Search

Effect of PI3K inhibitors on COX-2 expression in KUL98-MELA cells

Tumors use various mechanisms to avoid immune destruction. Cyclooxygenase-2 (COX-2) expression may be a driver of immune suppression in melanoma, but the mechanisms involved remain elusive. Here, we show that COX-2 expression drives constitutive expression of indoleamine 2,3-dioxygenase 1 (IDO1) in human tumor cells. IDO1 is an immunosuppressive enzyme that degrades tryptophan. In a series of seven human tumor lines, constitutive IDO1 expression depends on COX-2 and prostaglandin E2 (PGE2), which, upon autocrine signaling through the EP receptor, activates IDO1 via the PKC and PI3K pathways. COX-2 expression itself depends on the MAPK pathway, which therefore indirectly controls IDO1 expression. Most of these tumors carry PI3K or MAPK oncogenic mutations, which may favor constitutive IDO1 expression. Celecoxib treatment promoted immune rejection of IDO1-expressing human tumor xenografts in immunodeficient mice reconstituted with human allogeneic lymphocytes. This effect was associated with a reduced expression of IDO1 in those ovarian SKOV3 tumors and an increased infiltration of CD3+ and CD8+ cells. Our results highlight the role of COX-2 in constitutive IDO1 expression by human tumors and substantiate the use of COX-2 inhibitors to improve the efficacy of cancer immunotherapy, by reducing constitutive IDO1 expression, which contributes to the lack of T-cell infiltration in “cold” tumors, which fail to respond to immunotherapy. Cancer Immunol Res; 5(8); 695–709. ©2017 AACR.

Human tumor cell lines used in this study.

BackgroundIndoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan-dioxygenase (TDO) are enzymes catabolizing the essential amino acid tryptophan into kynurenine. Expression of these enzymes is frequently observed in advanced-stage cancers and is associated with poor disease prognosis and immune suppression. Mechanistically, the respective roles of tryptophan shortage and kynurenine production in suppressing immunity remain unclear. Kynurenine was proposed as an endogenous ligand for the aryl hydrocarbon receptor (AHR), which can regulate inflammation and immunity. However, controversy remains regarding the role of AHR in IDO1/TDO-mediated immune suppression, as well as the involvement of kynurenine. In this study, we aimed to clarify the link between IDO1/TDO expression, AHR pathway activation and immune suppression.MethodsAHR expression and activation was analyzed by qRT-PCR and western blot analysis in cells engineered to express IDO1/TDO, or cultured in medium mimicking tryptophan catabolism by IDO1/TDO.In vitrodifferentiation of naïve CD4+T cells into regulatory T cells (Tregs) was compared in T cells isolated from mice bearing differentAhralleles or a knockout ofAhr, and cultured in medium with or without tryptophan and kynurenine.ResultsWe confirmed that IDO1/TDO expression activated AHR in HEK-293-E cells, as measured by the induction of AHR target genes. Unexpectedly, AHR was also overexpressed upon IDO1/TDO expression. AHR overexpression did not depend on kynurenine but was triggered by tryptophan deprivation. Multiple human tumor cell lines overexpressed AHR upon tryptophan deprivation. AHR overexpression was not dependent on GCN2, and strongly sensitized the AHR pathway. As a result, kynurenine and other tryptophan catabolites, which are weak AHR agonists in normal conditions, strongly induced AHR target genes in tryptophan-depleted conditions. Tryptophan depletion also increased kynurenine uptake by increasing SLC7A5 (LAT1) expression in a GCN2-dependent manner. Tryptophan deprivation potentiated Treg differentiation from naïve CD4+T cells isolated from mice bearing an AHR allele of weak affinity similar to the human AHR.ConclusionsTryptophan deprivation sensitizes the AHR pathway by inducing AHR overexpression and increasing cellular kynurenine uptake. As a result, tryptophan catabolites such as kynurenine, more potently activate AHR, and Treg differentiation is promoted. Our results propose a molecular explanation for the combined roles of tryptophan deprivation and kynurenine production in mediating IDO1/TDO-induced immune suppression.SIGNIFICANCEIn preclinical models, tryptophan degradation by IDO1 or TDO was shown to induce tumoral resistance to immune rejection, by restricting inflammation and promoting T-cell tolerance to immunogenic tumor antigens. However, the mechanism that translates these metabolic changes into T-lymphocyte malfunction within the tumor microenvironment (TME) is still uncertain. It has been proposed that kynurenine, the main tryptophan catabolite, acts as an endogenous ligand for the aryl hydrocarbon receptor (AHR), leading to the suggestion that the IDO1/Kyn/AHR axis could play a key role in modulating inflammatory and immune responses. However, recent studies challenged the notion that kynurenine is a genuine and potent AHR agonistic ligand. Moreover, the relative role of tryptophan depletion versus kynurenine production in IDO1/TDO mediated immune suppression remains unknown.In this work, we further explored and clarified the association between IDO1/TDO activity and AHR activation. Unexpectedly, we observed that tryptophan depletion strongly increased AHR expression, thereby potentiating its activation by weak agonists such as kynurenine and derivatives. Tryptophan depletion thereby potentiated the induction of regulatory T cells. This was particularly true in mouse strains that express an Ahr allele of weak affinity, similar to the human AHR.Tryptophan depletion also increased cellular kynurenine uptake by increasing SLC7A5 (LAT1) expression in a GCN2-dependent manner, thereby also contributing to a better AHR activation by kynurenine upon tryptophan depletion.Altogether, our findings identify a new mechanism explaining IDO/TDO mediated AHR activation and immune suppression, based on the sensitization of the AHR pathway by tryptophan depletion, resulting in a higher AHR stimulation by weak agonists of the kynurenine pathway, and a better induction of regulatory T cells.

