ALBERT

Description: The extrusion of DNA traps contributes to a key mechanism in which innate immune cells clear pathogens or induce sterile inflammation. Here we provide evidence that CD4+ T cells, a critical regulator of adaptive immunity, release extracellular threads of DNA on activation. These DNA extrusions convey autocrine costimulatory signals to T lymphocytes and can be detected in lymph nodes isolated during the priming phase of experimental autoimmune encephalomyelitis (EAE), a CD4+ T cell-driven mouse model of multiple sclerosis. Pharmacologic inhibition of mitochondrial reactive oxygen species (mtROS) abolishes the extrusion of DNA by CD4+ T cells, reducing cytokine production in vitro and T cell priming against myelin in vivo. Moreover, mtROS blockade during established EAE markedly ameliorates disease severity, dampening autoimmune inflammation of the central nervous system. Taken together, these experimental results elucidate a mechanism of intrinsic immune costimulation mediated by DNA threads released by activated T helper cells, and identify a potential therapeutic target for such disorders as multiple sclerosis, neuromyelitis optica, and CD4+ T cell-mediated disorders.

Description: Antineutrophil cytoplasmic antibodies (ANCAs) target proteins normally retained within neutrophils, indicating that cell death is involved in the autoimmunity process. Still, ANCA pathogenesis remains obscure. ANCAs activate neutrophils inducing their respiratory burst and a peculiar form of cell death, named NETosis, characterized by formation of neutrophil extracellular traps (NETs), decondensed chromatin threads decorated with cytoplasmic proteins endorsed with antimicrobial activity. NETs have been consistently detected in ANCA-associated small-vessel vasculitis, and this association prompted us to test whether the peculiar structure of NET favors neutrophil proteins uploading into myeloid dendritic cells and the induction of ANCAs and associated autoimmunity. Here we show that myeloid DCs uploaded with and activated by NET components induce ANCA and autoimmunity when injected into naive mice. DC uploading and autoimmunity induction are prevented by NET treatment with DNAse, indicating that NET structural integrity is needed to maintain the antigenicity of cytoplasmic proteins. We found NET intermingling with myeloid dendritic cells also positive for neutrophil myeloperoxidase in myeloperoxidase-ANCA-associated microscopic poliangiitis providing a potential correlative picture in human pathology. These data provide the first demonstration that NET structures are highly immunogenic such to trigger adaptive immune response relevant for autoimmunity.

Description: In myeloid malignancies, the neoplastic clone outgrows normal hematopoietic cells toward BM failure. This event is also sustained by detrimental stromal changes, such as BM fibrosis and osteosclerosis, whose occurrence is harbinger of a dismal prognosis. We show that the matricellular protein SPARC contributes to the BM stromal response to myeloproliferation. The degree of SPARC expression in BM stromal elements, including CD146+ mesenchymal stromal cells, correlates with the degree of stromal changes, and the severity of BM failure characterizing the prototypical myeloproliferative neoplasm primary myelofibrosis. Using Sparc−/− mice and BM chimeras, we demonstrate that SPARC contributes to the development of significant stromal fibrosis in a model of thrombopoietin-induced myelofibrosis. We found that SPARC deficiency in the radioresistant BM stroma compartment impairs myelofibrosis but, at the same time, associates with an enhanced reactive myeloproliferative response to thrombopoietin. The link betwen SPARC stromal deficiency and enhanced myeloid cell expansion under a myeloproliferative spur is also supported by the myeloproliferative phenotype resulting from the transplantation of defective Apcmin mutant hematopoietic cells into Sparc−/− but not WT recipient BM stroma. Our results highlight a complex influence of SPARC over the stromal and hematopoietic BM response in myeloproliferative conditions.

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: Key PointsIn SMZL, the quality of BM stromal microenvironment predicts disease progression. CD40-CD40L-mediated interactions between mast cells and BM mesenchymal cells engender proinflammatory conditions within SMZL infiltrates.

