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Acetaminophen (APAP)-induced liver injury in humans is associated with robust coagulation cascade activation and thrombocytopenia. However, it is not known whether coagulation-driven platelet activation participates in APAP hepatotoxicity. Here, we found that APAP overdose in mice caused liver damage accompanied by significant thrombocytopenia and accumulation of platelets in the liver. These changes were attenuated by administration of the direct thrombin inhibitor lepirudin. Platelet depletion with an anti-CD41 antibody also significantly reduced APAP-mediated liver injury and thrombin generation, indicated by the concentration of thrombin-antithrombin (TAT) complexes in plasma. Compared with APAP-treated wild-type mice, biomarkers of hepatocellular and endothelial damage, plasma TAT concentration, and hepatic platelet accumulation were reduced in mice lacking protease-activated receptor (PAR)-4, which mediates thrombin signaling in mouse platelets. However, selective hematopoietic cell PAR-4 deficiency did not affect APAP-induced liver injury or plasma TAT levels. These results suggest that interconnections between coagulation and hepatic platelet accumulation promote APAP-induced liver injury, independent of platelet PAR-4 signaling. Moreover, the results highlight a potential contribution of nonhematopoietic cell PAR-4 signaling to APAP hepatotoxicity.

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 In response to IL-15, we observed selective activation of the PI3K/Akt/mTOR (4.2 fold increase CD56bright NK cells, 1.2 CD56dim NK cells, p Collectively, these results suggest that CD56bright NK cells play an under-appreciated anti-tumor role in settings of abundant IL-15, such as following lymphodepleting chemotherapy, during preparation for stem cell transplantation, at sites of inflammation, or after exogenous IL-15 administration. Since CD56bright NK cells have different in vivo tissue localization (secondary lymphoid organs), distinct inhibitory, activating, and chemokine receptor expression compared to CD56dim NK cells, and are thought to be the most abundant NK cell subset when considering all human tissues, this study identifies a promising NK cell subset to harness for cancer immunotherapy. Disclosures No relevant conflicts of interest to declare.

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.

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 To determine whether this aberrant localization could be attributed to dysregulated migration, we examined NK cell trafficking between lymphoid organs. PTEN-deficient NK cells egress more efficiently from the bone marrow and preferentially reside in sinusoidal compartments (mean sinusoidal fraction: 19% [control] vs. 34% [PTENΔ/Δ], p Given the inappropriate localization without PTEN, we further evaluated the NK cell requirement for PTEN during an anti-lymphoma response. PTENΔ/Δ mice challenged i.p. with the NK-sensitive RMA/S lymphoma had defective expansion of the peritoneal compartment (absolute peritoneal NK cells at 48 hours: 1.9x105 vs. 7.2x104, p=0.04). Furthermore, using an adoptive transfer model that requires NK cell trafficking to distal sites of lymphoma challenge, we found that PTEN-deficient NK cells had significant defects in their recruitment to localized tumors (18.4-fold vs 1.5-fold increase in recruited NK cells, p Conclusions In this study, we describe the first report of NK-cell intrinsic PTEN loss in vivo. Collectively, our data suggests that unopposed PI3K signaling in NK cells dominantly affects key events responsible for appropriate cell trafficking and distribution, which is distinct from the role of PTEN in related lymphocyte lineages. These data implicate PTEN as a critical mediator of NK cell recruitment to sites of lymphoma and suggest that PTEN dysregulation, as in the case of PTEN loss-of-function mutations and hamartoma tumor syndromes, may result in defective NK cell-mediated immunity. Figure 1 NK-specific PTEN-deficient mice re-distribute NK cells among NK cell compartments. Figure 1. NK-specific PTEN-deficient mice re-distribute NK cells among NK cell compartments. Figure 2 Inappropriate NK cell retention in the blood contributes to the NK cell re-distribution observed in PTENΔ/Δ mice. Blood mononuclear cells were isolated, i.v. transferred into WT recipients and sacrificed after 16 hours. Figure 2. Inappropriate NK cell retention in the blood contributes to the NK cell re-distribution observed in PTENΔ/Δ mice. Blood mononuclear cells were isolated, i.v. transferred into WT recipients and sacrificed after 16 hours. Figure 3 Intravenous adoptively transferred PTENΔ/Δ NK cells are unable to migrate to peritoneal lymphoma. Control or PTENΔ/Δ NK cells were i.v. transferred with RMA/S challenge i.p. into RAG2-/-γc-/- hosts. Peritoneal exudate cells (PECs) were isolated after 48 hours. Figure 3. Intravenous adoptively transferred PTENΔ/Δ NK cells are unable to migrate to peritoneal lymphoma. Control or PTENΔ/Δ NK cells were i.v. transferred with RMA/S challenge i.p. into RAG2-/-γc-/- hosts. Peritoneal exudate cells (PECs) were isolated after 48 hours. Disclosures No relevant conflicts of interest to declare.

