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Key Points The semaphorin receptor PLXNC1 is an IDO1-interacting gene and a strong predictor of survival in AML.An IDO1-related immune gene signature, including PLXNC1, predicts survival in AML., Visual Abstract, The contribution of the bone marrow (BM) immune microenvironment to acute myeloid leukemia (AML) development is well-known, but its prognostic significance is still elusive. Indoleamine 2,3-dioxygenase 1 (IDO1), which is negatively regulated by the BIN1 proto-oncogene, is an interferon-γ-inducible mediator of immune tolerance. With the aim to develop a prognostic IDO1-based immune gene signature, biological and clinical data of 982 patients with newly diagnosed, nonpromyelocytic AML were retrieved from public datasets and analyzed using established computational pipelines. Targeted transcriptomic profiles of 24 diagnostic BM samples were analyzed using the NanoString’s nCounter platform. BIN1 and IDO1 were inversely correlated and individually predicted overall survival. PLXNC1, a semaphorin receptor involved in inflammation and immune response, was the IDO1-interacting gene retaining the strongest prognostic value. The incorporation of PLXNC1 into the 2-gene IDO1-BIN1 score gave rise to a powerful immune gene signature predicting survival, especially in patients receiving chemotherapy. The top differentially expressed genes between IDO1low and IDO-1high and between PLXNC1low and PLXNC1high cases further improved the prognostic value of IDO1 providing a 7- and 10-gene immune signature, highly predictive of survival and correlating with AML mutational status at diagnosis. Taken together, our data indicate that IDO1 is pivotal for the construction of an immune gene signature predictive of survival in AML patients. Given the emerging role of immunotherapies for AML, our findings support the incorporation of immune biomarkers into current AML classification and prognostication algorithms.

In solid tumors and hematological malignancies, including acute myeloid leukemia, some chemotherapeutic agents, such as anthracyclines, have proven to activate an immune response via dendritic cell-based cross-priming of anti-tumor T lymphocytes. This process, known as immunogenic cell death, is characterized by a variety of tumor cell modifications, i.e., cell surface translocation of calreticulin, extracellular release of adenosine triphosphate and pro-inflammatory factors, such as high mobility group box 1 proteins. However, in addition to with immunogenic cell death, chemotherapy is known to induce inflammatory modifications within the tumor microenvironment, which may also elicit immunosuppressive pathways. In particular, DCs may be driven to acquire tolerogenic features, such as the overexpression of indoleamine 2,3-dioxygensase 1, which may ultimately hamper anti-tumor T-cells via the induction of T regulatory cells. The aim of this review is to summarize the current knowledge about the mechanisms and effects by which chemotherapy results in both activation and suppression of anti-tumor immune response. Indeed, a better understanding of the whole process underlying chemotherapy-induced alterations of the immunological tumor microenvironment has important clinical implications to fully exploit the immunogenic potential of anti-leukemia agents and tune their application.

Introduction: ELN intermediate-risk AML poses considerable challenges to clinicians both in terms of accurate prognostication and optimal treatment. Indoleamine 2,3-dioxygenase 1 (IDO1) plays a central role as a mediator of immune tolerance in AML through the increase of Treg cells. IDO1 activity is negatively regulated by the BIN1 proto-oncogene. Herein, we analyzed the correlation between BIN1 and IDO1 expression in AML, also focusing on IDO1-interacting genes, with the aim to identify a predictive gene signature for OS. Methods: Biological and clinical data of 732 patients with de novo AML were retrieved from public TCGA and HOVON datasets. Since details on chemotherapy regimens were not available in the HOVON dataset, we decided to exclude patients >= 65 years from survival analyses. IDO1-interacting genes were selected through a co-expression analysis performed on TCGA RNA-sequencing data accessed through cBioPortal. The best genes combination predicting overall survival was plotted in a gene expression score. Patients were split in three different groups using score quartiles as cut-off. Results: In the HOVON dataset, IDO1 and BIN1 mRNA expression were negatively correlated (r = -0.40, P Conclusions: Our study shows a negative correlation between IDO1 and BIN1 in AML, suggesting IDO1 inhibition by BIN1, and identifies for the first time PLXNC1, a receptor for semaphorines, as an IDO1-interacting gene potentially implicated in immune response regulation. This finding corroborates the role of IDO1 and its interacting genes in the promotion of a tolerogenic microenvironment in AML. Lastly, our gene expression score predicted OS in intermediate-risk AML patients not undergoing HSCT, a finding which has clinical implications for accurate patient stratification and for clinical decision making, i.e., bridging these patients to transplant. Figure Disclosures Papayannidis: Pfizer: Honoraria; Amgen: Honoraria; Incyte: Honoraria; Novartis: Honoraria; Shire: Honoraria; Teva: Honoraria. Cavo:celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; bms: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; novartis: Honoraria; takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Rutella:MacroGenics, Inc.: Research Funding; NanoString Technologies, Inc.: Research Funding; Kura Oncology: Research Funding.

