The Mechanism of Leukoregulin Enhancement of Target Cell Susceptibility to NK Cell Mediated Cytotoxicity in Humans

Natural killer (NK) cells are a main subset of innate lymphocytes that contribute to host immune protection against viruses and tumors by mediating target cell killing and secreting a wide array of cytokines. Their functions are finely regulated by a balance between activating and inhibitory receptors and involve also adhesive interactions. Mechanotransduction is the process in which physical forces sensed by mechanosensors are translated into chemical signaling. Herein, we report findings on the involvement of this mechanism that is mainly mediated by actin cytoskeleton, in the regulation of NK cell adhesion, migration, tissue infiltration and functions. Actin represents the structural basis for NK cell immunological synapse (NKIS) and polarization of secretory apparatus. NK-target cell interaction involves the formation of both uropods and membrane nanotubes that allow target cell interaction over long distances. Actin retrograde flow (ARF) regulates NK cell signaling and controls the equilibrium between activation versus inhibition. Activating NKIS is associated with rapid lamellipodial ARF, whereas lower centripetal actin flow is present during inhibitory NKIS where β actin can associate with the tyrosine phosphatase SHP-1. Overall, a better knowledge of mechanotransduction might represent a future challenge: Realization of nanomaterials tailored for NK cells, would be important to translate in vitro studies in in vivo new immunotherapeutic approaches.

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

Journal of Experimental Medicine ◽

10.1084/jem.178.3.961 ◽

1993 ◽

Vol 178 (3) ◽

pp. 961-969 ◽

Cited By ~ 57

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.

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Thermal stresses reduce natural killer cell cytotoxicity

Journal of Applied Physiology ◽

10.1152/jappl.1995.79.3.732 ◽

1995 ◽

Vol 79 (3) ◽

pp. 732-737 ◽

Cited By ~ 29

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.

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Evidence for a dithiol-activated signaling pathway in natural killer cell avidity regulation of leukocyte function antigen-1: structural requirements and relationship to phorbol ester- and CD16-triggered pathways

Blood ◽

10.1182/blood.v86.6.2288.bloodjournal8662288 ◽

1995 ◽

Vol 86 (6) ◽

pp. 2288-2301 ◽

Cited By ~ 23

Author(s):

BS Edwards ◽

MS Curry ◽

EA Southon ◽

AS Chong ◽

LH Jr Graf

Keyword(s):

Natural Killer ◽

Phorbol Ester ◽

Adhesion Molecule ◽

Target Cell ◽

Actin Polymerization ◽

Nk Cell ◽

Killer Cell ◽

Disulfide Reduction ◽

Leukocyte Function ◽

Beta 2

Dithiothreitol (DTT) activation of the adhesive function of several different integrins suggests the existence of a common DTT-sensitive integrin regulatory element. Ui11/E3, a natural killer (NK) cell- resistant murine target cell line genetically engineered to constitutively express human intercellular adhesion molecule-1 (ICAM-1; CD54) was used in a flow cytometric experimental model to evaluate DTT effects on the NK cell integrin adhesion molecule, leukocyte function antigen-1 (LFA-1; alpha L beta 2, CD11a/CD18). DTT and several structurally related dithiol compounds elicited a dramatic elevation in conjugate formation that was dependent on target cell ICAM-1 expression, was blocked by LFA-1 alpha L or beta 2 chain-specific antibodies, and occurred in the absence of Ui11/E3 target cell exposure to DTT or quantitative changes in NK cell membrane LFA-1 expression. This avidity modulation of LFA-1 by DTT required actin polymerization, was abrogated by the protein kinase C inhibitor calphostin C, involved activities of calyculin A- and okadaic acid-sensitive serine/threonine protein phosphatases PP-1 and/or PP-2A but not geldanamycin-sensitive tyrosine kinases, and differed with respect to kinetics and enzyme inhibitor sensitivity from LFA-1 activation promoted by cross-linking of NK cell CD16 or phorbol ester treatment. A key structural feature of DTT was the presence of two thiol groups, both reduced but not physically adjacent as in the nonstimulatory dithiol, 2,3- dimercaptopropanol. LFA-1 activation was not because of DTT chelation of Ca2+ or Zn2+. Immunoblotting studies identified multiple NK cell plasma membrane-associated proteins to be reduced by DTT under LFA-1- activating conditions, but similar effects were also promoted by reducing agent treatments that failed to alter adhesive function. Direct chemical modification of LFA-1 seemed an unlikely basis of activation because (1) DTT activated LFA-1 in HSB2 T cells without detectable disulfide reduction in LFA-1 alpha L or beta 2 chains immunoprecipitated from these cells and (2) DTT treatment of NK cells did not hinder binding of KIM127 and KIM185, monoclonal antibodies that recognize epitopes in the potentially DTT-susceptible cysteine-rich domain of the beta 2 chain. Thus, these results extended the range of DTT-activatible integrins to include NK cell LFA-1 and characterized for the first time signaling-associated enzymatic activities involved in DTT activation of NK cell LFA-1. Moreover, they suggested that structural features of DTT, particularly SH group spatial positioning, are important in LFA-activation for reasons other than cation chelation or disulfide reduction.(ABSTRACT TRUNCATED AT 400 WORDS)

