Stem cell transplantation in brain tumors: a new field for molecular imaging?

Neural stem cells have been proposed as a new and promising treatment modality in various pathologies of the central nervous system, including malignant brain tumors. However, the underlying mechanism by which neural stem cells target tumor areas remains elusive. Monitoring of these cells is currently done by use of various modes of molecular imaging, such as optical imaging, magnetic resonance imaging and positron emission tomography, which is a novel technology for visualizing metabolism and signal transduction to gene expression. In this new context, the microenvironment of (malignant) brain tumors and the blood-brain barrier gains increased interest. The authors of this review give a unique overview of the current molecular-imaging techniques used in different therapeutic experimental brain tumor models in relation to neural stem cells. Such methods for molecular imaging of gene-engineered neural stem/progenitor cells are currently used to trace the location and temporal level of expression of therapeutic and endogenous genes in malignant brain tumors, closing the gap between in vitro and in vivo integrative biology of disease in neural stem cell transplantation.

Tumor necrosis factor (TNF) signaling, but not TWEAK (TNF-like weak inducer of apoptosis)-triggered cIAP1 (cellular inhibitor of apoptosis protein 1) degradation, requires cIAP1 RING dimerization and E2 binding.

Cellular inhibitor of apoptosis (cIAP) proteins, cIAP1 and cIAP2, are important regulators of tumor necrosis factor (TNF) superfamily (SF) signaling and are amplified in a number of tumor types. They are targeted by IAP antagonist compounds that are undergoing clinical trials. IAP antagonist compounds trigger cIAP autoubiquitylation and degradation. The TNFSF member TWEAK induces lysosomal degradation of TRAF2 and cIAPs, leading to elevated NIK levels and activation of non-canonical NF-kappaB. To investigate the role of the ubiquitin ligase RING domain of cIAP1 in these pathways, we used cIAP-deleted cells reconstituted with cIAP1 point mutants designed to interfere with the ability of the RING to dimerize or to interact with E2 enzymes. We show that RING dimerization and E2 binding are required for IAP antagonists to induce cIAP1 degradation and protect cells from TNF-induced cell death. The RING functions of cIAP1 are required for full TNF-induced activation of NF-kappaB, however, delayed activation of NF-kappaB still occurs in cIAP1 and -2 double knock-out cells. The RING functions of cIAP1 are also required to prevent constitutive activation of non-canonical NF-kappaB by targeting NIK for proteasomal degradation. However, in cIAP double knock-out cells TWEAK was still able to increase NIK levels demonstrating that NIK can be regulated by cIAP-independent pathways. Finally we show that, unlike IAP antagonists, TWEAK was able to induce degradation of cIAP1 RING mutants. These results emphasize the critical importance of the RING of cIAP1 in many signaling scenarios, but also demonstrate that in some pathways RING functions are not required.

Heterogeneous T-cell response to MAGE-A10(254-262): high avidity-specific cytolytic T lymphocytes show superior antitumor activity.

MAGE-encoded antigens, which are expressed by tumors of many histological types but not in normal tissues, are suitable candidates for vaccine-based immunotherapy of cancers. Thus far, however, T-cell responses to MAGE antigens have been detected only occasionally in cancer patients. In contrast, by using HLA/peptide fluorescent tetramers, we have observed recently that CD8(+) T cells specific for peptide MAGE-A10(254-262) can be detected frequently in peptide-stimulated peripheral blood mononuclear cells from HLA-A2-expressing melanoma patients and healthy donors. On the basis of these results, antitumoral vaccination trials using peptide MAGE-A10(254-262) have been implemented recently. In the present study, we have characterized MAGE-A10(254-262)-specific CD8(+) T cells in polyclonal cultures and at the clonal level. The results indicate that the repertoire of MAGE-A10(254-262)-specific CD8(+) T cells is diverse both in terms of clonal composition, efficiency of peptide recognition, and tumor-specific lytic activity. Importantly, only CD8(+) T cells able to recognize the antigenic peptide with high efficiency are able to lyse MAGE-A10-expressing tumor cells. Under defined experimental conditions, the tetramer staining intensity exhibited by MAGE-A10(254-262)-specific CD8(+) T cells correlates with efficiency of peptide recognition so that "high" and "low" avidity cells can be separated by FACS. Altogether, the data reported here provide evidence for functional diversity of MAGE-A10(254-262)-specific T cells and will be instrumental for the monitoring of peptide MAGE-A10(254-262)-based clinical trials.