Background: Although the recent advances have allowed the discovery of new and less toxic forms of treatment for acute myeloid leukemia (AML), chemotherapy remains as one of the most used treatments. Some chemotherapeutic agents can induce a type of cell death - immunogenic cell death (ICD) - that can promote modifications in cancer cells, which activates immune system against leukemia cells. Even though this can help the immune system fighting the cancer, chemotherapy is also responsible for inducing tolerogenic mechanisms, that enable cancer cells to escape from immune system control. We previously demonstrated that enzyme indoleamine-2,3-dioxygenase-1 (IDO1) is involved in T regulatory cells (Tregs) induction in AML during ICD induced by chemotherapy. Dying leukemia cells release ATP which binds P2X7 receptor on dendritic cells (DCs), resulting in IDO1 up-regulation. In the same time, ATP can be catabolized by CD39 e CD73 ectonucleotidases expressed on DCs in adenosine with suppressive properties, thus resulting in the stabilization of Tregs phenotype and function. In the present work, we have investigated the role of ATP catabolism in Tregs induction after chemotherapy treatment in AML. Materials and methods: For in vitro experiments,CD14 and CD3 positive cells were obtained from peripheral blood mononuclear cells of healthy donors. HL-60 leukemia cell line cells, untreated or treated with daunorubicin (DNR; 500 ng/ml) or cytarabine (Ara-C; 20 µg/ml) were loaded in immature DCs, previously differentiated from monocytes. After 24 h, immature DCs and DCs matured by treated HL-60 cells or DCs treated with ATP in presence or absence of P2X7 inhibitor, were analyzed for phenotype and used to activate T cells. After 5 days, a detailed phenotype characterization of induced Tregs was performed by flow cytometry. For in vivo experiments, Balbc/J wt mice were subcutaneously injected with 2x106 WEHI-3B leukemia cells. DNR (3 mg/kg), Ara-C (50 mg/kg) or sterile PBS vehicle (placebo) were administered at post inoculum days 7 and 9, then the tumor infiltrating lymphocytes were purified from tumor mass and stained by flow cytometry. Results: Firstly, we in vitro characterized immune phenotype of DCs loaded with HL-60 cells, previously treated with DNR, Ara-C or medium alone. We identified DNR as a potent maturation stimulus as compared to DCs loaded with untreated cells or unloaded immature DCs. In particular, both DNR and Ara-C induced a significant up-regulation of CD83, CD80 and CD86, but only DNR induced a significant up-regulation of CCR7, which is required for DC migration to lymphonodes. Then, the effect of chemotherapy on CD39 and CD73 expression in DCs was also evaluated. DNR induced a significant up-regulation of CD73-the rate limiting step of ATP catabolism, whereas upon Ara-C treatment there was a trend in the up-regulation of both CD39 and CD73. Interestingly, DCs treated with ATP alone achieved similar maturation markers level as DNR, but showed neither CCR7 nor CD39/CD73 up-regulation, which suggests that ATP alone is not sufficient to activate the ATP catabolism pathway. The analysis of Tregs induced after 5 days of co-culture with DCs treated as previously described showed a significant up-regulation of "fitness markers", in particular OX40/CD39 after DNR and OX40/PD-1 after DNR and Ara-C treatment as compared to unloaded immature DCs. Mouse models confirmed the capacity of DNR and Ara-C to induce DCs maturation and activation of ATP catabolism pathways by significantly inducing CD39 and CD73 up-regulation. Moreover, chemotherapy treatment resulted in the stabilization of Tregs suppressive phenotype as demonstrated by the up-regulation of OX40 and ICOS fitness markers in comparison to placebo treatment. Conclusions: Our data suggest that different mechanisms operate in tumor microenvironment. IDO may induce Tregs in an ATP-dependent manner, whereas the ATP catabolism may be involved in the stabilization of Tregs suppressive phenotype. In this context, the specific role of the main bioproduct of ATP catabolism, adenosine, is under investigation. Our results can help to understand the mechanisms of tolerance induction after chemotherapy in AML. Disclosures Cavo: takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; bms: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; novartis: Honoraria.

