scholarly journals Chemiluminescence response of human natural killer cells. I. The relationship between target cell binding, chemiluminescence, and cytolysis.

1982 ◽  
Vol 156 (2) ◽  
pp. 492-505 ◽  
Author(s):  
S L Helfand ◽  
J Werkmeister ◽  
J C Roder
Keyword(s):  
Tumor Cell ◽  
Nk Cells ◽  
Natural Killer ◽  
Tumor Cells ◽  
Cell Lines ◽  
Target Cell ◽  
Nk Cell ◽  
Target Antigen ◽  
Target Cells ◽  
Tumor Cell Lines

The binding of tumor cells or fetal fibroblasts to human natural killer (NK) cells led to a rapid chemiluminescence response within seconds of target-effector interaction. The degree of chemiluminescence was dependent on the concentration of NK-enriched lymphocytes or target cells, and plasma membrane vesicles from K562 also induced a chemiluminescence response. Mild glutaraldehyde treatment of effector cells abrogated their ability to generate chemiluminescence, whereas K562 target cells treated in the same way were almost fully able to induce a chemiluminescence response to NK-enriched lymphocytes. These results show a directionality of response with NK as the responders and tumor cells as the stimulators. A survey of eight different tumor cell lines and fetal fibroblast lines revealed a striking correlation (r greater than 0.93, P less than 0.001) between the ability of a given line to bind to NK-enriched lymphocytes, induce chemiluminescence, and to be lysed. Three differentiated sublines of K562 grown in butyrate and cloned induced little chemiluminescence compared with the K562 parent, and they were selectively resistant to NK-mediated binding and cytolysis. In addition, treatment of K562 cells with higher concentrations of glutaraldehyde for longer periods led to varying degrees of target antigen preservation, as measured in cold target competition assays and in conjugate formation. The degree of NK target antigen preservation correlated directly with the ability of the cells to induce chemiluminescence (r greater than 0.95). The degree of NK activation was also important because interferon-pretreated effectors generated more chemiluminescence upon stimulation with K562 or MeWo targets. Monocytes or granulocytes did not contribute to the chemiluminescence induced by NK-sensitive targets. Some NK-resistant tumor cell lines were sensitive to monocyte-mediated cytolysis and also induced chemiluminescence in monocytes but not NK cells. These results show that the target structures recognized by the NK cell may play a role in NK activation because the degree of chemiluminescence was directly proportional to the ability of a given target cell line to bind to the NK cell and to be lysed.

Expression of rat complement control protein Crry on tumor cells inhibits rat natural killer cell–mediated cytotoxicity

Blood ◽  
2002 ◽  
Vol 100 (9) ◽  
pp. 3304-3310 ◽  
Author(s):  
Theresa A. Caragine ◽  
Masaki Imai ◽  
Alan B. Frey ◽  
Stephen Tomlinson
Keyword(s):  
Tumor Cell ◽  
Nk Cells ◽  
Natural Killer ◽  
Tumor Cells ◽  
Cell Lines ◽  
Nk Cell ◽  
Human Tumor ◽  
Mammary Adenocarcinoma ◽  
Tumor Cell Lines ◽  
Human Tumor Cell

Abstract Crry is a rodent membrane–bound inhibitor of complement activation and is a structural and functional analog of the human complement inhibitors decay-accelerating factor and membrane cofactor protein. We found previously that expression of rat Crry on a human tumor cell line enhances tumorigenicity in nude rats. In this study, we investigated the effect that rat Crry expressed on tumor cells has on rat cell–mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC). The expression of rat Crry on the surface of different human tumor cell lines inhibited ADCC mediated by rat natural killer (NK) cells. C3 opsonization is known to enhance NK cell–mediated cytolysis, and a potential mechanism for Crry-mediated inhibition of NK cell lysis is through Crry modulation of C3 deposition on target cells. However, the transfection of tumor cell lines with Crry enhanced their resistance to NK cell–mediated lysis in the absence of exogenous complement. The resistance of Crry-expressing tumor cells to NK cell–mediated ADCC could be reversed by treatment with anti–Crry F(ab)2. In addition, anti–Crry F(ab)2 enhanced the susceptibility of 13762 rat mammary adenocarcinoma cells (that endogenously express Crry) to ADCC mediated by allogeneic rat NK cells in the absence of added complement. We found no evidence that rat NK cells were a source of complement for target cell deposition during the in vitro cytolysis assay. These data suggest a novel function for rat Crry in tumor immune surveillance that may be unrelated to complement inhibition.

