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Non-muscle-invasive bladder cancer (NMIBC) is a highly recurrent tumor despite intravesical immunotherapy instillation with the bacillus Calmette-Guérin (BCG) vaccine. In a prospective longitudinal study, we took advantage of BCG instillations, which increase local immune infiltration, to characterize immune cell populations in the urine of patients with NMIBC as a surrogate for the bladder tumor microenvironment. We observed an infiltration of neutrophils, T cells, monocytic myeloid-derived suppressor cells (M-MDSCs), and group 2 innate lymphoid cells (ILC2). Notably, patients with a T cell-to-MDSC ratio of less than 1 showed dramatically lower recurrence-free survival than did patients with a ratio of greater than 1. Analysis of early and later time points indicated that this patient dichotomy existed prior to BCG treatment. ILC2 frequency was associated with detectable IL-13 in the urine and correlated with the level of recruited M-MDSCs, which highly expressed IL-13 receptor α1. In vitro, ILC2 were increased and potently expressed IL-13 in the presence of BCG or tumor cells. IL-13 induced the preferential recruitment and suppressive function of monocytes. Thus, the T cell-to-MDSC balance, associated with a skewing toward type 2 immunity, may predict bladder tumor recurrence and influence the mortality of patients with muscle-invasive cancer. Moreover, these results underline the ILC2/IL-13 axis as a targetable pathway to curtail the M-MDSC compartment and improve bladder cancer treatment.

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.

Background: The immunoregulatory enzyme indoleamine 2,3-dioxygenase, which catalyzes the conversion of tryptophan into kynurenine, is expressed in a significant subset of patients with acute myeloid leukemia, resulting in the inhibition of T-cell proliferation and the induction of regulatory T cells. Acute myeloid leukemia cells can be differentiated into dendritic cells, which have increased immunogenicity and have been proposed as vaccines against leukemia.\ud Design and Methods: Leukemic dendritic cells were generated from acute myeloid leukemia cells and used as stimulators in functional assays, including the induction of regulatory T cells. Indoleamine 2,3-dioxygenase expression in leukemic dendritic cells was evaluated at molecular, protein and enzymatic levels.\ud Results: We demonstrate that, after differentiation into dendritic cells, both indoleamine 2,3-dioxygenase-negative and indoleamine 2,3-dioxygenase-positive acute myeloid leukemia samples show induction and up-regulation of indoleamine 2,3-dioxygenase gene and protein, respectively. Indoleamine 2,3-dioxygenase-positive acute myeloid leukemia dendritic cells catabolize tryptophan into kynurenine metabolite and inhibit T-cell proliferation through an indoleamine 2,3-dioxygenase-dependent mechanism. Moreover, indoleamine 2,3-dioxygenase-positive leukemic dendritic cells increase the number of allogeneic and autologous CD4+CD25+ Foxp3+ T cells and this effect is completely abrogated by the indoleamine 2,3-dioxygenase-inhibitor, 1-methyl tryptophan. Purified CD4+CD25+ T cells obtained from co-culture with indoleamine 2,3-dioxygenase-positive leukemic dendritic cells act as regulatory T cells as they inhibit naive T-cell proliferation and impair the complete maturation of normal dendritic cells. Importantly, leukemic dendritic cell-induced regulatory T cells are capable of in vitro suppression of a leukemia-specific T cell-mediated immune response, directed against the leukemia-associated antigen, Wilms’ tumor protein.\ud Conclusions: These data identify indoleamine 2,3-dioxygenase-mediated catabolism as a tolerogenic mechanism exerted by leukemic dendritic cells and have clinical implications for the use of these cells for active immunotherapy of leukemia.

Innate Lymphoid Cells (ILCs) are a recently identified family of lymphocytes, divided into 3 different groups that mirror the functional specialization of helper CD4 T cells. However, it is now clear that the spectrum of ILC diversity is much broader than originally appreciated. ILCs have been shown to play important roles in inflammatory and autoimmune diseases, and their involvement in anti-tumor responses has also recently been evidenced by us and others. Here, we report on the identification of a previously undescribed human ILC subset, named hereafter “ILC-killer” (ILC-k), characterized by a Lineage negative, CD127+ cKIT- CRTh2- CD56+ phenotype, thus being distinct from conventional helper ILCs, e.g ILC1 (CD56-), ILC2 (CRTh2) and ILC3 (cKIT+). Transcriptomic profiling of ex-vivo highly pure ILC-k, conventional ILC subsets and conventional Natural Killer (NK) cell subsets showed closeness between ILC-k, ILC3 and CD56bright NK cells. However, ILC-k display a specific metabolism (low glucose uptake, high mitochondrial activity) and cytotoxic gene signature, also confirmed at protein level. By their functional evaluation, ILC-k showed specific ability to kill tumor cell lines in a KIR-independent, but NKp30- and Trail-dependent manner. Their relative frequency is decreased and both their metabolic fitness and cytotoxic potential are impaired in Acute Myeloid Leukemia (AML) patients at diagnosis (N=51). Upon remission, ILC frequencies and functions are restored to normal levels. Overall, we identified cytotoxic ILCs, distinct from conventional NK cells, that are functionally impaired in AML. Given their KIR-independence, ILC-k might represent promising therapeutic candidates to improve NK-cell based immunotherapy, that currently necessitates KIR-HLA mismatch between donor and recipient for full efficiency. Citation Format: Bérengère Salomé, Alejandra Gomez-Cadena, Romain Loyon-Bonato, Madeleine Suffiotti, Valentina Salvestrini, Antonio Curti, Paolo Tentorio, Domenico Mavilio, Carsten Riether, Adrian Ochsenbein, Emanuela Marcenaro, David Gfeller, Pedro Romero, Sara Trabanelli, Camilla Jandus. ILC-k: Human innate lymphoid cells displaying unique metabolic features and KIR-independent cytotoxicity, impaired in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3786.