Maintaining stable tryptophan levels is required to control neuronal and immune activity. We report that tryptophan homeostasis is largely controlled by the stability of tryptophan 2,3-dioxygenase (TDO), the hepatic enzyme responsible for tryptophan catabolism. High tryptophan levels stabilize the active tetrameric conformation of TDO through binding non-catalytic exosites, resulting in rapid catabolism of tryptophan. In low tryptophan, the lack of tryptophan binding in the exosites destabilizes the tetramer into inactive monomers and dimers, and unmasks a 4-amino-acid degron that triggers TDO polyubiquitination by SKP1-CUL1-F-box complexes, resulting in proteasome-mediated degradation of TDO and rapid interruption of tryptophan catabolism. The non-metabolizable analog alpha-methyl-tryptophan stabilizes tetrameric TDO, and thereby stably reduces tryptophanemia. Our results uncover a mechanism allowing a rapid adaptation of tryptophan catabolism to ensure quick degradation of excess tryptophan while preventing further catabolism below physiological levels. This ensures a tight control of tryptophanemia, as required for both neurological and immune homeostasis.

The proteasome is responsible for selective degradation of proteins. It exists in mammalian cells under four main subtypes, which differ by the combination of their catalytic subunits: the standard proteasome (β1–β2–β5), the immunoproteasome (β1i–β2i–β5i) and the two intermediate proteasomes (β1–β2–β5i and β1i–β2–β5i). The efficiency of the four proteasome subtypes to degrade ubiquitinated or oxidized proteins remains unclear. Using cells expressing exclusively one proteasome subtype, we observed that ubiquitinated p21 and c-­myc were degraded at similar rates, indicating that the four 26S proteasomes degrade ubiquitinated proteins equally well. Under oxidative stress, we observed a partial dissociation of 26S into 20S proteasomes, which can degrade non-ubiquitinated oxidized proteins. Oxidized calmodulin and hemoglobin were best degraded in vitro by the three β5i-containing 20S proteasomes, while their native forms were not degraded. Circular dichroism analyses indicated that ubiquitin-independent recognition of oxidized proteins by 20S proteasomes was triggered by the disruption of their structure. Accordingly, β5i-containing 20S proteasomes degraded unoxidized naturally disordered protein tau, while 26S proteasomes did not. Our results suggest that the three β5i-containing 20S proteasomes, namely the immunoproteasome and the two intermediate proteasomes, might help cells to eliminate proteins containing disordered domains, including those induced by oxidative stress.

Tumors use various mechanisms to avoid immune destruction. Cyclooxygenase-2 (COX-2) expression may be a driver of immune suppression in melanoma, but the mechanisms involved remain elusive. Here, we show that COX-2 expression drives constitutive expression of indoleamine 2,3-dioxygenase 1 (IDO1) in human tumor cells. IDO1 is an immunosuppressive enzyme that degrades tryptophan. In a series of seven human tumor lines, constitutive IDO1 expression depends on COX-2 and prostaglandin E2 (PGE2), which, upon autocrine signaling through the EP receptor, activates IDO1 via the PKC and PI3K pathways. COX-2 expression itself depends on the MAPK pathway, which therefore indirectly controls IDO1 expression. Most of these tumors carry PI3K or MAPK oncogenic mutations, which may favor constitutive IDO1 expression. Celecoxib treatment promoted immune rejection of IDO1-expressing human tumor xenografts in immunodeficient mice reconstituted with human allogeneic lymphocytes. This effect was associated with a reduced expression of IDO1 in those ovarian SKOV3 tumors and an increased infiltration of CD3+ and CD8+ cells. Our results highlight the role of COX-2 in constitutive IDO1 expression by human tumors and substantiate the use of COX-2 inhibitors to improve the efficacy of cancer immunotherapy, by reducing constitutive IDO1 expression, which contributes to the lack of T-cell infiltration in “cold” tumors, which fail to respond to immunotherapy. Cancer Immunol Res; 5(8); 695–709. ©2017 AACR.

Tryptophan catabolism mediated by indoleamine 2,3-dioxygenase (IDO1) is an important mechanism of peripheral immune tolerance contributing to tumoral immune resistance, and IDO1 inhibition is an active area of drug development. Tryptophan 2,3-dioxygenase (TDO) is an unrelated hepatic enzyme that also degrades tryptophan along the kynurenine pathway. Here, we show that enzymatically active TDO is expressed in a significant proportion of human tumors. In a preclinical model, TDO expression by tumors prevented their rejection by immunized mice. We developed a TDO inhibitor, which, upon systemic treatment, restored the ability of mice to reject TDO-expressing tumors. Our results describe a mechanism of tumoral immune resistance based on TDO expression and establish proof-of-concept for the use of TDO inhibitors in cancer therapy.

