Molecular and immunological evaluation of the expression of cancer/testis gene products in human colorectal cancer.

Tumor-specific gene products, such as cancer/testis (CT) antigens, constitute promising targets for the development of T cell vaccines. Whereas CT antigens are frequently expressed in melanoma, their expression in colorectal cancers (CRC) remains poorly characterized. Here, we have studied the expression of the CT antigens MAGE-A3, MAGE-A4, MAGE-A10, NY-ESO-1 and SSX2 in CRC because of the presence of well-described HLA-A2-restricted epitopes in their sequences. Our analyses of 41 primary CRC and 14 metastatic liver lesions confirmed the low frequency of expression of these CT antigens. No increased expression frequencies were observed in metastatic tumors compared to primary tumors. Histological analyses of CRC samples revealed heterogeneous expression of individual CT antigens. Finally, evidence of a naturally acquired CT antigen-specific CD8(+) T cell response could be demonstrated. These results show that the expression of CT antigens in a subset of CRC patients induces readily detectable T cell responses.

Essential role of the Wnt pathway effector Tcf-1 for the establishment of functional CD8 T cell memory.

Immune protection from intracellular pathogens depends on the generation of terminally differentiated effector and of multipotent memory precursor CD8 T cells, which rapidly regenerate effector and memory cells during recurrent infection. The identification of factors and pathways involved in CD8 T cell differentiation is of obvious importance to improve vaccination strategies. Here, we show that mice lacking T cell factor 1 (Tcf-1), a nuclear effector of the canonical Wingless/Integration 1 (Wnt) signaling pathway, mount normal effector and effector memory CD8 T cell responses to infection with lymphocytic choriomeningitis virus (LCMV). However, Tcf-1-deficient CD8 T cells are selectively impaired in their ability to expand upon secondary challenge and to protect from recurrent virus infection. Tcf-1-deficient mice essentially lack CD8 memory precursor T cells, which is evident already at the peak of the primary response, suggesting that Tcf-1 programs CD8 memory cell fate. The function of Tcf-1 to establish CD8 T cell memory is dependent on the catenin-binding domain in Tcf-1 and requires the Tcf-1 coactivators and Wnt signaling intermediates beta-catenin and gamma-catenin. These findings demonstrate that the canonical Wnt signaling pathway plays an essential role for CD8 central memory T cell differentiation under physiological conditions in vivo. They raise the possibility that modulation of Wnt signaling may be exploited to improve the generation of CD8 memory T cells during vaccination or for therapies designed to promote sustained cytotoxic CD8 T cell responses against tumors.

The neurobiological basis of prefrontal cortex impairment in the phencyclidine model of schizophrenia : convergent reduction of synaptic glutamate release, energy metabolism and cognitive performance

