Binding Of Natural Killer Cells Research Articles

P482: NOVEL BISPECIFIC INNATE CELL ENGAGER AFM28 FOR THE TREATMENT OF CD123 POSITIVE ACUTE MYELOID LEUKEMIA AND MYELODYSPLASTIC SYNDROME

Background: Novel treatments for patients with acute myeloid leukemia (AML) and high-risk myelodysplastic syndrome (HR-MDS) with relapsed or refractory (R/R), or measurable residual disease are urgently needed. Targeting of the surface antigen CD123 expressed on both leukemic blasts and stem cells (LSCs) of most patients allows for depletion of both compartments, which is considered a prerequisite for deep anti-tumor responses and induction of prolonged remission. AFM28 is a novel bispecific Innate Cell Engager (ICE®) designed for bivalent high-affinity binding of CD16A on natural killer (NK) cells, redirecting effector cell cytotoxicity to CD123+ tumor cells. Allogeneic NK cell therapy is emerging as a next-generation treatment with demonstrated activity in R/R AML and a very benign safety profile. Retargeting of NK cells with AFM28 may be a particularly effective treatment strategy in patients with CD123+ AML and HR-MDS. Aims: 1. Preclinical characterization of AFM28 pharmacology and toxicology in vitro and in vivo to support clinical development. 2. Assessment of binding and activation of NK cells by AFM28 to test suitability for combination development with allogeneic NK cell therapy. Methods: Flow cytometry (FC) was used to assess binding and retention of AFM28 on NK cells, NK cell activation (degranulation, IFNγ expression) and depletion of leukemic cells from AML and MDS patient specimens. Quantification of secreted cytokines in leukocyte cultures employed bead-based luminex methodology. In vivo efficacy data were acquired by BLI measurement from a murine AML model. Cynomolgus data were acquired from non-GLP dose range finding and a GLP toxicology study using standard methods (ELISA, IHC, FC). Results: AFM28 induced NK cell activation at low picomolar concentrations and mediated efficacious depletion of CD123+ tumor cell lines and primary leukemic cells, including cells with low CD123 expression. NK cell activation was induced more potently than by an effector-enhanced IgG1 and was accompanied by more pronounced degranulation and IFNγ production. Similarly, AFM28 induced depletion of leukemic cells in patient bone marrow samples, without lysis of CD34+/CD123− cells, suggesting sparing of healthy hematopoietic progenitors. Potent anti-tumor activity was confirmed in vivo in a murine tumor model. In preclinical toxicology models, AFM28-induced target cell depletion was associated with minimal release of inflammatory cytokines, including IL-6, TNFα and IFNγ from leukocytes in vitro, and only low-level release of IL-6 in cynomolgus monkeys, despite efficacious depletion of CD123+ basophils and plasmacytoid dendritic cells (pDCs). Characterization of NK cell binding revealed high-avidity interaction, long cell surface retention, and potent induction of NK cell-mediated tumor cell lysis with no impact on NK cell viability. Summary/Conclusion: These data demonstrate that AFM28 exhibits potent pharmacological activity in vitro and in vivo and efficaciously induces NK cell-mediated depletion of primary leukemic cells. Further, toxicology data suggest good tolerability with a low risk of cytokine release syndrome. Together, these findings support the clinical investigation of AFM28 as a potential novel treatment of CD123+ AML and HR-MDS. A first-in-human trial investigating safety and activity of single agent AFM28 in patients with R/R AML is currently in preparation. In addition, these data support the combination of AFM28 with allogeneic NK cell therapy, which holds potential as an effective, novel treatment strategy.

Assessment of FcgRIIIA single nucleotide polymorphisms (SNPs) on the efficacy of IgG1 monoclonal antibodies (mAbs) in PANGEA study patients (pts) with advanced gastroesophageal adenocarcinoma (aGEA).

