scholarly journals Immunocytokines with Target Cell-Restricted IL-15 Activity for Induction of NK Cell Reactivity Against Acute Myeloid Leukemia (AML)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 731-731
Author(s):  
Bastian J. Schmied ◽  
Latifa Zekri ◽  
Martin Pflügler ◽  
Melanie Märklin ◽  
Lothar Kanz ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Nk Cells ◽  
Target Cell ◽  
Nk Cell ◽  
Target Antigen ◽  
Cytokine Release ◽  
Cell Reactivity ◽  
Flow Cytometric ◽  
Patent Applications ◽  
Xenotransplantation Model

Abstract Introduction: The efficacy of monoclonal antibodies (mAbs), which have substantially improved treatment options for cancer patients, largely relies on their ability to induce antibody-dependent cellular cytotoxicity (ADCC) of NK cells. Recently, we have introduced Fc-optimized (SDIE modification) antibodies targeting CD133 and CD135 (FLT3) with improved capacity to induce NK cell reactivity against AML cells (Koerner et al, Leukemia 2017; Hofmann et al, Leukemia 2012). Our FLT3 mAb termed FLYSYN is currently clinically evaluated in AML patients with minimal residual disease (NCT02789254). Notably, NK cell reactivity can be substantially increased by the cytokine IL-15, but clinical application of truly effective doses is currently prevented by substantial side effects due to unspecific immune activation (Conlon et al, JCO 2015). To overcome this limitation and to strengthen therapeutic efficacy, we fused our Fc-optimized CD133 and CD135 mAbs to an IL-15 mutant with abolished binding to IL-15 receptor α (IL-15Rα). The resulting modified immunocytokines (MIC) should substitute trans-presentation of IL-15 by binding to their target antigens on leukemic cells which facilitates stimulation of IL-15Rβ/γ on NK cells. Methods: Comparative analysis of MIC133/MIC135 binding to target cells, target antigen expression and induction of antigen shift was performed by flow cytometry using primary AML cells and target antigen transfected cell lines. NK cell activation was monitored by flow cytometric analyses of activation markers such as CD69 and CD25. Cytokine release, in particular that of IFN-γ, was measured by ELISA. Target cell killing in cocultures of healthy peripheral blood mononuclear cells (PBMC) with primary AML cells or target antigen transfected cell lines was studied by Europium, Xcelligence and flow cytometry based assays. Toxicity against healthy FLT3 expressing cells was studied by flow cytometric analysis of monocytes, dendritic cells and CD34+ cells within healthy PBMC or bone marrow. For in vivo analysis, MIC135 was tested in a NOD.Cg-Prkdc(scid)IL2rg(tmWjl)/Sz xenotransplantation model by inducing leukemia with primary AML cells and polyclonal NK cells as effector cells. Results: Functional analyses confirmed target antigen-restricted binding of MIC133/MIC135 with saturating doses reached at approximately 1µg/ml. FLT3 was found to be expressed on primary AML cells with significantly higher extent and to be less susceptible to antigen shift compared to CD133. Analysis of activation and cytokine release, the latter being particularly relevant for side effects, demonstrated that MIC proteins stimulate NK cells in a target cell-restricted manner and to a profoundly greater extent than their Fc-optimized counterparts without IL-15. In line, target cell killing induced by either MIC was clearly superior to that of the respective Fc-optimized CD133 and FLT3 mAbs as revealed by various experimental systems using primary AML cells. MIC135, which was chosen for further development due to its superior characteristics described above, did not induce unwanted effects against healthy FLT3 expressing cells and potently reduced leukemic burden in a NSG xenotransplantation model with primary AML and polyclonal NK cells. Conclusion: In summary, MIC stimulate NK cells in a target cell-restricted manner, clearly outperform Fc-optimized antibodies and thus constitute a promising treatment option for AML. Disclosures Jung: Several patent applications: Patents & Royalties: e.g. EP3064507A1. Salih:Several patent applications: Patents & Royalties: e.g. EP3064507A1.

