NK Cells In Control Research Articles

Enhancing Dendritic Cell-based Immunotherapy with IL-2/Monoclonal Antibody Complexes for Control of Established Tumors.

U.S. Food and Drug Administration-approved high-dose IL-2 therapy and dendritic cell (DC) immunization offer time-tested treatments for malignancy, but with defined issues of short in vivo t1/2, toxicity, and modest clinical benefit. Complexes of IL-2 with specific mAbs (IL-2c) exhibit improved stability in vivo with reduced toxicity and are capable of stimulating NK cell and memory phenotype CD8 T cell proliferation. In this study, we demonstrate that IL-2c treatment in tumor-bearing mice can enhance NK cell and tumor-specific CD8 T cell numbers. Importantly, DC immunization coupled with stabilized IL-2c infusion drastically improves the tumor-specific effector CD8 T cell response. DC + IL-2c treatment enhances number, 41BB and GITR expression, granzyme B production, CTL/regulatory T cell ratio, and per-cell killing capacity of CD8 T cells without increasing inhibitory molecule expression. Notably, IL-2c treatment of anti-CD3-stimulated human CD8 T cells resulted in higher number and granzyme B production, supporting the translational potential of this immunotherapy strategy for human malignancy. DC + IL-2c treatment enhances both endogenous NK cell and tumor Ag-specific CD8 T cell immunity to provide a marked reduction in tumor burden in multiple models of pre-existing malignancy in B6 and BALB/c mice. Depletion studies reveal contributions from both tumor-specific CD8 T cells and NK cells in control of tumor burden after DC + IL-2c treatment. Together, these data suggest that combination therapy with DC and IL-2c may be a potent treatment for malignancy.

A Higher Frequency of NKG2A+ than of NKG2A- NK Cells Responds to Autologous HIV-Infected CD4 Cells irrespective of Whether or Not They Coexpress KIR3DL1.

Epidemiological and functional studies implicate NK cells in HIV control. However, there is little information available on which NK cell populations, as defined by the inhibitory NK cell receptors (iNKRs) they express, respond to autologous HIV-infected CD4(+) (iCD4) T cells. NK cells acquire antiviral functions through education, which requires signals received from iNKRs, such as NKG2A and KIR3DL1 (here, 3DL1), engaging their ligands. NKG2A interacts with HLA-E, and 3DL1 interacts with HLA-A/B antigens expressing the Bw4 epitope. HIV-infected cells downregulate HLA-A/B, which should interrupt negative signaling through 3DL1, leading to NK cell activation, provided there is sufficient engagement of activating NKRs. We examined the functionality of NK cells expressing or not NKG2A and 3DL1 stimulated by HLA-null and autologous iCD4 cells. Flow cytometry was used to gate on each NKG2A(+)/NKG2A(-) 3DL1(+)/3DL1(-) (NKG2A(+/-) 3DL1(+/-)) population and to measure the frequency of all possible combinations of CD107a expression and gamma interferon (IFN-γ) and CCL4 secretion. The highest frequency of functional NK cells responding to HLA-null cell stimulation was the NKG2A(+) 3DL1(+) NK cell population. The highest frequencies of functional NK cells responding to autologous iCD4 cells were those expressing NKG2A; coexpression of 3DL1 did not further modulate responsiveness. This was the case for the functional subsets characterized by the sum of all functions tested (total responsiveness), as well as by the trifunctional CD107a(+) IFN-γ(+) CCL4(+), CD107a(+) IFN-γ(+), total CD107a(+), and total IFN-γ(+) functional subsets. These results indicate that the NKG2A receptor has a role in NK cell-mediated anti-HIV responses. HIV-infected CD4 (iCD4) cells activate NK cells, which then control HIV replication. However, little is known regarding which NK cell populations iCD4 cells stimulate to develop antiviral activity. Here, we examine the frequency of NK cell populations, defined by the presence/absence of the NK cell receptors (NKRs) NKG2A and 3DL1, that respond to iCD4 cells. NKG2A and 3DL1 are involved in priming NK cells for antiviral functions upon encountering virus-infected cells. A higher frequency of NKG2A(+) than NKG2A(-) NK cells responded to iCD4 cells by developing antiviral functions such as CD107a expression, which correlates with NK cell killing, and secretion of gamma interferon and CCL4. Coexpression of 3DL1 on the NKG2A(+) and NKG2A(-) NK cells did not modulate responses to iCD4 cells. Understanding the mechanisms underlying the interaction of NK cells with iCD4 cells that lead to HIV control may contribute to developing strategies that harness NK cells for preventing or controlling HIV infection.

Acquisition of activation receptor ligand by trogocytosis renders NK cells hyporesponsive.