In acute myeloid leukemia (AML), alloreactive natural killer (NK) cells are crucial mediators of immune responses after haploidentical stem cell transplantation. Allogeneic NK cell infusions have been adoptively transferred with promising clinical results. We aimed at determining whether the composition of NK graft in terms of frequency of alloreactive NK cells influence the clinical response in a group of elderly AML patients undergoing NK immunotherapy. Purpose: In acute myeloid leukemia (AML), alloreactive natural killer (NK) cells are crucial mediators of immune responses after haploidentical stem cell transplantation. Allogeneic NK cell infusions have been adoptively transferred with promising clinical results. We aimed at determining whether the composition of NK graft in terms of frequency of alloreactive NK cells influence the clinical response in a group of elderly AML patients undergoing NK immunotherapy. Experimental Design: Seventeen AML patients, in first complete remission (CR; median age 64 years, range 53-73) received NK cells from haploidentical KIR-ligand-mismatched donors after fludarabine/cyclophosphamide chemotherapy, followed by IL2. To correlate donor NK cell activity with clinical response, donor NK cells were assessed before and after infusion. Results: Toxicity was moderate, although 1 patient died due to bacterial pneumonia and was censored for clinical follow-up. With a median follow-up of 22.5 months (range, 6-68 months), 9 of 16 evaluable patients (0.56) are alive disease-free, whereas 7 of 16 (0.44) relapsed with a median time to relapse of 9 months (range, 3-51 months). All patients treated with molecular disease achieved molecular CR. A significantly higher number of donor alloreactive NK cell clones was observed in responders over nonresponders. The infusion of higher number of alloreactive NK cells was associated with prolonged disease-free survival (0.81 vs. 0.14, respectively; P = 0.03). Conclusions: Infusion of purified NK cells is feasible in elderly AML patients as post-CR consolidation strategy. The clinical efficacy of adoptively transferred haploidentical NK cells may be improved by infusing high numbers of alloreactive NK cells.

e14543Background: The multi-targeted receptor tyrosine kinase inhibitors (TKIs) sunitinib and nivolumab (anti-PD-1) have shown remarkable antitumor activity. Preliminar clinical data on TKIs and an...

BACKGROUND: Overall survival of adult acute myeloid leukemia (AML) remains poor due to the lack of novel and effective therapies. The cancer cell death induced by some chemotherapeutic agents, especially anthracyclines, such as daunorubicin (DNR), named immunogenic cell death (ICD), is characterized by intra- and peri-cellular modifications, which favor the induction of anti-tumor T-cell immune response. Among them, the extracellular release of adenosine triphosphate (extracellular ATP, eATP) from dying tumor cells primes dendritic cells (DCs) by activating purinergic P2X7 receptors, thus eliciting the presentation of tumor antigens to T cell. DCs are key regulators of adaptive immunity, promoting or suppressing T-cell responses. One of the suppressive mechanisms involves the expression of indoleamine 2,3-dioxygenase 1 (IDO1), which plays a major role in the induction of T-cell tolerance through the expansion of regulatory T cells (Tregs). The present study aimed at evaluating the involvement of IDO-1 during ATP-driven ICD in AML. METHODS: AML patients were analyzed at diagnosis and after DNR-based chemotherapy. Ex vivo T cells were characterized by FACS and tested for their capacity to produce IFN-γ in response to autologous blasts. Then, CD8+IFN-γ-producing T cells were expanded and further characterized. ATP was used as an ICD representative model. In vitro, murine WEHI-3B and human HL-60 leukemic cell lines and primary blasts were tested for ATP release after DNR treatment. To in vivo investigate DNR-induced ICD, WEHI-3B cells stable transfected with luciferase PmeLUC were inoculated subcutaneously in BALB/c mice to measure ATP release directly from tumor mass. Tumor infiltrating DCs and T cells were characterized by FACS and immunohistochemistry after chemotherapy and plasma levels of cytokines were measured. In vitro DNR-treated AML cells were pulsed into immature DCs, previously generated from healthy donors. DCs maturation and IDO1 expression were examined (by FACS and western blot, respectively) and correlated with the presence of ATP in culture medium. IDO-driven Tregs induction was assessed. Finally, functional immunological tests were performed in vitro to test the ability of Tregs to inhibit leukemia antigen-specific IFN-γ production (FACS analysis) by ICD-activated T cells. RESULTS: After chemotherapy, 15/23 AML patients had an increase in leukemia-specific IFN-γ producing CD4+ and CD8+ T cells. Also an increase of Tregs was observed with a peak at day 21. CD8+ IFN-γ-producing T cells, which resulted in a skewing toward an effector memory phenotype, were activated and cytotoxic against autologous AML blasts but showed features of exhaustion and were defective in perforin production. In vitro and in vivo DNR induced ATP release from AML cells. In vivo the analysis of tumor-infiltrating T cells after treatment has shown an exhausted phenotype of cytotoxic CD8+ cells, increased IFN-γ+ Tregs and decreased TNF-α+ effector T cells. DNR treatment also increased in vivo plasma levels of cytokines IFN-γ, IL-1β, TNF-α, IL-12. Moreover, in DNR-treated mice we observed a significant increase of CD11c+ mature DCs which express IDO1 in tumor infiltrate. In vitro, loading of DNR-treated AML cells into DCs resulted in increased maturation, but also in IDO1 induction. Interestingly, extracellular ATP was directly involved in DCs maturation and IDO1 expression via purinergic receptor P2Y11. ICD-driven DCs were able to expand Tregs in an IDO-dependent manner. Finally, ICD both triggers a leukemia-specific IFN-γ production by CD8+T cells and induces Tregs, via IDO1-expressing DCs, which in turn inhibit leukemia-specific T cell. CONCLUSIONS: Overall, our data indicate that in AML chemotherapy-induced ICD has contrasting, and not fully elucidated, effects on T-cell immune response, resulting in the induction of leukemia-specific CTLs, albeit with defective features, and Tregs. In this scenario, the effects of ATP release from dying leukemia cells on DCs may be pivotal, as indicated by its capacity to concomitantly induce DC maturation and activation as well as tolerogenic function via IDO1. The combination of novel immunological drugs, such as IDO1 and/or checkpoint inhibitors, with conventional chemotherapy may represent an interesting approach to contrast tolerance induction and, then, fully exploit the immunogenic effect of chemotherapy. Disclosures Martinelli: Genentech: Consultancy; Amgen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Novartis: Speakers Bureau; Ariad: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; MSD: Consultancy; BMS: Speakers Bureau. Cavo:Celgene: Consultancy, Honoraria; Millennium: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Amgen: Consultancy, Honoraria.

