. Results In both the 14-week IV study and the 4-week SC study, BION-1301 was evaluated for safety, TK and PD. For TK and PD, BION-1301 levels, uncomplexed APRIL levels and immunoglobulin levels (IgA, IgG, IgM) were quantified in serum. Immunophenotyping was performed on peripheral blood to assess the impact of BION-1301 on the B cell compartment. In both studies BION-1301 was well tolerated with no test-article related findings. 14-week IV dosing as well as 4-week SC dosing of BION-1301 reduced serum APRIL levels as well as serum immunoglobulin levels. Conclusion Results of these nonclinical toxicity studies with toxicology, TK and PD endpoints demonstrate that BION-1301 is well tolerated for up to 14 weeks of dosing, corroborates pharmacodynamic proof-of-mechanism and support prolonged dosing and SC administration of BION-1301 in patients with IgA Nephropathy.

Longan Pulp Polysaccharide Protects Systemic Immunity and Intestinal Immunity in Mice Induced by Cyclophosphamide

Abstract Objectives This study aimed to explore the effect of longan pulp polysaccharide (LP) on the systemic immunity and intestinal mucosal immunity with immunosuppressive mice. The synthesis processing and secretion of intestinal secretory IgA (SIgA) were investigated. Methods Serum IgA, IgG, IgM and intestinal SIgA were detected by ELISA. Genes involved in the synthesis and secretion of SIgA were detected by Q-PCR and western blot. Results LP increased the thymus index, spleen index, and serum IgA level in cyclophosphamide (CTX)-treated mice. SIgA secretion in intestinal lumen was increased by LP as well. The underlying mechanism comes down to the facts as follows: LP increased intestinal cytokines expression and TGFβRII that is associated with pathways of IgA class switch recombination (CSR). By improving protein expression of mucosal address in cell-adhesion molecule-1 (MAdCAM-1) and integrin α4β7, LP was beneficial to gut homing of IgA + plasma cells. LP increased IgA, polymeric immunoglobulin receptor (pIgR), and secretory component (SC) to fortify the SIgA secretion. Conclusions This study suggested that moderate consumption of LP is helpful for improving systemic immunity and intestinal mucosal immunity via promotion of intestinal SIgA to strengthen the mucosal barrier. Funding Sources This work was supported by the National Key Research Project of China (2018YFC1602105, 2019YFD1002304), Guangdong Provincial Science and Technology Project (2018A050506050), President Foundation of Guangdong Academy of Agricultural Sciences (201812B).

Local eosinophils are associated with increased IgA subclass levels in the sinonasal mucosa of chronic rhinosinusitis with polyp patients

BackgroundChronic rhinosinusitis (CRS) describes an inflammatory condition affecting the sinonasal mucosa. As the immune system players such as immunoglobulins play prominent roles in the development of CRS, we aimed to investigate the expression of IgA subclasses and factors involved in IgA class switching in the sinonasal mucosa of CRS patients.MethodsSpecimens were collected from the sinonasal mucosa of the healthy controls and CRS patients. Histological assessments were performed by H&E and immunohistochemistry. Real-time PCR and ELISA methods were applied to measure gene expression and protein levels extracted from tissue samples, respectively.ResultsWe observed that total IgA and subclass-positive cells were higher in the patient groups than controls. There was a significant correlation between the number of eosinophils and total IgA and subclasses-positive cells (Pv < 0.0001). The expression of CXCL13, BAFF, AID, and germline transcripts were increased in CRSwNP patients. In contrast to IgA2 levels, IgA1 levels were significantly increased in the sinonasal tissue of CRSwNP patients (Pv < 0.01). TGF-β was significantly elevated in the sinonasal tissue of patients with CRSsNP.ConclusionsIncreased protein levels of IgA subclasses and related antibody-producing cells were associated with elevated eosinophils in CRSwNP patients which may result in eosinophil pathological functions. Several therapeutic approaches might be developed to modulate the IgA production to ameliorate the inflammatory mechanisms in CRSwNP patients.

Specific microbiota enhances intestinal IgA levels by inducing TGF-β in T follicular helper cells of Peyer's patches in mice.