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 Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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 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 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 Disclosures: No relevant conflicts of interest to declare.

Background NK cells are innate lymphocytes that are important for host defense against infections, and are potent anti-cancer immune effectors. In peripheral blood, human NK cells are categorized into developmentally related, but functionally distinct, subsets. CD56dim NK cells, thought to be developmentally more mature, are the major subset in peripheral blood (80-95%), express perforin and granzyme B at rest and exhibit degranulation, cytotoxicity and IFN-γ responses against tumor targets without prior stimulation. In contrast, CD56bright NK cells, are less mature, are the major subset in secondary lymphoid tissues, lack expression of perforin and granzyme B and are associated with minimal degranulation, cytotoxicity, and IFN-γ responses to tumor targets. IL-15 has been shown to support the survival and proliferation of CD56bright NK cells, but its impact on the anti-leukemia response has not been reported. Here we investigate the impact of brief exposure to human recombinant IL-15 on functional responses of CD56bright and CD56dim NK cells to leukemia target cells including primary AML blasts. Methods Normal human donor NK cells (>95% purity) were cultured with cytokine free media (control) or with 5 ng/ml of rhIL-15 (primed) for 16 hours, washed and then tested for functional responses after co-incubation with K562 cells or primary AML blasts for 6 hours. NK cell functional responses assessed include degranulation, cytokine production and cytotoxicity (using flow based killing assays). For the tumor:nk cell conjugate analyses, pre-stained NK cells were co-incubated with CFSE labeled K562 cells and then CD56bright conjugate formation assessed by gating on CFSE+CD56+CD16- cells. Results Anti-leukemia effector functions of human NK cells are classically attributed to the CD56dim subset, however after priming for 16 hours with rhIL-15 (5 ng/mL, a concentration that stimulates via the IL-2/15Rβγc), surprisingly, we observed that IL-15-primed CD56bright NK cells exhibited significantly greater degranulation (CD107a), cytokine (IFN-γ and TNF-α) and cytotoxic responses to both K562 leukemia cells (Figure 1) and to primary AML blasts (figure 2), compared to IL-15 primed CD56dim NK cells from the same donor. Further, we found a marked increase in the expression of perforin (70 ± 5% vs. 12 ± 6%, P< 0.0001), granzyme B (64 ± 5% vs. 12 ± 2.5%, P< 0.0001), and TRAIL (89 ± 2.5% vs. 6 ± 1%, P< 0.0001) in IL-15 primed CD56bright NK cells. We found an increased number of tumor conjugates with the IL-15 primed, compared to control, CD56bright cells at 5 minutes (19 ± 3% vs. 3.5 ± 1%, P= 0.02), 15 minutes (22 ± 3% vs. 8 ± 2%, P= 0.0003) or at 30 minutes (13 ± 2% vs. 3.5 ± 1%, P= 0.008) from the same donors. Further, there was a significant increase in the expression of NKG2D (MFI of 8.5 ± 2 vs. 3 ± 0.5, P= 0.03), NKp30 (65 ± 4% vs. 21 ± 3%, P< 0.0001), NKp44 (57 ± 3% vs. 15 ± 3%, P< 0.0001), CD2 (MFI of 25 ± 1.5 vs. 13 ± 1, P=0.004) and LFA-1/CD11a (MFI of 45 ± 1 vs. 29 ± 2, P=0.006) in the IL-15 primed CD56bright NK cells. Due to their known role in activating anti-tumor target responses by NK cells, NKG2D, NKp44, NKp30, CD2, and LFA-1 were evaluated for a non-redundant contribution to the anti-leukemia response of IL-15 primed CD56bright NK cells. Simultaneous blockade of these receptors caused almost complete abrogation of the enhanced anti-leukemic response by the IL-15 primed CD56bright NK cells (Figure 3). Conclusions CD56bright NK cells are traditionally considered to poorly respond to leukemia targets. Here we show that stimulation with IL-15 for a few hours markedly enhances their anti-leukemia properties including degranulation and cytotoxicity, as well as IFN-γ and TNF-α production, to a level significantly exceeding CD56dim NK cells. These functional enhancements are explained by multiple mechanisms, including increased cytotoxic effector proteins (perforin, granzyme B, TRAIL), improved leukemia cell conjugation, and enhanced activation requiring LFA-1, CD2 and NKG2D. These results suggest that CD56bright NK cells may play an under-appreciated anti-tumor role in settings of abundant IL-15, such as following lymphodepleting chemotherapy, preparation for stem cell transplantation, or exogenous IL-15 administration. Disclosures: No relevant conflicts of interest to declare.

