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Haematopoietic stem cells (HSCs) are characterized by their self-renewal potential associated to dormancy. Here we identify the cell surface receptor neogenin-1 as specifically expressed in dormant HSCs. Loss of neogenin-1 initially leads to increased HSC expansion but subsequently to loss of self-renewal and premature exhaustion in vivo. Its ligand netrin-1 induces Egr1 expression and maintains quiescence and function of cultured HSCs in a Neo1 dependent manner. Produced by arteriolar endothelial and periarteriolar stromal cells, conditional netrin-1 deletion in the bone marrow niche reduces HSC numbers, quiescence and self-renewal, while overexpression increases quiescence in vivo. Ageing associated bone marrow remodelling leads to the decline of netrin-1 expression in niches and a compensatory but reversible upregulation of neogenin-1 on HSCs. Our study suggests that niche produced netrin-1 preserves HSC quiescence and self-renewal via neogenin-1 function. Decline of netrin-1 production during ageing leads to the gradual decrease of Neo1 mediated HSC self-renewal., Haematopoietic stem cells (HSCs) are characterized by their self-renewal potential and associated dormancy. Here the authors show that niche produced netrin-1 preserves HSC quiescence and self-renewal via neogenin-1, and that decline of netrin-1 production during ageing leads to decreased Neo1 mediated HSC self-renewal.

INTRODUCTION: Recent evidence demonstrated that the treatment of acute myeloid leukemia (AML) cells with Daunorubicin (DNR) but not Cytarabine (Ara-C), results in efficient activation of anti-leukemia T cells. This process, named as immunogenic cell death (ICD), is characterized by some specific events. In the clinical setting, chemotherapy including anthracyclines and Ara-C remains a gold standard for AML treatment. However, the probability of relapse remains elevated, particularly in elderly or prognostically "high risk" patients In the last decade, Etoposide (Eto) and Fludarabine (Flu) have been added to the standard treatment for AML to potentiate its therapeutic effect, and tested in many trials. Regarding the immunogenicity of these two drugs, too few studies are reported in recent literature, and even fewer regarding AML. We therefore studied the immunogenic potential of Eto and Flu as compared to DNR and Ara-C. METHODS: AML cell lines HL-60 and KG-1, and primary AML cells were treated with all four drugs. Calreticulin and heat shock proteins 70/90 translocation, non-histone chromatin-binding protein high mobility group box 1 and adenosine triphosphate release were evaluated. The treated cells were then pulsed into dendritic cells and used forin vitroimmunological tests, in particular for T-cell proliferation and T regulatory cells (Tregs) induction. RESULTS: Collectively, our data indicate that, among the drugs that have been proposed to increase the efficacy of the conventional chemotherapy backbone including DNR and Ara-C, Eto has a similar and comparable capacity to DNR in inducing both early and late ICD events. On the contrary, Flu has a low if any effect, proving similar to Ara-C. Moreover, Eto treatment was the most powerful among the tested drugs in stimulating T-cell proliferation, thus suggesting a significant capacity to activate the immune response. On the contrary, Flu had weak immunogenic potential and can be considered a non-immunogenic chemotherapy drug. Interestingly, Flu was significantly more potent in inducing suppressive T regulatory cells compared to other drugs. CONCLUSIONS: Taken together, the present investigation expands the knowledge on the immunogenic and tolerogenic potential of the chemotherapy drugs commonly used in the therapy of AML. Among these, important differences have been observed, indicating that, particularly in an era when immunotherapy is being included in the clinical stage of AML treatment, the immunological perspective of chemotherapy should be taken into consideration in therapy decision-making. This research was funded by AIRC (Associazione Italiana per la Ricerca sul Cancro) 2017 IG20654. Bologna AIL (Associazione Italiana contro le Leucemie)/ Bologna Section. FATRO/Foundation Corrado and Bruno Maria Zaini-Bologna. Disclosures Cavo: Sanofi: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 accomodations, Speakers Bureau;GlaxoSmithKline:Honoraria, Speakers Bureau;Karyopharm:Honoraria;Novartis: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 accomodations, Speakers Bureau;AbbVie:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees;BMS:Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau;Amgen:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Chemotherapy-induced immunogenic cell death can favor dendritic cell (DC) cross-priming of tumor-associated antigens for T cell activation thanks to the release of damage-associated molecular patterns, including ATP. Here, we tested the hypothesis that in acute myeloid leukemia (AML), ATP release, along with its well-known immune stimulatory effect, may also contribute to the generation of an immune suppressive microenvironment. In a cohort of AML patients, undergoing combined daunorubicin and cytarabine chemotherapy, a population of T regulatory cells (Tregs) with suppressive phenotype, expressing the immune checkpoint programmed cell death protein 1 (PD-1), was significantly increased. Moving from these results, initial in vitro data showed that daunorubicin was more effective than cytarabine in modulating DC function toward Tregs induction and such difference was correlated with the higher capacity of daunorubicin to induce ATP release from treated AML cells. DCs cultured with daunorubicin-treated AML cells upregulated indoleamine 2,3-dioxygenase 1 (IDO1), which induced anti-leukemia Tregs. These data were confirmed in vivo as daunorubicin-treated mice show an increase in extracellular ATP levels with increased number of Tregs, expressing PD-1 and IDO1+CD39+ DCs. Notably, daunorubicin failed to induce Tregs and tolerogenic DCs in mice lacking the ATP receptor P2X7. Our data indicate that ATP release from chemotherapy-treated dying cells contributes to create an immune suppressive microenvironment in AML.

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.