In humans and mice, mucosal immune responses are dominated by IgA antibodies and the cytokine TGF-β, suppressing unwanted immune reactions but also targeting Ig class switching to IgA. It had been suggested that eosinophils promote the generation and maintenance of mucosal IgA-expressing plasma cells. Here, we demonstrate that not eosinophils, but specific bacteria determine mucosal IgA production. Co-housing of eosinophil-deficient mice with mice having high intestinal IgA levels, as well as the intentional microbiota transfer induces TGF-β expression in intestinal T follicular helper cells, thereby promoting IgA class switching in Peyer's patches, enhancing IgA+ plasma cell numbers in the small intestinal lamina propria and levels of mucosal IgA. We show that bacteria highly enriched for the genus Anaeroplasma are sufficient to induce these changes and enhance IgA levels when adoptively transferred. Thus, specific members of the intestinal microbiota and not the microbiota as such regulate gut homeostasis, by promoting the expression of immune-regulatory TGF-β and of mucosal IgA.

Commensal-bacteria-derived butyrate promotes the T-cell-independent IgA response in the colon.

Secretory immunoglobulin A (SIgA), the most abundant antibody isotype in the body, maintains a mutual relationship with commensal bacteria and acts as a primary barrier at the mucosal surface. Colonization by commensal bacteria induces an IgA response, at least partly through a T-cell-independent process. However, the mechanism underlying the commensal-bacteria-induced T-cell-independent IgA response has yet to be fully clarified. Here, we show that commensal-bacteria-derived butyrate promotes T-cell-independent IgA class switching recombination (CSR) in the mouse colon. Notably, the butyrate concentration in human stools correlated positively with the amount of IgA. Butyrate up-regulated the production of transforming growth factor β1 and all-trans retinoic acid by CD103+CD11b+ dendritic cells, both of which are critical for T-cell-independent IgA CSR. This effect was mediated by G-protein-coupled receptor 41 (GPR41/FFA3) and GPR109a/HCA2, and the inhibition of histone deacetylase. The butyrate-induced IgA response reinforced the colonic barrier function, preventing systemic bacterial dissemination under inflammatory conditions. These observations demonstrate that commensal-bacteria-derived butyrate contributes to the maintenance of the gut immune homeostasis by facilitating the T-cell-independent IgA response in the colon.

Triptolide inhibits tonsillar IgA production by upregulating FDC-SP in IgA nephropathy.

IgA nephropathy (IgAN) is primarily resulted of qualitative abnormality of IgA. The occurrence of IgAN is associated with affected tonsils which enhances the IgA production via IgA class switching and immuno-activation. Follicular dendritic cell-secreted protein (FDC-SP) was found to be a negative effect for IgA production in tonsil. The previous studies suggested that Triptolide might reduce IgA production by its immunosuppression role. Given this background, this study investigated the mechanisms underlying the role of Triptolide and FDC-SP in the generation of IgA and IgA class switching in tonsil of IgAN patients. Immunohistochemistry and reverse transcription-polymerase chain reaction revealed that the expression of FDC-SP was increased in the tonsils of IgAN patients with Triptolide treatment compared with those without treatment. Meanwhile, the expression of FDC-SP was negatively correlated with IgA inducing cytokines in the tonsils of IgAN patients treated with Triptolide, due to the significant decreased IgA-bearing cells. The expression of FDC-SP in tonsillar tissue was confirmed by double immunofluorescence. Importantly, Triptolide promoted FDC-SP secretion, and correlated negatively with decreased IgA production in isolated FDC-associated clusters, which had been isolated from patients without TW treatment previously. Our study demonstrated that Triptolide might have an impact on FDC-SP production and downregulation of IgA synthesis in the tonsils of IgAN patients, which could be a promising strategy for therapeutic intervention in IgAN patients.

CUL7 E3 Ubiquitin Ligase Mediates the Degradation of Activation-Induced Cytidine Deaminase and Regulates the Ig Class Switch Recombination in B Lymphocytes.

