Natural Killer Cell Cytolytic Activity Research Articles

Natural Killer Cell Activities Against iPSCs-Derived HLA-Knockout Platelets and Megakaryocytes Reveal Perfect Rejection Profiles for Allotransfusion

Background Platelet transfusion refractoriness (PTR) due to immune factors occurs in 5-15% of thrombocytopenic patients who have received transfusions. The dominant cause of immune PTR is the production of allo-antibodies to human leukocyte antigen (HLA) class I, which is expressed on platelets. In current clinical settings, transfusion of HLA-compatible platelets is the only practical strategy, but their supply is weak due to limited donor source, gives excessive burden on specific donors, and requires increased efforts and costs. To overcome these issues, we plan to produce HLA-knockout platelets from iPSCs-derived megakaryocytes (MKs) as an alternative solution, applicable to all HLA types. However, whether they would be attacked by natural killer (NK) cells has not been well-studied. NK cells are known to show cytotoxic activity against cells downregulated for HLA class I ("missing self" theory). Therefore we assessed the interaction between HLA-knockout platelets derived from induced pluripotent stem cells (iPSCs) and NK cells in allogeneic settings.Methods and Results Immortalized megakaryocyte progenitor cell lines (imMKCLs) were previously established from iPSCs as a source of platelet production with a robust proliferation potential (Nakamura, 2014). Beta 2-microglobulin gene was knocked-out by CRISPR/Cas9 system to obtain HLA-knockout imMKCLs and platelets. NK cells were prepared from peripheral blood of eleven healthy donors.After co-cultures of NK cells and target cells for 6 hours with IL-2, we examined the NK cell cytolytic activity marker CD107, and target cell damage marker Annexin V using flow cytometry. Positive rates of both markers were not enhanced by co-culture with either HLA-expressed or HLA-knockout platelets for all donors. Furthermore, addition of platelets showed minimal effect on high cytotoxic activity of NK cells against K562 cells. In contrast, coculture of imMKCLs with NK cells resulted in higher detection of CD107 and Annexin V staining in some NK cell donors. These data suggested that platelets are immunologically inert for NK cells irrespective of class I HLA expression, while imMKCLs can be potentially attacked. Accordingly, platelets did not express NK cell activating ligands, which were expressed on imMKCLs and K562 cells.To confirm the above-mentioned results in vivo, mice were transfused with NK cells and platelets and MKs together. In our preliminary data, the circulation of platelets was not different between HLA-expressed or HLA-knockout type. In contrast, MKs were shown to be attacked in some cases.Conclusion HLA-knockout platelets evaded attacked from NK cells, while imMKCLs possessed immunogenicity to NK cells. This study provides extended experimental evidence that HLA-knockout platelets produced from a single imMKCL clone are immunologically applicable to all HLA types including majority of patients with PTR. On the other hand, contaminating imMKCLs in imMKCL-derived platelet products can be rejected by NK cells, contributing to their enhanced safety profiles. Taken together, stage of HLA-deficiency in imMKCLs as a starting material of platelet supply shall lead to industrial production of HLA universal platelets. DisclosuresNo relevant conflicts of interest to declare.

Alemtuzumab treatment alters circulating innate immune cells in multiple sclerosis.

Objective:To characterize changes in myeloid and lymphoid innate immune cells in patients with relapsing-remitting multiple sclerosis (MS) during a 6-month follow-up after alemtuzumab treatment.Methods:Circulating innate immune cells including myeloid cells and innate lymphoid cells (ILCs) were analyzed before and 6 and 12 months after onset of alemtuzumab treatment. Furthermore, a potential effect on granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)–23 production by myeloid cells and natural killer (NK) cell cytolytic activity was determined.Results:In comparison to CD4+ T lymphocytes, myeloid and lymphoid innate cell subsets of patients with MS expressed significantly lower amounts of CD52 on their cell surface. Six months after CD52 depletion, numbers of circulating plasmacytoid dendritic cells (DCs) and conventional DCs were reduced compared to baseline. GM-CSF and IL-23 production in DCs remained unchanged. Within the ILC compartment, the subset of CD56bright NK cells specifically expanded under alemtuzumab treatment, but their cytolytic activity did not change.Conclusions:Our findings demonstrate that 6 months after alemtuzumab treatment, specific DC subsets are reduced, while CD56bright NK cells expanded in patients with MS. Thus, alemtuzumab specifically restricts the DC compartment and expands the CD56bright NK cell subset with potential immunoregulatory properties in MS. We suggest that remodeling of the innate immune compartment may promote long-term efficacy of alemtuzumab and preserve immunocompetence in patients with MS.