In a phase II clinical study, pretreated multiple myeloma patients with relapsing or stable disease received autologous anticancer vaccine containing dendritic cells loaded with Id-protein. Patients received a total of 6 vaccine doses intradermally in monthly intervals. No clinical responses were observed. During the follow-up with a median of 33.1 months (range: 11-43 months), the disease remained stable in 7/11 (64%) of patients. Immune responses measured by ELISpot were noted in 3/11 (27%) and DTH skin test for Id-protein was positive in 8/11 (73%) of patients; out of those, 1/11 (9%) and 5/11 (46%), respectively, had preexisting immune response to Id-protein before the vaccination began. Outcomes were compared to those of a control group of 13 patients. A trend to lower cumulative incidence of progression in the vaccinated group was observed at 12 months from the first vaccination (p= 0.099). More patients from the control group compared to vaccinated patients required active anticancer therapy [4/11 (36%) vs. 8/13 (62%)]. Vaccines based on dendritic cells loaded with Id-protein are safe and induce specific immune response in multiple myeloma patients. Our results suggest that the vaccination could stabilize the disease in approximately two-thirds of patients.dendritic cells, immunotherapy, anticancer vaccines, Id-protein, multiple myeloma.

Despite novel treatment strategies, multiple myeloma (MM) remains an incurable disease with low immunogenicity and multiple immune defects. We developed an ex vivo strategy for inducing myeloma-specific cytotoxic T lymphocytes (CTLs) and demonstrate the possibility of identification and long-term in vivo monitoring of individual myeloma-specific T-cell clones using the most sensitive clonotypic assay that is able to detect low frequencies of T-cell clones (1 clonotypic cell in 10(6) cells). Ten patients with MM were examined for the presence of tumour-reactive T cells using dendritic cells loaded with autologous tumour cells. All patients had detectable myeloma-reactive T cells in vitro. Expanded myeloma-reactive T cells demonstrated specific cytotoxic effects against autologous tumour cells in vitro (median 39.6% at an effector:target ratio of 40:1). The clonality of myeloma-specific T cells was studied with a clonotypic assay, which demonstrated both oligoclonal and monoclonal populations of myeloma-specific T cells. CD8(+) CTLs were the most immunodominant myeloma-specific T-cell clones and clinical responses were closely associated with the in vivo expansion and long-term persistence of individual CD8(+) T-cell clones, usually at very low frequencies (10(-3)-10(-6)). We conclude that the clonotypic assay is the most sensitive tool for immunomonitoring of low-frequency T cells.

Adoptive immunotherapy is a promising approach in the treatment of multiple myeloma. We have tested the identification, separation, and expansion of allogeneic myeloma-specific T cells in vitro. Irradiated myeloma cell line ARH 77 has been used to stimulate allogeneic CD4(+) and CD8(+) T lymphocytes. Activated myeloma-specific T cells that produced interferon-gamma were isolated using immunomagnetic beads and further expanded in vitro to numbers of up to 400 x 106 T cells. Specificity of the T lymphocytes was tested using a 5-(6-)carboxyfluoresceine diacetate succinimidyl ester (CFSE)-based cytotoxicity test. This study demonstrates the feasibility of identification and isolation of tumor-specific T cells from allogeneic donors that can be expanded in vitro to numbers useful for clinical applications.

Anticancer immunotherapy using dendritic cell-based vaccines is a strategy aimed at the induction and maintenance of immune responses against cancer cells. Clinical applications of dendritic cells (DCs) require stringent adherence to Good Manufacturing Practice (GMP) methods and rigorous standardization of DC-based vaccine preparation. Recently, closed systems for DC culture have been developed with a goal to minimize the risk of contamination. Here, we compare the yield, immunophenotype, and functional properties of DCs generated in Lifecell X-Fold culture bags and in plastic wells, both from adherence-selected monocytes, and review the current literature on closed systems for DC generation. We found that both the overall yield and the yield of CD83+ cells in cell culture bags was lower than in the standard culture method. No statistically significant differences were observed in the expression of DC immunophenotypic markers. The capability of DCs cultured in bags and in wells to induce the proliferation of allogeneic mononuclear cells were equivalent. The performance of DCs in mixed lymphocyte reaction correlated significantly (p = 0.005) with the CD83 expression but not with the CD80, CD86, HLA-DR, CD1a, and CD1c expression. We conclude that the immunophenotype and stimulatory properties of DCs cultured in closed cell culture bags are similar to those generated by conventional method using cell culture wells.

