IntroductionNatural killer (NK) cells are CD56 ϩ CD3 Ϫ large granular lymphocytes that comprise a key cellular compartment of the innate immune system. NK cells have been shown to exert antitumor activity against the malignant plasma cell clone in multiple myeloma (MM). 1-4 However, through several established mechanisms, the NK-cell versus MM effect is attenuated as the disease inexorably progresses. 5-9 MM is increasing in incidence and remains incurable despite the advent of potent novel therapies such as lenalidomide and bortezomib. 10 In fact, both lenalidomide and bortezomib have been shown to confer anti-MM activity, in part, through recovery or enhancement of the NK-cell versus MM effect. 11,12 The NK-cell versus MM effect is subject to modulation through intracellular signal transduction cascades initiated by activating and inhibitory receptors at the NK-cell surface interacting with ligands expressed on MM tumor cells. Programmed death 1 (PD-1), a member of the B7 family of cosignaling molecules, and its associated ligands PD-L1 and PD-L2 have been shown to play a key role in down-regulating the T-cell immune response. 13 The constitutive or inducible expression of PD-1 has been characterized on several immune cell subsets, including T, B, and dendritic cells; however, to date, comparatively little is known regarding PD-1 expression on NK cells and whether or not the PD-1/PD-L1 axis is involved in the NK-cell versus MM effect. 14 CT-011 (CureTech, LTD; previously CT-AcTibody or BAT) is a novel immunoglobulin G1 (IgG1) humanized monoclonal antibody (mAb) that modulates the immune response through interaction with PD-1, with previously demonstrated antitumor efficacy in experimental models of both solid and liquid tumors. [15][16][17] Several human malignancies, including MM, express cognate ligands for PD-1 (eg, PD-L1) and play a key role in tumor immunoevasion. 18,19 In a phase 1 clinical trial of patients with advanced hematologic malignancies including MM, CT-011 was demonstrated to be safe and well tolerated with evidence of single-agent clinical beneficial responses in 33% of the patients. 20 Given the results of this phase 1 study and the potential complementary mechanisms of action between CT-011 and lenalidomide, we hypothesized these agents in combination may represent a promising novel therapy for MM.Lenalidomide (Revlimid; Celgene) exerts efficacy in part through enhancement of the NK-cell versus MM effect, 11 an effect likely mediated through T-cell production of interleukin-2 (IL-2) in response to this agent. 21 The numbers of both T cells and NK cells are increased in patients receiving lenalidomide therapy 22 ; however, NK-cell killing is also enhanced, including antibodydependent cellular cytotoxicity and natural cytotoxicity. 23,24 Moreover, these events correlate with clinical responses to lenalidomide therapy in patients with MM. 22 In this report, we show that the PD-1/PD-L1 signaling axis mediates NK-cell activation and cytotoxicity against MM. We show that freshly isolated NK cells...

Human natural killer (NK) cells originate from CD34(+) hematopoietic progenitor cells, but the discrete stages of NK cell differentiation in vivo have not been elucidated. We identify and functionally characterize, from human lymph nodes and tonsils, four NK cell developmental intermediates spanning the continuum of differentiation from a CD34(+) NK cell progenitor to a functionally mature NK cell. Analyses of each intermediate stage for CD34, CD117, and CD94 cell surface expression, lineage differentiation potentials, capacity for cytokine production and natural cytotoxicity, and ETS-1, GATA-3, and T-BET expression provide evidence for a new model of human NK cell differentiation in secondary lymphoid tissues.

Signaling pathways between cell surface receptors and the BCL-2 family of proteins regulate cell death. Survival factors induce the phosphorylation and inactivation of BAD, a proapoptotic member. Purification of BAD kinase(s) identified membrane-based cAMP-dependent protein kinase (PKA) as a BAD Ser-112 (S112) site-specific kinase. PKA-specific inhibitors blocked the IL-3-induced phosphorylation on S112 of endogenous BAD as well as mitochondria-based BAD S112 kinase activity. A blocking peptide that disrupts type II PKA holoenzyme association with A-kinase-anchoring proteins (AKAPs) also inhibited BAD phosphorylation and eliminated the BAD S112 kinase activity at mitochondria. Thus, the anchoring of PKA to mitochondria represents a focused subcellular kinase/substrate interaction that inactivates BAD at its target organelle in response to a survival factor.

In humans, T cells differentiate in thymus and B cells develop in bone marrow (BM), but the natural killer (NK) precursor cell(s) and site(s) of NK development are unclear. The CD56bright NK subset predominates in lymph nodes (LN) and produces abundant cytokines compared to the cytolytic CD56dim NK cell that predominates in blood. Here, we identify a novel CD34dimCD45RA(+) hematopoietic precursor cell (HPC) that is integrin alpha4beta7bright. CD34dimCD45RA(+)beta7bright HPCs constitute <1% of BM CD34(+) HPCs and approximately 6% of blood CD34(+) HPCs, but >95% of LN CD34(+) HPCs. They reside in the parafollicular T cell regions of LN with CD56bright NK cells, and when stimulated by IL-15, IL-2, or activated LN T cells, they become CD56bright NK cells. The data identify a new NK precursor and support a model of human NK development in which BM-derived CD34dimCD45RA(+)beta7bright HPCs reside in LN where endogenous cytokines drive their differentiation to CD56bright NK cells in vivo.