The role of Malt1 proteolytic activity in T cell activation and lymphoma development

Abstract Long term contact with pathogens induces an adaptive immune response, which is mainly mediated by T and B cells. Antigen-induced activation of T and B cells is an important event, since it facilitates the transition of harmless, low proliferative lymphocytes into powerful and fast expanding cells, which can, if deregulated, be extremely harmful and dangerous for the human body. One of the most important events during lymphocyte activation is the induction of NF-xB activity, a transcription factor that controls not only cytokine secretion, but also lymphocyte proliferation and survival. Recent discoveries identified the CBM complex as the central regulator of NF-xB activity in lymphocytes. The CBM complex consists of the three proteins Carma1, Bcl10 and Malt1, in which Carma1 serves as recruitment platform of the complex and Bcl10 as an adaptor to recruit Malt1 to this platform. But exactly how Malt1 activates NF-x6 is still poorly understood. We discovered that Malt1 is a protease, which cleaves its interaction partner Bcl10 upon T and B cell stimulation. We mapped the Bcl10 cleavage site by single point mutations as well as by a proteomics approach, and used this knowledge to design a fluorogenic Malt1 reporter peptide. With this tool were we able to the first time demonstrate proteolytic activity of Malt1 in vitro, using recombinant Malt1, and in stimulated T cells. Based on similarities to a metacaspase, we designed a Malt1inhibitor, which allowed unto investigate the role of Malt1 activity in T cells. Malt1-inhibited T cells showed a clear defect in NF-xB activity, resulting in impaired IL-2 cytokine secretion levels. We also found a new unexpected role for Bcl10; the blockade of Bcl10 cleavage resulted in a strongly impaired capability of stimulated T cells to adhere to the extracellular matrix protein fibronectin. Because of the central position of the C8M complex, it is not surprising that different lymphomas show abnormal expressions of Carma1, Bcl10 and Malt1. We investigated the role of Malt1 proteolytic activity in the most aggressive subtype of diffuse large B cell lymphomas called ABC, which was described to depend on the expression of Carmal, and frequently carries oncogenic Carmal mutations. We found constitutive high Malt1 activity in all tested ABC cell lines visualized by detection of cleavage products of Malt1 substrates. With the use of the Malt1-inhibitor, we could demonstrate that Malt-inhibition in those cells had two effects. First, the tumor cell proliferation was decreased, most likely because of lower autocrine stimulation by cytokines. Second, we could sensitize the ABC cells towards cell death, which is most likely caused by reduced expression of prosurvival NF-xB target gens. Taken together, we identified Malt1 as a protease in T and B cells, demonstrated its importance for NF-xB signaling and its deregulation in a subtype of diffuse large B cell lymphoma. This could allow the development of a new generation of immunomodulatory and anti-cancer drugs. Résumé Un contact prolongé avec des pathogènes provoque une réponse immunitaire adaptative qui dépend principalement des cellules T et 8. L'activation des lymphocytes T et B, suite à la reconnaissance d'un antigène, est un événement important puisqu'il facilite la transition pour ces cellules d'un état de prolifération limitée et inoffensive à une prolifération soutenue et rapide. Lorsque ce mécanisme est déréglé ìl peut devenir extrêmement nuisible et dangereux pour le corps humain. Un des événement les plus importants lors de l'activation des lymphocytes est l'induction du facteur de transcription NFxB, qui organise la sécrétion de cytokines ainsi que la prolifération et la survie des lymphocytes. Le complexe CBM, composé des trois protéines Carmai, Bc110 et Malt1, a été récemment identifié comme un régulateur central de l'activité de NF-x8 dans les lymphocytes. Carma1 sert de plateforme de recrutement pour ce complexe alors que Bc110 permet d'amener Malt1 dans cette plateforme. Cependant, le rôle exact de Malt1 dans l'activation de NF-tcB reste encore mal compris. Nous avons découvert que Malt1 est une protéase qui clive son partenaire d'interaction BcI10 après stimulation des cellules T et B. Nous avons identifié le site de clivage de BcI10 par une série de mutations ponctuelles ainsi que par une approche protéomique, ce qui nous a permis de fabriquer un peptide reporteur fluorogénique pour mesurer l'activité de Malt1. Grâce à cet outil, nous avons démontré pour la première fois l'activité protéolytique de Malt1 in vitro à l'aide de protéines Malt1 recombinantes ainsi que dans des cellules T stimulées. La ressemblance de Malt1 avec une métacaspase nous a permis de synthétiser un inhibiteur de Malt1 et d'étudier ainsi le rôle de l'activité de Malt1 dans les cellules T. L'inhibition de Malt1 dans les cellules T a révélé un net défaut de l'activité de NF-x8, ayant pour effet une sécrétion réduite de la cytokine IL-2. Nous avons également découvert un rôle inattendu pour Bcl10: en effet, bloquer le clivage de Bcl10 diminue fortement la capacité d'adhésion des cellules T stimulées à la protéine fïbronectine, un composant de la matrice extracellulaire. En raison de la position centrale du complexe CBM, il n'est pas étonnant que le niveau d'expression de Carmai, Bcl10 et Malt1 soit anormal dans plusieurs types de lymphomes. Nous avons examiné le rôle de l'activité protéolytique de Malt1 dans le sous-type le plus agressif des lymphomes B diffus à grandes cellules, appelé sous-type ABC. Ce sous-type de lymphomes dépend de l'expression de Carmai et présente souvent des mutations oncogéniques de Carma1. Nous avons démontré que l'activité de Malt1 était constitutivement élevée dans toutes les lignées cellulaires de type ABC testées, en mettant en évidence la présence de produits de clivage de différents substrats de Malt1. Enfin, l'utilisation de l'inhibiteur de Malt1 nous a permis de démontrer que l'inhibition de Malt1 avait deux effets. Premièrement, une diminution de la prolifération des cellules tumorales, probablement dûe à leur stimulation autocrine par des cytokines fortement réduite. Deuxièmement, une sensibilisation des cellules de type ABC à ia mort cellulaire, vraisemblablement causée par l'expression diminuée de gènes de survie dépendants de NF-tcB. En résumé, nous avons identifié Malt1 comme une protéase dans les cellules T et B, nous avons mis en évidence son importance pour l'activation de NF-xB ainsi que les conséquences du dérèglement de l'activité de Malt1 dans un sous-type de lymphome B diffus à larges cellules. Notre étude ouvre ainsi la voie au développement d'une nouvelle génération de médicaments immunomodulateurs et anti-cancéreux.

