Macrophages escape inhibition of major histocompatibility complex class I-dependent antigen presentation by cytomegalovirus

The mouse cytomegalovirus (MCMV) m152- and m06-encoded glycoproteins gp40 and gp48, respectively, independently downregulate major histocompatibility complex (MHC) class I surface expression during the course of productive MCMV infection in fibroblasts. As a result, presentation of an immediate-early protein pp89-derived nonapeptide to H-2L(d)-restricted CD8(+) cytotoxic T cells is completely prevented in fibroblasts. Here we demonstrate that MCMV-infected primary bone marrow macrophages and the macrophage cell line J774 constitutively present pp89 peptides during permissive MCMV infection to cytotoxic T lymphocytes (CTL). In contrast to fibroblasts, expression of the m152 and m06 genes in macrophages does not affect surface expression of MHC class I. Assessment of pp89 synthesis and quantification of extracted peptide revealed a significantly higher efficiency of macrophages than of fibroblasts to process pp89 into finally trimmed peptide. The yield of pp89 peptide determined in MCMV-infected tissues of bone marrow chimeras confirmed that bone marrow-derived cells represent a prime source of pp89 processing in parenchymal organs. The finding that macrophages resist the viral control of MHC I-dependent antigen presentation reconciles the paradox of efficient induction of CMV-specific CD8(+) CTL in vivo despite extensive potential of CMVs to subvert MHC class I.     

Phagocytic processing of antigens for presentation by class II major histocompatibility complex molecules

Microbes and other particulate antigens (Ags) are internalized by phagocytosis and then reside in plasma membrane-derived phagosomes. The contribution of phagosomes to the degradation of Ags has long been appreciated. It has been unclear, however, whether peptides derived from these degraded antigens bind class II major histocompatibility complex (MHC-II) molecules within phagosomes or within endocytic compartments that receive Ag fragments from phagosomes. Recent experiments have demonstrated that phagosomes containing Ag- conjugated latex beads express a full complement of Ag-processing molecules, e.g. MHC-II molecules, invariant chain, H2-DM and proteases sufficient to degrade bead- associated Ag. These phagosomes mediate the formation of peptide–MHC-II complexes, which are transported to the cell surface and presented to T cells. Phagosomes acquire both newly synthesized and plasma membrane-derived MHC-II molecules, but the formation of peptide–MHC-II complexes in phagosomes primarily involves newly synthesized MHC-II molecules. The content and traffic of phagosomal proteins vary considerably with the type of Ag ingested. Pathogenic microbes can alter phagosome composition and function to reduce Ag processing. For example, Mycobacterium tuberculosis blocks the maturation of phagosomes and reduces the ability of infected cells to present exogenous soluble protein Ags.     

Generation of major histocompatibility complex class I antigens

Presentation of antigenic peptides by major histocompatibility complex (MHC) class I molecules on the surface of antigen-presenting cells is an effective extracellular representation of the intracellular antigen content. The intracellular proteasome-dependent proteolytic machinery is required for generating MHC class I-presented peptides. These peptides appear to be derived mainly from newly synthesized defective ribosomal products, ensuring a rapid cytotoxic T lymphocyte-mediated immune response against infectious pathogens. Here we discuss the generation of MHC class I antigens on the basis of the currently understood molecular, biochemical and cellular mechanisms.     

