Functional analysis of the antigen binding region of an MHC class II molecule

The MHC class II molecules bind antigenic peptides and present them to T cells. Their ability to carry out these functions depends, in a critical way, on the detailed structure of the membrane-distal α 1 and β l domains of these molecules. Using the I-Ak molecule and a series of hen egg lysozyme (HEL) peptide-specific, I-Ak-restricted T cell hybridomas as a model, we have examined the effect of altering essentially all of the polymorphic residues of the murine class II molecule on its ability to present Ag. Our results support the following conclusions: (1) both the location and the structural alteration introduced in a specific amino acid interchange are important in determining the effect the interchange will have on Ag presentation; and (2) changes in amino acids in the floor of the putative Ag binding cleft of the class II molecule can exert a major influence on the presentation of peptides to T cells. By carrying out direct binding experiments between the HEL(46-61) peptide and two mutant I-A molecules that fail to present HEL(46-61) to appropriate T cells, we were; able to assess, in a quantitative fashion, the role played by peptide binding in the failure to present Ag. Our results suggest that, in the two cases studied, the failure to bind the HEL(46-61) peptide was not primarily responsible for the failure of the mutant class II molecule to present that peptide. Specifically, an Aβ chain mutant that possesses d allelic residues at positions 65-67 in the second PMR of the Aκβ, chain actually binds HEL(46-61) at wild type (I-Ak) levels. In contrast, an A, chain chimera in which b allelic residues are inserted in the third PMR of the Akα chain, binds HEL(46-61) about three- to four-fold less well than wild type. While this decrease in binding affinity may be partially responsible for the inability of the latter chimeric molecule to present HEL(46-61), it can not be the total explanation because increasing the peptide concn even by an order of magnitude does not restore Ag presentation by APC expressing this chimeric molecule. These results are discussed in terms of the currently accepted model of the class II molecule.