Enhancement Of Natural Killer Activity Research Articles

372 IPSC-Derived NK cells Exhibit Potent In Vitro and In Vivo Tumorcidal Activity Against Patient-Derived Glioblastoma Stem Cells (GSCs)

INTRODUCTION: Natural Killer (NK) cells play pivotal roles in cancer immune surveillance and harbor potent anti-tumor activities. We have engineered an induced pluripotent stem cells (iPSCs) derived, NK cell (termed FT538) that express 1) a high-affinity non-cleavable CD16 Fc receptor to facilitate antibody engagement 2) a membrane-bound IL-15/IL-15 receptor fusion to enhance NK activity and persistence and 3) a knock-out of CD38 to enhance NK metabolic fitness. FT538 is produced in a GMP-compliant manner as an off-the-shelf immunotherapy. Clinical experiences with FT538 have shown evidence of durable anti-tumor immunity after single administration in select lymphoma patients. METHODS: Laboratory characterization and murine modeling. RESULTS: Without specific antibody to engage CD16, FT538 exhibited potent in vitro activity against ∼80% of a panel of isocitrate dehydrogenase wild-type patient derived xenograft (PDX) glioblastoma lines, including MGG8. When directly injected into the murine brain, 50% of the FT538 persisted for >2 weeks without addition of exogenous cytokine. Moreover, the mice exhibited no evidence of neurologic compromise, and brains harvested after injection show no signs of FT538 activation. In vivo intratumoral injection of FT538 into an orthotopically established MGG8 glioblastoma caused an ∼20 fold increase in granzyme B release by FT538, with a comparable increase of cleaved Caspase 3 in the tumor proper. In independent experiments, >90% of mice orthotopically implanted MGG8 survived beyond 100 days after intra-tumoral FT538 while all mice treated with mock injection died by 60 days. For glioblastoma PDX refractory to FT538, such as MGG4, tumoricidal activity of FT583 can be induced in vitro and in vivo by the addition of a tri-specific engager targeting B7H3, a surface protein highly expressed in glioblastomas. CONCLUSIONS: Our results suggest that FT538 great promise as a glioblastoma therapy, with plans for translation into a first-in-human clinical trial.

Dietary Supplementation with Biobran/MGN-3 Increases Innate Resistance and Reduces the Incidence of Influenza-like Illnesses in Elderly Subjects: A Randomized, Double-Blind, Placebo-Controlled Pilot Clinical Trial.

Influenza-like illness (ILI) remains a major cause of severe mortality and morbidity in the elderly. Aging is associated with a decreased ability to sense pathogens and mount effective innate and adaptive immune responses, thus mandating the development of protective nutraceuticals. Biobran/MGN-3, an arabinoxylan from rice bran, has potent anti-aging and immunomodulatory effects, suggesting that it may be effective against ILI. The objective of the current study was to investigate the effect of Biobran/MGN-3 on ILI incidence, natural killer (NK) cell activity, and the expressions of RIG-1 (retinoic acid-inducible gene 1), MDA5 (melanoma differentiation-associated protein 5), and their downstream signaling genes ISG-15 (interferon-stimulated genes 15) and MX1 (myxovirus (influenza) resistance 1, interferon-inducible). A double-blind, placebo-controlled clinical trial included eighty healthy older adults over 55 years old, 40 males and 40 females, who received either a placebo or Biobran/MGN-3 (500 mg/day) for 3 months during known ILI seasonality (peak incidence) in Egypt. The incidence of ILI was confirmed clinically according to the WHO case definition criteria. Hematological, hepatic, and renal parameters were assessed in all subjects, while the activity of NK and NKT (natural killer T) cells was assessed in six randomly chosen subjects in each group by the degranulation assay. The effect of Biobran/MGN-3 on RIG-1 and MDA5, as well as downstream ISG15 and MX1, was assessed in BEAS-2B pulmonary epithelial cells using flow cytometry. The incidence rate and incidence density of ILI in the Biobran/MGN-3 group were 5.0% and 0.57 cases per 1000 person-days, respectively, compared to 22.5% and 2.95 cases per 1000 person-days in the placebo group. Furthermore, Biobran/MGN-3 ingestion significantly enhanced NK activity compared to the basal levels and to the placebo group. In addition, Biobran/MGN-3 significantly upregulated the expression levels of RIG-1, MDA5, ISG15, and MX1 in the human pulmonary epithelial BEAS-2B cell lines. No side effects were observed. Taken together, Biobran/MGN-3 supplementation enhanced the innate immune response of elderly subjects by upregulating the NK activity associated with reduction of ILI incidence. It also upregulated the intracellular RIG-1, MDA5, ISG15, and MX1 expression in pulmonary epithelial tissue cultures. Biobran/MGN-3 could be a novel agent with prophylactic effects against a wide spectrum of respiratory viral infections that warrants further investigation.

