CD137 co-stimulation and blocking PD-1 enhances NK cell-mediated target cell lysis by CD30/CD16A TandAb AFM13.

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.

Download Full-text

Involvement of nitric oxide in target-cell lysis and DNA fragmentation induced by murine natural killer cells

Blood ◽

10.1182/blood.v87.12.5136.bloodjournal87125136 ◽

1996 ◽

Vol 87 (12) ◽

pp. 5136-5143 ◽

Cited By ~ 35

Author(s):

JG Filep ◽

C Baron ◽

S Lachance ◽

C Perreault ◽

JS Chan

Keyword(s):

Nitric Oxide ◽

Nk Cells ◽

Natural Killer ◽

Dna Fragmentation ◽

Target Cell ◽

Culture Medium ◽

Nk Cell ◽

Cell Lysis ◽

Specific Pattern ◽

Target Cells

Although it has been recognized for sometime that target cells destroyed by natural killer (NK) cells die largely by apoptosis, the underlying mechanisms are not fully understood. The aim of the present study was to examine the role of nitric oxide (NO) in mediating murine NK-cell-induced killing of YAC-1 lymphoma cells. NK calls induced extensive release of 125I-DNA and 51Cr from YAC-1 cells. The target killing ability of NK cells was associated with an increased production of NO as measured by concentrations of nitrite in the culture medium. That YAC-1 killing resulted, in part, from the production of NO was confirmed by the significant protection of cell lysis in L-arginine- depleted medium and by approximately 30 % attenuation of cell lysis and DNA fragmentation by an inhibitor of NO synthase, NG-nitro-L-arginine methyl ester (L-NAME) in a culture medium containing 1 mmol/L L- arginine. Fluorescence microscopic examination of YAC-1 cells showed the presence of changes in nuclear morphology characteristic for apoptosis. The percentage of apoptotic cells was markedly decreased by L-NAME. Further evidence for apoptosis is provided by the specific pattern of internucleosomal DNA fragmentation both in the absence and presence of L-NAME. During target-cell killing, an increased oxidation of intracellularly trapped dichlorofluorescein was observed in cells labeled with an antimouse NK-cell monoclonal antibody, as measured by flow cytometry. These increases were effectively prevented by L-NAME, but not W-13, an inhibitor of calmodulin. The ability of NO to induce cell lysis and DNA fragmentation in YAC-1 cells was further demonstrated by exposing tumor cells to chemically generated NO. Taken together, these observations suggest a role for NO as one of the mediators of NK-cell-mediated DNA fragmentation and cell lysis.

Download Full-text

Adapter Chimeric Antigen Receptor (aCAR)-Engineered NK-92 Cells: An Off-the-Shelf Cellular Therapeutic for Universal Tumor Targeting

Blood ◽

10.1182/blood-2018-99-116724 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3331-3331

Author(s):

Stefan Grote ◽

Christian M. Seitz ◽

Simon Diepold ◽

Markus Buchner ◽

Caroline Baden ◽

...

Keyword(s):