4526 Background: Targeted therapies (Ttx) have had limited efficacy in aGEA. The phase IIa PANGEA trial assessed the outcomes of pts treated with IgG1 mAbs targeting receptor tyrosine kinases (RTKs) or PD-1 based on predefined molecular groups. The fragment C (Fc) portion of mAbs binds to IgG receptors (FcgR) of immunologic effector cells such as natural killer (NK) cells, leading to antibody-dependent cell-mediated cytotoxicity (ADCC). The FcgR subclass, FcgRIIIA, has genetic variants with different Fc binding affinities. A single nucleotide polymorphism (SNP) in FcgRIIIA substitutes phenylalanine (F) with valine (V) at amino acid position 158, enhancing FcgR’s affinity for the IgG1 Fc domain. Pts with V/V or V/F FcgRIIIA allotypes have enhanced NK cell binding affinity compared to the homozygous F/F allotype. We evaluated the association of FcgRIIIA SNPs on Ttx outcomes amongst PANGEA pts and another cohort of aGEA pts treated with IgG1 mAbs. Methods: Whole-blood samples were identified from aGEA pts (N = 104), including 70/80 available PANGEA pts, who were treated with an IgG1 mAb (trastuzumab 24, anti-EGFR 21, anti-PD1 30, ramucirumab 48) in at least 1 Ttx line. After lymphocyte DNA extraction, FcgRIIIA genotyping was performed. The Cox proportional hazard model and log-rank tests, adjusted for pt age, were used to assess for an association of genotype with overall survival (OS). Results: Of 104 genotyped pts, the F/F, F/V & V/V genotypes were observed at a frequency of 32%, 51% and 17% respectively. There was no significant difference in median OS (mOS) between the F/F, F/V or V/V or comparing F/F vs V/F+V/V overall, nor in the PANGEA-only cohort. A trend of increased mOS was seen in 20 non-PANGEA pts harboring F/V or V/V compared to 14 F/F pts (mOS 43.4 vs 23.1 months, HR 0.41 [0.15-1.14] p = 0.09). However, 3-year OS rates trended higher in V/F+V/V pts (22%, 16/71) compared to F/F pts (7%, 2/33) (p = 0.09). At 3 years, 50% of V/V+V/F non-PANGEA pts were alive versus 13% of F/F pts (p = 0.04), while 13% of V/V+V/F PANGEA pts were alive versus 0% of F/F pts (p = 0.32). Conclusions: Amongst pts receiving IgG1 mAbs, the high affinity V FcgRIIIA SNP enriched for a subgroup of ‘extreme responders’ alive 3 years from diagnosis. Multivariate analyses accounting for baseline characteristics in a larger number of pts are ongoing to further elucidate the role of FcgRIIIA SNPs as predictive biomarkers. These findings may have implications on IgG1 mAb ADCC optimization.

Abstract 1520: IL-6 up-regulates pd-l1 and facilitates lung cancer escape from NK cell immune function through its downstream MEK-ERK signaling

Abstract Background: We previously reported that IL-6 was associated with therapy resistance to radiotherapy and cisplatin chemotherapy, the two most common treatments for non-small cell lung cancer (NSCLC). Emerging in vivo evidence shows that natural killer (NK) cells have an important role in immune defense. In this study, we examined the role of IL-6 modulation in lung cancer PD-L1 expression and the impact on NK cell cytotoxicity. We also revealed the key molecular signaling pathway involved in this process, which serves as the potential therapeutic target to improve clinical lung cancer treatments. Methods: We examined the prevalence of IL-6 in tumors of NSCLC in immunohistochemical staining. The susceptibility of IL-6 expressing scramble control (A549sc and H157sc) cells and IL-6 knocked down (A549siIL-6 and H157siIL-6) cells were investigated in in vitro and in vivo human tumor xenograft studies in mice. In in vivostudies, tumors were developed by luciferase tagged A549siIL-6 vs. A549sc cell injection, and primary human NK cells were injected intravenously at the early stage of tumor development. IL-6 regulation of PD-L1 at the transcriptional level was investigated and NK cell binding to tumor cells was also examined. Results: We found the ubiquitous presence of IL-6 in NSCLC. We found IL-6 expressing lung cancer cells were more resistant to NK cell cytotoxicity than IL-6 knocked-down cells. In addition, we found higher expressions of programmed death receptor 1 ligand (PD-L1) in IL-6 expressing lung cancer cells than IL-6 knocked-down cells. In excised human tumor xenografts and in human NSCLC tumors, co-localization of IL-6 signaling and PD-L1 expression was observed. Furthermore, we discovered that IL-6 promoted PD-L1 expression at the transcriptional level via the regulation of its downstream MEK/Erk signaling pathway. In addition, we found that IL-6-MEK/Erk signaling also contributed to diverting NK cells away from binding to tumor cells. Conclusions and Impact: In conclusion, we found that IL-6 develops the resistance to NK cell actions by inducing PD-L1 in NSCLC cells and diverts NK cells away from tumor cells via its downstream MEK/Erk signaling. NK cell-mediated immune function plays an important role in cancer surveillance, invasion, and metastasis, but its role in lung cancer is not clear. In in vivo studies, we proved the importance of NK cell-mediated immune reaction. Our discovery of revealing the IL-6 downstream signaling, MEK/Erk, in triggering the PD-L1 up-regulation and in blocking NK cell binding with lung cancer cells is novel. Data from this investigation support that blocking MEK/Erk signaling pathway has the potential to enhance NK cell immune function to NSCLC by overcoming IL-6 mediated NK cell resistance. Citation Format: Soo Ok Lee, Rongying Zhu, Xiang Xue, Donglai Cchen, Shanzhou Duan, Mingjing Shen, Feng Chen, Ying Tsai, Peter Keng, Yongbing Chen, Yuhchyau chen. IL-6 up-regulates pd-l1 and facilitates lung cancer escape from NK cell immune function through its downstream MEK-ERK signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1520.