scholarly journals Preferential Interaction of a Novel Tumor Surface Protein (p38.5) with Naive Natural Killer Cells

1997 ◽  
Vol 185 (10) ◽  
pp. 1735-1742 ◽  
Author(s):  
Ballabh Das ◽  
Mary O. Mondragon ◽  
Shi-Zhen Tao ◽  
Allen J. Norin
Keyword(s):  
T Lymphocytes ◽  
Membrane Protein ◽  
Tumor Cell ◽  
Nk Cells ◽  
Natural Killer ◽  
Cell Lines ◽  
Nk Cell ◽  
Surface Protein ◽  
Dependent Manner ◽  
Tumor Cell Lines

A receptor–ligand interaction exclusive to natural killer (NK) cell–mediated recognition and triggering of tumor cell destruction has not yet been identified. In contrast, molecules that are involved in cellular adhesion and regulation of NK cytolysis have been well studied. In this report, a novel tumor surface protein is identified that exhibits characteristics of a recognition structure for naive NK cells. A tagged ligand–cell adsorption technique revealed a 38.5-kD plasma membrane protein (p38.5) from a prototypical NK-susceptible cell line (K562) that preferentially bound to NK cells (CD3−CD5−CD16+) relative to T lymphocytes (CD3+CD5+ CD16−). The molecule was purified to apparent homogeneity for further characterization. An amino acid sequence of an 11-mer internal peptide of p38.5 did not exhibit homology to known proteins. Affinity-purified antibody generated against this peptide (anti-p38.5) reacted with a single protein of 38.5 kD on Western blots of whole cell extracts of K562. Flow cytometry and immunoprecipitation studies of surface-labeled tumor cells demonstrated expression of p38.5 on NK-susceptible tumor cell lines (K562, MOLT-4, Jurkat), whereas p38.5 was not detected on NK-resistant tumor cell lines (A549, Raji, MDA-MB-231). Significantly, p38.5 loss variants derived from wild-type Jurkat and Molt-4 cell lines exhibited decreased susceptibility to NK cell–mediated lysis demonstrating a strong association between cell surface expression of p38.5 and cytotoxicity. Purified p38.5 retained preferential binding to NK cells and inhibited NK activity in a dose-dependent manner, thereby providing direct evidence of a role in the lytic process. Binding studies identified a 70-kD membrane protein from NK cells as a possible receptor for the p38.5 tumor ligand. Consistent with cellular adsorption studies, the 70-kD, p38.5 binding protein was not detected on T lymphocytes. Based on studies demonstrating selective binding of p38.5 to NK cells, lack of expression on NK-resistant tumor cell lines and ability of the purified molecule to block cytolysis, we conclude that p38.5 may serve as a recognition/triggering ligand for naive human NK cells.

36 Molecular events regulating solid tumor cell responses to natural killer cells

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A37-A37
Author(s):  
Michal Sheffer ◽  
Constantine Mitsiades
Keyword(s):  
Tumor Cell ◽  
Antigen Presentation ◽  
Nk Cells ◽  
Tumor Cells ◽  
Cell Lines ◽  
Solid Tumor ◽  
Nk Cell ◽  
Tumor Cell Lines ◽  
Cell Responses ◽  
Genome Scale