We report that melanoma cell lines expressing the interleukin-1 receptor exhibit 4- to 10-fold lower levels of mRNA of microphthalmia-associated transcription factor (MITF-M) when treated with interleukin-1 beta. This effect is NF-kappa B and JNK-dependent. MITF-M regulates the expression of melanocyte differentiation genes such as MLANA, tyrosinase and gp100, which encode antigens recognized on melanoma cells by autologous cytolytic T lymphocytes. Accordingly, treating some melanoma cells with IL-1 beta reduced by 40-100% their ability to activate such antimelanoma cytolytic T lymphocytes. Finally, we observed large amounts of biologically active IL-1 alpha or IL-1 beta secreted by two melanoma cell lines that did not express MITF-M, suggesting an autocrine MITF-M downregulation. We estimate that similar to 13% of melanoma cell lines are MITF-M-negative and secrete IL-1 cytokines. These results indicate that the repression of melanocyte-differentiation genes by IL-1 produced by stromal cells or by tumor cells themselves may represent an additional mechanism of melanoma immune escape.

MAGE-A1 belongs to a group of germ line-specific genes that rely primarily on DNA methylation for repression in somatic tissues. In many types of tumors, the promoter of these genes becomes demethylated and transcription becomes activated. We showed previously that, although MZ2-MEL melanoma cells contain an active unmethylated MAGE-A1 gene, they lack the ability to induce demethylation of newly integrated MAGE-A1 transgenes that were methylated in vitro before transfection. In the same cells, unmethylated MAGE-A1 transgenes were protected against remethylation, and this appeared to depend on the level of transcriptional activity. We therefore proposed that hypomethylation of MAGE-A1 in tumors relies on a past demethylation event and on the presence of appropriate transcription factors that maintain the promoter unmethylated. Here, we tested this hypothesis further by examining whether induction of a transient demethylation phase in MZ2-MEL would suffice to convert a previously methylated MAGE-A1 transgene into a permanently hypomethylated and active one. For induction of the demethylation phase, we used antisense oligonucleotides targeting the three known human DNA methyltransferases. We found that down-regulation of DNMT1, but not of DNMT3A and DNMT3B, induces activation of the MAGE-A1 transgene, suggesting that DNMT1 has a predominant role for methylation maintenance in MZ2-MEL cells. By using a selectable MAGE-A1 transgene construct, we were able to isolate a cell population in which DNMT1 depletion had resulted in transgene activation. The promoter region of the transgene was almost completely unmethylated in these cells, and this active and unmethylated state was maintained for over 60 days after restoration of normal DNMT1 expression.

After vaccination of melanoma patients with MAGE antigens, we observed that even in the few patients showing tumor regression, the frequency of anti-vaccine T cells in the blood was often either undetectable or

Representational difference analysis, a cDNA subtraction approach, was used to identify genes that are expressed in acute leukemia but not in normal hematological tissues. We isolated a cDNA fragment from a cell line derived from a B cell acute lymphoblastic leukemia bearing two Philadelphia chromosomes. The cDNA derives from an orphan gene that was named BLACE. BLACE is located in region 7q36 and encodes a major 5.3-kb transcript and several alternatively spliced minor transcripts. Significant expression of BLACE was detected by RT-PCR and quantitative RT-PCR in bone marrow samples from B cell acute lymphoblastic leukemia patients. BLACE was not or was scarcely expressed in other types of hematological malignancies, in normal tissues, and in solid tumors.

A number of genes of the MAGE-A, B, and C families have been shown to code for antigens that are recognized on many human tumors by autologous cytolytic T lymphocytes. These antigens ought to be strictly tumor specific because the encoding MAGE genes are not expressed in normal adult cells except for male germline cells, which lack HLA expression. To identify new genes of this type, we performed representational difference analysis on a melanoma cell line by subtraction with a normal skin sample. This led to the identification of MAGE-C2, a new member of the MAGE-C family. A search for nucleotide sequences encoding MAGE-like proteins in public databases led to the identification of three additional MAGE genes, which were named MAGE-B5, MAGE-B6, and MAGE-C3. The four new MAGE genes are not expressed in normal tissues, except for testis, and are expressed in tumors of different histological origins. Therefore, like other MAGE genes expressed specifically in tumors, MAGE-B5, MAGE-B6, MAGE-C2, and MAGE-C3 ought to encode antigens that could be targets for cancer immunotherapy.

MAGE genes are frequently expressed in several types of human malignancy and code for antigens recognized by cytotoxic T lymphocytes. We have previously described a monoclonal antibody (MAb), named 6Cl, that recognizes the MAGE-1 protein and cross-reacts with a 72-kDa protein present in lysates of melanoma cells such as MZ2-MEL. To identify this protein, we have screened an expression library prepared from MZ2-MEL cells. Several clones that encoded a protein recognized by antibody 6Cl contained a sequence identical to that of MAGE-10, another member of the MAGE-A gene family. Full-length MAGE-10 cDNA clones, obtained after screening additional cDNA melanoma libraries, were found to be approximately 2.5 kb in length. In vitro translation and transient transfection experiments indicated that MACE-IO codes for a protein of approximately 72 kDa. This product was recognized by MAb 6CI as well as by a polyclonal serum raised against a MAGE-10 peptide, thus demonstrating its identity with MAGE-10. Analysis of MAGE-10 mRNA by RT-PCR confirmed its presence in testis and placenta but not in other normal tissues. Expression of MAGE-10 in melanoma tumors was found to parallel that of MAGE-1. Western blot analysis with the polyclonal anti-MAGE-10 antibody showed the presence of MAGE-10 in lysates of purified trophoblast cells. Immuno-cytochemistry of cultured melanoma cells indicated that MAGE-10 is a nuclear protein. (C) 1999 Wiley-Liss, Inc.

