TG2-gluten complexes as antigens for gluten-specific and transglutaminase-2 specific B cells in celiac disease
Fig 5
TG2-gluten complexes are efficient antigens for both DGP-specific and TG2-specific B cells and allow for help of T cells with many different specificities.
(A) Amino acid sequences of the rhodamine-labeled α33mer and the FITC-labeled ω34mer peptides. Glutamine residues expected to be targeted by TG2 are enlarged and given in bold. (B) Gradient SDS-PAGE (4–20%) showing incorporation of FITC-conjugated ω-34mer and Rhodamine-conjugated α33mer into both TG2 monomers, dimers and multimers. The gel shows TG2-gluten complexes separated into monomers (lanes 1, 4), dimers/ trimers (lanes 2, 5) and multimers (lanes 3, 6) made using a 2:1 (lanes 1–3) or 17:1 (lanes 4–6) molar ratio of α33mer: ω34mer peptide. Arrow indicates monomeric TG2 (78 kDa). (C) The ω34mer peptide is crosslinked to TG2 to a higher degree than the α33mer peptide. Protein concentration and fluorescent labels were quantified using a Nanodrop UV-Vis spectrophotometer, and the number of fluorescent peptides per TG2 molecule were calculated. The table shows the average values of monomers, dimers/trimers and multimers of one representative experiment. (D) Irradiated A20 B cells expressing HLA-DQ2.5 and a TG2-specific BCR (14E06), gluten-specific BCR (3B01) or non-specific BCR (2F02) were incubated with complexes of TG2 and FITC-labeled native ω34mer gliadin peptide and rhodamine B-labeled α33mer gliadin peptide, and their ability to present deamidated peptides to TCC specific for the DQ2.5-glia-ω1 (TCC1383.P.A.6), DQ2.5-glia-ω2 (TCC737.30), DQ2.5-glia-α1 epitope (TCC430.1.57) or DQ2.5-glia-α2 epitope (TCC436.5.3) was assessed by [3H] thymidine incorporation. Symbols represent mean counts per minute (CPM) +/- SEM of culture triplicates. Data are representative of 3 independent experiments.