Along with their immunogenic role, dendritic cells (DCs) are also critical in maintaining tolerance to self-antigens by inducing regulatory T cells (Tregs) via the expression of the immunomodulatory enzyme indoleamine 2,3-dioxygenase 1 (IDO1). In turn, Tregs modulate the maturation and/or function of DCs. In immune thrombocytopenia (ITP), the interaction between DCs and Tregs has never been investigated although decreased number/function of Tregs as well as altered DCs have been described. Here, we ask whether, in ITP: (1) IDO1 expression/activity is decreased in mature DCs; (2) IDO1-mediated Treg generation is impaired; and (3) DC maturation is abnormally modulated by Tregs. We found that in ITP, DCs show reduced capability of upregulating the expression/activity of IDO1. This finding results in the reduced ability of mature DCs of converting T cells into Tregs. In turn, Tregs are characterized by decreased interleukin-10 production and show lower ability of inhibiting DC maturation. In conclusion, these data point out the role of IDO1 in the impaired regulatory T cell development of ITP patients and suggest that the cross-talk between Tregs and DCs is hampered and plays a pathogenetic role.

Indoleamine 2,3-dioxygenase 1 (IDO1) is an immunosuppressive molecule expressed in some subsets of normal and neoplastic cells. Mature human dendritic cells (DCs) have been shown to express IDO1, but little is known about its expression and function during DC differentiation from bone marrow hematopoietic stem/progenitor cells (HSPCs). Here, we show that during invitro differentiation along the myeloid DC lineage, CD34+ HSPCs acquire IDO1 expression, which acts in a tolerogenic manner by inducing a population of fully functional CD4+CD25+ FOXP3+ T-regulatory cells. Phenotypically, CD1a+CD14- HPSC-derived DCs expressed IDO1, langerin, CD11b, and CD1c. Cell-sorting experiments demonstrated that IDO1 expression is found in a subset of CD1a+CD14-langerin+ cells, expressing CD103, which is capable of inducing T-regulatory cells in an IDO1-dependent manner. In conclusion, DC differentiation from CD34+ HSPCs results in the expression of a functionally active IDO1 protein in CD1a+langerin+, CD103-expressing DCs. These data point toward IDO1 expression as part of a tolerogenic signature during DC development.

Immune thrombocytopenia (ITP) is an autoimmune disorder in which, for still unknown reasons, platelet surface proteins become antigenic and stimulate the immune system to produce autoantibodies and self-reactive cytotoxic T lymphocytes. These findings result in immune-induced platelet destruction

Dendritic cells (DCs) are professional APCs that have a role in the initiation of adaptive immune responses and tolerance. Among the tolerogenic mechanisms, the expression of the enzyme IDO1 represents an effective tool to generate T regulatory cells. In humans, different DC subsets express IDO1, but less is known about the IDO1-related enzyme IDO2. In this study, we found a different pattern of expression and regulation between IDO1 and IDO2 in human circulating DCs. At the protein level, IDO1 is expressed only in circulating myeloid DCs (mDCs) and is modulated by PGE2, whereas IDO2 is expressed in both mDCs and plasmacytoid DCs and is not modulated by PGE2. In healthy subjects, IDO1 expression requires the presence of PGE2 and needs continuous transcription and translation, whereas IDO2 expression is constitutive, independent from suppressor of cytokine signaling 3 activity. Conversely, in patients suffering from inflammatory arthritis, circulating DCs express both IDO1 and IDO2. At the functional level, both mDCs and plasmacytoid DCs generate T regulatory cells through an IDO1/IDO2-dependent mechanism. We conclude that, in humans, whereas IDO1 provides an additional mechanism of tolerance induced by proinflammatory mediators, IDO2 is stably expressed in steady-state conditions and may contribute to the homeostatic tolerogenic capacity of DCs.

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.

Objectifs L’immunotherapie par le bacille de Calmette-Guerin est le traitement standard des cancers non musculo-invasifs de la vessie (NMIBC) a haut risque. L’efficacite therapeutique du traitement repose sur une forte inflammation locale conduisant au controle de la croissance tumorale. Cependant, 30–50 % des patients ne repondent pas au traitement. Nous avons donc investigue les parametres immunitaires possiblement impliques dans l’echec de traitement. Methodes Nous savons que divers facteurs immunosuppresseurs, tels que les cellules myeloides suppressives (MDSC), peuvent inhiber l’immunite anti-tumorale. Nous avons donc caracterise l’infiltration immunitaire locale dans l’urine de patients NMIBC et analyse si les sous-populations immunitaires effectrices versus regulatrices influencaient la recidive tumorale. Dans une etude prospective longitudinale incluant 28 patients NMIBC, les urines pre- et post-instillation BCG ont ete recoltees au cours des 6 semaines du traitement. Les lymphocytes-T, neutrophiles, monocytes, ainsi que les MDSC ont ete analyses par cytometrie-en-flux a 10 couleurs. Resultats Nous avons observe une augmentation progressive du nombre de toutes les sous-populations immunitaires testees, au cours du traitement BCG. Nous avons mis en evidence une infiltration importante de MDSC (CD14 + CD33 + CD11b + HLA-DRneg/low) chez la plupart des patients. Pour evaluer l’equilibre entre cellules effectrices et regulatrices, nous avons mesure le ratio « lymphocytes-T/MDSC », ce qui a permis la repartition des patients en 2 groupes : ceux presentant une majorite de lymphocytes-T (ratio > 1) et ceux presentant une majorite de MDSC (ratio p Conclusion L’equilibre entre lymphocytes-T et MDSC serait predictif de l’echec therapeutique du BCG. Cela met l’accent sur la necessite de developper de nouveaux outils capables de diriger les reponses immunitaires vers un profil moins tolerogene.

