B N Beck

Functional similarities of AeEα Ia molecules as determined by analysis with T-cell clones

Recognition of AeEα Ia antigens at the functional level was investigated using T-cell clones. The reactivities of an alloreactive and an antigen-reactive clone, both of which recognized AeEα Ia molecules, were compared on a panel of stimulator/antigen-presenting cells of various genotypes. The two clones recognized all tested AebEαxIa molecules, where x is a haplotype capable of expressing an Ia.7-bearing Eα polypeptide. Ia antigen recognition by either clone could be inhibited by the monoclonal antibody Y-17, which recognizes a combinatorial serologic determinant on certain AeEα molecules. There were no differences in the recognition of Ia by the alloreactive versus the antigen-reactive clone, suggesting that Ia antigens are recognized by the two clones in a fundamentally similar way. The recognition of these various Ia molecules by the two cloned T-cell lines provides evidence that the Eα polypeptides from H-2 haplotypes k, d, r, and u are functionally indistinguishable.

The contribution of beta-strand residues to serologic epitopes on the A ? k polypeptide

The receptor-ligand interaction that occurs between T helper lymphocytes and antigen presenting cells (APC) is unusual in that both receptor and ligand are variable. The T-cell receptors are clonally variable, dependent on the gene rearrangements that occur during T cell development. The ligand is an ill-defined complex of antigenic peptide and MHC class II molecule; the universe of peptides derived from both self and foreign proteins associate with the naturally polymorphic MHC molecules to create an enormous variety of ligands. To explore the relationship between the sequence diversity and ligand function of mouse MHC class II molecules, we have produced a series of variant cell lines expressing mutant A k molecules, using monoclonal antibody-based selection techniques (Beck et al. 1984). The analysis of serology, function, and sequence of four of these mutants has been described previously (Beck et al. 1984; Beck et al. 1987). In this report we complete the analysis of six additional mutants. These results indicate that, in addition to residues in the allelicly variable region spanning positions 61-70, predicted to be in the helices on the outer face of the molecule, residues predicted to lie on a ~-strand that runs beneath these helical regions also contribute to serologic epitopes. However, as might be expected from their positions in the molecule, these/3-strand residues appear not to participate in T-cell receptor interactions. The four mutant Ak-expressing cell lines described previously (derived from the B cell B lymphoma hybrid line TA3) carry mutations resulting in substitutions in the amino-terminal domain of the A~ polypeptide. Since that time, six additional A~ mutants have been isolated: two are unique, independent mutants, three are revertants of one of the first four mutants, and one is a double-mutant derived by subjecting a mutant cell line to selection for

Structure-Function Analysis of Serologically Selected Cell Lines Expressing Mutant A β k Polypeptides

A panel of twleve mutant A β k expressing cell lines has been produced from a mutagenized B cell hybridoma using a protocol of negative and positive immunoselection. Each of the serologically-defined epitopes on the A β k polypeptide has been altered in one or more of the mutant cell lines. Immunoselection techniques have been used to select also for secondary mutations in some cell lines, resulting either in reversion to the wild-type or near wild-type phenotype or in the alteration of additional A β k serologic epitopes. Characterization of the ability of the mutant cell lines to stimulate antigen and alloantigen-reactive T cell hybridomas has shown that they display distinct functional phenotypes ranging from nearly wild-type to almost completely defective. DNA sequence analysis of the A β k gene cloned from each mutant cell line has revealed the structural basis for the mutant serologic and functional phenotypes. To date, each β chain mutant has been found to have a single amino acid substitution (resulting from a single nucleotide substitution) in the β1 domain. The substitutions are clustered in or near the third allelicly-defined variable region of the Aβ polypeptide (residues 61–70). These results indicate that this region of the Aβ polypeptide comprises multiple serologically defined epitopes. Furthermore, specific residues within this region appear to be important for effective T cell activation.