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.

New Insights into the Mechanisms of Proteasome-Mediated Peptide Splicing Learned from Comparing Splicing Efficiency by Different Proteasome Subtypes.

By tying peptide fragments originally distant in parental proteins, the proteasome can generate spliced peptides that are recognized by CTL. This occurs by transpeptidation involving a peptide-acyl-enzyme intermediate and another peptide fragment present in the catalytic chamber. Four main subtypes of proteasomes exist: the standard proteasome (SP), the immunoproteasome, and intermediate proteasomes ß1-ß2-ß5i (single intermediate proteasome) and ß1i-ß2-ß5i (double intermediate proteasome). In this study, we use a tandem mass tag-quantification approach to study the production of six spliced human antigenic peptides by the four proteasome subtypes. Peptides fibroblast growth factor-5172-176/217-220, tyrosinase368-373/336-340, and gp10040-42/47-52 are better produced by the SP than the other proteasome subtypes. The peptides SP110296-301/286-289, gp100195-202/191or192, and gp10047-52/40-42 are better produced by the immunoproteasome and double intermediate proteasome. The current model of proteasome-catalyzed peptide splicing suggests that the production of a spliced peptide depends on the abundance of the peptide splicing partners. Surprisingly, we found that despite the fact that reciprocal peptides RTK_QLYPEW (gp10040-42/47-52) and QLYPEW_RTK (gp10047-52/40-42) are composed of identical splicing partners, their production varies differently according to the proteasome subtype. These differences were maintained after in vitro digestions involving identical amounts of the splicing fragments. Our results indicate that the amount of splicing partner is not the only factor driving peptide splicing and suggest that peptide splicing efficiency also relies on other factors, such as the affinity of the C-terminal splice reactant for the primed binding site of the catalytic subunit.

Parkinson's disease protein PARK7 prevents metabolite and protein damage caused by a glycolytic metabolite.

Cells are continuously exposed to potentially dangerous compounds. Progressive accumulation of damage is suspected to contribute to neurodegenerative diseases and aging, but the molecular identity of the damage remains largely unknown. Here we report that PARK7, an enzyme mutated in hereditary Parkinson's disease, prevents damage of proteins and metabolites caused by a metabolite of glycolysis. We found that the glycolytic metabolite 1,3-bisphosphoglycerate (1,3-BPG) spontaneously forms a novel reactive intermediate that avidly reacts with amino groups. PARK7 acts by destroying this intermediate, thereby preventing the formation of proteins and metabolites with glycerate and phosphoglycerate modifications on amino groups. As a consequence, inactivation of PARK7 (or its orthologs) in human cell lines, mouse brain, and Drosophila melanogaster leads to the accumulation of these damaged compounds, most of which have not been described before. Our work demonstrates that PARK7 function represents a highly conserved strategy to prevent damage in cells that metabolize carbohydrates. This represents a fundamental link between metabolism and a type of cellular damage that might contribute to the development of Parkinson's disease.

A case of convergent evolution: Several viral and bacterial pathogens hijack RSK kinases through a common linear motif.

Microbes have been coevolving with their host for millions of years, exploiting host resources to their own benefit. We show that viral and bacterial pathogens convergently evolved to hijack cellular mitogen-activated protein kinase (MAPK) p90-ribosomal S6-kinases (RSKs). Theiler's virus leader (L) protein binds RSKs and prevents their dephosphorylation, thus maintaining the kinases active. Recruitment of RSKs enables L-protein-mediated inhibition of eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2 or PKR) and stress granule formation. Strikingly, ORF45 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) and YopM protein of Yersinia use the same peptide motif as L to recruit and activate RSKs. All three proteins interact with a conserved surface-located loop of RSKs, likely acting as an allosteric regulation site. Some unrelated viruses and bacteria thus evolved to harness RSKs in a common fashion, yet to target distinct aspects of innate immunity. As documented for Varicella zoster virus ORF11, additional pathogens likely evolved to hijack RSKs, using a similar short linear motif.

