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Designing effective antileukemic immunotherapy will require understanding mechanisms underlying tumor control or resistance. Here, we report a mechanism of escape from immunologic targeting in an acute myeloid leukemia (AML) patient, who relapsed 1 year after immunotherapy with engineered T cells expressing a human leukocyte antigen A*02 (HLA-A2)–restricted T cell receptor (TCR) specific for a Wilms’ tumor antigen 1 epitope, WT1126–134(TTCR-C4). Resistance occurred despite persistence of functional therapeutic T cells and continuous expression of WT1 and HLA-A2 by the patient’s AML cells. Analysis of the recurrent AML revealed expression of the standard proteasome, but limited expression of the immunoproteasome, specifically the beta subunit 1i (β1i), which is required for presentation of WT1126–134. An analysis of a second patient treated with TTCR-C4demonstrated specific loss of AML cells coexpressing β1i and WT1. To determine whether the WT1 protein continued to be processed and presented in the absence of immunoproteasome processing, we identified and tested a TCR targeting an alternative, HLA-A2–restricted WT137–45epitope that was generated by immunoproteasome-deficient cells, including WT1-expressing solid tumor lines. T cells expressing this TCR (TTCR37–45) killed the first patients’ relapsed AML resistant to WT1126–134targeting, as well as other primary AML, in vitro. TTCR37–45controlled solid tumor lines lacking immunoproteasome subunits both in vitro and in an NSG mouse model. As proteasome composition can vary in AML, defining and preferentially targeting these proteasome-independent epitopes may maximize therapeutic efficacy and potentially circumvent AML immune evasion by proteasome-related immunoediting.

Supplementary figures and tables

Supplementary tables and figures

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 PDGFRβ 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

Monoclonal antibodies (mAbs) blocking immune checkpoints such as programmed death ligand 1 (PD-L1) have yielded strong clinical benefits in many cancer types. Still, the current limitations are the lack of clinical response in a majority of patients and the development of immune-related adverse events in some. As an alternative to PD-L1–specific antibody injection, we have developed an approach based on the engineering of tumor-targeting T cells to deliver intratumorally an anti–PD-L1 nanobody. In the MC38-OVA model, our strategy enhanced tumor control as compared with injection of PD-L1–specific antibody combined with adoptive transfer of tumor-targeting T cells. As a possible explanation for this, we demonstrated that PD-L1–specific antibody massively occupied PD-L1 in the periphery but failed to penetrate to PD-L1–expressing cells at the tumor site. In sharp contrast, locally delivered anti–PD-L1 nanobody improved PD-L1 blocking at the tumor site while avoiding systemic exposure. Our approach appears promising to overcome the limitations of immunotherapy based on PD-L1–specific antibodies.

Supplementary Figure from T Cell–Mediated Targeted Delivery of Anti–PD-L1 Nanobody Overcomes Poor Antibody Penetration and Improves PD-L1 Blocking at the Tumor Site

Supplementary Data from T Cell–Mediated Targeted Delivery of Anti–PD-L1 Nanobody Overcomes Poor Antibody Penetration and Improves PD-L1 Blocking at the Tumor Site

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

Table S2 shows a selectivity screen of PF-06840003 in in vitro pharmacological assays.

Supplementary Methods

Table S1 shows the plasma protein binding and blood to plasma ratio in mouse, rat, dog and human. Table S3 shows the plasma concentrations following a single oral dose of PF-06840003. Figure S1 shows that PF-06840003 does not impact cell viability. Figure S2 shows that PF-06840003 rescues T-cell proliferation in a co-culture assay. Figure S3 shows tumor growth inhibition of CT26 tumors at different dose levels. Figure S4 shows that IDO1 inhibitor mediated tumor growth inhibition depends on CD8+ T cells. Figure S5 shows a combination benefit with anti-PD-L1 treatment in CT26 tumors. Figure S6 shows IDO1i mediated tumor growth inhibition of MDA-MB-231 breast tumors in humanized mice. Figure S7 shows that PF-06840003 improves the TGI of avelumab in a humanized mouse breast tumor model. Figure S8 shows the fractions of CT26 tumor immune infiltrating cells.

