Beta2-microglobulin required for cell surface expression of blastocyst MHC

Blastocyst MHC is a mouse MHC class Ib gene that is selectively expressed in blastocysts and placenta like human HLA-G, which protect fetal trophoblasts and some tumor cells from NK cell attack, and in TAP-dependent expression on the cell surface. We expressed blastocyst MHC cDNA in beta2-deficient EL-4 S3 or beta2m-transfected EL-4 S3 cells. In parental EL-4 S3 cells, only 47-kDa blastocyst MHC protein was expressed and retained in the cytoplasm. However, additional 51-kDa blastocyst MHC protein was expressed on the surface of beta2m-transfected EL-4 S3 cells. The 51-kDa protein was resistant to Endo-H, whereas the 47-kDa protein was sensitive for Endo-H. The results suggested that beta2m as well as TAP was necessary for the transportation of blastocyst MHC from endoplasmic reticulum to cell surfaces through the Golgi apparatus, similar to other MHC class I molecules.     

Immunocytochemical localization of S-100 beta beta protein in olfactory and supporting cells of lamb olfactory epithelium

By immunocytochemistry, we have identified two novel cell types, olfactory and supporting cells of lamb olfactory epithelium, expressing S-100 beta beta protein. S-100 immune reaction product was observed on ciliary and plasma membranes, on axonemes and in the cytoplasm adjacent to plasma membranes and to basal bodies of olfactory vesicles. A brief treatment of olfactory mucosae with Triton X-100 before fixation is necessary for detection of S-100 beta beta protein within olfactory vesicles. In the absence of such a treatment, the immune reaction product is restricted to ciliary and plasma membranes. On the other hand, irrespective of pre-treatment of olfactory mucosae, S-100 beta immune reaction product in supporting cells is restricted to microvillar and plasma membranes. The anti-S-100 beta antiserum used in these studies does not bind to basal cells of the olfactory epithelium or to cells of the olfactory glands, whereas it binds to Schwann cells of the olfactory nerve. An anti-S-100 alpha antiserum does not bind to cellular elements of the olfactory mucosa, Schwann cells, or axons of the olfactory nerve. The present data provide, for the first time, evidence for the presence of S-100 beta beta protein in mammalian neurons (olfactory cells).     

Possible role of Ab b gene in mouse resistance to EL4 metastases

S (survivor) mutants were produced in mice for genetic analysis of host resistance to metastatic cancer. S-mutants S-27 and S-31 resist transplantation of lymphoma EL4 of parental C57BL/6J (B6) mice while they accept parental skin grafts. Mutant S-27 also resists formation of spontaneous metastases from intradermally growing EL4 tumor into lymp nodes; mutant S-31 is highly susceptible to EL4 metastases. Another mutant. H-2 ^bm26 (bm26), resists EL4 and rejects B6 skin grafts. Major histocompatibility complex (MHC) class I and class II gene expression was compared in these mutants and normal B6 mice. All three mutants tested, S-27, S-31, and bm26, expressed a low amount of K ^b mRNA in organspecific fashion. Mutant bm26 and S-31 expressed a low amount of Ab ^b mRNA and of A^b antigen on their spleen cells. Some oligonucleotide probes designed to hybridize to the second exon of the clss II MHC gene Ab ^b did not hybridize with DNA from all three mutants. These findings suggest extensive sequence alternations in the Ab ^b gene in mutants S-27, S-31, and bm26; they also suggest a major role of MHC in the control of host resistance to spontaneous metastases of the EL4 tumor. Address correspondence and offprint request to: I. K.. Egorov.     

Genetic variability of the major histocompatibility complex class I homologue encoded by human cytomegalovirus leads to differential binding to the inhibitory receptor ILT2

Human cytomegalovirus carries a gene, UL18, that is homologous to cellular major histocompatibility complex (MHC) class I genes. Like MHC class I molecules, the protein product of the UL18 gene associates with beta2-microglobulin, and the stability of this complex depends on peptide loading. UL18 protein binds to ILT2 (CD85j), an inhibitory receptor present on B cells, monocytes, dendritic cells, T cells, and NK cells that also recognizes classical and nonclassical MHC molecules. These observations suggest that UL18 may play a role in viral immune evasion, but its real function is unclear. Since this molecule has similarity with polymorphic MHC proteins, we explored whether the UL18 gene varied between virus isolates. We report here that the UL18 gene varies significantly between virus isolates: amino acid substitutions were found in the predicted alpha1, alpha2, and alpha3 domains of the UL18 protein molecule. We also studied the ability of several variant UL18 proteins to bind to the ILT2 receptor. All of the variants tested bound to ILT2, but there were marked differences in the affinity of binding to this receptor. These differences were reflected in functional assays measuring inhibition of the cytotoxic capacity of NK cells via interaction with ILT2. In addition, the variants did not bind other members of the CD85 family. The implications of these data are discussed.     

