Function and regulation of a steroidogenic CYP450 enzyme in the mitochondrion of Toxoplasma gondii.

As an obligate intracellular parasite, Toxoplasma gondii must import essential nutrients from the host cell into the parasitophorous vacuole. We previously reported that the parasite scavenges cholesterol from host endocytic organelles for incorporation into membranes and storage as cholesteryl esters in lipid droplets. In this study, we have investigated whether Toxoplasma utilizes cholesterol as a precursor for the synthesis of metabolites, such as steroids. In mammalian cells, steroidogenesis occurs in mitochondria and involves membrane-bound type I cytochrome P450 oxidases that are activated through interaction with heme-binding proteins containing a cytochrome b5 domain, such as members of the membrane-associated progesterone receptor (MAPR) family. Our LC-MS targeted lipidomics detect selective classes of hormone steroids in Toxoplasma, with a predominance for anti-inflammatory hydroxypregnenolone species, deoxycorticosterone and dehydroepiandrosterone. The genome of Toxoplasma contains homologs encoding a single type I CYP450 enzyme (we named TgCYP450mt) and a single MAPR (we named TgMAPR). We showed that TgMAPR is a hemoprotein with conserved residues in a heme-binding cytochrome b5 domain. Both TgCYP450 and TgMAPR localize to the mitochondrion and show interactions in in situ proximity ligation assays. Genetic ablation of cyp450mt is not tolerated by Toxoplasma; we therefore engineered a conditional knockout strain and showed that iΔTgCYP450mt parasites exhibit growth impairment in cultured cells. Parasite strains deficient for mapr could be generated; however, ΔTgMAPR parasites suffer from poor global fitness, loss of plasma membrane integrity, aberrant mitochondrial cristae, and an abnormally long S-phase in their cell cycle. Compared to wild-type parasites, iΔTgCYP450mt and ΔTgMAPR lost virulence in mice and metabolomics studies reveal that both mutants have reduced levels of steroids. These observations point to a steroidogenic pathway operational in the mitochondrion of a protozoan that involves an evolutionary conserved TgCYP450mt enzyme and its binding partner TgMAPR.

Inhibition by stabilization: targeting the Plasmodium falciparum aldolase-TRAP complex.

BACKGROUND: Emerging resistance of the malaria parasite Plasmodium to current therapies underscores the critical importance of exploring novel strategies for disease eradication. Plasmodium species are obligate intracellular protozoan parasites. They rely on an unusual form of substrate-dependent motility for their migration on and across host-cell membranes and for host cell invasion. This peculiar motility mechanism is driven by the 'glideosome', an actin-myosin associated, macromolecular complex anchored to the inner membrane complex of the parasite. Myosin A, actin, aldolase, and thrombospondin-related anonymous protein (TRAP) constitute the molecular core of the glideosome in the sporozoite, the mosquito stage that brings the infection into mammals. METHODS: Virtual library screening of a large compound library against the PfAldolase-TRAP complex was used to identify candidate compounds that stabilize and prevent the disassembly of the glideosome. The mechanism of these compounds was confirmed by biochemical, biophysical and parasitological methods. RESULTS: A novel inhibitory effect on the parasite was achieved by stabilizing a protein-protein interaction within the glideosome components. Compound 24 disrupts the gliding and invasive capabilities of Plasmodium parasites in in vitro parasite assays. A high-resolution, ternary X-ray crystal structure of PfAldolase-TRAP in complex with compound 24 confirms the mode of interaction and serves as a platform for future ligand optimization. CONCLUSION: This proof-of-concept study presents a novel approach to anti-malarial drug discovery and design. By strengthening a protein-protein interaction within the parasite, an avenue towards inhibiting a previously "undruggable" target is revealed and the motility motor responsible for successful invasion of host cells is rendered inactive. This study provides new insights into the malaria parasite cell invasion machinery and convincingly demonstrates that liver cell invasion is dramatically reduced by 95 % in the presence of the small molecule stabilizer compound 24.

