To study B-cell development from bone marrow (BM), we generated recombination-activating gene 1 (
Characterization of Ag-specific BCR repertoires is essential for understanding disease mechanisms involving humoral immunity. This is optimally done by interrogation of paired H chain V region (V) and L chain V region (V) sequences of individual and Ag-specific B cells. By applying single-cell high-throughput sequencing on gut lesion plasma cells (PCs), we have analyzed the transglutaminase 2 (TG2)-specific V:V autoantibody repertoire of celiac disease (CD) patients. Autoantibodies against TG2 are a hallmark of CD, and anti-TG2 IgA-producing gut PCs accumulate in patients upon gluten ingestion. Altogether, we analyzed paired V and V sequences of 1482 TG2-specific and 1421 non-TG2-specific gut PCs from 10 CD patients. Among TG2-specific PCs, we observed a striking bias in and gene usage, as well as pairing preferences with a particular presence of the pair. Selective and biased V:V pairing was particularly evident among expanded clones. In general, TG2-specific PCs had lower numbers of mutations both in V and V genes than in non-TG2-specific PCs. TG2-specific PCs using had particularly few mutations. Importantly, V segments paired with displayed proportionally low mutation numbers, suggesting that the low mutation rate among PCs is dictated by the BCR specificity. Finally, we observed selective amino acid changes in V and V and striking CDR3 length and J segment selection among TG2-specific pairs. Hence this study reveals features of a disease- and Ag-specific autoantibody repertoire with preferred V:V usage and pairings, limited mutations, clonal dominance, and selection of particular CDR3 sequences.
AimsIn the present study we have investigated the comparative switching propensity of murine peritoneal and splenic B cell subpopulations to IgA in presence of retinoic acid (RA) and TGF-β.Methods and ResultsTo study the influence of RA and TGF-β on switching of B cell subpopulations to IgA, peritoneal (B1a, B1b and B2 cells) and splenic (B1a, marginal zone, and B2) B cells from normal BALB/c mice were FACS purified, cultured for 4 days in presence of RA and TGF-β and the number of IgA producing cells was determined by ELISPOT assay or FACS analysis. In presence of TGF-β, peritoneal B1b cells switched to IgA more potently than other peritoneal B cell subpopulations. When TGF-β was combined with retinoic acid (RA), switching to IgA was even more pronounced. Under these conditions, “innate” B cells like peritoneal and splenic B1 cells and MZ B cells produced IgA more readily than B2 cells. Additionally, high frequency of nucleotide exchanges indicating somatic hypermutation in VH regions was observed. Besides IgA induction, RA treatment of sorted PEC and splenic B cells led to expression of gut homing molecules - α4β7 and CCR9. Intraperitoneal transfer of RA-treated B1 cells into Rag1-/- recipients resulted in IgA in serum and gut lavage, most efficiently amongst B1b cell recipients.ConclusionPresent study demonstrates the differential and synergistic effect of RA and TGF-β on switching of different B cell subpopulations to IgA and establishes the prominence of peritoneal B1b cells in switching to IgA under the influence of these two factors. Our study extends our knowledge about the existing differences among B cell subpopulations with regards to IgA production and indicates towards their differential contribution to gut associated humoral immunity.
B cells play important roles in autoimmune diseases through autoantibody production, cytokine secretion, or antigen presentation to T cells. In most cases, the contribution of B cells as antigen-presenting cells is not well understood. We have studied the autoantibody response against the enzyme transglutaminase 2 (TG2) in celiac disease patients by generating recombinant antibodies from single gut plasma cells reactive with discrete antigen domains and by undertaking proteomic analysis of anti-TG2 serum antibodies. The majority of the cells recognized epitopes in the N-terminal domain of TG2. Antibodies recognizing C-terminal epitopes interfered with TG2 cross-linking activity, and B cells specific for C-terminal epitopes were inefficient at taking up TG2-gluten complexes for presentation to gluten-specific T cells. The bias toward N-terminal epitopes hence reflects efficient T-B collaboration. Production of antibodies against N-terminal epitopes coincided with clinical onset of disease, suggesting that TG2-reactive B cells with certain epitope specificities could be the main antigen-presenting cells for pathogenic, gluten-specific T cells. The link between B cell epitopes, antigen presentation, and disease onset provides insight into the pathogenic mechanisms of a T cell-mediated autoimmune condition.