Characterization of a divergent non-classical MHC class I gene in sharks

Sharks are the most ancient group of vertebrates known to possess members of the major histocompatibility complex (MHC) gene family. For this reason, sharks provide a unique opportunity to gain insight into the evolution of the vertebrate immune system through comparative analysis. Two genes encoding proteins related to the MHC class I gene family were isolated from splenic cDNA derived from spiny dogfish shark ( Squalus acanthias). The genes have been designated MhcSqac-UAA*01 and MhcSqac-UAA*NC1. Comparative analysis demonstrates that the Sqac-UAA*01 protein sequence clusters with classical MHC class I of several shark species and has structural elements common to most classical MHC class I molecules. In contrast, Sqac-UAA*NC1 is highly divergent from all vertebrate classical MHC class I proteins, including the Sqac-UAA *01 sequence and those of other shark species. Although Sqac-UAA*NC1 is clearly related to the MHC class I gene family, no orthologous genes from other species were identified due to the high degree of sequence divergence. In fact, the Sqac NC1 protein sequence is the most divergent MHC class-I-like protein identified thus far in any shark species. This high degree of divergence is similar in magnitude to some of the MHC class-I-related genes found in mammals, such as MICA or CD1. These data support the existence of a class of highly divergent non-classical MHC class I genes in the most primitive vertebrates known to possess homologues of the MHC and other components of the adaptive immune system.     

Autocrine induction of major histocompatibility complex class I antigen expression results from induction of beta interferon in oncogene-transformed BALB/c-3T3 cells.

By varying growth conditions, we identified a novel mechanism of autocrine regulation of major histocompatibility complex (MHC) class I gene expression by induction of beta interferon gene expression in transformed BALB/c-3T3 cells. Low-serum conditions enhanced MHC class I antigen expression in v-rasKi- and v-mos-transformed BALB/c-3T3 cells but not in untransformed BALB/c-3T3 cells. Transformed and untransformed cells grown under standard serum conditions (10% bovine calf serum) expressed similar cell surface levels of MHC class I antigens. However, low-serum conditions (0.5% bovine calf serum) induced four- to ninefold increases in cell surface levels of MHC class I antigens in both v-rasKi- and v-mos-transformed cells but not in untransformed cells. These increases in MHC class I gene expression were seen at both the mRNA and cell surface protein levels and involved not only the heavy-chain component of the class I antigens but also beta 2 microglobulin. Beta 1 interferon mRNA and beta interferon-inducible 2',5'-oligoadenylate synthetase mRNA were induced by growth under low-serum conditions in transformed BALB/c-3T3 cells, and antibodies to beta interferon blocked the induction of MHC class I antigen expression by serum deprivation in these cells. These results demonstrate that growth under low-serum conditions leads to induction of beta interferon expression in oncogene-transformed cells which then directly mediates autocrine enhancement of MHC class I gene expression.     

Edmonston measles virus prevents increased cell surface expression of peptide-loaded major histocompatibility complex class II proteins in human peripheral monocytes

Gamma interferon (IFN-gamma) induces expression of the gene products of the major histocompatibility complex (MHC), whereas IFN-alpha/beta can interfere with or suppress class II protein expression. In separate studies, measles virus (MV) was reported to induce IFN-alpha/beta and to up-regulate MHC class II proteins. In an attempt to resolve this paradox, we examined the surface expression of MHC class I and class II proteins in MV-infected peripheral monocytes in the presence and absence of IFN-alpha/beta. Infection of purified monocytes with Edmonston B MV resulted in an apparent increase in cell surface expression of HLA-A, -B, and -C class I proteins, but it had no effect on the expression of HLA-DR class II proteins. MV-infected purified monocytes expressed IFN-alpha/beta, but no measurable IFN-gamma expression was detected in supernatant fluids. Class II protein expression could be enhanced by coculture of purified monocytes with uninfected peripheral blood mononuclear cell (PBMC) supernatant. MV infection of PBMCs also did not affect expression of class II proteins, but the expression of HLA-A, -B, and -C class I proteins was increased two- to threefold in most donor cells. A direct role for IFN-alpha/beta suppression of MHC class II protein expression was not evident in monocytes since MV suppressed class II protein expression in the absence of IFN-alpha/beta. Taken together, these data suggest that MV interferes with the expression of peptide-loaded class II complexes, an effect that may potentially alter CD4(+)-T-cell proliferation and the cell-mediated immune responses that they help to regulate.     