Abstract 5604: Novel biomarkers Ly6E/K play important role in drug resistance and tumor immune escape

Abstract Stem cell antigen Sca-1 is implicated in murine cancer stem cell biology and breast cancer models, but the role of its human homologs Ly6K and Ly6E in breast cancer are not established. Here we report increased expression of Ly6K/E in human breast cancer specimens correlates with poor overall survival, with an additional specific role for Ly6E in poor therapeutic outcomes. Increased expression of Ly6K/E also correlated with increased expression of the immune checkpoint molecules PDL1 and CTLA4, increased tumor-infiltrating T regulatory cells, and decreased natural killer (NK) cell activation. Mechanistically, Ly6K/E was required for TGFβ signaling and proliferation in breast cancer cells, where they contributed to phosphorylation of Smad1/5 and Smad2/3. Furthermore, Ly6K/E promoted cytokine-induced PDL1 expression and activation and binding of NK cells to cancer cells. Finally, we found that Ly6K/E promoted drug resistance and facilitated immune escape in this setting. Overall, our results establish a pivotal role for a Ly6K/E signaling axis involving TGFβ in breast cancer pathophysiology and drug response, and highlight this signaling axis as a compelling realm for therapeutic invention. Citation Format: Midar A. Hossiny, William R. Frazier, Noriko Steiner, Bhaskar Kallakury, Rakesh Kumar, Geeta Upadhyay. Novel biomarkers Ly6E/K play important role in drug resistance and tumor immune escape [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5604. doi:10.1158/1538-7445.AM2017-5604

Ly6E/K Signaling to TGFβ Promotes Breast Cancer Progression, Immune Escape, and Drug Resistance.

Stem cell antigen Sca-1 is implicated in murine cancer stem cell biology and breast cancer models, but the role of its human homologs Ly6K and Ly6E in breast cancer are not established. Here we report increased expression of Ly6K/E in human breast cancer specimens correlates with poor overall survival, with an additional specific role for Ly6E in poor therapeutic outcomes. Increased expression of Ly6K/E also correlated with increased expression of the immune checkpoint molecules PDL1 and CTLA4, increased tumor-infiltrating T regulatory cells, and decreased natural killer (NK) cell activation. Mechanistically, Ly6K/E was required for TGFβ signaling and proliferation in breast cancer cells, where they contributed to phosphorylation of Smad1/5 and Smad2/3. Furthermore, Ly6K/E promoted cytokine-induced PDL1 expression and activation and binding of NK cells to cancer cells. Finally, we found that Ly6K/E promoted drug resistance and facilitated immune escape in this setting. Overall, our results establish a pivotal role for a Ly6K/E signaling axis involving TGFβ in breast cancer pathophysiology and drug response, and highlight this signaling axis as a compelling realm for therapeutic invention. Cancer Res; 76(11); 3376-86. ©2016 AACR.

The anti-lung cancer activity of SEP is mediated by the activation and cytotoxicity of NK cells via TLR2/4 in vivo

