Detailed in vivo analysis of interferon-gamma induced major histocompatibility complex expression in the the central nervous system: astrocytes fail to express major histocompatibility complex class I and II molecules

To recognize and respond immunologically to foreign antigens, T lymphocytes require the presentation of foreign peptides by MHC molecules. To determine which cells of the central nervous system (CNS) are capable of expressing MHC molecules, we used confocal microscopy and dual immunofluorescence with cell-specific and MHC-specific antibodies to study brain sections of adult mice. We took advantage of transgenic mice that initiate CNS-specific expression of IFN-gamma at 8 weeks of age. This inflammatory cytokine is a strong inducer of MHC expression both in culture and in vivo. From this analysis, we clearly found MHC class I and II expression on endothelial, microglial, and oligodendrocyte cell types, but did not find astrocytes or neurons capable of expressing either MHC class I or II molecules under these conditions. This finding suggests that, although microglia and oligodendrocytes may participate in the antigen presentation process in the organism, we found no in vivo evidence to support the concept that astrocytes act as antigen-presenting cells.     

Peptide presentation by class-I major histocompatibility complex molecules

MHC class-I molecules express distinct peptide-binding pockets within their antigen-binding groove. These are critically involved in the binding of antigenic peptides. The amino acid composition of a pocket dictates the structure of a peptide which can be bound in it. This is evident as a consensus amino acid motif which has to exist within a peptide in order for it to bind to a particular MHC allele. Perturbation of a MHC pocket by amino acid substitution can result in the abolition of peptide binding. Less drastic mutations of the peptide-binding groove, particularly the ones away from the critical pocket, can subtly alter the conformation of bound peptide. Both types of substitution exert an influence on the TCR recognition of antigenic peptide. Peptides are also critically involved in the positive selection of the class-I-restricted TCR repertoire in the thymus. These self peptides act by mimicking their foreign antigens. This mimicking involves the binding of self peptides and foreign antigenic peptides to the same pockets of the MHC class-I-antigen binding groove. Consequently, MHC class-I polymorphism in the antigen binding groove controls the intrathymic positive selection and peripheral antigen presentation by the same mechanisms. The majority of positively selecting self peptides could well originate from the extracellular processing of circulating self proteins. Using the diverse, extracellularly generated self peptides and the different determinant density requirements for positive versus negative selection, the immune system can ensure the repertoire diversity, avoiding both the massive clonal deletion of the selected repertoire and the autoreactivity of its T cells.     

Interactions between peptides and major histocompatibility complex molecules]

T-cell receptors recognize foreign proteins as peptide fragments associated with major histocompatibility complex (MHC) molecules. Properties of antigenic peptides, methods for detecting peptide-MHC molecule combinations, and the characteristics of the interaction are reviewed. Possible explanations for graft rejection and autoimmune disease are suggested in the light of these data.     

Enterobacterial infection modulates major histocompatibility complex class I expression on mononuclear cells.

Major histocompatibility complex (MHC) class I expression is reduced in several viral infections, but it is not known whether the same happens during infections caused by intracellular enterobacteria. In this study, the expression of MHC class I antigens on peripheral blood mononuclear cells (PBMC) from 16 patients with Salmonella, Yersinia, or Klebsiella infection was investigated. During or after the acute infection, the expression of MHC class I antigens was markedly decreased in eight patients, all with genotype HLA-B27, and six out of eight with reactive arthritis (ReA). A significant decrease of monomorphic MHC class I was found in three patients, of HLA-B27 in eight (P<0.05) and of HLA-A2 in two. However, patients negative for the HLA-B27 genotype, or healthy HLA-B27-positive individuals, did not have a significant decrease of MHC class I antigens. During the decreased expression on the cell surface, intracellular retention of MHC class I antigens was observed, whereas HLA-B27 mRNA levels did not vary significantly. This is the first evidence that enterobacterial infection may down-regulate expression of MHC class I molecules in vivo and that down-regulation is predominant in patients with the HLA-B27 genotype.     