Both CD4+CD25+Foxp3+ Regulatory T Cells and CD3-CD56+ NK Cells Affect the Response of Newly Diagnosed Chronic Myeloid Leukemia Patients to Treatment with a Tyrosine Kinase Inhibitor

Background: Regulatory T cells (Treg) play a role in the maintenance of chronic myeloid leukemia (CML). In contrast, natural killer (NK) cells have spontaneous cytolytic activity against various tumor cells, including leukemic cells, and play an important role in immune surveillance against tumor cells. In this study, we examined changes in the number of circulating Treg and NK cells and the NK activity of patients with CML before and after treatment with a tyrosine kinase inhibitor (TKI). Our goal was to evaluate the clinical significance of this treatment on these two different cell types.Patients and Methods: Treg and NK cells were characterized and quantified by flow cytometry in 13 newly diagnosed CML patients, 5 newly diagnosed patients with other myeloproliferative neoplasms (MPN), and 15 healthy controls. We analyzed all CML patients diagnosed with chronic phase (CP) disease and MPN patients from January 2015 and June 2016. Patients with CML, who were initially treated with TKI between January 2015 and June 2016 and were followed up for more than one year, were enrolled in the study. The mRNA level of the BCR-ABL fusion gene was analyzed by real-time PCR analysis at approximately 6-month intervals during the first year and at 12-month intervals thereafter. After obtaining informed consent, blood was drawn from all patients at initial diagnosis and 12 months after the start of treatment. CD4+CD25+Foxp3+ Treg cells, CD3+CD4+T cells, CD3+CD8+ T cells, and CD3-CD56+ NK cells were analyzed using flow cytometry, and the absolute numbers of these cells were calculated. The NK activity was measured by the standard 51Cr release assay at effector: target (E: T) ratios of 20:1.Results: The median age for CML patients was 65 years (range: 47-84), and 9 of the patients were male. The median period of observation was 543 days. After 12 months, 8 CML patients (MMR-group) had a major molecular response (MMR), and 5 patients (NR-group) did have not a MMR. Before treatment, the absolute number of Treg cells was significantly lower in the MMR-group than that in the NR-group (27.6 ± 10 vs. 50.0 ± 24.0 /μL, p=0.037). Similarly, the absolute number of Treg cells after treatment was significantly lower in both the MMR-group (27.3±10.3 /μL) and the MPN-group (27.6±11.5 /μL) than that in the NR-group (40.0±9.9 /μL, p=0.05, p<0.01, respectively). Treg% values in peripheral lymphocytes before treatment in the NR (7.4±2.1%) were significantly higher than that in MMR group and in the healthy group (4.7±1.7%, 5.5±1.1%, p=0.024, p=0.016, respectively). The absolute number and percentages of CD3-CD56+ NK cells before treatment as well as after treatment did not differ between each group. However, NK activity before treatment in the NR-group (15±1.4%) was significantly lower than that in MMR-group (47.2± 11.6 vs 41.7±4.1%, p<0.01, p<0.01, respectively). With time, the number of CD8+ T cells in the NR group decreased significantly from 940±570 to 348± 167/μL (p=0.040), and the Treg% increased significantly from 0.18± 0.13 to 0.84± 0.29% (p<0.01). The relative percentage of CD3+CD4+ T cells (4.7±4.4%) in the 15 CML patients increased remarkably after treatment (12.8±4.3%, p<0.01) but the absolute number did not change.Conclusions: Treg cell numbers were significantly lower in the CML patients with a MMR compared to that in the patients without a MMR. Although the number of NK cells did not change in the MMR group compared to that in the NR group, the NK activity was significantly higher. While the mechanisms for these changes in cell populations are not completely understood, it is also important to note that we have not yet studied the cellular source and production of cytokines which enhance NK activity. The data suggest that some immunological mechanisms are involved in the response to TKI. Moreover, the increased levels of NK activity in patients with MMR suggest that not only Treg cell numbers but also NK activity may have clinical importance in evaluating the effects of TKI therapy. DisclosuresNo relevant conflicts of interest to declare.