Strongylocentrotus nudus egg polysaccharide (SEP) has been reported to display antitumor activity. However, the effects of SEP and its underlying mechanism in the treatment of lung cancer remain unclear, particularly with an immunodeficient mouse model of human non-small cell lung cancer (NSCLC). In the present study, we investigated the anti-lung cancer effects of SEP and its underlying mechanism of action in both Lewis lung cancer (LLC)-bearing C57/BL6J mice and human NSCLC H460-bearing nude mice. Although SEP showed no inhibitory effects on tumor cells in vitro, it markedly stimulated the percentage of CD3−NK1.1+ cells and natural killer (NK) cell cytotoxicity in the spleens of nude mice and C57/BL6J mice. In LLC-bearing mice, SEP not only inhibited tumor growth but also promoted NK-mediated cytotoxicity, the NK1.1+ cell population, and IL-2 and IFN-γ secretion. SEP significantly suppressed H460 growth in nude mice, which was abrogated by the selective depletion of NK cells via the intraperitoneal injection of anti-asialo GM-1 antibodies. Furthermore, anti-TLR2/4 antibodies blocked both SEP and NK cell binding and SEP-induced perforin secretion. SEP-induced proliferation and IFN-γ secretion by NK cells in wild type mice were partially impaired in TLR2 or TLR4 knockout mice. These results suggest that SEP-promoted NK cytotoxicity, which was partially mediated via TLR2 and TLR4, was the main contributing factor to lung cancer inhibition in vivo and that SEP may be a potential immunotherapy candidate for the treatment of lung cancer.

The effect of TBBPA on MAP3Ks in human natural killer cells

Natural killer (NK) cells are part of the innate immune system. Upon encountering a tumor cell, virally‐infected cell, or antibody‐coated cell, the NK cell releases cytotoxic granules which lead to the lysis of the target cell. Tetrabromobisphenol A (TBBPA) is an organic brominated flame retardant (BFR) known to have harmful effects on the environment. It is utilized as a heat resistant chemical in various industrial and commercial applications in order to retard the burning of textiles, buildings, plastics, among other things. Significant levels of TBBPA have been found in human serum, human milk and adipose tissue. Human exposure can result from the inhalation of polluted air and/or the ingestion of contaminated food. A previous study demonstrated that a twenty‐four hour exposure of NK cells to 5μM of TBBPA resulted in a >;95% decrease in cytotoxic function and exposure to 2.5 μM of TBBPA resulted in a 76% decrease in lytic function. TBBPA also caused a decrease in NK cell binding capacity by 70% at 5 μM. We have shown that exposureto this chemical leads to the activation of specific Mitogen‐Activated‐Protein‐Kinases(MAPKs) and MAPK‐Kinases (MAP2Ks) in human NK cells. The objective of this study is to examine whether there also exists a TBBPA‐induced activation of MAP3Ks, the upstream regulator MAP2Ks. Our results show significant activating phosphorylations of MAP3Ks after exposure of human NK cells to TBBPA.

Hexabromocyclododecane decreases tumor-cell-binding capacity and cell-surface protein expression of human natural killer cells.

Hexabromocyclododecane (HBCD) is a flame retardant that decreases the lytic function of human natural killer (NK) cells. NK cells defend against tumor cells and virally infected cells. Thus, HBCD has the potential to increase cancer incidence and viral infections. NK cells must bind to their targets for lysis to occur. Thus, concentrations of HBCD that decrease lytic function were examined for their ability to alter NK binding to tumor targets. Levels of HBCD that caused a loss of binding function were examined for effects on expression of cell surface proteins needed for binding. NK cells exposed to HBCD for 24 h, 48 h or 6 days or to HBCD for 1 h followed by 24 h, 48 h or 6 days in HBCD-free media were examined for binding function and cell surface protein expression. The results indicated that exposure of NK cells to 10 microM HBCD for 24 h (which caused a greater than 90% loss of lytic function) caused a very significant decrease in NK cell binding function (70.9%), and in CD16 and CD56 cell-surface protein expression (57.8 and 24.6% respectively). NK cells exposed to 10 microM HBCD for 1 h followed by 24 h in HBCD-free media (which caused a 89.3% loss of lytic function) showed decreased binding function (79.2%), and CD 16 expression (48.1%). Results indicate that HBCD exposures decreased binding function as well as cell-surface marker expression in NK cells and that these changes may explain the losses of lytic function induced by certain HBCD exposures.

Immune defects in families and patients with xeroderma pigmentosum and trichothiodystrophy.

