The JAK1/JAK2 Inhibitor Ruxolitinib Substantially Affects NK Cell Biology

Abstract Introduction We recently demonstrated that ruxolitinib (INCB018424), the first approved JAK1/JAK2 inhibitor for treatment of myelofibrosis (MF), exerts potent anti-inflammatory activity. This may at least in part explain higher infection rates observed in ruxolitinib-treated patients. NK cells are critical for cancer-immune surveillance and cytokine-mediated signals are central for proper NK cell activation. We here aimed to characterize in detail the effects of JAK1/2 inhibition on human NK cells. Methods Highly purified CD56+ NK cells were isolated from human peripheral buffy coats by magnetic bead isolation and subsequently exposed to increasing concentrations of ruxolitinib (0.1-10 µM). Cytokine (1000U/ml IL-2, 25ng/ml IL-15)-induced NK cell proliferation was analyzed by CFSE dilution. Phenotypic and functional NK cell activation markers (NKp46, NKG2D, Granzyme B, CD16, and CD69) were analyzed by flow cytometry (including CD107a expression for degranulation). NK cell function was tested by flow-cytometry-based killing assays and quantification of IFN-γ production upon stimulation with either MHC class I-deficient K562 target cells or cytokines (IL-12, IL-18). In addition, phenotypic and functional analyses were also tested during NK receptor activation via plate-bound activating NKp46 antibodies. Signaling events were analyzed by Western Blot analysis to detect phosphorylation of JAK1 and JAK2 as well as by applying phospho-flow technology to evaluate ruxolitinib-mediated changes of cytokine-dependent signalling cascades (pS6, pSTAT1, pSTAT3, pSTAT5, pERK, pAKT, pP38, and pZAP70). Results Our results demonstrate provide first evidence that ruxolitinib profoundly affects cytokine-induced NK cell activation. This includes a significant and dose-dependent reduction of NK cell proliferation, reduced induction of activation-associated surface markers (including NKp46, NKG2D, Granzyme B, CD16, CD69) as well as impaired killing activity against the classical NK target cell line K562. In addition, all main functional activities of NK cells are down-regulated as shown by reduced cytotoxic capacity, impaired degranulation and IFN-γ production. After wash-out, the inhibitory effects of ruxolitinib on NK cells are fully reversible, as shown by proper re-activation by cytokines. In contrast to cytokine-mediated NK cell activation, stimulation via the NK-specific receptor NKp46 are not affected by ruxolitinib. Of note, ruxolitinib does not affect NK cell viability. On a molecular level, phospho-flow analyses revealed that cytokine associated signaling events, such as phosphorylation of STAT5 and S6 were dose-dependently reduced by ruxolitinib in primary human NK cells. Conclusions Ruxolitinib strongly inhibits NK cell activation leading to impaired proliferation and functional activity. Experiments verifying these effects in patients are currently ongoing and will be presented at the meeting. Our findings may have important clinical implications, when considering the application of ruxolitinib as GvHD therapy, because NK cells are critically involved in the GvL effect after allogeneic stem cell transplantation. Disclosures: Wolf: Novartis: Honoraria, Research Funding.

Download Full-text

Dengue Virus-Infected Dendritic Cells, but Not Monocytes, Activate Natural Killer Cells through a Contact-Dependent Mechanism Involving Adhesion Molecules

mBio ◽

10.1128/mbio.00741-17 ◽

2017 ◽

Vol 8 (4) ◽

Cited By ~ 25

Author(s):

Vivian Vasconcelos Costa ◽

Weijian Ye ◽

Qingfeng Chen ◽

Mauro Martins Teixeira ◽

Peter Preiser ◽

...

Keyword(s):

