Antigenic competition at the level of peptide-Ia binding.

Abstract

We examined the direct binding of a hen egg white lysozyme peptide, , to membrane I-Ak (pro-tein encoded in the A locus of the I region) molecules in the presence of detergent. A number of synthetic peptide derivatives, which did not stimulate our T-cell reactive hybridomas, competed for the binding of HEL(46-61) to I-Ak and also inhibited the functional presentation ofHEL(46-61). Inhibitors included a peptide lacking a tyrosine at position 53 and a peptide corresponding to the autologous lysozyme peptide. Presentation was examined with cells or with supported planar phospholipid membranes bearing only I-Ak and HEL(46-61).Other peptides that did not compete for the binding did not inhibit functional presentation. We concluded that the binding of an immunogenic peptide to I-A is critical for presentation, that the I-A molecule does not discriminate between autologous and foreign related determinants but does recognize structurally different peptides. Our evidence suggests that our immunogenic peptide bears noncontiguous amino acids critical for contact I-A binding interspersed with amino acids critical for interaction with T cells.We have demonstrated that a class II histocompatibility protein, an I-region associated (Ia) molecule, directly binds to a defined synthetic immunogenic peptide derived from hen egg white lysozyme (HEL) (1). We measured the binding of a fluorescently labeled immunogenic HEL peptide, 4-nitrobenzo-2-oxa-1,3-diazole (NBD)-HEL (46-61), to the I-Ak molecule (protein encoded in the A locus ofthe Iregion), in detergent, by equilibrium dialysis. The peptide HEL-(46-61) contains the immunodominant epitope recognized by mice of the k haplotype (2). The binding was shown to be specific, with a Kd of approximately 2 ,uM. Furthermore, the binding was haplotyje specific, in that NBD-HEL(46-61) did not bind to the I-A molecule, a product of a nonresponder allele, when measured under identical conditions. These results may provide necessary insights to understand how Ia molecules can influence T-cell activation in an antigen specific manner.We have now addressed the question of whether synthetic peptide derivatives ofthe immunogenic peptide, generated by a series of amino acid deletions, substitutions, or truncations, can compete with NBD-HEL(46-61) for binding to I-Ak using our established binding assay system (1). At the same time we have studied the effect of some of these same peptides on the functional presentation of MATERIALS AND METHODS Purification of I-Ak. I-Ak was affinity purified on a large scale using an affinity matrix, the anti-I-Ak monoclonal antibody 10-2.16 coupled to CNBr-activated Sepharose 4B from plasma membranes derived from~-1011 TA3 hybridoma cells as described (3). Briefly, a plasma membrane-enriched fraction was isolated, washed in high salt (0.5 M NaCl, 10 mM EDTA), lysed in 0.5% Triton X-100 containing 15 mM triethanolamine, 0.2 mM phenylmethylsulfonyl fluoride, leupeptin at 10 gg/ml at pH 8.0, and passed through the anti-I-Ak affinity column. I-Ak was eluted wi...

Molecular characterization of an arthritogenic collagen peptide interacting with I‐Ar

“…[16][17][18] Thus, if the relevant class II molecules can bind and present certain self peptides the host becomes at risk of developing an autoimmune response. The relevant sequences of bovine and murine CII (445-453) differ only with a conservative substitution of a Ser (mouse) residue for an Ala (bovine) residue at position 447 which is an MHC anchor residue (Table 1).…”

Cellular mechanisms of antigen processing and the function of class I and II major histocompatibility complex molecules.

“…This is important, because MHC molecules bind many peptides with little apparent specificity or discrimination between self and foreign epitopes (Babbitt et al, 1986;Buus et al, 1988;Lorenz and Allen, 1988). Thus, many different peptides must be presented simultaneously to ensure that immunogenic epitopes necessary for a particular immune response are included in the repertoire of expressed peptide complexes.…”

“…6) Several algorithms predict sequences that are more likely to bind particular MHC molecules (DeLisi and Berzofsky, 1985;Rothbard and Taylor, 1988;Sette et al, 1988;Reyes et al, 1989), and 7) Peptides compete for binding to MHC molecules based on the affinity of each for the particular MHC allele. Clear competition among peptides for binding to the single MHC-11 binding site can be documented in vitro (Babbitt et al, 1986) and also in functional analyses using APC (Werdelin, 1982;Rock and Benacerraf, 1983).…”

“…2) These core epitopes usually consist of 10 amino acids, but peptides as short as 5 amino acids bind to MHC molecules (Reddehase et aL., 1989). 3) Peptides derived from both foreign antigens and self proteins bind to MHC molecules, i.e., these molecules do not discriminate between self and non-self (Babbitt et aL, 1986). This discrimination in the immune response is controlled by the repertoire of responding T cells, which is molded by events in the thymus and by mechanisms of peripheral tolerance.…”

“…Some peptides, once bound, will practically not dissociate unless the MHC-11 molecules are drastically denatured. Binding kinetics and competition studies with unlabeled peptides established that there is a single binding site (Babbitt et al, 1986;Buus et al, 1987).…”