It has been reported that ATP inhibits or stimulates lymphoid cell proliferation depending on the cellular subset analyzed. In this study, we show that ATP exerts strikingly opposite effects on anti-CD3/CD28–activated and regulatory CD4+ T cells (Tregs), based on nucleotide concentration. We demonstrate that physiological concentrations of extracellular ATP (1–50 nM) do not affect activated CD4+ T cells and Tregs. Conversely, higher ATP concentrations have a bimodal effect on activated CD4+ T cells. Whereas 250 nM ATP stimulates proliferation, cytokine release, expression of adhesion molecules, and adhesion, 1 mM ATP induces apoptosis and inhibits activated CD4+ T cell functions. The expression analysis and pharmacological profile of purinergic P2 receptors for extracellular nucleotides suggest that activated CD4+ T cells are induced to apoptosis via the upregulation and engagement of P2X7R and P2X4R. On the contrary, 1 mM ATP enhances proliferation, adhesion, migration, via P2Y2R activation, and immunosuppressive ability of Tregs. Similar results were obtained when activated CD4+ T cells and Tregs were exposed to ATP released by necrotized leukemic cells. Taken together, our results show that different concentrations of extracellular ATP modulate CD4+ T cells according to their activated/regulatory status. Because extracellular ATP concentration highly increases in fast-growing tumors or hyperinflamed tissues, the manipulation of purinergic signaling might represent a new therapeutic target to shift the balance between activated CD4+ T cells and Tregs.

BACKGROUND: Overall survival of adult acute myeloid leukemia (AML) is still poor due to the lack of novel and effective therapies. In different malignancies including AML, some chemotherapy agents, such as daunorubicin (DNR) but not cytarabine (Ara-C), activate the immune response via the cross-priming of anti-tumor T cells by dendritic cells (DCs). Such process, known as immunogenic cell death (ICD), is characterized by intracellular and pericellular modifications of tumor cells, such as the cell surface translocation of calreticulin (CRT) and heat shock proteins 70/90 (HSPs 70/90), the extracellular release of ATP and pro-inflammatory factor HMGB1. Alongside with ICD, 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, which may ultimately affect anti-tumor T-cell responses. In this study, we characterize ICD in AML to evaluate the involvement of some DC-related inhibitory pathways, such as the expression of indoleamine-2,3-dioxygenase 1 (IDO1) and the activation of PD-L1/PD-1 axis. METHODS: AML patients were analyzed at diagnosis.Before and after DNR-based chemotherapy, patient-derived T cells were extensively characterized by FACS and analyzed for their capacity to produce IFN-γ in response to autologous blasts. The AML cell line HL-60 and primary AML cells were then exposed, in vitro, to different drugs, including DNR and, as control drug, Ara-C. Dying cells were tested for the surface expression of CRT and HSPs 70/90, the release of HMGB1 and ATP. Functionally, immature DCs generated from healthy donors were pulsed with DNR-treated AML cells. Then, loaded DCs were tested for the expression of maturation-associated markers and of inhibitory pathways, such as IDO1 and PD-L1 and used to stimulate autologous CD3+ T cells. After co-culture, autologous healthy donor T cells were analyzed for IFN-g production, PD-1 expression and Tregs induction. A mouse model was set up to investigate in vivo the mechanism(s) underlying ICD in AML. The murine myelomonocytic leukemia cell line WEHI was transfected with luciferase PmeLUC probe, inoculated subcutaneously into BALB/c mice and used to measure in vivo ATP release after chemotherapy. Tumor-infiltrating T cells and DCs were characterized and correlated with ATP release. RESULTS: DNR treatment induced ICD-related modifications in both AML cell lines and primary blasts, including CRT, HSP70 and HSP90 exposure on cell surface, HMGB1 release from nucleus to cytoplasm and supernatant increase of ATP. Ex vivo, T-cell monitoring of DNR-treated AML patients displayed an increase in leukemia-specific IFN-g-producing CD4+ and CD8+ T cells in 20/28 evaluated patients. However, FACS analysis of CD8+ effector T cells emerging after chemotherapy showed a significant up-regulation of exhaustion marker such as LAG3 and PD-1, which paralleled with their reduced ability to produce active effector molecules, such as perforin and granzyme. Moreover, an increase of circulating Tregs was observed after DNR-based chemotherapy. In vitro, loading of chemotherapy-treated AML cells into DCs resulted not only in the induction of a maturation phenotype, but also in over-expression of inhibitory pathways, such as IDO1 and PD-L1. The silencing of IDO1 increased the capacity of DCs loaded with DNR-treated AML cells to induce leukemia-specific IFN-γ production by CD4+ and CD8+ T cells. In vivo, DNR therapy of mice inoculated with established murine AML cell line resulted in increased ATP release. Similarly to ex vivo and in vitro results, tumor-infiltrating DCs showed an increase in maturation status. Moreover, CD4+ and CD8+ T cells had increased IFN-γ production, but showed an exhausted phenotype. CONCLUSIONS: Our data confirm that chemotherapy-induced ICD may be active in AML and results in increased leukemia-specific T-cell immune response. However, a deep, ex vivo, in vitro and in vivo characterization of chemotherapy-induced T cells demonstrated an exhausted phenotype, which may be the result of the inhibitory pathways induction in DCs, such as IDO and PD-L1. The present data suggest that combination of chemotherapy with inhibitors of IDO1 and PD-L1 may represent an interesting approach to potentiate the immunogenic effect of chemotherapy, thus resulting in increased anti-leukemia immune response. Disclosures Cavo: Janssen-Cilag, Celgene, Amgen, BMS: Honoraria.