Deficiency of a Niemann-Pick, type C1-related protein in toxoplasma is associated with multiple lipidoses and increased pathogenicity.

Several proteins that play key roles in cholesterol synthesis, regulation, trafficking and signaling are united by sharing the phylogenetically conserved 'sterol-sensing domain' (SSD). The intracellular parasite Toxoplasma possesses at least one gene coding for a protein containing the canonical SSD. We investigated the role of this protein to provide information on lipid regulatory mechanisms in the parasite. The protein sequence predicts an uncharacterized Niemann-Pick, type C1-related protein (NPC1) with significant identity to human NPC1, and it contains many residues implicated in human NPC disease. We named this NPC1-related protein, TgNCR1. Mammalian NPC1 localizes to endo-lysosomes and promotes the movement of sterols and sphingolipids across the membranes of these organelles. Miscoding patient mutations in NPC1 cause overloading of these lipids in endo-lysosomes. TgNCR1, however, lacks endosomal targeting signals, and localizes to flattened vesicles beneath the plasma membrane of Toxoplasma. When expressed in mammalian NPC1 mutant cells and properly addressed to endo-lysosomes, TgNCR1 restores cholesterol and GM1 clearance from these organelles. To clarify the role of TgNCR1 in the parasite, we genetically disrupted NCR1; mutant parasites were viable. Quantitative lipidomic analyses on the ΔNCR1 strain reveal normal cholesterol levels but an overaccumulation of several species of cholesteryl esters, sphingomyelins and ceramides. ΔNCR1 parasites are also characterized by abundant storage lipid bodies and long membranous tubules derived from their parasitophorous vacuoles. Interestingly, these mutants can generate multiple daughters per single mother cell at high frequencies, allowing fast replication in vitro, and they are slightly more virulent in mice than the parental strain. These data suggest that the ΔNCR1 strain has lost the ability to control the intracellular levels of several lipids, which subsequently results in the stimulation of lipid storage, membrane biosynthesis and parasite division. Based on these observations, we ascribe a role for TgNCR1 in lipid homeostasis in Toxoplasma.

Syntaxin 11 marks a distinct intracellular compartment recruited to the immunological synapse of NK cells to colocalize with cytotoxic granules.

The syntaxin 11 (STX11) gene is mutated in a proportion of patients with familial haemophagocytic lymphohistiocytosis (FHL) and exocytosis of cytotoxic granules is impaired in STX11-deficient NK cells. However, the subcellular localization, regulation of expression and molecular function of STX11 in NK cells and other cytotoxic lymphocytes remain unknown. Here we demonstrate that STX11 expression is strictly controlled by several mechanisms in a cell-type-specific manner and that the enzymatic activity of the proteasome is required for STX11 expression in NK cells. In resting NKL cells, STX11 was localized in the cation-dependent mannose-6-phosphate receptor (CD-M6PR)-containing compartment, which was clearly distinct from cytotoxic granules or Rab27a-expressing vesicles. These subcellular structures appeared to fuse at the contact area with NK-sensitive target cells as demonstrated by partial colocalization of STX11 with perforin and Rab27a. Although STX11-deficent allo-specific cytotoxic T-lymphocytes efficiently lysed target cells and released cytotoxic granules, they exhibited a significantly lower extent of spontaneous association of perforin with Rab27a as compared with STX11-expressing T cells. Thus, our results suggest that STX11 promotes the fusion of Rab27a-expressing vesicles with cytotoxic granules and reveal an additional level of complexity in the spatial/temporal segregation of subcellular structures participating in the process of granule-mediated cytotoxicity.

IFN-gamma protects short-term ovarian carcinoma cell lines from CTL lysis via a CD94/NKG2A-dependent mechanism.