Natural Killer Cells In Pediatric Acute Lymphoblastic Leukemia Patients At Diagnosis Demonstrate An Inhibitory Phenotype and Reduced Cytolytic Capacity

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1397-1397 ◽  
Author(s):  
Rayne H. Rouce ◽  
Takuya Sekine ◽  
Gerrit Weber ◽  
Claude Chew ◽  
Katayoun Rezvani ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Nk Cells ◽  
Nk Cell ◽  
Lymphoblastic Leukemia ◽  
Target Cells ◽  
Healthy Controls ◽  
Cytokine Release ◽  
Absolute Count ◽  
Flow Cytometric ◽  
Pediatric All

Abstract Background Natural killer (NK) cells are a key component of innate immunity, with the potential to recognize and kill transformed malignant cells without prior sensitization. A balance between activating and inhibitory signals from cell surface receptors determines NK cell cytotoxicity and cytokine release. Therapeutic approaches to augmenting NK cell function are being explored in various malignancies. Little is known about NK phenotype and function in patients with childhood acute lymphoblastic leukemia (ALL), the most common childhood cancer. Here we describe an inhibitory phenotype and impaired cytolytic function in NK cells from pediatric ALL patients at diagnosis, compared with healthy pediatric controls. Restoring NK function may be a useful therapeutic approach in ALL. Methods Peripheral blood mononuclear cells (PBMCs) were isolated from 25 patients with newly diagnosed B-ALL, age 1-16 years, and 7 healthy controls, age 2-13 years, in order to compare NK cell frequency, immunophenotype, and functional activity. NK frequency was assessed by flow cytometric staining for CD56+CD3- cells. NK phenotype was assessed by surface expression of activating receptors NKp30, NKp44, NKp46 and NKG2D and inhibitory receptors KIR2DL1/S1, KIR2DL2/S2, KIR3DL1 and NKG2A. Functional activity was determined by incubation of NKs with target cells, followed by flow cytometric measurement of degranulation (surface CD107a) and cytokine release (intracellular IFNg and TNFa). Targets included the MHC class I deficient K562 cell line and, where available, autologous ALL blasts. Results ALL patients demonstrated significantly lower absolute NK cell counts compared with healthy controls (mean absolute count 168 vs. 406 cells/uL, p = 0.0002). They also exhibited significantly fewer NK cells expressing the activating marker NKp46 (mean absolute count 70 vs. 165, p = 0.016); and a significantly higher percentage of cells expressing the inhibitory marker NKG2A (mean 20.5% vs. 1.95% in controls, p = 0.012) (Fig 1A). In co-culture assays with K562 target cells, ALL patients' NK cells demonstrated inferior degranulation and cytokine release compared to healthy controls (representative data in Fig 1B; mean IFNγ production of 1.2% vs. 4.8%, p = 0.02; mean TNFα production of 1.8% vs. 3.8%, p = 0.06; and mean surface CD107a of 5.4% vs. 15.1%, p = 0.08). ALL samples (n = 3) demonstrated little to no cytokine release when incubated with autologous blasts compared with the response elicited by PMA-ionomycin (representative data in Fig 1C; mean CD107a 0.92% vs. 7.85%, p = 0.04; mean IFNγ 0.26% vs 40.47%, p = 0.10; mean TNFα 0.2% vs 41%, p = 0.008). Conclusion At diagnosis, pediatric ALL patients exhibit a lower frequency of NK cells, an inhibitory phenotype, and decreased cytolytic activity compared to healthy pediatric controls, particularly against autologous leukemic blasts. These results suggest that augmentation of the NK response may be useful therapeutically to improve outcomes in childhood ALL. Disclosures: No relevant conflicts of interest to declare.

