Retinoic acid induces blood-brain barrier development.

The blood-brain barrier (BBB) is crucial in the maintenance of a controlled environment within the brain to safeguard optimal neuronal function. The endothelial cells (ECs) of the BBB possess specific properties that restrict the entry of cells and metabolites into the CNS. The specialized BBB endothelial phenotype is induced during neurovascular development by surrounding cells of the CNS. However, the molecular differentiation of the BBB endothelium remains poorly understood. Retinoic acid (RA) plays a crucial role in the brain during embryogenesis. Because radial glial cells supply the brain with RA during the developmental cascade and associate closely with the developing vasculature, we hypothesize that RA is important for the induction of BBB properties in brain ECs. Analysis of human postmortem fetal brain tissue shows that the enzyme mainly responsible for RA synthesis, retinaldehyde dehydrogenase, is expressed by radial glial cells. In addition, the most important receptor for RA-driven signaling in the CNS, RA-receptor ß (RARß), is markedly expressed by the developing brain vasculature. Our findings have been further corroborated by in vitro experiments showing RA- and RARß-dependent induction of different aspects of the brain EC barrier. Finally, pharmacologic inhibition of RAR activation during the differentiation of the murine BBB resulted in the leakage of a fluorescent tracer as well as serum proteins into the developing brain and reduced the expression levels of important BBB determinants. Together, our results point to an important role for RA in the induction of the BBB during human and mouse development.

MicroRNAs regulate human brain endothelial cell-barrier function in inflammation: implications for multiple sclerosis.

Blood-brain barrier (BBB) dysfunction is a major hallmark of many neurological diseases, including multiple sclerosis (MS). Using a genomics approach, we defined a microRNA signature that is diminished at the BBB of MS patients. In particular, miR-125a-5p is a key regulator of brain endothelial tightness and immune cell efflux. Our findings suggest that repair of a disturbed BBB through microRNAs may represent a novel avenue for effective treatment of MS.

Astrocyte-derived retinoic acid: a novel regulator of blood-brain barrier function in multiple sclerosis.

Multiple sclerosis (MS) lesions are characterized by the presence of activated astrocytes, which are thought to actively take part in propagating lesion progression by secreting pro-inflammatory mediators. Conversely, reactive astrocytes may exert disease-dampening effects through the production of trophic factors and anti-inflammatory mediators. Astrocytic control of the blood-brain barrier (BBB) is crucial for normal brain homeostasis and BBB disruption is a well-established early event in MS lesion development. Here, we set out to unravel potential protective effects of reactive astrocytes on BBB function under neuroinflammatory conditions as seen in MS, where we focus on the role of the brain morphogen retinoic acid (RA). Immunohistochemical analysis revealed that retinaldehyde dehydrogenase 2 (RALDH2), a key enzyme for RA synthesis, is highly expressed by reactive astrocytes throughout white matter lesions compared to control and normal appearing white matter. In vitro modeling of reactive astrocytes resulted in increased expression of RALDH2, enhanced RA synthesis, and a protective role for astrocyte-derived RA on BBB function during inflammation-induced barrier loss. Furthermore, RA induces endothelial immune quiescence and decreases monocyte adhesion under inflammatory conditions. Finally, we demonstrated that RA attenuated oxidative stress in inflamed endothelial cells, through activation of the antioxidant transcription factor nuclear factor E2 related factor 2. In summary, RA synthesis by reactive astrocytes represents an endogenous protective response to neuroinflammation, possibly aimed at protecting the BBB against inflammatory insult. A better understanding of RA signaling in MS pathophysiology may lead to the discovery of novel targets to halt disease progression.

P-glycoprotein regulates trafficking of CD8(+) T cells to the brain parenchyma.