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.

Introduction Factor XIII (FXIII) deficiency is an extremely rare autosomal recessive bleeding disorder with a reported prevalence of 1 in 5 million people worldwide. The adult hemophilia clinic in St. John’s, NL, Canada manages 5 patients with severe FXIII deficiency and 1 heterozygote. 3 of the 5 severe FXIII patients are siblings. Another sister died of intracranial hemorrhage after trauma. These 3 FXIII patients were also found to have a FXII deficiency, and were described in 1970 in Pediatrics by Mary Hanna shortly after their diagnosis. All 5 of the patients in our cohort report a very strongly positive bleeding history prior to beginning prophylaxis with Fibrogammin P at a dose of 10 U/kg every 3-4 weeks. One heterozygote patient has so far had minimal bleeding. Here we report the bleeding scores of the 5 severely deficient patients prior to and after beginning prophylaxis with Fibrogammin P in 1996. A recombinant FXIII A-subunit concentrate was licensed in Canada in 2012. Therefore treating clinicians now have a choice of therapy for their patients. This product recommends prophylaxis at a dose of 35 U/kg. This product is considerably more expensive than the plasma-derived product currently used. Fibrogammin P is not currently licensed in Canada. Methods Bleeding scores are an important tool to quantify the bleeding history of a patient. The bleeding score that was used to score this FXIII cohort is the MCMDM–1VWD. A study by Bowman et al. calculated the bleeding scores of 100 healthy subjects (35 males, 65 females. These subjects were found to have a mean bleeding score of 0.16 with a standard deviation of 1.7. A bleeding score of equal to or greater than 4 is considered abnormal. Results The bleeding scores of our cohort ranged from 19 to 29 with a mean score of 23.7 prior to starting prophylaxis with Fibrogammin P. Four out of five patients reported significant umbilical cord bleeding, 1 or more episode of intracranial hemorrhage, hemarthrosis, or at least 1 major bleed post surgery prior to starting prophylaxis on Fibrogammi P. After starting Fibrogammin P, patients report very little bleeding, and score in the normal range for the period they have been on prophylaxis. These patients were exposed to hepatitis B and C, but not HIV through prior exposure to blood products. There were no further seroconversions since starting therapy with Fibrogammin P. Three of these patients underwent 7 surgeries, receiving either 10-20 U/kg of Fibrogammin either 1 or 2 days prior to surgery and 10 U/kg for 5 days post-surgery. Patients and physicians reported very satisfactory outcomes. Only one patient had a breakthrough bleed on prophylaxis. He required a transfusion of packed cells and an extra treatment of Fibrogammin P for significant rectal bleeding due to internal hemorrhoids. In this very small group of patients no clinically significant inhibitor to FXIII has been documented. Discussion Some studies reported in the literature suggest that it is important to keep FXIII levels above 10% to prevent spontaneous hemorrhage. Although our patients are doing well, we know their trough FXIII levels run from 0.2 to 0.5%. Choosing the optimal dose and optimal therapy for these patients is now more nuanced. As patients have had good outcomes to date on low dose Fibrogammin prophylaxis, switching to either high dose prophylaxis with Fibrogammin or recombinant FXIII to keep trough FXIII levels above 10% will result in approximately a 4 or 38.5-fold increase in cost respectively for prophylaxis alone. These estimations do not include any extra therapy required. On the other hand it is essential to make every effort to prevent intracranial hemorrhage. Disclosures: No relevant conflicts of interest to declare.