RÉSUMÉ : Le traitement répété à la phencyclidine (PCP), un bloqueur du récepteur NMDA (NMDAR), reproduit chez les rongeurs une partie de la symptomatologie typique de la schizophrénie. Le blocage prolongé du NMDAR par la PCP mime une hypofunction du NMDAR, une des principales altérations supposées exister dans les cerveaux des patients schizophréniques. Le but de notre étude était d'examiner les conséquences neurochimiques, métaboliques et fonctionnelles du traitement répété à la phencyclidine in vivo, au niveau du cortex préfrontal (cpf), une région cérébrale qui joue un rôle dans les déficits cognitifs observés chez les patients schizophréniques. Pour répondre à cette question, les rats ou les souris ont reçu chaque jour une injection soit de PCP (5 mg/kg), soit de solution saline, pendant 7 ou 14 jours. Les animaux ont ensuite été sacrifiés au moins 24 heures après le dernier traitement. Des tranches aiguës du cpf ont été préparées rapidement, puis stimulées avec une concentration élevée de KCI, de manière à induire une libération de glutamate à partir des terminaisons synaptiques excitatrices. Les résultats montrent que les tranches du cpf des animaux traités à la PCP ont libéré une quantité de glutamate significativement inférieure par rapport à celles des animaux contrôle. Ce déficit de libération a persisté 72 heures après la fin du traitement, tandis qu'il n'était pas observé dans le cortex visuel primaire, une autre région corticale. En outre, le traitement avec des antipsychotiques, l'halopéridol ou l'olanzapine, a supprimé le déficit induit par la PCP. Le même déficit de libération a été remarqué sur des synaptosomes obtenus à partir du cpf des animaux traités à la phenryclidine. Cette observation indique que la PCP induit une modification plastique adaptative du mécanisme qui contrôle la libération du glutamate dans les terminaisons synaptiques. Nous avons découvert que cette modification implique la sous-régulation d'un NMDAR présynaptique, qui serait doué d'un rôle d'autorécepteur stimulateur de la libération du glutamate. Grâce à des tests comportementaux conduits en parallèle et réalisés pour évaluer la fonctionnalité du cpf, nous avons observé chez les souris traitées à la PCP une flexibilité comportementale réduite lors d'un test de discrimination de stimuli visuels/tactiles. Le déficit cognitif était encore présent 4 jours après la dernière administration de PCP. La technique de l'autoradiographie quantitative du [14C]2-deoxyglucose a permis d'associer ce déficit à une réduction de l'activité métabolique cérébrale pendant le déroulement du test, particulièrement au niveau du cpf. Dans l'ensemble, nos résultats suggèrent que le blocage prolongé du NMDAR lors de l'administration répétée de PCP produit un déficit de libération du glutamate au niveau des terminaisons synaptiques excitatrices du cpf. Un tel déficit pourrait être provoqué par la sousrégulation d'un NMDAR présynaptique, qui aurait une fonction de stimulateur de libération; la transmission excitatrice du cpf s'en trouverait dans ce cas réduite. Ce résultat est en ligne avec l'activité métabolique et fonctionnelle réduite du cpf et l'observation de déficits cognitifs induits lors de l'administration de la PCP. ABSTRACT : Sub-chronic treatment with phencyclidine (PCP), an NMDA receptor (NMDAR) channel blocker, reproduces in rodents part of the symptomatology associated to schizophrenia in humans. Prolonged pharmacological blockade of NMDAR with PCP mimics NMDAR hypofunction, one of the main alterations thought to take place in the brains of schizophrenics. Our study was aimed at investigating the neurochemical, metabolic and behavioral consequences of repeated PCP administration in vivo, focusing on the functioning of the prefrontal cortex (pfc), a brain region highly relevant for the cognitive deficits observed in schizophrenic patients. Rats or mice received a daily administration of either PCP (5 mg/kg) or saline for 7 or 14 days. At least 24 hours after the last treatment the animals were sacrificed. Acute slices of the pfc were quickly prepared and challenged with high KCl to induce synaptic glutamate release. Pfc slices from PCP-treated animals released significantly less glutamate than slices from salinetreated animals. The deficit persisted 72 hours after the end of the treatment, while it was not observed in another cortical region: the primary visual cortex. Interestingly, treatment with antipsychotic drugs, either haloperidol or olanzapine, reverted the glutamate release defect induced by PCP treatment. The same release defect was observed in synaptosomes prepared from the pfc of PCP-treated animals, indicating that PCP induces a plastic adaptive change in the mechanism controlling glutamate release in the glutamatergic terminals. We discovered that such change most likely involves the down-regulation of a newly identified, pre-synaptic NMDAR with stimulatory auto-receptor function on glutamate release. In parallel sets of behavioral experiments challenging pfc function, mice sub-chronically treated with PCP displayed reduced behavioral flexibility (reversal learning) in a visual/tactile-cued discrimination task. The cognitive deficit was still evident 4 days after the last PCP administration and was associated to reduced brain metabolic activity during the performance of the behavioral task, notably in the pfc, as determined by [14C]2-deoxyglucose quantitative autoradiography. Clverall, our findings suggest that prolonged NMDAR blockade by repeated PCP administration results in a defect of glutamate release from excitatory afferents in the pfc, possibly ascribed to down-regulation of apre-synaptic stimulatory NMDAR. Deficient excitatory neurotransmission in the pfc is consistent with the reduced metabolic and functional activation of this area and the observed PCP-induced cognitive deficits.