The trafficking of cytotoxic CD8(+) T lymphocytes across the lining of the cerebral vasculature is key to the onset of the chronic neuro-inflammatory disorder multiple sclerosis. However, the mechanisms controlling their final transmigration across the brain endothelium remain unknown. Here, we describe that CD8(+) T lymphocyte trafficking into the brain is dependent on the activity of the brain endothelial adenosine triphosphate-binding cassette transporter P-glycoprotein. Silencing P-glycoprotein activity selectively reduced the trafficking of CD8(+) T cells across the brain endothelium in vitro as well as in vivo. In response to formation of the T cell-endothelial synapse, P-glycoprotein was found to regulate secretion of endothelial (C-C motif) ligand 2 (CCL2), a chemokine that mediates CD8(+) T cell migration in vitro. Notably, CCL2 levels were significantly enhanced in microvessels isolated from human multiple sclerosis lesions in comparison with non-neurological controls. Endothelial cell-specific elimination of CCL2 in mice subjected to experimental autoimmune encephalomyelitis also significantly diminished the accumulation of CD8(+) T cells compared to wild-type animals. Collectively, these results highlight a novel (patho)physiological role for P-glycoprotein in CD8(+) T cell trafficking into the central nervous system during neuro-inflammation and illustrate CCL2 secretion as a potential link in this mechanism.

Disturbed function of the blood-cerebrospinal fluid barrier aggravates neuro-inflammation.

Multiple sclerosis (MS) is a chronic neuro-inflammatory disorder, which is marked by the invasion of the central nervous system by monocyte-derived macrophages and autoreactive T cells across the brain vasculature. Data from experimental animal models recently implied that the passage of leukocytes across the brain vasculature is preceded by their traversal across the blood-cerebrospinal fluid barrier (BCSFB) of the choroid plexus. The correlation between the presence of leukocytes in the CSF of patients suffering from MS and the number of inflammatory lesions as detected by magnetic resonance imaging suggests that inflammation at the choroid plexus contributes to the disease, although in a yet unknown fashion. We here provide first insights into the involvement of the choroid plexus in the onset and severity of the disease and in particular address the role of the tight junction protein claudin-3 (CLDN3) in this process. Detailed analysis of human post-mortem brain tissue revealed a selective loss of CLDN3 at the choroid plexus in MS patients compared to control tissues. Importantly, mice that lack CLDN3 have an impaired BCSFB and experience a more rapid onset and exacerbated clinical signs of experimental autoimmune encephalomyelitis, which coincides with enhanced levels of infiltrated leukocytes in their CSF. Together, this study highlights a profound role for the choroid plexus in the pathogenesis of multiple sclerosis, and implies that CLDN3 may be regarded as a crucial and novel determinant of BCSFB integrity.

Roles for HB-EGF and CD9 in multiple sclerosis.

Early events in multiple sclerosis (MS) lesion formation are loss of blood-brain barrier (BBB) integrity, immune cell trafficking into the central nervous system, and demyelination. To date, the molecular mechanisms underlying these pathogenic events are poorly understood. Heparin-binding epidermal growth factor (HB-EGF) is a trophic factor that is induced by inflammatory stimuli and has previously been shown to interact with tetraspanins (TSPs), a family of transmembrane proteins that are involved in cellular migration and adhesion. Given the known roles of TSPs and HB-EGF, we hypothesized that HB-EGF and TSPs may play a role in the processes that underlie MS lesion formation. We examined the expression of HB-EGF and the TSPs CD9 and CD81 in MS brain and found that HB-EGF was highly induced in reactive astrocytes in active lesions. TSPs were constitutively expressed throughout normal appearing white matter and control white matter. In contrast, CD9 was reduced in demyelinated lesions and increased on blood vessels in lesion areas. In vitro studies revealed that expression of HB-EGF and TSPs is regulated during inflammation. Importantly, blocking either HB-EGF or CD9 significantly reduced the migration of monocytes across brain endothelial cell monolayers. Moreover, blocking CD9 strongly enhanced the barrier function of the BBB in vitro. Together, we demonstrate that these molecules are likely implicated in processes that are highly relevant for MS lesion formation, and therefore, HB-EGF and TSPs are promising therapeutic targets.

Reduced expression of PGC-1α partly underlies mitochondrial changes and correlates with neuronal loss in multiple sclerosis cortex.