Abstract 4534: A therapeutic humanized anti-carcinoma monoclonal antibody (mAb) can enhance NK activity and target immunosuppressive regulatory T cells

Abstract Background: NEO-201 is a therapeutic IgG1 humanized mAb reactive against many different carcinomas, but not reactive against most normal tissues. No reactivity was observed with NEO-201 in subsets of hematopoietic cells except CD15+ granulocytes. Functional analysis revealed that NEO-201 is capable of engaging in ADCC and CDC to kill tumor cells. Previous studies showed that NEO-201 attenuates growth of human tumor xenografts in mice and demonstrates safety/tolerability in non-human primates with a transient decrease in neutrophils being the only adverse effect observed. A first in human clinical trial evaluating NEO-201 in adults with chemo-resistant solid tumors is ongoing at the NIH clinical Center. NEO-201 recognizes tumor-associated variants of CEACAM5 and 6. CEACAM1 is a potent inhibitor of natural killer (NK) cell function; binding between CEACAM1 on NK cells and CEACAM1 or CEACAM5 on tumor cells inhibits activation signaling by NKG2D, which prevents NK cell cytolysis and permits tumor cells to evade NK killing. Preclinical evaluation showed that NEO-201 reacts against human regulatory T cells (Tregs). This study was designed to assess NK enhancing pathway and further investigated the phenotypic and functional effects of NEO-201 on human Tregs in vitro. Materials &amp; Methods: Various human tumor cell lines were used as target cells and NK-92 cells (CEACAM1+/CD16-) were used as effectors to assess the ability of NEO-201 to block the interaction between CEACAM5 on tumor cells and CEACAM1 on NK cells in order to enhance the in vitro killing of tumor cells. Phenotypic analysis was conducted by flow cytometry for Treg markers: CD4, CD25, CD127, FoxP3, CD15s, CD45RA, CCR4, NEO-201 antigen, CEACAM5 and CEACAM6. Results: Functional analysis revealed that NEO-201 is capable of engaging enhancing NK-92 activity to kill tumor cells. Expression profiling revealed that various tumor cell lines expressed different levels of CEACAM5+ and NEO-201+ cells. Addition of NEO-201 significantly enhanced NK-92 cell cytotoxicity against highly CEACAM5+/NEO-201+ cells, suggesting that its activity is correlated with the level of CEACAM5+/NEO-201+ tumor cells. Furthermore, the % of NEO-201+ cells in the population of CD4+CD25highCD127negFoxP3+CD15s+CCR4+Tregs ranged from 60%-80%. NEO-201 mAb mediated CDC activity against Tregs. Conclusions: This study demonstrates NEO-201 can block the interaction between tumor cell CEACAM5 and NK cell CEACAM1 to reverse CEACAM1-dependent inhibition of NK killing. NEO-201 can also target and eliminate human immunosuppressive regulatory T cells (Tregs). Citation Format: Massimo Fantini, Maria Pia Morelli, Christina M. Annunziata, Philip M. Arlen, Kwong Y. Tsang. A therapeutic humanized anti-carcinoma monoclonal antibody (mAb) can enhance NK activity and target immunosuppressive regulatory T cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4534.

Enhancement of natural killer activity and IFN-γ production in an IL-12-dependent manner by a Brassica rapa L.

Certain food components possess immunomodulatory effects. The aim of this study was to elucidate the mechanism of the immunostimulatory activity of Brassica rapa L. We demonstrated an enhancement of natural killer (NK) activity and interferon (IFN)-γ production in mice that were orally administered an insoluble fraction of B. rapa L. The insoluble fraction of B. rapa L. significantly induced IFN-γ production in mouse spleen cells in an interleukin (IL)-12-dependent manner, and NK1.1+ cells were the main cells responsible for producing IFN-γ. Additionally, the results suggested that the active compounds in the insoluble fraction were recognized by Toll-like receptor (TLR) 2, TLR4, and C-type lectin receptors on dendritic cells, and they activated signaling cascades such as MAPK, NF-κB, and Syk. These findings suggest that B. rapa L. is a potentially promising immuno-improving material, and it might be useful for preventing immunological disorders such as infections and cancers by activating innate immunity.

High-Risk Leukemia: Past, Present, and Future Role of NK Cells.

