Biomedical subjects
Publications and source records attributed to C Rabourdin-Combe.
Antigen presentation is now a central theme in molecular cell biology. A recent meeting focused on the developments in the field and their implications for future manipulation of the immune system. The following report was written from a round-table discussion involving a number of participants'.
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In order to determine the factors that influence the glycosylation of an integral membrane protein, we investigated the N-glycosylation of a molecule of the human major histocompatibility complex (MHC) class II, the HLA-DR antigen. This glycoprotein was studied in a human Epstein-Barr-virus-transformed B cell line and in a mouse fibroblastic cell line co-transfected with DR alpha and DR beta genes. We observed that the HLA-DR-antigen glycosylation pattern depends on the cell line in which processing takes place and is closely related to the glycosylation pattern of the overall cellular glycoproteins. Furthermore, when comparing the glycosylation of the separated alpha- and beta-chains, differences were noticed within the same molecule, showing the importance of the individual peptide backbone for the glycosylation process.
The hypothesis that self as well as foreign proteins are processed into peptides and presented by major histocompatibility complex antigens leads to a set of working principles that could govern cellular interactions in immune responses. In particular, "idiopeptides," derived from immunoglobulins and T-cell receptors and recognized by appropriate T cells, are expected to play an important regulatory role. We show here that these speculations fit into a consistent view of the immune system.
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Using a mouse cell line transformed with and expressing a single HLA DR-alpha and DR-beta chain gene, we present evidence that the product of the DR-beta chain gene carries a supertypic determinant, BR3, previously defined by serology. The amino acid sequence of this beta chain gene is determined from the DNA sequence. Another DR-associated supertypic specificity defined by monoclonal antibody MCS7 was not encoded by this DR-beta chain gene. This provides formal proof that a supertypic specificity can be associated with a product of a distinct DR-beta locus. We propose that haplotypes sharing such specificities are evolutionarily related.
Different cDNA clones have been isolated that encode each of the three chains of HLA-DR antigens: alpha, intermediate and beta, as well as another beta chain, most likely DC. Whereas the DR alpha and intermediate chains seem encoded by single genes, the DR and DC beta chains are most likely encoded by multiple genes; furthermore, their polymorphism can be readily detected by restriction analysis of cellular DNA. Several genomic DNA clones were isolated for the DR and DC beta chain genes and for the intermediate chain gene. The sum of all distinct cDNA clones and genomic DNA clones for HLA-DR beta chains, isolated from a heterozygous cell line, represent five genes. This implies the existence of at least three nonallelic DR beta chain genes in addition to the DC beta chain genes. The complete sequence of one of the DR beta chains is presented. A genomic DNA clone for a DR beta chain was transferred into mouse L cells and found to be expressed into RNA of the same size as DR beta mRNA. The finding, among the genes for class II antigens, of multiple genes for the beta chain of HLA-DR, distinct from those of other known subregions such as DC, emphasizes the importance of gene transfer experiments, where individual genes can be expressed and tested for their functional role in the immune response.
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The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate prevents the synthesis of antibodies directed against sheep red blood cells in an in vitro system. The inhibitory effect of 3 phorbol diesters on this immune response was positively correlated with their tumor-promoting activity. The effect did not appear to be mediated through the inhibition of cell proliferation. Results suggest that the tumor promoter may alter the differentiation of the precursor cells to antibody-producing cells.
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An immunoglobulin binding factor (IBF) produced by in vivo alloantigen-activated T cells suppresses in vitro antibody production to sheep red blood cells (SRBC) and probably represents the soluble form of the T cell Fc receptor. To study the relationship between IBF and the major histocompatibility complex (MHC), two types of experiments were undertaken. First, different IBF were prepared by injecting thymocytes from mice of H-2k, H-2b and H-2d haplotypes into irradiated, allogeneic recipient mice. Cells recovered in the spleens five days later were incubated for 2 h at 37 degrees C, and IBF was purified by affinity chromatography on insolubilized IgG. Purified IBF was then applied to immunosorbents prepared with anti-H-2 and anti-Ia anti sera. Acid eluates were then tested for their suppressive activity on in vitro antibody production to SRBC. Under these conditions, IBF was found to react with antisera raised against products of the whole MHC and of the I region of the MHC, but not with anti-H-2 D antisera. These data indicate that IBF bears, or is associated with Ia determinants. Second, IBF preparations from mice of H-2k, H-2b, H-2d haplotypes were tested for their capacity to suppress the in vitro antibody production to SRBC by spleen cells of a series of mouse strains. The data indicate that IBF is not genetically restricted in its function.
While the induction of antibody synthesis depends on antigen specific T cell factors, its magnitude is under control of non antigen specific T cell factors. In this respect, TRF ("T cell Replacing Factor") amplifies antibody responses while IBF ("Immunoglobulin Binding Factor") acts as a suppressor factor. Using cultures of spleen cells from nude mice, we show that both factors act sequentially, influencing the final differenciation of B cells to antibody producing cells. We have no evidence of direct interaction between TRF and IBF.
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The biochemical characteristics of immunoglobulin binding factor produced by activated cells (ATC) were investigated. For this purpose, supernatants of ATC were purified by affinity chromatography on insolubilized IgG and the eluted material was iodinated (125I), treated with mercaptoethanol; and run on SDS polyacrylamide gels. The radioactivity was found in two peaks corresponding to m.w. of 38,000 d and 18,000 d. This result extends and confirms our previous findings that IBF produced by ATC is identical to IBF produced by L-5178-Y internally labeled thymoma cells. The effect of various pH, temperatures, and proteolytic and glycolytic enzymes on the binding properties of 3H-leucine-or H-fucose-labeled IBF to IgG and on the polyacrylamide gel profiles was also studied. By all these criteria, IBF appeared to be a glycoprotein in which the presence of the 38,000 to 40,000 d chain is necessary for the binding to IgG. In the attempt to study the relationships between IBF and I-region products, purified IBF produced by ATC was incubated with anti-Ia immunoadsorbent, and the eluted material was iodinated and run on gels. The 38,000 d and 18,000 d chains characteristic of IBF were found to be specifically retained on the relevant immunoadsorbent. These data favor the hypothesis that IBF bears or is associated with Ia determinants.
The H-2 I region in the mouse and the HLA-D region in humans contain a set of polymorphic genes which encode Ia antigens and control the immune response. Cloned genes for the different polypeptide chains of one of the human Ia antigens, HLA-DR, have been used for the co-transformation of mouse L cells. Expression of HLA-DR antigens at the surface of transfected mouse cells has been documented with monoclonal antibodies.
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