Increased Susceptibility of the CD57– NK Cells Expressing KIR2DL2/3 and NKG2C to iCasp9 Gene Retroviral Transduction and the Relationships with Proliferative Potential, Activation Degree, and Death Induction Response

Nowadays, the use of genetically modified NK cells is a promising strategy for cancer immunotherapy. The additional insertion of genes capable of inducing cell suicide allows for the timely elimination of the modified NK cells. Different subsets of the heterogenic NK cell population may differ in proliferative potential, in susceptibility to genetic viral transduction, and to the subsequent induction of cell death. The CD57–NKG2C+ NK cells are of special interest as potential candidates for therapeutic usage due to their high proliferative potential and certain features of adaptive NK cells. In this study, CD57– NK cell subsets differing in KIR2DL2/3 and NKG2C expression were transduced with the iCasp9 suicide gene. The highest transduction efficacy was observed in the KIR2DL2/3+NKG2C+ NK cell subset, which demonstrated an increased proliferative potential with prolonged cultivation. The increased transduction efficiency of the cell cultures was associated with the higher expression level of the HLA-DR activation marker. Among the iCasp9-transduced subsets, KIR2DL2/3+ cells had the weakest response to the apoptosis induction by the chemical inductor of dimerization (CID). Thus, KIR2DL2/3+NKG2C+ NK cells showed an increased susceptibility to the iCasp9 retroviral transduction, which was associated with higher proliferative potential and activation status. However, the complete elimination of these cells with CID is impeded.

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Lack of proliferative capacity of human effector and memory T cells expressing killer cell lectinlike receptor G1 (KLRG1)

Blood ◽

10.1182/blood-2002-02-0657 ◽

2002 ◽

Vol 100 (10) ◽

pp. 3698-3702 ◽

Cited By ~ 209

Author(s):

David Voehringer ◽

Marie Koschella ◽

Hanspeter Pircher

Keyword(s):

T Cells ◽

Nk Cells ◽

Nk Cell ◽

Killer Cell ◽

Cell Subset ◽

Peripheral Blood Lymphocytes ◽

Proliferative Potential ◽

Proliferative Capacity ◽

Effector T Cell ◽

Proliferation Of Lymphocytes

Adaptive immunity necessitates the proliferation of lymphocytes. In the mouse, we have previously shown that antigen-experienced T cells that have lost their proliferative potential express the killer cell lectinlike receptor G1 (KLRG1). By using a newly generated monoclonal antibody specific for human KLRG1, we now demonstrate that expression of KLRG1 also identifies T cells in humans that are capable of secreting cytokines but that fail to proliferate after stimulation. Furthermore, our data show that proliferative incapacity of CD8 T cells correlates better with KLRG1 expression than with absence of the CD28 marker. In peripheral blood lymphocytes (PBLs) from healthy adult donors, KLRG1 was expressed on 44% ± 14% of CD8 and 18% ± 10% of CD4 T cells. KLRG1 expression was restricted to antigen-experienced T cells. Here, KLRG1+ cells were preferentially found in the CCR7− effector T-cell pool. Besides T cells, a significant portion (approximately 50%) of human natural killer (NK) cells expressed KLRG1. Interestingly, these KLRG1+ NK cells were found exclusively in the CD56dim NK-cell subset. Thus, the expression of KLRG1 identifies a subset of NK cells and antigen-experienced T cells in humans that lack proliferative capacity.

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Telomerase Reverse Transcriptase Increases Proliferation and Lifespan of Human NK Cells without Immortalization

Biomedicines ◽

10.3390/biomedicines9060662 ◽

2021 ◽

Vol 9 (6) ◽

pp. 662

Author(s):

Maria A. Streltsova ◽

Maria O. Ustiuzhanina ◽

Eugene V. Barsov ◽

Sofya A. Kust ◽

Rodion A. Velichinskii ◽

...