Natural killer (NK) cells are a population of cytotoxic innate lymphocytes that evolved prior to their adaptive counterparts and constitute one of the first lines of defense against infected/mutated cells. Several studies have shown that in patients with acute leukemia given haploidentical hematopoietic stem cell transplantation, donor-derived NK cells play a key role in the eradication of cancer cells. The antileukemic effect is mostly related to the presence of “alloreactive” NK cells, that is, mature KIR+ NK cells that express inhibitory KIR mismatched with HLA class I (KIR-L) of the patient. A genotypic analysis detecting KIR B haplotype and the relative B content is an additional donor selection criterion. These data provided the rationale for implementing phase I/II clinical trials of adoptive infusion of either selected or ex vivo-activated NK cells, often from an HLA-mismatched donor. In this review, we provide a historical perspective on the role played by NK cells in patients with acute leukemia, focusing also on the various approaches to adoptive NK cell therapy and the unresolved issues therein. In addition, we outline new methods to enhance NK activity, including anti-KIR monoclonal antibody, bi-/trispecific antibodies linking NK cells to cytokines and/or target antigens, and CAR-engineered NK cells.

Effect of heated naringenin on immunomodulatory properties and cellular antioxidant activity.

Naringenin is one of the most popular flavonoids derived from citrus. It has been reported to be an effective anti-inflammatory compound. Citrus fruit may be used raw, cooked, stewed, or boiled. The present study was conducted to investigate the effect of thermal processes on naringenin in its immunomodulatory and cellular antioxidant activities. The effects of flavonoids on B and T cell proliferation were assessed on splenocytes stimulated or not with mitogens. However, their effects on cytotoxic T lymphocyte (CTL) and natural killer (NK) activities were assessed in splenocytes co-incubated with target cells. The amount of nitric oxide production and the lysosomal enzyme activity were evaluated in vitro on mouse peritoneal macrophages. Cellular antioxidant activity in splenocytes and macrophages was determined by measuring the fluorescence of the dichlorofluorescin (DCF). Our findings revealed that naringenin induces B cell proliferation and enhances NK activity. The highest concentration of native naringenin exhibits a significant proliferation of T cells, induces CTL activity, and inhibits cellular oxidation in macrophages. Conversely, it was observed that when heat-processed, naringenin improves the cellular antioxidant activity in splenocytes, increases the cytotoxic activity of NK cells, and suppresses the cytotoxicity of T cells. However, heat treatment maintains the anti-inflammatory potency of naringenin.

Augmentation of natural killer cell activity in vitro and in vivo by sericin-derived oligopeptides

This study investigated the effects of sericin-derived oligopeptides on natural killer (NK) activity. In vitro exposure of human peripheral blood mononuclear cells with sericin-derived oligopeptides resulted in an augmentation of NK cell activity against K562 target cells and the effects appeared to be dose-related. Experiments designed to examine whether enhanced NK activity was due to direct or indirect activation of NK cells revealed that sericin oligopeptides did not induce activity of purified NK cells, and that sericin oligopeptides augmented NK activity indirectly by inducing the production of IL-2 and IFN-γ cytokines. In in vivo experimentation where mice were orally administered with sericin oligopeptides and splenic mononuclear cells tested against YAC-1 target cells, significant increase in NK activity was obtained compared to control mice. Elevated levels of IL-2 were also evident in all oligopeptides-treated groups. As demonstrated both in vitro and in vivo, these results indicate that sericin-derived oligopeptides have efficient NK-enhancing activity and suggest the potential therapeutic applications of such oligopeptides for functional improvement of NK cells, and possibly for treatment of tumor and infectious diseases in which NK activity contributes to host defense.

Combined Treatment of Herbal Mixture Extract H9 with Trastuzumab Enhances Anti-tumor Growth Effect

Extracts from Asian medicinal herbs are known to be successful therapeutic agents against cancer. In this study, the effects of three types of herbal extracts on anti-tumor growth were examined. Among the three types of herbal extracts, H9 showed stronger anti-tumor growth effects than H5 and H11 in vivo. To find the molecular mechanism by which H9 inhibited the proliferation of breast cancer cell lines, the levels of apoptotic markers were examined. Proapoptotic markers, including cleaved PARP and cleaved caspases 3 and 9, were increased, whereas the anti-apoptotic marker Bcl-2 was decreased by H9 treatment. Next, the combined effect of H9 with the chemotherapeutic drugs doxorubicin/cyclophosphamide (AC) on tumor growth was examined using 4T1-tumor-bearing mice. The combined treatment of H9 with AC did not show additive or synergetic anti-tumor growth effects. However, when tumor-bearing mice were co-treated with H9 and the targeted anti-tumor drug trastuzumab, a delay in tumor growth was observed. The combined treatment of H9 and trastuzumab caused an increase of natural killer (NK) cells and a decrease of myeloid-derived suppressor cells (MDSC). Taken together, H9 induces the apoptotic death of tumor cells while increasing anti-tumor immune activity through the enhancement of NK activity and diminishment of MDSC.