It is now well established that human melanoma cells express antigens that are recognised by cytolytic T lymphocytes derived from the tumour-bearing patient. The molecular definition of these antigens is progressing at an accelerated pace. The currently characterised melanoma antigens can be classified into three categories: differentiation antigens, antigens encoded by genes that are specifically expressed in tumours, and antigens encoded by mutated genes. Several of these antigens are sufficiently tumour-specific to qualify them as candidate anti-cancer vaccines in melanoma patients.

Recently, our group has shown that tryptophan 2,3 dioxygenase (TDO), a hepatic enzyme catalyzing the first step of tryptophan degradation, is expressed in many tumors, thereby contributing to tumoral immune resistance.1 The complementary role of tryptophan catabolites has been demonstrated by others.2We set out to develop new, improved TDO inhibitors using as the starting point the only (unoptimized) series previously known in the literature.3 Herein, we describe the syntheses and structure-activity studies around a series of 3-alkenyl indoles and their derivatives.4 The TDO inhibitory potency was evaluated and rationalized by molecular modeling studies, while solubility, stability and oral bioavailability were determined for selected compounds. The most promising candidate was evaluated in a preclinical model in mice where, upon systemic treatment, it indeed reversed TDO-mediated tumoral immune resistance.5

Tryptophan catabolism mediated by indoleamine 2,3-dioxygenase (IDO) is an important mechanism of peripheral immune tolerance contributing to tumoral immune resistance.1 IDO inhibition has been an active area of research in drug development for a number of years.2 Recently, our group has shown that tryptophan 2,3 dioxygenase (TDO), an unrelated hepatic enzyme also catalyzing the first step of tryptophan degradation, is as well expressed in many tumors preventing their rejection by locally depleting tryptophan.3 The role of tryptophan catabolites was demonstrated by another group.4 Herein, we report the syntheses and structure-activity studies around a series of 3-(2-(pyridyl)ethenyl)indoles.5 Some 80 novel heterocyclic compounds were synthesized. Their TDO inhibitory potency was evaluated and rationalized by molecular modeling studies. The best candidate in terms of potency, selectivity, solubility and oral bioavailability was evaluated in a preclinical model in mice. Upon systemic treatment, the compound reversed TDO-mediated tumoral immune resistance.6 References This work was supported in part by FNRS-Télévie (7.4.543.07). 1) Uyttenhove, C., et al, Nat. Med. 2003, 9, 1269-1274. 2) Macchiarulo, A., et al, Amino Acids 2009, 37, 219-229; Röhrig, U. F., et al, J. Med. Chem. 2010, 53, 1172-1189. 3) Van den Eynde, B., et al, WO2010008427, 2010. 4) Opitz, C. A., et al, Nature 2011, 478, 197-203. 5) Dolusic, E., et al, J. Med. Chem. 2011, 54, 5320-5334. 6) Pilotte, L., et al, Proc. Natl. Acad. Sci. USA 2012, 109, 2497 - 2502.

Several genes with specific expression in germ cells show aberrant activation in different types of tumors. These genes, termed cancer-germline (CG) genes, encode tumor-specific antigens, which represent potential targets for therapeutic vaccination against cancer. The germline-specific gene BORIS (Brother Of the Regulator of Imprinted Sites), which encodes an 11-zinc-fingers transcriptional regulator, was recently qualified as a new CG gene, as it was found to be activated in a variety of tumor samples. Moreover, it was suggested that BORIS might be responsible for the activation of most other CG genes, including gene MAGE-A1, in tumors. In the present study, we evaluated the frequency of BORIS activation in melanoma by quantitative RT-PCR. BORIS activation was detected in 27% (n = 63) melanoma tissue samples. Surprisingly, many melanoma samples expressed MAGE-A1 and other CG genes in the absence of BORIS activation, suggesting that BORIS is not an obligate factor for activation of these genes in melanoma. Consistently, forced expression of BORIS in melanoma cell lines did not induce expression of MAGE-A1. Our results indicate that BORIS may serve as a useful target for immunotherapy of melanoma. However, it appears that BORIS is neither necessary nor sufficient for the activation of other CG genes.