Natural Killer Cell Receptors and Cytotoxic Activity in Phosphomannomutase 2 Deficiency (PMM2-CDG).

BackgroundPMM2-CDG is the most common N-glycosylation defect and shows an increased risk of recurrent and/or severe, sometimes fatal, infections in early life. We hypothesized that natural killer (NK) cells, as important mediators of the immune response against microbial pathogens and regulators of adaptive immunity, might be affected in this genetic disorder.ObjectiveTo evaluate possible defects on PMM2-CDG NK peripheral blood cell number, killing activity and expression of membrane receptors.MethodsWe studied fresh and activated NK cells from twelve PMM2-CDG cells. The number and expression of lymphoid surface receptors were studied by flow cytometry. The NK responsiveness (frequency of degranulated NK cells) and killing activity against K562 target cells was determined in the NK cytotoxicity assay.ResultsWe found an increase of blood NK cells in three patients with a severe phenotype. Two of them, who had suffered from moderate/severe viral infections during their first year of life, also had reduced T lymphocyte numbers. Patient activated NK cells showed increased expression of CD54 adhesion molecule and NKG2D and NKp46 activating receptors. NKp46 and 2B4 expression was inversely correlated with the expression of NKG2D in activated PMM2-CDG cells. Maximal NK activity against K562 target cells was similar in control and PMM2-CDG cells. Interestingly, the NK cell responsiveness was higher in patient cells. NKG2D and specially CD54 increased surface expression significantly correlated with the increased NK cell cytolytic activity according to the modulation of the killer activity by expression of triggering receptors and adhesion molecules.ConclusionsOur results indicate that hypoglycosylation in PMM2-CDG altered NK cell reactivity against target cells and the expression of CD54 and NKG2D, NKp46 and 2B4 activating receptors during NK cell activation. This suggests a defective control of NK cell killing activity and the overall anti-viral immune response in PMM2-CDG patients. The present work improves our understanding of the immunological functions in PMM2-CDG and possibly in other CDG-I types.

Contradicting interplay between insulin-like growth factor-1 and miR-486-5p in primary NK cells and hepatoma cell lines with a contemporary inhibitory impact on HCC tumor progression

In this study, an impaired natural killer (NK) cell cytolytic activity in 135 hepatocellular carcinoma (HCC) patients parallel to a reduced expression level of insulin-like growth factor (IGF)-1 in NK cells of HCC patients has been revealed. Ectopic expression of miR-486-5p, a direct upstream regulator of IGF-1, restored the endogenous level of IGF-1 in NK cells of HCC patients, thus augmenting its cytolytic activity against Huh7 cells in an opposite manner to the IGF-1 siRNAs. Unorthodoxly, over-expression of miR-486-5p in target hepatocytes resulted in the repression of IGF-1, suppression of Huh7 cells proliferation and viability in a similar pattern to the IGF-1 siRNAs. Therefore, this study highlights a potential role of IGF-1 in modulating cytolytic potential of NK cells of HCC patients. miR-486-5p acts in a cell-specific manner, differentially modulating IGF-1 expression in NK cells and their target hepatocytes with a contemporary inhibitory impact on HCC progression.

A heterozygous RAB27A mutation causes delayed cytolytic granule polarization and hemophagocytic lymphohistiocytosis in 2 unrelated teenagers