Anti-influenza virus effects of both live and non-liveLactobacillus acidophilusL-92 accompanied by the activation of innate immunity

The antiviral effects of both a live and non-live Lactobacillus acidophilus strain L-92 (L-92) were investigated by oral administration (10 mg/mouse per d) daily for 21 d in a mouse model infected intranasally with influenza virus (H1N1). Virus titres in the lung of mice administered either live or non-live L-92 cells daily for 15 d were repressed 6 d after virus infection compared with the control group. Natural killer (NK) activity in the orally administered non-live L-92 group was higher compared with that of the control group before virus infection and on day 6. In contrast, NK activity in the live L-92 group compared with the control group was not significantly changed on both days, but was significantly higher on day 1. In contrast, live L-92 showed a greater repression of virus proliferation compared with non-live L-92, 6 d after the infection. Live L-92 decreased the number of neutrophils in the lung and suppressed lung weight, leading to the consequent deterioration of consolidation scores of the lung. These results indicated that pretreatment of live or non-live L-92 cells had protective effects against influenza virus infection. Among the measured cytokines and chemokines, eotaxin, macrophage colony-stimulating factor, IL-1b, RANTES (regulated on activation, normal T cell expressed and secreted) and interferon-a were significantly increased in the lung: IL-17 was significantly increased in Peyer’s patch of the live L-92 group compared with the control group. A mechanistic study suggested that the enhancement of NK activity in the lung caused by stimulating various antiviral cytokines and chemokines after the oral administration of L-92 cells might be important in protecting against virus infection.

Overcome the Impairment of NK Cells for Icon and Antibody Immunotherapy of Cancer

Natural killer (NK) cells play an important role in innate immunity and in mediating antibody and Icon (an antibody-like factor VII/IgG1 Fc immunoconjugate, which, to our best knowledge, was the first therapeutic agent for dual targeting of both the tumor cells and tumor angiogenic endothelial cells) for cancer immunotherapy. However, a common yet often neglected observation and challenge in antibody immunotherapy is that NK cells are often impaired in cancer patients. Here we hypothesize that the impairment of NK cells significantly contributes to host resistance to antibody immunotherapy for cancer. In order for antibody and Icon to achieve their optimal therapeutic efficacy, we briefly reviewed the current strategies to enhance NK activity, including infusion of cytokines, vaccines or NK cells, and the use of dietary supplements. Moreover, from our point of view we identified some remaining challenges and propose to combine these NK-enhancing strategies with Icon or antibody to overcome NK impairment and ultimately to optimize the efficacy of Icon and antibody immunotherapy for cancer.

Increased natural killer cell cytotoxicity and NKp30 expression protects against hepatitis C virus infection in high-risk individuals and inhibits replication in vitro

CD56(pos) natural killer (NK)/natural T (NT) cells are important innate effectors providing the first line of defense against viral infection. Enhanced NK activity has been shown to protect from human immunodeficiency virus-1 infection. However, the role played by these innate effectors in protection against or development of hepatitis C virus (HCV) infection is unknown. We characterized CD56(pos) populations in 11 injection drug users (IDUs) who remained uninfected despite being repeatedly exposed to HCV. NK profiles in exposed but uninfected (EU) individuals were compared with preinfection samples (median 90 days prior to HCV seroconversion) collected from 14 IDUs who were exposed and subsequently became infected (EI) and unexposed normal control subjects (n = 8). Flow cytometric analysis of CD56(pos) populations demonstrated that EUs had a higher proportion of CD56(low) mature (P = 0.0011) NK cells compared with EI subjects. Bead-isolated NKs ( > 90% purity) from EUs had significantly higher interleukin-2 (IL-2)-induced cytolytic activity against the NK-sensitive cell line K562 at an effector-to-target ratio of 10:1 (P < 0.0001). NKp30, a natural cytotoxicity receptor involved in NK activation, is highest on NK/NT cells in EUs relative to infected subjects. Using the JFH-1 infection system, we demonstrated that NKp30(high) cells in the absence of exogenous stimulation significantly reduce infection of hepatocytes. CD56(pos) populations in EUs are enriched for effector NKs displaying enhanced IL-2-induced cytolytic activity and higher levels of the natural cytotoxicity receptor NKp30-activating receptor. In addition, NKp30(high) cells are more effective in preventing infection of Huh-7.5 cells than their NKp30(low/neg) counterparts. These data support the hypothesis that NK cells contribute to anti-HCV defense in vivo in the earliest stages of infection, providing innate protection from HCV acquisition.