Xeroderma pigmentosum (XP) is a rare autosomal recessive disease characterized by photosensitivity, a high incidence of cancer in sun-exposed portions of the skin and a reduced capacity to repair the u.v.-induced DNA damage. One of the XP mutations (XP-D) has also been identified in patients affected by trichothiodystrophy (TTD), a rare autosomal recessive disease characterized by brittle hair, mental and physical retardation, peculiar face and ichthyosis. However, in these patients there is no evidence of increased skin tumour incidence. Since an impairment of cell-mediated immunity has been proposed as a co-factor in the cancer proneness of XP patients, we investigated the involvement of immune defect(s) in five XP patients, five TTD patients, their parents, and 24 TTD relatives. We evaluated the phenotype of circulating lymphocytes, natural killer (NK) cell lytic activity, target cell binding of NK cells at single cell level and the effect of interferons (IFN) alpha and beta on NK cell activity. The relative proportion of CD3+ and CD4+ circulating lymphocytes was reduced in XP but not in TTD patients. NK cell lytic activity was decreased in XP patients and their mothers, but their fathers showed normal lytic activity. NK activity varied among TTD families: four out of five patients and their relatives presented low NK cell activity, and one family was normal. In TTD family members, NK activity increased after incubation with IFN-alpha or IFN-beta, but never reached normal values. In contrast, in XP patients and their mothers, the defect was almost completely corrected after in vitro incubation with IFN-alpha or IFN-beta. Our study indicates impaired NK lytic activity in the majority of TTD and XP patients and that this defect is present also in members of their families. In addition, XP patients present a low number of circulating T cells. These multiple abnormalities, together with DNA repair defects, could be related to the increased cancer risk in XP patients.

NK-cell activation and antibody-dependent cellular cytotoxicity induced by rituximab-coated target cells is inhibited by the C3b component of complement

AbstractAntibody-dependent cellular cytotoxicity (ADCC) and complement fixation both appear to play a role in mediating antitumor effects of monoclonal antibodies (mAbs), including rituximab. We evaluated the relationship between rituximab-induced complement fixation, natural killer (NK)–cell activation, and NK cell–mediated ADCC. Down-modulation of NK- cell CD16 and NK-cell activation induced by rituximab-coated target cells was blocked by human serum but not heat-inactivated serum. This inhibition was also observed in the absence of viable target cells. C1q and C3 in the serum were required for these inhibitory effects, while C5 was not. An antibody that stabilizes C3b on the target cell surface enhanced the inhibition of NK-cell activation induced by rituximab-coated target cells. Binding of NK cells to rituximab-coated plates through CD16 was inhibited by the fixation of complement. C5-depleted serum blocked NK cell–mediated ADCC. These data suggest that C3b deposition induced by rituximab-coated target cells inhibits the interaction between the rituximab Fc and NK-cell CD16, thereby limiting the ability of rituximab-coated target cells to induce NK activation and ADCC. Further studies are needed to define in more detail the impact of complement fixation on ADCC, and whether mAbs that fail to fix complement will be more effective at mediating ADCC.

Alteration of an essential NK cell signaling pathway by low doses of tributyltin in human natural killer cells

Tributyltin (TBT), a toxic and widespread environmental contaminant, has been shown to inhibit natural killer (NK) cell cytotoxic function significantly. Inhibition of NK cell cytotoxic function has the potential to increase viral infections and tumor growth. Upon NK cell binding to lysis-sensitive tumor cells, an intracellular pathway is activated, which generally begins with activation of non-receptor protein tyrosine kinases (PTKs) and ends with mitogen-activated protein kinase (MAPK)-mediated release of lytic granules toward the contacted target cell. In the current studies, we used a cytotoxicity assay to examine how low doses (200 nM or lower) of TBT affect cytotoxic function. Additionally, we investigated how low doses of TBT modulate the signaling pathway that dictates lytic granule exocytosis. A 1 h exposure to 200, 100, 50 and 25 nM TBT significantly decreased cytotoxic function 6 d later. We also saw significant activation of p38 and p44/42 by as low as 50 nM TBT within ten minutes of exposure. The observed activation of MAPKs, p38 and p44/42, implicated their upstream activators MAPK kinases (MAP2Ks). On examining MAP2Ks, MKK3/6 and MEK1/2, activation was seen within ten minutes. However, when the most upstream signaling molecules in this pathway, non-receptor protein tyrosine kinases (PTKs) such as Syk, ZAP-70, Pyk2 and Src were examined, no significant activation was seen. These data imply that upstream activators of MAP2Ks, MAP2K kinases (MAP3Ks), are activated by TBT exposures and/or that MAP2K phosphatases are being inhibited by TBT. Taken together, these data suggest that TBT-induced activation of MAPKs, p38 and p44/42, is caused by their upstream activators MAP2Ks, MKK3/6 and MEK1/2, respectively.

