RT1-U: identification of a novel, active, class Ib alloantigen of the rat MHC.

In common with other mammalian species, the laboratory rat (Rattus norvegicus) expresses MHC class I molecules that have been categorized as either classical (class Ia) or nonclassical (class Ib). This distinction separates the class Ia molecules that play a conventional role in peptide Ag presentation to CD8 T cells from the others, whose function is unconventional or undefined. The class Ia molecules are encoded by the RT1-A region of the rat MHC, while the RT1-C/E/M region encodes up to 60 other class I genes or gene fragments, a number of which are known to be expressed (or to be expressible). Here we report upon novel MHC class Ib genes of the rat that we have expression cloned using new monoclonal alloantibodies and which we term RT1-U. The products detected by these Abs were readily identifiable by two-dimensional analysis of immunoprecipitates and were shown to be distinct from the class Ia products. Cellular studies of these molecules indicate that they function efficiently as targets for cytotoxic killing by appropriately raised polyclonal alloreactive CTL populations. The sequences of these class Ib genes group together in phylogenetic analysis, suggesting a unique locus or family. The combined serological, CTL, and sequence data all indicate that these products are genetically polymorphic.

Monoclonal antibodies raised against semi-purified preparations of human islets define subpopulations of pancreatic cells.

Monoclonal antibodies were produced from fusions between splenocytes from BALB/c mice immunised with purified human islets and the mouse myeloma cell line NS-0/Uncl. Supernatants from uncloned hybrids were screened by immunohistology on frozen sections of human pancreas. The range of specificities appeared to reflect the relative purity of the human islet preparations used. Eight monoclonal antibodies were investigated further, four of these bound to islet cells, two to acinar cells, one to ductal cells and one to occasional cells. The antigens recognised by these antibodies were characterised by immunohistology using a number of different tissues, as well as haemagglutination, immunoblotting and radioimmunoassay for insulin. Seven of the eight antibodies studied were IgM. One acinar cell antibody (IgG2a) precipitated proteins of 200Kd and 11OKd molecular weight. None of the antibodies bound directly to insulin. Seven of the antibodies appear to have defined previously unreported epitopes in the pancreas and will prove useful in further studies of human pancreatic cells.

Isolation of monoclonal antibodies reacting with peribacteriod membranes and other components of pea root nodules containing Rhizobium leguminosarum.

Plant and bacterial antigens contributing to nodule development and symbiosis in pea (Pisum sativum L.) roots were identified after isolation of a set of monoclonal antibody (McAb)-producing hybridoma lines. Rats were immunised with the peribacteriod material released by mild osmotic shock treatment from membrane-enclosed bacteroids of Rhizobium leguminosarum bv. viceae. In order to diversify the range of McAb specificities, this material was either used as immunogen directly (method 1), or after immunodepletion of a set of glycoprotein and lipopolysaccharide antigens (method 2), or after deglycosylation (method 3). After fusion and screening of cloned hybridoma lines, these three immunisation methods gave respectively 4, 2 and 1 classes of McAb with unique antigen specificities. Ultrastructural immunogold localisation studies showed four different antigens to be present on peribacteriod and plasma membranes (identified by MAC 64, 202, 206 or 209); in addition, a glycoprotein of plant origin but present in the infection-thread matrix was identified by MAC 204. Although none of the epitopes recognised by these McAb was nodule-specific, several were found to be more abundant in extracts of nodule tissue than in uninfected roots (MAC 64, 202, 204, 206). Two McAb reacted with new bacterial antigens: MAC 203 identified a bacterial antigen expressed upon infection but not in free-living cultures of Rhizobium, and MAC 115 identified a bacterial polypeptide (55 kdaltons) that was present in both free-living and bacteroid forms. There were also some McAb of broader specificity that react with antigens present in both plant and bacterial cytoplasms.

In vivo prevention of thyroid and pancreatic autoimmunity in the BB rat by antibody to class II major histocompatibility complex gene products.

Evidence is accumulating that the development of insulin-dependent diabetes mellitus involves autoimmune phenomena, both in the human and in the BB rat model. A strong association is observed in both cases with alleles of the class II major histocompatibility complex (MHC). Results of the present study show that autoimmune phenomena, as assessed by the presence of clinical diabetes or histological thyroiditis, are prevented by the injection of monoclonal antibodies to class II gene products in the BB rat. Immunosuppression was specifically obtained with a monoclonal antibody to the murine I-E equivalent, as opposed to the murine I-A equivalent, of the rat major histocompatibility complex. This represents indirect evidence for I-E subregion control of immune responses to islet cell and thyroid antigens in the BB rat model. The frequent occurrence of anaphylactic type deaths in young (1 month old) animals receiving more than six weekly injections of partially purified homologous (rat) monoclonal antibodies to rat class II gene products underscores the potential risks of this type of immunotherapy. The presumed immunologic mechanism (IgE antibody) and its specificity (anti-allotype, anti-idiotype, or anti-impurity) must be clarified to assess the risks and feasibility of this type of therapy.

Monoclonal antibodies to antigens in the peribacteroid membrane from Rhizobium-induced root nodules of pea cross-react with plasma membranes and Golgi bodies.

Three rat hybridoma lines that produced monoclonal antibodies reacting with the peribacteroid membrane from Pisum sativum were isolated, and these all appeared to recognize the same antigenic structure. Using one of these monoclonal antibodies, AFRC MAC 64, electron microscopy of immunogold-stained thin sections of nodule tissue revealed that the antigen, present in the peribacteroid membrane, was also found in the plant plasma membranes and in the Golgi bodies, but not in the endoplasmic reticulum. When peribacteroid membrane proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose by electro-blotting, it was found that MAC 64 bound to a series of protease-sensitive bands that migrated in the mol. wt. range 50-85 K. The epitope was sensitive to periodate oxidation and its structure may therefore involve the carbohydrate component of a membrane glycoprotein. We suggest that this structure originates in the Golgi apparatus and is subsequently transferred to the peribacteroid membranes and plasma membranes. The monoclonal antibody also reacted with peribacteroid membranes from nodules of Vicia and lupin, and with plasma membranes and Golgi membranes from uninfected plant cells, including root tip cells from onion (Allium cepa), indicating that the antigen is highly conserved in the plasma membranes of plant cells.

The alloantigenic organization of RT1Aa, a class I major histocompatibility complex molecule of the rat.

Over 300 monoclonal IgG alloantibodies have been prepared against RT1Aa , the class I major histocompatibility complex molecule of the DA rat. In this study a combination of techniques is exploited to show that all these antibodies can be allocated to 9 antigenic sites which form a continuous antigenic surface, that is, no site is completely isolated from the rest. The results suggest that techniques for the identification of antigenic sites using competitive inhibition of monoclonal antibody binding are generally valid, in the sense that competition between antibodies appears most commonly to represent competition between combining sites for a structural feature of the antigenic surface. From the distribution of antibodies between sites, it is clear that the RT1Aa molecule has three immunogenic areas against which nearly all the antibodies studied were directed. Of these areas one is both antigenically complex, consisting of four closely spaced sites, and remarkably immunodominant. Antibodies directed at sites between the major areas are extremely rare.