HLA-DQ2 and HLA-DQ8 Are Expressed on So-Called Antigen-Presenting Cells

<p>The function of antigen-presenting cells (APCs) is to bind peptides derived from pathogens and “present” these to the T cells of the immune system. T cells detect such HLA–peptide complexes through their T cell receptors. In celiac disease, T cells respond to gluten peptides bound to HLA-DQ2 or HLA-DQ8. Gluten-derived peptides (red diamond) bind only with low affinity to HLA-DQ2 and HLA-DQ8, but TG2 can modify such peptides, which turns them into high-affinity binders. Consequently, the HLA–gluten peptide complexes are more stable, which facilitates and enhances T cell responses to such peptides.</p

VSL#3 inhibits LPS-induced phosphorylation of STAT-1.

<p>(<b>A</b>) LPS-induced genes, significantly suppressed by VSL#3, were analyzed by using Metacore Network analysis (Transcription factor). STAT-1 was predicted to be a key driver of this cluster. DC were stimulated for 3 hours, and phosphorylation of NF-κBp65 (<b>B</b>) and STAT-1 (<b>C</b>) was quantified in nuclear extracts using the Trans-am assay. Absorbance values (mean ± SD) of 5 individual donors are shown. Rank-Wilcoxon paired test: * p<0.05, ** p<0.01.</p

mRNA expression levels of 84 chemokines and cytokines after 4 hours of stimulation with LPS, VSL#3 or a combination of both, as determined by qPCR arrays.

<p>mRNA expression levels of 84 chemokines and cytokines after 4 hours of stimulation with LPS, VSL#3 or a combination of both, as determined by qPCR arrays.</p

Peptides generated from the 26-mer and 33-mer.

<p>Numbers and letters refer to peaks in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128065#pone.0128065.g002" target="_blank">Fig 2</a></p><p>Sequences that contain epitopes are underlined</p><p>*peptides shorter than eight amino acids are considered too short to contain an epitope</p><p>Peptides generated from the 26-mer and 33-mer.</p

Ineffective detoxification by digestive enzyme supplements.

<p>33-mer was incubated for 60 minutes with 1 capsule equivalent of digestive enzyme supplements, or 1/10 capsule equivalent of AN-PEP as a control. The peptide reaction products were deamidated using tissue transglutaminase, purified by C18 solid phase extraction and tested for T-cell activation. As a negative control, T-cells were incubated without the 33-mer peptide or digestive enzyme supplements (blanc). As a positive control, T-cells were incubated with 33-mer peptide. The enzyme preparations, when tested separately, were not toxic for the T-cells (not shown). T-cells stimulated with control peptide (LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF) incorporated 40,000 cpm. T-cell proliferation ([³H]thymidine incorporation in cpm) is shown as a function of the input of 33-mer and its degradation products. Error bars represent standard deviation of triplicate experiments. The result shown is representative for two independent experiments.</p

Epitopes contained in the gastrointestinal-enzyme resistant 26-mer and 33-mer gluten peptides.

<p>The sequences of the 26-mer and 33-mer peptide are shown. Underneath, the position of the immunogenic epitopes is denoted</p><p>^*the epitopes DQ8-glia-γ1a and DQ8-glia-γ1b are identical to epitopes DQ2.5-glia-γ4c respectively DQ2.5-glia-γ3</p><p>Epitopes contained in the gastrointestinal-enzyme resistant 26-mer and 33-mer gluten peptides.</p

Mass spectrometric analysis of the degradation products of 26-mer and 33-mer peptide by digestive enzyme supplements.

<p>Peptide was incubated at 37°C for 30 minutes at pH 6.0 in the presence of 1 capsule equivalent digestive enzyme supplements. As a control, 1/100 capsule equivalent of AN-PEP at pH 5.0 was also incubated. Peptide reaction products were analyzed by Q-TOF LC-MS and their MS spectra shown in this Figure. The identity of the prominent peptides 1–6 and a-g are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128065#pone.0128065.t004" target="_blank">Table 4</a>. In the case of AN-PEP, small amounts of epitope-containing peptides were observed under these conditions, but they disappear at higher enzyme concentration or prolonged incubation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128065#pone.0128065.g003" target="_blank">Fig 3</a>). The digestive enzyme supplements display comparable activities and remove at most two amino acids from the N-terminus of the 26-mer (peptide 1 (26-mer) is degraded to peptide 2, and then to peptide 3), and three amino acids at most from the 33-mer peptide (peptide a (33-mer) is degraded to b, to c, and then to peptide d).</p

Protein compositon of digestive enzyme supplements.

<p>20 μg of digestive enzyme supplement was acid-inactivated, reduced, alkylated and digested with 2 μg of trypsin as specified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128065#pone.0128065.s008" target="_blank">S2 Text</a> Methods.Tryptic peptides were analyzed by LC-MS and the proteins identified using Mascot search engine. The numbers represent the number of unique peptides matching the protein.</p><p>¹Uniprot database</p><p>²alternative name, Lactase-N</p><p>Protein compositon of digestive enzyme supplements.</p

Identification of an Epitope in Avenin Recognized by Intestinal T-Cells of Celiac Disease Patients

<div><p>(A) Reverse-phase HPLC of a pepsin-trypsin digest of avenin. Peptides in fraction 25, obtained from gel filtration, were eluted by an acetonitrile gradient (straight line), and 41 fractions were collected. Fractions recognized by T-cell lines in subsequent experiments are indicated by the numbers above the peaks.</p> <p>(B) T-cell recognition of fractions obtained by reverse-phase HPLC. All 41 fractions obtained in (A) were tested for recognition by the intestinal T-cell line 422.2.4 (derived from an oat-intolerant celiac disease patient). The fractions were incubated with DR3-DQ2 homozygous antigen-presenting cells overnight before the T-cell line was added. Specific T-cell responses were measured by ³H-thymidine incorporation. Pepsin-trypsin-digested avenins, both TG2-treated and untreated, were used as control antigens.</p> <p>(C) Sequences of the peptides in the stimulatory fractions from reverse-phase HPLC identified by tandem mass spectrometry and overlapping synthetic peptides used for T-cell assays. For better comparison, the amino acid sequence of the avenin precursor protein JQ1047 (gi82331) is taken as a consensus sequence, and deviating residues are underlined.</p></div

Reactivity of an HLA-DQ2-Restricted T-Cell Clone Derived from a T-Cell Line (CD496.2.3) Established by Avenin Stimulation of an Intestinal Biopsy of Patient CD496

<div><p>T-cell responses were measured by ³H-thymidine incorporation and are represented as SIs.</p> <p>(A) The clone specifically recognizes the avenin peptide Av-α9B after treatment with TG2. The peptides were tested at 10 μM.</p> <p>(B) The clone recognizes avenin but not gluten antigen after treatment with TG2.</p></div