In patients with celiac disease, inflammatory T cell responses to HLA-DQ2-bound gluten peptides are thought to cause disease. Two types of HLA-DQ2 molecules exist, termed HLA-DQ2.5 and HLA-DQ2.2. Whereas HLA-DQ2.5 predisposes to celiac disease, HLA-DQ2.2 does not. We now provide evidence that the diseaseassociated HLA-DQ2.5 molecule presents a large repertoire of gluten peptides, whereas the non-disease-associated HLA-DQ2.2 molecule can present only a subset of these. C eliac disease (CD) is the most common food-induced enteropathy in humans. Patients display a permanent intolerance toward the gluten proteins in wheat. About 95% of CD patients are HLA-DQ2 ϩ (DQA1*0501, DQB1*0201, termed HLA-DQ2.5 hereafter). HLA-DQ molecules bind and present peptides to antigen-specific T cells. It is now commonly accepted that HLA-DQ2.5 can bind and present gluten peptides and that these HLA-DQ-peptide complexes induce inflammatory T cell responses, causing disease. The HLA-DQ2.5 molecule preferentially binds peptides with negatively charged amino acids at anchor positions (1, 2). Whereas gluten peptides contain few negative charges, these charges can be introduced by the enzyme tissue transglutaminase (tTG) that selectively deamidates glutamine residues in gluten peptides (3-5).A second HLA-DQ2 molecule ex ists (DQA1*0201, DQB1*0202, termed HLA-DQ2.2 hereafter) with peptidebinding properties that are virtually identical with the properties of HLA-DQ2.5 (6). In fact, HLA DQ2.2 has been shown to bind and present a gluten peptide to T cells (7). Yet, HLA-DQ2.2 does not predispose for CD unless it is expressed together with HLA-DQ2.5 (8). Individuals homozygous for the diseaseassociated HLA-DQ2.5 genotype or HLA-DQ2.2/2.5 heterozygous have the highest risk for developing CD. In contrast, HLA-DQ2.5/non-DQ2.2 heterozygous individuals have an only slightly increased risk (9-11). To determine the mechanism underlying these observations we have now compared the gluten-specific T cell response in the context of the HLA-DQ2.5 and HLA-DQ2.2 molecules. Materials and MethodsSynthetic Peptides. Peptides were synthesized by standard fluorenylmethoxycarbonyl (Fmoc) chemistry on a SyroII peptide synthesizer (MultiSynTech, Witten, Germany). The integrity of the peptides was checked by RP-HPLC and mass spectrometry. The gluten epitopes were synthesized with glutamic acid residues at positions that are deamidated by tTG.

Background & Aims Invariant natural killer T (iNKT) cells undergo canonical, Vα14–Jα18 rearrangement of the T-cell receptor (TCR) in mice; this form of the TCR recognizes glycolipids presented by CD1d. iNKT cells mediate many different immune reactions. Their constitutive activated and memory phenotype and rapid initiation of effector functions after stimulation indicate previous antigen-specific stimulation. However, little is known about this process. We investigated whether symbiotic microbes can determine the activated phenotype and function of iNKT cells. Methods We analyzed the numbers, phenotypes, and functions of iNKT cells in germ-free mice, germ-free mice reconstituted with specified bacteria, and mice housed in specific pathogen-free (SPF) environments. Results SPF mice, obtained from different vendors, have different intestinal microbiota. iNKT cells isolated from these mice differed in TCR Vβ7 frequency and cytokine response to antigen, which depended on the environment. iNKT cells isolated from germ-free mice had a less mature phenotype and were hypo-responsive to activation with the antigen α-galactosylceramide. Intra-gastric exposure of germ-free mice to Sphingomonas bacteria, which carry iNKT cell antigens, fully established phenotypic maturity of iNKT cells. In contrast, reconstitution with Escherichia coli, which lack specific antigens for iNKT cells, did not affect the phenotype of iNKT cells. The effects of intestinal microbes on iNKT cell responsiveness did not require toll-like receptor signals, which can activate iNKT cells independently of TCR stimulation. Conclusions Intestinal microbes can affect iNKT cell phenotypes and functions in mice.