BackgroundNatural killer (NK) cells exhibit potent activity in pre-clinical models of diverse hematologic malignancies and solid tumors and infusion of high numbers of NK cells, either autologous or allogeneic, after their ex vivo expansion and activation, has been feasible and safe in clinical studies.MethodsTo systematically define molecular features in human tumor cells which determine their degree of sensitivity to human allogeneic NK cells, we quantified the NK cell responsiveness of hundreds of molecularly-annotated ‘DNA-barcoded’ solid tumor cell lines in multiplexed format (PRISM; Profiling Relative Inhibition Simultaneously in Mixtures approach),1 correlating cytotoxicity scores for each cell line with the CCLE transcriptional data2 (RNA-seq), to reveal genes that are associated with resistance or sensitivity to NK cells. In addition, we applied genome-scale CRISPR-based gene editing screens in several solid tumor cell lines to interrogate, at a functional level, which genes regulate tumor cell response to NK cells.3 4Figure 1 schematically depicts the two screens.ResultsBased on these orthogonal studies, NK sensitive tumor cells tend to exhibit high levels of the NK cell-activating ligand B7-H6 (NCR3LG1); low levels of the inhibitory ligand HLA-E; microsatellite instability (MSI) status; high transcriptional signature for chromatin remodeling complexes and low antigen presentation machinery genes. Treatment with HDAC inhibitor reduced the sensitivity of SW620 colon cancer cells, increased antigen presentation machinery, including HLA-E, and reduced B7-H6. Importantly, transcriptional signatures of NK cell-sensitive tumor cells correlate with immune checkpoint inhibitor resistance in clinical samples. Widespread analysis of CCLE transcriptional signatures revealed that cell lines with mesenchymal-like program tend to be more sensitive to NK cells, compared with epithelial-like cell lines. Indeed, mesenchymal tumors tend to have lower expression of antigen presentation machinery in both CCLE and TCGA.Abstract 36 Figure 1Overview of PRISM and CRISPR studies a, Schematic depiction of PRISM study. b, Schematic depiction of CRISPR screens. c, Histogram of gene fold changes (z-scores). Listed are selected genes with most prominent p-values across more than one screen.ConclusionsThis study provides a comprehensive map of mechanisms regulating tumor cell responses to NK cells, with implications for future biomarker-driven applications of NK cell immunotherapies. The integration of PRISM and CRISPR identified potential regulators of tumor cell response to NK cell, which upon further validation, may serve as biomarkers in future NK cell-based studies. Moreover, NK cells may complement T-cells, killing tumor cells that do not respond to immune checkpoint inhibitors.AcknowledgementsThis work was supported by Stand Up To Cancer (SU2C) Convergence 2.0 Grant; SU2C Phillip A. Sharp Award for Innovation in Collaboration; Claudia Adams Barr Program in Innovative Basic Cancer Research; Human Frontier Science Program Fellowship; and Leukemia and Lymphoma Society Scholar Award.ReferencesYu C, et al., High-throughput identification of genotype-specific cancer vulnerabilities in mixtures of barcoded tumor cell lines. Nat Biotechnol 2016. 34(4): p. 419–23.Ghandi M, et al. Next-generation characterization of the cancer cell line encyclopedia. Nature 2019. 569(7757): p. 503–508.Doench JG, et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol 2016. 34(2): p. 184–191.Shalem O, et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 2014;343(6166): p. 84–87.

JAK1 and JAK2 Modulate Tumor Cell Susceptibility To Natural Killer (NK) Cells Through Regulation Of PDL1 Expression

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3472-3472
Author(s):  
Roberto Bellucci ◽  
Allison Martin ◽  
Davide Bommarito ◽  
Kathy S. Wang ◽  
Gordon J Freeman ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Nk Cells ◽  
Tumor Cells ◽  
Cell Lines ◽  
Primary Tumor ◽  
Nk Cell ◽  
Cell Lysis ◽  
Cell Killing ◽  
Tumor Cell Lines ◽  
Ifn Γ

