The irreversible Bruton's tyrosine kinase (Btk) inhibitor ibrutinib has shown efficacy against B-cell tumors such as chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma. Fc␥ receptors (Fc␥R) on immune cells such as macrophages play an important role in tumor-specific antibody-mediated immune responses, but many such responses involve Btk. Here we tested the effects of ibrutinib on Fc␥R-mediated activities in monocytes. We found that ibrutinib did not affect monocyte Fc␥R-mediated phagocytosis, even at concentrations higher than those achieved physiologically, but suppressed Fc␥R-mediated cytokine production. We confirmed these findings in macrophages from Xid mice in which Btk signaling is defective. Because calcium flux is a major event downstream of Btk, we tested whether it was involved in phagocytosis. The results showed that blocking intracellular calcium flux decreased Fc␥R-mediated cytokine production but not phagocytosis. To verify this, we measured activation of the GTPase Rac, which is responsible for actin polymerization. Results showed that ibrutinib did not inhibit Rac activation, nor did the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid tetrakis(acetoxymethyl ester). We next asked whether the effect of ibrutinib on monocyte Fc␥R-mediated cytokine production could be rescued by IFN␥ priming because NK cells produce IFN␥ in response to antibody therapy. Pretreatment of monocytes with IFN␥ abrogated the effects of ibrutinib on Fc␥R-mediated cytokine production, suggesting that IFN␥ priming could overcome this Btk inhibition. Furthermore, in monocyte-natural killer cell co-cultures, ibrutinib did not inhibit Fc␥R-mediated cytokine production despite doing so in single cultures. These results suggest that combining ibrutinib with monoclonal antibody therapy could enhance chronic lymphocytic leukemia cell killing without affecting macrophage effector function.

Fcγ receptors (FcγR) are critical mediators of monoclonal antibody cancer therapies, as they drive cytotoxic processes upon binding of effector cells to opsonized targets. Along with natural killer (NK) cells, monocytes are also known to destroy antibody-coated targets via antibody-dependent cellular cytotoxicity (ADCC). However, the precise mechanisms by which monocytes carry out this function have remained elusive. Here, we show that human monocytes produce the protease Granzyme B upon both FcγR and Toll-like Receptor (TLR) 8 activation. Treatment with TLR8 agonists elicited Granzyme B and also enhanced FcγR-mediated Granzyme B production in an additive fashion. Furthermore, monocyte-mediated ADCC against cetuximab-coated tumor targets was enhanced by TLR8 agonist treatment, and this enhancement of ADCC required Granzyme B. Hence, we have identified Granzyme B as an important mediator of FcγR function in human monocytes and have uncovered another mechanism by which TLR8 agonists may enhance FcγR-based therapies.

Edited by Peter CresswellAcute myeloid leukemia (AML) is characterized by the proliferation of immature myeloid lineage blasts. Due to its heterogeneity and to the high rate of acquired drug resistance and relapse, new treatment strategies are needed. Here, we demonstrate that IFN␥ promotes AML blasts to act as effector cells within the context of antibody therapy. Treatment with IFN␥ drove AML blasts toward a more differentiated state, wherein they showed increased expression of the M1-related markers HLA-DR and CD86, as well as of Fc␥RI, which mediates effector responses to therapeutic antibodies. Importantly, IFN␥ was able to up-regulate CD38, the target of the therapeutic antibody daratumumab. Because the antigen (CD38) and effector receptor (Fc␥RI) were both simultaneously up-regulated on the AML blasts, we tested whether IFN␥ treatment of the AML cell lines THP-1 and MV4-11 could stimulate them to target one another after the addition of daratumumab. Results showed that IFN␥ significantly increased daratumumab-mediated cytotoxicity, as measured both by 51 Cr release and lactate dehydrogenase release assays. We also found that the combination of IFN␥ and activation of Fc␥R led to the release of granzyme B by AML cells. Finally, using a murine NSG model of subcutaneous AML, we found that treatment with IFN␥ plus daratumumab significantly attenuated tumor growth. Taken together, these studies show a novel mechanism of daratumumab-mediated killing and a possible new therapeutic strategy for AML.

