Celiac disease (CD) is an immune mediated disorder in which mucosal autoantibodies to the enzyme transglutaminase 2 (TG2)1 are generated in response to the exogenous antigen gluten2 in individuals who are HLA-DQ2 or HLA-DQ83. We assessed in a comprehensive and non-biased manner the IgA anti-TG2 response by expression cloning of the antibody repertoire on ex vivo isolated intestinal antibody-secreting cells (ASCs). We found that TG2-specific plasma cells are hugely expanded in patients with active CD, representing on average 10% of ASCs within the duodenal mucosa. Surprisingly, anti-TG2 antibodies were of high affinity and yet showed little adaptation by somatic mutations. Unlike infection-induced peripheral blood plasmablasts4, the TG2-specific ASCs had neither recently proliferated nor were they short-lived ex vivo. Altogether these observations demonstrate that there is a germline repertoire with high affinity for TG2 that may favour massive generation of autoreactive B cells. Anti-TG2 antibodies did not block enzymatic activity and served as substrates for TG2-mediated crosslinking when expressed as IgD or IgM, but not as IgA1 or IgG1. This could result in preferential recruitment of plasma cells from naïve IgD/IgM-expressing B cells, thus possibly explaining why the anti-TG2 response bears signs of a primary immune response despite the disease chronicity.

SUMMARY The B cell response to Salmonella typhimurium (STm) occurs massively at extrafollicular sites, without notable germinal centers (GCs). Little is known in terms of its specificity. To expand the knowledge of antigen targets, we screened plasmablast (PB)-derived monoclonal antibodies (mAbs) for Salmonella specificity, using ELISA, flow cytometry, and antigen microarray. Only a small fraction (0.5%–2%) of the response appeared to be Salmonella-specific. Yet, infection of mice with limited B cell receptor (BCR) repertoires impaired the response, suggesting that BCR specificity was important. We showed, using laser microdissection, that somatic hypermutation (SHM) occurred efficiently at extrafollicular sites leading to affinity maturation that in turn led to detectable STm Ag-binding. These results suggest a revised vision of how clonal selection and affinity maturation operate in response to Salmonella. Clonal selection initially is promiscuous, activating cells with virtually undetectable affinity, yet SHM and selection occur during the extrafollicular response yielding higher affinity, detectable antibodies.

The gluten-sensitive enteropathy celiac disease is tightly associated with the production of autoantibodies specific for the enzyme transglutaminase 2 (TG2)5. The mechanisms underlying the activation of autoreactive B cells, however, are not well defined. To gain more insight into this autoimmune response we have characterized the binding of TG2 by a panel of human monoclonal antibodies generated by expression cloning of immunoglobulin genes from single plasma cells of the celiac disease lesion. The antibodies were highly specific to TG2 and bound preferentially to the “open”, Ca2+-activated enzyme conformation. Epitope mapping revealed that they recognize few distinct conformational epitopes that cluster in the N-terminal half of the enzyme. Two of the epitopes were overlapping with the fibronectin binding site in TG2, and none of the epitopes was accessible when TG2 was in a cell surface-bound form. Based on our findings we propose that the autoantibodies are generated against the soluble, catalytically active enzyme, whereas antibodies reactive with cell surface-associated TG2 are absent from the response due to negative selection of B cells recognizing membrane-bound self-antigen. The findings give insight into the mechanisms controlling the formation of anti-TG2 autoantibodies in celiac disease.

Analyses of somatic hypermutation (SHM) patterns in B-cell immunoglobulin (Ig) sequences have important basic science and clinical applications, but are often confounded by the intrinsic biases of SHM targeting on specific DNA motifs (i.e., hot- and cold-spots). Modeling these biases has been hindered by the difficulty in identifying mutated Ig sequences in vivo in the absence of selection pressures, which skew the observed mutation patterns. To generate a large number of unselected mutations, we immunized B1-8 heavy-chain transgenic mice with nitrophenyl (NP) to stimulate NP-specific λ+ germinal center B cells, and sequenced the unexpressed κ light chains using next-generation methods. Most of these κ sequences had out-of-frame junctions and were presumably uninfluenced by selection. Despite being non-functionally rearranged, they were targeted by SHM and displayed a higher mutation frequency than functional sequences. We used 39,173 mutations to construct a quantitative SHM targeting model. The model showed targeting biases that were consistent with classic hot- and cold-spots, yet revealed additional highly mutable motifs. We observed comparable targeting for functional and non-functional sequences, suggesting similar biological processes operate at both loci. However, we observed species-specific and chain-specific targeting patterns, demonstrating the need for multiple SHM targeting models. Interestingly, the targeting of C/G bases and the frequency of transition mutations at C/G bases was higher in mice compared with humans, suggesting lower levels of DNA repair activity in mice. Our models of SHM targeting provide insights into the SHM process and support future analyses of mutation patterns.

