Rachel Ehrlich

Human cytomegalovirus protein US2 interferes with the expression of human HFE, a nonclassical class I major histocompatibility complex molecule that regulates iron homeostasis.

ABSTRACT HFE is a nonclassical class I major histocompatibility complex (MHC) molecule that is mutated in the autosomal recessive iron overload disease hereditary hemochromatosis. There is evidence linking HFE with reduced iron uptake by the transferrin receptor (TfR). Using a panel of HFE and TfR monoclonal antibodies to examine human HFE (hHFE)-expressing cell lines, we demonstrate the expression of stable and fully glycosylated TfR-free and TfR-associated hHFE/β2m complexes. We show that both the stability and assembly of hHFE complexes can be modified by the human cytomegalovirus (HCMV) viral protein US2, known to interfere with the expression of classical class I MHC molecules. HCMV US2, but not US11, targets HFE molecules for degradation by the proteasome. Whether this interference with the regulation of iron metabolism by a viral protein is a means of potentiating viral replication remains to be determined. The reduced expression of classical class I MHC and HFE complexes provides the virus with an efficient tool for altering cellular metabolism and escaping certain immune responses.

HFE—A Novel Nonclassical Class I Molecule that Is Involved in Iron Metabolism

R. E. is supported by the German-Israeli Foundation (GIF) and by the Israel Health Ministry. R. E. and F. L. are supported by the European Community Contract Number QLG1-CT-1999–00665.

Transferrin receptor co-localizes and interacts with the hemochromatosis factor (HFE) and the divalent metal transporter-1 (DMT1) in trophoblast cells

Iron uptake and storage are tightly regulated to guarantee sufficient iron for essential cellular processes and to prevent the production of damaging free radicals. The placenta is the entry site for iron, which is delivered to the developing embryo. Iron is taken up by syncytiotrophoblast cells and is transported unidirectionally from mother to fetus against a concentration gradient. Several iron transporters and regulators were recently characterized, including DMT1 and ferroportin/Ireg1 that transport iron through membranes, and HFE that regulates TfR‐mediated iron uptake. In this study we demonstrate that in a differentiated choriocarcinoma cell line BeWo, HFE, and TfR are localized mainly in recycling endosomes and a small percentage of these complexes is observed in late endosomes with DMT1 while in trophoblast cells, the level of TfR is significantly lower and it is detected with HFE and DMT1 mainly in late endosomes. Most interestingly, TfR and HFE, as well as TfR and DMT1 interact in placental trophoblast cells. Based on previous and these data we suggest that the level of intracellular iron may regulate both TfR expression (on the post‐transcriptional and the post‐translational levels) and TfR trafficking/transcytosis in polarized cells.

Modulation of Antigen Processing and Presentation by Persistent Virus Infections and in Tumors

Cell-mediated immunity is effective against cells harboring active virus replication and is critical for the elimination of ongoing infections, opposing tumor progression, and reducing or preventing the reactivation of persistent viruses and tumor metastasis. The capacity of persistent viruses and tumor cells to maintain a long-term relationship with their host presupposes mechanisms for circumventing antiviral or antitumor defenses. By suppressing the expression of molecules associated with antigen processing and presentation, abrogation of the major immune mechanism that deals with the elimination of infected and transformed cells is achieved. This is accomplished in tumors predominantly by transcriptional downregulation of genes encoding class I major histocompatibility complex antigens, peptide transporter molecules, and the proteasome-associated low molecular mass protease subunits, and in cells expressing viral proteins by interfering with peptide transport and the assembly/transport of class I complexes. In addition, virus-infected cells and selected tumor cells express mainly nonimmunogenic or antagonistic peptide epitopes. This review describes mechanisms used by viruses and in transformed cells for interference with antigen processing and presentation and addresses their significance for in vivo viral persistence and tumor progression.

Organization and Functional Analysis of the Mouse Transporter Associated with Antigen Processing 2 Promoter

In accordance with the key role of MHC class I molecules in the adaptive immune response against viruses, they are expressed by most cells, and their expression can be enhanced by cytokines. The assembly and cell surface expression of class I complexes depend on a continuous peptide supply. The peptides are generated mainly by the proteasome and are transported to the endoplasmic reticulum by a peptide transport pump consisting of two subunits, TAP1 and TAP2. The proteasome low molecular weight polypeptide (2 and 7), as well as TAP (1 and 2) genes, are coordinately regulated and are induced by IFNs. Despite this coordinate regulation, examination of tumors shows that these genes can be discordantly down-regulated. In pursuing a molecular explanation for these observations, we have characterized the mouse TAP2 promoter region and 5′-flanking sequence. We show that the 5′ untranslated regions of TAP2 genes have a characteristic genomic organization that is conserved in both the mouse and the human. The mouse TAP2 promoter belongs to a class of promoters that lack TATA boxes but contain a MED1 (multiple start site element downstream) sequence. Accordingly, transcription is initiated from multiple sites within a 100-nucleotide window. An IFN regulatory factor 1 (IRF1)/IRF2 binding site is located in this region and is involved in both basal and IRF1-induced TAP2 promoter activity. The implication of the extensive differences found among the promoters of class I heavy chain, low molecular weight polypeptide, and TAP genes, all encoding proteins involved in Ag presentation, is discussed.