Gene expression profiling of isolated tumour cells from anaplastic large cell lymphomas: insights into its cellular origin, pathogenesis and relation to Hodgkin lymphoma.

Anaplastic large cell lymphoma (ALCL) is a main type of T-cell lymphomas and comprises three distinct entities: systemic anaplastic lymphoma kinase (ALK) positive, systemic ALK(-) and cutaneous ALK(-) ALCL (cALCL). Little is known about their pathogenesis and their cellular origin, and morphological and immunophenotypical overlap exists between ALK(-) ALCL and classical Hodgkin lymphoma (cHL). We conducted gene expression profiling of microdissected lymphoma cells of five ALK(+) and four ALK(-) systemic ALCL, seven cALCL and sixteen cHL, and of eight subsets of normal T and NK cells. The analysis supports a derivation of ALCL from activated T cells, but the lymphoma cells acquired a gene expression pattern hampering an assignment to a CD4(+), CD8(+) or CD30(+) T-cell origin. Indeed, ALCL display a down-modulation of many T-cell characteristic molecules. All ALCL types show significant expression of NFkappaB target genes and upregulation of genes involved in oncogenesis (e.g. EZH2). Surprisingly, few genes are differentially expressed between systemic and cALCL despite their different clinical behaviour, and between ALK(-) ALCL and cHL despite their different cellular origin. ALK(+) ALCL are characterized by expression of genes regulated by pathways constitutively activated by ALK. This study provides multiple novel insights into the molecular biology and pathogenesis of ALCL.

Dissecting TCR-MHC/peptide complex interactions with A2/peptide multimers incorporating tumor antigen peptide variants: crucial role of interaction kinetics on functional outcomes.

Soluble peptide/MHC-class-I (pMHC) multimers have recently emerged as unique reagents for the study of specific interactions between the pMHC complex and the TCR. Here, we assessed the relative binding efficiency of a panel of multimers incorporating single-alanine-substituted variants of the tumor-antigen-derived peptide MAGE-A10(254-262) to specific CTL clones displaying different functional avidity. For each individual clone, the efficiency of binding of multimers incorporating MAGE-A10 peptide variants was, in most cases, in good although not linear correlation with the avidity of recognition of the corresponding variant. In addition, we observed two types of discrepancies between efficiency of recognition and multimer binding. First, for some peptide variants, efficient multimer binding was detected in the absence of measurable effector functions. Some of these peptide variants displayed antagonist activity. Second, when comparing different clones we found clear discrepancies between the dose of peptide required to obtain half-maximal lysis in CTL assays and the binding efficiency of the corresponding multimers. These discrepancies, however, were resolved when the differential stability of the TCR/pMHC complexes was determined. For individual clones, decreased recognition correlated with increased TCR/pMHC off-rate. TCR/pMHC complexes formed by antagonist ligands displayed off-rates faster than those of TCR/pMHC complexes formed with weak agonists. In addition, when comparing different clones, the efficiency of multimer staining correlated better with relative multimer off-rates than with half-maximal lysis values. Altogether, the data presented here reconcile and extend our previous results on the impact of the kinetics of interaction of TCR with pMHC complexes on multimer binding and underline the crucial role of TCR/pMHC off-rates for the functional outcome of such interactions.

T cells dampen innate immune responses through inhibition of NLRP1 and NLRP3 inflammasomes.