The gut mucosal surface is efficiently protected by Abs, and this site represents one of the richest compartments of Ab-secreting cells in the body. A simple and effective method to generate Ag-specific human monoclonal Abs (hmAbs) from such cells is lacking. In this paper, we describe a method to generate hmAbs from single Ag-specific IgA- or IgM-secreting cells of the intestinal mucosa. We found that CD138-positive plasma cells from the duodenum expressed surface IgA or IgM. Using eGFP-labeled virus-like particles, we harnessed the surface Ig expression to detect rotavirus-specific plasma cells at low frequency (0.03–0.35%) in 9 of 10 adult subjects. Single cells were isolated by FACS, and as they were viable, further testing of secreted Abs by ELISPOT and ELISA indicated a highly specific selection procedure. Ab genes from single cells of three donors were cloned, sequenced, and expressed as recombinant hmAbs. Of 26 cloned H chain Ab genes, 22 were IgA and 4 were IgM. The genes were highly mutated, and there was an overrepresentation of the VH4 family. Of 10 expressed hmAbs, 8 were rotavirus-reactive (6 with Kd < 1 × 10−10). Importantly, our method allows generation of hmAbs from cells implicated in the protection of mucosal surfaces, and it can potentially be used in passive vaccination efforts and for discovery of epitopes directly relevant to human immunity.

To understand the biology of Ab-secreting cells in the human small intestine, we examined Ab production of intestinal biopsies kept in culture. We found sustained IgA and IgM secretion as well as viable IgA- or IgM-secreting cells after >4 wk of culture. The Ab-secreting cells were nonproliferating and expressing CD27 and CD138, thus having a typical plasma cell phenotype. Culturing of biopsies without tissue disruption gave the highest Ab production and plasma cell survival suggesting that the environment regulates plasma cell longevity. Cytokine profiling of the biopsy cultures demonstrated a sustained presence of IL-6 and APRIL. Blocking of the activity of endogenous APRIL and IL-6 with BCMA–Fc and anti-human IL-6 Ab demonstrated that both these factors were essential for plasma cell survival and Ab secretion in the biopsy cultures. This study demonstrates that the human small intestine harbors a population of nonproliferating plasma cells that are instructed by the microenvironment for prolonged survival and Ab secretion.

We have developed a high-throughput protein expression and interaction analysis platform that combines cDNA phage display library selection and massive gene sequencing using the 454 platform. A phage display library of open reading frame (ORF) fragments was created from mRNA derived from different tissues. This was used to study the interaction network of the enzyme transglutaminase 2 (TG2), a multifunctional enzyme involved in the regulation of cell growth, differentiation and apoptosis, associated with many different pathologies. After two rounds of panning with TG2 we assayed the frequency of ORFs within the selected phage population using 454 sequencing. Ranking and analysis of more than 120 000 sequences allowed us to identify several potential interactors, which were subsequently confirmed in functional assays. Within the identified clones, three had been previously described as interacting proteins (fibronectin, SMOC1 and GSTO2), while all the others were new. When compared with standard systems, such as microtiter enzyme-linked immunosorbant assay, the method described here is dramatically faster and yields far more information about the interaction under study, allowing better characterization of complex systems. For example, in the case of fibronectin, it was possible to identify the specific domains involved in the interaction.

A hallmark of celiac disease is autoantibodies to transglutaminase 2 (TG2). By visualizing TG2-specific antibodies by antigen staining of affected gut tissue, we identified TG2-specific plasma cells in the lamina propria as well as antibodies in the sub-epithelial layer, inside the epithelium, and at the brush border. The frequency of TG2-specific plasma cells were found not to correlate with serum antibody titers, suggesting that antibody production at other sites may contribute to serum antibody levels. Upon commencement of a gluten-free diet, the frequency of TG2-specific plasma cells in the lesion dropped dramatically within 6 months, yet some cells remained. The frequency of TG2-specific plasma cells in the celiac lesion is thus dynamically regulated in response to gluten exposure. Laser microdissection of plasma cell patches, followed by antibody gene sequencing, demonstrated that clonal cells were seeded in distinct areas of the mucosa. This was confirmed by immunoglobulin heavy chain repertoire analysis of plasma cells isolated from individual biopsies of 2 untreated patients, both for TG2-specific and non TG2-specific cells. Our results shed new light on the processes underlying the B-cell response in celiac disease, and the approach of staining for antigen specific antibodies should be applicable to other antibody-mediated diseases.