Abstract NK cells are the primary effectors of the innate immune response against infections pathogens and malignant transformation through their efficient cytolytic activity and cytokine secretion. Nevertheless, tumor cells have developed mechanisms to evade innate immune surveillance and the molecular basis for target resistance to NK cell-mediated lysis is not yet completely understood. To identify novel pathways that modulate tumor cell resistance to NK cells, we previously developed a cell-cell interaction based screening approach using a large sub-set of a lentiviral shRNA library containing multiple independent shRNAs targeting more than 1,000 human genes. Using this approach we found that silencing JAK1 and JAK2 significantly increased secretion of INF-γ from NK cells and increased tumor cell susceptibility to NK cell lysis. To examine the role of the JAK signaling pathway in the modulation of tumor cell susceptibility to NK lysis, we analyzed down-stream signaling pathways in several cell lines (IM9, U937, K562, RPMI, MM1S KM12BM) and primary tumor cells (AML, MM, ALL). In the absence of NK cells, silencing JAK1 or JAK2 did not affect the basal activation of STAT proteins (STAT1(pY701), STAT1(pS727), STAT3(pY705), STAT3(pS727), STAT4(pY693), STAT5(pY694), STAT6(pY641)) or AKT(pS473) and ERK1/2(pT202/pY204) or expression of activating or inhibitory ligands on tumor cells. Because JAK1 and JAK2 transduce signals downstream of the IFN-γ receptor, we hypothesized that JAKs may play a role in tumor cell evasion of NK cell activities such as cytolysis and IFN-γ secretion. To test this hypothesis we pre-incubated various tumor cell lines or primary tumor cells with activated NK supernatant or recombinant human IFN-γ. Tumor cell activation in this fashion resulted in activation of STAT1 (pSTAT1(pY701)) but none of the other STATs, ERK or AKT. As expected, STAT1 activation was blocked when JAK1 or JAK2 were silenced or inhibited by a JAK inhibitor. Silencing of STAT1 with 2 independent shRNAs also resulted in increased tumor susceptibility to NK cell cytolysis in 3 different tumor cell lines tested. To confirm that IFN-γ secreted by activated NK cells induced resistance in tumor cell targets we used a blocking IFN-γ antibody (D9D10). 10μg/ml D9D10 completely blocked STAT1 phosphorylation and in different experiments using U937, IM-9, KM12BM, MM1S and RPMI we found that D9D10 significantly increased specific NK target cell lysis by 51.8%, 78.5%, 25.1%, 20.6% and 28.5% compared to IgG1 isotype controls. Similar results were obtained whit different primary tumor cells. To determine whether IFN-γ stimulation affected expression of ligands involved in NK cell recognition of tumor cells, we analyzed the effect of activated NK supernatant or IFN-γ on the expression of MHC Class I, β2M, HLA-C, HLA-A2, NKG2D, NKP44, NKP46, NKP30 ligands using chimeric FC proteins, MICA/B, DNAM-1 ligands (CD112, CD155), 2B4 ligand (CD48), TRAIL ligands (TRAIL-R1, TRAIL-R2), Fas ligand (CD95) and PD1 ligands (PDL1, PDL2, B7H3, B7H4). The basal expression of these ligands varied among the various tumor cell lines or primary tumors tested but the only ligand that was significantly up-regulated in every tumor sample tested was PDL1. PDL1 expression by tumor cells is known to inhibit T cell immunity. To test whether increased levels of PDL1 could also inhibit NK cell killing, we co-cultured primary NK cells with U937, IM9, KM12BM, RPMI, K562, MM1S, primary MM, AML and ALL cells with or without 10μg/ml anti-PDL1 antibody (recombinant mab with Fc mutated to eliminate FcR-mediated effects). Blocking PDL1 significantly increased NK cell killing of U937, IM9, KM12BM, RPMI, MM, AML and ALL (p=0.03, p=0.02, p=0.03, p=0.005, p=0.009, p=0.03 and p=0.02 respectively). NK cell killing activity did not further increase when a JAK inhibitor was added to the co-culture. These results show that NK cell secretion of IFN-γ results in IFN receptor signaling and activation of JAK1, JAK2 and STAT1 in the tumor cell targets, followed by rapid up-regulation of PDL1 expression and increased resistance to NK cell lysis. Blockade of JAK pathway activation prevents subsequent PDL1 up-regulation resulting in increased susceptibility of tumor cells to NK cell activity suggesting that JAK pathway inhibitors may work synergistically with other immunotherapy regimens by eliminating IFN-induced PDL1 mediated immunoinhibition. Disclosures: Freeman: Bristol-Myers-Squibb/Medarex: Patents & Royalties; Roche/Genentech: Patents & Royalties; Merck: Patents & Royalties; EMD-Serrono: Patents & Royalties; Boehringer-Ingelheim: Patents & Royalties; Amplimmune: Patents & Royalties; CoStim Pharmaceuticals: Patents & Royalties; Costim Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees.

Enhancement of Tumor Cell Susceptibility to NK Cell Activity Through Inhibition of the PI3K Signaling Pathway.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2152-2152
Author(s):  
Davide Bommarito ◽  
Allison Martin ◽  
Maria-Dorothea Nastke ◽  
Jerome Ritz ◽  
Roberto Bellucci
Keyword(s):  
Tumor Cell ◽  
Nk Cells ◽  
Tumor Cells ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Cell Activity ◽  
Hematologic Malignancies ◽  
Target Cells ◽  
Tumor Susceptibility

