Ralph T. Kubo

An antibody-secreting B cell hybridoma, KJ1-26.1, has been prepared from mice immunized with the T cell hybridoma DO-11.10, which recognizes chicken ovalbumin in association with I-Ad (cOVA/I-Ad). KJ1-26.1 blocks I-restricted antigen recognition by DO-11.10 and a subclone of this T cell hybridoma, DO-11.10.24, which has the same specificity for cOVA/I-Ad as its parent. KJ1-26.1 does not block I-restricted antigen recognition by any other T cell hybridoma tested, including a number of T cell hybridomas closely related to DO-11.10, with similar, but not identical, specificities for antigen/I. Moreover, KJ1-26.1 binds to DO-11.10 and DO-11.10.24, but not to any other T cell hybridomas tested, including three subclones of DO-11.10 that have lost the ability to recognize cOVA/I-Ad. Thus, in every regard KJ1-26.1 appears to be binding to all or part of the receptors for antigen/I on the T cell hybridoma DO-11.10. KJ1-26.1 appears to bind to approximately 15,000 molecules/cell on the surface of DO-11.10. The antibody precipitates an 80,000 dimer from the cells, which on reduction migrates as 40-44,000 monomers. The receptor(s) for antigen/I on DO-11.10 therefore includes molecules with these properties.

The relationship between binding affinity for HLA class I molecules and immunogenicity of discrete peptide epitopes has been analyzed in two different experimental approaches. In the first approach, the immunogenicity of potential epitopes ranging in MHC binding affinity over a 10,000-fold range was analyzed in HLA-A*0201 transgenic mice. In the second approach, the antigenicity of approximately 100 different hepatitis B virus (HBV)-derived potential epitopes, all carrying A*0201 binding motifs, was assessed by using PBL of acute hepatitis patients. In both cases, it was found that an affinity threshold of approximately 500 nM (preferably 50 nM or less) apparently determines the capacity of a peptide epitope to elicit a CTL response. These data correlate well with class I binding affinity measurements of either naturally processed peptides or previously described T cell epitopes. Taken together, these data have important implications for the selection of epitopes for peptide-based vaccines, and also formally demonstrate the crucial role of determinant selection in the shaping of T cell responses. Because in most (but not all) cases, high affinity peptides seem to be immunogenic, our data also suggest that holes in the functional T cell repertoire, if they exist, may be relatively rare.

Research on the specificities, functions, and maturation of T cells would be greatly aided by a collection of monoclonal antibodies which distinguishes different types of TCR. With this end in mind hamsters were immunized and tested for production of pan-reactive anti-mouse alpha beta TCR antibodies. In this report we describe the properties and uses of a mAb, H57-597, produced from one of these animals. The mAb reacts with surface receptors on all alpha beta TCR-bearing cells and does not react with receptors on gamma delta+ T cells. In an immobilized form, this antibody can directly activate T cells bearing alpha beta TCR. It can be used in immunoprecipitation reactions to precipitate receptor from the appropriate cell types. In combination with anti-CD3, the antibody can be used in cytofluorographic analyses to measure numbers of CD3+, alpha beta+, and CD3+, gamma delta+ cells in the thymus and periphery.

The peptide binding specificities of HLA-DRB1*0401, DRB1*0101, and DRB1*0701 have been analyzed by the use of large collections of synthetic peptides corresponding to naturally occurring sequences. The results demonstrated that nearly all peptides binding to these DR molecules bear a motif characterized by a large aromatic or hydrophobic residue in position 1 (Y, F, W, L, I, V, M) and a small, noncharged residue in position 6 (S, T, C, A, P, V, I, L, M). In addition, allele-specific secondary effects and secondary anchors were defined, and these parameters were utilized to derive allele-specific motifs and algorithms. By the combined use of such algorithms, peptides capable of degenerate DRB1*0101, DRB1*0401, and DRB1*0701 binding were identified. Additional experiments utilizing a panel of quantitative assays specific for nine additional common DR molecules identified a large set of DR molecules, which includes at least the DRB1*0101, DRB1*0401, DRB1*0701, DRB5*0101, DRB1*1501, DRB1*0901, and DRB1*1302 allelic products, characterized by overlapping peptide-binding repertoires. These results have implications for understanding the molecular interactions involved in peptide-DR binding, as well as the genetic and structural basis of MHC polymorphism. These results also have potential practical implications for the development of epitope-based prophylactic and therapeutic vaccines.