There is growing evidence that mitochondrial dysfunction and associated reactive oxygen species (ROS) formation contribute to neurodegenerative processes in multiple sclerosis (MS). Here, we investigated whether alterations in transcriptional regulators of key mitochondrial proteins underlie mitochondrial dysfunction in MS cortex and contribute to neuronal loss. Hereto, we analyzed the expression of mitochondrial transcriptional (co-)factors and proteins involved in mitochondrial redox balance regulation in normal-appearing grey matter (NAGM) samples of cingulate gyrus and/or frontal cortex from 15 MS patients and nine controls matched for age, gender and post-mortem interval. PGC-1α, a transcriptional co-activator and master regulator of mitochondrial function, was consistently and significantly decreased in pyramidal neurons in the deeper layers of MS cortex. Reduced PGC-1α levels coincided with reduced expression of oxidative phosphorylation subunits and a decrease in gene and protein expression of various mitochondrial antioxidants and uncoupling proteins (UCPs) 4 and 5. Short-hairpin RNA-mediated silencing of PGC-1α in a neuronal cell line confirmed that reduced levels of PGC-1α resulted in a decrease in transcription of OxPhos subunits, mitochondrial antioxidants and UCPs. Moreover, PGC-1α silencing resulted in a decreased mitochondrial membrane potential, increased ROS formation and enhanced susceptibility to ROS-induced cell death. Importantly, we found extensive neuronal loss in NAGM from cingulate gyrus and frontal cortex of MS patients, which significantly correlated with the extent of PGC-1α decrease. Taken together, our data indicate that reduced neuronal PGC-1α expression in MS cortex partly underlies mitochondrial dysfunction in MS grey matter and thereby contributes to neurodegeneration in MS cortex.

Myc-associated zinc finger protein (MAZ) is regulated by miR-125b and mediates VEGF-induced angiogenesis in glioblastoma.

In patients with glioblastomas, vascular endothelial growth factor (VEGF) is a key mediator of tumor-associated angiogenesis. Glioblastomas are notorious for their capacity to induce neovascularization, driving continued tumor growth. Here we report that miR-125b is down-regulated in glioblastoma-associated endothelial cells, resulting in increased expression of its target, myc-associated zinc finger protein (MAZ), a transcription factor that regulates VEGF. The down-regulation of miR-125b was also observed on exposure of endothelial cells to glioblastoma-conditioned medium or VEGF, resulting in increased MAZ expression. Further analysis revealed that inhibition of MAZ accumulation by miR-125b, or by MAZ-specific shRNAs, attenuated primary human brain endothelial cell migration and tubule formation in vitro, phenomena considered to mimick angiogenic processes in vitro. Moreover, MAZ expression was elevated in brain blood vessels of glioblastoma patients. Altogether these results demonstrate a functional feed-forward loop in glioblastoma-related angiogenesis, in which VEGF inhibits the expression of miR-125b, resulting in increased expression of MAZ, which in its turn causes transcriptional activation of VEGF. This loop is functionally impeded by the VEGF receptor inhibitor vandetanib, and our results may contribute to the further development of inhibitors of tumor-angiogenesis.

NADPH oxidase expression in active multiple sclerosis lesions in relation to oxidative tissue damage and mitochondrial injury.

Multiple sclerosis is a chronic inflammatory disease of the central nervous system, associated with demyelination and neurodegeneration. The mechanisms of tissue injury are poorly understood, but recent data suggest that mitochondrial injury may play an important role in this process. Mitochondrial injury can be triggered by reactive oxygen and nitric oxide species, and we recently provided evidence for oxidative damage of oligodendrocytes and dystrophic axons in early stages of active multiple sclerosis lesions. In this study, we identified potential sources of reactive oxygen and nitrogen species through gene expression in carefully staged and dissected lesion areas and by immunohistochemical analysis of protein expression. Genome-wide microarrays confirmed mitochondrial injury in active multiple sclerosis lesions, which may serve as an important source of reactive oxygen species. In addition, we found differences in the gene expression levels of various nicotinamide adenine dinucleotide phosphate oxidase subunits between initial multiple sclerosis lesions and control white matter. These results were confirmed at the protein level by means of immunohistochemistry, showing upregulation of the subunits gp91phox, p22phox, p47phox, nicotinamide adenine dinucleotide phosphate oxidase 1 and nicotinamide adenine dinucleotide phosphate oxidase organizer 1 in activated microglia in classical active as well as slowly expanding lesions. The subunits gp91phox and p22phox were constitutively expressed in microglia and were upregulated in the initial lesion. In contrast, p47phox, nicotinamide adenine dinucleotide phosphate oxidase 1 and nicotinamide adenine dinucleotide phosphate oxidase organizer 1 expression were more restricted to the zone of initial damage or to lesions from patients with acute or early relapsing/remitting multiple sclerosis. Double labelling showed co-expression of the nicotinamide adenine dinucleotide phosphate oxidase subunits in activated microglia and infiltrated macrophages, suggesting the assembly of functional complexes. Our data suggest that the inflammation-associated oxidative burst in activated microglia and macrophages plays an important role in demyelination and free radical-mediated tissue injury in the pathogenesis of multiple sclerosis.

