This project works in part to support laboratory wide efforts on use of the rabbit as a model for infection with human retroviruses and, in addition, original investigations on the MHC products are being conducted. Current efforts involve definition of the diversity of the rabbit class II DQ alpha genes by sequence analysis. Studies to define the regions of diversity and to determine whether all haplotypes contain equal numbers of DQA genes are underway. Other efforts concern the nonpolymorphic class II gene DOB. While a role has been assigned to the other nonpolymorphic antigen HLA-DM in promoting peptide binding to class II molecules in endocytic compartments the function of HLA-DO remains uncertain. As a first step towards an analysis of HLA-DO function, a polyclonal antiserum was raised against the HLA-DOb chain expressed in transfected cells. A DR/DO chimeric b chain was expressed as a heterodimer with the DRa chain and could be detected at the cell surface of transfected mouse fibroblasts using a mAb specific for the DRa chain. The mouse polyclonal antiserum raised against the chimeric DR/DO heterodimer reacted preferentially with HLA-DO and could be used to detect HLA-DO molecules after biosynthetic labeling of B cell lines. However, in contrast to the DR/DO chimeric molecule expressed on transfected fibroblasts, HLA-DO molecules were not detectable at the surface of B cell lines by either flow cytometry or surface iodination. Subcellular fractionation and radioiodination were used to show that HLA-DO was present in late endocytic compartments of B cell lines. Specificity of HLA-DO detection was confirmed by use of mutant B cell lines lacking HLA-DR or HLA-DO, respectively. The MHC defined rabbit colony is being aggressively bred to maintain known haplotypes, especially those that are rare. Information on the haplotypes of the progenitors of the are being verified by RFLP analysis. Continued attempts to inbreed progeny of known haplotypes have been unsuccessful. Therefore, the male progeny are being outbred to a group of ten successfully breeding females. Thirty pups have been produced to date.