Induction of NK Cell Reactivity Against AML Cells by Fc-Engineered RANK-Ig Fusion Proteins.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2625-2625
Author(s):  
Tina Nuebling ◽  
Benjamin J Schmiedel ◽  
Miyuki Azuma ◽  
Pascal Schneider ◽  
Ludger Grosse-Hovest ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Nk Cells ◽  
Fusion Proteins ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Residual Disease ◽  
Target Antigen ◽  
Dependent Manner ◽  
Cell Reactivity ◽  
Allogenic Stem Cell Transplantation

Abstract Abstract 2625 NK cells are cytotoxic lymphocytes that play an important role in anti-tumor immunity. A clinically important feature of NK cells is their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) upon application of anti-tumor antibodies. In acute myeloid leukemia (AML) NK cells largely contribute to the therapeutic efficacy allogenic stem cell transplantation (SCT). Recently we demonstrated that AML cells functionally express the TNF family member RANK ligand (RANKL) which impairs NK cell anti-leukemia reactivity (Schmiedel et al., ASH annual meeting 2011). Here we developed a strategy to combine blocking of the NK inhibitory effects of RANKL with targeting of AML cells for NK cell ADCC. To this end we generated fusion proteins consisting of the extracellular domain of RANK and a human IgG1 Fc part that was modified by amino acid exchange. Compared to wild type RANK-Fc fusion protein (RANK-Fc-WT), our mutant RANK-Fc-ADCC (S239D/I332E) displayed highly enhanced affinity to FcγRIIIa (CD16) on NK cells. Primary AML cells expressed substantial levels of RANKL in 53 of 78 (68%) investigated patient cases, and our RANK-Ig fusion proteins bound to AML cells in a target antigen-specific manner. Treatment with both RANK-Fc-WT and RANK-Fc-ADCC clearly reduced the release of RANKL-induced immunomodulatory factors like TNF, IL-6, IL-8 and IL-10 by AML cells. When the effects of the fusion proteins on NK cell ADCC were studied we found that treatment with RANK-Fc-WT only slightly enhanced NK cell reactivity against RANKL-positive patient AML cells. However, RANK-Fc-ADCC potently induced NK cell ADCC and cytokine production in response to AML targets in a target antigen-dependent manner due to the functional properties of its engineered Fc moiety. Taken together, our Fc-engineered RANK-Fc-ADCC fusion protein may serve to modulate the cytokine milieu involved in AML pathophysiology and target RANKL-expressing leukemia cells for NK anti-tumor reactivity. Thus, RANK-Fc-ADCC constitutes an attractive immunotherapeutic means for the treatment of AML, e.g. for elimination of minimal residual disease after conventional therapy including SCT. Disclosures: No relevant conflicts of interest to declare.

Engineering of Trispecific Molecule That Induces Natural Killer Cell Expansion While Mediating CD133-Specific Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3518-3518
Author(s):  
Jörg Uwe Schmohl ◽  
Martin Felices ◽  
Jeffrey S. Miller ◽  
Dan Vallera
Keyword(s):  
Flow Cytometry ◽  
Nk Cells ◽  
Board Of Directors ◽  
Cell Expansion ◽  
Nk Cell ◽  
Hematologic Malignancies ◽  
Cytokine Release ◽  
Advisory Committees ◽  
Anti Cancer ◽  
Ifn Γ

