ALBERT

Description: 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.

Description: 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.

Description: 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.

Description: 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.

Description: 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.

Description: 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.

Description: 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.

Description: 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.

Description: 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.

Description: 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.