We have described a post-PCR detection method for the 12 MAGE-A sequences on a DNA microarray (1) and compared the results with a method using pairs of primers unique for each sequence (2). The microarray assay did not differentiate between MAGE-A3 and MAGE-A6 amplicons, which differed by only 1 nucleotide, and could not distinguish between PCR products amplified from mRNA and those amplified from genomic DNA. In addition, the assay could not identify false-negative results related to RNA degradation or to enzyme inhibition during reverse transcription-PCR. Here we describe an assay that is designed to overcome these drawbacks and appears to be more sensitive. We used 3 primer pairs: 1 for amplification of the 12 MAGE-A sequences (1); 1 for specific amplification of MAGE-A3 ; and 1 for amplification of an endogenous, ubiquitously expressed control gene ( MAGE-D2 ) (3). The low-density microarray includes new capture probes for MAGE-A3 and - D2 . The assay involved DNase treatment of total RNA, reverse transcription of mRNA with oligo(dT) primer, triplex PCR amplification in the presence of biotin-dATP/biotin-dCTP, and hybridization of the resulting amplicons on a DNA microarray (see the Experimental Protocol in the Data Supplement that accompanies the online version of this Letter at http://www.clinchem.org/content/vol51/issue12). The new capture probe for MAGE-A3 corresponded to a sequence downstream from the location of the reverse primer DPASCONB4, and a primer pair (DPSA3 and DPASA3) defining a MAGE-A3 amplicon encompassing the new probe sequence …

Melanoma patients have high frequencies of T cells directed against antigens of their tumor. The frequency of these antitumor T cells in the blood is usually well above that of the anti-vaccine T cells observed after vaccination with tumor antigens. In a patient vaccinated with a MAGE-3 antigen presented by HLA-A1, we measured the frequencies of anti-vaccine and antitumor T cells in several metastases to evaluate their respective potential contribution to tumor rejection. The frequency of anti-MAGE-3.A1 T cells was 1.5 x 10(-5) of CD8 T cells in an invaded lymph node, sixfold higher than in the blood. An antitumor cytotoxic T lymphocyte (CTL) recognizing a MAGE-C2 antigen showed a much higher enrichment with a frequency of approximately 10%, 1,000 times higher than its blood frequency. Several other antitumor T clonotypes had frequencies >1%. Similar findings were made on a regressing cutaneous metastasis. Thus, antitumor T cells were approximately 10,000 times more frequent than anti-vaccine T cells inside metastases, representing the majority of T cells present there. This suggests that the anti-vaccine CTLs are not the effectors that kill the bulk of the tumor cells, but that their interaction with the tumor generates conditions enabling the stimulation of large numbers of antitumor CTLs that proceed to destroy the tumor cells. Naive T cells appear to be stimulated in the course of this process as new antitumor clonotypes arise after vaccination.

MAGE-A1 belongs to a family of 12 genes that are active in various types of tumors and silent in normal tissues except in male germ-line cells. The MAGE-encoded antigens recognized by T cells are highly tumor-specific targets for T cell-oriented cancer immunotherapy. The function of MAGE-A1 is currently unknown. To analyze it, we attempted to identify protein partners of MAGE-A1. Using yeast two-hybrid screening, we detected an interaction between MAGE-A1 and Ski Interacting Protein (SKIP). SKIP is a transcriptional regulator that connects DNA-binding proteins to proteins that either activate or repress transcription. We show that MAGE-A1 inhibits the activity of a SKIP-interacting transactivator, namely the intracellular part of Notch1. Deletion analysis indicated that this inhibition requires the binding of MAGE-A1 to SKIP. Moreover, MAGE-A1 was found to actively repress transcription by binding and recruiting histone deacetylase 1 (HDAC1). Our results indicate that by binding to SKIP and by recruiting HDACs, MAGE-A1 can act as a potent transcriptional repressor. MAGE-A1 could therefore participate in the setting of specific gene expression patterns for tumor cell growth or spermatogenesis.

"Cancer-germline" genes such as those of the MAGE family are expressed in many tumors and in male germline cells, but are silent in normal tissues. They encode shared tumor-specific antigens, which have been used in therapeutic vaccination trials of cancer patients. MAGE-6 is expressed in more than 70% of metastatic melanomas and more than 50% of carcinomas of the lung, esophagus, bladder, and head and neck. We report here the identification of a new MAGE-6 antigenic peptide, which is recognized by a tumor-specific cytolytic T lymphocyte clone isolated from a melanoma patient. The peptide, ISGGPRISY, corresponds to positions 293 to 301 of the MAGE-6 protein sequence and is presented by HLA-Cw1601 molecules.

Background: The MAGE-A genes encode antigens that are of particular interest for antitumor immunotherapy because they are strictly tumor specific and are shared by many tumors. We developed a rapid method to identify the MAGE-A genes expressed in tumors. Methods: A low-density DNA microarray was designed to discriminate between the 12 MAGE-A cDNAs amplified by PCR with only one pair of consensus primers. The assay involved reverse transcription of total RNA with oligo(dT) primer, followed by PCR amplification and hybridization on a microarray. Amplification in the presence of Biotin-16-dUTP allowed subsequent detection of the amplicons on the microarray carrying 12 capture probes, each being specific for a MAGE-A gene. Probe–amplicon hybrids were detected by a streptavidin-based method. Results: PCR conditions were optimized for low detection limits and comparable amplification efficiencies among all MAGE-A nucleotide sequences. The microarray assay was validated with a panel of 32 samples, by comparison with well-established reverse transcription-PCR assays relying on amplification with primers specific for each gene. Virtually identical results were obtained with both methods, except for MAGE-A3 and MAGE-A5. Detection of MAGE-A5 was more sensitive with the microarray assay. Detection of MAGE-A3 was hampered by the presence of MAGE-A6, which is 98% identical: the MAGE-A3 capture probe cross-hybridized with MAGE-A6 amplicons because these sequences differed by only a single base. Conclusions: This post-PCR microarray assay could be useful to evaluate MAGE expression in tumors before therapeutic vaccinations with MAGE-A gene products.