Inflammation is a protective attempt by the host to remove injurious stimuli and initiate the tissue healing process. The inflammatory response must be actively terminated, however, because failure to do so can result in 'bystander' damage to tissues and diseases such as arthritis or type-2 diabetes. Yet the mechanisms controlling excessive inflammatory responses are still poorly understood. Here we show that mouse effector and memory CD4(+) T cells abolish macrophage inflammasome-mediated caspase-1 activation and subsequent interleukin 1beta release in a cognate manner. Inflammasome inhibition is observed for all tested NLRP1 (commonly called NALP1) and NLRP3 (NALP3 or cryopyrin) activators, whereas NLRC4 (IPAF) inflammasome function and release of other inflammatory mediators such as CXCL2, interleukin 6 and tumour necrosis factor are not affected. Suppression of the NLRP3 inflammasome requires cell-to-cell contact and can be mimicked by macrophage stimulation with selected ligands of the tumour necrosis factor family, such as CD40L (also known as CD40LG). In a NLRP3-dependent peritonitis model, effector CD4(+) T cells are responsible for decreasing neutrophil recruitment in an antigen-dependent manner. Our findings reveal an unexpected mechanism of inflammasome inhibition, whereby effector and memory T cells suppress potentially damaging inflammation, yet leave the primary inflammatory response, crucial for the onset of immunity, intact.

Light-induced retinal degeneration correlates with changes in iron metabolism gene expression, ferritin level, and aging.

PURPOSE: Retinal degeneration is associated with iron accumulation in several rodent models in which iron-regulating proteins are impaired. Oxidative stress is catalyzed by unbound iron. METHODS: The role of the heavy chain of ferritin, which sequesters iron, in regulating the thickness of the photoreceptor nuclear layer in the 4- and 16-month-old wild-type H ferritin (HFt(+/+)) and heterozygous H ferritin (HFt(+/-)) mice was investigated, before and 12 days after exposure to 13,000-lux light for 24 hours. The regulation of gene expression of the various proteins involved in iron homeostasis, such as transferrin, transferrin receptor, hephaestin, ferroportin, iron regulatory proteins 1 and 2, hepcidin, ceruloplasmin, and heme-oxygenase 1, was analyzed by quantitative (q)RT-PCR during exposure (2, 12, and 24 hours) and 24 hours after 1 day of exposure in the 4-month-old HFt(+/+) and HFt(+/-) mouse retinas. RESULTS: Retinal degeneration in the 4-month-old HFt(+/-) mice was more extensive than in the HFt(+/+) mice. Yet, it was more extensive in both of the 16-month-old mouse groups, revealing the combined effect of age and excessive light. Injury caused by excessive light modified the temporal gene expression of iron-regulating proteins similarly in the HFt(+/-) and HFt(+/+) mice. CONCLUSIONS: Loss of one allele of H ferritin appears to increase light-induced degeneration. This study highlighted that oxidative stress related to light-induced injury is associated with major changes in gene expression of iron metabolism proteins.

Catabolite repression control of pyocyanin biosynthesis at an intersection of primary and secondary metabolism in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, the catabolite repression control (Crc) protein repressed the formation of the blue pigment pyocyanin in response to a preferred carbon source (succinate) by interacting with phzM mRNA, which encodes a key enzyme in pyocyanin biosynthesis. Crc bound to an extended imperfect recognition sequence that was interrupted by the AUG translation initiation codon.

Developmentally regulated expression of P-glycoprotein (multidrug resistance) activity in mouse thymocytes.

P-glycoprotein (P-gly) is the transmembrane efflux pump responsible for multidrug resistance in tumor cells. The activity of P-gly in mature peripheral lymphocytes is lineage specific, with CD8+ T cells and natural killer (NK) cells expressing high levels as compared to CD4+ T cells and B cells. We have now investigated P-gly activity in immature and mature subsets of mouse thymocytes. Our data indicate that P-gly activity is undetectable in immature CD4-8- and CD4+8+ thymocyte subsets. Among mature thymocytes, P-gly activity is absent in the CD4+ subset but present in the more mature (HSAlow) fraction of CD8+ cells. Furthermore, while thymic CD4-8- T cell receptor (TCR) gamma delta cells have little P-gly activity, a minor subset of CD4-8- or CD4+ TCR alpha beta + thymocytes bearing the NK1.1 surface marker expresses high levels of P-gly activity. Collectively, our results indicate that P-gly activity arises late during thymus development and is expressed in a lineage-specific fashion.