Abstract Background: Selective cancer stem cell (CSC) elimination in solid as well as in and hematologic malignancies is a critical goal in immunotherapy since CSC cause drug refractory relapse. Furthermore this small cell group is known for tumor initiation and self-renewal capabilities and was already described to be a valuable target in breast, head and neck as well as in ovarian cancer. CSCs are also important for the development of hematologic malignancies and might be an excellent cell to target, currently not addressed through current therapies, in order to prevent relapse. The design of state-of-art anti-cancer immune engagers should address three important issues including ADCC, CSC targeting, and rapid effector cell expansion that sustains a potent anti-cancer response. In order to improve the current conventional bispecific immune-engager platform, we synthesized a 16133 BiKE consisting of scFvs binding FcγRIII (CD16) on Natural Killer (NK) cells and CD133 on CSC and then introduced a modified IL-15 crosslinker capable of stimulating NK effector cells. Methods: DNA shuffling and ligation techniques were used to assemble and synthesize the 1615133 trispecific NK cell engager (TriKE) (Figure 1A). The construct was tested for specificity using flow cytometry, cytotoxicity using chromium release assays, and lytic degranulation. IL-15-mediated expansion was measured using flow based proliferation assays. Also, Interferon (IFN)-γ release was measured by flow cytometry since it is important in the anti-cancer response. Results: The new TriKE showed specific and efficient induction of NK cell related cytotoxicity as seen in 51chromium release assays with Caco-2 cells, which express high levels of CD133. Activity was superior compared to 16133 BiKE as seen in effector:target ratios of 20:1 (21.9 ± 0.8% vs. 17.9 ± 2.2%), 10:1 (9.4 ± 0.3% vs. 7.9 ± 2.4%) and 5:1 (4.3 ± 0.2% vs. 5.4 ± 1.5%) (Figure 1B). Proliferation and NK expansion with the 1615133 TriKE was far greater than that achieved with the BiKE devoid of IL-15 as tested with purified NK cells exposed to both drugs for 7 days and stained with a reactive dye (Proliferation index 1.7 ± 0.3 vs. 1.2 ± 0.01, p=<0.0001, n=5). Drug binding and induction of cytotoxic degranulation was CD133+ specific as proved with Caco-2 and Raji cells as positive and negative controls (respectively). In Caco-2 cell targets the BiKE as well as the TriKE showed significant superior activity compared to, NCI IL-15, anti-CD16 scFv and anti-CD133 scFv controls (CD107a expression 37.5 ± 0.2% and 36.9 ± 0.2% vs. 19.6 ± 0.1, 18.3 ± 0.7, 12.6 ± 0.4, p<0.001, n=3), (Figure 1C). NK cell related cytokine release measured via IFN-γ detection was higher in the TriKE compared the BiKE (38.3 ± 0.2 % vs. 13.1 ± 0.3 %, p<0.001, n=3) and higher than all other controls. NK cell related cytokine release studies showed that although IFN-γ levels were elevated, they did not approach the levels achieved with IL-12/IL-18 (38.3 ± 0.2 % vs. 60 ± 0.2 %, p<0.001, n=3) indicating that the release induced with the TriKE was not at supraphysiologic levels. Conclusion: 1615133 TriKE showed specific and improved anti-cancer activity over BiKE and provides a self-sustaining mechanism via induction of IL-15 signaling on NK cells. Inclusion of IL-15 might be a promising platform technology since CD133 can be substituted by other promising tumor associated antigens to create a highly specific and efficient drug. By improving NK cell performance, the new TriKE represents a highly active drug against drug refractory relapse inducing CSC with an encouraging safety profile. Disclosures Miller: Oxis Biotech Scientific Advisory Board: Membership on an entity's Board of Directors or advisory committees. Vallera:Oxis Biotech: Consultancy, Membership on an entity's Board of Directors or advisory committees.

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.

542 Expansion of cytotoxic NK Cells from PBMCs using individualized cytokine combination

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A578-A578
Author(s):  
Andreia Maia ◽  
Joana Lerias ◽  
Markus Maeurer ◽  
Mireia Castillo-Martin
Keyword(s):  
Flow Cytometry ◽  
Nk Cells ◽  
Natural Killer ◽  
Peripheral Blood ◽  
Blood Donors ◽  
Nk Cell ◽  
Tumour Cells ◽  
List Type ◽  
Mhc I ◽  
Cytokine Combination

