Murine class II (Ia) molecules associate with human invariant chain

Polymorphic class II (Ia) major histocompatibility complex (MHC) gene products associate intracytoplasmically with a third nonpolymorphic class II molecule, the invariant chain (Ii), which is encoded by gene(s) unlinked to the MHC. Although the role of the Ii chain in the expression of cell surface Ia molecules is unclear, it has been suggested that the Ii chain helps in the assembly and intracellular transport of class II antigens. In this study, we demonstrate that the murine polymorphic class II antigens of an interspecies mouse-human hybrid, which has segregated the murine invariant chain gene, associates with the human invariant chain gene intracytoplasmically. The murine Ia antigens are expressed on the cell surface and can function as restriction elements in antigen presentation to T cells. The biochemical analysis demonstrates that the regions of the Ii gene that are critical to its interaction with Ia molecules are conserved between species.     

Fluorogenic probes for monitoring peptide binding to class II MHC proteins in living cells

A crucial step in the immune response is the binding of antigenic peptides to major histocompatibility complex (MHC) proteins. Class II MHC proteins present their bound peptides to CD4(+) T cells, thereby helping to activate both the humoral and the cellular arms of the adaptive immune response. Peptide loading onto class II MHC proteins is regulated temporally, spatially and developmentally in antigen-presenting cells. To help visualize these processes, we have developed a series of novel fluorogenic probes that incorporate the environment-sensitive amino acid analogs 6-N,N-dimethylamino-2-3-naphthalimidoalanine and 4-N,N-dimethylaminophthalimidoalanine. Upon binding to class II MHC proteins these fluorophores show large changes in emission spectra, quantum yield and fluorescence lifetime. Peptides incorporating these fluorophores bind specifically to class II MHC proteins on antigen-presenting cells and can be used to follow peptide binding in vivo. Using these probes we have tracked a developmentally regulated cell-surface peptide-binding activity in primary human monocyte-derived dendritic cells.     

Differences in DNA sequence specificity among MHC class II X box binding proteins

The class II (Ia) MHC Ag are integral membrane proteins whose expression is limited to specific cell types. A pair of consensus sequences, X and Y, is found upstream from all class II genes and deletion of each of these sequences eliminates expression of transfected genes. Cells that express Ia demonstrate a coordinate response to lymphokines and other stimuli. These conserved sequences might, therefore, play a role in tissue specificity or lymphokine inducibility of Ia gene expression. The X box sequence of the murine class II A alpha gene diverges much more substantially from the X consensus than does the Y box motif of this gene. We demonstrate that this X box motif is nonetheless recognized by sequence-specific DNA-binding proteins, as is the more closely conserved Y box. Gel retardation assays and DNase I footprints were compared for a panel of Ia+ and Ia- cells as well as for cells stimulated with the Ia-inducing lymphokines IL-4 and IFN-gamma. The level, retardation pattern and region of DNA contact were comparable in all instances. Thus the availability of active DNA-binding X and Y box factors cannot alone account for the regulation of A alpha expression. To test whether the same set of proteins binds all class II MHC conserved motifs, oligonucleotide probe binding and cross-competition experiments with X box sequences from A alpha, E alpha, and E beta genes were performed. These studies demonstrated A alpha, E alpha, and E beta DNA-protein complexes with unique mobilities and specificities. In addition, all three X box oligonucleotide probes generated one faint complex with an affinity profile of E beta greater than E alpha much greater than A alpha. These three complexes comigrated and thus may represent a communal binding protein. The data are most consistent with the conclusion that multiple proteins bind class II MHC X boxes. For A alpha, the predominant complexes represent different specificities from the predominant E alpha and E beta X box binding proteins.     

The major histocompatibility complex of the rat,RT 1

Recombinational analysis has shown that the rat MHC,RT1 is divided into two regions:RT1.A, which codes for class I (transplantation) antigens, andRT1.B, which controls the humoral immune response and proliferative response to allogeneic cells as well as the expression of class II (Ia) antigens. Serological and sequence studies suggest that there might be more than one antigen-coding locus within theRT1.A region. Results obtained by sequential immunoprecipitation reveal that both regions code for at least two gene products. By implication, theRT1 complex must therefore harbor at least four loci;RT1.A andD coding for class I glycoproteins (45,000 daltons); andRT1.B andE coding for class II (Ia) glycoproteins (35,000 and 28,000 daltons).     

Regulation of class II gene expression in a murine pre-B cell line

Class II major histocompatibility complex (MHC) gene expression has been studied in an Abelson virus-transformed pre-B cell line R8, and its Ia-negative variant R8205. These variant cells contained barely detectable levels of RNA specific for all class II genes, including the nonpolymorphic invariant chain gene (Ii), and did not express cell surface Ia. Fusion of this murine Ia-negative cell line to the human Ia-positive Raji cell produced an interspecies hybridoma that expressed the murine Ia. These data are further evidence for the existence of trans-acting factors that can regulate class II gene expression. Furthermore, the T cell-derived lymphokine B cell stimulatory factor 1 (BSF-1) induced expression of class II genes in the R8205 cells. Exposure of R8205 cells to an antibody that has been shown to mimic BSF-1 activity on normal B cells also resulted in expression of class II genes. These data demonstrate that three distinct signals--a lymphokine, an alloantibody binding to membrane structures, and an interspecies trans-acting factor--can induce expression of class II genes.     