Because NK cells secrete cytotoxic granules and cytokines that can destroy surrounding cells and help shape the subsequent immune response, they must be kept under tight control. Several mechanisms, at different levels, are in place to control NK cell function. In this study, we describe a novel mechanism regulating NK cell function in which NK cells acquire ligands for activating receptors from target cells by trogocytosis, rendering the NK cells hyporesponsive. In this model, murine NK cells acquire m157, the murine CMV-encoded ligand for the Ly49H-activating receptor, from target cells both in vitro and in vivo. Although acquisition of m157 requires cell-to-cell contact, it does not require the expression of the Ly49H receptor by the NK cell. Acquired m157 protein is expressed on the NK cell surface with a glycosylphosphatidylinisotol linkage and interacts with the Ly49H receptor expressed on the NK cell. This interaction results in blocking the Ly49H receptor that prevents the NK cells from recognizing m157-expressing targets and continuous engagement of the Ly49H-activating receptor, which results in the hyporesponsiveness of the Ly49H(+) NK cell to stimulation through other activating receptors. Thus, NK cell acquisition of a ligand for an activation receptor by trogocytosis renders them hyporesponsive. This mechanism, by which mature NK cell function can be altered, has important implications in regard to how NK cells respond to tumors in specific microenvironments as well as the use of expanded NK cells in treating various malignancies.

Altered NK cell function in obese healthy humans.

BackgroundObesity is associated with an elevated risk for several types of cancer and thus a major health hazard. However, the mechanism between overweight and cancer susceptibility is still elusive. Leptin, mainly produced by adipocytes links food intake and energy expenditure. In addition, recent studies have shown an immunomodulatory impact of leptin on NK cells. The purpose of the present study was to investigate whether leptin stimulation of NK cells from obese humans leads to altered functions as compared to NK cells from lean subjects. On the basis of body mass index 20 healthy individuals were classified in two groups: normal weight (<25 kg/m2) and obese (>30 kg/m2). Peripheral blood mononuclear cells (PBMC) were isolated from blood samples. We used flow cytometry to assess differences in phenotype and activity markers (CD107a, CD178 and TRAIL) of PBMCs between both groups. Furthermore, we determined after short-term in vitro leptin stimulation the phosphorylation of JAK2, downstream target of the intracellular signaling cascade of the leptin receptor, by Western Blotting and numbers of NK-cell-tumor-cell-conjugates as well as Granzyme+ and IFN-γ+ NK cells by flow cytometry. Finally, the proliferative capacity of control and long-term (7 days) leptin-stimulated NK cells was examined.ResultsAs opposed to similar NK cell counts, the number of CD3+CD56+ cells was significantly lower in obese compared to lean subjects. Human NK cells express the leptin receptor (Ob-R). For further determination of Ob-R, intracellular target proteins of PBMCs were investigated by Western Blotting. Phosphorylation of JAK2 was lower in obese as compared to normal weight subjects. Furthermore, significantly lower levels of TNF-related apoptosis-inducing ligand (TRAIL) as an NK cell functional marker in obese subjects were found. In vitro leptin stimulation resulted in a higher production of interferon-γ in NK cells of normal weight subjects. Interestingly, long-term leptin stimulation had no significant influence on numbers of proliferating NK cells.ConclusionsNK cells from obese healthy humans show functional deficits and altered responses after in vitro leptin challenge.

Human Cytokine-Induced Memory-like (CIML) NK Cells Are Active Against Myeloid Leukemia in Vitro and in Vivo