Abstract Frequently fatal, primary hemophagocytic lymphohistiocytosis (HLH) occurs in infancy resulting from homozygous mutations in natural killer (NK) and CD8 T cell cytolytic pathway genes. Secondary HLH presents after infancy and may be associated with heterozygous mutations in HLH genes. We report 2 unrelated teenagers with HLH and an identical heterozygous RAB27A mutation (259 G>C). The contribution of this Rab27A missense (A87P) mutation on NK cell cytotoxicity was studied by cloning it into a lentiviral expression vector prior to introduction into the human NK-92 cell line. NK cell degranulation (CD107a expression), target cell conjugation, and K562 target cell lysis was compared between mutant and wild-type (WT) transduced NK-92 cells. Polarization of granzyme B to the immunologic synapse and interaction of mutant Rab27A (A87P) with Munc13-4 were explored by confocal microscopy and proximity ligation assay (PLA), respectively. Over-expression of the RAB27A mutation had no effect on cell conjugate formation between the NK and target cells but decreased NK cell cytolytic activity and degranulation. Moreover, the mutant Rab27A protein was predicted to disrupt binding to WT Munc13-4 by crystal structure modelling, and decreased interaction of Rab27A (A87P) with WT Munc13-4 was shown by PLA in situ and by co-immunoprecipiation in vitro. Finally, Rab27A (A87P) over-expression in NK-92 cells delayed granzyme B polarization toward the immunologic synapse, as noted by confocal microscopy, with a resulting increase in interferon-γ production, a cytokine responsible for HLH. This heterozygous RAB27A mutation blurs the genetic distinction between primary and secondary HLH by contributing to HLH via a partial dominant-negative effect.

Assessment of ixekizumab, an interleukin-17A monoclonal antibody, for potential effects on reproduction and development, including immune system function, in cynomolgus monkeys.

The reproductive and developmental toxicity of ixekizumab, a selective inhibitor of interleukin-17A (IL-17A), was assessed in the following studies in cynomolgus monkeys: fertility (3-month dosing), embryo-fetal development (EFD; dosing from gestation day (GD) 20 through 139), and pre-postnatal development (PPND; dosing from GD 20 through parturition). Because IL-17A has functional roles in innate and humoral immunity, immune system modulation was evaluated in the EFD and PPND studies; immunological evaluations in infants comprised peripheral blood immunophenotyping, Natural Killer cell cytolytic activity, and T-cell-dependent antibody (IgG and IgM) primary and secondary responses to antigen challenge. Ixekizumab exposure was sustained during the dosing periods in most adult monkeys. Fetal exposure at Cesarean section (GD 140-142; EFD study) was 18-25% of maternal exposure and ixekizumab was present in infants for up to 29 weeks postpartum. There were no adverse effects attributed to ixekizumab in any study. Importantly, immune system development and maturation were unaffected.

Natural Killer p46 Controls Hepatitis B Virus Replication and Modulates Liver Inflammation.

Natural killer (NK) cells play an important role in hepatitis B virus (HBV) infection control, and are regulated by a complex network of activating and inhibitory receptors. However, NK cell activity in HBV patients remains poorly understood. The objective of this study was to investigate the phenotypic and functional characteristics of circulating NK cells in patients during different chronic hepatitis B (CHB) infection stages. We investigated NK cell phenotypes, receptor expression and function in 86 CHB patients and 20 healthy controls. NK cells were purified and NK cell subsets were characterized by flow cytometry. Cytotoxic activity (CD107a) and interferon-gamma (IFN-γ) secretion were examined, and Natural Killer p46 (NKP46) blockade and spontaneous NK cell cytolytic activity against K562, HepG2 and HepG2.215 cell lines was studied. Activating NKp46 receptor expression was higher in inactive HBsAg carriers when compared with other groups (p = 0.008). NKp46 expression negatively correlated with HBV DNA (R = -0.253, p = 0.049) and ALT (R = -0.256, p = 0.045) levels. CD107a was higher in immune-activated groups when compared with immune-tolerant groups (p = 0.039). CD107a expression was related to viral load (p = 0.02) and HBeAg status (p = 0.024). In vitro NKp46 blockade reduced NK cell cytolytic activity against HepG2 and HepG2.215 cell lines (p = 0.02; p = 0.039). Furthermore, NK cells from high viral load CHB patients displayed significantly lower specific cytolytic activity against anti-NKp46-loaded K562 targets (p = 0.0321). No significant differences were observed in IFN-γ secretion (p > 0.05). In conclusion, NKp46 expression regulates NK cell cytolytic function. NKp46 may moderate NK cell activity during HBV replication suppression and HBV-associated liver damage and may be critical for NK cell activity during CHB infection.

Naringenin enhances NK cell lysis activity by increasing the expression of NKG2D ligands on Burkitt's lymphoma cells.