Tumors use indoleamine 2,3-dioxygenase-1 (IDO1) as a major mechanism to induce an immunosuppressive microenvironment. IDO1 expression is upregulated in many cancers and considered to be a resistance mechanism to immune checkpoint therapies. IDO1 is induced in response to inflammatory stimuli such as IFNγ and promotes immune tolerance by depleting tryptophan and producing tryptophan catabolites, including kynurenine, in the tumor microenvironment. This leads to effector T-cell anergy and enhanced Treg function through upregulation of FoxP3. As a nexus for the induction of key immunosuppressive mechanisms, IDO1 represents an important immunotherapeutic target in oncology. Here, we report the identification and characterization of the novel selective, orally bioavailable IDO1 inhibitor EOS200271/PF-06840003. It reversed IDO1-induced T-cell anergy in vitro. In mice carrying syngeneic tumor grafts, PF-06840003 reduced intratumoral kynurenine levels by over 80% and inhibited tumor growth both in monotherapy and, with an increased efficacy, in combination with antibodies blocking the immune checkpoint ligand PD-L1. We demonstrate that anti–PD-L1 therapy results in increased IDO1 metabolic activity thereby providing additional mechanistic rationale for combining PD-(L)1 blockade with IDO1 inhibition in cancer immunotherapies. Supported by these preclinical data and favorable predicted human pharmacokinetic properties of PF-06840003, a phase I open-label, multicenter clinical study (NCT02764151) has been initiated.

Monoclonal antibodies (mAbs) blocking immune checkpoints such as programmed death ligand 1 (PD-L1) have yielded strong clinical benefits in many cancer types. Still, the current limitations are the lack of clinical response in a majority of patients and the development of immune-related adverse events in some. As an alternative to PD-L1–specific antibody injection, we have developed an approach based on the engineering of tumor-targeting T cells to deliver intratumorally an anti–PD-L1 nanobody. In the MC38-OVA model, our strategy enhanced tumor control as compared with injection of PD-L1–specific antibody combined with adoptive transfer of tumor-targeting T cells. As a possible explanation for this, we demonstrated that PD-L1–specific antibody massively occupied PD-L1 in the periphery but failed to penetrate to PD-L1–expressing cells at the tumor site. In sharp contrast, locally delivered anti–PD-L1 nanobody improved PD-L1 blocking at the tumor site while avoiding systemic exposure. Our approach appears promising to overcome the limitations of immunotherapy based on PD-L1–specific antibodies.

SUMMARYLimited supply and catabolism restrict the essential amino acid tryptophan (Trp) in tumors. How tumors sustain translation under Trp stress remains unclear. Unlike other amino acids, Trp stress activates the EGFR, which enhances macropinocytosis and RAS signaling to the MTORC1 and p38/MAPK kinases, sustaining translation. The AHR forms part of the Trp stress proteome and promotes autophagy to sustain Trp levels, and ceramide biosynthesis. Thus, Trp restriction elicits pro-translation signals enabling adaptation to nutrient stress, placing Trp into a unique position in the amino acid-mediated stress response. Our findings challenge the current perception that Trp restriction inhibits MTORC1 and the AHR and explain how both cancer drivers remain active. A glioblastoma patient subgroup with enhanced MTORC1 and AHR displays an autophagy signature, highlighting the clinical relevance of MTORC1-AHR crosstalk. Regions of high Trp or high ceramides are mutually exclusive, supporting that low Trp activates the EGFR-MTORC1-AHR axis in glioblastoma tissue.HIGHLIGHTSUnder Trp stress,EGFR-RAS signaling activates macropinocytosis, MTORC1 and p38.MTORC1 and p38 driven translation induces AHR levels and activity.AHR enhances ceramides and autophagy, sustaining intracellular Trp.In glioblastoma, ceramides localize to low Trp areas, and high AHR associates with MTORC1 activity and autophagy.