Abstract 1047 Indoleamine 2,3-dioxygenase (IDO) enzymes (IDO1 and IDO2) critically regulate the rate-limiting step of tryptophan degradation along kynurenine pathway in several cellular subsets, including dendritic cells (DCs). In particular, it is known that IDO1 expression in DCs is induced by inflammatory mediators, especially PGE2, and enhances DCs tolerogenic capacity, such as the induction of T regulatory cells (Tregs). In absence of exogenous stimuli, IDO1 is driven to proteasomal degradation by SOCS3. Conversely, few and contrasting results are available about IDO2 expression and function in human DCs. We, then, besides IDO1, investigated the expression of IDO2 in human DC subsets, obtained from blood samples of healthy volunteers. Circulating myeloid and plasmacytoid DCs were purified and analyzed for the expression of both IDO enzymes. Our data demonstrate that myeloid DCs express higher levels of both IDO1 and IDO2 mRNA in comparison to plasmacytoid DCs. Moreover, PGE2 modulates IDO1 and IDO2 mRNA expression only in myeloid DCs and has no effect in plasmacytoid DCs. At protein level, IDO1 is expressed only in myeloid DCs and is modulated by PGE2, whereas IDO2 is expressed in both myeloid and plasmacytoid DCs and is not modulated by PGE2. Interestingly, when gene transcription or protein translation are inhibited, IDO2 protein expression persists independently from PGE2, while IDO1 expression requires PGE2 presence and needs continuous transcription and translation. Such discrepancy might be derived by a different binding of IDO1 and IDO2 to SOCS3, which in absence of pro-inflammatory stimuli is known to bind IDO1 and to drive it to proteasomal degradation. Indeed, SOCS3 immunoprecipitation and proteasomal inhibition experiments show that SOCS3 does not bind IDO2 which is not, therefore, driven to proteasomal degradation and is stably expressed. At the functional level, both myeloid and plasmacytoid DCs generate Tregs through an IDO-dependent mechanism, since the addition of IDO inhibitors 1-MT-D and 1-MT-L abrogate Tregs generation. More specifically, the silencing of IDO2 through its specific siRNA downregulates kynurenine production and Tregs generation, thus demonstrating that IDO2 is functionally active in DCs. In conclusion, our data demonstrate that, besides IDO1, human DCs also express IDO2, which contributes to tolerance by degrading tryptophan into kynurenine and by inducing Tregs. However, unlike IDO1, IDO2 expression is independent from inflammatory mediators, such as PGE2, and does not require continuous synthesis. These findings indicate IDO pathway as a critical check-point in the fine-tuning of DC-mediated tolerance induction. In particular, while IDO2 expression may confer a homeostatic tolerogenic capacity to DCs upon steady-state conditions, inflammation results in the induction of additional mechanisms of immune tolerance, such as the up-regulation of IDO1. Disclosures: Metz: New Link Genetics Corporation: Employment.

Abstract 2190 Adenosine 5'-triphosphate (ATP) plays a pivotal role in several cellular processes, through specific cell membrane purinergic P2 receptors (P2Rs). During inflammation and tumor cell growth, cell necrosis causes the release of intracellular ATP into the extracellular space, thus increasing from low (1–10 nM) to high (5–10 mM) the concentration of extracellular ATP. For this reason, variations in the extracellular ATP concentration might activate/inhibit the immune system. Here we investigated the role of ATP on CD4+ T-cell functions. We first demonstrated the expression of P2Rs for extracellular nucleotides in human activated CD4+ T cells and regulatory T cells (Tregs) We then show that physiological concentrations of extracellular ATP (i.e. 1–50 nM) do not affect both activated CD4+ T cells and Tregs. Conversely, supraphysiological concentrations of ATP show a bimodal effect on activated CD4+ T cells. Whereas 250 nM of ATP stimulates proliferation, cytokine release, expression of adhesion molecules and adhesion, high ATP concentration (i.e. 1 mM) induces apoptosis and inhibits activated CD4+ T-cell functions. On the contrary, at the same high concentration, ATP enhances the proliferation, adhesion, migration and immunosuppressive ability of Tregs. Similar results are obtained when activated CD4+ T cells and Tregs are exposed to ATP released by necrotized leukemic blasts. The present results provide evidence that different concentrations of extracellular ATP modulate T cells according to their activation status. Therefore, high concentrations of ATP, compatible with fast-growing tumors or hyper-inflamed tissues, may have a key role in killing activated CD4+ T cells and in expanding Tregs. Disclosures: No relevant conflicts of interest to declare.