Natural killer (NK) cells are capable of identifying and killing tumor cells as well as virus infected cells without pre-sensitization. NK cells express activating and inhibitory receptors, and can distinguish between normal and tumor cells. The present study was designed to demonstrate the importance of the expression level of NKG2D ligands on the Burkitt's lymphoma cell line, Raji, in enhancing NK cell cytolytic activity. Various flavonoids were used as stimulants to enhance the expression of NKG2D ligands. NK cell lysis activity against Raji was not changed by pre-treatment of Raji with luteolin, kaempferol, taxifolin and hesperetin. However, treatment of Raji with naringenin showed increased sensitivity to NK cell lysis than untreated control cells. The activity of naringenin was due to enhanced NKG2D ligand expression. These results provide evidence that narigenin's antitumor activity may be due to targeting of NKG2D ligand expression and suggests a possible immunotherapeutic role for cancer treatment.

The Progesterone (P) Receptor Antagonist Mifepristone Does Not Lower Serm Progeserone Induced Blocking Factor (PIBF) in the Presence of P

Exposure to progesterone (P), whether endogenous or exogenous, leads to a rapid rise of the immunomodulatory protein the progesterone induced blocking factor (PIBF). Exposure of natural killer (NK) cells to PIBF causes stabilization of perforin granules, thus inhibiting NK cell cytolytic activity. The circulating PIBF immunomodulatory protein is believed to cause a shift from thymic helper (TH) 1 cytokine dominance to TH2 cytokine dominance. This may help the fetal semi-allograft to escape immune surveillance.

Abstract LB-268: Entinostat increases susceptibility of osteosarcoma cells to NK cell lysis without suppressing NK cell cytolytic activity

Abstract Introduction: Osteosarcoma (OS) is the most common primary malignant tumor of the bone in pediatric patients. Lungs are the most common site of metastasis. Patients with recurrent and metastatic OS are resistant to chemotherapy and have very poor survival rates. Our goal is to find an alternate more effective therapy for OS lung metastasis. Allogeneic natural killer (NK) cells are emerging as a promising treatment strategy for cancers. Although data from our lab and other labs show that OS cells are susceptible to NK cell cytotoxicity, NK cell therapy by itself may not be effective enough to cure OS since tumor cells can escape immunotherapy. One way to augment cytotoxicity of NK cell is using HDAC inhibitors (HDACi). Several studies have shown that HDACis (including entinostat) sensitize tumor cells for cytotoxic effects of NK cells mostly by inducing expression of ligands for NK cells on tumor cells. In this study we sought to investigate whether entinostat would augment the cytotoxic effects NK cells on OS cells. Methods: In our in vitro study we investigated whether entinostat would increase expression of ligands for NK cell receptors on OS cells and as a result would enhance OS cells’ susceptibility to the cytotoxic effects of NK cells. Since the combination of NK cells and entinostat will be used in our in vivo study (a mouse model with OS lung metastasis) we determined the effect of the drug on NK cell viability, receptor expression, and cytotoxicity. NK cells were isolated from healthy donor buffy coats and were expanded ex vivo for 4 weeks in RPMI 1640 media supplemented with artificial antigen-presenting cells (K562) and IL-2. Results: We demonstrated that 2μM entinostat (≤ IC50) for 48hr upregulates expression of NK cell ligands (ULBP1, ULBP2/5/6, and MIC A/B) on OS cell lines (LM7, KRIB, CCHOSD, and CCHOSO). Upregulated ligands on OS cells are stable for at least 24hr after drug removal from the cultured media. Moreover, entinostat treatment increased susceptibility of OS cell lines to cytotoxic killing by NK cells. By using blocking antibodies for NK receptors we confirmed that NK cell cytotoxicity toward OS cells is dependent on receptor and ligand interaction. Treatment of NK cells with up to 2μM entinostat did not affect the viability of NK cells. This drug did not affect the expression of NK cell receptors (NKG2D, NKp30, NKp44, NKp46, and DNAM-1) within the first 24hr; however, after 48hr 1μM and 2μM of the drug decreased expression of receptors except DNAM-1. Pre-treatment of NK cells with entinostat did not decrease cytotoxicity of NK cell to OS cell lines. Conclusion: Our results demonstrate that entinostat immunosensitizes OS cells to NK cell lysis by inducing upregulation of ligands for NK cells on OS cells. We also show that up to 2μM treatment of entinostat for 24 hr does not have any adverse effect on NK cells. These results tell us the time point at which to administer NK cells and entinostat in combination to mice in our in vivo. These results support our hypothesis, saying that combining entinostat with NK cell therapy would augment the cytotoxicity effect of NK cells on osteosarcoma lung metastasis. Citation Format: Simin Kiany, Ling Yu, Michele Guindani, Jimin Wu, John Stewart, Eugenie S Kleinerman. Entinostat increases susceptibility of osteosarcoma cells to NK cell lysis without suppressing NK cell cytolytic activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-268. doi:10.1158/1538-7445.AM2014-LB-268