The genes of the MAGE-A family code for antigens that are strictly tumor-specific and are shared by many human tumors. Melanoma patients have been immunized against these antigens and some tumor regressions have been observed. However, no unequivocal evidence of cytolytic T cell responses has been obtained by analyzing the blood lymphocytes of these patients. Hence it was considered worthwhile to examine in mouse systems whether or not immunization against antigens derived from the mouse Mage homologs can produce cytolytic T cell responses. We have identified an antigenic peptide encoded by mouse gene Mage-a2, and here we show that immunization of DBA/2 mice with a recombinant adenovirus containing either just the sequence encoding this peptide or a large part of the Mage-a2 coding sequence produces strong cytolytic T cell responses. The Mage-a2 system should prove useful for the comparison of vaccination modalities that could be applied to human patients in therapeutic vaccination trials with MAGE antigens.

From melanoma patient LB1751, cytolytic T lymphocytes (CTL) were generated that lysed specifically autologous tumor cells. To establish whether these CTL recognized one of the Ags that had previously been defined, a CTL clone was stimulated with cells expressing various MAGE genes. It produced TNF upon stimulation with target cells expressing MAGE-A10. The Ag was found to be nonapeptide GLYDGMEHL (codons 254–262), which is presented by HLA-A2.1. This is the first report on the generation of anti-MAGE CTL by autologous mixed lymphocyte-tumor cell culture (MLTC) from a melanoma patient other than patient MZ2, from whom the first MAGE gene was identified. MAGE genes are expressed in many tumors but not by normal tissues except male germline cells and placenta, which do not express HLA molecules. Therefore, the identification of an antigenic peptide derived from MAGE-A10 adds to the repertoire of tumor-specific shared Ags available for anti-tumoral vaccination trials.

The human MAGE genes are expressed in a wide variety of tumors but not in normal cells, with the exception of the male germ cells, placenta, and, possibly, cells of the developing embryo. These genes encode tumor-specific antigens recognized by cytolytic T lymphocytes. The MAGE genes are located on the X chromosome, in three clusters denoted MAGE-A, B, and C, mapping at q28, p21.3, and q26, respectively. The function of these genes remains unknown. Because mice offer many advantages for the study of genes that may be involved in embryonic development, we looked for the murine equivalents of the 12 human MAGE-A genes. Using a MAGE-A probe, we isolated 8 new murine genes that are homologous to the MAGE genes. On average, the open reading frames (ORFs) of these 8 closely related genes display a slightly higher degree of nucleotide identity with the MAGE-A ORFs than with the MAGE-B or MAGE-C ORFs. Furthermore, like MAGE-A genes, they encode acidic proteins, whereas the MAGE-B genes encode basic proteins. Accordingly, these 8 murine genes were named Mage-a1 to 8 (approved symbols Magea1 to 8). Mage-a genes were mapped in two different loci on the mouse X chromosome. Mage-a4 and Mage-a7 are located in a region that is syntenic to either Xp21 or Xq28. The 6 other genes are arranged in a cluster located in a region syntenic to Xp22. Like their human counterparts, Mage-a genes were found to be transcribed in adult testis, but not in other tissues. Expression of some Mage-a genes was also detected in tumor cell lines. Two Mage-a genes were found to be expressed in blastocysts.

Representational difference analysis was used to identify genes that are expressed in a human melanoma cell line and not in normal skin. A cDNA clone that appeared to be specific for tumors was obtained and the corresponding gene was sequenced. This new gene was named LAGE-I. Using a LAGE-I probe to screen a cDNA library from the same melanoma cell line, we identified a closely related gene, which proved to be identical to NY-ESO-I, a gene recently reported to code for an antigen recognized by autologous antibodies in an esophageal squamous cell carcinoma. Gene LAGE-I maps to Xq28. It comprises 3 exons. Alternative splicing produces 2 major transcripts encoding polypeptides of 210 and 180 residues, respectively. Expression of LAGE-I was observed in 25-50% of tumor samples of melanomas, non-small-cell lung carcinomas, bladder, prostate and head and neck cancers. The only normal tissue that expressed the gene was testis. As for MAGE-AI, expression of LAGE-I is induced by deoxy-azacytidine in lymphoblastoid cells, suggesting that tumoral expression is due to demethylation. The expression of LAGE-I is strongly correlated with that of NY-ESO-I. It is also clearly correlated with the expression of MAGE genes.