白假丝酵母激活的巨噬细胞自噬促进MHCⅡ抗原提呈

目的 探究白假丝酵母（Candida albicans）激活的Raw264.7细胞自噬在主要组织相容性复合体Ⅱ类分子（MHCⅡ）抗原提呈以及协同刺激分子表达中的作用。方法 C. albicans刺激Dectin-1单克隆抗体封闭或白皮杉醇阻断的Raw264.7细胞,Western blot法检测LC3Ⅱ表达量,RT-PCR检测CD80与CD86的表达。免疫荧光实验观察有无3-MA预处理的GFP-LC3-Raw264.7细胞与C. albicans共孵育后MHCⅡ与LC3在胞浆的分布,ELISA法检测有无封闭Dectin-1或3-MA预处理的Raw264.7细胞与C. albicans共孵育不同时间段后IL-6的分泌。结果 阻断Dectin-1或Sky后C. albicans诱导的LC3Ⅱ表达降低。LC3、MHCⅡ与胞内C. albicans存在显著的共定位关系,阻断自噬后C. albicans与MHCⅡ的共定位明显减弱。C. albicans引发Raw264.7细胞表达CD80与CD86 mRNA,封闭Dectin-1或阻断自噬后二者转录水平降低。C. albicans通过Dectin-1引发Raw264.7细胞分泌IL-6,阻断自噬对IL-6分泌无显著影响。结论 C. albicans通过Dectin-1/Sky通路激活巨噬细胞自噬,自噬体的构建促进MHCⅡ招募至胞内C. albicans,并促进协同刺激分子的表达。     

Biochemical characterization of activation-associated bovine class I major histocompatibility complex antigens

Utilizing a 'sandwich' ELISA assay we have been able to demonstrate that mAb W6/32, B1G6 and IL-A19 are reactive with three different monomorphic determinants on bovine class I major histocompatibility complex (MHC) molecules. Sequential immunoprecipitations performed with the mAb revealed that class I molecules on PBM comprise a single population with respect to reactivity with the mAb in that the beta 2m-associated proteins bear all three epitopes. By contrast, TCGF-driven lymphoblasts and cells transformed by Theileria parva (Tp) additionally express molecules of Mr 45000 bound to beta 2m which are recognized by mAb B1G6 and IL-A19 but not by W6/32. These two subclasses of molecules were further distinguished on the basis that, when tunicamycin was added to cultures in the preparation of cells for analysis, mAb W6/32 precipitated class I heavy chains of Mr 39000 while the extra molecules detected only by mAb B1G6 and IL-A19 were of Mr 37000 and 39000. On thymocytes, the mAb W6/32-non-reactive class I molecules are present in low amounts and are expressed by cells in the medulla area, unlike BoT1 (analogous to human CD1) molecules which are expressed by the cortical cells. Our studies also revealed that the supposed beta 2m-specific mAb B1G6 does not recognize the beta 2m-associated molecules (BoT1) precipitated by mAb TH97A and thus the specificity of mAb B1G6 in cattle is for an epitope on bovine beta 2m which is strongly influenced by the nature of the heavy chain with which the beta 2m is associated.     

Biochemical characterization of activation-associated bovine class I major histocompatibility complex antigens

Summary. Utilizing a 'sandwich' ELISA assay we have been able to demonstrate that mAb W6/32, B1G6 and IL-A19 are reactive with three different monomorphic determinants on bovine class I major histocompatibility complex (MHC) molecules. Sequential immunoprecipitations performed with the mAb revealed that class I molecules on PBM comprise a single population with respect to reactivity with the mAb in that the β2m-associated proteins bear all three epitopes. By contrast, TCGF-driven lymphoblasts and cells transformed by Theileria parva (Tp) additionally express molecules of Mr 45000 bound to β2m which are recognized by mAb B1G6 and IL-A19 but not by W6/32. These two subclasses of molecules were further distinguished on the basis that, when tunicamycin was added to cultures in the preparation of cells for analysis, mAb W6/32 precipitated class I heavy chains of Mr 39000 while the extra molecules detected only by mAb B1G6 and IL-A19 were of Mr 37000 and 39000. On thymocytes, the mAb W6/32-non-reactive class I molecules are present in low amounts and are expressed by cells in the medulla area, unlike BoT1 (analogous to human CD1) molecules which are expressed by the cortical cells. Our studies also revealed that the supposed β2m-specific mAb B1G6 does not recognize the β2m-associated molecules (BoT1) precipitated by mAb TH97A and thus the specificity of mAb B1G6 in cattle is for an epitope on bovine β2m which is strongly influenced by the nature of the heavy chain with which the β2m is associated.     

