Tryptophan depletion sensitizes the AHR pathway by increasing AHR expression and GCN2/LAT1-mediated kynurenine uptake, and potentiates induction of regulatory T lymphocytes.

BACKGROUND: Indoleamine 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. METHODS: AHR expression and activation was analyzed by RT-qPCR and western blot analysis in cells engineered to express IDO1/TDO, or cultured in medium mimicking tryptophan catabolism by IDO1/TDO. In vitro differentiation of naïve CD4+ T cells into regulatory T cells (Tregs) was compared in T cells isolated from mice bearing different Ahr alleles or a knockout of Ahr, and cultured in medium with or without tryptophan and kynurenine. RESULTS: We 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 on IDO1/TDO expression. AHR overexpression did not depend on kynurenine but was triggered by tryptophan deprivation. Multiple human tumor cell lines overexpressed AHR on tryptophan deprivation. AHR overexpression was not dependent on general control non-derepressible 2 (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. CONCLUSIONS: Tryptophan 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.

Systemic tryptophan homeostasis.

Tryptophan is an essential amino acid, which is not only a building block for protein synthesis, but also a precursor for the biosynthesis of co-enzymes and neuromodulators, such as NAD/NADP(H), kynurenic acid, melatonin and serotonin. It also plays a role in immune homeostasis, as local tryptophan catabolism impairs T-lymphocyte mediated immunity. Therefore, tryptophan plasmatic concentration needs to be stable, in spite of large variations in dietary supply. Here, we review the main checkpoints accounting for tryptophan homeostasis, including absorption, transport, metabolism and elimination, and we discuss the physiopathology of disorders associated with their dysfunction. Tryptophan is catabolized along the kynurenine pathway through the action of two enzymes that mediate the first and rate-limiting step of the pathway: indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO). While IDO1 expression is restricted to peripheral sites of immune modulation, TDO is massively expressed in the liver and accounts for 90% of tryptophan catabolism. Recent data indicated that the stability of the TDO protein is regulated by tryptophan and that this regulation allows a tight control of tryptophanemia. TDO is stabilized when tryptophan is abundant in the plasma, resulting in rapid degradation of dietary tryptophan. In contrast, when tryptophan is scarce, TDO is degraded by the proteasome to avoid excessive tryptophan catabolism. This is triggered by the unmasking of a degron in a non-catalytic tryptophan-binding site, resulting in TDO ubiquitination by E3 ligase SKP1-CUL1-F-box. Deficiency in TDO or in the hepatic aromatic transporter SLC16A10 leads to severe hypertryptophanemia, which can disturb immune and neurological homeostasis.

Tryptophanemia is controlled by a tryptophan-sensing mechanism ubiquitinating tryptophan 2,3-dioxygenase.

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 noncatalytic 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 four-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 nonmetabolizable 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.

Efficiency of the four proteasome subtypes to degrade ubiquitinated or oxidized proteins.

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 (ß1-ß2-ß5), the immunoproteasome (ß1i-ß2i-ß5i) and the two intermediate proteasomes (ß1-ß2-ß5i and ß1i-ß2-ß5i). 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 ß5i-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, ß5i-containing 20S proteasomes degraded unoxidized naturally disordered protein tau, while 26S proteasomes did not. Our results suggest that the three ß5i-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.

microRNA-155, induced by interleukin-1ß, represses the expression of microphthalmia-associated transcription factor (MITF-M) in melanoma cells.

Loss of expression of surface antigens represents a significant problem for cancer immunotherapy. Microphthalmia-associated transcription factor (MITF-M) regulates melanocyte fate by driving expression of many differentiation genes, whose protein products can be recognized by cytolytic T lymphocytes. We previously reported that interleukin-1ß (IL-1ß) can downregulate MITF-M levels. Here we show that downregulation of MITF-M expression by IL-1ß was paralleled by an upregulation of miR-155 expression in four melanoma lines. We confirmed that miR-155 was able to target endogenous MITF-M in melanoma cells and demonstrated a role for miR-155 in the IL-1ß-induced repression of MITF-M by using an antagomiR. Notably, we also observed a strong negative correlation between MITF-M and miR-155 levels in a mouse model of melanoma. Taken together, our results indicate that MITF-M downregulation by inflammatory stimuli might be partly due to miR-155 upregulation. This could represent a novel mechanism of melanoma immune escape in an inflammatory microenvironment.