BackgroundAdoptive immunotherapy relies on the use of T-cells to target tumour cells, through Major Histocompatibility Complex (MHC) Class I recognition(1). However, many tumours display alterations in the MHC-I pathway, a well-described immune evasion mechanism(2). Natural Killer (NK) cells recognize transformed cells independently from the presence of MHC-I and may be a reliable therapeutic option for patients with altered tumour MHC-I expression. The source of NK cells may be autologous or allogeneic and NK cells are also clinically relevant recipients of transgenic receptors (TCRs or antibodies) targeting tumour cells. NK cells have been categorized according to their CD56 and CD16 surface expression into different subpopulations: cytotoxic (CD56+CD16+) and regulatory (CD56brightCD16-)(3). Expanding cytotoxic NK cells is challenging, since the frequency of NK cells is low in peripheral blood(4) and there is also – at this point – not an optimal expansion protocol available.The goal of this project is to determine the best cytokine combination that facilitates expansion of cytotoxic NK cells that either target tumor cells directly or serve as recipients for transgenic receptors.MethodsPeripheral Blood Mononuclear Cells (PBMCs) were extracted using Ficoll methodology from blood donors and cultured in T25 flasks with Cell Genix Medium supplemented with 10% human serum and antibiotics. NK cells were expanded supplemented with feeder cells (ratio 1:1) and different cytokine combinations (1000 U/mL of IL-2, 10 U/ml of IL-12, 180 U/mL of IL-15 and/or 1 U/mL of IL-21) during 20 days. The immunophenotype of expanded NK cells was analyzed at days 0, 5, 10, 15 and 20 by flow cytometry. The cytotoxicity of NK cells was measured by a CD107a Assay or by a Total Cytotoxicity and Apoptosis Assay at days 10 and 20. Thirteen different cytokine combinations were tested.Results4/13 cytokine combinations produced a statistically significant increase of the absolute number of NK cells with a higher percentage of cytotoxic NK cells (figure 1). However, induction of cytotoxicity was not associated with a strong NK cell expansion. The regulatory NK cells subset (CD56brightCD16-) showed the highest percentage of CD107a-expressing cells, more than the CD56+CD16+, the most cytotoxic subpopulation of NK cells.Abstract 542 Figure 1Representative percentage of NK cells in total lymphocytes (A), CD56+CD16+ subpopulation in total NK cells (B), and CD56brightCD16- subpopulation amongst total NK cells (C) at different time points (5, 10, 15 and 20 days) expanded from PBMCs* p-value < 0.05ConclusionsThis work shows that we are able to grow and efficiently expand NK cells from PBMCs with different cytokine combinations leading to clinically relevant NK cell numbers as well as cytotoxic functions. This enables to produce NK cell products for therapy and as recipients for transgenic tumor antigen-specific receptors.AcknowledgementsThe authors would like to thank the Champalimaud Foundation Biobank, the Vivarium Facility and the Flow Cytometry Platform of the Champalimaud Centre for the Unknown.Ethics ApprovalThis study was approved by the Champalimaud Foundation Ethics Committee and by the Ethics Research Committee of NOVA Medical School of NOVA University of Lisbon.ConsentWritten informed consent was obtained from the blood donors to use their samples for research purposes.ReferencesRosenberg SA, Restifo NP, Yang JC, Morgan RA, Mark E. Adoptive cell transfer: a clinical path to effective cancer immunotherapy. Nat Rev Cancer 2008;8(4):299–308.Aptsiauri N, Ruiz-Cabello F, Garrido F. The transition from HLA-I positive to HLA-I negative primary tumors: the road to escape from T-cell responses. Curr Opin Immunol 2018;51:123–32.Di Vito C, Mikulak J, Mavilio D. On the way to become a natural killer cell. Front Immunol. 2019;10(August):1–15.Zotto G Del, Antonini F, Pesce S, Moretta F, Moretta L. Comprehensive phenotyping of human PB NK Cells by Flow Cytometry. 2020;1–9.

scholarly journals Glucocorticoid-induced TNFR-related protein (GITR) ligand modulates cytokine release and NK cell reactivity in chronic lymphocytic leukemia (CLL)

Leukemia ◽  
2011 ◽  
Vol 26 (5) ◽  
pp. 991-1000 ◽  
Author(s):  
C Buechele ◽  
T Baessler ◽  
S Wirths ◽  
J U Schmohl ◽  
B J Schmiedel ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Nk Cell ◽  
Lymphocytic Leukemia ◽  
Cytokine Release ◽  
Related Protein ◽  
Cell Reactivity

scholarly journals Mechanosensation and Mechanotransduction in Natural Killer Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Giorgio Santoni ◽  
Consuelo Amantini ◽  
Matteo Santoni ◽  
Federica Maggi ◽  
Maria Beatrice Morelli ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Target Cell ◽  
Nk Cell ◽  
Tyrosine Phosphatase ◽  
Cell Interaction ◽  
Structural Basis ◽  
Adhesive Interactions

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.

