Using alloantisera prepared by planned immunization in cynomolgus macaques, it was possible to partially define the polymorphism of the major histocompatibility complex (MHC) in Macaca fascicularis, which is designated CyLA. These reagents define more than 40 class I CyLA alloantigens and at least 10 class II alloantigens. Six variants of fact B (Bf), an MHC-linked protein of the alternate complement pathway, can be recognized by isoelectric focusing and immunization. The goal of the proposed study is to extend the CyLA definition to the molecular and genetic level. A series of B-lymphoblastoid cell lines will be established using Epstein-Barr virus to provide a reference cell panel for biochemical analysis of the most commonly occurring class I and class II antigens. Radioisotopically-labelled CyLA antigens will be immunoprecipitated with monomorphic monoclonal HLA antigens known to cross-react with certain CyLA-A, B, C antigens. Neuraminidase-digested samples will be analyzed by one- and two-dimensional isoelectric focusing. This type of analysis will provide independent conformation of proposed serologic subdivisions and may establish the existence of biochemically distinct CyLA forms that are not identifiable by the available serologic reagents. Polymorphism of genes for components of the classical complement pathway (C2, C4A, C4B) will be studied by electrophoretic techniques to further characterize the class III gene products. DNA probes will be developed to study the genetic organization and structure of the CyLA gene family. Complementary DNA (cDNA) from mRNA of class I and class I expressing cells will be used to probe the DNA polymorphism. These DNAs will be sequenced and subsequently used to isolate their counterparts from a genomic DNA library. The genomic DNA library will be probed for additional MHC genes which are not represented in the cDNA library. Cloned genes to CyLA molecules will be transfected into recipient cells for structural and functional analyses beginning with the CyLA-A8.2 gene, whose product is recognized with anti-HLA monoclonal antibodies. The results of this investigation will include knowledge of the basis for and extent of CyLA polymorphism. The reagents and probes will enable investigators to undertake studies of the phylogenetic development of the MHC and can be immediately applied to the development of macaque models of disease and improved colony management.