IFN-gamma regulates the immunogenicity of target cells by increasing their expression of HLA class I molecules. This facilitates the T cell receptor-mediated recognition by CD8(+) T cells but decreases target cell sensitivity to lysis by NK cells due to engagement of inhibitory NK receptors. In this study, short-term tumor cell lines from patients with advanced ovarian carcinomas were established. We demonstrate the paradoxical finding that IFN-gamma treatment of these short-term ovarian carcinoma cell lines (OVACs) resulted in resistance of tumor cells to lysis by peptide- and allospecific CD8(+) T cells. Blocking experiments revealed that this phenomenon was dependent on enhanced inhibitory signalling via CD94/NKG2A receptors expressed on the effector cells. This was associated with increased expression of HLA-E mRNA and HLA-G at the protein level in IFN-gamma-treated OVACs. Furthermore, pulsing of untreated OVACs with the leader sequence peptide of HLA-G protected these cells from lysis by CTLs, thus mimicking the inhibitory effect of IFN-gamma. This study provides evidence that CD94/NKG2A receptors play an important role in regulating T cell activity against tumors and shows that IFN-gamma modulation of target cells may shift the balance of triggering and inhibitory signals to T cells, turning off their cytolytic activity.

Autocrine secretion of Fas ligand shields tumor cells from Fas-mediated killing by cytotoxic lymphocytes.

Mechanisms responsible for resistance of tumors to death receptor-mediated damage by cytotoxic lymphocytes are not well understood. Uveal melanoma cells expressed Fas but were insensitive to Fas triggering induced by bystander cytotoxic T lymphocytes or a Fas-specific agonistic antibody; this could not be ascribed to tumor counterattack against T cells or general resistance of the tumors to apoptosis. Treatment with inhibitors of metalloproteases rendered uveal melanomas sensitive to Fas-mediated cytotoxicity. Metalloprotease inhibitors did not affect the expression of Fas but increased the surface expression of Fas ligand (FasL), which correlated with the disappearance of soluble FasL from culture supernatants of tumor cells. FasL eluted from the surface of uveal melanomas specifically inhibited cytotoxic T lymphocyte lysis of tumor cells pretreated with an inhibitor of metalloproteases. In addition to uveal melanomas, a number of other tumor cell lines of various cellular origins were sensitized to Fas-mediated cytotoxicity by metalloprotease inhibitors. Our results show that autocrine secretion of FasL shields tumor cells from Fas-mediated killing by cytotoxic lymphocytes. This defines a novel mechanism of tumor escape from immune surveillance.

Retinoids act as multistep modulators of the major histocompatibility class I presentation pathway and sensitize neuroblastomas to cytotoxic lymphocytes.

The current therapeutic modalities achieve low response rates in human neuroblastoma, a frequent extracranial malignancy of the early childhood. We have assessed the effect of retinoids, used presently for the treatment of neuroblastoma, on the discrete steps of the MHC class I processing machinery and susceptibility of neuroblastoma cells to CTL-mediated killing. We demonstrate that retinoic acid derivatives induce the expression of proteolytic and regulatory subunits of the immunoproteasome, increase the half-life of MHC class I complexes, and enhance the sensitivity of neuroblastoma cells to both MHC class I-restricted peptide-specific and HLA nonrestricted lysis by CTLs. Importantly, effects of retinoids on the MHC class I pathway appear to be independent of IFN-gamma and/or TNF-alpha as intermediate messengers. To our knowledge, this is the first demonstration of inflammation-unrelated biological molecules that induce systemic modulation of antigen presentation in nonprofessional antigen presenting cells. Our findings suggest that the application of retinoids and T cell-based immunotherapy may be an effective combination for the treatment of neuroblastoma.

Virtual Screening and Experimental Validation Identify Novel Inhibitors of the Plasmodium falciparum Atg8-Atg3 Protein-Protein Interaction.