Intramuscular electroporation of a P1A-encoding plasmid vaccine delays P815 mastocytoma growth.

This study aimed to construct DNA vaccines encoding the mouse P1A tumor antigen and to generate a protective immune response against the P815 mastocytoma, as a model for vaccines against human MAGE-type tumor antigens. DNA vaccines were constructed and delivered to mice by intramuscular electroporation before tumor challenge. Immunization with a plasmid coding for the full-length P1A significantly delayed tumor growth and mice survived at least 10 days longer than untreated controls. 10% of the mice completely rejected the P815 tumors while 50% of them showed a regression phase followed by tumor regrowth. Mice immunized by electroporation of a P1A(35-43) minigene-encoding plasmid failed to reject tumor and even delay tumor growth. The P1A(35-43)-encoding plasmid was modified and helper epitope sequences were inserted. However, these modified plasmids were not able to improve the response against P815 mastocytoma. Consistent with these results, a 12-fold higher CTL activity was observed when the plasmid coding for full-length P1A was delivered as compared to the plasmid encoding the P1A(35-43) epitope. Our results demonstrated that electroporation is an efficient method to deliver DNA vaccines against P815 and suggested the superiority of full-length as compared to minigene constructs for DNA vaccines.

Reversal of tumoral immune resistance by inhibition of tryptophan 2,3-dioxygenase.

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.

Interleukins 1alpha and 1beta secreted by some melanoma cell lines strongly reduce expression of MITF-M and melanocyte differentiation antigens.

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-1beta. This effect is NF-kappaB 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-1beta reduced by 40-100% their ability to activate such antimelanoma cytolytic T lymphocytes. Finally, we observed large amounts of biologically active IL-1alpha or IL-1beta secreted by two melanoma cell lines that did not express MITF-M, suggesting an autocrine MITF-M downregulation. We estimate that approximately 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.

Expression of BORIS in melanoma: lack of association with MAGE-A1 activation.

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.

Transient down-regulation of DNMT1 methyltransferase leads to activation and stable hypomethylation of MAGE-A1 in melanoma cells.

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.

High frequency of antitumor T cells in the blood of melanoma patients before and after vaccination with tumor antigens.

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 <10(-5) of CD8 T cells. This frequency being arguably too low for these cells to be sole effectors of rejection, we reexamined the contribution of T cells recognizing other tumor antigens. The presence of such antitumor T cells in melanoma patients has been widely reported. To begin assessing their contribution to vaccine-induced rejection, we evaluated their blood frequency in five vaccinated patients. The antitumor cytotoxic T lymphocyte (CTL) precursors ranged from 10(-4) to 3 x 10(-3), which is 10-10,000 times higher than the anti-vaccine CTL in the same patient. High frequencies were also observed before vaccination. In a patient showing nearly complete regression after vaccination with a MAGE-3 antigen, we observed a remarkably focused antitumoral response. A majority of CTL precursors (CTLp's) recognized antigens encoded by MAGE-C2, another cancer-germline gene. Others recognized gp100 antigens. CTLp's recognizing MAGE-C2 and gp100 antigens were already present before vaccination, but new clonotypes appeared afterwards. These results suggest that a spontaneous antitumor T cell response, which has become ineffective, can be reawakened by vaccination and contribute to tumor rejection. This notion is reinforced by the frequencies of anti-vaccine and antitumor CTLs observed inside metastases, as presented by Lurquin et al. (Lurquin, C., B. Lethe, V. Corbiere, I. Theate, N. van Baren, P.G. Coulie, and T. Boon. 2004. J. Exp. Med. 201:249-257).

Contrasting frequencies of antitumor and anti-vaccine T cells in metastases of a melanoma patient vaccinated with a MAGE tumor antigen.

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 interacts with adaptor SKIP and the deacetylase HDAC1 to repress transcription.

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.

A gene expressed exclusively in acute B lymphoblastic leukemias.

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 new tumor-specific antigenic peptide encoded by MAGE-6 is presented to cytolytic T lymphocytes by HLA-Cw16.

"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.

DNA microarray to monitor the expression of MAGE-A genes.

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.