Dendritic Cell/NK Cell Interaction in RNA Virus Infection

Natural killer (NK) cells constitute a major component of the innate immune system and have key roles in early immune responses to pathogens. Although NK cells can be directly activated by pathogens, other sensor cells which recognize pathogen-associated pattern molecules are required for the full activation of NK cells. The NK-activating capacity is observed in myeloid dendritic cells (mDCs), plasmacytoid DCs, macrophages and monocytes. The tropism of the pathogen and the route of invasion influence which sensor cells participate in the NK activation. Influenza virus, measles virus and respiratory syncytial virus can infect human mDCs and induce the ligands of the NK activating receptor NKG2D. Up-regulated NKG2D ligands on mDCs contribute to the mDC-mediated NK activation. By contrast, HCV does not replicate in mDCs and is not an immunostimulatory agent against mDCs. In this case, mDCs are stimulated after detecting dsRNA in the HCV-infected apoptotic hepatocytes via TLR3 and elicit NK activation though a direct cell/cell contact. The TLR3 signal seems to lead the up-regulation of key molecules on the surface membrane of mDCs to enhance NK activity by direct linkage. Keywords: Dendritic cell, NK cell, RNA virus, hepatitis C virus, dsRNA, Toll like receptor 3

Improvement of deficient natural killer activity and delayed bactericidal activity by a thiol proteinase inhibitor, E-64-d, in leukocytes from Chediak–Higashi syndrome patients in vitro

We previously reported that administration of a potent calpain inhibitor, E-64-d, which protects protein kinase C (PKC) from proteolysis, in a mouse model of Chediak–Higashi syndrome (CHS) (beige mice), decreases its susceptibility to Staphylococcus aureus infection. In the present study, we examined the in vitro effect of E-64-d on both deficient natural killer (NK) and delayed bactericidal activities of leukocytes from six CHS patients. Our results showed that pretreatment of peripheral blood mononuclear cells (PBMCs) obtained from CHS patients with E-64-d (1 μg/ml) significantly enhanced NK activity against K562 cells. The delayed bactericidal activity of polymorphonuclear cells (PMNs) against S. aureus also showed marked improvement. This was recovered to almost normal levels when PMNs were pretreated with E-64-d (1 μg/ml). On the other hand, the same concentration of E-64-d did not affect either the NK or bactericidal activity of normal controls. In addition, we confirmed that following E-64-d treatment, the abnormal down-regulation of PKC activity after concanavalin A (Con A) stimulation was eliminated in PBMCs obtained from CHS patients. To examine whether PKC is involved in the NK cell-mediated cytolysis and bactericidal activity of PMNs, two potent PKC inhibitors, chelerythrin and GÖ6976, were used. We found that chelerythrin inhibits NK activity of normal PBMCs in a dose-dependent manner, and GÖ6976 inhibits NK activity at doses that inhibit Ca 2+-dependent PKC isozymes. These inhibitors also suppressed the bactericidal activity of PMNs against S. aureus. Taken together, our findings suggested that E-64-d improved the compromised NK and bactericidal activity of leukocytes from CHS patients by reversing the down-regulation of PKC activity.

Aureobasidium-Derived Soluble Branched (1,3-1,6) β-Glucan (Sophy β-glucan) Enhances Natural Killer Activity in Leishmania amazonensis-Infected Mice

The beta-glucans derived from yeast cell walls have been reported for having many immunomodulatory activities in vivo and in vitro. In this study, Aureobasidium-derived soluble branched (1,3-1,6) beta-glucan (Sophy beta-glucan) was checked for natural killer (NK) activity and for the production of IFN-gamma and IL-4 in Leishmania amazonensis infection. The main experiment was performed with a group of female C57BL/6 and BALB/c mice, orally supplemented with 5% of Sophy beta-glucan and infected with promastogotes of L. amazonensis (1 x 10(7)) into the footpad. Increase in the footpad thickness with time was observed in BALB/c mice in spite of the oral Sophy beta-glucan supplement, but it was less in C57BL/6 mice. The difference in overall mean footpad thickness between 'infection only' versus 'infection + glucan' groups was statistically significant (P < 0.001). High NK activity in C57BL/6 than BALB/c mice was observed in 'glucan only' group compared to the control group and also in 'infection + glucan' group compared to 'infection only' group. The difference in the NK activity among these groups was significant (P < 0.05). The IFN-gamma level increased at weeks 7 and 8 post-infection in C57BL/6 mice and was significantly high in 'infection + glucan' group compared to the 'infection only' group (P < 0.05). IL-4 levels did not increase up to detectable levels throughout the study. The results led a conclusion that Sophy beta-glucan enhances NK activity and cellular immunity in L. amazonensis-infected mice.