Activation-induced cytidine deaminase (AID) initiates class switch recombination and somatic hypermutation in Ig genes. The activity and protein levels of AID are tightly controlled by various mechanisms. In this study, we found that CUL7 E3 ubiquitin ligases specifically mediated AID ubiquitination. CUL7 overexpression or knockdown influenced the decay of AID, affecting AID protein levels and subsequently IgA class switching in CH12F3 cells, a mouse B lymphocyte cell line. Further analysis indicated that CUL7 mediated AID ubiquitination by forming a complex with FBXW11. In a CUL7 fl/fl CD19 cre+ mouse model, we demonstrated that CUL7 knockout significantly enhanced AID protein levels in B cells in the germinal center and increased both the IgG1 and IgA class switching. Collectively, our results reveal a subtle regulation mechanism for tightly controlling AID protein levels. The manipulation of this pathway may be useful for regulating AID abundance and efficiency of Ig class switching and is therefore a potential target for developing immunologic adjuvants for vaccines of various pathogens such as HIV-1 and influenza viruses.

Endonuclease and redox activities of human apurinic/apyrimidinic endonuclease 1 have distinctive and essential functions in IgA class switch recombination

The base excision repair (BER) pathway is an important DNA repair pathway and is essential for immune responses. In fact, it regulates both the antigen-stimulated somatic hypermutation (SHM) process and plays a central function in the process of class switch recombination (CSR). For both processes, a central role for apurinic/apyrimidinic endonuclease 1 (APE1) has been demonstrated. APE1 acts also as a master regulator of gene expression through its redox activity. APE1's redox activity stimulates the DNA-binding activity of several transcription factors, including NF-κB and a few others involved in inflammation and in immune responses. Therefore, it is possible that APE1 has a role in regulating the CSR through its function as a redox coactivator. The present study was undertaken to address this question. Using the CSR-competent mouse B-cell line CH12F3 and a combination of specific inhibitors of APE1's redox (APX3330) and repair (compound 3) activities, APE1-deficient or -reconstituted cell lines expressing redox-deficient or endonuclease-deficient proteins, and APX3330-treated mice, we determined the contributions of both endonuclease and redox functions of APE1 in CSR. We found that APE1's endonuclease activity is essential for IgA-class switch recombination. We provide evidence that the redox function of APE1 appears to play a role in regulating CSR through the interleukin-6 signaling pathway and in proper IgA expression. Our results shed light on APE1's redox function in the control of cancer growth through modulation of the IgA CSR process.

Human milk induces IgA class switch recombination in cord blood B-cells

Human milk induces IgA class switch recombination in cord blood B-cells

Triptolide Inhibits Tonsillar IgA Class Switching by Upregulating FDC-SP in IgA Nephropathy

IgA nephropathy (IgAN) is characterized by qualitative abnormality of IgA in affected tonsils, where present enhancement of IgA production by IgA class switching and immuno-activation. Follicular dendritic cell-secreted protein (FDC-SP) was found to be effective in tonsillar IgA production of IgAN. Triptolide, a core medicinal component of Tripterygium wilfordii, has been suggested to control IgA production by its' immunosuppression effect. Given this background, this study investigated the mechanisms underlying the role of Triptolide and FDC-SP in the generation of IgA and IgA class switching in tonsil of IgAN patients. Immunohistochemistry and reverse transcription-polymerase chain reaction revealed that the expression of FDC-SP were increased and was correlated negatively with decreased IgA inducing cytokines in the tonsils of IgAN patients with Triptolide treatment compared with those without treatment, followed by significantly decreased of IgA-bearing cells. The location of FDC-SP in tonsillar tissue was confirmed by double immunofluorescence. Importantly, Triptolide promoted FDC-SP secretion, and correlated negatively with decreased IgA production in isolated FDC-associated clusters. Our study demonstrated that Triptolide may be involved in IgA production in the tonsils of IgAN patients, inhibiting IgA class switching in IgAN patients through the cooperative roles of FDC-SP, highlighting a promising strategy for therapeutic intervention in IgAN. Funding Statement: This work was supported by the National Nature Science Foundation of China (81600539), Natural Science Foundation of Heilongjiang Province of China (QC2012C041, LC2016038), Nn10 program of Harbin Medical University Cancer Hospital (Nn10 2017-03), Postdoctoral Science Start-up Foundation of Heilongjiang Province (Hongxue Meng), Harbin Special fund project for Science and technology innovation(2016RAQXJ203), Youth elite training Foundation of Harbin Medical University Cancer Hospital (JY2016-06) and Outstanding Youth Foundation of Harbin Medical University Cancer Hospital (JCQN-2018-05)，Clinical Scientific Research Foundation of China Medical and Health Development Foundation (Hongxue Meng), Harbin Medical University Postgraduate Research and Practice Innovation Project (Huining Li). Declaration of Interests: All authors have read the journal's policy on disclosure of potential conflicts of interest and have none to declare. Ethics Approval Statement: This research was carried out according to the principles of the Declaration of Helsinki. Written informed consent was obtained from all participants in this study. Approval for this study was obtained from the Medical Ethics Committees of First Affiliated Hospital of Hei Longjiang University of Chinese Medicine (HZYLLBA201714).