Investigation of chalcogen bioisosteric replacement in a series of heterocyclic inhibitors of tryptophan 2,3-dioxygenase.

Selenium is an underexplored element that can be used for bioisosteric replacement of lower molecular weight chalcogens such as oxygen and sulfur. More studies regarding the impact of selenium substitution in different chemical scaffolds are needed to fully grasp this element's potential. Herein, we decided to evaluate the impact of selenium incorporation in a series of tryptophan 2,3-dioxygenase (TDO2) inhibitors, a target of interest in cancer immunotherapy. First, we synthesized the different chalcogen isosteres through Suzuki-Miyaura type coupling. Next, we evaluated the isosteres' affinity and selectivity for TDO2, as well as their lipophilicity, microsomal stability and cellular toxicity on TDO2-expressing cell lines. Overall, chalcogen isosteric replacements did not disturb the on-target activity but allowed for a modulation of the compounds' lipophilicity, toxicity and stability profiles. The present work contributes to our understanding of oxygen/sulfur/selenium isostery towards increasing structural options in medicinal chemistry for the development of novel and distinctive drug candidates.

Rational Design of Original Fused-Cycle Selective Inhibitors of Tryptophan 2,3-Dioxygenase.

Tryptophan 2,3-dioxygenase (TDO2) is a heme-containing enzyme constitutively expressed at high concentrations in the liver and responsible for l-tryptophan (l-Trp) homeostasis. Expression of TDO2 in cancer cells results in the inhibition of immune-mediated tumor rejection due to an enhancement of l-Trp catabolism via the kynurenine pathway. In the study herein, we disclose a new 6-(1H-indol-3-yl)-benzotriazole scaffold of TDO2 inhibitors developed through rational design, starting from existing inhibitors. Rigidification of the initial scaffold led to the synthesis of stable compounds displaying a nanomolar cellular potency and a better understanding of the structural modulations that can be accommodated inside the active site of hTDO2.

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.

IDO1 and TDO inhibitory evaluation of analogues of the marine pyrroloiminoquinone alkaloids: Wakayin and Tsitsikammamines.

Indoleamine 2,3-dioxygenase (IDO1) and tryptophane 2,3-dioxygenase (TDO) are two heme-containing enzymes which catalyze the conversion of tryptophan to N-formylkynurenine. Both enzymes are well establish therapeutic targets as important factors in the tumor immune evasion mechanism. A number of analogues of the marine pyrroloquinoline alkaloids tsitsikammamines or wakayin have been synthesized, two of them were synthesized using an original method to build the bispyrroloquinone framework. All the derivatives were evaluated in a cellular assay for their capacity to inhibit the enzymes. Six compounds have shown a significant potency on HEK 293-EBNA cell lines expressing hIDO1 or hTDO.

Tryptophan 2,3-Dioxygenase Expression Identified in Murine Decidual Stromal Cells Is Not Essential for Feto-Maternal Tolerance.

Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO) catalyze the rate-limiting step of tryptophan catabolism along the kynurenine pathway, which has important immuno suppressive properties, particularly in tumor cells and dendritic cells. The prominent expression of IDO1 in the placenta also suggested a role in preventing immune rejection of fetal tissues, and pharmacological inhibition of IDO1 induced abortion of allogeneic fetuses in mice. However, this was later challenged by the lack of rejection of allogeneic fetuses in IDO1-KO mice, suggesting that other mechanisms may compensate for IDO1 deficiency. Here we investigated whether TDO could contribute to feto-maternal tolerance and compensate for IDO1 deficiency in IDO1-KO mice. Expression of TDO mRNA was previously detected in placental tissues. We developed a new chimeric rabbit anti-TDO antibody to confirm TDO expression at the protein level and identify the positive cell type by immunohistochemistry in murine placenta. We observed massive TDO expression in decidual stromal cells, starting at day E3.5, peaking at day E6.5 then declining rapidly while remaining detectable until gestation end. IDO1 was also induced in decidual stromal cells, but only at a later stage of gestation when TDO expression declined. To determine whether TDO contributed to feto-maternal tolerance, we mated TDO-KO and double IDO1-TDO-KO females with allogeneic males. However, we did not observe reduced fertility. These results suggest that, despite its expression in decidual stromal cells, TDO is not a dominant mechanism of feto-maternal tolerance able to compensate for the absence of IDO1. Redundant additional mechanisms of immunosuppression likely take over in these KO mice. The massive expression of TDO during decidualization might suggest a role of TDO in angiogenesis or vessel tonicity, as previously described for IDO1.

