Ibrutinib Targets Nurse-like Cells Supporting an Immunosuppressive Phenotype in Chronic Lymphocytic Leukemia
Authors: Fiorcari Stefania , Maffei Rossana , Audrito Valentina , Martinelli Silvia , Ten Hacken Elisa , Grisendi Giulia , Zucchini Patrizia , Quadrelli Chiara , Luppi Mario , Burger Jan A. , Deaglio Silvia , Marasca Roberto 
Published: October 2019

Abstract
Introduction: In lymphoid organs, nurse-like cells (NLCs) show properties of tumor-associated macrophages with a M2-like phenotype, associated to prominent immunosuppressive functions, and play a crucial role in CLL survival. Ibrutinib is a potent inhibitor of BTK and ITK kinases and is able to counteract the microenvironmental-derived pro-survival effects in CLL cells. One of the main effects of ibrutinib treatment is attenuated retention and homing of CLL cells to tissue compartments, which ultimately leads to mobilization of CLL cells from microenvironmental niches into the peripheral blood.
Aim: Here, we investigated whether ibrutinib treatment may have an effect on the immunosuppressive properties of NLCs.
Methods: NLCs were generated in complete medium after 12 days of in vitro culture of CLL-PBMCs. Cell activation status, cell surface markers, real time PCR and functional studies were performed after 1h or 24h of ibrutinib treatment, to test whether ibrutinib influenced M1 or M2-polarization of NLCs.
Results: We first observed that, after ibrutinib treatment, the activation status and viability of NLCs were not affected, the morphology of NLCs in vitro analyzed by flow-cytometric FCS and SSC plots was not modified and the number of NLCs generated after 12 days of culture was preserved. Furthermore, treatment with ibrutinib stimulated the expression of the M2-associated markers hemoglobin scavenger receptor CD163 and the mannose receptor C type 1 CD206, and induced expression of the monocyte differentiation antigen CD14. At the gene expression level, ibrutinib induced strong expression of genes involved in M2 polarization such as CD163 (293%, p<0.01), IL10 (279%, p<0.05), MRC1/CD206 (180%, p<0.05) and CCL18 (271%, p<0.05), and concomitantly decreased the expression of M1 polarization genes such as IL1 (53%, p<0.01), TNFα (57%, p<0.01) and IL2 (11%, p<0.01), as compared to the untreated control (100%). Of interest, treatment with ibrutinib stimulated also the expression of the enzyme nicotinamide phosphoribosyltransferase (NAMPT) to 141% (p<0.05) and the programmed death-ligand 1 (PD-L1) to 133% (p<0.01) in NLCs, both important in the induction of an immunosuppressive and protective microenvironment in CLL. Overall these data suggest that ibrutinib could stimulate features related to suppression of the immune system. Next, we tested the ability of ibrutinib to regulate actin cytoskeletal reorganization, which is an essential process during macrophages phagocytosis. Upon ibrutinib stimulation, NLCs showed low actin polymerization levels, since F-actin formation decreased to 66% (p<0.05) as compared to unstimulated control (100%). In addition, ibrutinib impaired the phagocytic activity of NLCs and functionally affected the expression of MAC-1 (CD11b/CD18), which is required for optimal phagocytosis, from 100% to 86% (p<0.01).
Conclusions: Collectively, these preliminary data provide new insights into the effects of ibrutinib treatment on the modulation of immune elements in CLL tissue microenvironment. Noteworthy, our data demonstrate that ibrutinib further promotes M2-polarization features of NLCs, suggesting that this drug not only has an effect on the CLL clone but also extensively influences the cellular components of the CLL microenvironment, whether these effects may be related to ibrutinib-resistance warrants further investigations.

Disclosures
Burger: Boehringer Ingelheim Pharma: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Noxxon: Membership on an entity's Board of Directors or advisory committees; Portola Pharmaceuticals: Research Funding; Gilead: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding. Marasca:Janssen: Honoraria, Other: support for travel to congresses.