New therapies are needed against malaria, a parasitic infection caused by Plasmodium falciparum, as drug resistance emerges against the current treatment, artemisinin. We previously characterized the Atg8-Atg3 protein-protein interaction (PPI), which is essential for autophagy and parasite survival. Herein we illustrate the use of virtual library screening to selectively block the PPI in the parasite without inhibiting the homologous interaction in humans by targeting the A-loop of PfAtg8. This A-loop is important for Atg3 binding in Plasmodium, but is absent from the human Atg8 homologues. In this proof-of-concept study, we demonstrate a shift in lipidation state of PfAtg8 and inhibition of P. falciparum growth in both blood- and liver-stage cultures upon drug treatment. Our results illustrate how in silico screening and structure-aided drug design against a PPI can be used to identify new hits for drug development. Additionally, as we targeted a region of Atg8 that is conserved within apicomplexans, we predict that our small molecule will have cross-reactivity against other disease-causing apicomplexans, such as Toxoplasma, Cryptosporidium, Theileria, Neospora, Eimeria, and Babesia.

Flow Cytometry Based Detection and Isolation of Plasmodium falciparum Liver Stages In Vitro.

Malaria, the disease caused by Plasmodium parasites, remains a major global health burden. The liver stage of Plasmodium falciparum infection is a leading target for immunological and pharmacological interventions. Therefore, novel approaches providing specific detection and isolation of live P. falciparum exoerythrocytic forms (EEFs) are warranted. Utilizing a recently generated parasite strain expressing green fluorescent protein (GFP) we established a method which, allows for detection and isolation of developing live P. falciparum liver stages by flow cytometry. Using this technique we compared the susceptibility of five immortalized human hepatocyte cell lines and primary hepatocyte cultures from three donors to infection by P. falciparum sporozoites. Here, we show that EEFs can be detected and isolated from in vitro infected cultures of the HC-04 cell line and primary human hepatocytes. We confirmed the presence of developing parasites in sorted live human hepatocytes and characterized their morphology by fluorescence microscopy. Finally, we validated the practical applications of our approach by re-examining the importance of host ligand CD81 for hepatocyte infection by P. falciparum sporozoites in vitro and assessment of the inhibitory activity of anti-sporozoite antibodies. This methodology provides us with the tools to study both, the basic biology of the P. falciparum liver stage and the effects of host-derived factors on the development of P. falciparum EEFs.

HLA expression in uveal melanoma: there is no rule without some exception.

A decrease in the expression of HLA antigens is considered a characteristic of tumor progression and is considered an important tumor-escape mechanism. In general, HLA Class I expression is even further decreased on metastases. Tumor cells that loose their HLA Class I antigens become less susceptible to lysis by specific T cells, but may become more sensitive to Natural Killer cells. Loss of HLA Class I can be observed at different levels, i.e. total loss of Class I, loss of expression of one locus or one haplotype, or even one specific allele. We studied HLA expression on human uveal melanoma and observed that loss of expression of a locus or one or more alleles is a common phenomenon. However, in contrast with the commonly accepted paradigm, loss of HLA Class I expression on the uveal melanoma was not associated with tumor cell escape and a worse survival, but with a better survival of the patients involved. We hypothesize that this is due to the route of metastases formation: in uveal melanoma, spreading of metastases is purely hematogeneous, and it is quite possible that NK-cell mediated surveillance of tumor cells in the blood is the underlying mechanism. This is supported by our finding that metastases of uveal melanoma have a high HLA Class I expression, leading to our conclusion that uveal melanoma is an exception to the general rule regarding HLA Class I expression in tumor immunology.

Priming of CD8(+) T Cell Responses to Liver Stage Malaria Parasite Antigens.

While the role of malaria parasite-specific memory CD8(+) T cells in the control of exo-erythrocytic stages of malaria infection is well documented and generally accepted, a debate is still ongoing regarding both the identity of the anatomic site where the activation of naive pathogen-specific T cells is taking place and contribution of different antigen-presenting cells (APCs) into this process. Whereas some studies infer a role of professional APCs present in the lymph nodes draining the site of parasite injection by the mosquito, others argue in favor of the liver as a primary organ and hepatocytes as stimulators of naïve parasite-specific T cell responses. This review aims to critically analyze the current knowledge and outline new lines of research necessary to understand the induction of protective cellular immunity against the malaria parasite.