Clinical Trials ◽

Target Cell ◽

Research Funding ◽

Effector Cell ◽

Immune Cell ◽

Nk Cell ◽

Cell Lysis ◽

Specific Lysis ◽

Effector Cells ◽

On Demand

Abstract Chimeric antigen receptor (CAR) expressing T cells (CAR-Ts) have demonstrated tremendous clinical success, especially when targeted against the B-phenotypic antigens CD19 and CD22 in ALL, CLL as well as NHL. Despite the recent success, production of CAR-Ts still requires an extensive and time consuming manufacturing process. Moreover, CAR-Ts have to be prepared individually for each patient. Especially in heavily pretreated and rapidly progressing patients, which often lack sufficient numbers of healthy T cells for CAR-T production, alternatives are a significant clinical need. NK cells might represent a promising alternative effector cell source. The continuously expandable and well established NK cell line NK-92 can provide a safe and consistent way to produce NK effector cells in a GMP-compliant and cost-effective way. Irradiated NK-92 CARs as an "off-the-shelf on-demand" cell therapeutic are currently tested in pre-clinical and early-phase clinical trials. Furthermore, NK-92 can be redirected by CARs to mediate direct antigen specific lysis. We have recently developed a universal adapter CAR (aCAR) system. By splitting antigen recognition and CAR-immune cell activation, introducing adapter molecules (AMs), the system allows precise quantitative (on-/off-switch) as well as qualitative (change and combination of target antigens) regulation of immune cell function. aCARs are based on the unique properties of a novel scFv targeting a "neo"-epitope-like structure consisting of the endogenous vitamin biotin in the context of a specific linker, referred to as linker-label-epitope (LLE). LLEs can be easily conjugated on novel or preexisting AM formats like monoclonal antibodies (mAbs) or mAb fragments in a GMP-compliant manner. In the present study, we intended to combine the universal and flexible targeting as well as controllability of the aCAR with the "off-the-shelf" properties of NK-92 cells. NK-92 was obtained from ATCC and transduced with aCARs containing either CD28 or 4-1BB co-stimulatory plus CD3-ζ signaling domains. Importantly, only CD28 containing aCARs sufficiently mediated specific target cell lysis in the presence of biotinylated antibodies (LLE-AMs). Using single cell sorting, aCAR NK-92 clones with the highest CAR expression were selected and demonstrated significantly improved target cell lysis, in a LLE-AMs dependent manner. Both, LLE-AMs against CD19 and CD20, were capable of inducing significant NK-92-mediated lysis against the NHL cell lines Raji, Daudi and JeKo-1. Cytotoxicity experiments using aCAR NK-92 cells and primary lymphoma cells are ongoing. Specificity of the LLE-AM directed effector cell elimination was further proven using a JeKo-1 CD19 and/or CD20 knock out (KO) antigen-loss model. aCAR NK-92 mediated antigen specific lysis only in the presence of the target antigen and the specific LLE-AM. Moreover, combinations of anti-CD19 and anti-CD20 LLE-AMs are capable of avoiding antigen evasion. To test the universal applicability of aCAR NK-92, specific target cell lysis against a variety of different tumor entities was demonstrated using LLE-conjugated therapeutic antibodies. Importantly, irradiation of effector cells, as required in all active clinical trials using NK-92, prior to testing had no observable effect on target cell lysis. Finally, the investigation of potential on-target off-tumor reactivity against healthy B cells showed no cytotoxic effects of aCAR NK-92 cells in combination with LLE-AMs against CD19 or CD20. In conclusion, we have generated an NK cell line, aCAR NK-92, whose effector function can be tightly regulated and redirected against one or multiple antigens allowing tunable and universal targeting. Moreover, aCAR NK-92 cells can be manufactured as an "off-the-shelf on-demand" standardized product improving the practicality of NK CAR therapy combined with the possibility of tailoring a specific LLE-AM platform for patient-individualized treatment. Disclosures Seitz: Miltenyi Biotec: Patents & Royalties, Research Funding. Mittelstaet:Miltenyi Biotec: Employment, Patents & Royalties. Kaiser:Miltenyi Biotec: Employment, Patents & Royalties. Schlegel:Miltenyi Biotec: Patents & Royalties, Research Funding. Handgretinger:Miltenyi Biotec: Patents & Royalties: Co-patent holder of TcR alpha/beta depletion technologies, Research Funding.

Download Full-text

Cytolysis by Ca-permeable transmembrane channels. Pore formation causes extensive DNA degradation and cell lysis.

Journal of Experimental Medicine ◽

10.1084/jem.169.3.765 ◽

1989 ◽

Vol 169 (3) ◽

pp. 765-777 ◽

Cited By ~ 72

Author(s):

A Hameed ◽

K J Olsen ◽

M K Lee ◽

M G Lichtenheld ◽

E R Podack

Keyword(s):

Target Cell ◽

Time Course ◽

Nk Cell ◽

Cell Lysis ◽

Dna Degradation ◽

Alpha Toxin ◽

Membrane Pore ◽

Course Of Action ◽

Intracellular Ca ◽

Dna Release

This study investigates the effect of the purified membrane pore formers, staphylococcal alpha-toxin and CTL perforin, on target cell lysis as measured by 51Cr release and on nuclear damage as measured by DNA degradation and 125IUdR release. Both pore formers cause dose-dependent cell lysis, which is accompanied by DNA release. The ratio of DNA/Cr release depends on the nature of target cell and shows the same pattern as the ratio of release of the two markers reported for CTL-mediated lysis of the same targets. DNA degradation is dependent on the presence of intracellular Ca in the target cell and is totally blocked if Ca is chelated by Quin 2 intracellularly and EGTA extracellularly. DNA degradation, in addition, is inhibited by the lysosomotropic agents NH4Cl, chloroquine, and monensin. rTNF doubles the degree of DNA degradation mediated by alpha-toxin in 3-h assays. We conclude that pore formers alone can mediate DNA degradation. In addition, they may promote the uptake of other factors and thereby accelerate their time course of action. DNA degradation by pore formers requires active target participation in a pathway that is dependent on intracellular Ca and lysosomes. These aspects of target lysis resemble CTL- and NK cell-mediated cytolysis.

Download Full-text

CD56 regulates human NK cell cytotoxicity through Pyk2

10.1101/2020.03.19.998427 ◽

2020 ◽

Author(s):

Justin T. Gunesch ◽

Amera L. Dixon ◽

Tasneem Ebrahim ◽

Melissa Berrien-Elliott ◽

Swetha Tatineni ◽

...