Impaired natural and CD16-mediated NK cell cytotoxicity in patients with WAS and XLT: ability of IL-2 to correct NK cell functional defect

AbstractIn this study we show that Wiskott-Aldrich syndrome protein (WASp), a critical regulator of actin cytoskeleton that belongs to the Scar/WAVE family, plays a crucial role in the control of natural killer (NK) cell cytotoxicity. Analysis of NK cell numbers and cytotoxic activity in patients carrying different mutations in the WASP coding gene indicated that although the percentage of NK cells was normal or increased, natural cytotoxicity and antibody-mediated NK cell cytotoxicity were inhibited in all patients with the classical WAS phenotype and in most patients carrying mutations associated with the X-linked thrombocytopenia (XLT) phenotype. The inhibition of NK cell-mediated cytotoxicity was associated with the reduced ability of WAS and XLT NK cells to form conjugates with susceptible target cells and to accumulate F-actin on binding. Treatment with interleukin-2 (IL-2) corrected the functional defects of NK cells by affecting their ability to bind to sensitive target cells and to accumulate F-actin. In addition, we provide information on the molecular mechanisms that control WASp function, demonstrating that binding of NK cells to sensitive targets or triggering through CD16 by means of reverse antibody-dependent cellular cytotoxicity (ADCC) rapidly activates Cdc42. We also found that WASp undergoes tyrosine phosphorylation upon CD16 or β2-integrin engagement on NK cells. (Blood. 2004;104:436-443)

Rolling adhesion of human NK cells to porcine endothelial cells mainly relies on CD49d-CD106 interactions.

Acute vascular rejection in pig-to-primate xenotransplantation involves recognition and damage of porcine (po) endothelial cells (EC) by human (hu) leukocytes, probably including natural killer (NK) cells. To study such interactions we analyzed rolling and static adhesion of hu NK cells to po EC. The effects of blocking hu and po adhesion molecules on the adhesion hu NK cells to po EC monolayers was analyzed under shear stress (10 min, 37 degrees C, 0.7 dynes/cm2) or under static conditions (10 min, 37 degrees C). All used cell populations were phenotypically characterized by flow cytometry. Blocking of CD106 on po EC or its ligand CD49d on hu NK cells decreased rolling adhesion of both fresh and activated hu NK cells by more than 75%. Masking of CD62L on fresh but not activated hu NK resulted in a 44% decrease in rolling adhesion, in line with the diminished cell surface expression of CD62L upon activation. Antibodies to CD31, CD54, CD62E, and CD62P on EC or CD11a, CD18, and CD162 on NK cells had only minor effects on rolling adhesion. The adhesion of the FcgammaRIII- hu NK cell line NK92 to po EC was inhibited by 95% after masking po CD106 whereas antibodies to po CD31, CD54, CD62E, or CD62P had no effect, thereby excluding effects of Fc-receptor-dependent binding of hu NK cells to po EC. Static adhesion of activated NK cells was reduced by approximately 60% by blocking either CD49d or CD106, by 47% by blocking CD11a, and by 82% upon simultaneous blocking of CD11a and CD49d. Interactions between hu CD49d and po CD106 are crucial for both rolling and firm adhesion of hu NK cells to po EC and thus represent attractive targets for specific therapeutic interventions to prevent NK cell-mediated responses against po xenografts.

Phenyltin Inhibition of the Cytotoxic Function of Human Natural Killer Cells

Phenyltin (PT) contamination has been reported in water, sediment, and fish. However, the role of PT in weakening human immune function mediated through natural killer (NK) lymphocytes has not been elucidated. In this study, we report the effects of in vitro exposure to triphenyltin (TPT), diphenyltin (DPT), and monophenyltin (MPT) on the function of human NK cells. Exposure to TPT (1 μM, for 1h) inhibited the tumor killing capacity of NK cells by 85%. Exposure of NK cells to DPT for 1 h (5 μM) and 24 h (1.5 μM) reduced tumor lysis by greater than 90%. A 24-h exposure of NK cells to 5 μM MPT reduced tumor lysis by greater than 80%. Assays assessing the ability of NK cells to bind to tumor cells showed that a 24-h pretreatment with TPT, DPT, or MPT reduced NK cell binding to tumor cells by greater than 50%. The toxic potential of the PTs followed the order TPT>DPT>MPT. In comparison with butyltins (BTs), in vitro effects of PTs revealed that these compounds are relatively less toxic to NK cells than BTs. The results of this study provide evidence that phenyltin compounds are immunotoxic to human NK cells under in vitro experimental conditions.