Identification of an Atg8-Atg3 protein-protein interaction inhibitor from the medicines for Malaria Venture Malaria Box active in blood and liver stage Plasmodium falciparum parasites.

Atg8 is a ubiquitin-like autophagy protein in eukaryotes that is covalently attached (lipidated) to the elongating autophagosomal membrane. Autophagy is increasingly appreciated as a target in diverse diseases from cancer to eukaryotic parasitic infections. Some of the autophagy machinery is conserved in the malaria parasite, Plasmodium. Although Atg8's function in the parasite is not well understood, it is essential for Plasmodium growth and survival and partially localizes to the apicoplast, an indispensable organelle in apicomplexans. Here, we describe the identification of inhibitors from the Malaria Medicine Venture Malaria Box against the interaction of PfAtg8 with its E2-conjugating enzyme, PfAtg3, by surface plasmon resonance. Inhibition of this protein-protein interaction prevents PfAtg8 lipidation with phosphatidylethanolamine. These small molecule inhibitors share a common scaffold and have activity against both blood and liver stages of infection by Plasmodium falciparum. We have derivatized this scaffold into a functional platform for further optimization.

Dynamics of the major histocompatibility complex class I processing and presentation pathway in the course of malaria parasite development in human hepatocytes: implications for vaccine development.

Control of parasite replication exerted by MHC class I restricted CD8+ T-cells in the liver is critical for vaccination-induced protection against malaria. While many intracellular pathogens subvert the MHC class I presentation machinery, its functionality in the course of malaria replication in hepatocytes has not been characterized. Using experimental systems based on specific identification, isolation and analysis of human hepatocytes infected with P. berghei ANKA GFP or P. falciparum 3D7 GFP sporozoites we demonstrated that molecular components of the MHC class I pathway exhibit largely unaltered expression in malaria-infected hepatocytes until very late stages of parasite development. Furthermore, infected cells showed no obvious defects in their capacity to upregulate expression of different molecular components of the MHC class I machinery in response to pro-inflammatory lymphokines or trigger direct activation of allo-specific or peptide-specific human CD8+ T-cells. We further demonstrate that ectopic expression of circumsporozoite protein does not alter expression of critical genes of the MHC class I pathway and its response to pro-inflammatory cytokines. In addition, we identified supra-cellular structures, which arose at late stages of parasite replication, possessed the characteristic morphology of merosomes and exhibited nearly complete loss of surface MHC class I expression. These data have multiple implications for our understanding of natural T-cell immunity against malaria and may promote development of novel, efficient anti-malaria vaccines overcoming immune escape of the parasite in the liver.

The microenvironment of human neuroblastoma supports the activation of tumor-associated T lymphocytes.

Tumor infiltration by lymphocytes has been linked to improved clinical outcome in children with neuroblastoma (NB) but T-cell activation has never been demonstrated to occur within the NB microenvironment. Here we show that tumor-associated lymphocytes (TALs) obtained from lesions representing all genetic subsets of NB and autologous peripheral blood lymphocytes (PBLs) analyzed on the day of tumor excision differed in composition, phenotype and functional characteristics. The NB microenvironment appeared to promote the accumulation of CD3+CD8+ T cells and contained a larger proportion of T cells expressing the interleukin-2 receptor α chain (CD25) and manifesting an effector memory (CCR7-CD45RA-) phenotype. Accordingly, the stimulation of PBLs with autologous tumor cells in short-term cultures increased the proportion of effector memory T cells, upregulated CD25, stimulated the expression of the TH1 cytokines interferon γ and tumor necrosis factor α, and reduced the expression of transforming growth factor ß. In situ proliferation as well as a characteristic pattern of T-cell receptor aggregation at the contact sites with malignant cells was revealed by the immunohistochemical staining of TALs in primary tumors, indicating that the NB milieu is compatible with the activation of the immune system. Our results are compatible with the hypothesis that CD8+ T cells are specifically activated within the NB microenvironment, which appears to be permissive for effector memory responses.