Abstract 3894 Introduction: human NK cells are large, granular cells derived from lymphocyte lineage. They are critical mediators of the innate immunity as they rapidly respond to pathogens infected and tumour cells through cytotoxic and cytokines-producing responses. Indoleamine 2,3-dioxygenase (IDO1) is an enzyme catalyzing the degradation of the essential amino acid L-tryptophan into kynurenines. Several cell types, including dendritic cells, have been shown to express IDO1, which acts as a potent immunosuppressive agent. Recently, in addiction to IDO1, the new variant IDO2 was described but its immunosuppressive role is still under investigations. The aim of the present work is to study the expression and the role of IDO1 and IDO2 in NK cells. Methods: CD3-CD56+NK cells were immunomagnetically purified from healthy donors. IFN-gamma production was measured by ELISA assay. IDO1 and DAP12 transcript levels were normalized on ABL. IDO2 expression was measured by semi-quantitative PCR. Kynurenine levels were evaluated by colorimetric assay. Results: peripheral blood NK cells were treated with IL-2 for 16h, 40h, 88h and 160h. At the end of each IL-2 stimulation, supernatants were collected and IFN-gamma production was measured by colorimetric assay. IDO1 expression in NK cells was evaluated by quantitative real-time PCR and Western blotting. Our data show that, during IL-2-mediated activation, IDO1 is up-regulated at mRNA and protein levels. This effect is maximum after an overnight incubation and it decreases at later time points. IDO1 expression is clearly correlated with IFN-gamma production by NK cells whereas IFN-gamma receptor expression is not affected. Culturing NK cells with a combination of IL-2 and anti-IFN-gamma antibody demonstrated that IDO1 expression is regulated, at least in part, by IFN-gamma. Interestingly, IDO1 mRNA upregulation is associated with downregulation of DAP12, a gene whose expression has been showed to be inversely correlated to IDO1 in dendritic cells. IDO2 expression seems to be not affected by IL-2 stimulation. Treatment with other inflammatory cytokines demonstrated that IDO1 is modulated by different stimuli. Overnight incubation with IL-12, IFN-gamma and a combination of IL-2 and IL-6 showed IDO1 induction. IFN-gamma showed the highest IDO1 induction at transcript and protein level and, surprisingly, of IDO2 expression. IDO1 expression by NK cells resulted in increased production of kynurenines and this effect was abrogated by the addition of the IDO1 inhibitor 1-methyl tryptophan. Conclusion: our results demonstrate that NK cells upregulate IDO1 expression during cytokine-mediated activation and that IDO1 expression is regulated, at least in part, by IFN-gamma. Similarly to DCs, NK cells express IDO1 gene in association with DAP12, thus suggesting a common regulatory pathway among different cell subsets. Disclosures: Baccarani: NOVARTIS: Honoraria; BRISTOL MYERS SQUIBB: Honoraria.

Abstract 278 Indoleamine 2,3-dioxigenase (IDO) is the rate-limiting enzyme in tryptophan catabolism along the kynurenine pathway. IDO expression by different cell subsets inhibits T-cell activation, proliferation and survival and induces regulatory T cells (Tregs). Although human monocyte-derived dendritic cells have been shown to express IDO, little is known about its expression in other subsets of human DCs, including those generated from CD34+ hematopoietic progenitors (CD34+-derived DCs). In particular, no data are currently available for IDO expression in CD34+-derived DC subsets, such as dermal DCs and epidermal Langherans cells (LCs), which are physiologically resident DCs in the skin and act as the main sentinels against pathogens. In the present study, we performed a full characterization of IDO expression and function by human DCs generated from CD34+ cells. CD34+-derived DCs were generated from purified CD34+ cells after 7 days of culture with GM-CSF and TNF-alpha. In some experiments, day 7-DCs were sorted into different subsets and tested for IDO expression and function. Alternatively, day 7-DCs were cultured with GM-CSF and IL-4 and then matured with a cytokine cocktail of maturation stimuli (IL-1beta, TNF-alpha, IL-6, PGE2). After culture, DCs were analyzed for IDO expression by real-time PCR and western immunoblot, kynurenine production, inhibition of allogenic proliferation and Tregs induction. CD34+ cells did not express IDO mRNA, without significant differences among different progenitor cell sources (cord blood, mobilized peripheral blood, bone marrow). During DC differentiation, IDO mRNA expression was observed at day 7, but not at day 14. At day 14, maturation stimuli induced a marked up-regulation of IDO mRNA and protein, which resulted in increased kynurenine production and inhibition of T-cell proliferation. Flow cytometry analysis of day-7 cells revealed a double population, which comprised CD14+CD1a− dermal DCs and CD14−CD1a+ LCs. Interestingly, IDO mRNA and protein were observed only in dermal DCs, but not in LCs. IDO expression by dermal DCs resulted in increased production of kynurenine and in reduced allostimulatory capacity of T-cell proliferation. Dermal DCs were shown to induce a population of CD4+CD25+Foxp3+ which acted as Tregs by inhibiting allogeneic T-cell proliferation. This effect was abrogated by the addition of the IDO inhibitor 1-methyl tryptophan. Interestingly, dermal DCs and LCs showed a differential expression pattern of chemokine receptors. In particular, while both LCs and dermal DC expressed CCR7, CXCR4 expression segregated only in IDO-expressing dermal DCs. These data may correlate to different trafficking features of CD34+-derived DC subsets and may be associated to IDO expression. In conclusion, DC differentiation of CD34+ cells results in the expression of a functionally active IDO protein in dermal DCs, but not in LCs. Given the role of IDO in regulating immune tolerance, our data may suggest that within the complex skin microenvironment dermal DCs are intrinsically committed to function as regulatory DCs, whereas LCs are devoted to act as activating DCs. These data have implications for a better understanding of the development of the immune response during inflammation/infection. Disclosures: No relevant conflicts of interest to declare.