Thirteen patients with acute myeloid leukemia, 5 with active disease, 2 in molecular relapse, and 6 in morphologic complete remission (CR; median age, 62 years; range, 53-73 years) received highly purified CD56+CD3− natural killer (NK) cells from haploidentical killer immunoglobulin-like receptor–ligand mismatched donors after fludarabine/cyclophosphamide immunosuppressive chemotherapy, followed by IL-2. The median number of infused NK cells was 2.74 × 106/Kg. T cells were < 105/Kg. No NK cell–related toxicity, including GVHD, was observed. One of the 5 patients with active disease achieved transient CR, whereas 4 of 5 patients had no clinical benefit. Both patients in molecular relapse achieved CR that lasted for 9 and 4 months, respectively. Three of 6 patients in CR are disease free after 34, 32, and 18 months. After infusion, donor NK cells were found in the peripheral blood of all evaluable patients (peak value on day 10). They were also detected in BM in some cases. Donor-versus-recipient alloreactive NK cells were shown in vivo by the detection of donor-derived NK clones that killed recipient's targets. Adoptively transferred NK cells were alloreactive against recipient's cells, including leukemia. In conclusion, infusion of purified NK cells is feasible in elderly patients with high-risk acute myeloid leukemia. This trial was registered at www.clinicaltrial.gov as NCT00799799.

The regulation of the interaction between the immune system and antigens, which may lead to the induction of immune tolerance, is critical both under physiologic conditions and in different pathological settings. In the past few years, major strides have been made in our understanding of the molecular and cellular bases of this process. Novel pathways have been identified and several novel therapeutic agents are currently under clinical investigation for those diseases in which the normal balance between activation and suppression of the immune response is altered. The tryptophan catabolic enzyme, indoleamine 2,3-dioxygenase (IDO), is one of the key players involved in the inhibition of cell proliferation, including that of activated T cells. Recent works have demonstrated a crucial role for IDO in the induction of immune tolerance during infection, pregnancy, transplantation, autoimmunity, and neoplasias, including hematologic malignancies. In this review, the role of IDO in the induction of immunologic tolerance is addressed with a specific focus on its recently discovered effect on hematologic malignancies.

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 696 Regulatory T cells (Tregs) are a subset of T cells involved in the maintenance of peripheral self-tolerance. Specifically, Tregs modulate the maturation and/or function of dendritic cells (DCs). In turn, along with their immunogenic role, DCs are also critical in maintaining tolerance to self-antigens by inducing Tregs via the expression of the immunomodulatory enzyme indoleamine 2,3-dioxygenase 1 (IDO1). IDO1-expressing DCs may play a role in preventing the initiation of autoimmune disorders. Immune Thrombocytopenia (ITP) is an autoimmune disorder in which platelet surface proteins become antigenic and stimulate the immune system. However, in ITP, the interaction between DCs and Tregs has never been investigated, although decreased numbers of Tregs as well as altered DCs have been described. Therefore, in the present study, we investigated whether, in ITP: 1) IDO1 expression/activity is decreased in monocyte-derived DCs (Mo-DCs); 2) the mechanism of Tregs generation is impaired; 3) Mo-DCs maturation is abnormally modulated by Tregs. We studied 54 patients with active ITP. Nineteen patients were newly diagnosed and 35 patients had persistent (25 cases) or chronic (10 cases) ITP. At the time of samples collection, all patients with persistent or chronic ITP were out of any treatment for at least two months and none of the patients received previous treatment with rituximab or were splenectomized. We demonstrated that in mature Mo-DCs from ITP patients the absolute number of IDO1 transcripts was significantly reduced as compared with healthy donors (973.000±771.000 vs 2.409.000±1.592.000 (IDO1 copy number/ABL1 copy number)*10.000; p In summary, this study demonstrates that in ITP low IDO1 expression/activity in DCs results in the reduced ability of generating Tregs. In turn, circulating Tregs with impaired IL-10 production show decreased capacity of inhibiting DCs maturation. Taken together these data suggest that in ITP the cross-talk between Tregs and DCs is hampered and plays a pathogenetic role. As a consequence, DCs are less “tolerogenic” and Tregs are defective in their regulatory function. Disclosures: No relevant conflicts of interest to declare.

