Tissue-specific Regulation of Immune Responses to Dietary Proteins

__Abstract__ The term oral (or mucosal) tolerance has been defined as the suppression of T- and B-cell responses to an antigen by prior administration of the antigen by the oral route . From this definition, it can be inferred that mucosal tolerance is not just the ignorance of antigens that are applied via the mucosa or the absence of an immune response, but quite the contrary, mucosal tolerance refers to a dynamic process that depends on the active suppression of antigen-specific immune responses. Although such induction of immunological tolerance is not exclusive for antigens encountered via the mucosa and can be achieved outside the mucosal environment as well, it is well established that specific features of mucosal tissues favor the induction of tolerance. Here we discuss the mechanisms that are required for the induction of mucosal tolerance and the unique properties of the mucosal microenvironment that allow for intestinal homeostasis and the development of mucosal tolerance

Changes in Natural Foxp3+Treg but Not Mucosally-Imprinted CD62LnegCD38+Foxp3+Treg in the Circulation of Celiac Disease Patients

Background:Celiac disease (CD) is an intestinal inflammation driven by gluten-reactive CD4+ T cells. Due to lack of selective markers it has not been determined whether defects in inducible regulatory T cell (Treg) differentiation are associated with CD. This is of importance as changes in numbers of induced Treg could be indicative of defects in mucosal tolerance development in CD. Recently, we have shown that, after encounter of retinoic acid during differentiation, circulating gut-imprinted T cells express CD62LnegCD38+. Using this new phenotype, we now determined whether alterations occur in the frequency of natural CD62L+Foxp3+ Treg or mucosally-imprinted CD62LnegCD38+Foxp3+ Treg in peripheral blood of CD patients. In particular, we compared pediatric CD, aiming to select for disease at onset, with adult CD.Methods:Cell surface markers, intracellular Foxp3 and Helios were determined by flow cytometry. Foxp3 expression was also detected by immunohistochemistry in duodenal tissue of CD patients.Results:In children, the percentages of peripheral blood CD4+Foxp3+ Treg were comparable between CD patients and healthy age-matched controls. Differentiation between natural and mucosally-imprinted Treg on the basis of CD62L and CD38 did not uncover differences in Foxp3. In adult patients on gluten-free diet and in refractory CD increased percentages of circulating natural CD62L+Foxp3+ Treg, but normal mucosally-imprinted CD62LnegCD38+Foxp3+ Treg frequencies were observed.Conclusions:Our data exclude that significant numeric deficiency of mucosally-imprinted or natural Foxp3+ Treg explains exuberant effector responses in CD. Changes in natural Foxp3+ Treg occur in a subset of adult patients on a gluten-free diet and in refractory CD patients

CTR (4032)

Mass spectrometry QExactive RAW data file; tryptic digest of non-crosslinked TG

Data from: Enhanced B-cell receptor recognition of the autoantigen transglutaminase 2 by efficient catalytic self-multimerization

A hallmark of the gluten-driven enteropathy celiac disease is autoantibody production towards the enzyme transglutaminase 2 (TG2) that catalyzes the formation of covalent protein-protein cross-links. Activation of TG2-specific B cells likely involves gluten-specific CD4 T cells as production of the antibodies is dependent on disease-associated HLA-DQ allotypes and dietary intake of gluten. IgA plasma cells producing TG2 antibodies with few mutations are abundant in the celiac gut lesion. These plasma cells and serum antibodies to TG2 drop rapidly after initiation of a gluten-free diet, suggestive of extrafollicular responses or germinal center reactions of short duration. High antigen avidity is known to promote such responses, and is also important for breakage of self-tolerance. We here inquired whether TG2 avidity could be a feature relevant to celiac disease. Using recombinant enzyme we show by dynamic light scattering and gel electrophoresis that TG2 efficiently utilizes itself as a substrate due to conformation-dependent homotypic association, which involves the C-terminal domains of the enzyme. This leads to the formation of covalently linked TG2 multimers. The presence of exogenous substrate such as gluten peptide does not inhibit TG2 self-cross-linking, but rather results in formation of TG2-TG2-gluten complexes. The celiac disease autoantibody epitopes, clustered in the N-terminal part of TG2, are conserved in the TG2-multimers as determined by mass spectrometry and immunoprecipitation analysis. TG2 multimers are superior to TG2 monomer in activating A20 B cells transduced with TG2-specific B-cell receptor, and uptake of TG2-TG2-gluten multimers leads to efficient activation of gluten-specific T cells. Efficient catalytic self-multimerization of TG2 and generation of multivalent TG2 antigen decorated with gluten peptides suggest a mechanism by which self-reactive B cells are activated to give abundant numbers of plasma cells in celiac disease. Importantly, high avidity of the antigen could explain why TG2-specific plasma cells show signs of an extrafollicular generation pathway