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Specificity of HLA class I antigen recognition by human NK clones: evidence for clonal heterogeneity, protection by self and non-self alleles, and influence of the target cell type.

Journal of Experimental Medicine ◽

10.1084/jem.178.4.1321 ◽

1993 ◽

Vol 178 (4) ◽

pp. 1321-1336 ◽

Cited By ~ 117

Author(s):

V Litwin ◽

J Gumperz ◽

P Parham ◽

J H Phillips ◽

L L Lanier

Keyword(s):

Nk Cells ◽

Mhc Class I ◽

Target Cell ◽

Nk Cell ◽

Hla Class I ◽

Class I ◽

Target Cells ◽

Clonal Heterogeneity ◽

Multiple Alleles ◽

Wide Range

Prior studies using polyclonal populations of natural killer (NK) cells have revealed that expression of certain major histocompatibility complex (MHC) class I molecules on the membrane of normal and transformed hematopoietic target cells can prevent NK cell-mediated cytotoxicity. However, the extent of clonal heterogeneity within the NK cell population and the effect of self versus non-self MHC alleles has not been clearly established. In the present study, we have generated more than 200 independently derived human NK cell clones from four individuals of known human histocompatibility leukocyte antigens (HLA) type. NK clones were analyzed for cytolytic activity against MHC class I-deficient Epstein Barr virus (EBV) transformed B lymphoblastoid cell lines (B-LCL) stably transfected with several HLA-A, -B, or -C genes representing either self or non-self alleles. All NK clones killed the prototypic HLA-negative erythroleukemia K562 and most lysed the MHC class I-deficient C1R and 721.221 B-LCL. Analysis of the panel of HLA-A, -B, and -C transfectants supported the following general conclusions. (a) Whereas recent studies have suggested that HLA-C antigens may be preferentially recognized by NK cells, our findings indicate that 70% or more of all NK clones are able to recognize certain HLA-B alleles and many also recognize HLA-A alleles. Moreover, a single NK clone has the potential to recognize multiple alleles of HLA-A, HLA-B, and HLA-C antigens. Thus, HLA-C is not unique in conferring protection against NK lysis. (b) No simple patterns of HLA specificity emerged. Examination of a large number of NK clones from a single donor revealed overlapping, yet distinct, patterns of reactivity when a sufficiently broad panel of HLA transfectants was examined. (c) Both autologous and allogeneic HLA antigens were recognized by NK clones. There was neither evidence for deletion of NK clones reactive with self alleles nor any indication for an increased frequency of NK clones recognizing self alleles. (d) With only a few exceptions, protection conferred by transfection of HLA alleles into B-LCL was usually not absolute. Rather a continuum from essentially no protection for certain alleles (HLA-A*0201) to very striking protection for other alleles (HLA-B*5801), with a wide range of intermediate effects, was observed. (e) Whereas most NK clones retained a relatively stable HLA specificity, some NK clones demonstrated variable and heterogeneous activity over time. (f) NK cell recognition and specificity cannot be explained entirely by the presence or absence of HLA class I antigens on the target cell.(ABSTRACT TRUNCATED AT 400 WORDS)