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 2770 Recent studies suggest a bi-directional interaction between regulatory T cells (Tregs) and dendritic cells (DCs). Despite their role as inducers of immunity, DCs may also be critical in maintaining tolerance to self-antigens by inducing Tregs. Specifically, Indoleamine 2,3-dioxygenase 1 (IDO 1) expressing DCs expand Tregs. In turn, Tregs, which express CTLA4, engage its receptors B7-1(CD80)/B7-2(CD86), thus inducing the expression of IDO1 enzyme in DCs. IDO1 expression leads to tryptophan depletion and immunosuppressive kynurenine enrichment in the tissue. Both factors drive the de novo generation of Tregs from CD4+ T cells. In addition, Tregs may modulate the maturation and/or function of DCs, which are required for the activation of effector T cells. Tregs out compete with naïve T cells in aggregating around DCs. After forming aggregates, Tregs specifically downregulate the expression of CD80 and CD86 on DCs. The result of the interaction between Tregs and DCs is the impaired capacity of the antigen presenting cells to establish long lasting interactions with effector T cells. Therefore, given the crucial role of DCs and Tregs in initiating, regulating, maintaining or repressing immune responses, investigations on their interaction is of great importance for better elucidating the pathogenesis of ITP. Of note, in ITP this interaction and its pathogenetic role have never been investigated in depth. In this paper, we enumerated and functionally characterized Treg subsets, exploring whether in ITP abnormal Tregs interactions with DCs and/or effector T cells might play a pathogenetic role. Specifically, we studied whether, in ITP patients: 1) DCs maturation is differentially modulated by Tregs from ITP patients as compared with healthy donors; 2) the mechanism of Tregs generation is altered. Forty adult ITP patients, newly diagnosed (14 cases) or with persistent (20 cases) or chronic ITP (9 cases) were studied after informed consent. At the time of the study, patients with persistent or chronic ITP were out off therapy by at least two months. None of the patients were splenectomized. The median platelet count at the time of the study was 53×109/L (range 8–99). Allogeneic Mixed Leukocyte Reaction (MLR) was performed to test the suppressive activity of Tregs. To analyze the in vitro ability of highly purified CD4+CD25+ T cells to inhibit DCs maturation, normal immature CD14-derived DCs were cultured alone and with allogeneic CD4+CD25+ T cells from healthy subjects or ITP patients in the presence of Lipopolysaccaryde. CD80 and CD86 expression of CD14-derived DCs was then tested at flow cytometry. To evaluate the in vitro conversion of non Tregs into Tregs, CD4+CD25- T cells were cultured alone and with autologous mature CD14-derived DCs from ITP patients and healthy subjects. The percentages of CD4+CD25highFoxP3+ T cells were quantitated at flow cytometry. mRNA IDO1 expression of immature and mature CD14-derived DCs was then evaluated by Real-time RT-PCR. To determine IDO1 enzyme activity kynurenine levels were measured in the supernantants of mature CD14-derived DCs. We found that in ITP Tregs show lower ability to suppress T cell proliferation and to inhibit CD14-derived DCs maturation because they do not affect the expression of the costimulatory molecules CD80 and CD86. We show that the absolute number of Tregs was significantly decreased in ITP patients in comparison to healthy subjects (CD4+CD25highFoxp3+ T cells (5.5±4.3 vs 11.6±6.9 cells/microL; p below 0.01) and CD4+CD25highCD127low/negative T cells (50.9±27.3 vs 80.5±37.7 cells/microL; p below 0.02)). In addition, in ITP patients we document that the low number of circulating Tregs may be due to the reduced ability of mature CD14-derived DCs to convert non-Treg cells (CD4+CD25-) into Tregs (CD4+CD25highFoxP3+ Tregs). This is related to reduced expression and activity of the IDO1 enzyme in mature CD14-derived DCs from ITP patients, since DCs expressing IDO1 favour Treg generation. In conclusion, taken together our data demonstrate that in ITP the cross-talk between Tregs and DCs is impaired and plays a pathogenetic role. As a consequence, we found the generation of more immunogenic DCs and defective Tregs. Disclosures: No relevant conflicts of interest to declare.

Abstract 4287 Purpose: To evaluate safety, feasibility and anti-leukemia potential of haploidentical KIR-L mismatched natural killer (NK) cell infusion in elderly high risk acute myeloid leukemia (AML) patients. Patients and Methods: Thirteen patients (5 active disease, 2 molecular relapse and 6 complete remissions) with median age 62 years (range 53–73) received NK cell infusion after immunosuppressive chemotherapy (fludarabine/cyclophosphamide), followed by interleukin-2. Highly purified CD56+CD3- NK cells from haploidentical KIR-L mismatched donors were used. The median number of infused NK cells was 2.74 × 106/Kg. T cells were less than 105/Kg. NK cell chimerism, phenotyping, and functional assays were performed. Results: No significant toxicity, including graft versus host disease, related to NK cell infusion was observed. Among patients with active disease, 1/5 obtained transient complete remission (CR), whereas 4/5 patients had no clinical benefit. Both patients in molecular relapse obtained CR, which lasted 9 and 4 months. Three/6 patients in morphologic CR are disease-free after 34, 32 and 18 months. Donor NK cells were demonstrated in the peripheral blood (PB) of all evaluable patients with a peak at day 10 after infusion and, in some cases, also in the bone marrow (BM). NK alloreactivity was demonstrated in vivo by the detection of donor-derived postinfusion NK clones capable of killing recipient targets. Conclusion: Infusion of purified CD56+CD3- NK cells is feasible and safe in elderly high risk AML patients. Adoptively transferred NK cells, which can be detected in PB and BM after infusion, are alloreactive against recipient cells and may induce an anti-leukemic activity. 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.

In this issue of Blood , Jasperson and colleagues show that indoleamine 2,3-dioxygenase (IDO) is a novel component of the inhibition of T-cell activation after allogeneic stem-cell transplantation, and offer a rationale for innovative strategies in the management of GVHD targeting this pathway.

Indoleamine 2,3-dioxygenase (IDO) enzyme, which catalyzes the conversion of tryptophan into kynurenine, has been identified as a novel immunosuppressive agent by inhibiting T-cell proliferation and is involved in tolerance induction to tumors. We have recently shown that IDO protein is constitutively expressed in a significant subset of newly diagnosed acute myeloid leukemia (AML) patients, resulting in tryptophan catabolism along the kynurenine pathway and in the inhibition of allogeneic T-cell proliferation. Moreover, we demonstrated that IDO-expressing AML cells are capable to promote the differentiation of new CD4+CD25+Foxp3+ T regulatory cells (Treg cells). AML cells may be differentiated into leukemic dendritic cells (AML-DCs) which have increased immunogenicity and may be used as vaccine against leukemia. We in vitro generated DCs from 7 AML samples according to standard procedures and tested IDO expression and activity. At baseline, 5/7 AML samples expressed IDO, whereas 2/7 did not. After differentiation into DCs, IDO+ AML samples showed an up-regulation of IDO mRNA and protein, and IDO− AML cells turned positive. IDO-expressing AML-DCs were capable to catabolize tryptophan into kynurenine metabolite and, functionally, they inhibited allogeneic T-cell proliferation through an IDO-dependent mechanism. Similarly to undifferentiated IDO+ AML blasts, IDO-expressing AML-DCs induced a population of CD4+CD25+Foxp3+ Treg cells, which were capable to suppress in vitro allogeneic naïve T-cell proliferation. These data identify IDO-mediated catabolism as a general tolerogenic mechanism in AML cells, including AML-DCs and raise several concerns for the use of AML-DCs as cellular vaccine against leukemia.