Invasive equine trophoblast expresses conventional class I major histocompatibility complex antigens

Monoclonal antibodies and alloantisera were used in an indirect immunohistochemical assay to determine the expression of class I and class II Major Histocompatibility Complex (MHC) antigens by equine placental cells and the endometrial tissues at the fetal-maternal interface. MHC class I antigens were expressed at high density on the surface of the trophoblast cells of the chorionic girdle at days 32-36, just prior to their invasion of the endometrium. The mature gonadotrophin-secreting cells of the endometrial cups, which are derived from the chorionic girdle cells, had greatly reduced levels of MHC class I antigen expression while no MHC class I antigens were detectable on the non-invasive trophoblast cells of the allantochorion, except in small isolated patches. MHC class I antigens immunoprecipitated from chorionic girdle cells with either monoclonal antibodies or alloantisera had a relative molecular mass of 44,000, which was identical to that of MHC class I antigens precipitated from lymphocytes with the same reagents. MHC class II antigens were not detected on any trophoblast cells, although they were expressed at high levels by the endometrial glandular and lumenal epithelium immediately bordering the endometrial cups. MHC class I antigens were also expressed at high levels by endometrial tissues in the area of the cups. The high level of MHC class I antigen expression by endometrial glands within and bordering the cups was in sharp contrast to the greatly reduced class I antigen expression by the mature endometrial cup cells themselves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Asparagine deamidation perturbs antigen presentation on class II major histocompatibility complex molecules

Post-translational protein modifications can be recognized by B and T lymphocytes and can potentially make "self"-proteins appear foreign to the immune system. Such modifications may directly affect major histocompatibility complex-restricted T cell recognition of processed peptides or may perturb the processing events that generate such peptides. Using the tetanus toxin C fragment protein as a test case, we show that spontaneous deamidation of asparagine residues interferes with processing by the enzyme asparagine endopeptidase (AEP) and contributes to diminished antigen presentation. Deamidation inhibits AEP action either directly, when asparagine residues targeted by AEP are modified, or indirectly, when adjacent Asn residues are deamidated. Thus, deamidation of long-lived self-proteins may qualitatively or quantitatively affect the spectrum of self-peptides displayed to T cells and may thereby contribute to the onset or exacerbation of autoimmune disease.     

Tumor antigens and proteomics from the point of view of the major histocompatibility complex peptides

The major histocompatibility complex (MHC) peptide repertoire of cancer cells serves both as a source for new tumor antigens for development of cancer immunotherapy and as a rich information resource about the protein content of the cancer cells (their proteome). Thousands of different MHC peptides are normally displayed by each cell, where most of them are derived from different proteins and thus represent most of the cellular proteome. However, in contrast to standard proteomics, which surveys the cellular protein contents, analyses of the MHC peptide repertoire correspond more to the rapidly degrading proteins in the cells (i.e. the transient proteome). MHC peptides can be efficiently purified by affinity chromatography from membranal MHC molecules, or preferably following transfection of vectors for expression of recombinant soluble MHC molecules. The purified peptides are resolved and analyzed by capillary high-pressure liquid chromatography-electrospray ionization-tandem mass spectrometry, and the data are deciphered with new software tools enabling the creation of large databanks of MHC peptides displayed by different cell types and by different MHC haplotypes. These lists of identified MHC peptides can now be used for searching new tumor antigens, and for identification of proteins whose rapid degradation is significant to cancer progression and metastasis. These lists can also be used for identification of new proteins of yet unknown function that are not detected by standard proteomics approaches. This review focuses on the presentation, identification and analysis of MHC peptides significant for cancer immunotherapy. It is also concerned with the aspects of human proteomics observed through large-scale analyses of MHC peptides.     

Crystallization of murine major histocompatibility complex class I H-2Kb with single peptides.