Trypanosoma cruzi infection modulates in vivo expression of major histocompatibility complex class II molecules on antigen-presenting cells and T-cell stimulatory activity of dendritic cells in a strain-dependent manner

A striking feature of Chagas' disease is the diversity of clinical presentations. Such variability may be due to the heterogeneity among Trypanosoma cruzi isolates or to the host immune response. Employing two strains which differ in their virulence, we investigated the effect of in vivo infection on professional antigen-presenting cells (APC). Acute infection with the virulent RA strain downregulated the expression of major histocompatibility complex (MHC) class II on splenic dendritic cells (DC) and inhibited its induction on peritoneal macrophages and splenic B cells. It also impaired the ability of DC to prime allogeneic T cells and to form homotypic clusters, suggesting a low maturation state of these cells. In contrast, the low-virulence K98 strain maintained the expression of MHC class II on DC or stimulated it on peritoneal macrophages and B cells and preserved DC's T-cell priming capacity and homotypic clustering. DC from RA-infected mice elicited a lower activation of T. cruzi-specific T-cell proliferation than those from K98-infected mice. APC from RA-infected mice that reached the chronic phase of infection restored MHC class II levels to those found in K98-infected mice and upregulated costimulatory molecules expression, suggesting that the immunosuppression caused by this strain is only transient. Taken together, the results indicate that in vivo infection with T. cruzi modulates APC functionality and that this is accomplished in a strain-dependent manner.     

Isoelectric focusing of bovine major histocompatibility complex class I molecules

The products of the major histocompatibility complex (MHC) loci regulate an individual's immune response to pathogens. Cattle provide an important model to study the relationship between disease susceptibility and MHC haplotype since large half-sibling families are common. The definitive demonstration, however, of a firm relationship between MHC phenotype and disease susceptibility in cattle will require a precise definition of the bovine MHC allelic products. Available reagents for serological characterization of the bovine MHC gene products have not been adequate for these purposes. We have shown that existing mouse monoclonal antibodies and rabbit anti-human antisera precipitate bovine class I molecules, that these structures separate well by one-dimensional isoelectric focusing (1-D IEF), and that immunoprecipitation followed by 1-D IEF allows the detection of bovine class I MHC allelic products. Through this technique, we have identified previously undetected class I products. This approach will facilitate a detailed characterization of the bovine MHC class I gene products.     

Properties and applications of single-chain major histocompatibility complex class I molecules

Stable major histocompatibility complex (MHC) class I molecules at the cell surface consist of three separate, noncovalently associated components: the class I heavy chain, the β(2)-microglobulin light chain, and a presented peptide. These three components are assembled inside cells via complex pathways involving many other proteins that have been studied extensively. Correct formation of disulfide bonds in the endoplasmic reticulum is central to this process of MHC class I assembly. For a single specific peptide to be presented at the cell surface for possible immune recognition, between hundreds and thousands of peptide-containing precursor polypeptides are required, so the overall process is relatively inefficient. To increase the efficiency of antigen presentation by MHC class I molecules, and for possible therapeutic purposes, single-chain molecules have been developed in which the three, normally separate components have been joined together via flexible linker sequences in a single polypeptide chain. Remarkably, these single-chain MHC class I molecules fold up correctly, as judged by functional recognition by cells of the immune system, and more recently by X-ray crystallographic structural data. This review focuses on the interesting properties and potential of this new type of engineered MHC class I molecule.     

Isoelectric focusing of bovine major histocompatibility complex class II molecules

Serological approaches have been relatively unsuccessful in defining the allelic products of the bovine major histocompatibility (MHC) class II loci. We demonstrate that bovine class II allelic products can be characterized by precipitation with a polyclonal antiserum and separation using one-dimensional isoelectric focusing. Polymorphic beta chains were present in immunoprecipitates from both biosynthetically and surface-labeled lectin-stimulated bovine T cells. Precipitates from biosynthetically labeled but not surface-labeled T cells contained a basic invariant chain and a non-polymorphic structure. The non-polymorphic structure appears to be a beta chain. The polymorphic class II beta chain co-segregated with bovine MHC class I allelic products in a half-sibling family, providing evidence for linkage between bovine class I and class II loci. This approach to the biochemical analysis of the bovine class II structures should facilitate the investigation of the association between the bovine products and disease susceptibility.     

Natural killer cell receptors for major histocompatibility complex class I and related molecules in cytomegalovirus infection

Downmodulation of major histocompatibility complex (MHC) class I molecules by cytomegalovirus (CMV) impairs the engagement of specific leucocyte-inhibitory receptors, rendering infected cells vulnerable to natural killer (NK) cells. Members of the murine Ly49 and human KIR families, CD85j (ILT2 or leucocyte Ig-like receptor-1), as well as the CD94/NKG2A-inhibitory killer lectin-like receptor (KLR) fulfil this surveillance role. On the other hand, NK-activating receptors specific to ligands expressed on virus-infected cells may overcome the control by inhibitory receptors. In this regard, NKG2D and Ly49H lectin-like molecules trigger NK-cell functions recognizing, respectively class I-related stress-inducible molecules and the m157 murine CMV glycoprotein. Among a variety of immune evasion strategies, CMV promotes the synthesis of class I surrogates and selectively preserves the expression of some class I molecules in infected cells; moreover, CMV interferes with the expression of ligands for NKG2D. We herein review these aspects of the host-pathogen interaction, discussing a number of open issues.     