The expression of the catalytic enzyme of tryptophan, indoleamine 2,3 dioxygenase (IDO) has been identified as a T-cell inhibitor effector pathway in different normal and neoplastic cells. We have recently shown that normal bone marrow (BM) cells, including hematopoietic CD34+ cells, express IDO mRNA only upon IFN-γ stimulation, whereas in a subset of human acute myeloid leukemia cells (AML) IDO is constitutively expressed both at molecular and protein level and induces immunological escape by promoting the generation of T-reg cells. To investigate whether the IDO transcript can be used as a marker for minimal residual disease (MRD) detection, we used a sensitive real-time reverse transcription-polymerase reaction assay (qRT-PCR) to quantify IDO mRNA levels in peripheral blood (PB) and BM samples of newly diagnosed AML patients. The level of IDO transcript was evaluated as IDO copy number/104 ABL copies. As control samples, we used normal PB and BM mononuclear cells (MNCs). Our data showed that normal BM and PB cells expressed minimal amount of IDO mRNA (range 100–1000). Among AML samples, we identified three subsets of patients according to IDO mRNA expression: 1) 33/74 (44.6%) IDO negative (i.e. range < 100), 28/74 (37.84%) IDO low (range 100–1000) and 13/74 (17.6%) IDO high (i.e. range > 1000). BM and PB AML blasts gave similar results. Assessment of protein expression and enzymatic activity was in accordance with molecular results. Some patients were evaluated for IDO mRNA expression before and after induction chemotherapy and the IDO levels were found to correlate with the reduction of BM blasts. Taken together, our qRT-PCR data demonstrate that normal PB and BM cells are negative for IDO mRNA expression, which, in turn, is significantly up-regulated in a subset of AML patients (IDO high) and IDO mRNA expression correlates with tumor burden, thus suggesting its possible role in the detection of MRD in IDO high patients.

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.

Introduction: This is a Phase 1b monocentric clinical trial, aimed to evaluate the safety, feasibility and preliminary clinical efficacy of alloreactive natural killer (NK) cell infusions after immunosuppressive regimen in elderly acute myeloid leukemia (AML) patients, who achieved first complete remission (CR) after induction/consolidation chemotherapy. Methods: Seventeen AML patients, in first morphological CR (3 patients showed molecular disease) (median age 64 years, range 53-73) received highly purified CD56+CD3- NK cells from haploidentical KIR-ligand mismatched donors after fludarabine/cyclophosphamide (Flu/Cy) immunosuppressive chemotherapy, followed by interleukin-(IL-) 2. Results: No signs of NK cell-related toxicity, including graft-versus-host disease, were observed. Myelosuppression was moderate although 1 patient died due to overwhelming bacterial pneumonia and was censored for clinical follow-up. With a median follow-up of 22.5 months (range, 6-68 months), 9/16 evaluable patients (0.56) are alive disease-free, whereas 7/16 (0.44) relapsed with a median time to relapse of 9 months (range, 3-51 months). Among relapsed patients, 2 individuals showed a very prolonged CR phase of 24 and 51 months, respectively, in absence of any concomitant anti-leukemia treatments. Three patients, treated with molecular disease, achieved molecular CR lasting 9 and 4 months in 2 cases and 8+ months in the third patient. These results have been compared to that obtained in a cohort of patients, who achieved CR after induction/consolidation chemotherapy, but did not undergo NK cell immunotherapy due to the absence of a KIR-L mismatched donor (Fig. 1). Disease-free survival (DFS) in the control cohort was 0.39, indicating a trend towards better DFS under the NK therapy (0.56; p=0.07). To correlate the donor NK activity with the clinical response, donor NK cells, as evaluated both as the total number of infused NK cells and as the frequency of alloreactive NK clones, were assessed before NK cell infusion. No statistically significant correlation between the total number of infused NK cells and clinical response was observed. On the contrary, we observed a higher number of donor-derived alloreactive NK cell clones in the group of responders as compared to that of non-responders (Fig. 2A). Accordingly to statistical analysis, a threshold of 8 over 100 NK clones was chosen as the cut-off level discriminating responders versus non-responders (Fig. 2A). Importantly, the infusion of higher number of donor alloreactive NK cells (more than 8/100 clones) was associated with prolonged DFS (Fig.2B). In particular, for patients who received more than 8/100 alloreactive NK cell clones the probability of DFS was 0.81, whereas patients who received less than 8/100 clones had a DFS of 0.14. Such difference was statistically significant (p=0.03) and demonstrates that the number of alloreactive NK cell clones in the donor may discriminate patients receiving NK cell therapy as for response evaluation. Interestingly, the DFS of the group of patients who received less than 8/100 donor alloreactive NK cell clones was comparable to that of the historical control group (Fig. 2B). This finding suggests that the therapeutical effect of the whole procedure, which comprises chemotherapy (Flu/Cy) plus NK immunotherapy, mainly relies on the anti-leukemia activity of alloreactive NK cells. Noteworthy, the increased anti-leukemia effect in patients receiving the higher number of donor alloreactive NK cells was not associated with increased myelosuppression, as shown by comparable data of hematological recovery after NK cell infusion in responders and non-responders.Conclusions: infusion of purified NK cells is feasible in elderly patients with AML as post-CR consolidation strategy and donor NK alloreactivity has a predictive role on the clinical outcome of treated patients. Supported by: Italian Leukemia Association (BolognAIL), Section of Bologna and Fondazione Carisbo, Bologna, ELN, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