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.

BackgroundDespite their revolutionary success in cancer treatment over the last decades, immunotherapies encounter limitations in certain tumor types and patients. The efficacy of immunotherapies depends on tumor antigen-specific CD8 T-cell viability and functionality within the immunosuppressive tumor microenvironment, where oxygen levels are often low. Hypoxia can reduce CD8 T-cell fitness in several ways and CD8 T cells are mostly excluded from hypoxic tumor regions. Given the challenges to achieve durable reduction of hypoxia in the clinic, ameliorating CD8 T-cell survival and effector function in hypoxic condition could improve tumor response to immunotherapies.MethodsActivated CD8 T cells were exposed to hypoxia and metformin and analyzed by fluorescence-activated cell sorting for cell proliferation, apoptosis and phenotype. In vivo, metformin was administered to mice bearing hypoxic tumors and receiving either adoptive cell therapy with tumor-specific CD8 T cells, or immune checkpoint inhibitors; tumor growth was followed over time and CD8 T-cell infiltration, survival and localization in normoxic or hypoxic tumor regions were assessed by flow cytometry and immunofluorescence. Tumor oxygenation and hypoxia were measured by electron paramagnetic resonance and pimonidazole staining, respectively.ResultsWe found that the antidiabetic drug metformin directly improved CD8 T-cell fitness in hypoxia, both in vitro and in vivo. Metformin rescued murine and human CD8 T cells from hypoxia-induced apoptosis and increased their proliferation and cytokine production, while blunting the upregulation of programmed cell death protein 1 and lymphocyte-activation gene 3. This appeared to result from a reduced production of reactive oxygen species, due to the inhibition of mitochondrial complex I. Differently from what others reported, metformin did not reduce tumor hypoxia, but rather increased CD8 T-cell infiltration and survival in hypoxic tumor areas, and synergized with cyclophosphamide to enhance tumor response to adoptive cell therapy or immune checkpoint blockade in different tumor models.ConclusionsThis study describes a novel mechanism of action of metformin and presents a promising strategy to achieve immune rejection in hypoxic and immunosuppressive tumors, which would otherwise be resistant to immunotherapy.

Glioblastoma is an extremely aggressive and difficult cancer to treat, which may partly be due to its limited ability to induce T-cell responses. However, combining viral vector vaccines with other therapies to generate tumor-specific T cells may provide a meaningful benefit to patients. Here, we investigated whether heterologous prime-boost vaccination with chimpanzee-derived adenoviral vector ChAdOx1 and modified vaccinia Ankara (MVA) vaccines could generate therapeutically effective CD8+ T-cell responses against a model antigen P1A, a mouse homolog of human tumor-associated Melanoma Antigen GenE (MAGE)-type antigens, expressed by a BGL-1 mouse glioblastoma cell line. We demonstrated that heterologous prime-boost vaccination with ChAdOx1/MVA vaccines targeting P1A generated a high magnitude of CD8+ T cells specific for the P1A35-43 epitope presented by the MHC class I molecule H-2Ld. Prophylactic vaccination with ChAdOx1/MVA-P1A significantly prolonged the survival of syngeneic mice subcutaneously challenged with P1A-expressing BGL-1 tumors. Furthermore, different vaccination schedules significantly impact the magnitude of antigen-specific CD8+ T-cell responses and may impact protective efficacy. However, the substantial induction of myeloid-derived suppressor cells (MDSCs) by this tumor model presents a significant challenge in the therapeutic setting. Future work will investigate the efficacy of this vaccination strategy on intracranial P1A-expressing BGL-1 models. Citation Format: Emily E. Steffke, James McAuliffe, Vinnycius Pereira Almeida, Amanda Wicki, Laurine Noblecourt, Gary Kohanbash, Hideho Okada, Carol Sze Ki Leung, Masaki Terabe, Benoit J. Van Den Eynde. Heterologous prime-boost viral vector vaccines for the treatment of a P1A-expressing BGL-1 glioblastoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 682.