B cell receptor triggering sensitizes human B cells to TRAIL-induced apoptosis.

TRAIL is known to cause death in tumor cells, but physiological regulation of its activity remains poorly characterized. We demonstrate that BCR triggering sensitizes transformed centroblast-like BL cells and peripheral blood memory B cells to TRAIL-mediated apoptosis. The sensitization correlated with surface down-regulation and intracellular retention of TRAIL-R4, along with changes in the expression of several Bcl-2 protein family members. Although enhancing FAS-mediated cell death, CD40 activation protected B cells from TRAIL-induced apoptosis. Combination of Ig cross-linking with CD40 ligation did not prevent TRAIL-R4 down-regulation but induced changes in the mitochondria-regulated pathway of apoptosis that are known to be associated with resistance to TRAIL. Human CD5(+) B cells, presumably stimulated by reactivity to self without immunological help, exhibited very high ex vivo sensitivity to TRAIL. Our results define the first B-lymphocyte-specific physiological signal that increases cellular sensitivity to TRAIL. This may be important for our understanding of TRAIL involvement in the control of B cell responses and aid in designing TRAIL-based therapies for B cell lymphomas.

Soluble factors released by activated cytotoxic T lymphocytes interfere with death receptor pathways in neuroblastoma.

Neuroblastoma (NB) is often described as an unfavorable target for both HLA-restricted and death receptor-mediated elimination by cytotoxic T lymphocytes (CTLs) due to low or absent HLA class I and caspase-8 expression. We investigated the effects of soluble factors released by CTLs activated by TCR triggering (named as activated supernatant; AS) on the levels and composition of cell surface molecules involved in HLA-restricted and HLA-independent NB cell recognition (surface immune phenotype). Using a panel of long-term propagated NB cell lines and freshly isolated primary human NB cells, we analyzed surface expression of the (1) cognate receptors for TNFalpha, Fas and TRAIL; (2) HLA class I and II heterodimers; (3) adhesion molecules; (4) the intracellular expression and activation of caspase-8, as well as (5) the susceptibility of NB cells to death receptor-mediated killing prior to and after exposure to AS. The exposure of NB cells to soluble factors released by activated CTLs skewed the surface immune phenotype of both long term cultured and primary NB cells, induced the expression and activation of caspase-8 and increased the susceptibility of tumor cells to lysis by TRAIL and Fas-agonistic antibody. Blocking experiments identified IFNgamma and TNFalpha as main factors responsible for modulating the surface antigens of NB cells by AS. Our data suggest that recruitment of CTLs activated on third party targets into the vicinity of the NB tumor mass, may override the "silent" immune phenotype of NB cells via the action of soluble factors.

Differentiation induced by physiological and pharmacological stimuli leads to increased antigenicity of human neuroblastoma cells.

Sympathetic neuronal differentiation is associated with favorable prognosis of neuroblastoma (NB), the most common extra-cranial solid tumor of early childhood. Differentiation agents have proved useful in clinical protocols of NB treatment, but using them as a sole treatment is not sufficient to induce tumor elimination in patients. Therefore, complementary approaches, such as immunotherapy, are warranted. Here we demonstrate that differentiation of NB cell lines and ex vivo isolated tumor cells in response to physiological or pharmacological stimuli is associated with acquisition of increased antigenicity. This manifests as increased expression of surface major histocompatibility class I complexes and ICAM-1 molecules and translates into increased sensitivity of NB cells to lysis by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. The latter is paralleled by enhanced ability of differentiated cells to form immune conjugates and bind increased amounts of granzyme B to the cell surface. We demonstrate, for the first time, that, regardless of the stimulus applied, the differentiation state in NBs is associated with increased tumor antigenicity that enables more efficient elimination of tumor cells by cytotoxic lymphocytes and paves the way for combined application of differentiation-inducing agents and immunotherapy as an auxiliary approach in NB patients.