Mesenteric CD103+DCs Initiate Switched Coxsackievirus B3 VP1-Specific IgA Response to Intranasal Chitosan-DNA Vaccine Through Secreting BAFF/IL-6 and Promoting Th17/Tfh Differentiation.

Intranasal chitosan-formulated DNA vaccination promotes IgA secretion in the intestine. However, the mechanism whereby chitosan-DNA skews IgA class switch recombination (CSR) of B cells in the Gut-associated lymph tissue (GALT) is not fully resolved. In this study, we investigated the effects of nasally administered chitosan-DNA (pcDNA3.1-VP1 plasmid encoding VP1 capsid protein of Coxsackievirus B3) on IgA production, DC activation and Tfh/Th17 response in the intestine. Compared to DNA immunization, intranasal chitosan-DNA vaccination induced antigen-specific IgA production in feces, a pronounced switching of antigen-specific IgA+ plasmablast B cells in the mesenteric lymph nodes (MLNs) and an enhanced expression of post-recombination Iα-CH transcripts/IgA germline transcript (αGT) as well as activation-induced cytidine deaminase (AID) in MLN B cells. MLN Tfh frequency was markedly enhanced by chitosan-DNA, and was associated with VP1-specific IgA titer. 24 h after immunization, intranasal chitosan-DNA induced a recruitment of CD103+DCs into the MLN that paralleled a selective loss of CD103+DCs in the lamina propria (LP). In vivo activated MLN-derived CD103+DCs produced high levels of IL-6 and BAFF in response to chitosan-DNA, which up-regulated transmembrane activator and CAML interactor (TACI) expression on MLN B cells. Upon co-culture with IgM+B in the presence of chitosan-DNA, MLN CD103+DCs induced IgA production in a T-dependent manner; and this IgA-promoting effect of CD103+DC was blocked by targeting TACI and, to a lower extent, by blocking IL-6. MLN CD103+DCs displayed an enhanced capacity to induce an enhanced CD4+Th17 response in vivo and in vitro, and IL-17A deficient mice had a pronounced reduction of specific intestinal IgA following immunization. Taken together, mesenteric CD103+DCs are indispensable for the adjuvant activity of chitosan in enhancing DNA vaccine-specific IgA switching in gut through activating BAFF-TACI and IL-6-IL-6R signaling, and through inducing Th17/Tfh differentiation in the MLN.

Identification of a Transcriptionally Forward α Gene and Two υ Genes within the Pigeon (Columba livia) IgH Gene Locus.

Compared with mammals, the bird Ig genetic system relies on gene conversion to create an Ab repertoire, with inversion of the IgA-encoding gene and very few cases of Ig subclass diversification. Although gene conversion has been studied intensively, class-switch recombination, a mechanism by which the IgH C region is exchanged, has rarely been investigated in birds. In this study, based on the published genome of pigeon (Columba livia) and high-throughput transcriptome sequencing of immune-related tissues, we identified a transcriptionally forward α gene and found that the pigeon IgH gene locus is arranged as μ-α-υ1-υ2. In this article, we show that both DNA deletion and inversion may result from IgA and IgY class switching, and similar junction patterns were observed for both types of class-switch recombination. We also identified two subclasses of υ genes in pigeon, which share low sequence identity. Phylogenetic analysis suggests that divergence of the two pigeon υ genes occurred during the early stage of bird evolution. The data obtained in this study provide new insight into class-switch recombination and Ig gene evolution in birds.

B lymphocytes confer immune tolerance via cell surface GARP-TGF-β complex.