An increased level of IL-6 suppresses NK cell activity in peritoneal fluid of patients with endometriosis via regulation of SHP-2 expression.

Is the decreased natural killer (NK) cell cytolytic activity in the peritoneal fluid (PF) of endometriosis patients due to primary cytokine activity? An increased level of interleukin-6 (IL-6) in the PF of patients with endometriosis suppresses NK cell cytolytic activity by down-regulating cytolytic granule components, such as granzyme B and perforin, through the modulation of Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2) expression. Endometriosis is known to be related to a defect in NK cell cytolytic activity. Additionally, the levels of inflammatory cytokines are elevated in the PF of women with endometriosis. The effects of PF on the differentiation and functional activity of NK cells were investigated in patients with or without endometriosis, and cytokines that reduce NK cell cytolytic activity in endometriosis patients were examined. The study included women who underwent laparoscopic examination for the diagnosis of endometriosis from August 2012 to July 2013 (33 women with, and 15 women without, endometriosis). Women of reproductive age (20-40 years old) who underwent laparoscopic examination for endometriosis were included. Cytokines present in the PF were identified by enzyme-linked immunosorbent assay. The cytolytic activity of NK cells in the PF was also analyzed using a calcein-acetoxy methyl ester (AM) release assay. PF from patients with endometriosis suppressed the differentiation and cytotoxicity of NK cells compared with PF from controls (P < 0.05). Increased levels of IL-6 were also found in the PF of patients with endometriosis (P < 0.01), and IL-6 levels were negatively correlated with the cytolytic activity of NK cells (rs = -0.558, P = 0.03). Furthermore, IL-6 reduced the cytolytic activity of NK cells, concomitantly with the down-regulation of granzyme B and perforin (P < 0.05), by modulating SHP-2. Importantly, the addition of anti-IL-6 to the PF of endometriosis patients restored the activity of NK cells (P < 0.01), suggesting that IL-6 plays a crucial role in the reduction of NK cell activity in the PF of patients with endometriosis. PF contains various inflammatory cytokines in addition to IL-6 and so it is possible that other cytokines may affect the differentiation and activity of NK cells. Our results imply that the suppression of IL-6 using an anti-IL-6 antibody or soluble IL-6 receptor could rescue the impairment of NK cell activity in patients with endometriosis. This work was supported by the KRIBB Creative Research Program (KCS3051312); the STP project (DTM0111221) of the Ministry of Knowledge & Economy and the Basic Science Research Program (RBM0271312) of the National Research Foundation of Korea (NRF) from the Ministry of Education, Science & Technology. There are no conflicts of interest.

Modulation of natural killer cell antitumor activity by the aryl hydrocarbon receptor

The aryl hydrocarbon receptor (AhR) has become increasingly recognized for its role in the differentiation and activity of immune cell subsets; however, its role in regulating the activity of natural killer (NK) cells has not been described. Here, we show that AhR expression is induced in murine NK cells upon cytokine stimulation. We show that in the absence of AhR, NK cells have reduced cytolytic activity and reduced capacity to control RMA-S tumor formation in vivo, despite having normal development and maturation markers. Although AhR was first identified to bind the xenobiotic compound dioxin, AhR is now known to bind a variety of natural exogenous (e.g., dietary) and endogenous ligands. We show that activation of AhR with an endogenous tryptophan derivative, 6-formylindolo[3,2-b]carbazole, potentiates NK cell IFN-γ production and cytolytic activity. Further, administration of 6-formylindolo[3,2-b]carbazole in vivo enhances NK cell control of tumors in an NK cell- and AhR-dependent manner. Finally, similar effects on NK cell potency occur with AhR dietary ligands, potentially explaining the numerous associations that have been observed in the past between diet and NK cell function. Our studies introduce AhR as another regulator of NK cell activity in vivo.