Lipopolysaccharide-Induced Enhancement of Natural Killer Cell Cytotoxicity

Abstract The n-6 and n-3 families of fatty acids serve as precursors in the formation of mediators observed in inflammation. Eicosanoids from the cyclooxygenase and lipoxygenase pathways have been shown to influence natural killer (NK) cell activity. In this study, rats were fed diets either deficient in essential fatty acids (EFAD), or diets that contained marine oil (15% menhaden, MEN) or safflower oil (15% safflower, SAF). Rats were then subjected to either in vivolipopolysaccaride (LPS) or a sham procedure. LPS treated animals had higher (p < 0.05) NK activity than those of the sham group. EFAD-fed animals had higher (p < 0.05) NK activity than animals fed diets containing lipids. Dietary treatment and LPS interactions were not significant, indicating that major shifts in cell lipid concentrations did not alter endotoxin-induced enhancement of NK activity. Rats fed EFAD diets had enhanced NK activity in both sham and LPS-treated animals.

Cells with natural killer activity in human rabies.

Cytotoxic lymphocyte function in 13 patients with rabies was studied by counting the number of CD56 cells and assessing natural killer (NK) cell activity. There was no significant difference in the number of killer cells between rabies patients and 31 normal controls (P greater than 0.05). Two of six non-fatal encephalitic patients due to causes other than rabies had reduced CD56 numbers. Base-line NK cell responses versus K562 cell targets did not differ among the normal control and rabies groups (P greater than 0.05). Study of the non-rabies encephalitis group showed heterogeneous results with wide variation. Significant enhancement of NK activity was seen in four rabies patients and in 10 normal control subjects tested after interferon-alpha (IFN-alpha) and IL-2. None of the four patients with encephalitis due to causes other than rabies showed such enhancement. Our results suggest that NK cells of rabies patients are not fully stimulated and that this might contribute to the virulence of rabies. The cause of this phenomenon remains unknown.

Combination chemo-immunotherapy: kinetics of in vivo and in vitro generation of natural killer cells and lymphokine-activated killer cells in the rat

Rats received a single high dose of cyclophosphamide (Cy) (150 mg/kg), followed 48 h later (on day 0) by immunization with a T cell-dependent soluble antigen, ovalbumin in Freund's complete adjuvant (FCA). The effect of this treatment on lymphoid cell subpopulations in the spleen, natural killer (NK) cell and interleukin-2 (IL-2) induced lymphokine-activated killer (LAK) cell activity was examined. Cy (with and without ovalbumin) caused a large relative increase (by day 14) in splenic OX8+, OX19- cells with NK morphology. A marked relative increase in fresh NK cell activity was noted after Cy + ovalbumin, but not consistently after Cy alone. Elevated NK activity was Cy dose- and time-dependent, was evident within 7 days post Cy/ovalbumin and persisted for at least 28 days. Pooled splenic mononuclear cells (MNC), obtained 14 days after Cy/ovalbumin, lost all cytolytic activity against YAC-1 cells when cultured in the absence of human recombinant IL-2 (rIL-2). In contrast, similarly maintained cells from normal rats displayed NK activity higher than normal 'fresh' levels. Upon culture in medium containing 500 U/ml rIL-2, however, 'augmented' NK activity was equivalent, on a per-cell basis, in both normal and Cy/ovalbumin-pretreated groups. LAK activity generated in vitro (i.e. against NK-resistant target cells) was significantly lower in the latter group, and the overall yield of cells was reduced. By day 21 after Cy/ovalbumin, augmented NK activity was significantly greater than controls, on a per-cell and total culture yield basis. Moreover, LAK activity was now similar between groups. It is concluded that the chemotherapy/immunization protocol which we have used can greatly enhance NK activity in vivo and that these cells are responsive to induction of LAK activity by IL-2 in vitro.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) counteracts the inhibiting effect of monocytes on natural killer (NK) cells.