Description: 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.

Description: Introduction. Mesenchymal stromal cells (MSCs) substantially contribute to the creation of hematopoietic niche by regulating hematopoietic stem cell (HSC) fate and have a unique immune-modulating capacity. In the leukemic milieu, the presence of MSCs constitutes a side effect, since MSCs not only favor leukemic cell survival, but they can also generate an immune-tolerant environment. Although recent findings have outlined a putative MSC role in hematological malignancy development, MSC-dependent mechanisms potentially supporting leukemia remain unclear. We hypothesize that leukemic cells can shape bone marrow (BM) MSCs by inducing functional changes, able to convert the BM microenvironment from hostile to permissive for leukemia. Methods. We isolated acute myeloid leukemia (AML) cells and generated AML-MSCs from the BM of AML patients. Next, we set up AML-MSC/AML cell co-culture experiments and we investigated gene expression in AML-MSCs and AML cells before and after co-cultures. Results. Our microarray data on BM isolated cells (AML patients, N=61; healthy donors, N=7) indicated Interferon(IFN)-γ as un up-regulated gene in almost 40% of AML samples. Furthermore, multivariate analysis, showed that IFN-g-positive AML patients had a better overall survival. Thus, we decided to deepen IFN-ϒ-dependent modifications in leukemic milieu through in vitro studies. In AML-MSC/AML cell co-culture experiments, we confirmed microarray data and we found that AML cells produced IFN-γ. We next demonstrated that indoleamine 2,3-dioxygenase (IDO)1 enzyme, a master regulator of MSC immune suppressive functions, is up-regulated in AML-MSCs after co-culture with IFN-γ-producing AML cells. Such effect was abrogated by adding to cell cultures an IFN-γ neutralizing antibody. Finally, we found that AML-MSCs, after co-culture with IFN-γ-producing AML cells, were able to induce regulatory T cell in a IDO1-dependent manner. To gain further insight in AML cell-dependent MSC modifications, we analyzed MSC expression of IFN-γ-stimulated genes (ISGs) such as Programmed death-ligand (PDL)-1 and Nitric Oxide synthase (NOS)-2 which are known to regulate immunity and promote tolerance. In particular, we tested the ISG expression in MSCs after co-cultures with IFN-ϒ positive or IFN-γ negative AML cells. We found that IFN-γ positive, but not IFN-γ negative AML cells, were able to induce PDL-1 and NOS2 in AML-MSCs. Thus, ISG expression profile in AML-MSCs after co-cultures with IFN-γ positive AML cells was similar as that observed in MSCs after exposure to recombinant IFN-γ. Conclusions. Our data suggest that inflammatory signals produced by AML cells are able to modify MSC functions, thus favoring an immune-tolerant and leukemia supporting milieu. Overall, our results would likely contribute to unravel MSC-dependent mechanisms promoting leukemia and will help to provide novel applications for drugs already under experimentation (e.g. IDO-inhibitors, Checkpoint inhibitors) to translate into more effective therapies in AML patients. Disclosures Cavo: AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Description: Background Acute myeloid leukemia (AML) has been considered for a long time exclusively driven by critical mutations in hematopoietic stem cells (HSCs). Recently, the contribution of bone marrow (BM) microenvironment has gained increasing attention, challenging the evidence that AML derives exclusively from leukemic cell-intrinsic defects. Mesenchymal stromal cells (MSCs) are a key component of the BM microenvironment by regulating HSC fate and having a unique immune-modulatory capacity mostly mediated by interferon (IFN)-γ-induced indoleamine 2,3-dioxygenase (IDO)-1 enzyme activity. New studies demonstrated that the alterations of MSCs are able (e.g. by promoting an inflammatory/genotoxic microenvironment) to induce hematological diseases in mice models and humans. Moreover, AML cells seem to exploit MSC-dependent pro-survival signals to their advantage. All these concepts converge to