Statins affect cell-surface expression of major histocompatibility complex class II molecules by disrupting cholesterol-containing microdomains

Statins, the main therapy for hypercholesterolemia, are currently considered as possible immunomodulatory agents. Statins inhibit the production of proinflammatory cytokines and reduce the expression of several immunoregulatory molecules, including major histocompatibility complex class II (MHC-II) molecules. In this study, we investigated the mechanism by which simvastatin reduces the membrane expression of MHC-II molecules on several human cell types. We demonstrate that the reduction of MHC-II membrane expression by simvastatin correlates with disruption of cholesterol-containing microdomains, which transport and concentrate MHC-II molecules to the cell surface. In addition, we demonstrate that statins reduce cell-surface expression of other immunoregulatory molecules, which include MHC-I, CD3, CD4, CD8, CD28, CD40, CD80, CD86, and CD54. Our observations indicate that the downregulation of MHC-II at the cell surface contributes to the immunomodulatory properties of statins and is achieved through disruption of cholesterol-containing microdomains, which are involved in their intracellular transport.     

Lipid rafts,major histocompatibility complex molecules,and immune regulation

Glycolipid-enriched membrane microdomains ("rafts") are critical sites for signal transduction and other processes such as intracellular transport. While the participation of T-cell rafts in the formation of the immunological synapse is well established, the role of rafts on antigen-presenting cells (APCs) as well as the relationship between these domains and major histocompatibility complex (MHC) molecules is less clearly defined. We therefore investigated whether MHC class I or II molecules are found in rafts of the human macrophage-monocytic cell line U937. We detected the preferential localization of MHC class II, but not class I, molecules in rafts. Furthermore, raft disruption resulted in a decrease in constitutive protein tyrosine phosphorylation events in U937 cells. Our findings are reviewed in the context of results from other groups who also found important associations of MHC class II molecules with APC rafts. Additional, and at times contradictory, findings by others regarding the relationship between rafts and MHC molecules are also discussed. It is concluded that class II MHC molecules can localize in rafts of APCs and that this localization may be relevant for APC function and thus immune regulation.     

Polarized expression of major histocompatibility complex class I molecules in human endometrial and endocervical epithelial cells.

The localization of major histocompatibility complex (MHC) class I molecules in human endometrial and endocervical epithelial cells was studied with an immunogold technique on ultrathin cryosections. At the cell surface, MHC class I molecules are delivered solely to the basolateral plasma membrane in human epithelia. Therefore, only the basolateral domain of epithelial cells is capable of presenting antigen to MHC class I-restricted cytotoxic T cells.     

Applications of major histocompatibility complex class I molecules expressed as single chains

Generation of CD8 T-cell responses to pathogens and tumors requires optimal expression of class I major histocompatibility complex/peptide complexes, which, in turn, is dependent on host cellular processing events and subject to interference by pathogens. To create a stable structure that is more immunogenic and resistant to immune evasion pathways, we have engineered class I molecules as single-chain trimers (SCTs), with flexible linkers connecting peptide, beta2m, and heavy chain. Herein we extend our earlier studies with SCTs to the K(b) ligand derived from vesicular stomatitis virus (VSV) to characterize further SCTs as probes of immune function as well as their potential in immunotherapy. The VSVp-beta2m-K(b) SCTs were remarkably stable at the cell surface, and immunization with DNA encoding SCTs elicited complex-specific antibody. In addition, SCTs were detected by cytotoxic T-lymphocytes specific for the native molecule, and the covalently bound peptide was highly resistant to displacement by exogenous peptide. SCTs can also prime CD8 T-cells in vivo that recognize the native molecule. Furthermore, SCTs were resistant to downregulation by the immune evasion protein mK3 of gamma herpesvirus 68. Moreover, owing to their preassembled nature, SCTs should be resistant to other immune evasion proteins that restrict peptide supply. Thus, SCTs possess therapeutic potential both for prophylactic treatment and for the treatment of ongoing infection.