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.

Within the tumor immunology community, the topic of proteasomal spliced peptides (PSP) has generated a great deal of controversy. In the earliest reports, careful biological validation led to the conclusion that proteasome-catalyzed peptide splicing was a rare event. To date, six PSPs have been validated biologically. However, the advent of algorithms to identify candidate PSPs in mass spectrometry data challenged this notion, with several studies concluding that the frequency of spliced peptides binding to MHC class I was quite high. Since this time, much debate has centered around the methodologies used in these studies. Several reanalyses of data from these studies have led to questions about the validity of the conclusions. Furthermore, the biological and technical validation that should be necessary for verifying PSP assignments was often lacking. It has been suggested therefore that the research community should unite around a common set of standards for validating candidate PSPs. In this review, we propose and highlight the necessary steps for validation of proteasomal splicing at both the mass spectrometry and biological levels. We hope that these guidelines will serve as a foundation for critical assessment of results from proteasomal splicing studies.

Four proteasome subtypes are commonly present in mammalian tissues: standard proteasomes, which contain the standard catalytic subunits β1, β2 and β5; immunoproteasomes containing the immuno-subunits β1i, β2i and β5i; and two intermediate proteasomes, containing a mix of standard and immuno-subunits. Recent studies revealed the expression of two tissue-specific proteasome subtypes in cortical thymic epithelial cells and in testes: thymoproteasomes and spermatoproteasomes. In this review, we describe the mechanisms that enable the ATP- and ubiquitin-dependent as well as the ATP- and ubiquitin-independent degradation of proteins by the proteasome. We focus on understanding the role of the different proteasome subtypes in maintaining protein homeostasis in normal physiological conditions through the ATP- and ubiquitin-dependent degradation of proteins. Additionally, we discuss the role of each proteasome subtype in the ATP- and ubiquitin-independent degradation of disordered proteins. We also discuss the role of the proteasome in the generation of peptides presented by MHC class I molecules and the implication of having different proteasome subtypes for the peptide repertoire presented at the cell surface. Finally, we discuss the role of the immunoproteasome in immune cells and its modulation as a potential therapy for autoimmune diseases.

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 β1-β2-β5i (single intermediate proteasome) and β1i-β2-β5i (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.