Abstract Abstract 2152 NK cells are the primary effectors of the innate immune response against cells that have undergone malignant transformation. Unlike T cells, NK cells recognize their targets through a complex array of activating and inhibitory receptors, which regulate the intensity of the NK response against individual cells. Tumor cells have developed ways to escape killing by NK cells through a variety of mechanisms that are poorly understood. Using shRNA libraries, we previously demonstrated that several common signaling pathways modulate susceptibility of various tumor cells to NK cell activity. In this study we focused on one of the PI3K sub-units (PI3KCB, p110β isoform) that was identified in this genetic screen. The PI3K pathway has been linked to diverse cellular functions including cell growth, proliferation, survival and intracellular trafficking but PI3K signaling has not previously been associated with susceptibility to NK cells. To examine the mechanisms of PI3KCB involvement in tumor susceptibility to NK cell lysis, we tested 4 tumor cell lines representing different hematologic malignancies: IM9-multiple myeloma, U937-acute myeloid leukemia, K562-chronic myeloid leukemia and Jurkat-acute T cell leukemia. Stable cell lines were established expressing 4 independent shRNAs (P1, P2, P3, P4-shRNA) and 2 irrelevant shRNAs as controls. Western blot and RT-PCR showed different silencing activity of individual PI3KCB-shRNAs with efficient silencing of PI3KCB by 3 of 4 shRNAs in IM9 (P1, P2, P4-shRNA), 2 of 4 in K562 and U937 (P1, P2-shRNA) and 2 of 4 in Jurkat (P2, P4-shRNA). No PI3KCB gene silencing or protein down regulation was observed with P3-shRNA that was used as an additional negative control. PI3KCB silencing did not affect the proliferative capacity of the 4 tumor cell lines tested over a 5 day period. When compared with irrelevant shRNAs or P3-shRNA, effective silencing of PI3KCB in IM-9, K562 and U937 induced increased IFNγ secretion when target cells were incubated with primary purified NK effector cells and 2 different human NK cell lines (NKL and NK-92). Silencing of PI3KCB in Jurkat target cells did not affect IFNγ secretion by NK cells. To determine whether increased IFNγ secretion by NK cells correlated with increased lytic activity, we incubated target cells with NK effectors overnight and assessed cytotoxicity using AnnexinV/7AAD. Lysis of IM9-PI3KCB-P1, P2 and P4-shRNA was increased by 10%-15% compared to irrelevant controls (P=0.04, P=0.04 and P=0.03 respectively); lysis of U937-PI3KCB-P1 and P2-shRNA was increased by 20% and 22% (P=0.02 and P=0.04 respectively); and lysis of K562-PI3KCB-P1 and P2-shRNA was increased by 10% and 20% (P=0.04 and P=0.04, respectively). Correlating with IFNγ assays, increased lysis was not detected for the Jurkat-PI3KCB-silenced cell lines. To define mechanisms responsible for increased susceptibility to NK cells, we examined the expression of several activating/inhibitory ligands on the membrane of PI3KCB-silenced tumor cells. Flow cytometry demonstrated increased expression of MICB (NKG2D ligand) and CD48 (2B4 ligand) in IM9-PI3KCB-KO lines; increased expression of MICA, MICB, CD48 and CD112/CD115 (DNAM-1 ligands) in K562-PI3KCB-KO lines; and increased expression of CD48, CD112 in U937-PI3KCB-KO lines. Reduced expression of MHC class I was also found in IM9 and U937 lines after PI3KCB silencing. Importantly, none of the activating/inhibitory ligands tested were modulated in Jurkat cells after PI3KCB silencing. Co-culture experiments with NK cells and blocking antibodies targeting NKG2D significantly reduced NK IFNγ secretion against IM9-PI3KCB-KO and K562-PI3KCB-KO targets when compared to controls. In contrast, DNAM-1 blocking in U937-PI3KCB-KO produced a uniform decrease of IFNγ secretion in U937-PI3KCB-KO and controls suggesting that in this target, modulation of other NK ligands could also be involved. These data demonstrate that the beta sub-unit of PI3K (PI3KCB) can modulate tumor susceptibility in different hematologic malignancies and that this effect is, at least in part, due to modulation of several activating/inhibitory ligands. These findings identify a new and important role of PI3KCB in modulating tumor cell susceptibility to NK cells and open the way to future combined target immunotherapies. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Recognition of virus-infected cells by natural killer cell clones is controlled by polymorphic target cell elements.