IFN-γ Affects Both the Stability and the Intracellular Transport of Class I MHC Complexes

In accordance with the key role of major histocompatibility complex (MHC) class I molecules in the adaptive immune response against viruses, their expression can be enhanced by the potent cytokine interferon-gamma (IFN-gamma), which upregulates the expression of multiple components in the pathway of class I-restricted antigen presentation. In this study, we analyzed the effect of IFN-gamma treatment on class I formation, peptide editing, trafficking, and cell surface expression. We show that IFN-gamma treatment promotes significantly the assembly and cell surface expression of stable class I complexes. Yet the existence of large intracellular pools of both free class I heavy chains and suboptimal class I complexes indicates that the optimal peptide supply limits cell surface expression levels of class I complexes. Unexpectedly, we found that IFN-gamma appears generally to slow the maturation rates of both class I complexes and transferrin receptors. Apparently, IFN-gamma causes prolonged retention of molecules in the endoplasmic reticulum (ER) because it regulates the expression of ER-residing proteins that participate in protein maturation. Consequently, it induces more rigorous ER quality control. The significance of these effects of IFN-gamma for in vivo immune responses is discussed.

Expression of allogeneic MHC class I antigens by transgenic mouse trophoblast does not interfere with the normal course of pregnancy.

Mammalian embryos express paternal histocompatibility antigens which make them potential targets for maternal immune responses. Yet, the histoincompatible fetus survives and develops normally. Down regulation of classical MHC antigen expression by trophoblast cells which are in direct contact with maternal circulation has been repeatedly shown. The trophoblast cells are unable to function properly in antigen presentation and do not induce allogeneic rejection reactions. In the present study we have created transgenic mice that express an allogeneic class I transgene whose transcription is controlled by the transferrin receptor promoter. The expression patterns of the transgene product mice from a single transgenic line were studied in each of the typical placental subpopulations. The allogeneic class I antigen was expressed in the allantoic plate region of the trophoblast, and this expression was not restricted to the endothelial region but extended also to the spongiotrophoblast, as well as the major blood vessels and in the endodermal sinuses. In contrast to the normal class I expression, prominent levels of allogeneic H-2 antigens were detected in the labyrinthine trophoblast. The fetal resorption rate in females mated with these transgenic males was not higher then the normal rate, and the embryos survived and developed normally. These data imply that the unusual expression of allogeneic class I antigens in certain trophoblast subpopulations does not affect fetal development.

Manipulation of iron to determine survival: Competition between host and pathogen

Iron is an essential nutrient that can determine cellular survival. Many organisms have evolved sophisticated mechanisms for iron uptake and transport to support their growth. The dual dependence on iron of both the host and invading pathogen initiates a competition for this nutrient following infection. Microorganisms have developed various strategies to acquire iron from the host. These are counter-balanced by an iron-withholding strategy that the host deploys as part of its defense system. This strategy, involving many iron-regulatory proteins, mediates iron depletion at the mucosal surfaces, in the extracellular environment, and within the cells. Iron is sequestered into storage by the host in order to deprive the pathogens of this factor and to prevent their proliferation. This system can be compromised. In particular, new evidence is emerging that suggests that viruses are able to specifically target and regulate proteins involved in iron homeostasis. This review focuses on the procedures employed by the host and viruses to regulate iron as a means of defense and survival, respectively.

Selective Mechanisms Utilized by Persistent and Oncogenic Viruses to Interfere with Antigen Processing and Presentation

Cell-mediated immunity is effective against cells harboring active virus replication, and is critical for the elimination of ongoing infections, regression of virus-associated tumors, and reducing or preventing the reactivation of persistent viruses. The capacity of persistent and oncogenic viruses to maintain a long-term relationship with their host presupposes viral mechanisms for circumventing antiviral defenses. By suppressing the expression of molecules associated with antigen processing and presentation, viruses abrogate the major immune mechanism that deals with the elimination of infected and tumor cells. This is accomplished either by transcriptional downregulation of genes encoding class I MHC antigens, peptide transporter molecules, and the proteasome-associated LMP subunits, or by interfering with transport of class I molecules to the cell surface. In some cases viruses shut off the expression of most viral proteins during latency or express mainly non-immunogenic or antagonistic peptide epitopes. This review describes selective mechanisms utilized by viruses for interference with antigen processing and presentation, and addresses their significance for in vivo viral persistence and tumor progression.

Some characteristics of natural cytostatic mouse splenocytes

Murine B16 melanoma cells and metastasis variants of this tumor are resistant to NK activity mediated by normal splenocytes. The B16 cells are, however, sensitive to splenocyte-mediated cytostasis. Cytostasis was measured by a [125I]UDR incorporation-inhibition [(125I]UDR I-I) assay. The main characteristics of the [125I[UDR I-I assay and of the cells mediating it are as follows: The activity is mediated by splenocytes but not by thymocytes, it is not syngeneically restricted and it does not decrease with age. The presence of effector cells is required as splenocyte supernatants or supernatants of effector-target cell mixtures do not cause [125I]UDR I-I. The activity is probably mediated by at least 2 populations of non-phagocytic splenocytes. The first population adheres to plastic surfaces and to Sephadex G-10 columns while the other does not. The sensitivity to [125I]UDR I-I of the high metastasis B16 variant was similar to that of the low metastasis variant.