Opisthorchis viverrini antigens up-regulates the expression of CD80 and MHC class II in JAWSII mouse dendritic cells and promotes IL-10 and TGF-β secretions

Dendritic cells (DCs) are antigen-presenting cells (APC) involved in the initiation of immune responses. Maturation of DCs is characterized by the high expression of major histocompatibility complex (MHC) class II and co-stimulatory clusters of differentiation (CD) 40, CD80, and CD86 molecules. Matured DCs are required for T cell differentiation and proliferation. However, the response of DCs to Opisthorchis viverrini antigens has not yet been understood. Therefore, this study sought to determine the expression of surface molecules of JAWSII mouse DCs stimulated by crude somatic (CS) and excretory-secretory (ES) antigens of O. viverrini. ES antigen significantly induced only mRNA expression of CD80 and MHC class II in JAWSII mouse DCs, while CS antigen promoted up-regulation of both mRNA and protein levels of CD80 and MHC class II, indicating relative maturation of JAWII mouse DCs. Moreover, the secreted cytokines from the co-cultures of O. viverrini antigens stimulated JAWSII DC with naïve CD4⁺ T cells was determined. Significantly increased levels of immunosuppressive cytokines interleukin (IL)-10 and transforming growth factor beta (TGF-β) were found. The up-regulation of these cytokines may indicate the response of regulatory T cells (Treg) to CS antigen-stimulated JAWSII DC. These findings may lead to a better understanding of the role that DCs play in O. viverrini infection.     

Increased expression and altered subunit composition of proteasomes induced by continuous proteasome inhibition establish apoptosis resistance and hyperproliferation of Burkitt lymphoma cells

The proteasome is the main protease for extralysosomal protein degradation in eukaryotic cells, and constitutes a sophisticated high molecular mass proteinase complex underlying a tightly coordinated expression and assembly of multiple subunits and subcomplexes. Here we show that continuous inhibition of proteasomal chymotrypsin-like peptidase activity by the proteasome inhibitor bortezomib induces in human Namalwa Burkitt lymphoma cells increased de novo biogenesis of proteasomes accompanied by increased expression of the proteasome maturation protein POMP, increased expression of 19S-20S-19S proteasomes, and abrogation of expression of beta 1i, beta 2i and beta 5i immunosubunits and PA28 in favor of increased expression of constitutive proteolytic beta1, beta2 and beta 5 subunits and 19S regulatory complexes. These alterations of proteasome expression and subunit composition are accompanied by an increase in proteasomal caspase-like, trypsin-like and chymotrypsin-like peptidase activities, not inhibitable by high doses of bortezomib. Cells harboring these proteasomal alterations display rapid proliferation and cell cycle progression, and acquire resistance to apoptosis induced by proteasome inhibitors, gamma-irradiation and staurosporine. This acquired apoptosis resistance is accompanied by de novo expression of anti-apoptotic Hsp27 protein and the loss of ability to accumulate and stabilize pro-apoptotic p53 protein. Thus, increased expression, altered subunit composition and increased activity of proteasomes constitute a hitherto unknown adaptive and autoregulatory feedback mechanism to allow cells to survive the lethal challenge of proteasome inhibition and to establish a hyperproliferative and apoptosis-resistant phenotype.     

Recognition of classical and heavy chain forms of HLA-B27 by leukocyte receptors

As an MHC class I protein, the disease association of HLA-B27 with inflammatory arthritis has been widely assumed to imply a role for the T cell antigen receptor (TCR) in disease. However, in addition to their classical antigen-presenting role, HLA class I proteins are recognised by members of the killer immunoglobulin receptor (KIR) and leukocyte immunoglobulin-like receptor (LILR/ILT/LIR) families. Unusual properties of HLA-B27 include an ability of free heavy chains (FHC) to reach the cell surface in the absence of beta2m and to maintain their peptide-binding groove in vitro. This review describes immunomodulatory receptors that recognise HLA class I, and the recognition of HLA-B27 in both the classical beta2m-associated and beta2m-independent forms by members of the KIR and LILR families. Alternative recognition of different forms of HLA-B27 by leukocyte receptors could influence the function of cells from both innate and adaptive immune systems, and may indicate a role for various leukocyte populations in HLA-B27-associated inflammatory disease.     