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 (Mo-DCs) have been shown to express IDO, little is known about the expression of IDO in other subsets of human DCs, including those generated from CD34+ hematopoietic progenitors (CD34+-derived DCs) In the present study, we performed a full characterization of IDO expression and function by human dendritic cells including Mo-DCs and CD34+-derived DCs. Mo-DCs were generated from purified CD14+ monocytes after culture with GM-CSF and IL-4 and then matured with CD40L, LPS alone, LPS plus IFN-gamma and a cytokine cocktail (IL-1beta, TNF-alpha, IL-6, PGE2). CD34+-derived DCs were generated from purified CD34+ cells after 7 days of culture with GM-CSF and TNF-alpha, followed by 7 day-treatment with GM-CSF and IL-4 and then matured with the same stimuli used for MoDCs. After culture, DCs were analyzed for IDO expression by real-time PCR and western immunoblot, kynurenine production, inhibition of allogenic proliferation and Tregs induction. Monocytes lack IDO expression. Immature Mo-DCs have little if any expression of IDO. During maturation, the cytokine cocktail is the most effective in up-regulating IDO, both at mRNA and protein level, which is paralleled with higher kynurenine production and inhibition of allogeneic T cell proliferation. PGE2 has a crucial role in inducing IDO expression, while IL6 has an opposite effect. Mature Mo-DCs are shown to generate a population of CD4+CD25+FOXP3+ Tregs which are capable of suppressing allogeneic T-cell proliferation. This inhibitory effect is abrogated by the addition of the IDO inhibitor 1-methyl tryptophan (1-MT). CD34+ cells lack IDO mRNA expression. During DC differentiation, IDO expression is observed at day 7, but not at day 14. Flow cytometry analysis of day 7 cells reveal a population of CD34−, CD14+, CD1a+/− cells. Similarly to Mo-DCs, maturation stimuli induce a marked up-regulation of IDO mRNA. Similar results are observed when IDO protein is measured. Production of kynurenine, inhibition of allogeneic T cell proliferation and generation of Tregs from normal CD3+ T-cells are also paralleled with IDO expression. In conclusion, in human DCs the maturation status is associated with a differential expression of IDO both in Mo-DCs and CD34+-derived DCs. Differentiation of CD34+ cells into DCs results in a transient and early expression of IDO at precursor level. Given its functional capacity to inhibit T-cell-mediated immune response, the expression of IDO along DC differentiation may indicate a protective role of the pool of precursor cells which are committed to DC lineage.

Abstract 3901 Adenosine 5'-triphosphate (ATP) is emerging as an extracellular signaling molecule playing a pivotal role in several cellular processes, through specific cell membrane purinergic P2 receptors (P2Rs). Under physiological conditions, ATP is present in the extracellular space at low concentrations (1-10 nM), whereas during inflammation and tumor cell growth ATP is present in the extracellular space at high concentrations, when 5–10 mM of ATP are quickly released from cytoplasm following plasma membrane damage or membrane stretching. For these reasons, extracellular ATP, via activation of P2Rs, might be an important regulator of inflammatory and immune response. CD4+ T cells are often exposed to different ATP concentrations in healthy or in injured/inflamed tissues. In the present study, we investigated the expression of purinergic P2 receptors (P2Rs) on human activated and regulatory CD4+ T cells and tested the lymphocyte functions in presence of low (1-10 nM), intermediate (250 nM) and high (1 mM) concentration of extracellular ATP. We assessed CD4+ T cells proliferation, apoptosis, phenotype, cytokine release, migration and matrix/cells adhesion. We show that activated CD4+ T cells express all P2Rs subtypes, whereas Tregs do not express P2X6 and P2Y2. At a functional level, low concentrations of extracellular ATP do not modulate CD4+ T cell functions. An increase in ATP concentration (250 nM) stimulates CD4+ T cells during activation: activated CD4+ T cells enhance their proliferation, the secretion of several cytokines critical for T cell functions (IL-2, IL-1b, IFN-g, IL-8), the expression of adhesion molecules (CD49d and CD54) and the capacity to adhere to cellular matrix or to other cells. Tregs seem to be unaffected by 250 nM of ATP. In contrast, high concentrations of ATP (1 mM) “turn off” activated CD4+ T cells and “turn on” Tregs. 1 mM of ATP inhibits activation of CD4+ T cells, by enhancing apoptosis and diminishing proliferation, cell-adhesion and the release of pro-inflammatory cytokines. Conversely, 1 mM of ATP attracts Tregs and stimulates their proliferation and their capacity to adhere to other cells. Moreover, Tregs cultured in presence of 1 mM of extracellular ATP are more efficient in inhibiting T cell proliferation. In summary, the present data show that the concentration of extracellular ATP regulates CD4+ T cell functions. Low ATP concentrations, as in physiological conditions, do not affect CD4+ T cell functions, whereas any enhancement of ATP concentration alters CD4+ T cell behavior. Specifically, a small increase stimulates CD4+ T cell activation, whereas a high increase inhibits CD4+ T cell activation and promotes the immunosuppression Tregs-mediated. We propose that the present in vitro data might explain how in vivo ATP regulates the behavior of activated CD4+ T cells and Tregs in case of inflammation or tumor cell growth. A small enhancement of ATP concentration occurs at the beginning of an inflammatory state or at the first stages of tumor growth; these ATP concentrations alert CD4+ T cells to the presence of a possible damage, which does not yet require Tregs involvement. In contrast, in case of severe inflammation, high ATP concentrations might prevent a further involvement of activated CD4+ T cells and promotes Tregs recruitment, avoiding hyper-inflammation. In case of advanced stages of tumorigenesis, high ATP concentration might be a tumor-escape mechanism, by killing activated CD4+ T cells and by attracting Tregs to surround the tumor. Disclosures: No relevant conflicts of interest to declare.