For patients with inoperable esophageal adenocarcinoma (EAC), prognosis on conventional chemotherapy (CTX) remains poor. In 2021, the FDA approved two αPD-1 immune checkpoint inhibitors (ICI) for addition to fluoropyrimidine/platinum-containing CTX in this first-line setting. As ICI+CTX enters the clinic, understanding ICI responses and predicting which patients will benefit from ICI addition are key challenges. To address these challenges, we assessed clinical and molecular profiles from the experimental LUD2015-005 trial (NCT02735239, EudraCT 2015-005298-19). Treatment consisted of an initial four-week ICI-only window with durvalumab (αPD-L1) with or without a single dose of tremelimumab (αCTLA-4), followed by 6 cycles of ICI+CTX (CapOx). 38 inoperable patients received treatment (35 EAC; 3 ESCC); median overall survival (OS) and progression-free survival (PFS) were 13.4 and 9.3 months, respectively. All patients reported at least one treatment emergent adverse event (TEAE), with 29 (76.3%) reporting grade 3 or higher TEAEs. EAC patients with available samples (n = 33) were taken forward for biomarker analysis, using tumor and adjacent normal biopsies collected at pre-treatment (PreTx), after four weeks of ICI-only (ICI-4W), and at the end of ICI+CTX (PostTx). Transcriptomic comparison of paired PreTx and ICI-4W EAC biopsies (n = 28) revealed ICI-induced upregulation of a novel T-cell inflammation signature (termed INCITE). Stronger INCITE upregulation correlated with greater tumor shrinkage during the ICI-only window, and tumors with minimal INCITE upregulation showed markers of ICI resistance, including Innate PD-1 Resistance (IPRES). Despite correlation with ICI-only responses, INCITE changes were not associated with overall ICI+CTX outcomes. To find predictive biomarkers of ICI+CTX outcomes, we conducted comprehensive genomic and transcriptomic profiling of PreTx EAC biopsies (n = 33). First, we generated a novel 65,000 cell scRNA-seq dataset and designed a deconvolution workflow to resolve tumor cell composition. Unexpectedly, monocyte composition was strongly linked with greater overall survival (OS) (HR: 0.40 [0.23-0.69]; p = 0.001; FDR = 0.047). Coding tumor mutational burden (TMB) was also associated with improved OS (HR: 0.50 [0.28-0.89]; p = 0.019). Multivariate modelling suggested monocyte composition and TMB were independent and complementary predictors of outcomes. Neither factor was associated with outcomes in a TCGA cohort of EAC patients not treated with ICI, suggesting these biomarkers may be specific to ICI or ICI+CTX. Our findings suggest monocyte composition and TMB may identify EAC patients likely to benefit from ICI+CTX. INCITE upregulation may also serve as a useful monitor of ICI efficacy. These timely findings further our understanding of ICI response and resistance and may help inform patient selection for ICI+CTX. Citation Format: Thomas M. Carroll, Joseph A. Chadwick, Richard P. Owen, Michael J. White, Joseph Kaplinsky, Iliana Peneva, Anna Frangou, Jaeho Chang, Phil F. Xie, Andrew Roth, Bob Amess, Hantao Lou, Katy J. McCann, Georgina Berridge, Roman Fischer, Chansavath Phetsouphanh, Ayo O. Omiyale, Brittany-Amber Jacobs, David Ahern, Simon R. Lord, Stewart Norris-Bulpitt, Sam T. Dobbie, Lucinda Griffiths, Kristen Aufiero Ramirez, Toni Ricciardi, Mary J. Macri, Aileen Ryan, Ralph R. Venhaus, Benoit J. Van den Eynde, Ioannis Karydis, Benedikt M. Kessler, Benjamin Schuster-Böckler, Mark R. Middleton, Xin Lu. Comprehensive molecular profiling to predict first-line immunochemotherapy outcomes in inoperable esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1247.

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. ispartof: FRONTIERS IN IMMUNOLOGY vol:11 ispartof: location:Switzerland status: published

