Mechanisms of Tolerance Induction through T Regulatory Cells during Chemotherapy-Mediated Immunogenic Cell Death in Acute Myeloid Leukemia
Authors: Ocadlikova Darina , Casella Serenella , Pintao Ines , Adinolfi Elena , Sangaletti Sabina , De Marchi Elena , Pegoraro Anna , Orioli Elisa , Colombo Mario Paolo , Di Virgilio Francesco , Cavo Michele , Curti Antonio 
Published: November 2019

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

Disclosures
Cavo: takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; bms: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; novartis: Honoraria.