Gene MAGE-4 (HGMW-approved symbol MAGE4) is expressed in several types of tumors, but not in normal tissues, except testis and placenta, The 5' end of this gene contains eight homologous exons spread over a 5.8-kb region, These exons are alternatively spliced to a unique second exon and a unique third exon, which encodes a protein of 317 amino acids. The analysis of transcripts found in testis, placenta, and a sarcoma cell line showed that each of the alternative first exons is used in at least one of these tissues, Various regions of the promoter of the fifth alternative exon (1.5) were cloned in a luciferase reporter plasmid, and the constructs were transfected in a sarcoma cell. line that expresses MAGE-4, Two Ets motifs located between positions -70 and -29 relative to the transcription start site were found to drive 55% of the promoter activity, A region containing a Sp1 consensus binding site located upstream of the two Ets motifs was found to be responsible for 44% of the transcriptional activity. MAGE-4a promoters 1.4 and 1.6, which also contain the Sp1 and the two Ets binding motifs, supported a level of transcription comparable to that of promoter 1.5, whereas promoter 1.1, which contains only one Ets binding site, was sixfold less active. In line with observations made with gene MAGE-1 (HGMW-approved symbol MAGE1), we found that promoter 1.5 stimulated a high level of transcription in a melanoma cell line that does not express MAGE-4. This suggests that the tumor-specific expression of MAGE genes is not determined by the presence of specific transcription factors. (C) 1997 Academic Press.

We reported previously that human gene MAGE-1 directs the expression of a tumor antigen recognized on a melanoma by autologous cytolytic T lymphocytes. Probing cosmid libraries with a MAGE-1 sequence, we identified 11 closely related genes. The analysis of hamster-human somatic cell hybrids indicated that the 12 MAGE genes are located in the q terminal region of chromosome X. Like MAGE-1, the 11 additional MAGE genes have their entire coding sequence located in the last exon, which shows 64%-85% identity with that of MAGE-1. The coding sequences of the MAGE genes predict the same main structural features for all MAGE proteins. In contrast, the promoters and first exons of the 12 MAGE genes show considerable variability, suggesting that the existence of this gene family enables the same function to be expressed under different transcriptional controls. The expression of each MAGE gene was evaluated by reverse transcription and polymerase chain reaction amplification. Six genes of the MAGE family including MAGE-1 were found to be expressed at a high level in a number of tumors of various histological types. None was expressed in a large panel of healthy tissues, with the exception of testis and placenta.

To characterize the proteins P91Ap and P198p, of which mutants generate the tum- antigens P91A and P198, respectively, rabbit antisera were raised with ovalbumin-coupled synthetic peptides that correspond to their respective C terminus. In immunoadsorption tests using immobilized protein A the antisera recognized the translation products synthesized by rabbit reticulocyte lysates programmed with the SP6 polymerase transcripts of the P91A and P198 cDNA. The presence of the two proteins was demonstrated by SDS-PAGE and immunoblotting in all the mouse cells and organs examined. P91Ap is a constituent of the cytosol; despite a remarkable homology to the Drosophila diphenol oxidase DOX-A2, it separates from murine catechol oxidase activity in rate zonal sedimentation analysis. P198p is a ribosomal constituent, or a factor firmly linked to both the free and membrane-bound ribosomes. These subcellular localizations strengthen other evidence that the antigens presented to T lymphocytes by class I products of the major histocompatibility complex derive from proteins of the cytosol, or in direct contact with it.

New antigens recognized on mouse tumor cells by syngeneic cytolytic T lymphocytes (CTL) can arise as a result of point mutations. Such mutations were observed in the genome of antigenic variants (“turn- variants”) obtained by mutagenic treatment of mouse tumor cells. Three unrelated mutated genes coding for three different turn” antigens have been identified (De Plaen et al. 1988); Sibille et al. 1990; Szikora et al. 1990). The normal equivalents of these genes are expressed in normal tissues. In all instances, the turn- mutation is located in the sequence that encodes the antigenic peptide and it results into a change of one amino-acid in the peptide. For two antigens, this confers to the antigenic peptide the capability to bind to the presenting class I MHC molecule (Lurquin et al. 1989).

Mutagen treatment of mouse P815 tumor cells produces immunogenic mutants that express new transplantation antigens (tum- antigens) recognized by cytolytic T cells. The gene encoding tum- antigen P91A comprises 12 exons and a mutation located in exon 4 is responsible for the production of a new antigenic peptide. Transfection experiments showed that the expression of the antigen could be transferred not only by the entire gene but also by gene segments comprising only the mutated exon and parts of the surrounding introns. This was observed with subgenic regions that were not cloned in expression vectors. Antigen expression did not require the integration of the transfected gene segment into a resident P91A gene by homologous recombination. It also occurred when the subgenic segment was transfected without the usual selective gene, which comprises an eucaryotic promoter, and also without plasmid sequences, which are known to contain weak promoters. When a stop codon was introduced at the beginning of exon 4, the expression of the antigen was maintained and evidence was obtained that an ATG codon located in this region served as initiation site for the translation of the antigenic peptide. But we have not obtained evidence indicating that antigenic peptides are direct translation products rather than degradation products of entire proteins.

We have constructed a pTZ19R-derived vector which allows efficient positive selection of recombinant plasmids. The system uses the EcoK restriction activity of Escherichia coli K-12. to select against non-recombinant plasmids. The vector contains an EcoK site which, if deleted or disrupted by ligating a DNA fragment, yields recombinant plasmids that are no longer susceptible to EcoK restriction when transformed into a restriction-proficient E. coli host.