Cell-based computational modeling of vascular morphogenesis using Tissue Simulation Toolkit.

Computational modeling has become a widely used tool for unraveling the mechanisms of higher level cooperative cell behavior during vascular morphogenesis. However, experimenting with published simulation models or adding new assumptions to those models can be daunting for novice and even for experienced computational scientists. Here, we present a step-by-step, practical tutorial for building cell-based simulations of vascular morphogenesis using the Tissue Simulation Toolkit (TST). The TST is a freely available, open-source C++ library for developing simulations with the two-dimensional cellular Potts model, a stochastic, agent-based framework to simulate collective cell behavior. We will show the basic use of the TST to simulate and experiment with published simulations of vascular network formation. Then, we will present step-by-step instructions and explanations for building a recent simulation model of tumor angiogenesis. Demonstrated mechanisms include cell-cell adhesion, chemotaxis, cell elongation, haptotaxis, and haptokinesis.

A cell-based model of extracellular-matrix-guided endothelial cell migration during angiogenesis.

Angiogenesis, the formation of new blood vessels sprouting from existing ones, occurs in several situations like wound healing, tissue remodeling, and near growing tumors. Under hypoxic conditions, tumor cells secrete growth factors, including VEGF. VEGF activates endothelial cells (ECs) in nearby vessels, leading to the migration of ECs out of the vessel and the formation of growing sprouts. A key process in angiogenesis is cellular self-organization, and previous modeling studies have identified mechanisms for producing networks and sprouts. Most theoretical studies of cellular self-organization during angiogenesis have ignored the interactions of ECs with the extra-cellular matrix (ECM), the jelly or hard materials that cells live in. Apart from providing structural support to cells, the ECM may play a key role in the coordination of cellular motility during angiogenesis. For example, by modifying the ECM, ECs can affect the motility of other ECs, long after they have left. Here, we present an explorative study of the cellular self-organization resulting from such ECM-coordinated cell migration. We show that a set of biologically-motivated, cell behavioral rules, including chemotaxis, haptotaxis, haptokinesis, and ECM-guided proliferation suffice for forming sprouts and branching vascular trees.

Activation of a dendritic cell-T cell axis by Ad5 immune complexes creates an improved environment for replication of HIV in T cells.