Abstract 4298 Indoleamine 2,3-dioxygenase (IDO1) and indoleamine 2,3-dioxygenase-like (IDO2) are enzymes involved in the tryptophan catabolism along the kynurenine pathway. While it is established that IDO1-expressing dendritic cells (DCs) contribute to tolerance in a number of biological settings, little is known about the expression and function of IDO2 in DCs. Human DCs can be generated in vitro to obtain immunogenic antigen-presenting cells (APC), used as cellular vaccines. In the clinical setting, DCs are commonly matured with a cytokine cocktail (CC) which includes TNF-a, IL-1b, IL-6 and PGE2. In particular, PGE2 enhances APC function of DCs by increasing IL-12 production and facilitating DC migration to lymph nodes. However, PGE2 is also a strong IDO1 inducer, which by this route can also limit the anti-tumor activity of DC-based immunotherapies. Thus, understanding the roles of IDO1 and IDO2 in DCs may impact the development of vaccines or DC-based immunotherapies. In the present study, we fully characterized IDO1 and IDO2 expression and function in human monocyte-derived dendritic cells (Mo-DCs). Mo-DCs were generated from purified CD14+ monocytes after culture with GM-CSF and IL-4 and then matured with CD40L, LPS alone, LPS plus IFN-g and the CC. We observed that immature Mo-DCs had little if any expression of both IDO1 and IDO2, whereas mature Mo-DCs exhibited upregulation of both enzymes. Among the different maturation stimuli, CC was the most effective in upregulating IDO1 and IDO2, both at the message and protein levels. This effect was associated also with the highest kynurenine production. By means of IDO1 and IDO2 expression, mature Mo-DCs were inhibited in stimulating allogeneic T cell proliferation and generated a population of CD4+CD25+FOXP3+ Tregs which highly suppressed allogeneic and autologous T-cell proliferation. On the basis of evidence that IDO1 is preferentially inhibited by the L-isoform of 1 methyl-tryptophan (1-MT) and IDO2 by the D-isoform, we performed functional enzyme tests in presence of both isoforms. Notably, both isoforms exhibited inhibitory effects, although we observed a stronger effect of L-1-MT than with D-1-MT suggesting a greater contribution of IDO1 than IDO2. These results offer direct evidence that Mo-DCs express functional IDO1 and IDO2 proteins. During the maturation phase, Mo-DCs enhance their tolerogenic qualities, and in particular the capacity to induce Tregs, through the upregulation of both IDO1 and IDO2. Beside the critical role of IDO1 in enhancing the immunosuppressive capacity of DCs, we show, for the first time, that IDO2 is involved also. Our findings imply that, from a clinical standpoint, to improve the efficacy of DC-based vaccines mature DCs should be combined with molecules that can inhibit the activity of both IDO1 and IDO2. Disclosures: Metz: NewLink Genetics: Employment. Prendergast:New Link Genetics Corp: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Abstract 3511 Poster Board III-448 CD4+CD25+ regulatory T cells (Tregs) are critical in maintaining self-tolerance and preventing organ-specific autoimmune diseases. However, the role of Tregs in the pathogenesis of immune thrombocytopenia (ITP), an immune disorder in which increased platelet clearance is caused by antiplatelet autoantibodies, has not yet been clarified. The purpose of the present study was to investigate whether the interaction between dendritic cells (DCs) and regulatory T cells (Tregs) may play a pathogenetic role in ITP. Forty patients with active disease and 35 healthy subjects were enrolled into the study. We firstly characterized the number (by flow cytometry) and the suppressive activity (by modified allogeneic mixed leukocyte reaction) of Tregs in ITP. We found that the absolute number of Tregs was significantly decreased in ITP patients in comparison to healthy subjects (CD4+CD25highFoxp3+ T cells (5.5±4.3 vs 11.6±6.9 cells/microL; p below 0.01) and CD4+CD25highCD127low-negative T cells (50.9±27.3 vs 80.5±37.7 cells/microL; p below 0.02)). We documented also that in ITP suppressive activity of Tregs was defective as compared to healthy subjects. In parallel experiments we studied the in vitro conversion of CD4+CD25- T cells, either from ITP patients or healthy subjects, into CD4+CD25+Foxp3+ Tregs by co-coltures with mature autologous/allogeneic DCs. The flow cytometry analysis showed that in ITP the low number of circulating Tregs may be partly due to the reduced ability of DCs to convert non-Treg cells into Tregs. We then explored the in vitro capability of CD4+CD25+ Tregs, either from ITP patients or healthy subjects, to inhibit allogeneic DCs maturation by flow cytometry evaluation of the expression of the costimulatory molecules CD80 and CD86. We demonstrated that in ITP patients CD4+CD25+ Tregs show lower ability to inhibit DCs maturation because they do not affect the expression of CD80 and CD86 molecules. This finding may be related to the lower level of Interleukin-10 and Interleukin-6 in the cocoltures. Taken together, these findings document that the interaction between DCs and Tregs is altered in ITP and suggest that this dysfunction may play a pathogenetic role. Supported in part by BolognaAIL (Italian association against Leukemia, Bologna section) 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.

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.