Immunotoxicity of Environmentally Relevant Concentrations of Butyltins on Human Natural Killer Cells in Vitro

The widespread environmental contamination, bio-accumulation, and toxic effects of butyltins (BTs) in wildlife is well documented, but the role of BTs in debilitating human immune function mediated through natural killer (NK) lymphocytes (a primary immune defense against tumor and virally infected cells) has not been described. In this study, we assessed the effects of in vitro exposure to a range of concentrations (encompassing environmentally relevant concentrations) of MBT, DBT, and TBT on human natural killer lymphocytes obtained from adult male and female donors. TBT inhibited the tumor-killing capacity of NK cells when the NK cells were pretreated in vitro at 200 nM for as little as 1 h. Inhibition of NK cytotoxic function ranged from 40 to greater than 90%. The toxic potential of butyltins followed the order of TBT > DBT > MBT. Conjugation assays revealed that after a 24-h exposure to TBT, there was about a 50% decrease in NK cell binding to tumor cells, indicating alteration of the NK cell receptors for tumor cells. Analysis of whole-blood samples for BTs revealed the presence of detectable concentrations of MBT, DBT, and TBT in all of the donors, indicating possible exposure of NK cells to BTs in the blood. The results of this study provide evidence that butyltin compounds significantly inhibit NK cell function and possible NK cell-mediated immunotoxic potential in humans.

Kinetics of in vitro natural killer activity against K562 cells as detected by flow cytometry.

Natural killer (NK) cells bind to K562 tumor target cells in vitro and kill them. The binding and cytotoxic activities of NK cells are tightly related to each other: degranulation of the cytotoxic effector is the basis for target cell damage and a consequence of effector-target recognition and binding. However, the two phases of NK activity, binding and killing, have always been measured separately by various methodologies and under different experimental conditions, because of the lack of a comprehensive methodology able to measure both of them at one time. Here we describe the simultaneous measurement of the binding and killing activities against K562 of resting and cytokine (IL-2 or IL-12)-stimulated NK cells by flow cytometry. NK, K562 and conjugates can be identified and measured by flow cytometry on the basis of NK mAb staining and target cells autofluorescence (Binding Plot). Within each population of the binding plot, killed targets can be identified and measured by their scatter characteristics (Cytotoxicity Plot). We show that i) the conjugate formation is enhanced in cytokine-stimulated cells, even at relatively short co-incubation times; ii) the conjugate release is also accelerated by cytokines; iii) the conjugate release is always quicker than the induction of the morphological changes in the target cell that generate its modified scattering properties.

Control of lytic function by mitogen-activated protein kinase/extracellular regulatory kinase 2 (ERK2) in a human natural killer cell line: identification of perforin and granzyme B mobilization by functional ERK2.

The signal pathways that control effector function in human natural killer (NK) cells are little known. In this study, we have identified the critical role of the mitogen-activated protein kinase (MAPK) pathway in NK lysis of tumor cells, and this pathway may involve the mobilization of granule components in NK cells upon interaction with sensitive tumor target cells. Evidence was provided by biological, biochemical, and gene transfection methods. NK cell binding to tumor cells for 5 min was sufficient to maximally activate MAPK/extracellular signal-regulatory kinase 2 (ERK2), demonstrated by its tyrosine phosphorylation and by its ability to function as an efficient kinase for myelin basic protein. MAPK activation was achieved in NK cells only after contact with NK-sensitive but not NK-resistant target cells. In immunocytochemical studies, cytoplasmic perforin and granzyme B were both maximally redirected towards the tumor contact zone within 5 min of NK cell contact with tumor cells. A specific MAPK pathway inhibitor, PD098059, could block not only MAPK activation but also redistribution of perforin/granzyme B in NK cells, which occur upon target ligation. PD098059 also interfered with NK lysis of tumor cells in a 5-h 51Cr-release assay, but had no ability to block NK cell proliferation. Transient transfection studies with wild-type and dominant-negative MAPK/ERK2 genes confirmed the importance of MAPK in NK cell lysis. These results document a pivotal role of MAPK in NK effector function, possibly by its control of movement of lytic granules, and clearly define MAPK involvement in a functional pathway unlinked to cell growth or differentiation.

IL-15 is chemotactic for natural killer cells and stimulates their adhesion to vascular endothelium.