Transforming growth factor beta 1 production by CD4+ CD25+ regulatory T cells in peripheral blood mononuclear cells from healthy subjects stimulated with Leishmania guyanensis.

Transforming growth factor beta (TGF-beta) has been shown to be a central immunomodulator used by leishmaniae to escape effective mechanisms of protection in human and murine infections with these parasites. However, all the information is derived from studies of established infection, while little is known about TGF-beta production in response to Leishmania stimulation in healthy subjects. In this study, TGF-beta1 production was demonstrated in peripheral blood mononuclear cells from healthy subjects never exposed to leishmaniae in response to live Leishmania guyanensis, and the TGF-beta1-producing cells were described as a distinct subpopulation of CD4(+) CD25(+) regulatory T cells. The suppressive properties of CD4(+) CD25(+) T cells were demonstrated in vitro by their inhibition of production of interleukin 2 (IL-2) and IL-10 by CD4(+) CD25(-) T cells in the presence of either anti-CD3 or L. guyanensis. Although neutralization of TGF-beta1 did not reverse the suppressive activity of CD4(+) CD25(+) T cells activated by anti-CD3, it reversed the suppressive activity of CD4(+) CD25(+) T cells activated by L. guyanensis. Altogether our data demonstrated that TGF-beta1 is involved in the suppressive activity of L. guyanensis-stimulated CD4(+) CD25(+) T cells from healthy controls.

Ectopic human telomerase catalytic subunit expression maintains telomere length but is not sufficient for CD8+ T lymphocyte immortalization.

Like most somatic human cells, T lymphocytes have a limited replicative life span. This phenomenon, called senescence, presents a serious barrier to clinical applications that require large numbers of Ag-specific T cells such as adoptive transfer therapy. Ectopic expression of hTERT, the human catalytic subunit of the enzyme telomerase, permits fibroblasts and endothelial cells to avoid senescence and to become immortal. In an attempt to immortalize normal human CD8(+) T lymphocytes, we infected bulk cultures or clones of these cells with a retrovirus transducing an hTERT cDNA clone. More than 90% of transduced cells expressed the transgene, and the cell populations contained high levels of telomerase activity. Measuring the content of total telomere repeats in individual cells (by flowFISH) we found that ectopic hTERT expression reversed the gradual loss of telomeric DNA observed in control populations during long term culture. Telomere length in transduced cells reached the levels observed in freshly isolated normal CD8(+) lymphocytes. Nevertheless, all hTERT-transduced populations stopped to divide at the same time as nontransduced or vector-transduced control cells. When kept in IL-2 the arrested cells remained alive. Our results indicate that hTERT may be required but is not sufficient to immortalize human T lymphocytes.

The biochemistry of drug metabolism-an introduction: part 7. Intra-individual factors affecting drug metabolism.

This review on intra-individual factors affecting drug metabolism completes our series on the biochemistry of drug metabolism. The article presents the molecular mechanisms causing intra-individual differences in enzyme expression and activity. They include enzyme induction by transcriptional activation and enzyme inhibition on the protein level. The influencing factors are of physiological, pathological, or external origin. Tissue characteristics and developmental age strongly influence enzyme-expression patterns. Further influencing factors are pregnancy, disease, or biological rhythms. Xenobiotics, drugs, constituents of herbal remedies, food constituents, ethanol, and tobacco can all influence enzyme expression or activity and, hence, affect drug metabolism.

Delta-like 4 is the essential, nonredundant ligand for Notch1 during thymic T cell lineage commitment.