NK cells are innate lymphoid cells that mediate anti-leukemia responses. The ability of MHC-haploidentical NK cells to recognize and eliminate AML blasts have been established in the setting of stem cell transplantation and early phase adoptive NK cell immunotherapy trials. However, the optimal approach to prepare human NK cells for maximal anti-leukemia capacity is unclear. As one form of innate NK cell memory, cytokine-induced memory-like (CIML) NK cells are induced by a brief (16 hour) pre-activation of human NK cells with the combination of IL-12, IL-15, and IL-18, while control NK cells from the same donor are activated by IL-15 only. In published work, this combined IL-12, IL-15, and IL-18 pre-activation results in enhanced proliferation and augmented IFN-gamma responses to cytokine or activating receptor-based re-stimulation following a rest period of 1 – 6 weeks. We hypothesized that CIML NK cells exhibit improved anti-leukemia properties compared to control NK cells from the same individual. Purified primary human CIML NK cells [both CD56bright and CD56dim subsets] produce more IFN-gamma, compared to control NK cells, upon re-stimulation with K562 cells or primary AML blasts after 7 days of rest (p<0.05 and p<0.001, N=5). CIML NK cells also exhibit higher granzyme B protein expression (p<0.01; N=8), and increased cytotoxicity against K562 leukemia targets in vitro (p<0.001, 2.5:1 and 5:1 E:T ratios). We next established a NOD-SCID-gamma-c-/- (NSG) xenograft model to investigate primary human CIML NK cell responses in vivo, with survival supported by low dose IL-2 administered every other day. Seven days following injection of 4 million NK cells / mouse, human CIML NK cells traffic to the bone marrow, spleen, liver and blood, and exhibited better in vivo expansion and persistence, compared to control NK cells (p=0.05 in the blood and bone marrow). Further, the characteristic enhanced functionality of CIML compared to control NK cells when restimulated with K562 targets was retained when assessed ex vivo 7 days post-transfer (p<0.05). Next, we investigated the ability of CIML versus control NK cells from the same donor to clear K562 AML cells in vivo. First, luciferase expressing K562 cells (1 million / mouse) were engrafted into sub-lethally irradiated (250 cGy) NSG mice. On day 3 after K562 challenge, primary human CIML or control NK cells from the same donor (4 million / mouse) were injected, which were supported in vivo using low dose IL-2. CIML NK cells exhibited significantly improved in vivo leukemia clearance as evidenced by whole mouse bioluminescence imaging (see Figure, P=0.03, N=7 mice per group). Thus, human CIML NK cells exhibit enhanced in vitro and in vivo anti-leukemia effects, compared to control NK cells. Based on these findings, a first-in-human phase 1 study of CIML NK cells in relapsed/refractory AML is currently underway. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The ap-2 clathrin adaptor mediates endocytosis of an inhibitory killer cell Ig-like receptor in human NK cells.

Stable surface expression of human inhibitory killer cell Ig-like receptors (KIRs) is critical for controlling NK cell function and maintaining NK cell tolerance toward normal MHC class I(+) cells. Our recent experiments, however, have found that Ab-bound KIR3DL1 (3DL1) readily leaves the cell surface and undergoes endocytosis to early/recycling endosomes and subsequently to late endosomes. We found that 3DL1 internalization is at least partially mediated by an interaction between the μ2 subunit of the AP-2 clathrin adaptor complex and ITIM tyrosine residues in the cytoplasmic domain of 3DL1. Disruption of the 3DL1/μ2 interaction, either by mutation of the ITIM tyrosines in 3DL1 or mutation of μ2, significantly diminished endocytosis and increased surface expression of 3DL1 in human primary NK cells and cell lines. Furthermore, we found that the 3DL1/AP-2 interaction is diminished upon Ab engagement with the receptor, as compared with untreated cells. Thus, we have identified AP-2-mediated endocytosis as a mechanism regulating the surface levels of inhibitory KIRs through their ITIM domains. Based on our results, we propose a model in which nonengaged KIRs are internalized by this mechanism, whereas engagement with MHC class I ligand would diminish AP-2 binding, thereby prolonging stable receptor surface expression and promoting inhibitory function. Furthermore, this ITIM-mediated mechanism may similarly regulate the surface expression of other inhibitory immune receptors.

Loss of cytotoxicity and gain of cytokine production in murine tumor-activated NK cells.

NK cells are able to form a functional memory suggesting that some NK cells are surviving the activation process. We hypothesized that NK cell activation causes the development of a distinct NK cell subset and studied the fate of murine post-activation NK cells. Activation was achieved by in vivo and in vitro exposures to the melanoma tumor cell line B16 that was followed by differentiation in IL-2. When compared with control NK cells, post-activation CD25+ NK cells expressed little granzyme B or perforin and had low lysis activity. Post-activation NK cells expressed CD27, CD90, CD127, and were low for CD11b suggesting that tumor-induced activation is restricted to an early NK cell subset. Activation of NK cells led to decreases of CD16, CD11c and increases of CD62L and the IL-18 receptor. In vivo activated but not control NK cells expressed a variety of cytokines that included IFNγ, TNFα, GM-CSF and IL-10. These data suggest that the exposure of a subset of peripheral NK cells to the B16 tumor environment caused an exhaustion of their cytolytic capacity but also a gain in their ability to produce cytokines.

Klebsiella pneumoniae alleviates influenza-induced acute lung injury via limiting NK cell expansion.