A Novel KRAS Antibody Highlights a Regulation Mechanism of Post-Translational Modifications of KRAS during Tumorigenesis.

KRAS is a powerful oncogene responsible for the development of many cancers. Despite the great progress in understanding its function during the last decade, the study of KRAS expression, subcellular localization, and post-translational modifications remains technically challenging. Accordingly, many facets of KRAS biology are still unknown. Antibodies could be an effective and easy-to-use tool for in vitro and in vivo research on KRAS. Here, we generated a novel rabbit polyclonal antibody that allows immunolabeling of cells and tissues overexpressing KRAS. Cell transfection experiments with expression vectors for the members of the RAS family revealed a preferential specificity of this antibody for KRAS. In addition, KRAS was sensitively detected in a mouse tissue electroporated with an expression vector. Interestingly, our antibody was able to detect endogenous forms of unprenylated (immature) and prenylated (mature) KRAS in mouse organs. We found that KRAS prenylation was increased ex vivo and in vivo in a model of KRASG12D-driven tumorigenesis, which was concomitant with an induction of expression of essential KRAS prenylation enzymes. Therefore, our tool helped us to put the light on new regulations of KRAS activation during cancer initiation. The use of this tool by the RAS community could contribute to discovering novel aspects of KRAS biology.

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.

Knock-in of murine Calr del52 induces essential thrombocythemia with slow-rising dominance in mice and reveals key role of Calr exon 9 in cardiac development.

Frameshifting mutations (-1/+2) of the calreticulin (CALR) gene are responsible for the development of essential thrombocythemia (ET) and primary myelofibrosis (PMF). The mutant CALR proteins activate the thrombopoietin receptor (TpoR) inducing cytokine-independent megakaryocyte progenitor proliferation. Here, we generated via CRISPR/Cas9 technology two knock-in mouse models that are heterozygous for a type-I murine Calr mutation. These mice exhibit an ET phenotype with elevated circulating platelets compared with wild-type controls, consistent with our previous results showing that murine CALR mutants activate TpoR. We also show that the mutant CALR proteins can be detected in plasma. The phenotype of Calr del52 is transplantable, and the Calr mutated hematopoietic cells have a slow-rising advantage over wild-type hematopoiesis. Importantly, a homozygous state of a type-1 Calr mutation is lethal at a late embryonic development stage, showing narrowed ventricular myocardium walls, similar to the murine Calr knockout phenotype, pointing to the C terminus of CALR as crucial for heart development.

Inhibition of Tryptophan-Dioxygenase Activity Increases the Antitumor Efficacy of Immune Checkpoint Inhibitors.

