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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.

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.

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.

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.

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.

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.

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.

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.

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.