Multiple myeloma has been considered a weakly immunogenic malignancy that can cause profound defects in the immune system. An important issue for the immunotherapy of myeloma is the identification of appropriate tumor-associated antigens (TAAs). Recently, hTERT (human telomerase reverse transcriptase) was detected on a majority of human malignancies. In the studies reported here, we studied antigen-specific and HLA-A2-restricted cytotoxic activity against an ARH77 myeloma cell line in vitro. An HLA-A2-specific hTERT-derived nonapeptide ((540)ILAKFLHWL(548)) was used as a TAA. Myeloma-specific cytotoxic activity of hTERT-reactive cytotoxic lymphocytes (CTLs) was established by repeated stimulation of the CTLs via dendritic cells loaded with hTERT-derived nonapeptide. These studies were able to demonstrate that hTERT-reactive T-lymphocytes can be identified and expanded using relatively simple in vitro techniques consisting of antigen-specific stimulation, immunomagnetic sorting, and then induction of rapid expansion.

Abstract 4787 Indoleamine 2,3-dioxygenase (IDO) is a heme-containing enzyme that catalyzes the first and rate-limiting step in tryptophan degradation along the kynurenine pathway. IDO is able to inhibit T-cell function and to induce the transformation of T-cells into regulatory T-cells. Several studies demonstrated that IDO expression is involved in immune tolerance induction during pregnancy, infection, transplantation, autoimmune diseases and neoplasias, including acute myeloid leukemia (AML). In particular, our and other groups demonstrated that IDO is expressed in a significant proportion of AML patients and that it increases along with disease progression. Here, we addressed the correlation between IDO expression by AML cells, risk factors at diagnosis and patients' outcome. Adult AML patients from the Hematology Institute “L. and A. Seràgnoli” in Bologna were analyzed for risk characteristics at diagnosis and for IDO expression by RT-PCR and by Western-Blot analysis. Patients were stratified according to age at diagnosis, de novo or secondary disease (pre-existing myelodysplastic syndrome or radio-chemotherapy), leucocytosis, cytogenetics (on the basis of cytogenetic characteristics patients were divided into low, intermediate and high risk groups) and FLT3 and NPM mutational status. Fifty-two patients with AML at diagnosis were analyzed for IDO expression both at gene and protein level. According to IDO transcript levels, patients were divided into IDO-negative (21%) and IDO positive (79%). Positive patients were further subdivided into three different subgroups according to IDO level: IDO-low expression (78%), IDO-intermediate expression (10%) and IDO-high expression (12%) patients. When IDO protein was assessed, we found a correlation between IDO mRNA level and the detection of IDO protein. In particular, IDO protein was detectable only in IDO-high-expressing patients. No statistically significant differences in the recurrence of prognostic characteristics at diagnosis among the groups considered were observed, even though IDO-negative and IDO-low expressing patients showed a higher median age at diagnosis than IDO-intermediate and IDO-high expressing patients and an increased frequency of high-risk cytogenetics was found in IDO-high expressing patients. Response to induction chemotherapy regimen was then analyzed among the four groups of patients. Only patients who received cytotoxic chemotherapy were evaluated for response. Intriguingly, we found that refractory patients were 60% among patients who express IDO at high level and 27% among IDO-negative patients. In conclusion, IDO-high expressing patients show an increased proportion of refractory disease than IDO negative patients. To support our preliminary findings, a multivariate analysis on a larger cohort of patients is currently ongoing. Disclosures: No relevant conflicts of interest to declare.

Abstract 3231 The indoleamine 2,3-dioxygenases IDO1 and IDO2 participate in tryptophan catabolism along the kynurenine pathway and are specifically inhibited by 1-methyltryptophan (1-MT). IDO1 expression in dendritic cells (DCs) is upregulated by various maturation stimuli. In particular, PGE2 plays a crucial role in inducing IDO1-expressing DCs. While it is well established that IDO1-expressing DCs contribute to immunological tolerance by a number of mechanisms, little is known about the expression, function and regulation of IDO2 in DCs. Here we show that immature monocyte-derived DCs (Mo-DCs) express IDO2 which, like IDO1, is also upregulated upon maturation with a prostaglandin-E2 (PGE2)-containing cytokine cocktail. However, while IDO1 upregulation is strictly dependent on PGE2 during maturation, IDO2 expression level is not affected by PGE2. We show that IDO2 expressed by Mo-DCs is enzymatically active, producing L-kynurenine, and that this activity affects T-cell functions by inhibiting allogeneic and autologous T-cell proliferation and by inducing formation of a population of T regulatory cells (Tregs). Taken together, our findings establish different regulatory pathways for IDO2 and IDO1 in Mo-DCs, where IDO2 is functionally active, and they demonstrate that the tolerogenic function of Mo-DCs relies in part on PGE2-independent expression of IDO2. Disclosures: Metz: New Link Genetics Corporation: Employment. Prendergast:New Link Genetics Corporation: Employment.