A protective effect induced by bacterial preinfection upon a subsequent lethal influenza virus infection has been observed, but the underlying immune mechanisms have not yet been fully elucidated. In this study, we used a mouse model of Klebsiella pneumoniae preinfection to gain insight into how bacterial preinfection influences the subsequent lethal influenza virus infection. We found that K. pneumoniae preinfection significantly attenuated lung immune injury and decreased mortality during influenza virus infection, but K. pneumoniae-specific immunity was not involved in this cross-protection against influenza virus. K. pneumoniae preinfection limited NK cell expansion, which was involved in influenza-induced immune injury and death. Furthermore, K. pneumoniae preinfection could not control NK cell expansion and death during influenza virus infection in Rag1(-/-) mice, but adoptive transfer of T cells from wild-type mice was able to restore this protective effect. Our data suggest that the adaptive immune response activated by bacterial infection limits the excessive innate immune response induced by a subsequent influenza infection, ultimately protecting mice from death.

Mir-15/16 antagonizes c-Myb to control natural killer cell maturation (LYM7P.714)

Abstract NK cells develop in the bone marrow and complete maturation in peripheral organs, but the factors controlling maturation are incompletely understood. The miR-15/16 family is highly expressed in NK cells, and to study its role in NK cells, we generated a novel mouse model of NK-specific miR-15/16 deficiency (15aKO). 15aKO NK cells have a deficiency of mature (CD11b+CD27-) NK cells, with no alterations in total NK cell numbers or death, consistent with a block in maturation. The transcription factor Myb is expressed highly in immature NK cells, nearly absent in mature NK cells, and has been implicated in NK maturation. We found that miR-15/16 represses Myb, and Myb levels are increased in 15aKO NK cells. To directly investigate the role of Myb in NK cells, we developed a novel NK maturation system in which immature primary NK cells are transduced, adoptively transferred, and matured in vivo. As in the 15aKO mouse, transferred 15aKO NK cells had defective maturation. However, both 1) restoration of miR-15/16, or 2) knockdown of Myb, rescued maturation of the 15aKO NK cells, demonstrating that miR-15/16’s repression of Myb is critical to proper NK cell maturation. Furthermore, WT NK cells overexpressing Myb did not mature as well as control (WT NK+GFP) cells. Thus, the regulation of Myb by miR-15/16 is essential for NK cell maturation, revealing a novel mechanism controlling NK cell development.

Adaptor protein Crk-like (CrkL) controls natural killer cell function via regulating the accumulation of activating receptors and phosphorylated Vav-1 molecules at immunological synapses (INM2P.433)

Abstract Very few studies have addressed how CrkL, a ubiquitous adaptor protein, regulates the immune response, especially in NK cells. Previous studies indicate that CrkL is required for NKG2D-mediated cellular cytotoxicity (NC) and CD16 signaling triggered antibody mediated cellular cytotoxicity (ADCC). However, how CrkL protein controls NK cell cytotoxicity remains unclear. Here we used lentiviral inducible shRNA to silence CrkL in human NK cell line and primary NK cells. Inducible knockdown of CrkL significantly reduced NK-target conjugation, degranulation, and cytotoxicity in both NC- and ADCC-mediated immune responses. Further investigation on the function and signaling transduction at the NK cytotoxic immunological synapse (IS) reveals that polarization of lytic granules, and accumulation of both F-actin and phosphorylated Vav-1 at the IS were greatly reduced in CrkL-silenced NK cells. NK cell activation involves in actin-dependent activating receptor clustering. CrkL silencing resulted in reduced accumulation of ligands for CD16 and NKG2D on the lipid bilayer. Similar results were obtained in NK cells from DiGeorge Syndrome patients (CrkL gene deletion). We thus propose a new model for adaptor protein CrkL function in NK cell activation: CrkL controls NK cell cytotoxicity via promoting F-actin accumulation and lytic granule polarization by modulating the accumulation of activating receptors and phosphorylated Vav-1 molecules at the IS.

Role of NKG2D in the induction of mouse natural killer cell exhaustion (IRC8P.473)

Abstract Natural Killer (NK) cells have been used as a cancer therapy against multiple cancers. Despite initial promises, NK-based immunotherapy has resulted in limited success and therefore, new approaches are needed. Our group described the induction of NK cell exhaustion (NCE) upon tumor exposure that resulted in reduced cytotoxic function associated with lower expression of several molecules such as NKG2D and the transcription factors Eomesodermin and T-bet. Here we show that NCE induced by tumor cells is MHC-I independent and is not mediated by the expression of NKG2D ligands (NKG2DL) on tumor cells. However, when NKG2D-deficient NK cells were exposed to tumor cells limited proliferation and higher IFNg production was observed suggesting a role for NKG2D in NCE. Moreover, NCE patters were observed at later stages of in vitro IL2-activation that were reduced in NKG2D-deficient NK cells. Interestingly, concomitant downregulation of NKG2D and upregulation of NKG2DL (MULT1, Rae-1) was also detected. Upregulation of NKGDL expression was particularly seen after the cells were acid-stripped which was shown to disrupt MHC-I-Ly49s cis-interaction complexes. NCE was also observed after prolonged in vivo IL2 stimulation that impaired NK cell maturation. In conclusion, we demonstrated a role for NKG2D in the induction of NCE that could represent a self-regulatory mechanism important in controlling NK cell activation utilized by tumor cells to prevent NK cell anti-tumor responses.