Congenital immunodeficiency with a regulatory defect in MHC class II gene expression lacks a specific HLA-DR promoter binding protein, RF-X

The expression of MHC class II genes is tightly regulated. One form of congenital severe combined immunodeficiency (SCID) is characterized by a regulatory defect that precludes expression of HLA class II genes. B lymphocyte cell lines from such SCID patients provide a tool for identifying putative regulatory proteins that bind to class II gene promoters. We have identified three proteins binding to specific segments of the HLA-DRA promoter, two of which interact to form the predominant DNA-protein complex observed. One of these proteins, defined as an X box binding protein (RF-X), is specifically missing in cells from class II deficient SCID patients. We propose that the molecular defect in this congenital HLA class II regulatory deficiency is a lack of RF-X and that this factor plays an important role in the normal regulation of MHC class II gene expression.     

Interferon-gamma induces enhanced expression of Ia and H-2 antigens on B lymphoid, macrophage, and myeloid cell lines

The levels of class II major histocompatibility complex (MHC) antigens (la antigens) on cells of a cultured B lymphoma line (WEHI-279) were significantly increased after 24 hr incubation with medium conditioned by concanavalin A-stimulated mouse or rat spleen cells, or by an azobenzenearsonate- (ABA) specific T cell clone that had been stimulated with ABA-coupled spleen cells or concanavalin A. The levels and properties of the la-inducing activity correlated with those of interferon-gamma (IFN-gamma) measured by inhibition of virus plaque formation. Both the la-inducing activity and the IFN-gamma from the T cell clone had an apparent m.w. of 40,000 determined by gel filtration, were sensitive to treatment with trypsin or exposure to pH 2, but were stable to heat (56 degrees C, 1 hr). The induction of la antigens on WEHI-279 cells was dose-dependent, and the maximum response occurred at a concentration corresponding to 1 to 2 U/ml of antiviral activity. This T cell-derived IFN-gamma-like molecule also increased the expression of cell surface la antigens on another B cell line (WEHI-231), and cell lines of macrophage (J774) and myeloid (WEHI-3B and WEHI-265) origin. Furthermore, in all cases the levels of class I MHC (H-2K or H-2D) antigens were also increased. Similar patterns of induction of Ia and H-2 antigens were obtained with supernatants containing IFN-gamma produced by a monkey cell line (COS) that had been transfected with a plasmid bearing the cloned murine IFN-gamma gene. This activity was sensitive to pH 2 and was not present in the supernatant from COS cells that were not transfected with the murine IFN-gamma gene. These results established that IFN-gamma is the T cell-derived molecule that induces the enhanced expression of Ia and H-2 antigens on B cells and macrophages. A major physiologic role of IFN-gamma may be to regulate immune function through the enhanced expression of MHC antigens.     

Two-dimensional gel analysis of swine histocompatibility antigens

Miniature swine MHC antigens from three inbred herds were examined by two-dimensional gel electrophoresis. These antigens were found to constitute a series of complex glycoproteins displaying haplotype-specific patterns that allowed the distinction of both class I and class II molecules among the three haplotypes. Selected outbred pig antisera reacted with a subset of class I antigens, suggesting the presence of at least two distinct molecular species among these antigens. Similarly, alloantisera reacting with mouse Ia antigens and a monoclonal anti-human DR were shown to immunoprecipitate a subset of class II molecules. Examination of the cells from two recombinant haplotypes demonstrated that both independent recombinational events took place between the class I and class II genes.     

A lipopolysaccharide-induced DNA-binding protein for a class II gene in B cells is distinct from NF-kappa B.

Class II (Ia) major histocompatibility complex molecules are cell surface proteins normally expressed by a limited subset of cells of the immune system. These molecules regulate the activation of T cells and are required for the presentation of antigens and the initiation of immune responses. The expression of Ia in B cells is determined by both the developmental stage of the B cell and by certain external stimuli. It has been demonstrated previously that treatment of B cells with lipopolysaccharide (LPS) results in increased surface expression of Ia protein. However, we have confirmed that LPS treatment results in a significant decrease in mRNA encoding the Ia proteins which persists for at least 18 h. Within the upstream regulatory region of A alpha k, an NF-kappa B-like binding site is present. We have identified an LPS-induced DNA-binding protein in extracts from athymic mice whose spleens consist predominantly of B cells. Binding activity is present in low levels in unstimulated spleen cells and is increased by LPS treatment. This protein binds to two sites in a regulatory region of the Ia A alpha k gene, one of which contains the NF-kappa B-like binding site. DNA fragments containing these sites cross-compete for protein binding. Analysis by DNase I footprinting identified a target binding sequence, named the LPS-responsive element. Although this target sequence contains an NF-kappa B-like binding site, competition with a mutant oligonucleotide demonstrated that bases critical for NF-kappa B binding are not required for binding of the LPS-inducible protein. Therefore, we hypothesized that this inducible protein represents a new mediator of LPS action, distinct from NF-kappa B, and may be one mechanism to account for the decrease in mRNA encoding the Ia proteins.     

Immunoenzyme analysis of the change in the expression of class II antigens of the major histocompatibility complex under the influence of interferon preparations

The interferons (IFN's) are natural regulators of the immune response. This quality of IFN depends to a large extent on their capacity to change the expression of the antigens of the second (II) class of the Major Histocompatibility Complex. The most effective modulator of the expression of the antigens of the class II MHC is beta-IFN. The aim of this work is to explore with the aid of enzyme-linked immunosorbent assay (ELISA) peculiarities of an action of the preparations alpha- and beta-IFN's of a man on the expression of the antigens of the second class on macrophages of mice with using monoclonal antibodies to the products of subregions I-A and I-E MHC. As a result of exploration we have the following: both preparations IFN's (leukinterferon and beta-IFN) reinforce the expression of the antigens of the second class on the macrophages of mice spleens. With combined influence preparation alpha-IFN and beta-IFN show interweakened effect on the expression of the antigens of the class MHC. Exposed regularities of the expression have the same type for products of subregions I-A and I-E H-2 of mice complex. ELISA is the sensible method of determination of a level of the expression of the antigens of the class II MHC on the cell's populations.