X-ray quality crystals of a soluble murine class I H-2Kb molecule complexed with three different peptide antigens were grown in several forms by streak seeding and macroseeding methods. Co-crystals with VSV-8 (RGYVYGQL), OVA-8 (SIINFEKL) and SEV-9 (FAPGNYPAL) peptides were grown either from NaH2PO4/HPO4 or from polyethylene glycol 4000 within the pH range 5.0 to 7.5, with the use of 4-methyl-2-pentane diol (MPD) as an additive. The VSV-8 crystals grew in space groups P1, with cell dimensions a = 63.1 A, b = 69.1 A, c = 72.0 A, alpha = 89.9 degrees, beta = 77.1 degrees, gamma = 123.3 degrees and P2(1)2(1)2, with a = 138.1 A, b = 88.6 A, c = 45.7 A, and diffract to 2.9 and 2.3 A, respectively. Crystals of the SEV-9 complex grew from similar crystallization conditions to those of the orthorhombic VSV-8 complex with similar cell parameters and diffract to at least 2.5 A resolution. Crystals of the OVA-8 complex were obtained from either phosphate (space group C2, a = 118.7 A, b = 61.6 A, c = 85.3 A, beta = 108.4 degrees) or polyethylene glycol (space group P1, a = 64.5 A, b = 71.0 A, c = 66.3 A, alpha = 89.7 degrees, beta = 95.7 degrees, gamma = 123.3 degrees) and diffract to 3 A resolution. The crystallization procedures used here significantly increased the rate and production of X-ray quality crystals.     

Production and characterization of alloantisera specific for bovine class II major histocompatibility complex antigens

Ten alloantisera defining five major histocompatibility complex (MHC) class II specificities of the bovine lymphocyte antigen (BoLA) complex were produced and characterized. Eight antisera defining four of the specificities were generated by immunizing cattle with class I compatible-class II incompatible lymphocytes. The alloantiserum defining the fifth class II specificity was produced by skin implant immunization. A pregnancy serum specific for one of the class II specificities was also identified. The class II antigens recognized by these antisera were designated 'Dx' antigens to indicate that they are BoLA-D region antigens encoded by one or more undetermined class II loci. The molecules identified by the alloantisera are heterodimers composed of a 34-kd alpha and a 26- to 28-kd beta chain, and are expressed on B-lymphocytes but not on resting T-lymphocytes. In family studies the BoLA-Dx antigens segregated in linkage with the BoLA-A locus alleles. Most of the BoLA-A alleles present in the Cornell Holstein herd at a high frequency were found to exist in gametic association with two or more serologically defined class II haplotypes. On the basis of a population study it was determined that three pairs of class I and class II alleles (w10-Dx4, w31-Dx5, and c3-Dx2) were present in the Cornell herd at significantly increased frequencies.     

The influence of major histocompatibility complex class I antigens on tumor growth and metastasis

The work described here demonstrates the importance of major histocompatibility complex class I antigens for the control of tumor growth and metastasis by the host's immune system. In certain murine tumor cells which have lost expression of H-2 class I antigens, a de novo expression of H-2 can be achieved by transfection with syngeneic class I genes. In contrast to the parental cells the transfected tumors do not grow any more in syngeneic mice, or in other cases they do not form metastases. The studies suggest that the de novo expression of the H-2 antigens renders the tumors highly immunogenic and leads to effective recognition of a tumor-associated antigen in conjunction with the transfected H-2 antigen. These conclusions were confirmed in other tumor systems. For example, separation of a heterogeneous tumor into clones expressing high or low amounts of H-2 showed that only the tumor cell with low H-2 grew well in syngeneic mice, whereas the H-2 high tumor clones were rejected. In other studies in vitro induction by IFN-gamma of H-2 antigen on H-2 negative tumors led to reduced tumor growth in vivo which was due to the increased immunogenicity. About 10% of human tumors are also low or defective for HLA class I expression and often these tumors appear to be more malignant. The class I negative tumors could either have arisen from class I low or negative tissues or are HLA loss variants which escaped the attack of the immune system. Altogether, our studies and the data of other laboratories demonstrate the important role of class I antigens for anti-tumor immunity and they suggest that modulation of class I expression by gene transfection or by induction with soluble mediators could be a useful tool for the manipulation of tumor immunity.     