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.

Umbilical Mesenchymal Stem Cell-Derived Extracellular Vesicle Conditioning Has an Immunosuppressive Effect on NK Cells

2021 ◽  
Author(s):  
G. Dostert ◽  
V. Jouan-Hureaux ◽  
H. Louis ◽  
É. Velot
Keyword(s):  
Flow Cytometry ◽  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Nk Cells ◽  
Cell Activation ◽  
Nk Cell ◽  
Cell Communication ◽  
K562 Cells ◽  
Cell Cytotoxicity ◽  
Nk Cell Cytotoxicity

Background: In peripheral blood, human natural killer (NK) cells are immunological cells that nearly don’t express the ectonucleotidase CD73 on their plasma membrane. When exposed to mesenchymal stem cells (MSCs), NK cells are able to acquire CD73. MSCs are known to be CD73-positive (CD73+) and also to modulate the immune system, e.g. through adenosynergic pathway by ectonucleosidases, such as CD73. Extracellular vesicles (EVs) are involved in cell-to-cell communication. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have emerged as paracrine mediators that are part of MSC immunomodulatory effects including immunosuppressive properties and immune privilege. Objective: The aim of our work was to study if CD73 could be acquired by NK cells through cell-to-cell communication with MSC-EVs as cell culture additives. We also hypothesised that MSC-EVs would act as tolerance inducers to attenuate NK cell cytotoxicity. Methods: Cell isolation was made from human umbilical cords for MSCs and from human peripheral blood for NK cells. MSC-EVs were isolated by ultracentrifugation and filtration, then characterized by nanoparticle tracking assay and flow cytometry (CD9, 63, 81 and 73). MSC-EV interaction with NK cells was monitored by PKH67 staining. NK cell activation was followed by measuring the expression of CD73 and NK-activating receptor natural-killer group 2, member D (NKG2D) by flow cytometry. The cytotoxicity of NK cells or EV-conditioned NK cells was evaluated after co-culture with K562 cells. Results: We showed that MSC-EVs are nanoparticles able to express CD73 and interact with NK cells. MSC-EV conditioned NK cells seem to increase CD73 and decrease NKG2D through an EV-mediated mechanism. MSC-EVs have an immunosuppressive effect on NK cells by preventing NK cell activation and NK cell cytotoxicity towards K562 cells. Conclusions: Our results demonstrate that MSC-EVs could influence NK cell behaviour and act as immunosuppressant cell-based products.

scholarly journals CD161 Is Expressed in a Subset of T-Cell Prolymphocytic Leukemia Cases and Is Useful for Disease Follow-up

2019 ◽  
Vol 152 (4) ◽  
pp. 471-478
Author(s):  
Scott R Gilles ◽  
Sophia L Yohe ◽  
Michael A Linden ◽  
Michelle Dolan ◽  
Betsy Hirsch ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Nk Cells ◽  
Retrospective Review ◽  
T Cell Subsets ◽  
Prolymphocytic Leukemia ◽  
Flow Cytometry Data ◽  
Flow Cytometric ◽  
Flow Cytometric Evaluation

AbstractObjectivesCD161 (NKRP1) is a lectin-like receptor present on NK cells and rare T-cell subsets. We have observed CD161 expression in some cases of T-cell prolymphocytic leukemia (T-PLL) and found it to be useful in follow-up and detection of disease after treatment.MethodsRetrospective review of T-PLL cases with complete flow cytometry data including CD161.ResultsWe identified 10 cases of T-PLL with flow cytometric evaluation of CD161 available. Six of these cases were positive for CD161 expression. All CD161-positive cases were positive for CD8 with variable CD4 expression, whereas all CD161-negative cases were negative for CD8. In a case with two neoplastic subsets positive and negative for CD8, only the former expressed CD161.ConclusionsThese novel results suggest that CD161 is often aberrantly expressed in a defined subset of T-PLL positive for CD8. We are showing the utility of this immunophenotype in diagnosis and follow-up.

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