Introduction: Despite tremendous progress in cancer immunotherapy, most patients fail to benefit because of poorly characterized immune resistance mechanisms. Among these, expression of the tryptophan-catabolizing enzyme Indoleamine 2,3-dioxygenase (IDO) has been found in several tumors and associated with local immune suppression, notably by inhibiting T cell functions. However, the exact molecular pathways making T cells sensitive to IDO are still unclear. We propose to exploit the power of whole-genome single-guide RNA (sgRNA) CRISPR screens to uncover novel mechanisms of IDO-mediated T cell dysfunction. Methods: Cas9-expressing transgenic mice were crossed with mice expressing a transgenic T cell receptor (TCR) specifically recognizing a tumor antigen called P1A. Primary Cas9xTCRP1A CD8+ T cells were isolated from these mice and stimulated with P1A-expressing tumor cells. Cells were then transduced with the Teichmann retroviral genome wide CRISPR knockout library. Seven days after the first stimulation, CD8+ T cells were re-stimulated in a control (Tryptophanhigh, kynureninelow) or selective (Tryptophanlow, kynureninehigh) medium, mimicking the function of IDO in vitro. After four days of screening selection, genomic DNA was extracted from remaining living cells and sequencing libraries were prepared. sgRNA representation was then assessed by next-generation sequencing. Results: In vitro CRISPR knockout screening pipeline was successfully set up and validated. This includes: (1.) Successful transduction of the CRISPR knockout library, as well as efficient gene knockout in Cas9xTCRP1A primary CD8+ T cells. (2.) Confirming the strong inhibition of T cell proliferation and survival in the selective medium as compared to the control medium, thereby validating the screening selection strategy. (3.) Optimizing genomic DNA extraction procedure, as well as sequencing library preparation. Finally, the proposed in vitro CRISPR knockout screening was successfully launched, and analysis of potential candidate genes is ongoing. Conclusions and perspectives: Our study proposes to perform genome wide CRISPR knockout screens in T cells exposed to conditions mimicking IDO activity in vitro. Enriched sgRNA at the end of the selection should reveal genes whose inhibition improved T cell survival in Tryptophanlow/kynureninehigh medium. Resulting potential candidate targets will then be validated by knocking them out in T cells and assessing T cell functions under conditions mimicking IDO-mediated immune suppression in vitro. Similar validation of top candidate genes will also be performed in vivo in a model of adoptive cell transfer of target-knockout T cells in mice bearing IDO-expressing tumors. The identified genes/pathways involved in T cell sensitivity to Tryptophan shortage/Kynurenine enrichment should reveal new relevant mechanisms of IDO-mediated immune suppression in the tumor microenvironment. Citation Format: Raphaële Bombart, Jingjing Zhu, Benoit J. Van den Eynde. Uncovering molecular actors of IDO-mediated T cell dysfunction with genome-wide CRISPR/Cas9 knockout screens [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P025.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy and is refractory to available treatments. Delineating the regulatory mechanisms of metabolic reprogramming, a key event in pancreatic cancer progression, may identify candidate targets with potential therapeutic significance. We hypothesized that inflammatory signaling pathways regulate metabolic adaptations in pancreatic cancer. Metabolic profiling of tumors from PDAC patients with a high- (>median, n = 31) and low-NOS2 (inducible nitric oxide synthase

Supplemental material, Supplementary_Figures_cx1 for Induction of tryptophan 2,3-dioxygenase expression in human monocytic leukemia/lymphoma cell lines THP-1 and U937 by Delia Hoffmann, Luc Pilotte, Vincent Stroobant and Benoit J Van den Eynde in International Journal of Tryptophan Research

Tryptophan is required for T lymphocyte effector functions. Its degradation is one of the mechanisms selected by tumors to resist immune destruction. Two enzymes, tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase 1 (IDO1), control tryptophan degradation through the kynurenine pathway. A third protein, indoleamine 2,3-dioxygenase 2 (IDO2), was identified more recently. All three enzymes were reported to be expressed in tumors, and are candidate targets for pharmacological inhibition aimed at restoring effective anti-tumoral immunity. In this review, we compare these three enzymes in terms of structure, activity, regulation and expression in healthy and cancerous tissues, in order to appreciate their relevance to tumoral immune resistance.

The immunogenicity of malignant cells has recently been acknowledged as a critical determinant of efficacy in cancer therapy. Thus, besides developing direct immunostimulatory regimens, including dendritic cell-based vaccines, checkpoint-blocking therapies, and adoptive T-cell transfer, researchers have started to focus on the overall immunobiology of neoplastic cells. It is now clear that cancer cells can succumb to some anticancer therapies by undergoing a peculiar form of cell death that is characterized by an increased immunogenic potential, owing to the emission of the so-called "damage-associated molecular patterns" (DAMPs). The emission of DAMPs and other immunostimulatory factors by cells succumbing to immunogenic cell death (ICD) favors the establishment of a productive interface with the immune system. This results in the elicitation of tumor-targeting immune responses associated with the elimination of residual, treatment-resistant cancer cells, as well as with the establishment of immunological memory. Although ICD has been characterized with increased precision since its discovery, several questions remain to be addressed. Here, we summarize and tabulate the main molecular, immunological, preclinical, and clinical aspects of ICD, in an attempt to capture the essence of this phenomenon, and identify future challenges for this rapidly expanding field of investigation. ispartof: Frontiers in Immunology vol:6 issue:NOV ispartof: location:Switzerland status: published