Nurse like cells (NLCs) 3 are tumor-nurturing cells derived from CD14 ϩ monocytes in chronic lymphocytic leukemia (CLL) patients. In vitro studies have classified NLCs as CLLspecific, tumor-associated macrophage-like cells functioning as immune regulators and also possible inducers of emerging drug resistance (1-3). It has been shown recently that, in patients with diffuse large B cell lymphoma, a high density of CD68 ϩ /CD163 ϩ tumor-associated macrophages was significantly correlated with unfavorable prognosis and poor clinical outcome (4). Interferons have been widely accepted as modulators of macrophage plasticity and activation, and it is known that IFN␥ is capable of promoting the differentiation of monocytic cells (7). With regard to therapeutic use, Miller et al. (8) have shown that IFN␥ is beneficial for treating immune disorders such as systemic sclerosis and that it displays antitumor and antiangiogenic effects both in vitro and in vivo . IFN␥ treatment has also been shown to induce antineoplastic immune responses by sensitizing tumor cells to apoptosis via up-regulation of both MHC class I and II molecules and by enhancing antitumor immune activity while decreasing M2 characteristics in immune cells (9, 10). IFN␥ has been successfully used in cases of ovarian cancer, multiple myeloma (11), and bladder carcinoma and, recently, in malignant gliomas (12).Here we examined the effects of IFN␥ on the phenotype and function of NLCs. We found that IFN␥ significantly increased the expression of the M1-related markers CD86 and HLA-DR as well as the phagocytic receptor Fc␥RI. Concurrently, the prosurvival ligand CD31 was down-regulated. Consistent with this, IFN␥-treated NLCs showed superior phagocytic ability toward both opsonized sheep RBCs (SRBCs) and rituximab-coated CLL cells as well as withdrawal of support for CLL cell survival. These results show that IFN␥ can reprogram NLCs to function as immune effectors and suggest that therapies that enhance IFN␥ production locally may be valuable treatments for CLL, particularly when combined with monoclonal antibodies such as rituximab. Experimental ProceduresPatient Samples-Peripheral blood was collected from CLL patients with informed consent in accordance with the Declaration of Helsinki and under approval from the Institutional Review Board of Ohio State University.NLC Culture-Peripheral blood mononuclear cells were isolated from CLL patient blood by density gradient centrifugation over Ficoll-Hypaque (Nycomed, Oslo, Norway) and resus-

Monocytes and macrophages are critical for the effectiveness of monoclonal antibody therapy. Responses to antibody-coated tumor cells are largely mediated by Fc␥ receptors (Fc␥Rs), which become activated upon binding to immune complexes. Fc␥RIIb is an inhibitory Fc␥R that negatively regulates these responses, and it is expressed on monocytes and macrophages. Therefore, deletion or down-regulation of this receptor may substantially enhance therapeutic outcomes. Here we screened a panel of Toll-like receptor (TLR) agonists and found that those selective for TLR4 and TLR8 could significantly down-regulate the expression of Fc␥RIIb. Upon further examination, we found that treatment of monocytes with TLR4 agonists could lead to the ubiquitination of Fc␥RIIb protein. A search of our earlier microarray database of monocytes activated with the TLR7/8 agonist R-848 (in which Fc␥RIIb was down-regulated) revealed an up-regulation of membrane-associated ring finger (C3HC4) 3 (MARCH3), an E3 ubiquitin ligase. Therefore, we tested whether LPS treatment could up-regulate MARCH3 in monocytes and whether this E3 ligase was involved with LPS-mediated Fc␥RIIb down-regulation. The results showed that LPS activation of TLR4 significantly increased MARCH3 expression and that siRNA against MARCH3 prevented the decrease in Fc␥RIIb following LPS treatment. These data suggest that activation of TLR4 on monocytes can induce a rapid down-regulation of Fc␥RIIb protein and that this involves ubiquitination.Monocytes and macrophages play an important role in the innate immune response by phagocytosing IgG-opsonized infectious particles (1) and are major mediators in the destruction of tumor cells (2-5). Indeed, the importance of monocytes in clearing antibody-targeted tumor cells following the administration of therapeutic mAbs used in oncology indications has been well established (6 -8). However, despite showing statistically significant effects, the low rates of complete remission combined with the relatively high relapse rate suggest strongly that there is much room for improvement (9 -11). Therapeutic mAbs themselves are being improved, with the goal of increasing affinity toward Fc␥ receptors in some cases (12, 13). Along with this, immune modulators such as interferons (14, 15), interleukins (16 -20), synthetic compounds (21-23), and CpG oligonucleotides (16) are being explored as potential enhancers of antibody therapy.Antibody-dependent destruction of target cells is largely mediated by Fc␥ receptors (Fc␥Rs) 4 (5, 24, 25). Human monocytes and macrophages express at least four different functional Fc␥Rs: Fc␥RI, Fc␥RIIa, Fc␥RIIb, and Fc␥RIIIa (26). Of these, Fc␥RI, Fc␥RIIa, and Fc␥RIIIa are activating receptors that drive cellular responses to antibodies. These receptors either contain, within their cytoplasmic tails, an immune receptor tyrosine-based activation motif (ITAM), as in the case of Fc␥RIIa (27), or are associated with the ␥-chain homodimer that has an ITAM (28). The association of the ␥-chain is critical not only for surfa...