The STEP HIV vaccine trial, which evaluated a replication-defective adenovirus type 5 (Ad5) vector vaccine, was recently stopped. The reasons for this included lack of efficacy of the vaccine and a twofold increase in the incidence of HIV acquisition among vaccinated recipients with increased Ad5-neutralizing antibody titers compared with placebo recipients. To model the events that might be occurring in vivo, the effect on dendritic cells (DCs) of Ad5 vector alone or treated with neutralizing antiserum (Ad5 immune complexes [IC]) was compared. Ad5 IC induced more notable DC maturation, as indicated by increased CD86 expression, decreased endocytosis, and production of tumor necrosis factor and type I interferons. We found that DC stimulation by Ad5 IC was mediated by the Fcgamma receptor IIa and Toll-like receptor 9 interactions. DCs treated with Ad5 IC also induced significantly higher stimulation of Ad5-specific CD8 T cells equipped with cytolytic machinery. In contrast to Ad5 vectors alone, Ad5 IC caused significantly enhanced HIV infection in DC-T cell cocultures. The present results indicate that Ad5 IC activates a DC-T cell axis that, together with the possible persistence of the Ad5 vaccine in seropositive individuals, may set up a permissive environment for HIV-1 infection, which could account for the increased acquisition of HIV-1 infection among Ad5 seropositive vaccine recipients.

Direct analysis of peptide binding to cell-associated MHC class I molecules.

Exogenously added synthetic peptides can mimic endogenously produced antigenic peptides recognized on target cells by MHC class I-restricted cytolytic T lymphocytes. While it is assumed that exogenous peptides associate with class I molecules on the target cell surface, direct binding of peptides to cell-associated class I molecules has been difficult to demonstrate. Using a newly developed binding assay based on photoaffinity labeling, we have investigated the interaction of two antigenic peptides, known to be recognized in the context of H-2Kd or H-2Db, respectively, with 20 distinct class I alleles on living cells. None of the class I alleles tested, with the exception of H-2Kd or H-2Db, bound either of the peptides, thus demonstrating the exquisite specificity of peptide binding to class I molecules. Moreover, peptide binding to cell-associated H-2Kd was drastically reduced when metabolic energy, de novo protein synthesis or protein egress from the endoplasmic reticulum was inhibited. It is thus likely that exogenously added peptides do not associate with the bulk of class I molecules expressed at the cell surface, but rather bind to short-lived molecules devoid of endogenous peptides.

Manipulating keratinocyte stem cell proliferation and differentiation using RNA interference

ABSTRACT : The epidermis, the outermost compartment of the skin, is a stratified and squamous epithelium that constantly self-renews. Keratinocytes, which represent the main epidermal population, are responsible for its cohesion and barrier function. Epidermal renewal necessitates a fine equilibrium between keratinocyte proliferation and differentiation. The keratinocyte stem cell, located in the basal cell layer, is responsible for epidermal homeostasis and regeneration during the wound healing process. The transcription factor p63 structurally belongs to the p53 superfamily. It is expressed in the basal and supra-basal cell layers of stratified epithelia and is thought to be important for the renewal or the differentiation of keratinocyte stem cells (Yang et al., 1999; Mills et al., 1999). In order to better understand its function, we established an in vitro model of p63 deficient human keratinocyte stem cells using a shp63 mediated RNA interference. Knockdown of endogenous p63 induces downregulation of cell-adhesion genes as previously described (Carroll et al., 2006). Interestingly, the replating of attached p63-knockdown keratinocytes on a feeder layer results in a loss of attachment and proliferation. They are no longer clonogenic. However, if the same population are replated in a fibrin matrix, extended fibrinolysis is reported, a common process in wound healing, suggesting that p63 regulates the fibrinolytic pathway. This result was confirmed by Q-PCR and shows that the urokinase pathway, which mediates fibrinolysis, is upregulated. Altogether, these findings suggest a mechanism in which the fine tuning of p63 expression promotes attachment or release of the keratinocyte stem cell from the basement membrane by inducing genes of adhesion and/or of fibrinolysis. This mechanism may be important for epidermal self-renewal, differentiation as well as wound healing. Its misregulation may be partly responsible for the p63 knockout phenotype. The downregulation of p63 also induces a decrease in LEKTI expression. LEKTI (lymphoepithelial Kazal-type serine protease inhibitor) is a serine protease inhibitor encoded by the Spink5 gene. It is expressed and secreted in the uppermost differentiated layers of stratified epithelia and plays a role in the desquamation process. When this gene is disrupted, humans develop the Netherton syndrome (Chavanas et al., 2000b). It is a dermatosis characterized by hair dysplasias, ichtyosiform erythroderma and impairment in epidermal barrier function promoting inflammation similarly as in psoriasis with inflammatory infiltrate in excess. TNFα (tumor necrosis factor alpha) and EDA1 (ectodysplasin A1) are two transmembraneprecursors that belong to the TNF superfamily, which is involved in immune and inflammation regulation (Smahi et al., 2002). We suggest that the secreted serine protease inhibitor LEKTI plays a role in the regulation of TNFα and EDA1 precursor cleavage and absence of LEKTI induces excess of inflammation. To investigate this hypothesis, we induced downregulation of Spink5 expression in rat keratinocyte stem cells by using a shSpink5 mediated RNA interference approach. Interestingly, expression of TNFα and EDA1 is modified after knockdown of Spink5 by Q-PCR. Moreover, downregulation of Spink5 induces loss of cohesiveness between keratinocytes and colonies adopt a scattered phenotype. Altogether, these preliminary data suggest that downregulation of LEKTI may play a role in the inflammatory response in Netherton syndrome patients, by regulating TNFα expression.