Differential phosphorylation of murine class I major histocompatibility antigens

Recent studies have shown that the H-2K and H-2D transplantation antigens are expressed differentially in different tissues of mouse. Our previous investigations also established that in thioglycolate-stimulated peritoneal macrophages the H-2D^k antigen exists in distinct cell surface and intracellular forms. These two forms are glycosylated differently. In this report, we have found that (1) H-2D^k antigen is phosphorylated whereas H-2K^k antigen is not, and (2) only the cell surface form of H-2D^k antigen is phosphorylated in thioglycolate-stimulated macrophages derived from C3H/Heha mice. This differential phosphorylation of H-2 antigens will provide a model system for further studies on the molecular mechanism and function of phosphrrylation of H-2 antigens.     

Enhanced immune presentation of a single-chain major histocompatibility complex class I molecule engineered to optimize linkage of a C-terminally extended peptide

Major histocompatibility complex class I molecules can be expressed as single polypeptides wherein the antigenic peptide, beta2-microglobulin, and heavy chain are attached by flexible linkers. These molecules, single-chain trimers (SCTs), are remarkably stable at the cell surface compared with native (noncovalently attached) class I molecules. In this study, we used a structure-based approach to engineer an F pocket variant SCT of the murine class I molecule Kb that presents the SIINFEKL epitope of ovalbumin. Mutation of heavy chain residue Tyr84 (Y84A) in the SCT resulted in enhanced serological and cytolytic CD8 T cell recognition of the covalently linked peptide due to better accommodation of the linker extending from the C terminus of the peptide. These SCTs exhibit significant cell-surface stability, which we hypothesize is rendered by their ability to continuously and efficiently rebind the covalently attached peptide. In addition, we demonstrate that SCT technology can be applied to tetramer construction using recombinant SCTs expressed in Escherichia coli. SCT-based tetramers could have applications for the enumeration of T and natural killer cells that recognize peptide.class I complexes prone to dissociation.     

The use of paramagnetic beads for the detection of major histocompatibility complex class I and class II antigens

Specific immunoadsorbents were prepared using paramagnetic particles (Dynabeads), and their ability to immunoprecipitate major histocompatibility complex (MHC) Class I and Class II antigens compared with conventional protein A Sepharose immunoadsorbents. Lysates of lymphoblastoid cells provided the antigen source which were visualized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Dynabeads were found to be as effective as protein A Sepharose immunoadsorbents at immunoprecipitating MHC Class I and Class II antigens, but had a much lower nonspecific binding capacity resulting in fewer interference bands and lower backgrounds.     

Immunohistochemical detection of strain-specific major histocompatibility complex class I antigens in paraffin-embedded rat osteochondral tissue

Summary An immunohistochemical method using formalin-fixed, paraffin wax-embedded sections is described for detecting strain-specific major histocompatibility complex class I antigens in knee-joint tissue from DA and Lewis strains of rat. The fixed osteochondral tissues were additionally decalcified in formic acid before processing for paraffin wax embedding. For immunohistochemistry, two monoclonal antibodies, one specific for DA class I allele RT1Aa and the other for Lewis class I allele RT1A1, were used together with the avidin-biotin immunoperoxidase procedure. It was necessary to use strain-specific normal rat serum as a diluent for the antibodies to suppress cross-strain recognition. DA-specific antibody stained positively only on DA rat sections, not on Lewis rat sections, and Lewis-specific antibody stained positively only on Lewis rat sections, and not on DA. Positive staining was localized in the bone marrow, osteochondral cells and endothelium. We propose that the use of a decalcification medium may have enhanced the immunoreactivity of the tissue. The method described can be used on sections of allografts from the two strains of rat to assess morphologically the extent of cellular replacement of the graft by the host's cells.