Multiple myeloma (MM) has been considered as low immunogenic incurable disease. The attempts has been made to invert the immune status to recognize myeloma cells by T cells or other cells of the immune system. Here we studied biology of myeloma-specific T cells in vitro. Irradiated myeloma cell line ARH 77 has been used as tumor antigen to stimulate peripheral blood mononuclear cells (PBMC) of 8 healthy volunteers. Dendritic Cells loaded by irradiated autologous MM cells has been used to stimulate PBMC of 10 MM patients. Activated responder T cells have been immunomagnetically separated based on surface expression of interferon gamma (Miltenyi Biotech) and expanded in 5 cases by phytohemaglutinin and repeated high-doses of interleukin 2. Cytotoxicity against the original myeloma cells has been tested after the expansion using propidium iodide or 7-amino actinomycin D. Responder T cells were labeled by CFSE to distinguish them from myeloma cells. Third-party PBMC and interferon gamma negative fraction of T cells served as controls. The percentage of interferon gamma positive cells in healthy donors has been enriched from 2.8±0.9 and 2.6±0.8 to 48.6±23.4 and 73.2±25.9 of CD3+CD4+ and CD3+CD8+ T cells, respectively, by immunomagnetic separation. Interferon gamma positive T cells have been further expanded in vitro from 0.54x106 ± 0.05x106 to 214.00x106 ± 103.46x106 within 4 weeks. A specific cytotoxicity has been tested after expansion. The killing of myeloma cells by expanded IFN g+ T cells reached 68.1±14.2%, while interferon gamma negative fraction killed only 0.8±0.3% of myeloma cells. As another control, killing of third-party PBMC by expanded interferon gamma positive T cells was 6.9±2.5%. Similar observations were made in MM patients and will be presented at the meeting. These data demonstrate a specific cytotoxic effect of expanded interferon gamma positive T cells against myeloma cell line ARH 77 and open the possibility for clinical use of tumor-specific T cells in MM.

Abstract 3233 Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme in tryptophan catabolism along the kynurenine pathway. IDO expressed by different cell subsets inhibits T-cell activation, proliferation and survival and induces regulatory T cells (Tregs), thus mediating immunological tolerance. Although human monocyte-derived dendritic cells (DCs) have been shown to express IDO, little is known about its expression in other subsets of human DCs, including those generated from CD34+ hematopoietic progenitors (CD34+-derived DCs). In particular, no data are currently available for IDO expression in CD34+-derived DC subsets. CD34+-derived DCs were generated from healthy donors from purified CD34+ cells after 7 days of culture with GM-CSF and TNF-a. Then, DCs were separated into CD1a−CD14+ and CD1a+CD14− cells. DCs subsets were analyzed for IDO expression by real-time PCR and western immunoblot, kynurenine production, inhibition of allogeneic proliferation and Tregs induction. CD34+ cells did not express IDO mRNA expression regardless of the progenitor cell sources (cord blood, mobilized peripheral blood, bone marrow). During DC differentiation, IDO expression and function, evaluated by enzymatic and immunological tests, was markedly induced at day 7. Interestingly, the expression of IDO was shown to be 10 times higher in the CD1a+ compartment as compared to CD1a- cell fraction. IDO expression resulted in increased production of kynurenine and in reduced allostimulatory capacity of T-cell proliferation. Moreover, CD1a+ cells were shown to induce a population of CD4+CD25+Foxp3+ which acted as Tregs by inhibiting allogeneic T cell proliferation. This effect was abrogated by the addition of the IDO inhibitor 1-methyl tryptophan. Phenotypically, IDO-expressing CD1a+ cells expressed typical marker of Langerhans cells such as CD207, CD11b, CD1c, as well as CD103, which has been recently identified as a marker for tolerogenic DCs. Importantly, IDO expression was mainly detected in the CD103+ CD207+ fraction, which induces Tregs through an IDO-dependent manner. In conclusion, DC differentiation of CD34+ cells results in the expression of a functionally active IDO protein in CD103-expressing DCs. Given the role of IDO in regulating immune tolerance, a subset of bone marrow-derived DCs, expressing CD103, may be intrinsically committed to function as regulatory DCs. These data point toward IDO expression as part of a tolerogenic signature during DC development. Disclosures: No relevant conflicts of interest to declare.