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Preclinical Development of Edit-201, a Multigene Edited Healthy Donor NK Cell with Enhanced Anti-Tumor Function and Superior Serial Killing Activity in an Immunosuppressive Environment

Blood ◽

10.1182/blood-2020-139988 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 33-33

Author(s):

Christopher M Borges ◽

Kevin Wasko ◽

Jared M Nasser ◽

Kelly Donahue ◽

Amanda Pfautz ◽

...

Keyword(s):

Cell Growth ◽

Nk Cells ◽

Target Cell ◽

Nk Cell ◽

Hematologic Malignancies ◽

Target Cells ◽

Publicly Traded ◽

Killing Assay ◽

Serial Killing

Natural killer (NK) cells distinguish tumor from healthy tissue via multiple mechanisms, including recognition of stress ligands and loss of MHC class I expression. For example, KIR mismatching enables allogenic NK cells to respond to MHC positive tumors in a similar manner to MHC negative tumors, making allogeneic NK cell therapy a promising approach for broad oncology indications. Accordingly, allogenic human HD-NK cells, including gene-modified cells, have demonstrated an impressive safety and efficacy profile when administered to patients with advanced hematologic malignancies. However, effector function of allogeneic NK cells can be diminished by the lack of functional persistence, as well as tumor-intrinsic immunosuppressive mechanisms, such as production of TGF-β. To this end, we developed a next-generation allogeneic NK cell therapy using CRISPR-Cas12a gene editing to enhance NK cell function through knockout of the genes CISH and TGFBR2. Both single and simultaneous targeting (DKO) of TGFBR2 and CISH in NK cells using CRISPR-Cas12a produced in/dels at both targets in greater than 80% of NK cells, with greater than 90% of edited NK cells viable at 72 hours post-editing. Importantly, we find that DKO NK cells do not phosphorylate the SMAD2/3 protein downstream of the TGF-b receptor complex and demonstrate increased phosphorylation of pSTAT3 and pSTAT5 upon IL-15 stimulation, consistent with protein level knockout of TGFBR2 and CISH. To determine whether DKO NK cells exhibited superior function relative to control NK cells, we first measured the ability of DKO NK cells to kill Nalm6 cells (adult B cell ALL) relative to unedited control NK cells. We find that in the presence of exogenous TGF-b, DKO NK cells demonstrate improved cytotoxicity against Nalm6 tumor targets by delaying tumor re-growth in comparison to control NK cells. To better characterize the ability of DKO NK cells to kill tumor cells, we developed an in vitro serial killing assay. In this long-duration assay, up to 30 days, control and DKO NK cells (grown in the presence of IL-15) were challenged every 48 hours with a new bolus of Nalm6 tumor targets. Both DKO and unedited NK cells control Nalm6 target cell growth for greater than 18 days (9 additions of new Nalm6 target cells), demonstrating a surprising ability for the same NK cells to serially kill new Nalm6 target cells for a prolonged period of time in vitro. We find that DKO NK cells produce higher levels of IFN-γ and TNF-α relative to control NK cells over the duration of the entire serial killing assay, suggesting that DKO NK cells can continue to produce these inflammatory cytokines even after serial killing. As many tumors, including hematologic malignancies, have high concentrations of TGF-β in their microenvironments, we next tested the ability of DKO NK cells to control the growth of Nalm6 cells in our serial killing assay in the presence of TGF-b. 10ng/mL TGF-β was added at the start of the assay as well as at each addition of new Nalm6 target cells. We observed that control NK cells fail to restrict Nalm6 target cell growth beyond 4 days (after 1 addition of new Nalm6 target cells) whereas DKO NK cells control Nalm6 target cell growth for greater than 18 days (after 9 additions of new Nalm6 target cells). Similar to the serial killing assay without TGF-b, we find that DKO NK cells produce higher concentrations of IFN-γ and TNF-α relative to control NK cells over the duration of the entire serial killing assay. Broadening our repertoire of target cells beyond B cell malignancies is now in progress, including the AML-like cell lines HL-60 and THP-1, the multiple myeloma cell line RPMI 8226, and various solid tumor targets. In summary, using CRISPR-Cas12a we demonstrated highly efficient gene editing of primary human NK cells at two unique targets designed to augment NK cell anti-tumor activity across a variety of malignancies. Most significantly, we demonstrate sustained anti-tumor serial-killing activity in the presence of the potent immunosuppressive cytokine TGF-β. Together, the increased overall effector function of CISH/TGFBR2 DKO primary human NK cells and their ability to serial kill, support their development as a potent allogeneic cell-based medicine for cancer. This potential medicine, termed EDIT-201, is being advanced to clinical study. Disclosures Borges: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Wasko:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Nasser:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Donahue:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Pfautz:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Antony:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Leary:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Sexton:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Morgan:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Wong:Editas Medicine: Current Employment, Current equity holder in publicly-traded company.