GM-CSF is known to accelerate haematopoietic recovery following allogeneic bone marrow transplantation (BMT). In addition, it may restore and enhance both granulocyte and monocyte functions. Stimulation of monocyte functions may induce a direct or an indirect anti-leukaemic activity due to an increase of cellular cytotoxicity and production of cytokines which may result in a reduction of the relapse rate after BMT. NK cells may play a crucial role in this activity. Therefore we studied the influence of monocytes on NK activity in combination with GM-CSF. Lymphocytes and monocytes were isolated from buffy coats of healthy individuals by counterflow centrifugation elutriation (CCE). NK activity was exerted by CD3-CD56+ cell populations and could be enhanced by IL-2 incubation overnight. Incubation of CD3-CD56+ cells with GM-CSF in the presence or absence of IL-2 hardly influenced NK activity of the lymphocyte population. Low amounts of monocytes enhanced NK activity. NK activity in lymphocyte population in the presence of equivalent numbers of monocytes with or without IL-2 was strongly decreased irrespective of the effector:target ratio (ETR). This appeared not to result from sterical hindrance effects of the present number of cells. However, addition of GM-CSF abrogated the inhibition of NK activity by monocytes in the presence of IL-2. In monocyte fractions neither IL-2 nor GM-CSF yielded NK activity. Our findings indicate that GM-CSF can affect NK activity by counteracting the suppressing effects of monocytes, and hence may improve the outcome after BMT.

Anti-KIR (1-7F9): A Fully Human Monoclonal Antibody (mAb) That Blocks KIR2DL1, −2 and −3, Promoting Natural Killer (NK) Cell-Mediated Lysis of Tumor Cells In Vitro and In Vivo.

In patients with acute myeloid leukemia (AML), haplo-identical stem cell transplantation (SCT) can lead to expansion and activation of Killer Immunoglobulin-like Receptor (KIR)-HLA class I mismatched NK cells, resulting in reduced rates of leukemia relapse and no graft-versus-host disease (Ruggeri et al. Science 2002). However, this SCT is not available to the majority of AML patients who are elderly. To explore the feasibility of achieving similar NK-mediated anti-leukemia activity by a pharmacological approach, we generated fully human anti-KIR mAbs that block the interactions of inhibitory KIR2DL receptors with their HLA-C ligands, thereby enhancing NK activity. Here we describe one such therapeutic candidate anti-KIR mAb, designated 1-7F9. As distinct HLA-C allotypes are recognized by KIR2DL1 or −2/3, only mAbs that cross-react with these KIRs would be expected to work in the entire population. Hence, 1-7F9 was initially selected based on its ability to bind soluble, recombinant KIR2L1, −2 and −3. By Biacore analysis, the bivalent affinities for KIR2DL1 and −3 were 0.43 × 10−9 M and 0.025 × 10−9 M, respectively. In experimental systems and in normal human blood, 1-7F9 bound KIR2DL1, −2 and −3, and −2DS1 and −2, but not to KIR2DS3 or −4. 1-7F9 dose-dependently inhibited the binding of soluble KIR2DL1-Fc to cell surface HLA-Cw4. 1-7F9 augmented the lysis of 721.221-Cw4 B-EBV cells by an NK cell line transfected with KIR2DL1 (YTS-2DL1) from 5% lysis in absence of mAb to a maximal 55% lysis at 5 ug/ml of mAb, but did not affect lysis by KIR-negative NK cells. Lysis of PHA-stimulated blasts and primary AML blasts by autologous IL-2 activated NK cells (E:T=6:1) was 10 and 15%, respectively, in absence of mAb vs 80% and 55% in presence of 1-7F9. Incubation of IL-2 activated blood mononuclear cells with 1-7F9 resulted in expression of the activation marker CD107 on about 10% of KIR2D-positive NK cells, which increased to 20% upon addition of HLA-C-positive B-EBV targets, suggesting that 1-7F9 preferentially induces activation of NK cells in presence of transformed cells. The isotype of 1-7F9 is IgG4; accordingly, it did not cause depletion of KIR positive cells in vitro or in vivo in KIR-transgenic mice despite long-lived KIR-occupancy. As KIR are not found in mice, in vivo activity was tested in a NOD-SCID mouse model where inoculation of in vitro-expanded NK cells (80% of NK cells KIR2D-positive) and autologous human B-EBV cells (E:T=1:3) resulted in death of all mice by day 26. A single injection of 1-7F9 (125 ug/mouse) resulted in long-term survival, with 100% of treated mice alive beyond day 60; in contrast, 60 ug/mouse of the mAb was ineffective. Similarly, ex vivo pre-incubation of NK cells with 1-7F9 (37,3 ug/106 NK cells) prior to inoculation in mice resulted in elimination of the autologous transformed B cells in vivo and survival of 100% of the treated animals. These data show that 1-7F9 augments NK-mediated tumor killing in vitro and in vivo, and that it exhibits long-lived KIR binding in vivo, providing a preclinical basis for initiating phase 1 clinical trials with the mAb.