EAT-2, a SAP-like adaptor, controls NK cell activation through phospholipase Cγ, Ca++, and Erk, leading to granule polarization

EAT-2, a SAP-like adaptor, controls NK cell activation through phospholipase Cγ, Ca⁺⁺, and Erk, leading to granule polarization

Tissue-Specific Microvascular Endothelial Cells Show Distinct Capacity To Activate NK Cells: Implications for the Pathophysiology of Granulomatosis with Polyangiitis

The relevance of tissue specificity of microvascular endothelial cells (MECs) in the response to inflammatory stimuli and sensitivity to immune cell-mediated injury is not well defined. We hypothesized that such MEC characteristics might shape their interaction with NK cells through the use of different adhesion molecules and NK cell receptor ligands or the release of different soluble factors and render them more or less vulnerable to NK cell injury during autoimmune vasculitis, such as granulomatosis with polyangiitis (GPA). To generate a comprehensive expression profile of human MECs of renal, lung, and dermal tissue origin, we characterized, in detail, their response to inflammatory cytokines and to proteinase 3, a major autoantigen in GPA, and analyzed the effects on NK cell activation. In this study, we show that renal MECs were more susceptible than lung and dermal MECs to the effect of inflammatory signals, showing upregulation of ICAM-1 and VCAM-1 on their surface, as well as release of CCL2, soluble fractalkine, and soluble VCAM-1. Proteinase 3-stimulated renal and lung MECs triggered CD107a degranulation in control NK cell. Notably, NK cells from GPA patients expressed markers of recent in vivo activation (CD69, CD107a), degranulated more efficiently than did control NK cells in the presence of renal MECs, and induced direct killing of renal MECs in vitro. These results suggest that, upon inflammatory conditions in GPA, renal MECs may contribute to the recruitment and activation of NK cells in the target vessel wall, which may participate in the necrotizing vasculitis of the kidney during this disease.

NK-cell dysfunction in human renal carcinoma reveals diacylglycerol kinase as key regulator and target for therapeutic intervention

The relevance of NK cells in tumor control is well established in mouse models and human hematologic malignancies; however, their contribution to the control of human solid tumors remains disputed due to problems with in situ detection and reports of functional inactivity in the tumor milieu. In this study, we established a reliable in situ detection method for NK cells. Moreover, we performed analysis to elucidate mechanisms that impair NK-cell function in the tumor milieu and thereby identify therapeutic targets that allow recovery of NK-cell functionality. It was observed that NK cells from clear cell renal cell carcinoma (ccRCC), compared to NK cells from nontumor kidney and peripheral blood lymphocytes (PBLs), displayed conjoint phenotypic alterations and dysfunction induced by the tumor milieu, which were associated mechanistically with high levels of signaling attenuator diacylglycerol kinase (DGK)-α and blunted mitogen-activated protein kinase pathway activation (ERK1/2, Jun kinase). Reinstating NK-cell functionality was possible by DGK inhibition or brief IL-2 culture, interventions that de-repressed the ERK pathway. The extent of alteration and magnitude of recovery could be linked to NK-cell frequency within ccRCC-infiltrating lymphocytes, possibly explaining the observed survival benefit of patients with NK(high) tumors. In conclusion, DGK-mediated dampening of the ERK pathway ensuing in NK-cell dysfunction was identified as an important escape mechanism in ccRCC. DGK and the ERK pathway thus emerge as promising therapeutic targets to restore suppressed NK-cell activity for the improvement of antitumor immunity.

Human Cytokine-Induced Memory-Like (CIML) NK Cells Exhibit Potent Anti-Leukemia Cytotoxicity and Maintain Memory-Like Functionality After Adoptive Transfer Into Immunodeficient NOD-SCID-Gc-/- (NSG) Mice