Keyword(s):

Life Span ◽

Nk Cells ◽

Cancer Cell ◽

Cell Cultures ◽

Nk Cell ◽

K562 Cells ◽

Proliferative Potential ◽

Cell Therapies ◽

Hla Dr

NK cells are the first line of defense against viruses and malignant cells, and their natural functionality makes these cells a promising candidate for cancer cell therapy. The genetic modifications of NK cells, allowing them to overcome some of their inherent limitations, such as low proliferative potential, can enable their use as a therapeutic product. We demonstrate that hTERT-engineered NK cell cultures maintain a high percentage of cells in the S/G2 phase for an extended time after transduction, while the life span of NK cells is measurably extended. Bulk and clonal NK cell cultures pre-activated in vitro with IL-2 and K562-mbIL21 feeder cells can be transduced with hTERT more efficiently compared with the cells activated with IL-2 alone. Overexpressed hTERT was functionally active in transduced NK cells, which displayed upregulated expression of the activation marker HLA-DR, and decreased expression of the maturation marker CD57 and activating receptor NKp46. Larger numbers of KIR2DL2/3+ cells in hTERT-engineered populations may indicate that NK cells with this phenotype are more susceptible to transduction. The hTERT-modified NK cells demonstrated a high natural cytotoxic response towards K562 cells and stably expressed Ki67, a proliferation marker. Overall, our data show that ectopic hTERT expression in NK cells enhances their activation and proliferation, extends in vitro life span, and can be a useful tool in developing NK-based cancer cell therapies.

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Natural Killer Cells Are Present in Rag1−/− Mice and Promote Tissue Damage During the Acute Phase of Ischemic Stroke

Translational Stroke Research ◽

10.1007/s12975-021-00923-3 ◽

2021 ◽

Author(s):

Leoni Rolfes ◽

Tobias Ruck ◽

Christina David ◽

Stine Mencl ◽

Stefanie Bock ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Adoptive Transfer ◽

Immune Cell ◽

Nk Cell ◽

Cell Subset ◽

Neurological Deficits ◽

In Vitro Assays ◽

Experimental Stroke ◽

Transfer Model

AbstractRag1−/− mice, lacking functional B and T cells, have been extensively used as an adoptive transfer model to evaluate neuroinflammation in stroke research. However, it remains unknown whether natural killer (NK) cell development and functions are altered in Rag1−/− mice as well. This connection has been rarely discussed in previous studies but might have important implications for data interpretation. In contrast, the NOD-Rag1nullIL2rgnull (NRG) mouse model is devoid of NK cells and might therefore eliminate this potential shortcoming. Here, we compare immune-cell frequencies as well as phenotype and effector functions of NK cells in Rag1−/− and wildtype (WT) mice using flow cytometry and functional in vitro assays. Further, we investigate the effect of Rag1−/− NK cells in the transient middle cerebral artery occlusion (tMCAO) model using antibody-mediated depletion of NK cells and adoptive transfer to NRG mice in vivo. NK cells in Rag1−/− were comparable in number and function to those in WT mice. Rag1−/− mice treated with an anti-NK1.1 antibody developed significantly smaller infarctions and improved behavioral scores. Correspondingly, NRG mice supplemented with NK cells were more susceptible to tMCAO, developing infarctions and neurological deficits similar to Rag1−/− controls. Our results indicate that NK cells from Rag1−/− mice are fully functional and should therefore be considered in the interpretation of immune-cell transfer models in experimental stroke. Fortunately, we identified the NRG mice, as a potentially better-suited transfer model to characterize individual cell subset-mediated neuroinflammation in stroke.

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Reduced frequency of cytotoxic CD56dim CD16+ NK cells leads to impaired antibody-dependent degranulation in EBV-positive classical Hodgkin lymphoma

Cancer Immunology Immunotherapy ◽

10.1007/s00262-021-02956-x ◽

2021 ◽

Author(s):

Elena Pánisová ◽

Anna Lünemann ◽

Simone Bürgler ◽

Monika Kotur ◽

Julien Lazarovici ◽

...