Brain endothelial barrier passage by monocytes is controlled by the endothelin system.

Homeostasis of the brain is dependent on the blood-brain barrier (BBB). This barrier tightly regulates the exchange of essential nutrients and limits the free flow of immune cells into the CNS. Perturbations of BBB function and the loss of its immune quiescence are hallmarks of a variety of brain diseases, including multiple sclerosis (MS), vascular dementia, and stroke. In particular, diapedesis of monocytes and subsequent trafficking of monocyte-derived macrophages into the brain are key mediators of demyelination and axonal damage in MS. Endothelin-1 (ET-1) is considered as a potent pro-inflammatory peptide and has been implicated in the development of cardiovascular diseases. Here, we studied the role of different components of the endothelin system, i.e., ET-1, its type B receptor (ET(B)) and endothelin-converting enzyme-1 (ECE-1) in monocyte diapedesis of a human brain endothelial cell barrier. Our pharmacological inhibitory and specific gene knockdown studies point to a regulatory function of these proteins in transendothelial passage of monocytes. Results from this study suggest that the endothelin system is a putative target within the brain for anti-inflammatory treatment in neurological diseases.

Adenosine triphosphate-binding cassette transporters mediate chemokine (C-C motif) ligand 2 secretion from reactive astrocytes: relevance to multiple sclerosis pathogenesis.

Adenosine triphosphate-binding cassette efflux transporters are highly expressed at the blood-brain barrier and actively hinder passage of harmful compounds, thereby maintaining brain homoeostasis. Since, adenosine triphosphate-binding cassette transporters drive cellular exclusion of potential neurotoxic compounds or inflammatory molecules, alterations in their expression and function at the blood-brain barrier may contribute to the pathogenesis of neuroinflammatory disorders, such as multiple sclerosis. Therefore, we investigated the expression pattern of different adenosine triphosphate-binding cassette efflux transporters, including P-glycoprotein, multidrug resistance-associated proteins-1 and -2 and breast cancer resistance protein in various well-characterized human multiple sclerosis lesions. Cerebrovascular expression of P-glycoprotein was decreased in both active and chronic inactive multiple sclerosis lesions. Interestingly, foamy macrophages in active multiple sclerosis lesions showed enhanced expression of multidrug resistance-associated protein-1 and breast cancer resistance protein, which coincided with their increased function of cultured foamy macrophages. Strikingly, reactive astrocytes display an increased expression of P-glycoprotein and multidrug resistance-associated protein-1 in both active and inactive multiple sclerosis lesions, which correlated with their enhanced in vitro activity on astrocytes derived from multiple sclerosis lesions. To investigate whether adenosine triphosphate-binding cassette transporters on reactive astrocytes can contribute to the inflammatory process, primary cultures of reactive human astrocytes were generated through activation of Toll-like receptor-3 to mimic the astrocytic phenotype as observed in multiple sclerosis lesions. Notably, blocking adenosine triphosphate-binding cassette transporter activity on reactive astrocytes inhibited immune cell migration across a blood-brain barrier model in vitro, which was due to the reduction of astrocytic release of the chemokine (C-C motif) ligand 2. Our data point towards a novel (patho)physiological role for adenosine triphosphate-binding cassette transporters, suggesting that limiting their activity by dampening astrocyte activation may open therapeutic avenues to diminish tissue damage during multiple sclerosis pathogenesis.

TCR gene transfer: MAGE-C2/HLA-A2 and MAGE-A3/HLA-DP4 epitopes as melanoma-specific immune targets.