Background Allogeneic NK cells are anti-leukemia immune effector lymphocytes, with evidence of activity in patients in adoptive transfer clinical studies. What is the optimal approach to prepare allogeneic NK cells for maximal effector function remains an open question for adoptive NK cell therapy. Recently described cytokine-induced memory-like (CIML) NK cells are generated following a brief (16 hour) pre-activation with a combination of IL-12+IL-15+IL-18 in both mice and humans. Following weeks or months of rest, CIML NK cells exhibit an enhanced recall IFN-g response when restimulated with K562 cells or cytokines. However, their anti-leukemic cytotoxic activity and identification of key supporting cytokines for survival and sustained functionality have not been reported. We hypothesized that CIML NK cells may have enhanced effector function against AML, providing a potential rationale for future clinical studies of CIML NK cells in AML patients. To test this hypothesis we investigated the CIML NK cell response to myeloid leukemia, including primary AML blasts, and evaluated their function following transfer into NSG mice. Methods Normal human donor NK cells (&gt;95% purity) were cultured with low dose (1 ng/mL) IL-15 alone (control) or pre-activated with IL-12 (10 ng/ml) + IL-15 (1 ng/ml) + IL-18 (50 ng/ml) for 16 hours. After washing, the cells were cultured for 7 days in low dose IL-15 (to support survival). Following this prolonged rest period in vitro, NK cell responses were assessed after 6-hour re-stimulation with K562 leukemia cells or primary AML blasts. NK cell functional responses assessed include IFN-g production and cytotoxicity (using flow based killing assays). For adoptive transfer experiments, 5-8 x 106 human CIML NK cells or control cells were injected into sub-lethally irradiated NSG mice, and assessed for persistence, expansion and function of the adoptively transferred CIML NK or control NK cell. Additional experiments included evaluating CIML NK cells for cytokine receptor expression and effector proteins after the 7 day rest period. Results As described previously, CIML NK cells had a significantly increased IFN-g response to K562 leukemia cells (15.5 ± 3% vs. 7 ± 1%, P=0.03). CIML NK cells also exhibited a more potent IFN-g response to primary blasts from untreated, newly diagnosed AML patients (N=4 AML samples, P&lt; 0.0001). Further, CIML NK cells demonstrated a significantly greater cytotoxic response, compared to control NK cells, upon co-incubation with K562 leukemia cells (Figure 1). Consistent with this enhanced cytotoxicity, CIML NK cells had significantly increased expression of granzyme A (P=0.005) and granzyme B (P=0.006) proteins. Further, we noted a marked induction of CD25 (IL-2Ra) after IL-12+IL-15+IL-18 pre-activation, which via the IL-2Rabg resulted in enhanced functional responses to picomolar concentrations of IL-2. This included enhanced cytotoxicity against leukemia cells, IFN-g production in response to co-stimulation with IL-12, and proliferation. To assess persistence and expansion upon adoptive transfer, NSG mice were injected with control or CIML NK cells. After 7 days (during which 75,000IU of IL-2 was injected qOD) there was a preferential expansion of the CIML NK cells in blood (11±2.6 vs. 5±1.3, P=0.01) and bone marrow (0.6±0.14 vs. 0.21±0.06, P= 0.03) in these mice as assessed by the ratio of human to mouse CD45 positive cells. Further, CIML NK cells supported in vivo in NSG mice exhibited enhanced IFN-g responses upon re-stimulation with K562 leukemia cells (10±1.5% vs. 2.5±1% IFN-g positive, P= 0.03) or IL-12+IL-15 (15±2% vs. 2±0.5%, P= 0.001). Conclusions Brief (16 hour) pre-activation with a combination of IL-12+IL-15+IL-18 results in the generation of CIML NK cells that have an enhanced IFN-g and cytotoxic response to K562 leukemia cells and primary allogeneic AML blasts. Further, CD25 is induced on CIML NK cells, which in the context of the high affinity IL-2Rabg confers selective responsiveness to low concentrations of IL-2 for proliferation, enhanced cytotoxicity, and enhanced IFN-g production. CIML NK cells may develop in vivo in NSG xenografts, and CIML NK cells appear to be selectively supported by exogenous low dose IL-2 in this context. These pre-clinical data support CIML NK cells as a novel optimization approach for NK cell adoptive immunotherapy. Disclosures: No relevant conflicts of interest to declare.

Mir-15/16 Antagonizes Myb To Control Natural Killer Cell Differentiation and Maturation