Keyword(s):

Nk Cells ◽

Hodgkin Lymphoma ◽

Nk Cell ◽

Cell Subset ◽

Adjunctive Treatment ◽

Classical Hodgkin Lymphoma ◽

Barr Virus ◽

Class 1 ◽

Epstein Barr

AbstractAround 30–50% of classical Hodgkin lymphoma (cHL) cases in immunocompetent individuals from industrialized countries are associated with the B-lymphotropic Epstein-Barr virus (EBV). Although natural killer (NK) cells exhibit anti-viral and anti-tumoral functions, virtually nothing is known about quantitative and qualitative differences in NK cells in patients with EBV+ cHL vs. EBV- cHL. Here, we prospectively investigated 36 cHL patients without known immune suppression or overt immunodeficiency at diagnosis. All 10 EBV+ cHL patients and 25 out 26 EBV- cHL were seropositive for EBV antibodies, and EBV+ cHL patients presented with higher plasma EBV DNA levels compared to EBV- cHL patients. We show that the CD56dim CD16+ NK cell subset was decreased in frequency in EBV+ cHL patients compared to EBV- cHL patients. This quantitative deficiency translates into an impaired CD56dim NK cell mediated degranulation toward rituximab-coated HLA class 1 negative lymphoblastoid cells in EBV+ compared to EBV- cHL patients. We finally observed a trend to a decrease in the rituximab-associated degranulation and ADCC of in vitro expanded NK cells of EBV+ cHL compared to healthy controls. Our findings may impact on the design of adjunctive treatment targeting antibody-dependent cellular cytotoxicity in EBV+ cHL.

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Direct Binding of Human NK Cell Natural Cytotoxicity Receptor NKp44 to the Surfaces of Mycobacteria and Other Bacteria

Infection and Immunity ◽

10.1128/iai.00870-07 ◽

2008 ◽

Vol 76 (4) ◽

pp. 1719-1727 ◽

Cited By ~ 91

Author(s):

Semih Esin ◽

Giovanna Batoni ◽

Claudio Counoupas ◽

Annarita Stringaro ◽

Franca Lisa Brancatisano ◽

...

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Cell Subset ◽

Surface Expression ◽

Binding Assays ◽

Igg Antibody ◽

Direct Stimulation ◽

Activating Receptors ◽

Accessory Cells

ABSTRACT Our previous studies demonstrated that Mycobacterium bovis bacillus Calmette-Guérin (BCG) can directly interact with human NK cells and induce the proliferation, gamma interferon production, and cytotoxic activity of such cells without the need for accessory cells. Thus, the aim of the present study was to identify the putative receptor(s) responsible for the recognition of BCG by human NK cells and potentially involved in the activation of NK cells. To this end, we first investigated the surface expression of three NK cell-activating receptors belonging to the natural cytoxicity receptor (NCR) family on highly purified human NK cells upon in vitro direct stimulation with BCG. An induction of the surface expression of NKp44, but not of NKp30 or NKp46, was observed after 3 and 4 days of in vitro stimulation with live BCG. The NKp44 induction involved mainly a particular NK cell subset expressing the CD56 marker at high density, CD56bright. In order to establish whether NKp44 could directly bind to BCG, whole BCG cells were stained with soluble forms of the three NCRs chimeric for the human immunoglobulin G (IgG) Fc fragment (NKp30-Fc, NKp44-Fc, NKp46-Fc), followed by incubation with a phycoerythrin (PE)-conjugated goat anti-human IgG antibody. Analysis by flow cytometry of the complexes revealed a higher PE fluorescence intensity for BCG incubated with NKp44-Fc than for BCG incubated with NKp30-Fc, NKp46-Fc, or negative controls. The binding of NKp44-Fc to the BCG surface was confirmed with immunogold labeling using transmission electron microscopy, suggesting the presence of a putative ligand(s) for human NKp44 on the BCG cell wall. Similar binding assays performed on a number of gram-positive and gram-negative bacteria revealed a pattern of NKp44-Fc binding restricted to members of the genus Mycobacterium, to the mycobacterium-related species Nocardia farcinica, and to Pseudomonas aeruginosa. Altogether, the results obtained indicate, for the first time, that at least one member of the NCR family (NKp44) may be involved in the direct recognition of bacterial pathogens by human NK cells.