Adoptive therapy with TCR gene-engineered T cells provides an attractive and feasible treatment option for cancer patients. Further development of TCR gene therapy requires the implementation of T-cell target epitopes that prevent "on-target" reactivity towards healthy tissues and at the same time direct a clinically effective response towards tumor tissues. Candidate epitopes that meet these criteria are MAGE-C2(336-344)/HLA-A2 (MC2/A2) and MAGE-A3(243-258)/HLA-DP4 (MA3/DP4). We molecularly characterized TCRαß genes of an MC2/A2-specific CD8 and MA3/DP4-specific CD4 T-cell clone derived from melanoma patients who responded clinically to MAGE vaccination. We identified MC2/A2 and MA3/DP4-specific TCR-Vα3/Vß28 and TCR-Vα38/Vß2 chains and validated these TCRs in vitro upon gene transfer into primary human T cells. The MC2 and MA3 TCR were surface-expressed and mediated CD8 T-cell functions towards melanoma cell lines and CD4 T-cell functions towards dendritic cells, respectively. We intend to start testing these MAGE-specific TCRs in phase I clinical trial.

Clearance of genotype 1b hepatitis C virus in chimpanzees in the presence of vaccine-induced E1-neutralizing antibodies.

Accumulating evidence indicates that neutralizing antibodies play an important role in protection from chronic hepatitis C virus (HCV) infection. Efforts to elicit such responses by immunization with intact heterodimeric E1E2 envelope proteins have met with limited success. To determine whether antigenic sites, which are not exposed by the combined E1E2 heterodimer structure, are capable of eliciting neutralizing antibody responses, we expressed and purified each as separate recombinant proteins E1 and E2, from which the immunodominant hypervariable region (HVR-1) was deleted. Immunization of chimpanzees with either E1 or E2 alone induced antigen-specific T-helper cytokines of similar magnitude. Unexpectedly, the capacity to neutralize HCV was observed in E1 but not in animals immunized with E2 devoid of HVR-1. Furthermore, in vivo only E1-vaccinated animals exposed to the heterologous HCV-1b inoculum cleared HCV infection.

T lymphocytes impair P-glycoprotein function during neuroinflammation.

The ATP-binding cassette (ABC) transporter P-glycoprotein (P-gp; ABCB1) is highly expressed at the blood-brain barrier (BBB). P-gp actively secretes and keeps the central nervous system (CNS) safe from body-born metabolites, but also from drugs and food components, emphasising the importance of its optimal function to maintain brain homeostasis. Here we demonstrate that vascular P-gp expression and function are strongly decreased during neuroinflammation. In vivo, the expression and function of brain endothelial P-gp in experimental allergic encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), were significantly impaired. Strikingly, vascular P-gp expression was decreased in both MS and EAE lesions and its disappearance coincided with the presence of perivascular infiltrates consisting of lymphocytes. Our data strongly suggest that activated CD4(+) T cells induce impaired function of brain endothelial P-gp. Notably, lymphocyte interaction through endothelial intracellular adhesion molecule -1 (ICAM-1) resulted in activation of a nuclear factor kappa B (NF-kappaB) signaling pathway, which resulted in endothelial P-gp malfunction. Our study provides first evidence that CD4(+) T cells are able to affect endogenous molecular protection mechanisms of brain endothelium. Loss of vascular P-gp function during neuroinflammation may disturb brain homeostasis and thereby aggravate disease progression via exposure of vulnerable CNS cells to detrimental compounds.

In vivo MR imaging of intercellular adhesion molecule-1 expression in an animal model of multiple sclerosis.

Upregulation of intercellular adhesion molecule 1 (ICAM-1) is an early event in lesion formation in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Monitoring its expression may provide a biomarker for early disease activity and allow validation of anti-inflammatory interventions. Our objective was therefore to explore whether ICAM-1 expression can be visualized in vivo during EAE with magnetic resonance imaging (MRI) using micron-sized particles of iron oxide (MPIO), and to compare accumulation profiles of targeted and untargeted MPIO, and a gadolinium-containing agent. Targeted αICAM-1-MPIO/untargeted IgG-MPIO were injected at two model-characteristic phases of EAE (in myelin oligodendrocyte glycoprotein35-55 -immunized C57BL/6 J mice), that is, at the peak of the acute phase (14 ± 1 days post-immunization) and during the chronic phase (26 ± 1 days post-immunization), followed by T2 *-weighted MRI. Blood-brain barrier (BBB) permeability was measured using gadobutrol-enhanced MRI. Cerebellar microvessels were analyzed for ICAM-1 mRNA expression using quantitative PCR (qPCR). ICAM-1 and iron oxide presence was examined with immunohistochemistry (IHC). During EAE, ICAM-1 was expressed by brain endothelial cells, macrophages and T-cells as shown with qPCR and (fluorescent) IHC. EAE animals injected with αICAM-1-MPIO showed MRI hypointensities, particularly in the subarachnoid space. αICAM-1-MPIO presence did not differ between the phases of EAE and was not associated with BBB dysfunction. αICAM-1-MPIO were associated with endothelial cells or cells located at the luminal side of blood vessels. In conclusion, ICAM-1 expression can be visualized with in vivo molecular MRI during EAE, and provides an early tracer of disease activity.