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KIR Gene and HLA-Cw Mismatches Affect on NK Cell Killer Activity

Blood ◽

10.1182/blood.v112.11.4926.4926 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4926-4926

Author(s):

Xiangshan Cao ◽

Jianyong Li

Keyword(s):

Cell Surface ◽

Nk Cells ◽

Target Cell ◽

Nk Cell ◽

Leukemic Cells ◽

Target Cells ◽

Cytotoxicity Assays ◽

Control Target ◽

Nk Cytotoxicity ◽

Kir Gene

Abstract The KIRs were knew as natural killer (NK) cell inhibitory receptors with specificity for HLA molecules on their cellular targets. We investigated NK cell activation on the number of matches between cell killer immunoglobulin-like receptor (KIR) gene and HLA-Cw, and the level of inhibitory KIRs expressed on NK cell surface and the cytotoxicity of NK cell against AML leukemic cells in vitro. NK cell were isolated and purified from 27 healthy donors by isolation kit, Target cells were blasts derived from bone marrow of 30 patients with AML.Inhibitory KIRs expression knew as CD158a, CD158b was analyzed by flow cytometry to estimate the percentage of NK cells that could be inhibited by the HLA-Cw ligands..KIR and HLA gene typing were performed by PCR –SSP. from donors and patients respectively. NK cytotoxicity against AML leukemic cells demonstrated by MTT which showed the correlation between NK cytotoxicity and the number of KIR/HLA matches. the NK-susceptible K562 cell line which lacks HLA class I expression, was used as a positive control target in all cytotoxicity assays, autologous non-NK cell was used as negative control target cell. the cytotoxicity assays was performed in E:T 50:1 20:1 10:1 5:1 2.5:1. Results demonstrated the less number of KIR/HLA-Cw matches, the more NK cells are activated..0 match of NK cell/target cell KIR/HLA-Cw, cytotoxicity was (50.66±8.40)%,1 match and 2 matches were (38.28±6.71)%, (19.74±4.15)%, F=20.226, P<0.001. NK cells expressed KIRs also had relationship with cytotoxicity, inhibitory KIRs expressed >50%, the cytotoxicity is 10%, inhibitory KIRs expressed 20%–50%, the cytotoxicity is 20%, inhibitory KIRs <25%, the cytotoxicity is 55%, F=16.276,p<0.001. Therefore these data indicate NK cell kill AML leukemic cells mechanism follow KIR/HLA-Cw mismatch theory, the level of inhibitory KIRs expressed on NK cell surface showed the percentage of NK cells that could be inhibited by the HLA-Cw ligands. Key words: KIR NK cell CD158 HLA-Cw

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

Blood ◽

10.1182/blood.v118.21.4034.4034 ◽

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.

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