1993 ◽  
Vol 178 (3) ◽  
pp. 961-969 ◽  
Author(s):  
M S Malnati ◽  
P Lusso ◽  
E Ciccone ◽  
A Moretta ◽  
L Moretta ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Target Cell ◽  
Nk Cell ◽  
Class I ◽  
Target Cells ◽  
Cell Recognition ◽  
Infected Cells ◽  
Nk Cell Recognition

Natural killer (NK) cells provide a first line of defense against viral infections. The mechanisms by which NK cells recognize and eliminate infected cells are still largely unknown. To test whether target cell elements contribute to NK cell recognition of virus-infected cells, human NK cells were cloned from two unrelated donors and assayed for their ability to kill normal autologous or allogeneic cells before and after infection by human herpesvirus 6 (HHV-6), a T-lymphotropic herpesvirus. Of 132 NK clones isolated from donor 1, all displayed strong cytolytic activity against the NK-sensitive cell line K562, none killed uninfected autologous T cells, and 65 (49%) killed autologous T cells infected with HHV-6. A panel of representative NK clones from donors 1 and 2 was tested on targets obtained from four donors. A wide heterogeneity was observed in the specificity of lysis of infected target cells among the NK clones. Some clones killed none, some killed only one, and others killed more than one of the different HHV-6-infected target cells. Killing of infected targets was not due to complete absence of class I molecules because class I surface levels were only partially affected by HHV-6 infection. Thus, target cell recognition is not controlled by the effector NK cell alone, but also by polymorphic elements on the target cell that restrict NK cell recognition. Furthermore, NK clones from different donors display a variable range of specificities in their recognition of infected target cells.

Thermal stresses reduce natural killer cell cytotoxicity

1995 ◽  
Vol 79 (3) ◽  
pp. 732-737 ◽  
Author(s):  
S. J. Won ◽  
M. T. Lin
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Target Cell ◽  
Nk Cell ◽  
Cell Activity ◽  
Cortisol Concentration ◽  
Target Cells ◽  
Nk Cell Activity ◽  
Colonic Temperature ◽  
Effector Target

The effects of different ambient temperatures (Ta) on the splenic natural killer (NK) cell activity, effector-target cell conjugation activity, and NK cell numbers were assessed in male inbred C3H/HeNCrj mice (7–10 wk old). The splenic NK cytotoxic activities were examined in a 4-h 51Cr release assay in mouse spleen cells that were obtained 1, 2, 4, 8, or 16 days after exposure to Ta of 22, 4, or 35 degrees C. The percentage of conjugating lymphocytes was calculated by counting the number of single lymphocytes bound to single target cells per 400 effector cells. The numbers of NK cells were expressed by the percentage of 5E6-positive cells. The 5E6 identifies only a subset of NK cells. It was found that the splenic NK cell activity, the effector-target cell conjugation activity, or the NK cell number began to fall 1 day after cold (Ta 4 degrees C) or heat (Ta 35 degrees C) stress. After a 16-day period of either cold or heat exposure, the fall in the splenic NK cell activity, the effector-target cell conjugation activity, or the number of 5E6-positive subsets of NK cells was still evident. Compared with those of the control group (Ta 22 degrees C), the cold-stressed mice had higher adrenal cortisol concentration and lower colonic temperature, whereas the heat-stressed animals had higher adrenal cortisol concentration and higher colonic temperature during a 16-day period of thermal exposure. However, neither cold nor heat stress affected both the body weight gain and the spleen weight in our mice.

Pluripotent Stem Cell-Derived Human Natural Killer Cells with Potent Anti-Multiple Myeloma Activity,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4034-4034
Author(s):  
David A. Knorr ◽  
Zhenya Ni ◽  
Allison Bock ◽  
Vijay G. Ramakrishnan ◽  
Shaji Kumar ◽  
...  
Keyword(s):  
Stem Cells ◽  
Multiple Myeloma ◽  
Stem Cell ◽  
Nk Cells ◽  
Natural Killer ◽  
Tumor Cells ◽  
Adoptive Immunotherapy ◽  
Target Cell ◽  
Peripheral Blood ◽  
Nk Cell