Mutagen treatment of mouse P815 tumor cells produces immunogenic mutants that express new transplantation antigens (tum- antigens) recognized by cytolytic T cells. We found that the gene conferring expression of tum- antigen P91A contains 12 exons, encoding a 60 kd protein lacking a typical N-terminal signal sequence. The sequence shows no significant similarity with sequences in current data bases. A mutation that causes expression of the antigen is located in exon 4; it is the only apparent difference between the normal and the antigenic alleles. A short synthetic peptide corresponding to a region of exon 4 located around this mutation makes P815 cells sensitive to lysis by anti-P91A cytolytic T cells. The mutation creates a strong aggretope enabling the peptide to bind the H-2 Ld molecule. Several secondary tumor cell variants that no longer express tum- antigen P91A were found to carry deletions in the gene.

A tk- cell line derived from mouse mastocytoma P815 was transfected with a plasmid carrying a thymidine kinase gene. The tk+ cells were obtained at a frequency of 10(-6). From some of these tk+ transfectants it was possible to select tk- cells with BrdU so that these cells could be submitted again to transfection with the thymidine kinase gene. By repeating cycles of transfection and tk+ selection followed by reverse selection with BrdU, it was possible to obtain a stable variant having a 100-fold increase in transfection efficiency. This HTR (high transfection) variant shows a high efficiency of transfection (10(-4)) for the neomycin-resistance gene as well as for the thymidine kinase gene.

Mutagen treatment of mouse mastocytoma P815 produces highly inununogenic “tum−” variants. Most of these variants express potent transplantation antigens which are not present on the original P815 tumor cells. These tum− antigens, which appear to be specific for each variant, elicit a strong cytolytic T lymphocyte (CTL) response, but do not seem to induce a specific antibody response. As a first step in the isolation of the gene of a tum− antigen, we attempted DNA-mediated gene transfer. As a DNA recipient cell we used P1.HTR, a highly transfectable P815 cell line, whose selection has been previously described. For the detection of antigen-expressing cells in transfected populations we developed a procedure that relies on the ability of these cells to stimulate the proliferation of the relevant CTL. Using DNA from tum− variant P91 mixed with a plasmid carrying an antibiotic resistance gene, we obtained several independent transfectants expressing a tum− antigen, at a frequency of approximately 1 in 13 000 antibiotic-resistant transfectants. These transfectants express only one of the two tum− antigens that were identified on P91, suggesting that these tum− antigens correspond to different genes. We expect that the detection procedure described here will be-suitable for the identification of transfectants for any gene that determines the expression of an antigen recognized by CTL.

Overgrowth of hybridomas by proliferating spleen T cells often hinders the production of monoclonal antibodies to certain antigens. To overcome this problem we derived neomycin-resistant fusion partner SP2 neoR.1 by transfection of SP2/0-Ag14 cells with plasmid pSVTK neo beta. Hybridomas obtained with SP2 neoR.1 grew optimally in the presence of the neomycin analog G418 at concentrations which blocked the proliferative response of T cells to mitogenic stimuli. The advantage of using SP2 neoR.1 and G418 under conditions where spleen cell proliferation occurs after fusion was demonstrated with hybridomas derived from a rat immunized with mouse helper T cell lines.

It has been known for a long time that some experimental tumors express antigens that constitute targets for immune rejection by the syngencic host. The existence of these tumor-specific transplantation antigens (TSTA) was first demonstrated with chemically induced mouse sarcomas: each independent tumor was found to express a different antigen (Prchn 1957). Later, these findings were extended to ultraviolet-induced tumors (Kripke 1981). The generality of the existence of transplantation antigens specific for each tumor was however put under serious question when spontaneous mouse tumors were found to be completely incapable of eliciting an immune rejection response (Hewitt 1976). But further, experiments demonstrated that even these tumors express weak TSTA that arc potential targets for immune rejection (Van Pel 1983).

Mouse Ltk − (H-2 k ) cells that became reactive with anti-H-2 d monoclonal antibodies (McAb) after transfection with DNA from H-2 d -positive cells were isolated by an indirect panning method. The cells were transfected with DNA from a plasmid containing the selective thymidine kinase (tk) gene in addition to total cell DNA, so that a first selection could be carried out in hypoxanthine/aminopterin/thymidine (HAT) medium. The HAT-selected tk + transfectants were incubated with anti-H-2 d McAb, washed and transferred to dishes coated with purified anti-immunoglobulin (anti-Ig). This 2-step method of panning has the advantage that only the anti-Ig reagent requires purification. Transfected cells clearly reactive with either anti-L d /R d or anti-K d McAb were isolated after only 1 or2 cycles of panning.

Cytolytic T lymphocyte (CTL) clones directed against spontaneous mouse leukemia LEC have been obtained. By transfecting a cosmid library into cells which were then tested for their ability to stimulate the CTL, we identified the gene coding for the antigen recognized by one of these CTL clones. It is the gag gene of an endogenous defective retrovirus that belongs to the intracisternal A particle (IAP) family. A gag-encoded nonapeptide presented by the H-2 D-k molecule caused recognition by the anti-LEC CTL clone. Southern blot and polymerase chain reaction analyses indicated that the expression of the antigen by the LEC tumor cell line resulted from the transposition of an IAP sequence into a new genomic location.