indicate a fundamental bi-directional interaction among malignant cells and BM microenvironment contributing to AML onset and progression. The mechanisms underlying this crosstalk have just been started to get unraveled. Among signals potentially driving the remodeling of the BM microenvironment, inflammation, a hallmark of cancer, seems to play a role. We hypothesize that 'inflammatory features' of leukemic cells can shape MSCs by inducing functional changes able to create a permissive/self-reinforcing niche favorable to escape therapy and immune response. Methods We isolated acute myeloid leukemia (AML) cells and generated AML-MSCs from the BM of AML patients. Gene expression profile (GEP) (AML, N=61; healthy donors, HDs N=7) and NanoString analysis (AML, N=24) on BM-derived cells were also done. Next, we set up AML-MSC/AML cell co-culture experiments and we investigated gene expression in AML-MSCs and AML cells before and after co-cultures. We also set up a murine model in which the IFN-γ expressing C1498 AML cells was knock down (KD) for the IFN-γ gene by RNA interference. BM infiltrate was analyzed in mice and AML patients. Results In a GEP-screening, we found that almost 40% of AML samples showed an IFN-γ expression higher than the median level of IFN-γ expression in HDs. NanoString data and pathway analysis indicated that IFN-γ high AML cells (above the median level) presented an inflammatory/immune modulating signature clearly distinct from IFN-γ low AML cells (below the median level). Moreover, IFN-γ expression in AML samples correlated with the up-regulation of IFN-γ-stimulated genes (ISGs) (e.g. IDO-1, Programmed death-ligand (PDL)-1 and Nitric Oxide synthase (NOS)-2), which are known to regulate immunity and tolerance. Thus, we aimed to gain insights into IFN-γ-dependent modifications in the leukemic milieu. In AML-MSC/AML cell co-culture experiments, we detected that AML cells produced IFN-γ. To gain insight in AML cell-dependent MSC modifications, we analyzed ISG expression in MSCs, after co-cultures with IFN-γ high or IFN-γ low AML cells. We found that IFN-γ high, but not IFN-γ low AML cells, were able to induce IDO-1, PDL-1 and NOS-2 in AML-MSCs. Moreover, ISG upregulation was abrogated by an IFN-γ neutralizing antibody. We also found that AML-MSCs, after co-culture with IFN-γ high AML cells, were able to induce regulatory T cells (Tregs) in an IDO1-dependent manner. In vivo experiments showed a higher percentage of engraftment in immunocompetent mice injected with parental IFN-γ expressing cells compared to mouse injected with the KD counterpart. The take of parental C1498 cells was associated to an increased frequency of Tregs in the BM. Furthermore, the BM microenvironment of mice injected with IFN-γ KD-C1498 cells showed a significant reduction of PD-L1 expressing cells. Consistently, BM infiltrate analysis in AML patients showed that the percentage of Tregs was correlated with the percentage of AML IFN-γ-positive cells in the BM. Conclusion Our data suggest that interferon-γ-dependent inflammatory signals produced by AML cells are able to modify MSC functions, thus favoring an immune modulating and leukemia-supporting milieu. Overall, our results unravel MSC-dependent mechanisms that might promote leukemia resistance to therapy, therefore informing the delivery of novel therapies targeting the AML microenvironment such as IDO inhibitors and immune checkpoint blockade. Disclosures Martinelli: Novartis: Consultancy, Other: trial grant; Daiichi Sankyo: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria, Other: trial grant; Incyte: Consultancy, Other: trial grant; Janssen: Consultancy, Other: trial grant; Roche: Consultancy, Other: trial grant; Amgen: Consultancy, Other: trial grant; Ariad: Consultancy, Other: trial grant; Celgene: Consultancy, Honoraria, Other: trial grant; Pfizer: Consultancy, Other: trial grant. Rutella:Kura Oncology: Research Funding; NanoString Technologies, Inc.: Research Funding; MacroGenics, Inc.: Research Funding. Cavo:bms: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; novartis: Honoraria.