Abstract 490: Clinical improvement of symptomatic advancing chronic lymphocytic leukemia following mifepristone therapy despite normal serum levels of the immunomodulatory protein the progesterone induced blocking factor (PIBF).

Abstract Recently a sensitive ELISA assay been created to detect serum levels of the immunomodulatory protein, the PIBF. One of the 34 kDa forms of PIBF increases markedly after exposure to progesterone (P) and inhibits natural killer (NK) cytolytic activity by stabilizing perforin granules and causes of shift from thymic helper (TH)1 to TH2 dominance thus diminishing the activity of the cellular immune system. The 34 kDa form is secreted by gamma/delta T cells and is considered a vital step in preventing immune destruction of the fetal semi-allograft. Evaluation of mRNA for PIBF and PIBF protein found that 29 of 29 human leukemia cell lines over-expressed mRNA for PIBF but only 3 of the lines expressed the PIBF protein. Adding P to the culture up-regulated PIBF protein expression whereas adding the P receptor antagonist mifepristone caused down-regulation of PIBF expression. Mifepristone treatment has been demonstrated to show significant palliative effects on a wide variety of murine and human cancers many of which are not known to be P receptor positive. The hypothesized mechanism is that these tumors either secrete PIBF or direct secretion by gamma/delta T cells in the tumor microenvironment and thus inhibit NK cell cytolytic activity. The inhibition is abrogated by inhibiting P receptors and thus a local form of P secretion by the tumor is hypothesized. The objective of this study was to determine if mifepristone will only help inhibit cancer growth in those individuals who have an increased PIBF level present in their serum. An 81 year old woman with chronic lymphocytic leukemia (CLL) started more rapid advancement and became very symptomatic. She refused chemotherapy when she almost had a lethal complication to oral chlorambucil. She requested mifepristone. Her serum PIBF level of 34.9 ng/mL was in the same range as controls without cancer. However within a month of taking 200mg daily of mifepristone orally she showed a dramatic improvement in her CLL with her white blood cell count dropping from 28,000 to 8,000. Her platelet count increased from 40,000 to 240,000 and 2 lung nodules thought to represent possible primary lung cancer (but possibly nodules from her CLL) completely disappeared on repeat CT scan. Also her energy markedly improved and her persistent cough stopped. The serum PIBF did not decrease with mifepristone therapy - 48.3 ng/mL one month later. These data demonstrated that failure to see a marked increase in serum PIBF in a patient with cancer does not predict failure to respond to mifepristone. Though mifepristone has ameliorated murine leukemia in AKR mice this is the first case report of this drug improving human leukemia. The 34-36 kDa PIBF found in cytoplasm of cancer cells may either not be secreted in the serum or may be immunologically different from serum PIBF found after P exposure. Citation Format: Jerome H. Check, Ann DiAntonio, Diane Check, Alex Jaffe, Rachael Cohen, Mojirayo Sarumi, Maya Srivastava. Clinical improvement of symptomatic advancing chronic lymphocytic leukemia following mifepristone therapy despite normal serum levels of the immunomodulatory protein the progesterone induced blocking factor (PIBF). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 490. doi:10.1158/1538-7445.AM2013-490

Bispecific and Trispecific Killer Cell Engagers Directly Activate Human NK Cells through CD16 Signaling and Induce Cytotoxicity and Cytokine Production

