ALBERT

Description: Phosphatase and tensin homolog (PTEN) is a critical negative regulator of the phosphoinositide-3 kinase pathway, members of which play integral roles in natural killer (NK) cell development and function. However, the functions of PTEN in NK cell biology remain unknown. Here, we used an NK cell-specific PTEN-deletion mouse model to define the ramifications of intrinsic NK cell PTEN loss in vivo. In these mice, there was a significant defect in NK cell numbers in the bone marrow and peripheral organs despite increased proliferation and intact peripheral NK cell maturation. Unexpectedly, we observed a significant expansion of peripheral blood NK cells and the premature egress of NK cells from the bone marrow. The altered trafficking of NK cells from peripheral organs into the blood was due to selective hyperresponsiveness to the blood localizing chemokine S1P. To address the importance of this trafficking defect to NK cell immune responses, we investigated the ability of PTEN-deficient NK cells to traffic to a site of tumor challenge. PTEN-deficient NK cells were defective at migrating to distal tumor sites but were more effective at clearing tumors actively introduced into the peripheral blood. Collectively, these data identify PTEN as an essential regulator of NK cell localization in vivo during both homeostasis and malignancy.

Description: Natural killer (NK) cells mediate anti-AML responses and previously published clinical trials of adoptive allogeneic NK cell therapy provide proof-of-principle that NK cells may eliminate leukemia cells in patients. However, complete remissions occur in 30-50% of patients with active AML and are typically of limited duration. Thus, improvements are needed for this promising cellular immunotherapy strategy. Following paradigm-shifting studies in mice, it was established that human NK cells exhibit an innate 'memory-like' responses following a brief, combined pre-activation with IL-12, -15, and -18 (Romee R et. al., Blood, 2012). These long-lived memory-like NK cells have an enhanced ability to produce IFN-g in response to restimulation with cytokines or activating receptor ligation, even following extensive proliferation. We hypothesized that memory-like NK cells exhibit enhanced responses to myeloid leukemia. Compared to control NK cells from the same donor, IL-12/15/18-induced memory-like NK cells produced significantly increased IFN-g upon co-culture with primary AML blasts in vitro (P

Description: 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

Description: Introduction Phosphatase and tensin homolog (PTEN) is the principal negative regulator of the PI3-kinase pathway, members of which are essential for regulating natural killer (NK) cell activation and effector functions (Tassi et al Immunity 2007, Kim et al Blood 2007). However, the role of PTEN in NK cell biology remains unknown, in part hampered by the embryonic lethality of global PTEN loss. Thus, we hypothesized that disruption of PTEN would uniquely impact NK cell developmental and functional pathways. To evaluate whether PTEN was required for normal NK cell functions, we generated and evaluated a mouse model of NK cell-specific PTEN deficiency (Ncr1iCre knockin x PTENflox; PTENΔ/Δ). Results In contrast to T and B lymphocytes with conditional PTEN loss, we discovered that PTEN primarily acts to regulate NK cell distribution, but not their development. PTEN deletion resulted in a significant loss of NK cells (Fig. 1) in the bone marrow (48% reduction, p

Description: Abstract 357 NK cells are innate immune lymphocytes important for early host defense against infectious pathogens and malignant transformation. MicroRNAs (miRNAs) are small regulatory RNAs that control a wide variety of cellular processes by specific targeting of mRNA 3'UTRs. The Dicer1 gene encodes a conserved enzyme essential for miRNA processing, and Dicer1 deficiency leads to a global defect in miRNA biogenesis. While miRNA expression and regulation of adaptive T and B lymphocytes are well established, their role in the regulation of NK cell biology remains unclear. We postulated that miRNAs serve an essential role in orchestrating NK cell development and activation. To test this hypothesis, we combined lymphocyte-restricted hCD2-Cre transgenic, Rosa26-YFP-Cre-reporter, and Dicer1 ‘floxed' mice. In this model, 25–50% of Dicer1 wt/wt NK cells are YFP+ marking expression of Cre. As expected, YFP+ NK cells from Dicer1 fl/fl and fl/wt mice were confirmed to excise Dicer1, and exhibit decreased total miRNA content based on Nanostring profiling and real-time qPCR (Dicer1 fl/fl: P