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Medulloblastoma rendered susceptible to NK-cell attack by TGFβ neutralization

Journal of Translational Medicine ◽

10.1186/s12967-019-2055-4 ◽

2019 ◽

Vol 17 (1) ◽

Cited By ~ 4

Author(s):

Allison B. Powell ◽

Sridevi Yadavilli ◽

Devin Saunders ◽

Stacey Van Pelt ◽

Elizabeth Chorvinsky ◽

...

Keyword(s):

Nk Cells ◽

Transforming Growth Factor ◽

Nk Cell ◽

Transforming Growth Factor Β ◽

In Vitro Models ◽

Dominant Negative ◽

Conditioned Media ◽

Retroviral Transduction ◽

Effector Cells

Abstract Background Medulloblastoma (MB), the most common pediatric brain cancer, presents with a poor prognosis in a subset of patients with high risk disease, or at recurrence, where current therapies are ineffective. Cord blood (CB) natural killer (NK) cells may be promising off-the-shelf effector cells for immunotherapy due to their recognition of malignant cells without the need for a known target, ready availability from multiple banks, and their potential to expand exponentially. However, they are currently limited by immune suppressive cytokines secreted in the MB tumor microenvironment including Transforming Growth Factor β (TGF-β). Here, we address this challenge in in vitro models of MB. Methods CB-derived NK cells were modified to express a dominant negative TGF-β receptor II (DNRII) using retroviral transduction. The ability of transduced CB cells to maintain function in the presence of medulloblastoma-conditioned media was then assessed. Results We observed that the cytotoxic ability of nontransduced CB-NK cells was reduced in the presence of TGF-β-rich, medulloblastoma-conditioned media (21.21 ± 1.19% killing at E:T 5:1 in the absence vs. 14.98 ± 2.11% in the presence of medulloblastoma-conditioned media, n = 8, p = 0.02), but was unaffected in CB-derived DNRII-transduced NK cells (21.11 ± 1.84% killing at E:T 5:1 in the absence vs. 21.81 ± 3.37 in the presence of medulloblastoma-conditioned media, n = 8, p = 0.85. We also observed decreased expression of CCR2 in untransduced NK cells (mean CCR2 MFI 826 ± 117 in untransduced NK + MB supernatant from mean CCR2 MFI 1639.29 ± 215 in no MB supernatant, n = 7, p = 0.0156), but not in the transduced cells. Finally, we observed that CB-derived DNRII-transduced NK cells may protect surrounding immune cells by providing a cytokine sink for TGF-β (decreased TGF-β levels of 610 ± 265 pg/mL in CB-derived DNRII-transduced NK cells vs. 1817 ± 342 pg/mL in untransduced cells; p = 0.008). Conclusions CB NK cells expressing a TGF-β DNRII may have a functional advantage over unmodified NK cells in the presence of TGF-β-rich MB, warranting further investigation on its potential applications for patients with medulloblastoma.

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The Ly-49D Receptor Activates Murine Natural Killer Cells

Journal of Experimental Medicine ◽

10.1084/jem.184.6.2119 ◽

1996 ◽

Vol 184 (6) ◽

pp. 2119-2128 ◽

Cited By ~ 135

Author(s):

L.H. Mason ◽

S.K. Anderson ◽

W.M. Yokoyama ◽

H.R.C. Smith ◽

R. Winkler-Pickett ◽

...