Characterization and mapping of the human SOX4 gene

The SOX genes comprise a large family related by homology to the HMG-box region of the testis-determining gene SRY. We have cloned and sequenced the human SOX4 gene. The open reading frame encodes a 474 amino acid protein, which includes an HMG-box. The non-box sequence is particularly rich in serine residues and has several polyglycine and polyalanine stretches. With somatic cell hybrids, human SOX4 has been mapped to Chromosome (Chr) 6p distal to the MHC region. There is no evidence for clustering of other members of the SOX1,-2, and-3 or SOX4 gene families around the SOX4 locus.     

Cell-surface expression and alloantigenic function of a human nonclassical class I molecule (HLA-E) in transgenic mice

We have introduced the gene (E*01033) encoding the heavy chain of the human nonclassical MHC class I Ag, HLA-E, into the mouse genome. Two founder mice carry a 21-kb fragment, the others bear an 8-kb fragment. Each of the founder mice was mated to mice of an already established C57BL/10 transgenic line expressing human beta2-microglobulin (beta2m). Cell surface HLA-E was detected on lymph node cells by flow cytometry only in the presence of endogenous human beta2m. However, HLA-E-reactive mouse CTL (H-2-unrestricted) lysed efficiently the target cells originating from HLA-E transgenic mice without human beta2m, showing that the HLA-E protein can be transported to the cell surface in the absence of human beta2m, presumably by association with murine beta2m. Rejection of skin grafts from HLA-E transgenic mice demonstrates that HLA-E behaves as a transplantation Ag in mice. HLA-E transgenic spleen cells are effective in stimulating an allogeneic CTL response in normal and human classical class I (HLA-B27) transgenic mice. Furthermore, results from split-well analysis indicate that the majority of the primary in vivo-induced CTL recognizes HLA-E as an intact molecule (H-2-unrestricted recognition) and not as an HLA-E-derived peptide presented by a mouse MHC molecule, although a small fraction (ranging from 4 to 21%) of the primary in vivo-induced CTL is able to recognize HLA-E in an H-2-restricted manner. Based on these observations, we conclude that HLA-E exhibits alloantigenic properties that are indistinguishable from classical HLA class I molecules when expressed in transgenic mice.     

Immunohistochemical study on the distribution of alpha and beta subunits of S-100 protein in human neoplasm and normal tissues

The immunohistochemical distribution and localization of the alpha and beta subunits of S-100 protein in human neoplasms and normal tissues were studied by the PAP method using monospecific rabbit antibodies against each subunit. Beta subunit immunoreactivity was detected in all S-100-positive cells and tumors reported previously. In contrast alpha subunit immunoreactivity was absent from Schwann cells, schwannomas, neurofibromas, granular cell myoblastomas, pituicytes of the neurohypophysis, Langerhans cells, interdigitating reticulum cells, and histiocytosis X cells. Interestingly, only the alpha subunit was detected in neurons of both central and peripheral nervous system, and in lymph node macrophages. Human S-100-positive cells are divided into three groups; the first is composed of cells containing only the beta subunit (probably S-100b; beta beta), the second consists of cells containing both the alpha and beta subunits, and the third is composed of cells containing only the alpha subunit (probably S- 100ao ; alpha alpha). The ontogentic relationships between S-100-positive cells and tumors are discussed in the light of these findings.     

High-resolution structure of HLA-A*0201 in complex with a tumour-specific antigenic peptide encoded by the MAGE-A4 gene

The heterotrimeric complex of the human major histocompatibity complex (MHC) molecule HLA-A*0201, beta2-microglobulin and the decameric peptide GVYDGREHTV derived from the melanoma antigen (MAGE-A4 protein has been determined by X-ray crystallography at 1.4 A resolution. MAGE-A4 belongs to a family of genes that are specifically expressed in a variety of tumours. MAGE-A4-derived peptides are presented by MHC molecules at the cell surface to cytotoxic T-lymphocytes. As the HLA-A*0201:MAGE-A4 complex occurs only on tumour cells, it is considered to be an appropriate target for immunotherapy. The structure presented here reveals potential epitopes specific to the complex and indicates which peptide residues could be recognised by T-cell receptors. In addition, as the structure could be refined anisotropically, it was possible to describe the movements of the bound peptide in more detail.