The long term goal of this laboratory is to define the molecular sites at which the effector arms of the immune response interact with class I MHC molecules by investigating the structural features of mutants in the H-2Kk molecule. Known mutations occur as two distinct sets. The first set are antibody-selected H-2Kk/d cell line derived mutants: LDHBll-4.1r31TGn, a mutant which is no longer recognized by the anti-H-2Kk monoclonal antibody 11-4.1; and LDHBll-4.1rev3.7.3, a mutant which was previously resistant, but has now regained sensitivity to 11-4.1. This """"""""revertant"""""""" differs from the standard H-2Kk molecule because it has lost reactivity with other anti-H-2Kk monoclonals. The second set is the in vivo mutant, H-2Kkml, which was selected by tissue graft rejection between inbred mice. This mutation is thought to represent a cell-mediated immunoselection. Thus, by defining the sites of mutation in these molecules, sites of interaction for antibodies and cytotoxic T-cells (CTL's) with the H-2Kk antigen will be defined. The specific means for achieving these goals include several methods. First, H-2Kk-like molecules from the LDHB11-4.1r31TGr, LDHBll-4.1rev 3.7.3 and H-2Kkml will be compared to the standard H-2Kk molecule and to one another by means of tryptic peptide mapping. Second, the mutant peptides will be isolated, purified, and sequenced using the techniques of radiochemical microsequencing. Preliminary information from peptide mapping studies of H-2Kkml suggests that microsequence analysis for H-2Kkml can begin shortly. In addition, in order to augment and eventually replace the cumbersome protein methods, recombinant DNA technology is being applied to elucidate the H-2Kkml structure and will be applied to analysis of other mutants as appropriate. A transformed cell line of H-2Kkml/d origin is also being derived in order to select new mutants at H-2Kkml. Preliminary data on one such cell line, however, suggests that the antigen of interest, H-2Kkml, is not expressed, while one other class I antigen, H-2Ld, is expressed. This """"""""expression"""""""" mutant will be further investigated. Finally, a CTL-mediated system for selection of new mutations from an H-2Kkml or H-2Kk cell line will be developed, with alternate plans for antibody-mediated selection. These mutants will be analyzed in the same way as existing mutants. These data will give a comprehensive picture of the sites of interaction between a class I MHC molecule and immune response effectors and may also add to information concerning generation of polymorphism in class I antigens.
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