Generation of an HLA-DQ2.5 knock-in mouse

The human MHC class II molecule HLA-DQ2.5 is implicated in multiple autoimmune disorders, including celiac disease, type 1 diabetes, and systemic lupus erythematosus. The pathogenic contribution of HLA-DQ2.5 in many of these disorders is not fully understood. There is thus a need for an HLA-DQ2.5 humanized mouse model with physiological expression of this MHC molecule that can be integrated into disease models. In this article, we report the generation of an HLA-DQ2.5 knock-in mouse strain on a C57BL/6 background in which sequences encoding the extracellular moieties of mouse MHC class II H2-IAa and H2-IAb1 have been replaced with those of HLA-DQA1*05:01 and HLA-DQB1*02:01. In heterozygous knock-in mice, the expression of HLA-DQ2.5 is superimposable with the expression of H2-IA. This was not the case in a regular untargeted HLA-DQ2.5 transgenic mouse. HLA-DQ2.5 in the knock-in animals is functional for T cell development and for Ag presentation to HLA-DQ2.5–restricted and gluten-specific T cells. Because C57BL/6 mice do not express H2-IEa, the only functional MHC class II molecule in homozygous HLA-DQ2.5 knock-in mice is the knock-in gene product. This alleviates the need for crossing with MHC class II knockout mice to study the isolated function of the MHC transgene. Our novel mouse strain provides an important tool to study the involvement of HLA-DQ2.5 in models of diseases with association to this HLA allotype

TG2-gluten complexes as antigens for gluten-specific and transglutaminase-2 specific B cells in celiac disease

A hallmark of celiac disease is the gluten-dependent production of antibodies specific for deamidated gluten peptides (DGP) and the enzyme transglutaminase 2 (TG2). Both types of antibodies are believed to result from B cells receiving help from gluten-specific CD4 + T cells and differentiating into antibody-producing plasma cells. We have here studied the collaboration between DGP- and TG2-specific B cells with gluten-specific CD4 + T cells using transgenic mice expressing celiac patient-derived T-cell and B-cell receptors, as well as between B-cell transfectants and patient-derived gluten-specific T-cell clones. We show that multivalent TG2-gluten complexes are efficient antigens for both TG2-specific and DGP-specific B cells and allow both types of B cells to receive help from gluten-specific T cells of many different specificities

Trimer plus (III+) (4048)

Mass spectrometry QExactive RAW data file; tryptic digest of self-crosslinked TG2 (trimer +, size exclusion fraction III+

Enhanced B-Cell Receptor Recognition of the Autoantigen Transglutaminase 2 by Efficient Catalytic Self-Multimerization

<div><p>A hallmark of the gluten-driven enteropathy celiac disease is autoantibody production towards the enzyme transglutaminase 2 (TG2) that catalyzes the formation of covalent protein-protein cross-links. Activation of TG2-specific B cells likely involves gluten-specific CD4 T cells as production of the antibodies is dependent on disease-associated HLA-DQ allotypes and dietary intake of gluten. IgA plasma cells producing TG2 antibodies with few mutations are abundant in the celiac gut lesion. These plasma cells and serum antibodies to TG2 drop rapidly after initiation of a gluten-free diet, suggestive of extrafollicular responses or germinal center reactions of short duration. High antigen avidity is known to promote such responses, and is also important for breakage of self-tolerance. We here inquired whether TG2 avidity could be a feature relevant to celiac disease. Using recombinant enzyme we show by dynamic light scattering and gel electrophoresis that TG2 efficiently utilizes itself as a substrate due to conformation-dependent homotypic association, which involves the C-terminal domains of the enzyme. This leads to the formation of covalently linked TG2 multimers. The presence of exogenous substrate such as gluten peptide does not inhibit TG2 self-cross-linking, but rather results in formation of TG2-TG2-gluten complexes. The celiac disease autoantibody epitopes, clustered in the N-terminal part of TG2, are conserved in the TG2-multimers as determined by mass spectrometry and immunoprecipitation analysis. TG2 multimers are superior to TG2 monomer in activating A20 B cells transduced with TG2-specific B-cell receptor, and uptake of TG2-TG2-gluten multimers leads to efficient activation of gluten-specific T cells. Efficient catalytic self-multimerization of TG2 and generation of multivalent TG2 antigen decorated with gluten peptides suggest a mechanism by which self-reactive B cells are activated to give abundant numbers of plasma cells in celiac disease. Importantly, high avidity of the antigen could explain why TG2-specific plasma cells show signs of an extrafollicular generation pathway.</p></div

Trimer (III) (4044)

Mass spectrometry QExactive RAW data file; tryptic digest of self-crosslinked TG2 (trimer, size exclusion fraction III