The interdependence of posttranscriptional gene regulation via miRNA and transcriptional regulatory networks in lymphocyte development is poorly understood. Here, we identified miR-191 as direct upstream modulator of a transcriptional module comprising the transcription factors Foxp1, E2A, and Egr1. Deletion as well as ectopic expression of miR-191 resulted in developmental arrest in B lineage cells, indicating that fine tuning of the combined expression levels of Foxp1, E2A, and Egr1, which in turn control somatic recombination and cytokine-driven expansion, constitutes a prerequisite for efficient Bcell development. In conclusion, we propose that miR-191 acts as a rheostat in B-cell development by fine tuning a key transcriptional program.Keywords: B cells r Lymphocyte development r miRNA r miR-191 r Transcriptional factors Additional supporting information may be found online in the Supporting
IntroductionIgA is the major class of Ab present in mucosal tissues of mammals. IgA constitutes a key defense mechanism against invasion by inhaled or ingested pathogens. IgA is also found at significant concentrations in the serum of many species, where it mediates the elimination of pathogens that have breached the mucosa [1].Among the various MALTs, IgA-producing cells are present in highest numbers in GALTs constituted by Peyer's patches (PPs), isolated lymphoid follicles (ILFs), and solitary intestinal lymphoid Correspondence: Dr. Bishnudeo Roy e-mail: Bishnudeo.Roy@helmholtz-hzi.de tissue (SILT) [2,3]. B cells, present in the B-cell follicles of such organized structures, form GCs upon Ag encounter. Under the influence of various cellular and molecular factors -T cells and cytokines, for instance -these B cells are thought to switch from IgM to IgA [4].Multiple pathways of IgA induction have been reported. The T-cell-dependent pathway of IgA induction is well known and, it is clear that IgA can also be induced in T-cell-independent (TI) manner (e.g. in T-cell-deficient mice, CD40 −/− mice, CD28 −/− mice, etc.) [5][6][7]. However, the precise contribution of T-cell-dependent and TI pathways to IgA production is not known.An important contribution of B-1 cells to TI responses has been suggested [6,8,9]. Peritoneal cavity (PEC) B-1 cells have also been claimed to contribute significantly to IgA-producing plasma cell (PC) Eur. J. Immunol. 2013Immunol. . 43: 2023Immunol. -2032. Importantly, according to phenotype, origin, and function, PEC B-1 cells can be divided into B-1a and B-1b subpopulations [12,13]. Phenotypically, B-1a cells are characterized as B220 lo CD19 hi IgM hi IgD lo CD43 + Mac-1 + CD5 int . B-1b cells share all the aforementioned markers with B-1a cells except CD5. With respect to differential contribution of these two B-1-cell subtypes to IgA production, recently, we could show that most of the IgA-secreting cells in the PEC of unmanipulated mice belonged to B-1b-cell subpopulation [14]. Additionally, IgA-derived VH regions from B-1b cells also contained frequent single nucleotide exchanges indicative of somatic hypermutation (SHM) [14]. Thus, a contribution of PEC B-1b cells to IgA production and hence to gut-associated immunity is quite likely. However, in spite of evidential suggestions for the contribution of B-1 cells to the intestinal PC pool, their contribution under unmanipulated conditions remains uncertain. This is partly due to the lack of appropriate markers to distinguish PCs according to their origin. In this context, a sequence-based approach to address this question should become very useful.In the present work, to investigate the participation of PEC B-1 cells in the gut-associated IgA production we have made use of the transgenic mouse model known as the L2 mouse line [15]. Mice of this line are characterized by the expression of a transgenic λ light chain obtained from the plasmacytoma MOPC315 (λ2 315 ). PEC of these mice contains almost exclusively CD5 + B-1a cells, whi...
Taken together, we present an in vivo model to study the role of MAP infection in CD. Our results confirm the hypothesis that MAP is able to exacerbate existing intestinal inflammation.
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