Tumor antigen-specific FOXP3+ CD4 T cells identified in human metastatic melanoma: peptide vaccination results in selective expansion of Th1-like counterparts.

We have previously shown that vaccination of HLA-A2 metastatic melanoma patients with the analogue Melan-A(26-35(A27L)) peptide emulsified in a mineral oil induces ex vivo detectable specific CD8 T cells. These are further enhanced when a TLR9 agonist is codelivered in the same vaccine formulation. Interestingly, the same peptide can be efficiently recognized by HLA-DQ6-restricted CD4 T cells. We used HLA-DQ6 multimers to assess the specific CD4 T-cell response in both healthy individuals and melanoma patients. We report that the majority of melanoma patients carry high frequencies of naturally circulating HLA-DQ6-restricted Melan-A-specific CD4 T cells, a high proportion of which express FOXP3 and proliferate poorly in response to the cognate peptide. Upon vaccination, the relative frequency of multimer+ CD4 T cells did not change significantly. In contrast, we found a marked shift to FOXP3-negative CD4 T cells, accompanied by robust CD4 T-cell proliferation upon in vitro stimulation with cognate peptide. A concomitant reduction in TCR diversity was also observed. This is the first report on direct ex vivo identification of antigen-specific FOXP3+ T cells by multimer labeling in cancer patients and on the direct assessment of the impact of peptide vaccination on immunoregulatory T cells.

Identification of MAGI1 as a tumor-suppressor protein induced by cyclooxygenase-2 inhibitors in colorectal cancer cells.

Cyclooxyganase-2 (COX-2), a rate-limiting enzyme in the prostaglandin synthesis pathway, is overexpressed in many cancers and contributes to cancer progression through tumor cell-autonomous and paracrine effects. Regular use of non-steroidal anti-inflammatory drugs or selective COX-2 inhibitors (COXIBs) reduces the risk of cancer development and progression, in particular of the colon. The COXIB celecoxib is approved for adjunct therapy in patients with Familial adenomatous polyposis at high risk for colorectal cancer (CRC) formation. Long-term use of COXIBs, however, is associated with potentially severe cardiovascular complications, which hampers their broader use as preventive anticancer agents. In an effort to better understand the tumor-suppressive mechanisms of COXIBs, we identified MAGUK with Inverted domain structure-1 (MAGI1), a scaffolding protein implicated in the stabilization of adherens junctions, as a gene upregulated by COXIB in CRC cells and acting as tumor suppressor. Overexpression of MAGI1 in CRC cell lines SW480 and HCT116 induced an epithelial-like morphology; stabilized E-cadherin and β-catenin localization at cell-cell junctions; enhanced actin stress fiber and focal adhesion formation; increased cell adhesion to matrix proteins and suppressed Wnt signaling, anchorage-independent growth, migration and invasion in vitro. Conversely, MAGI1 silencing decreased E-cadherin and β-catenin localization at cell-cell junctions; disrupted actin stress fiber and focal adhesion formation; and enhanced Wnt signaling, anchorage-independent growth, migration and invasion in vitro. MAGI1 overexpression suppressed SW480 and HCT116 subcutaneous primary tumor growth, attenuated primary tumor growth and spontaneous lung metastasis in an orthotopic model of CRC, and decreased the number and size of metastatic nodules in an experimental model of lung metastasis. Collectively, these results identify MAG1 as a COXIB-induced inhibitor of the Wnt/β-catenin signaling pathway, with tumor-suppressive and anti-metastatic activity in experimental colon cancer.