Tryptophan 2,3-dioxygenase (TDO) is an enzyme that degrades tryptophan into kynurenine and thereby induces immunosuppression. Like indoleamine 2,3-dioxygenase (IDO1), TDO is considered as a relevant drug target to improve the efficacy of cancer immunotherapy. However, its role in various immunotherapy settings has not been fully characterized. Here, we described a new small-molecule inhibitor of TDO that can modulate kynurenine and tryptophan in plasma, liver, and tumor tissue upon oral administration. We showed that this compound improved the ability of anti-CTLA4 to induce rejection of CT26 tumors expressing TDO. To better characterize TDO as a therapeutic target, we used TDO-KO mice and found that anti-CTLA4 or anti-PD1 induced rejection of MC38 tumors in TDO-KO, but not in wild-type mice. As MC38 tumors did not express TDO, we related this result to the high systemic tryptophan levels in TDO-KO mice, which lack the hepatic TDO needed to contain blood tryptophan. The antitumor effectiveness of anti-PD1 was abolished in TDO-KO mice fed on a tryptophan-low diet that normalized their blood tryptophan level. MC38 tumors expressed IDO1, which could have limited the efficacy of anti-PD1 in wild-type mice and could have been overcome in TDO-KO mice due to the high levels of tryptophan. Accordingly, treatment of mice with an IDO1 inhibitor improved the efficacy of anti-PD1 in wild-type, but not in TDO-KO, mice. These results support the clinical development of TDO inhibitors to increase the efficacy of immunotherapy of TDO-expressing tumors and suggest their effectiveness even in the absence of tumoral TDO expression.See article by Hoffmann et al., p. 19.

Tryptophan 2,3-Dioxygenase Expression Identified in Human Hepatocellular Carcinoma Cells and in Intratumoral Pericytes of Most Cancers.

Tryptophan catabolism is used by tumors to resist immune attack. It can be catalyzed by indoleamine 2,3-dioxygenase (IDO1) and tryptophan 2,3-dioxygenase (TDO). IDO1 is frequently expressed in tumors and has been widely studied as a potential therapeutic target to reduce resistance to cancer immunotherapy. In contrast, TDO expression in tumors is not well characterized. Several human tumor cell lines constitutively express enzymatically active TDO. In human tumor samples, TDO expression has previously been detected by transcriptomics, but the lack of validated antibodies has precluded detection of the TDO protein and identification of TDO-expressing cells. Here, we developed novel TDO-specific monoclonal antibodies and confirmed by immunohistochemistry the expression of TDO in the majority of human cancers. In all hepatocarcinomas (10/10), TDO was expressed by most tumor cells. Some glioblastomas (10/39) and kidney carcinomas (1/10) also expressed TDO in tumor cells themselves but only in focal tumor areas. In addition, all cancers tested contained foci of nontumoral TDO-expressing cells, which were identified as pericytes by their expression of PDGFRß and their location in vascular structures. These TDO-expressing pericytes belonged to morphologically abnormal tumor vessels and were found in high-grade tumors in the vicinity of necrotic or hemorrhagic areas, which were characterized by neoangiogenesis. We observed similar TDO-expressing pericytes in inflammatory pulmonary lesions containing granulation tissue, and in chorionic villi, two tissue types that also feature neoangiogenesis. Our results confirm TDO as a relevant immunotherapeutic target in hepatocellular carcinoma and suggest a proangiogenic role of TDO in other cancer types.See article by Schramme et al., p. 32.

Two isoprenylated flavonoids from Dorstenia psilurus activate AMPK, stimulate glucose uptake, inhibit glucose production and lower glycemia.

Root extracts of a Cameroon medicinal plant, Dorstenia psilurus, were purified by screening for AMP-activated protein kinase (AMPK) activation in incubated mouse embryo fibroblasts (MEFs). Two isoprenylated flavones that activated AMPK were isolated. Compound 1 was identified as artelasticin by high-resolution electrospray ionization mass spectrometry and 2D-NMR while its structural isomer, compound 2, was isolated for the first time and differed only by the position of one double bond on one isoprenyl substituent. Treatment of MEFs with purified compound 1 or compound 2 led to rapid and robust AMPK activation at low micromolar concentrations and increased the intracellular AMP:ATP ratio. In oxygen consumption experiments on isolated rat liver mitochondria, compound 1 and compound 2 inhibited complex II of the electron transport chain and in freeze-thawed mitochondria succinate dehydrogenase was inhibited. In incubated rat skeletal muscles, both compounds activated AMPK and stimulated glucose uptake. Moreover, these effects were lost in muscles pre-incubated with AMPK inhibitor SBI-0206965, suggesting AMPK dependency. Incubation of mouse hepatocytes with compound 1 or compound 2 led to AMPK activation, but glucose production was decreased in hepatocytes from both wild-type and AMPKß1-/- mice, suggesting that this effect was not AMPK-dependent. However, when administered intraperitoneally to high-fat diet-induced insulin-resistant mice, compound 1 and compound 2 had blood glucose-lowering effects. In addition, compound 1 and compound 2 reduced the viability of several human cancer cells in culture. The flavonoids we have identified could be a starting point for the development of new drugs to treat type 2 diabetes.