IntroductionNatural Killer (NK) cells are lymphocytes that are important for early host defense against infectious pathogens and malignant transformation. NK cells differentiate from the CLP in the bone marrow, where they are identified by markers such as CD56 and NKp46 in humans, and NK1.1, CD122, and NKp46 in mice. NK cells further mature in the periphery, and this maturation is essential for NK cell function, as both NK cell cytotoxicity and IFN-g production are dependent upon maturation. NK cell maturation is distinguished by surface marker transitions, including CD56bright to CD56dim in humans, and loss of CD27 expression in mice. However, the factors controlling NK cell differentiation and maturation are incompletely understood. We hypothesized that the transcription factor Myb had a role in this process, due to its high expression in immature NK cells and subsequent loss upon maturation.miRNAs are a family of small RNA molecules that control a wide variety of cellular processes via binding to target sites in the 3'UTR of messenger RNAs and downregulate protein production. The miR-15/16 family is very highly expressed in NK cells, and directly targets the 3'UTR of Myb. We hypothesized that a miR-15a/16-1KO mouse would have NK cell-intrinsic alterations in Myb levels, and would serve as a model of Myb upregulation. Here, we use lentiviral overexpression in primary human and mouse NK cells, as well as an in vitro human NK cell differentiation system, to demonstrate that Myb has critical roles in the NK cell differentiation and maturation processes. Furthermore, we generate a novel mouse model of miR-15/16 deficiency, and show that miR-15/16 is critically important for the regulation of Myb levels, and disruption of miR-15/16 prevents appropriate NK cell maturation. Results and ConclusionsIn order to investigate the role of Myb in NK cells, we transduced human NK cells, and cultured them in vitro. After 5 days of culture, GFP+ NK cells overexpressing Myb remained CD56bright (84±3 v. 6±2%, p<0.01), whereas NK cells expressing GFP only had differentiated to CD56dim (16±2 v. 94±3%, p<0.001). Mouse CD27+ NK cells were transduced with the same viruses, and adoptively transferred and allowed to mature for 7 days in their new hosts. 0% of NK cells overexpressing Myb matured to CD27-, while 11% of GFP only matured, and 22% of NK cells with knockdown of Myb matured to CD27-. Thus, cells overexpressing Myb have a block in maturation, and Myb downregulation is essential for complete NK cell maturation.To further investigate the role of Myb, we lentivirally transduced and cultured CD34+ progenitors in NK cell differentiation conditions. We found that cells overexpressing Myb had an increased percentage of immature CD56bright NK cells, which arose with more rapid kinetics (91±8 v. 28±16%, p<0.001 at day 14) [Fig. 1]. However, at later time points, cells overexpressing Myb failed to differentiate from CD56bright to the more mature CD56dim NK cells (8±6 v. 64±11%, p<0.01 at day 21). In contrast, CD34 cells transduced with an shRNA directed against Myb, differentiated to CD56dim NK cells more rapidly than control cells (90±7 v. 65±11, p<0.05 at day 21). Therefore, Myb drives initial NK cell differentiation, but prevents final maturation of NK cells. [Display omitted] We found that Myb is a direct target of miR-15/16, as overexpression of miR-15/16 reduces the signal of luciferase fused to the 3'UTR of Myb by 50% (p<0.001), while a sponge directed against miR-15/16 increases signal by 40% (p<0.001). Therefore, we generated a novel mouse model of NK cell-specific miR-15a/16-1 knockout driven by NKp46 (Ncr1), and confirmed that Myb expression was increased in miR-15a/16-1KO NK cells (9-fold in CD27+ NK cells, p<0.05). No early differentiation phenotype was observed, because Cre is expressed later, after NK cell lineage determination. In contrast, these mice lacked mature NK cells (31±4 v 62±6 %CD27- of splenic NK, p<0.01, Fig. 2). Additionally, miR-15a/16-1 overexpression in human CD34+ cells recapitulates the phenotype of Myb knockdown, establishing a direct link between miR-15/16 and Myb [Fig. 1]. Therefore, miR-15/16 controls Myb expression in a cell-intrinsic manner, and thereby directs NK cell differentiation and maturation. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Multiparametric Analysis of Host Response to Murine Cytomegalovirus in MHC Class I–Disparate Mice Reveals Primacy of Dk-Licensed Ly49G2+ NK Cells in Viral Control

MHC class I D(k) and Ly49G2 (G2) inhibitory receptor-expressing NK cells are essential to murine CMV (MCMV) resistance in MA/My mice. Without D(k), G2(+) NK cells in C57L mice fail to protect against MCMV infection. As a cognate ligand of G2, D(k) licenses G2(+) NK cells for effector activity. These data suggested that D(k)-licensed G2(+) NK cells might recognize and control MCMV infection. However, a role for licensed NK cells in viral immunity is uncertain. We combined classical genetics with flow cytometry to visualize the host response to MCMV. Immune cells collected from individuals of a diverse cohort of MA/My × C57L offspring segregating D(k) were examined before infection and postinfection, including Ly49(+) NK subsets, receptor expression features, and other phenotypic traits. To identify critical NK cell features, automated analysis of 110 traits was performed in R using the Pearson correlation, followed with a Bonferroni correction for multiple tests. Hierarchical clustering of trait associations and principal component analyses were used to discern shared immune response and genetic relationships. The results demonstrate that G2 expression on naive blood NK cells was predictive of MCMV resistance. However, rapid G2(+) NK cell expansion following viral exposure occurred selectively in D(k) offspring; this response was more highly correlated with MCMV control than all other immune cell features. We infer that D(k)-licensed G2(+) NK cells efficiently detected missing-self MHC cues on viral targets, which elicited cellular expansion and target cell killing. Therefore, MHC polymorphism regulates licensing and detection of viral targets by distinct subsets of NK cells required in innate viral control.