Keyword(s):

Nk Cells ◽

Cell Line ◽

Cell Lines ◽

Target Cell ◽

Immune Cells ◽

Nk Cell ◽

Cell Lysis ◽

Cytokine Secretion ◽

Functional Link ◽

Cytotoxic Function

Natural killer (NK) cells are innate immune cells that control viral infection and tumorigenic cell growth through targeted cell lysis and cytokine secretion. Human NK cells are classically defined as CD56+CD3− in peripheral blood. CD56 is neural cell adhesion molecule (NCAM1), and despite its ubiquitous expression on human NK cells, the role of CD56 in human NK cell cytotoxic function has not been fully explored. In non-immune cells, NCAM can induce signaling, mediate adhesion, and promote exocytosis, in part through interactions with focal adhesion kinase (FAK). Here we describe the generation and use of CD56-deficient human NK cell lines to define a novel requirement for CD56 in target cell lysis. Namely, we demonstrate that deletion of CD56 on the NK92 cell line led to impaired cytotoxic function against multiple susceptible target cell lines. Deletion of CD56 in a second NK cell line, YTS cells, led to a less severe cytotoxicity defect but impairment in cytokine secretion. Confocal microscopy of wild-type and CD56-KO NK92 cells conjugated to susceptible targets revealed that CD56-KO cells failed to polarize during immunological synapse (IS) formation and had severely impaired exocytosis of lytic granules at the IS. Phosphorylation of the FAK family member Pyk2 at tyrosine 402 was decreased in NK92 CD56-KO cells, demonstrating a functional link between CD56 and IS formation and signaling in human NK cells. Cytotoxicity, lytic granule exocytosis, and the phosphorylation of Pyk2 were rescued by the reintroduction of NCAM140 (CD56), into NK92 CD56-KO cells. These data highlight a novel functional role for CD56 in stimulating exocytosis and promoting cytotoxicity in human NK cells.

Download Full-text

Identifying NK-Cell Donors for Cell Therapy Based on Functional Phenotype.

Blood ◽

10.1182/blood.v110.11.3271.3271 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3271-3271

Author(s):

Lenka Hurton ◽

R. Iram Siddik ◽

Harjeet Singh ◽

Simon Olivares ◽

Brian Rabinovich ◽

...

Keyword(s):

Nk Cells ◽

Target Cell ◽

Nk Cell ◽

Hla Class I ◽

Cell Lysis ◽

Cell Killing ◽

Hla Typing ◽

Class I ◽

Target Cells ◽

Functional Assay

Abstract Donor natural killer (NK) cells after haploidentical hematopoietic stem-cell transplantation (HSCT) and infusion of haploidentical NK-cells have demonstrated a therapeutic effect. NK alloreactivity resulting from appropriate Killer cell Ig-like receptor (KIR)-ligand disparity in human-leukocyte-antigen (HLA)-haplotype mismatched HSCT has resulted in improved engraftment and decreased incidence of leukemia relapse. Yet, not all patient-donor pairs benefit for an allogeneic NK-cell effect. To identify NK-cell donors with a suitable KIR-ligand mismatch, we have developed a functional assay to measure NK-cell killing through KIR-ligand interactions. NK-cell lysis of target cells is blocked by inhibitory KIR that recognize classical HLA class I allotypes and HLA mismatches of an altered allelic repertoire, as in haploidentical HSCT, leading to KIR-ligand mismatch and alloreactive NK cell-mediated target killing (Figure 1A). A cytotoxicity assay was developed based on the NK-cell target HLAnull 721.221 cells, and a panel of targets with enforced expression of HLA genes recognized by KIR. After the killing assay was optimized for high throughput and sensitivity, we used the panel of targets to determine whether bulk populations of donor NK cells could be predicted to kill based on KIR and HLA typing. The results demonstrate patterns of target-cell lysis for the KIR repertoires corresponding, for some donors, with predicted donor-versus-recipient NK-cell alloreactivity (Figure 1B). A relative inhibition of HLA+ target-cell lysis of >30% was associated with binding of KIR to introduced HLA class I molecules. The benefit of this assay to transplant physicians is a tool to actually measure phenotype (lysis), rather than relying on predictive models based on genotype. This assay will be combined with typing data to help identify donors with NK-cell killing function for recipients of haploidentical HSCT and infusion of haploidentical NK cells. Figure 1. (A) Schematic of alloreactivity generated between NK cells that are KIR-ligand mismatched with targets. (B) Observed lysis of 721.221 cells, with enforced expression of HLA class I, by KIR-typed donar(box). Figure 1. (A) Schematic of alloreactivity generated between NK cells that are KIR-ligand mismatched with targets. (B) Observed lysis of 721.221 cells, with enforced expression of HLA class I, by KIR-typed donar(box).

Download Full-text