This study evaluates the mechanism by which bispecific and trispecific killer cell engagers (BiKEs and TriKEs) act to trigger human natural killer (NK) cell effector function and investigates their ability to induce NK cell cytokine and chemokine production against human B-cell leukemia. We examined the ability of BiKEs and TriKEs to trigger NK cell activation through direct CD16 signaling, measuring intracellular Ca²⁺ mobilization, secretion of lytic granules, induction of target cell apoptosis, and production of cytokine and chemokines in response to the Raji cell line and primary leukemia targets. Resting NK cells triggered by the recombinant reagents led to intracellular Ca²⁺ mobilization through direct CD16 signaling. Coculture of reagent-treated resting NK cells with Raji targets resulted in significant increases in NK cell degranulation and target cell death. BiKEs and TriKEs effectively mediated NK cytotoxicity of Raji targets at high and low effector-to-target ratios and maintained functional stability after 24 and 48 hours of culture in human serum. NK cell production of IFN-γ, TNF-α, granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-8, macrophage inflammatory protein (MIP)-1α, and regulated and normal T cell expressed and secreted (RANTES) was differentially induced in the presence of recombinant reagents and Raji targets. Moreover, significant increases in NK cell degranulation and enhancement of IFN-γ production against primary acute lymphoblastic leukemia and chronic lymphocytic leukemia targets were induced with reagent treatment of resting NK cells. In conclusion, BiKEs and TriKEs directly trigger NK cell activation through CD16, significantly increasing NK cell cytolytic activity and cytokine production against tumor targets, showing their therapeutic potential for enhancing NK cell immunotherapies for leukemias and lymphomas.

Human Natural Killer (NK) Cells: Differential Expression of Phosphatase and Tensin Homologue Deleted On Chromosome Ten (PTEN) During NK Cell Development Regulates Its Cytolytic Activity Against Leukemic Target Cells

AbstractAbstract 254Human NK cells are CD56(+)CD3(-) large granular lymphocytes characterized by the ability to directly kill virally infected or malignantly transformed cells. Five stages of human NK cell development can be identified in secondary lymphoid tissue. Stage 4 NK cells are immature CD56bright and have poor cytolytic activity against sensitive leukemic target cells at rest while stage 5 NK cells are mature CD56dim and have potent cytolytic activity against the same leukemic cells at rest. Both stage 4 CD56bright and stage 5 CD56dim NK cells can be found circulating in blood. We sought to determine the mechanism responsible for this different cytolytic activity by exploring changes in gene expression between CD56bright NK cells and CD56dim NK cells. We first observed that CD56bright NK cells expressed ∼5 fold greater amounts of PTEN protein over CD56dim NK cells by western blot (n=5, p < 0.04). Given that PTEN is a lipid phosphatase that opposes the PI3K/AKT pathway, we hypothesized that it may negatively regulate NK cell cytolytic activity. In order to test this, we used lentiviral infection to overexpress PTEN in the human NK cell line NK-92. Relative to cells infected with an empty lentiviral vector, NK-92 cells with overexpression of PTEN showed decreased cytotoxicity against sensitive leukemic target cells by at least 25% at all effecter:target ratios (n=4, p < 0.02). Next, we infected primary human NK cells with the same vector and showed an approximately 30% reduction in degranulation of cytolytic mediators as determined by CD107a mean fluorescent intensity (M.F.I.) when cultured with sensitive leukemic target cells (n=4, p<.08). Consistent with this, murine NK cells were isolated from FVB mice with a heterozygous germline deficiency in Pten (Pten+/−) and showed at least a 25% increase in cytotoxicity against sensitive lymphoma target cells (n=4, p<.0001). At the molecular level, when NK-92 cells overexpressing PTEN were bound to their sensitive leukemic target cells, they showed decreased activation along the AKT and ERK1/2 signaling pathways, which are known as positive regulators of NK cell cytotoxicity. Collectively, we identify a functional role for the tumor suppressor PTEN in normal human NK cell development. We demonstrate differential expression of PTEN in immature and mature human NK cells, show that it is a negative regulator of NK cell cytolytic activity, and suggest that this differential expression may in fact account for the difference in baseline cytolytic activity found in the CD56bright and CD56dim human NK cell subsets, possibly via the AKT and ERK1/2 pathways. Disclosures:No relevant conflicts of interest to declare.

Progesterone induced blocking factor (PIBF), an immunosuppressive protein that inhibits natural killer (NK) cell cytolytic activity, detected 3 days after embryo transfer (ET)

PIBF is an immunosuppressive protein found in pregnant women and is known to inhibit NK cell cytolytic activity. The objective of this study was to determine if PIBF is present at the peri-implantation time.

