Identification of Immunodominant Epitopes of -Gliadin in HLA-DQ8 Transgenic Mice following Oral Immunization (1)
A major feature of celiac disease is the inap- propriate intestinal T cell activation in HLA-DQA1*05-DQB1*02 (DQ2) and HLA-DQA1*03-DQB1*0302 (DQ8) patients, trig- gered by peptides from wheat gliadin and related prolamins from barley and rye. A specific gliadin peptide has been described to be immuno-dominant in DQ2 adults. This peptide is part of an immuno- active 33-mer gliadin peptide found to be resistant to digestion by gastric and pancreatic enzymes. Two HLA tg mice models have been developed based on the expression of HLA-DQ8 genes in the absence of their endogenous counterparts.
Swiss Prot entry Q9ZP09 was the closest in identity (99%) to the consensus sequence with only two mismatches (K3N; T3A). The HPLC profile obtained from the purified protein exhibited a major sharp peak. The expected molecular mass of the r-a-gliadin with and without the methionine residue at the N terminus was determined.
Peptides p10, p14, p16, p17, p18, p24, p25, and p26 were devoid of any deamidation, while peptides p11 and p12 were insoluble in the buffer used for the enzymatic treatment and hence were not further analyzed.
The cells were stimulated in vitro with each individual peptide of the panel to identify the amino acid sequences responsible for the immune response. We found that only peptides p13 and p23 and their corresponding deamidated forms induced significant in vitro proliferation of spleen and MLN cells. The response to gliadin was almost completely inhibited by anti-DQ, but not anti-DR Abs. Similar results were found for MLN cells.
The p123–132 peptide failed to induce a strong T cell proliferation of CD4+ spleen cells. Similar results were obtained for CD4+ MLN cells. The p13 residues Q122 and D130 fit respectively in P1 and P9 pockets of DQ8.
After three rounds of Ag stimulation, the peptide-specific T cell responses were characterized by a predominant IFN-g secretion in both compartments, whereas r-a-gliadin-derived T cells showed increased expression of IL-4 and IL-10.
Q9ZP09, representing an a-gliadin from spelt wheat. This protein includes sequence LGQQQPFPPQQPY correspond- ing to peptide p31–43, previously recognized to induce damage both in vitro and in vivo in celiac disease, and sequence LQLQPFPQPQLP of the DQ2-restricted a9 peptide p57–68. However, it does not contain DQ2-restricted a2 peptide p62–75 (FPQPQLPYPQPQLP) or the multivalent 33-mer peptide p57– 89 (LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF). Gliadin antigenicity is increased fol- lowing in vitro deamidation of glutamine residues to glutamic acid by tTG. The type of adjuvant and route of administration influence the immune response. In particular, mucosal coimmunization with CT predominantly enhances Th2 responses to coad- ministered Ag. Intranasal administration of gliadin in mice can down-regulate the systemic immune response to this Ag.