Fusion SL (Analis Devices) was used as per manufacturer instructions and chemiluminescent captured using Fusion SL (Analis Devices). sensitivity to existing therapeutic antibodies used in CLL. This is the first study to show that activation of the CD47:SIRP innate immune checkpoint contributes to ADP resistance in NLCs from CLL patients. Keywords:nurse-like-cells, chronic lymphocytic leukemia, macrophages, antibody dependent phagocytosis, antibody resistance 2, macrophage == Introduction == Macrophages are a cellular effector of antibody-dependent phagocytosis (ADP) (14). Indeed, reports suggest that macrophages may be the major immune effector to clear tumor cells in response to therapeutic antibodies such as obinutuzumab (3,57). Therapeutic antibodies targeting CD20, such as obinutuzumab and rituximab, are used in first- and second-line treatments for CLL (812). Although of confirmed clinical value in CLL treatment, acquired resistance to antibody therapies remains a serious issue and contributes to relapse and treatment failure (13). Culture of peripheral blood monocytes from chronic lymphocytic leukemia (CLL) patients gives rise to macrophage-like cells referred to as nurse like cells (NLCs). NLCs display profound ADP activity against CLL cells incubated with obinutuzumab or rituximab (4,14). In this regard, Cephalexin monohydrate NLCs provide a clinically relevantex vivomodel of ADP responses and mechanisms of resistance in CLL (1,2,4,14,15). For example, NLCs derived from patients with stable/early disease mount a strong ADP response in which 3060% of the NLCs participate in ADP against obinutuzumab or rituximab-opsonised CLL cells (1,14). We refer to these NLCs as sensitive (1,14). In contrast, NLCs derived Cephalexin monohydrate from patients with progressive/relapsed/refractory disease requiring treatment display a muted ADP response to obinutuzumab or rituximab (1,14). Typically, less than 10% of NLCs participate in ADP responses to antibody-opsonized CLL cells (1,14). We refer to these NLCs as resistant. This resistance to therapeutic antibodies is usually reversible and actionable (1,14). For example, the use of FcRIIB blocking antibodies, HDAC7-selective inhibitors, and SHIP1 inhibitors have all been shown to enhance ADP responses in phenotypically resistant NLCs (1,16,17). ADP responses are initiated when opsonized targets bind an Fc Receptor (FcR) complex on immune effectors such as NLCs (15). FcRs are a family of receptors comprising activating FcRs (e.g. FcR1,2A,3 and 4 in humans) and an inhibitory FcR2B (15). Once ligated, in the context of CLL, FcR induce ADPviaactivation of Cephalexin monohydrate either a SYK/BTK dependent signaling pathway or a PI3K/p110 signaling pathway (1,2). The SYK/BTK pathway is usually muted in phenotypically resistant NLCs whereas the PI3K/p110/AKT pathway is usually unaltered between phenotypically sensitive or resistant NLCs (2). The SYK/BTK mediated resistance can be reversed with Ship1 inhibitors (1). We recently discovered that histone deacetylase 7 (HDAC7) directly suppresses BTK activation in phenotypically resistant NLCs which can be reversed by treatment with HDAC7-selective inhibitors (17). Thus, ADP resistance is usually actionable and appears to be mediated exclusively by SYK and BTK signaling. Moreover, it is able to be altered with clinically available brokers. Recent evidence suggests that phagocytosis of opsonized or non-opsonized targets occur in the context of an immune synapse-like structure involving the clustering of activating and inhibitory immune receptors (18). Within the FcR family, there are activating and inhibitory FcRs which cluster prior to phagocytosis (1922). Moreover, external to the FcRs are coregulatory receptors such as SIRP which are Cephalexin monohydrate expressed on macrophages and activated by ligation with ligands such Cephalexin monohydrate as CD47 expressed on a target cell (18,23). There is growing evidence that this CD47/SIRP axis may inhibit phagocytosis of tumor cells (7,2325). A typical scenario would see CD47 (dont eat me signal) expressed on a malignancy cell bind to SIRP on an immune effector (e.g. macrophage) causing an inhibitory signal in the effector cell which dampens phagocytosis (7,23). Thus, the CD47:SIRP axis is referred to as an innate immune checkpoint (23) and is being targeted in a number of clinical trials in various malignancy types including AML, breast malignancy, CLL, lymphoma and head neck skin malignancy (26,27). Early data emerging from IL8 these trials suggests that blood cancers are more sensitive than solid tumors to anti-CD47:SIRP therapies (15). However, it has also emerged that targeting ubiquitously expressed CD47 has the potential for inducing unwanted side effects such as anaemia (23). Since.