Notch1 functions as a tumor suppressor in mouse skin.

Notch proteins are important in binary cell-fate decisions and inhibiting differentiation in many developmental systems, and aberrant Notch signaling is associated with tumorigenesis. The role of Notch signaling in mammalian skin is less well characterized and is mainly based on in vitro studies, which suggest that Notch signaling induces differentiation in mammalian skin. Conventional gene targeting is not applicable to establishing the role of Notch receptors or ligands in the skin because Notch1-/- embryos die during gestation. Therefore, we used a tissue-specific inducible gene-targeting approach to study the physiological role of the Notch1 receptor in the mouse epidermis and the corneal epithelium of adult mice. Unexpectedly, ablation of Notch1 results in epidermal and corneal hyperplasia followed by the development of skin tumors and facilitated chemical-induced skin carcinogenesis. Notch1 deficiency in skin and in primary keratinocytes results in increased and sustained expression of Gli2, causing the development of basal-cell carcinoma-like tumors. Furthermore, Notch1 inactivation in the epidermis results in derepressed beta-catenin signaling in cells that should normally undergo differentiation. Enhanced beta-catenin signaling can be reversed by re-introduction of a dominant active form of the Notch1 receptor. This leads to a reduction in the signaling-competent pool of beta-catenin, indicating that Notch1 can inhibit beta-catenin-mediated signaling. Our results indicate that Notch1 functions as a tumor-suppressor gene in mammalian skin.

Low doses of ionizing radiation promote tumor growth and metastasis by enhancing angiogenesis.

Radiotherapy is a widely used treatment option in cancer. However, recent evidence suggests that doses of ionizing radiation (IR) delivered inside the tumor target volume, during fractionated radiotherapy, can promote tumor invasion and metastasis. Furthermore, the tissues that surround the tumor area are also exposed to low doses of IR that are lower than those delivered inside the tumor mass, because external radiotherapy is delivered to the tumor through multiple radiation beams, in order to prevent damage of organs at risk. The biological effects of these low doses of IR on the healthy tissue surrounding the tumor area, and in particular on the vasculature remain largely to be determined. We found that doses of IR lower or equal to 0.8 Gy enhance endothelial cell migration without impinging on cell proliferation or survival. Moreover, we show that low-dose IR induces a rapid phosphorylation of several endothelial cell proteins, including the Vascular Endothelial Growth Factor (VEGF) Receptor-2 and induces VEGF production in hypoxia mimicking conditions. By activating the VEGF Receptor-2, low-dose IR enhances endothelial cell migration and prevents endothelial cell death promoted by an anti-angiogenic drug, bevacizumab. In addition, we observed that low-dose IR accelerates embryonic angiogenic sprouting during zebrafish development and promotes adult angiogenesis during zebrafish fin regeneration and in the murine Matrigel assay. Using murine experimental models of leukemia and orthotopic breast cancer, we show that low-dose IR promotes tumor growth and metastasis and that these effects were prevented by the administration of a VEGF receptor-tyrosine kinase inhibitor immediately before IR exposure. These findings demonstrate a new mechanism to the understanding of the potential pro-metastatic effect of IR and may provide a new rationale basis to the improvement of current radiotherapy protocols.

Amino acid identity and/or position determines the proteasomal cleavage of the HLA-A*0201-restricted peptide tumor antigen MAGE-3271-279.

The proteasome plays a crucial role in the proteolytic processing of antigens presented to T cells in the context of major histocompatibility complex class I molecules. However, the rules governing the specificity of cleavage sites are still largely unknown. We have previously shown that a cytolytic T lymphocyte-defined antigenic peptide derived from the MAGE-3 tumor-associated antigen (MAGE-3(271-279), FLWGPRALV in one-letter code) is not presented at the surface of melanoma cell lines expressing the MAGE-3 protein. By using purified proteasome and MAGE-3(271-279) peptides extended at the C terminus by 6 amino acids, we identified predominant cleavages after residues 278 and 280 but no detectable cleavage after residue Val(279), the C terminus of the antigenic peptide. In the present study, we have investigated the influence of Pro(275), Leu(278), and Glu(280) on the proteasomal digestion of MAGE-3(271-285) substituted at these positions. We show that positions 278 and 280 are major proteasomal cleavage sites because they tolerate most amino acid substitutions. In contrast, the peptide bond after Val(279) is a minor cleavage site, influenced by both distal and proximal amino acid residues.