Keyword(s):

Gene Family ◽

Nk Cells ◽

Natural Killer ◽

Cell Function ◽

Nk Cell ◽

Cell Subset ◽

Class I ◽

Target Cells ◽

Color Flow ◽

Functional Capabilities

Proteins encoded by members of the Ly-49 gene family are predominantly expressed on murine natural killer (NK) cells. Several members of this gene family have been demonstrated to inhibit NK cell lysis upon recognizing their class I ligands on target cells. In this report, we present data supporting that not all Ly-49 proteins inhibit NK cell function. Our laboratory has generated and characterized a monoclonal antibody (mAb) (12A8) that can be used to recognize the Ly-49D subset of murine NK cells. Transfection of Cos-7 cells with known members of the Ly-49 gene family revealed that 12A8 recognizes Ly-49D, but also cross-reacts with the Ly-49A protein on B6 NK cells. In addition, 12A8 demonstrates reactivity by both immunoprecipitation and two-color flow cytometry analysis with an NK cell subset that is distinct from those expressing Ly-49A, C, or G2. An Ly-49D+ subset of NK cells that did not express Ly49A, C, and G2 was isolated and examined for their functional capabilities. Tumor targets and concanovalin A (ConA) lymphoblasts from a variety of H2 haplotypes were examined for their susceptibility to lysis by Ly-49D+ NK cells. None of the major histocompatibility complex class I–bearing targets inhibited lysis of Ly-49D+ NK cells. More importantly, we demonstrate that the addition of mAb 12A8 to Ly-49D+ NK cells can augment lysis of FcγR+ target cells in a reverse antibody-dependent cellular cytotoxicity–type assay and induces apoptosis in Ly49D+ NK cells. Furthermore, the cytoplasmic domain of Ly-49D does not contain the V/IxYxxL immunoreceptor tyrosine-based inhibitory motif found in Ly-49A, C, or G2 that has been characterized in the human p58 killer inhibitory receptors. Therefore, Ly-49D is the first member of the Ly-49 family characterized as transmitting positive signals to NK cells, rather than inhibiting NK cell function.

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P03.24 Calreticulin exposure on malignant blasts correlates with improved NK cell-mediated cytotoxicity in AML patients

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-itoc7.62 ◽

2020 ◽

Vol 8 (Suppl 2) ◽

pp. A32.1-A32

Author(s):

I Truxova ◽

L Kasikova ◽

C Salek ◽

M Hensler ◽

D Lysak ◽

...

Keyword(s):

Nk Cells ◽

Cell Activation ◽

Nk Cell ◽

Type I ◽

Danger Signals ◽

Hla Dr ◽

Patient Prognosis ◽

Molecular Patterns ◽

Damage Associated Molecular Patterns

In some settings, cancer cells responding to treatment undergo an immunogenic form of cell death that is associated with the abundant emission of danger signals in the form of damage-associated molecular patterns. Accumulating preclinical and clinical evidence indicates that danger signals play a crucial role in the (re-)activation of antitumor immune responses in vivo, thus having a major impact on patient prognosis. We have previously demonstrated that the presence of calreticulin on the surface of malignant blasts is a positive prognostic biomarker for patients with acute myeloid leukemia (AML). Calreticulin exposure not only correlated with enhanced T-cell-dependent antitumor immunity in this setting but also affected the number of circulating natural killer (NK) cells upon restoration of normal hematopoiesis. Here, we report that calreticulin exposure on malignant blasts is associated with enhanced NK cell cytotoxic and secretory functions, both in AML patients and in vivo in mice. The ability of calreticulin to stimulate NK-cells relies on CD11c+CD14high cells that, upon exposure to CRT, express higher levels of IL-15Rα, maturation markers (CD86 and HLA- DR) and CCR7. CRT exposure on malignant blasts also correlates with the upregulation of genes coding for type I interferon. This suggests that CD11c+CD14high cells have increased capacity to migrate to secondary lymphoid organs, where can efficiently deliver stimulatory signals (IL-15Rα/IL- 15) to NK cells. These findings delineate a multipronged, clinically relevant mechanism whereby surface-exposed calreticulin favors NK-cell activation in AML patients.Disclosure InformationI. Truxova: None. L. Kasikova: None. C. Salek: None. M. Hensler: None. D. Lysak: None. P. Holicek: None. P. Bilkova: None. M. Holubova: None. X. Chen: None. R. Mikyskova: None. M. Reinis: None. M. Kovar: None. B. Tomalova: None. J.P. Kline: None. L. Galluzzi: None. R. Spisek: None. J. Fucikova: None.

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