Abstract Abstract 4034 Natural Killer (NK) cells are lymphocytes of the innate immune system with anti-viral and anti-cancer activity. Over the past decade, they have gained interest as a promising cellular source for use in adoptive immunotherapy for the treatment of cancer. Most notably, NK cells play an important role in the graft-vs-tumor effect seen in allogeneic hematopoietic stem cell transplantation (allo-HSCT), and a better understanding of NK cell biology has translated into improved transplant outcomes in acute myelogenous leukemia (AML). Small studies have demonstrated a role for NK cell activity in multiple myeloma (MM) patients receiving allo-HSCT. Investigators have also utilized haplo-identical killer immunoglobulin-like receptor (KIR) mismatched NK cells for adoptive immunotherapy in patients with multiple myeloma (MM). Our group has focused on the development of NK cells from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) as a novel starting source of lymphocytes for immunotherapy. We have previously demonstrated potent anti-tumor activity of hESC-derived NK cells in vitro and in vivo against a variety of different targets. We have also shown that iPSC-derived NK cells from a variety of different somatic cell starting sources posses potent anti-tumor and anti-viral activity. Here, we demonstrate hESC- and iPSC-derived NK cell development in a completely defined, feeder-free system that is amenable to clinical scale-up. These cultures contain a pure population of mature NK cells devoid of any T or B cell contamination, which are common adverse bystanders of cellular products isolated and enriched from peripheral blood. Our cultures are homogenous for their expression of CD56 and express high levels of effector molecules known to be important in anti-MM activity, including KIR, CD16, NKG2D, NKp46, NKp44, FasL and TRAIL. We have now tested the activity of hESC- and iPSC-derived NK cells against MM tumor cells in order to provide a universal source of lymphocytes for adoptive immunotherapy in patients with treatment refractory disease. We find that similar to peripheral blood NK cells (PB-NK), hESC- and iPSC-derived NK cells are cytotoxic against 3 distinct MM cell lines in a standard chromium release cytotoxicity assay. Specifically, activated PB-NK cells killed 48.5% of targets at 10 to 1 effector to target ratios, whereas hESC (46.3%) and iPSC (42.4%) derived NK cells also demonstrated significant anti-MM activity. Also, hESC- and iPSC-derived NK cells secrete cytokines (IFNγ and TNFα) and degranulate as demonstrated by CD107a surface expression in response to MM target cell stimulation. When tested against freshly isolated samples from MM patients, hESC- and IPSC-derived NK cells respond at a similar level as activated PB-NK cells, the current source of NK cells used in adoptive immunotherapy trials. These MM targets (both cell lines and primary tumor cells) are known to express defined ligands (MICA/B, DR4/5, ULBP-1, BAT3) for receptors expressed on NK cells as well as a number of undefined ligands for natural cytotoxicity receptors (NCRs) and KIR. As these receptor-ligand interactions drive the anti-MM activity of NK cells, we are currently evaluating expression of each of these molecules on the surface of both the effector and target cell populations. Not only do hESC- and iPSC-derived NK cells provide a unique, homogenous cell population to study these interactions, they also provide a genetically tractable source of lymphocytes for improvement of the graft-vs-myeloma effect and could be tailored on a patient specific basis using banks of hESC-or iPSC-derived NK cells with defined KIR genotypes for use as allogeneic or autologous effector cells. Disclosures: No relevant conflicts of interest to declare.

Natural Killer Cells with NKDG2D Cytotoxicity but NKp30-Dull Phenotype Predominate in Myelodysplastic Syndrome (MDS).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3436-3436
Author(s):  
Fanqi Bai ◽  
Jeffrey S. Painter ◽  
Cantor Alan ◽  
Zou JianXiang ◽  
Sheng Wei ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Cell Line ◽  
Cell Lines ◽  
Nk Cell ◽  
B Cell Lymphoma ◽  
Target Cells ◽  
Nkg2d Ligands ◽  
Human Stress ◽  
Normal Controls