Interleukin-15 (IL-15) is a recently described cytokine with IL-2-like stimulating activities on T lymphocytes and natural killer (NK) cells. IL-15 mediates its function through the beta- and gamma-chains of the IL-2 receptor. In this work, we have investigated the effect of IL-15 on the directional migration of NK cells in chemotaxis assays and on the ability of NK cells to bind to vascular endothelium. IL-15 (10-20 ng/mL) had chemotactic effects on freshly isolated resting NK cells as well as on long-termed IL-2-cultured NK cells. A checkerboard experiment demonstrated that migration in response to IL-15 was observed only in the presence of a positive gradient (chemotaxis). Overnight treatment of freshly isolated NK cells with IL-15 (10-20 ng/mL) augmented their binding to cultured endothelial cells (EC) in vitro, especially to resting EC. IL-15-activated NK cells bound to resting and tumor necrosis factor-activated EC by use of LFA-1/ICAM-1 and VLA-4/VCAM-1 adhesion pathways, essentially as untreated NK cells do. The fact that IL-15 increased NK cell binding to ICAM-1-transfected NIH-3T3 fibroblasts, together with the finding that IL-15 did not increase binding to extracellular matrix proteins, where the major molecules involved are VLA proteins, indicated that IL-15 primarily stimulates LFA-1-dependent adhesion. By increasing NK cell adhesion to vascular endothelium and migratory response, IL-15 is an important determinant of NK cell recruitment in tissues.

Human natural killer lymphocytes directly recognize evolutionarily conserved oligosaccharide ligands expressed by xenogeneic tissues.

In discordant xenogeneic species combinations, vascularized transplants are hyperacutely rejected, due to binding of xenoreactive natural antibodies (XNA) to selected tissues of the graft, followed by activation of the complement and coagulation cascades. A major epitope recognized by human XNA is the terminal disaccharide Gal alpha(1,3)Gal. Poorly defined, early cell-mediated events also contribute to recognition and rejection of discordant xenografts, and we have suggested a role of natural killer (NK) lymphocytes in this process. Human NK cells were used as effectors in functional assays of adhesion to and lysis of xenogeneic discordant endothelial cells in vitro. Adhesion and lysis inhibition experiments were performed using a large panel of carbohydrates, as well as F(ab')2 fragments of human XNA. COS cells transduced with the porcine alpha-galactosyltransferase were also used as targets for NK cell adhesion. We demonstrate that XNA-reactive carbohydrate epitopes expressed by xenogeneic cells, including Gal alpha(1,3)Gal, are also directly recognized by human NK cells. First, selected carbohydrates in solution displace with comparable efficiency both XNA and NK cell binding to xenogeneic endothelium; second, XNA F(ab')2 fragments selectively inhibit human NK cell adhesion to porcine endothelium, but not to human endothelium; third, unstimulated NK lymphocytes adhere selectively to COS-7 cells expressing the porcine glycosyltransferase that encodes the Gal alpha(1,3)Gal epitope. Collectively, our findings suggest that humoral and cellular components of the natural immune response against heterologous species independently evolved recognition patterns directed against overlapping carbohydrate determinants.

Expression and function of alpha 4/beta 7 integrin on human natural killer cells.

In this report we have analysed the expression and function of the alpha 4/beta 7 heterodimer in human natural killer (NK) cells. The expression of alpha 4 beta 7 is induced in NK cells upon activation as the anti alpha 4 beta 2 ACT-1 monoclonal antibody (mAb) family stained a minority of peripheral blood NK cells, whereas it strongly reacted with in vitro long-term interleukin-2 (IL-2)-activated NK cells. Incubation with ACT-1 on its F(ab) fragments induced a strong homotypic adhesion of NK cells, comparable to than stimulated by the anti-alpha 4 HPI 7 mAb. Cell cell interaction induced by the ACT-1 mAb was only prevented by another anti-alpha 4 mAb (HP2.1) that recognizes a different epitope. In alpha 4 beta 7-mediated cell aggregation the alpha 4 beta 7 heterodimer was redistributed to intercellular contact sites thus, suggesting a direct involvement of this integrin in the formation of cell clusters. In NK cells attached to Fibronectin (FN38) or vascular cell adhesion molecule-1 (VCAM-1), both alpha 4 beta 7 and alpha 4 beta 7 integrins were redistributed at the ventral cellular membrane forming discrete contact sites. The ACT-1 mAb only partially blocked NK cell binding to FN38, but in combination with the anti-beta 7 mAb LIAI 2, NK cell binding to FN38 was completely inhibited. In contrast. ACT-1 did not modify NK cell adhesion to VCAM-1 thus supporting the theory that the alpha 4 beta 7 binding sites for both ligands appear to be different. Our results indicate that upon IL-2-activation, expression of functional alpha 4 beta-integrin is induced on NK cells potentially participating in their interaction with both extracellular matrix and endothelial cells.