An Altered gp100 Peptide Ligand with Decreased Binding by TCR and CD8α Dissects T Cell Cytotoxicity from Production of Cytokines and Activation of NFAT.

Altered peptide ligands (APLs) provide useful tools to study T cell activation and potentially direct immune responses to improve treatment of cancer patients. To better understand and exploit APLs, we studied the relationship between APLs and T cell function in more detail. Here, we tested a broad panel of gp100280-288 APLs with respect to T cell cytotoxicity, production of cytokines, and activation of Nuclear Factor of Activated T cells (NFAT) by human T cells gene-engineered with a gp100-HLA-A2-specific TCRαß. We demonstrated that gp100-specific cytotoxicity, production of cytokines, and activation of NFAT were not affected by APLs with single amino acid substitutions, except for an APL with an amino acid substitution at position 3 (APL A3), which did not elicit any T cell response. A gp100 peptide with a double amino acid mutation (APL S4S6) elicited T cell cytotoxicity and production of IFNγ, and to a lesser extent TNFα, IL-4, and IL-5, but not production of IL-2 and IL-10, or activation of NFAT. Notably, T cell receptor (TCR)-mediated functions showed decreases in sensitivities for S4S6 versus gp100 wild-type (wt) peptide, which were minor for cytotoxicity but at least a 1000-fold more prominent for the production of cytokines. TCR-engineered T cells did not bind A3-HLA-A2, but did bind S4S6-HLA-A2 although to a lowered extent compared to wt peptide-HLA-A2. Moreover, S4S6-induced T cell function demonstrated an enhanced dependency on CD8α. Taken together, most gp100 APLs functioned as agonists, but A3 and S4S6 peptides acted as a null ligand and partial agonist, respectively. Our results further suggest that TCR-mediated cytotoxicity can be dissected from production of cytokines and activation of NFAT, and that the agonist potential of peptide mutants relates to the extent of binding by TCR and CD8α. These findings may facilitate the design of APLs to advance the study of T cell activation and their use for therapeutic applications.

Nrf2 and DJ1 are consistently upregulated in inflammatory multiple sclerosis lesions.

Oxidative stress plays a major role in multiple sclerosis (MS), a chronic inflammatory central nervous system (CNS) disease. Invading leukocytes contribute to cell damage and demyelination by producing excessive amounts of cytotoxic mediators, including reactive oxygen species (ROS). To counteract the damaging effects of ROS the CNS is endowed with a repertoire of endogenous antioxidant enzymes, which are regulated by the transcription factor NF-E2-related factor 2 (Nrf2). Upon exposure to ROS, Nrf2 translocates to the nucleus allowing transcriptional activation of various antioxidant enzymes. DJ1 is a protein that is involved in the stabilization of Nrf2 and hence acts as a positive regulator of Nrf2-driven antioxidant protection. Here, we investigate the (sub)cellular localization of Nrf2 and DJ1 in various MS lesion stages and show that Nrf2 is strikingly upregulated in active MS lesions, in both the nucleus and the cytoplasm of infiltrating macrophages and to a lesser extent in reactive astrocytes. Simultaneously, DJ1 protein expression is predominantly increased in astrocytes in both active and chronic inactive MS lesions compared to control brain tissue and normal-appearing white matter. Together, our findings suggest that persistent Nrf2-mediated transcription occurs in active MS lesions, but that this endogenous response is insufficient to prevent ROS-induced cellular damage, which is abundant in inflammatory MS lesions.

P-glycoprotein acts as an immunomodulator during neuroinflammation.