Abstract Natural Killer (NK) function in patients with MDS as measured by non-MHC-restricted cytotoxicity and activation-dependent cell cytotoxicity (ADCC) are reduced in patients with MDS, however, the mechanisms of the functional impairment are not known. Tumor cytolysis occurs through orchestrated control by inhibitory NK receptors (NKRs) and activating NKRs, which control signaling events that lead to polarized movement of perforin-containing granules toward the NK-tumor contact area. We found that NK cells from 23 out of 35 patients with MDS (66%) displayed reduced lysis of K562 tumor cells compared to age-matched normal controls (p<0.01). To better characterize this defect, we evaluated patient NK function against differential tumor targets including the MDS1 cell line established from an MDS patients. We found that MDS1 incited non-MHC-restricted lysis. Unactivated PBMCs, unactivated NK cells, NK cell lines (NK92 and NKL) but not purified unactivated T cells from normal donors killed MDS1 in 4-hr 51Cr-release assays. Normal NK cells and NK cell lines were also found to rapidly redistrubute perforin granules after exposure to MDS1suggesting that a perforin-dependent lytic pathway was activated. We then performed simultaneous cytolytic assays with K562, MDS1, and the 721.221 B cell lymphoma cell line as target cells. We found that NK cells from MDS patients had greater lytic activity against MDS1 (average 24% vs. average 8% at 50:1 Effector:Target ratio, respectively, p<0.01) Antibody-blocking experiments demonstrated that the NKL cell line and PBMCs from 8 out of 10 MDS patients predominantly used the NKG2D activating receptor to kill MDS1. Consistent with this finding, we showed that MDS1 cells express the major human stress-inducible endogenous proteins MICA and MICB, which are NKG2D ligands. In contast, lysis by NK92 cells and normal PBMCs was not appreciably reduced by NKG2D blocking antibodies suggesting that other unidentified NKR(s) also mediate lysis. To identify the NKRs expressed in MDS patients, we performed immunophenotyping for both the activating NKRs and inhibitory NKRs compared to age-matched normal controls. We found that two activating receptors, NKp30 and CD244 (2B4), were significantly reduced on NK cells from all MDS patients regardless of their ability to lyse NK targets. Inhibitory NKR expression and function were normal. Interestingly, NKG2D expression correlated with reduced cytolytic function. Similar to studies on normal NK cells with low NKp30 and NKp46 (NCRdull) phenotypes, these results suggest that low NKp30 expression leads to predominant NKG2D utilization for tumor cell lysis, which is reduced in MDS patients with defective NK function. Our findings provide critical information about potential importance for immunosurviellance through NKG2D-NKG2D ligands.

scholarly journals Protection from lysis by natural killer cells of group 1 and 2 specificity is mediated by residue 80 in human histocompatibility leukocyte antigen C alleles and also occurs with empty major histocompatibility complex molecules.

1996 ◽  
Vol 184 (3) ◽  
pp. 913-922 ◽  
Author(s):  
O Mandelboim ◽  
H T Reyburn ◽  
M Valés-Gómez ◽  
L Pazmany ◽  
M Colonna ◽  
...  
Keyword(s):  
Major Histocompatibility Complex ◽  
Nk Cells ◽  
Natural Killer ◽  
Target Cell ◽  
Nk Cell ◽  
Cell Lysis ◽  
Target Cells ◽  
Major Histocompatibility ◽  
Leukocyte Antigen ◽  
Histocompatibility Complex

Recognition of major histocompatibility complex class I molecules by natural killer (NR) cells leads to inhibition of target cell lysis. Based on the capacity of different human histocompatibility leukocyte antigen (HLA)-C and HLA-B molecules to inhibit target cell lysis by NK lines and clones, three NK allospecificities have been defined: NK1 and NK2 cells are inhibited by different HLA-C allotypes and NK3 cells by some HLA-B allotypes. The NK1 and NK2 inhibitory ligands on target cells correspond to a dimorphism of HLA-C at residues 77 and 80 in the alpha 1 helix: Asn77-Lys80 in NK1 and Ser77-Asn80 in NK2 inhibitory ligands. It has been reported that protection from NK1 killers depended on the presence of the Lys residue at position 80, an upward pointing residue near the end of the alpha 1 helix (and not on Asn77), whereas inhibition of NK2 effector cells required Ser77, a residue deep in the F pocket and interacting with the peptide (and not Asn80). As part of ongoing experiments to investigate the structural requirements for NK cell inhibition by HLA-C locus alleles, we also examined the effects of mutations at residues 77 and 80 on the ability of HLA-C alleles to confer protection from NK lysis. We present data confirming that the NK1 specificity depended on Lys80 (and not on Asn77); however recognition of NK2 ligands by NK cells was also controlled by the amino acid at position 80 (Asn), and mutation of Ser77 had no effect. Furthermore, bound peptide was shown to be unnecessary for the inhibition of NK cell-mediated lysis since HLA-C molecules assembled in the absence of peptide in RMA-S cells at 26 degrees C were fully competent to inhibit NK cells specifically. The implications of these data for peptide-independent recognition of HLA-C by NK receptors are discussed.

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