Celiac disease is a T cell-driven intolerance to wheat gluten. The gluten-derived T cell epitopes are proline-rich and thereby highly resistant to proteolytic degradation within the gastrointestinal tract. Oral supplementation with prolyl oligopeptidases has therefore been proposed as a potential therapeutic approach. The enzymes studied, however, have limitations as they are irreversibly inactivated by pepsin and acidic pH, both present in the stomach. As a consequence, these enzymes will fail to degrade gluten before it reaches the small intestine, the site where gluten induces inflammatory T cell responses that lead to celiac disease. We have now determined the usefulness of a newly identified prolyl endoprotease from Aspergillus niger for this purpose. Gluten and its peptic/tryptic digest were treated with prolyl endoprotease, and the destruction of the T cell epitopes was tested using mass spectrometry, T cell proliferation assays, ELISA, reverse-phase HPLC, SDS-PAGE, and Western blotting. We observed that the A. niger prolyl endoprotease works optimally at 4-5 pH, remains stable at 2 pH, and is completely resistant to digestion with pepsin. Moreover, the A. niger-derived enzyme efficiently degraded all tested T cell stimulatory peptides as well as intact gluten molecules. On average, the endoprotease from A. niger degraded gluten peptides 60 times faster than a prolyl oligopeptidase. Together these results indicate that the enzyme from A. niger efficiently degrades gluten proteins. Future studies are required to determine if the prolyl endoprotease can be used as an oral supplement to reduce gluten intake in patients.

SummaryMost bacterial proteins destined to leave the cytoplasm are exported to extracellular compartments or imported into the cytoplasmic membrane via the highly conserved SecA-YEG pathway. In the present studies, the subcellular distributions of core components of this pathway, SecA and SecY, and of the secretory protein pre-AmyQ, were analysed using green fluorescent protein fusions, immunostaining and/or immunogold labelling techniques. It is shown that SecA, SecY and (pre-)AmyQ are located at specific sites near and/or in the cytoplasmic membrane of Bacillus subtilis. The localization patterns of these proteins suggest that the Sec machinery is organized in spiral-like structures along the cell, with most of the translocases organized in specific clusters along these structures. However, this localization appears to be independent of the helicoidal structures formed by the actin-like cytoskeletal proteins, MreB or Mbl. Interestingly, the specific localization of SecA is dynamic, and depends on active translation. Moreover, reducing the phosphatidylglycerol phospholipids content in the bacterial membrane results in delocalization of SecA, suggesting the involvement of membrane phospholipids in the localization process. These data show for the first time that, in contrast to the recently reported uni-ExPortal site in the coccoïd Streptococcus pyogenes , multiple sites dedicated to protein export are present in the cytoplasmic membrane of rod-shaped B. subtilis .

Constitutive NF-κB activation in IECs induces inflammatory cytokines and chemokines in the lamina propria, but does not result in overt tissue damage unless acute inflammatory insults are present, causing TNF-dependent destruction and barrier disruption.

Summary Coreceptor CD4 and CD8αβ double negative (DN) TCRαβ+ intraepithelial T cells, although numerous, have been greatly overlooked and their contribution to the immune response is not known. Here we used T cell receptor (TCR) sequencing of single cells combined with retrogenic expression of TCRs, to study the fate and the major histocompatibility complex (MHC) restriction of DN TCRαβ+ intraepithelial T cells. The data show that commitment of thymic precursors to the DN TCRαβ+ lineage is imprinted by their TCR specificity. Moreover, the TCRs they express display a diverse and unusual pattern of MHC restriction that is non-overlapping with that of CD4+ or CD8αβ+ T cells, indicating that they sense antigens that are not recognized by the conventional T cell subsets. The new insights indicate that DN TCRαβ+ T cells form a third lineage of TCRαβ T lymphocytes expressing a variable TCR repertoire, which serve non-redundant immune functions.

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Celiac disease is an enteropathy caused by intolerance to dietary gluten. The disorder is strongly associated with DQA1*0501/DQB1*0201 (HLA-DQ2) as ∼95% of celiac patients express this molecule. HLA-DQ2 has unique Ag-binding properties that allow it to present a diverse set of gluten peptides to gluten-reactive CD4+ T cells so instigating an inflammatory reaction. Previous work has indicated that the presence of negatively charged amino acids within gluten peptides is required for specific binding. This, however, only partly explains the scale of the interaction. We have now characterized 432 natural ligands of HLA-DQ2 representing length variants of 155 distinct sequences. The sequences were aligned and the binding cores were inferred. Analysis of the amino acid distribution of these cores demonstrated that negatively charged residues in HLA-DQ2-bound peptides are favored at virtually all positions. This contrasts with a more restricted presence of such amino acids in T cell epitopes from gluten. Yet, HLA-DQ2 was also found to display a strong preference for proline at several anchor and nonanchor positions that largely match the position of proline in gluten T cell epitopes. Consequently, the bias for proline at p6 and p8 facilitates the enzymatic conversion of glutamine into glutamic acid in gluten peptides at p4 and p6, two important anchor sites. These observations provide new insights in the unique ability of HLA-DQ2 to bind a large repertoire of glutamine- and proline-rich gluten peptides. This knowledge may be an important asset in the development of future treatment strategies.