Determination of HLA-B7 T-cell epitopes

Abstract

A series of peptides derived from the alpha 1 and alpha 2 regions of the HLA-B7 and HLA-A2 molecules was synthesized with an automatic peptide synthesizer using the FMOC (9-fluorenylmethoxycarbonyl) technique. Peptides were analyzed and purified by reversed-phase high pressure liquid chromatography (HPLC). Peptide purity was > 96%. The effect of B7 peptides on mixed lymphocyte cultures was tested in vivo. The alloresponse in the cultures with B7 peptides was strongly enhanced. The peptides that were most effective inhibited cytotoxic T-lymphocyte (CTL) cytolysis. The data show that the peptides are immunogenic and that they are recognised by both the direct and indirect pathways. Further, the mechanism of peptide recognition was studied. We coupled peptide B7 (residues 62-70 in HLA-B7) and peptide A2 (residues 62-70 in HLA-B2) covalently to fluorescein isothiocyanate (FITC). B7-specific CTLs were incubated with these peptides at 37 degrees C for 90 min and cell fluorescence measured by flow cytometry. The B7 peptide was bound by 60% of the CTLs whereas the A2 peptide, as a negative control, bound only 10%. The molecular size of the ligands to which the peptides bind are being characterized by immunoprecipitation.

HLA-B7 β-pleated sheet-derived synthetic peptides are immunodominant T-cell epitopes regulating alloresponses

“…12,13,28 On this basis, several HLA-B7-derived peptides covering residues 56 to 120 were synthesized using the FMOC technique, as we previously described in more detail. 29 Peptides synthesized and used in these experiments are shown on the sequence alignment in Figure 1 and Table 1. After synthesis, peptides were cleaved from the resin, and all side chain-protecting groups were removed using a peptide cleavage solution constituting 8.8 mL trifluoro-acetic acid (Millipore, Bedford, MA), 0.5 g phenol (Riedel-de Haen, Seelzel, Germany), 0.2 mL tri-isopropyl silane (Sigma-Aldrich, Steinheim, Germany), and 0.5 mL distilled water.…”