Modulation of the tumor cell phenotype by IFN-gamma results in resistance of uveal melanoma cells to granule-mediated lysis by cytotoxic lymphocytes.

IFN-gamma, a pleiotropic immune regulator, is implicated in both tumor immune surveillance and selection of tumor variants resistant to immune control, i.e., immunoediting. In uveal melanoma patients, elevated serum levels of IFN-gamma correlate with the spread of metastasis and represent a negative prognostic marker. Treatment with IFN-gamma boosted the MHC class I presentation machinery in uveal melanoma cells but suppressed their MHC class I-restricted CTL lysis. Tumor cells exposed to IFN-gamma efficiently activated specific CTL but were less susceptible to permeabilization by perforin and exhibited a decreased capacity to bind and incorporate granzyme B. These results define a novel mechanism of resistance to granule-mediated CTL lysis in human tumors. Furthermore, the data suggest that immunoediting is not limited to genetic or epigenetic changes resulting in stable cellular phenotypes but also involves an inducible modulation of tumor cells in response to a microenvironment associated with immune activation.

Inhibition of heavy chain and beta2-microglobulin synthesis as a mechanism of major histocompatibility complex class I downregulation during Epstein-Barr virus replication.

The mechanisms of major histocompatibility complex (MHC) class I downregulation during Epstein-Barr virus (EBV) replication are not well characterized. Here we show that in several cell lines infected with a recombinant EBV strain encoding green fluorescent protein (GFP), the virus lytic cycle coincides with GFP expression, which thus can be used as a marker of virus replication. EBV replication resulted in downregulation of MHC class II and all classical MHC class I alleles independently of viral DNA synthesis or late gene expression. Although assembled MHC class I complexes, the total pool of heavy chains, and beta2-microglobulin (beta2m) were significantly downregulated, free class I heavy chains were stabilized at the surface of cells replicating EBV. Calnexin expression was increased in GFP+ cells, and calnexin and calreticulin accumulated at the cell surface that could contribute to the stabilization of class I heavy chains. Decreased expression levels of another chaperone, ERp57, and TAP2, a transporter associated with antigen processing and presentation, correlated with delayed kinetics of MHC class I maturation. Levels of both class I heavy chain and beta2m mRNA were reduced, and metabolic labeling experiments demonstrated a very low rate of class I heavy chain synthesis in lytically infected cells. MHC class I and MHC class II downregulation was mimicked by pharmacological inhibition of protein synthesis in latently infected cells. Our data suggest that although several mechanisms may contribute to MHC class I downregulation in the course of EBV replication, inhibition of MHC class I synthesis plays the primary role in the process.

Retinoic acid elicits cytostatic, cytotoxic and immunomodulatory effects on uveal melanoma cells.

The current therapy of uveal melanoma (UM) metastases remains inefficient, which warrants the development of new treatment modalities. For the first time we investigated the effects of retinoic acid (RA) on a panel of UM cell lines and found that RA induces morphological changes compatible with differentiation, suppresses proliferation and causes apoptosis in these cells. RA treatment resulted in an increase of p21, p27 and p53 protein levels and G1 arrest in UM cells, which correlated with significant down-modulation of surface Her2/neu proto-oncogene expression. In addition, RA-treated UM cells exhibited increased sensitivity to both MHC class I-restricted killing by cytotoxic T lymphocytes and NK cell-mediated lysis that were accompanied by more efficient conjugate formation between UM cells and killer lymphocytes. Taken together, our results implicate UM as a new target for treatment with retinoids and suggest that retinoids and T- or NK-cell based immunotherapy can have mutually enhancing effects in UM patients.