ATP-Dependent Induction of IDO1 Expression in Dendritic Cells Is Controlled through Non-Canonical NF-Kb Signaling Via P2Y11R and Leads to T Regulatory Cells Induction

Introduction: We previously demonstrated that chemotherapy-induced release of ATP from dying acute myeloid leukemia (AML) cells drives dendritic cells (DCs) to upregulate indoleamine 2,3-dyoxigenase 1 (IDO1), which in turn is involved in T regulatory cells (Tregs) induction. ATP released from chemotherapy-treated AML cells binds purinergic receptors on DCs thus inducing inflammasome activation (through P2X7R), DC maturation and IDO1 upregulation (both through P2Y11R). Since IDO1 expression was recently associated with non-canonical NF-kB activation, we hypothesized the same pathway as a principal intracellular mechanism through which ATP-dependent IDO1 expression in DCs is induced via P2Y11 activation. Moreover, we investigated a potential involvement of inflammasome machinery through P2X7R in IDO1 regulation. Finally, we evaluated the role of ATP catabolism in regulation of IDO1expression in DCs treated with ATP.

Methods: CD14⁺- derived DCs were cultured with ATP in presence or absence of P2X7R and P2Y11R antagonists (AZ 10606120 dihydrochloride and NF 340, respectively) and inflammasome inhibitors (Bay 11-7082 and Ac-YVAD-cmk), and then used for flow cytometry, western blot or real-time PCR analyses to evaluate DC phenotype, in particular CD73, CD39 and IDO1 expression, or release of IL-1beta as response pattern of activated inflammasome. Non-canonical NF-kB transcription factors such as Rel-B and p52 were evaluated in the nuclei of treated DCs by western blot. Adenosine release from DCs treated with ATP was evaluated by luminescence. Furthermore, DCs treated with ATP in presence of AZ 10606120 dihydrochloride and NF 340, respectively were treated also with antagonists of adenosine receptors A2A and A2B and then evaluated for IDO1 expression. Results: Our results confirmed that ATP induces DC maturation and IDO1 expression through P2Y11R, while inflammasome activation is mediated by P2X7R. Furthermore, analysis of IDO1 expression in DCs treated with ATP in presence of P2X7R and P2Y11R antagonists revealed a differential regulation of ATP-dependent IDO1 upregulation via P2Y11R (direct positive effect) and P2X7R (indirect negative effect). However, we did not confirm an involvement of inflammasome in inhibiting IDO1 regulation via P2X7R. Interestingly, we found a positive correlation of cytoplasmic IDO1 expression with the presence of Rel-B and p52, the most important transcriptional factors of non-canonical NF-kB pathway. As for ATP catabolism mediated by the expression of ATP ectonucleotidases CD39 and CD73 in DCs, ATP induced a significant up-regulation of CD73-the rate limiting step of ATP catabolism and an increase of adenosine production. Accordingly, DCs treated with ATP in the presence of P2X7R and P2Y11R antagonists and adenosine receptors A2AR and A2BR antagonists downregulated IDO1 expression. Conclusions: Our data suggest that different mechanisms operate in tumor microenvironment. 1. ATP-dependent IDO1 upregulation in DCs which is induced through non-canonical NF-kB signaling by P2Y11 activation while ATP-dependent inflammasome activation via P2X7R is not involved in IDO1 regulation. 2. ATP catabolism and its main product adenosine may be involved in modulation of IDO1 expression in DCs. A more-in-depth understanding of the intracellular mechanisms of IDO1 regulation has clinical implications for the development of IDO1 inhibitors, especially in combination with immunotherapy such as anti-CD73 or adenosine receptor agonist and immunogenic chemotherapy.

key words: AML; chemotherapy; ATP; IDO1; DCs; Tregs

Cavo:AbbVie, Amgen, Bristol Myers Squibb/Celgene, Pfizer, GlaxoSmithKline, Sanofi, Roche, Takeda: Consultancy, Honoraria; Janssen: Honoraria, Speakers Bureau. Curti:Novartis: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Membership on an entity's Board of Directors or advisory committees.