Description: Natural killer (NK) cells are lymphocytes that play an important role in the immune response to infection and malignancy. Recent studies in mice have shown that stimulation of NK cells with cytokines or in the context of a viral infection results in memory-like properties. We hypothesized that human NK cells exhibit such memory-like properties with an enhanced recall response after cytokine preactivation. In the present study, we show that human NK cells preactivated briefly with cytokine combinations including IL-12, IL-15, and IL-18 followed by a 7- to 21-day rest have enhanced IFN-γ production after restimulation with IL-12 + IL-15, IL-12 + IL-18, or K562 leukemia cells. This memory-like phenotype was retained in proliferating NK cells. In CD56dim NK cells, the memory-like IFN-γ response was correlated with the expression of CD94, NKG2A, NKG2C, and CD69 and a lack of CD57 and KIR. Therefore, human NK cells have functional memory-like properties after cytokine activation, which provides a novel rationale for integrating preactivation with combinations of IL-12, IL-15, and IL-18 into NK cell immunotherapy strategies.

Description: Abstract 3291 Introduction. How NK cells acquire enhanced functionality against infection or malignancy remains an important aspect of NK cell biology. Recent studies in mice have shown that activation of NK cells can lead to memory-like properties. The NK cell memory-like response is characterized by an initial activation event, a subsequent return to the resting state, and then enhanced functionality (e.g. IFN-γ production) upon re-stimulation. The discovery of memory-like NK cells has important implications for NK cell-based therapeutics by providing new strategies to enhance and prolong NK cell responses in patients. We hypothesized that cytokines can induce human memory-like NK cells with enhanced ‘recall’ function. Methods. Normal donor PBMCs, purified NK cells (〉95% purity) and flow-sorted NK cell subsets (〉99% purity) were cultured with low dose (1 ng/mL) IL-15 alone (control) or activated with combinations of IL-12 (10 ng/ml), IL-15 (100 ng/ml) and IL-18 (50 ng/ml) or by cross-linking CD16 (plate-bound anti-CD16 mAb) ± cytokines for 16 hours. After washing, the cells were then cultured for an additional 7–42 days in low dose IL-15 (to support survival). Following this prolonged rest period in vitro, NK cell responses (IFN-γ, degranulation) were assessed after 6-hour re-stimulation with cytokines or K562 leukemia cells. For proliferation experiments, NK cells were labeled with CFSE (5 uM, Sigma) for 5 minutes. Results. Initial stimulation with IL-12 + IL-18 for 16 hours resulted in the majority of NK cells producing IFN-γ protein, returning to baseline with no spontaneous IFN-γ production after several days of rest. However, after 7 days of rest the IL-12 + IL-18 pre-activated NK cells exhibited significantly increased IFN-γ response upon re-stimulation with IL-12 + IL-15 compared to controls, by both CD56bright (68 ± 6% vs. 38 ± 5%, p

Description: 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 (〉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〈 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.

Description: Introduction Natural 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 Conclusions In 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

Description: Natural killer (NK) cells are innate lymphoid cells that mediate anti-tumor responses via cytotoxicity and effector cytokine production. Human NK cells are divided into two subsets based on relative expression of CD56 (CD56bright and CD56dim) that classically participate in distinct functions. Cytotoxic CD56dim NK cells respond to tumor targets without prior stimulation, resulting in target cell death and transient secretion of effector cytokines (e.g. IFN-γ). In contrast, immunoregulatory CD56bright NK cells secrete abundant IFN-γ and other cytokines in response to cytokine receptor stimulation, but respond minimally to tumor target-based triggering. As a result of this dichotomy, translational strategies to enhance NK cell function for cancer immunotherapy have focused exclusively on the CD56dim subset. Based upon studies in mouse NK cells, we hypothesized IL-15 priming would enhance CD56bright anti-tumor functionality. Primary human NK cells from healthy donors were purified (〉95% CD56+CD3-), cultured overnight in medium alone (control) or medium with 5 ng/mL rhIL-15 (primed), washed, and assayed for anti-tumor responses. IL-15 priming significantly enhanced multiple CD56bright NK cell functional responses to the prototypical AML target cell line K562 (CD107a+: control 20% vs. primed 59%, p