Importance of NKG2D-NKG2D ligands interaction for cytolytic activity of natural killer cell

In this study, we investigate the relationship between natural killer (NK) cell susceptibility and the surface markers of cancer cells. Through phenotypic analysis, we found evidence that more susceptible cancer cell lines (K562 and Jurkat) express more NKG2D ligands. Major histocompatibility complex (MHC) class I chain-related A/B (MIC-A/B) and UL16 binding protein (ULBP) 1–5 molecules are typical ligands of NKG2D. The high killing activity of NK cells against K562 was abolished through the addition of a NKG2D blocking antibody. Upon in vitro stimulation with quercetin, low susceptible cancer cells increased NKG2D ligand expression, leading to enhancement of NK cell cytolytic activity. These results suggested that the anti-cancer activity of NK cells is not dependent on the origin and growth style of the target cells, but is dependent on the surface markers of the target cells.

The histone deacetylase inhibitor, romidepsin, suppresses cellular immune functions of cutaneous T‐cell lymphoma patients

Romidepsin is the second histone deacetylase inhibitor (HDACi) approved for the treatment of advanced stages of cutaneous T-cell lymphoma (CTCL). Recent in vitro data suggest that HDACis suppress immune function although these findings have not been confirmed in patients. Thus, we serially examined the cellular immune function of eight CTCL patients undergoing treatment with three cycles of romidepsin. We measured the patients' natural killer (NK) and dendritic cell (DC) function and performed an in vitro terminal deoxynucleotidyl transferase dUTP nick end labeling assay to measure cellular apoptosis. Patients' NK cell cytolytic activity decreased from baseline to the third cycle of treatment (P = 0.018) but stimulation with a toll-like receptor (TLR) agonist increased this activity (P = 0.018). At baseline, a TLR agonist could both activate patients' DC (P = 0.043) and stimulate interleukin-12 protein production (P = 0.043) but both were suppressed after the first cycle of romidepsin. Finally, we observed increased specificity for romidepsin-induced CD4+ tumor cell apoptosis and dose-dependent increases in cellular apoptosis of healthy cells in multiple lineages (P < 0.05). These findings raise concern that HDACis suppress immune function in CTCL patients and they support the concurrent use of multiple immune stimulatory agents to preserve the host immune response.

Changes of natural killer cell number and cytolytic activity during first trimester of pregnancy in recurrent spontaneous abortion patients and fertile control

Results %NK cells among peripheral blood lymphocyte in RSA patients before pregnancy was significantly higher than that of nonpregnant normal control. NK cell cytolytic activity in non-pregnant RSA patients was significantly higher than that of non-pregnant fertile group. After success to get pregnancy, NK cell cytolytic activity in RSA patients were decreased but were not significantly different compared to that of NK cell cytolytic activity checked at non-pregnant status. NK cell cytolytic activity in pregnant RSA patients was significantly higher than that of pregnant normal control.

Polysaccharide-K augments docetaxel-induced tumor suppression and antitumor immune response in an immunocompetent murine model of human prostate cancer

Advanced castration-resistant prostate cancer has high mortality rates and limited treatment options. Novel therapies are needed to better contend with this disease. Polysaccharide-K® (PSK), an extract of the mushroom Trametes versicolor, has immunomodulatory and tumor suppressive activities. PSK is used in Asia as a cancer immunotherapy. However, its benefit in combination with taxanes for prostate cancer is unknown. We examined whether PSK would enhance docetaxel-induced apoptosis and augment anti-tumor immune responses in orthotopic tumors using transgenic adenocarcinoma of the mouse prostate (TRAMP)-C2-bearing mice. Combining PSK with docetaxel induced significantly higher tumor suppression than either treatment alone (p<0.05), including a reduction in tumor proliferation and enhanced apoptosis. Combined PSK and docetaxel treatment led to a lower decrease in number of white blood cells than docetaxel alone, an effect accompanied by increased numbers of tumor-infiltrating CD4+ and CD8+ T cells. PSK with or without docetaxel significantly enhanced mRNA expression of IFN-γ compared to control, but did not significantly alter T-regulatory FoxP3 mRNA expression in tumors. PSK also augmented docetaxel-induced splenic natural killer cell cytolytic activity against YAC-1 target cells (p=0.045). This study is the first to show that PSK enhances docetaxel-induced prostate cancer tumor suppression, apoptosis and antitumor responses.