GARP, a cell surface docking receptor for binding and activating latent TGF-β, is highly expressed by platelets and activated Tregs. While GARP is implicated in immune invasion in cancer, the roles of the GARP-TGF-β axis in systemic autoimmune diseases are unknown. Although B cells do not express GARP at baseline, we found that the GARP-TGF-β complex is induced on activated human and mouse B cells by ligands for multiple TLRs, including TLR4, TLR7, and TLR9. GARP overexpression on B cells inhibited their proliferation, induced IgA class-switching, and dampened T cell-independent antibody production. In contrast, B cell-specific deletion of GARP-encoding gene Lrrc32 in mice led to development of systemic autoimmune diseases spontaneously as well as worsening of pristane-induced lupus-like disease. Canonical TGF-β signaling more readily upregulates GARP in Peyer patch B cells than in splenic B cells. Furthermore, we demonstrated that B cells are required for the induction of oral tolerance of T cell-dependent antigens via GARP. Our studies reveal for the first time to our knowledge that cell surface GARP-TGF-β is an important checkpoint for regulating B cell peripheral tolerance, highlighting a mechanism of autoimmune disease pathogenesis.

Genome-Wide Association Study on Immunoglobulin G Glycosylation Patterns.

Immunoglobulin G (IgG), a glycoprotein secreted by plasma B-cells, plays a major role in the human adaptive immune response and are associated with a wide range of diseases. Glycosylation of the Fc binding region of IgGs, responsible for the antibody’s effector function, is essential for prompting a proper immune response. This study focuses on the general genetic impact on IgG glycosylation as well as corresponding subclass specificities. To identify genetic loci involved in IgG glycosylation, we performed a genome-wide association study (GWAS) on liquid chromatography electrospray mass spectrometry (LC–ESI-MS)—measured IgG glycopeptides of 1,823 individuals in the Cooperative Health Research in the Augsburg Region (KORA F4) study cohort. In addition, we performed GWAS on subclass-specific ratios of IgG glycans to gain power in identifying genetic factors underlying single enzymatic steps in the glycosylation pathways. We replicated our findings in 1,836 individuals from the Leiden Longevity Study (LLS). We were able to show subclass-specific genetic influences on single IgG glycan structures. The replicated results indicate that, in addition to genes encoding for glycosyltransferases (i.e., ST6GAL1, B4GALT1, FUT8, and MGAT3), other genetic loci have strong influences on the IgG glycosylation patterns. A novel locus on chromosome 1, harboring RUNX3, which encodes for a transcription factor of the runt domain-containing family, is associated with decreased galactosylation. Interestingly, members of the RUNX family are cross-regulated, and RUNX3 is involved in both IgA class switching and B-cell maturation as well as T-cell differentiation and apoptosis. Besides the involvement of glycosyltransferases in IgG glycosylation, we suggest that, due to the impact of variants within RUNX3, potentially mechanisms involved in B-cell activation and T-cell differentiation during the immune response as well as cell migration and invasion involve IgG glycosylation.

Murine γδ T Cells Render B Cells Refractory to Commitment of IgA Isotype Switching.

γδ T cells are abundant in the gut mucosa and play an important role in adaptive immunity as well as innate immunity. Although γδ T cells are supposed to be associated with the enhancement of Ab production, the status of γδ T cells, particularly in the synthesis of IgA isotype, remains unclear. We compared Ig expression in T cell receptor delta chain deficient (TCRδ−/−) mice with wild-type mice. The amount of IgA in fecal pellets was substantially elevated in TCRδ−/− mice. This was paralleled by an increase in surface IgA expression and total IgA production by Peyer's patches (PPs) and mesenteric lymph node (MLN) cells. Likewise, the TCRδ−/− mice produced much higher levels of serum IgA isotype. Here, surface IgA expression and number of IgA secreting cells were also elevated in the culture of spleen and bone marrow (BM) B cells. Germ-line α transcript, an indicator of IgA class switch recombination, higher in PP and MLN B cells from TCRδ−/− mice, while it was not seen in inactivated B cells. Nevertheless, the frequency of IgA+ B cells was much higher in the spleen from TCRδ−/− mice. These results suggest that γδ T cells control the early phase of B cells, in order to prevent unnecessary IgA isotype switching. Furthermore, this regulatory role of γδ T cells had lasting effects on the long-lived IgA-producing plasma cells in the BM.