Thymic T cell lineage commitment is dependent on Notch1 (N1) receptor-mediated signaling. Although the physiological ligands that interact with N1 expressed on thymic precursors are currently unknown, in vitro culture systems point to Delta-like 1 (DL1) and DL4 as prime candidates. Using DL1- and DL4-lacZ reporter knock-in mice and novel monoclonal antibodies to DL1 and DL4, we show that DL4 is expressed on thymic epithelial cells (TECs), whereas DL1 is not detected. The function of DL4 was further explored in vivo by generating mice in which DL4 could be specifically inactivated in TECs or in hematopoietic progenitors. Although loss of DL4 in hematopoietic progenitors did not perturb thymus development, inactivation of DL4 in TECs led to a complete block in T cell development coupled with the ectopic appearance of immature B cells in the thymus. These immature B cells were phenotypically indistinguishable from those developing in the thymus of conditional N1 mutant mice. Collectively, our results demonstrate that DL4 is the essential and nonredundant N1 ligand responsible for T cell lineage commitment. Moreover, they strongly suggest that N1-expressing thymic progenitors interact with DL4-expressing TECs to suppress B lineage potential and to induce the first steps of intrathymic T cell development.

Impaired glucose metabolism in the heart of obese Zucker rats after treatment with phorbol ester.

OBJECTIVE: To investigate the influence of obesity on the regulation of myocardial glucose metabolism following protein kinase C (PKC) activation in obese (fa/fa) and lean (Fa/?) Zucker rats. DESIGN: Isolated hearts obtained from 17-week-old lean and obese Zucker rats were perfused with 200 nM phorbol 12-myristate 13-acetate (PMA) for different time periods prior to the evaluation of PKC and GLUT-4 translocation. For metabolic studies isolated hearts from 48 h starved Zucker rats were perfused with an erythrocytes-enriched buffer containing increased concentrations (10-100 nM) of PMA. MEASUREMENTS: Immunodetectable PKC isozymes and GLUT-4 were determined by Western blots. Glucose oxidation and glycolysis were evaluated by measuring the myocardial release of 14CO2 and 3H2O from [U-14C]glucose and [5-3H]glucose, respectively. RESULTS: PMA (200 nM) induced maximal translocation of ventricular PKCalpha from the cytosol to the membranes within 10 min. This translocation was 2-fold lower in the heart from obese rats when compared to lean rats. PMA also induced a significant translocation of ventricular GLUT-4 from the microsomal to the sarcolemmal fraction within 60 min in lean but not in obese rats. Rates of basal cardiac glucose oxidation and glycolysis in obese rats were approximately 2-fold lower than those of lean rats. Perfusion with increasing concentrations of PMA (10-100 nM) led to a significant decrease of cardiac glucose oxidation in lean but not in obese rats. CONCLUSION: Our results show that in the heart of the genetically obese Zucker rat, the impairment in PKCalpha activation is in line with a diminished activation of GLUT-4 as well as with the lack of PMA effect on glucose oxidation.

Activity-dependent regulation of energy metabolism by astrocytes: an update.

Astrocytes play a critical role in the regulation of brain metabolic responses to activity. One detailed mechanism proposed to describe the role of astrocytes in some of these responses has come to be known as the astrocyte-neuron lactate shuttle hypothesis (ANLSH). Although controversial, the original concept of a coupling mechanism between neuronal activity and glucose utilization that involves an activation of aerobic glycolysis in astrocytes and lactate consumption by neurons provides a heuristically valid framework for experimental studies. In this context, it is necessary to provide a survey of recent developments and data pertaining to this model. Thus, here, we review very recent experimental evidence as well as theoretical arguments strongly supporting the original model and in some cases extending it. Aspects revisited include the existence of glutamate-induced glycolysis in astrocytes in vitro, ex vivo, and in vivo, lactate as a preferential oxidative substrate for neurons, and the notion of net lactate transfer between astrocytes and neurons in vivo. Inclusion of a role for glycogen in the ANLSH is discussed in the light of a possible extension of the astrocyte-neuron lactate shuttle (ANLS) concept rather than as a competing hypothesis. New perspectives offered by the application of this concept include a better understanding of the basis of signals used in functional brain imaging, a role for neuron-glia metabolic interactions in glucose sensing and diabetes, as well as novel strategies to develop therapies against neurodegenerative diseases based upon improving astrocyte-neuron coupled energetics.