The Rac activator DOCK2 regulates natural killer cell–mediated cytotoxicity in mice through the lytic synapse formation

AbstractNatural killer (NK) cells play an important role in protective immunity against viral infection and tumor progression, but they also contribute to rejection of bone marrow grafts via contact-dependent cytotoxicity. Ligation of activating NK receptors with their ligands expressed on target cells induces receptor clustering and actin reorganization at the interface and triggers polarized movement of lytic granules to the contact site. Although activation of the small GTPase Rac has been implicated in NK cell–mediated cytotoxicity, its precise role and the upstream regulator remain elusive. Here, we show that DOCK2, an atypical guanine nucleotide exchange factor for Rac, plays a key role in NK cell–mediated cytotoxicity. We found that although DOCK2 deficiency in NK cells did not affect conjugate formation with target cells, DOCK2-deficienct NK cells failed to effectively kill leukemia cells in vitro and major histocompatibility complex class I–deficient bone marrow cells in vivo, regardless of the sorts of activating receptors. In DOCK2-deficient NK cells, NKG2D-mediated Rac activation was almost completely lost, resulting in a severe defect in the lytic synapse formation. Similar results were obtained when the Rac guanine nucleotide exchange factor activity of DOCK2 was selectively abrogated. These results indicate that DOCK2-Rac axis controls NK cell–mediated cytotoxicity through the lytic synapse formation.

Diverse novel functions of neutrophils in immunity, inflammation, and beyond.

Neutrophils have long been considered simple suicide killers at the bottom of the hierarchy of the immune response. That view began to change 10–20 yr ago, when the sophisticated mechanisms behind how neutrophils locate and eliminate pathogens and regulate immunity and inflammation were discovered. The last few years witnessed a new wave of discoveries about additional novel and unexpected functions of these cells. Neutrophils have been proposed to participate in protection against intracellular pathogens such as viruses and mycobacteria. They have been shown to intimately shape the adaptive immune response at various levels, including marginal zone B cells, plasmacytoid dendritic cells and T cell populations, and even to control NK cell homeostasis. Neutrophils have been shown to mediate an alternative pathway of systemic anaphylaxis and to participate in allergic skin reactions. Finally, neutrophils were found to be involved in physiological and pathological processes beyond the immune system, such as diabetes, atherosclerosis, and thrombus formation. Many of those functions appear to be related to their unique ability to release neutrophil extracellular traps even in the absence of pathogens. This review summarizes those novel findings on versatile functions of neutrophils and how they change our view of neutrophil biology in health and disease.

Soluble IP4 limits NK cell effector functions by controlling PI3K signaling (P1198)

Abstract NK cells have important functions in cancer immunosurveillance, bone marrow allograft rejection, fighting infections and reproduction. NK cell-based therapies are promising blood cancer treatments. Overcoming their currently limited efficacy requires a better understanding of the molecular mechanisms controlling NK cell development and dampening their effector functions. NK cells recognize pathogen-infected or tumor cells through invariant NK cell receptors (NKR), and then kill such stressed cells. Two second-messenger pathways downstream of NKRs are required for NK cell maturation and effector responses: PIP3-generation by PI3K, and generation of DAG and IP3 by PLCγ. IP3 plays a key signaling role by mobilizing Calcium, but can also be converted into soluble inositol(1,3,4,5)tetrakisphosphate (IP4). We and others previously showed that IP4 can act as a soluble analog of the PI3K lipid-product phosphatidylinositol(3,4,5) trisphosphate (PIP3) and control PIP3-mediated signaling protein membrane recruitment and activation in thymocytes and granulocytes. Here, we show data which suggest a novel IP4 function in promoting NK cell terminal differentiation and acquisition of a mature NKR repertoire. However, in mature NK cells, IP4 limits NKR induced IFNγ secretion, granule exocytosis and target-cell clearance, in part by inhibiting the PIP3 effector-kinase Akt. This identifies IP4 as an important novel regulator of NK cell development and function, and expands our understanding of the therapeutically important mechanisms dampening NK cell responses. Our results further suggest that PI3K regulation by soluble IP4 is a broadly important signaling paradigm.