BACKGROUND: Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system in which autoreactive myelin-specific T cells cause extensive tissue damage, resulting in neurological deficits. In the disease process, T cells are primed in the periphery by antigen presenting dendritic cells (DCs). DCs are considered to be crucial regulators of specific immune responses and molecules or proteins that regulate DC function are therefore under extensive investigation. We here investigated the potential immunomodulatory capacity of the ATP binding cassette transporter P-glycoprotein (P-gp). P-gp generally drives cellular efflux of a variety of compounds and is thought to be involved in excretion of inflammatory agents from immune cells, like DCs. So far, the immunomodulatory role of these ABC transporters is unknown. METHODS AND FINDINGS: Here we demonstrate that P-gp acts as a key modulator of adaptive immunity during an in vivo model for neuroinflammation. The function of the DC is severely impaired in P-gp knockout mice (Mdr1a/1b-/-), since both DC maturation and T cell stimulatory capacity is significantly decreased. Consequently, Mdr1a/1b -/- mice develop decreased clinical signs of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. Reduced clinical signs coincided with impaired T cell responses and T cell-specific brain inflammation. We here describe the underlying molecular mechanism and demonstrate that P-gp is crucial for the secretion of pro-inflammatory cytokines such as TNF-alpha and IFN-gamma. Importantly, the defect in DC function can be restored by exogenous addition of these cytokines. CONCLUSIONS: Our data demonstrate that P-gp downmodulates DC function through the regulation of pro-inflammatory cytokine secretion, resulting in an impaired immune response. Taken together, our work highlights a new physiological role for P-gp as an immunomodulatory molecule and reveals a possible new target for immunotherapy.

Vaccine-induced early control of hepatitis C virus infection in chimpanzees fails to impact on hepatic PD-1 and chronicity.

UNLABELLED: Broad T cell and B cell responses to multiple HCV antigens are observed early in individuals who control or clear HCV infection. The prevailing hypothesis has been that similar immune responses induced by prophylactic immunization would reduce acute virus replication and protect exposed individuals from chronic infection. Here, we demonstrate that immunization of naïve chimpanzees with a multicomponent HCV vaccine induced robust HCV-specific immune responses, and that all vaccinees exposed to heterologous chimpanzee-adapted HCV 1b J4 significantly reduced viral RNA in serum by 84%, and in liver by 99% as compared to controls (P=0.024 and 0.028, respectively). However, despite control of HCV in plasma and liver in the acute period, in the chronic phase, 3 of 4 vaccinated animals developed persistent infection. Analysis of expression levels of proinflammatory cytokines in serial hepatic biopsies failed to reveal an association with vaccine outcome. However, expression of IDO, CTLA-4 [corrected] and PD-1 levels in liver correlated with clearance or chronicity. CONCLUSION: Despite early control of virus load, a virus-associated tolerogenic-like state can develop in certain individuals independent of vaccination history.

CD8 alpha coreceptor to improve TCR gene transfer to treat melanoma: down-regulation of tumor-specific production of IL-4, IL-5, and IL-10.

Therapeutic success of TCR gene transfer to treat tumors depends on the ability of redirected T cells to become activated upon tumor recognition in vivo. Help provided by tumor-specific Th1 cells is reported to relieve T cells from an anergized state and to induce tumor regression. We recently demonstrated the ability to generate melanoma-specific Th1 cells by genetic introduction of both a CD8-dependent TCR and the CD8alpha coreceptor into CD4+ T cells. In this study, we analyzed a TCR that binds Ag independently of CD8, a property generally preferred to induce tumor-specific T cell responses, and addressed the contribution of CD8alpha following introduction into TCR-transduced CD4+ T cells. To this end, primary human CD4+ T cells were gene transferred with a high-avidity TCR, and were shown not only to bind peptide/MHC class I, but also to effectively kill Ag-positive tumor cells in the absence of CD8alpha. The introduction of CD8alpha up-regulates the tumor-specific production of TNF-alpha and IL-2 to some extent, but significantly down-regulates production of IL-4, IL-5, and IL-10 in CD4+ T cells. The introduction of a mutated cysteine motif in CD8alpha, which prevents its binding to LCK and linker for activation of T cells, did not adversely affect expression and T cell cytotoxicity, but counteracted the CD8alpha-mediated down-regulation of IL-4 and IL-5, but not IL-10. In conclusion, CD8alpha down-regulates the production of major Th2-type cytokines, in part mediated by LCK and/or linker for activation of T cells, and may induce differentiation of tumor-specific Th1 cells, which makes this coreceptor an interesting candidate to improve the clinical potential of TCR gene transfer to treat cancer.