Identifying Antigens Recognized by Cytolytic T Lymphocytes on Tumors.

Identification of antigenic peptides recognized by cytolytic T lymphocytes (CTL) is a prerequisite for the development of targeted cancer immunotherapy approaches. This chapter provides a global approach for the identification of peptides recognized by CTL. It implies the identification of the HLA molecule presenting the peptide as well as the design and screening of a cDNA library derived from the tumor cells. Methods used for the identification of spliced peptides on tumors are also described.

Production of spliced peptides by the proteasome.

CD8+ cytolytic T lymphocytes are essential players of anti-tumor immune responses. On tumors, they recognize peptides of about 8-to-10 amino acids that generally result from the degradation of cellular proteins by the proteasome. Until a decade ago, these peptides were thought to solely correspond to linear fragments of proteins that were liberated after the hydrolysis of the peptide bonds located at their extremities. However, several examples of peptides containing two fragments originally distant in the protein sequence challenged this concept and demonstrated that proteasome could also splice peptides together by creating a new peptide bond between two distant fragments. Unexpectedly, peptide splicing emerges as an essential way to increase the peptide repertoire diversity as these spliced peptides were shown to represent up to 25% of the peptides presented on a cell by MHC class I. Here, we review the different steps that led to the discovery of peptide splicing by the proteasome as well as the lightening offered by the recent progresses of mass spectrometry and bioinformatics in the analysis of the spliced peptide repertoire.

The Vacuolar Pathway of Long Peptide Cross-Presentation Can Be TAP Dependent.

The intracellular pathway of cross-presentation, which allows MHC class I-restricted presentation of peptides derived from exogenous Ags, remains poorly defined and may vary with the nature of the exogenous Ag and the type of APC. It can be cytosolic, characterized by proteasome and TAP dependency, or vacuolar, usually believed to be proteasome and TAP independent. Cross-presentation is particularly effective with long synthetic peptides, and we previously reported that the HLA-A2-restricted cross-presentation of a long peptide derived from melanoma Ag gp100 by human monocyte-derived immature dendritic cells occurred in a vacuolar pathway, making use of newly synthesized HLA-A2 molecules that follow a nonclassical secretion route. In this article, we show that the HLA-A1-restricted cross-presentation of a long peptide derived from tumor Ag MAGE-A3 by human monocyte-derived immature dendritic cells also follows a vacuolar pathway. However, as opposed to the HLA-A2-restricted peptide, cross-presentation of the HLA-A1-restricted peptide is TAP dependent. We show that this paradoxical TAP-dependency is indirect and reflects the need for TAP to load HLA-A1 molecules with peptides in the endoplasmic reticulum, to allow them to escape the endoplasmic reticulum and reach the vacuole, where peptide exchange with the cross-presented peptide likely occurs. Our results confirm and extend the involvement of the vacuolar pathway in the cross-presentation of long peptides, and indicate that TAP-dependency can no longer be used as a key criterion to distinguish the cytosolic from the vacuolar pathway of cross-presentation. They also stress the existence of an alternative secretory route for MHC class I, which will be worthy of further studies.