Dendritic Cells ◽

Virus Infection ◽

Dengue Virus ◽

Nk Cells ◽

Adhesion Molecules ◽

Cell Activation ◽

Nk Cell ◽

Denv Infection ◽

Ifn Γ

ABSTRACT Natural killer (NK) cells play a protective role against dengue virus (DENV) infection, but the cellular and molecular mechanisms are not fully understood. Using an optimized humanized mouse model, we show that human NK cells, through the secretion of gamma interferon (IFN-γ), are critical in the early defense against DENV infection. Depletion of NK cells or neutralization of IFN-γ leads to increased viremia and more severe thrombocytopenia and liver damage in humanized mice. In vitro studies using autologous human NK cells show that DENV-infected monocyte-derived dendritic cells (MDDCs), but not monocytes, activate NK cells in a contact-dependent manner, resulting in upregulation of CD69 and CD25 and secretion of IFN-γ. Blocking adhesion molecules (LFA-1, DNAM-1, CD2, and 2β4) on NK cells abolishes NK cell activation, IFN-γ secretion, and the control of DENV replication. NK cells activated by infected MDDCs also inhibit DENV infection in monocytes. These findings show the essential role of human NK cells in protection against acute DENV infection in vivo, identify adhesion molecules and dendritic cells required for NK cell activation, and delineate the sequence of events for NK cell activation and protection against DENV infection. IMPORTANCE Dengue is a mosquito-transmitted viral disease with a range of symptoms, from mild fever to life-threatening dengue hemorrhagic fever. The diverse disease manifestation is thought to result from a complex interplay between viral and host factors. Using mice engrafted with a human immune system, we show that human NK cells inhibit virus infection through secretion of the cytokine gamma interferon and reduce disease pathogenesis, including depletion of platelets and liver damage. During a natural infection, DENV initially infects dendritic cells in the skin. We find that NK cells interact with infected dendritic cells through physical contact mediated by adhesion molecules and become activated before they can control virus infection. These results show a critical role of human NK cells in controlling DENV infection in vivo and reveal the sequence of molecular and cellular events that activate NK cells to control dengue virus infection. IMPORTANCE Dengue is a mosquito-transmitted viral disease with a range of symptoms, from mild fever to life-threatening dengue hemorrhagic fever. The diverse disease manifestation is thought to result from a complex interplay between viral and host factors. Using mice engrafted with a human immune system, we show that human NK cells inhibit virus infection through secretion of the cytokine gamma interferon and reduce disease pathogenesis, including depletion of platelets and liver damage. During a natural infection, DENV initially infects dendritic cells in the skin. We find that NK cells interact with infected dendritic cells through physical contact mediated by adhesion molecules and become activated before they can control virus infection. These results show a critical role of human NK cells in controlling DENV infection in vivo and reveal the sequence of molecular and cellular events that activate NK cells to control dengue virus infection.

Download Full-text

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

Stem Cells and Regenerative Medicine - Biomedical and Health Research ◽

10.3233/bhr210028 ◽

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.

Download Full-text

Aberrant anti-viral response of natural killer cells in severe asthma

European Respiratory Journal ◽

10.1183/13993003.02422-2018 ◽

2020 ◽

Vol 55 (5) ◽

pp. 1802422

Author(s):

Justine Devulder ◽

Cécile Chenivesse ◽

Valérie Ledroit ◽

Stéphanie Fry ◽

Pierre-Emmanuel Lobert ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Severe Asthma ◽

Cell Activation ◽

Mononuclear Cells ◽

Nk Cell ◽

Peripheral Blood Mononuclear ◽

Healthy Donors ◽

Asthma Patients ◽

Ifn Γ

Rhinovirus infections are the main cause of asthma exacerbations. As natural killer (NK) cells are important actors of the antiviral innate response, we aimed at evaluating the functions of NK cells from severe asthma patients in response to rhinovirus-like molecules or rhinoviruses.Peripheral blood mononuclear cells from patients with severe asthma and healthy donors were stimulated with pathogen-like molecules or with the rhinoviruses (RV)-A9 and RV-2. NK cell activation, degranulation and interferon (IFN)-γ expression were analysed.NK cells from severe asthma patients were less cytotoxic than those from healthy donors in response to toll-like receptor (TLR)3, TLR7/8 or RV-A9 but not in response to RV-2 stimulation. Furthermore, when cultured with interleukin (IL)-12+IL-15, cytokines which are produced during viral infections, NK cells from patients with severe asthma were less cytotoxic and expressed less IFN-γ than NK cells from healthy donors. NK cells from severe asthmatics exhibited an exhausted phenotype, with an increased expression of the checkpoint molecule Tim-3.Together, our findings indicate that the activation of NK cells from patients with severe asthma may be insufficient during some but not all respiratory infections. The exhausted phenotype may participate in NK cell impairment and aggravation of viral-induced asthma exacerbation in these patients.

Download Full-text

Natural-killer cells and dendritic cells: “l'union fait la force”

Blood ◽

10.1182/blood-2005-03-1154 ◽

2005 ◽

Vol 106 (7) ◽

pp. 2252-2258 ◽

Cited By ~ 386

Author(s):

Thierry Walzer ◽

Marc Dalod ◽

Scott H. Robbins ◽

Laurence Zitvogel ◽

Eric Vivier

Keyword(s):

Dendritic Cells ◽

Nk Cells ◽

Natural Killer ◽

Cell Activation ◽

Nk Cell ◽

Interleukin 12 ◽

Cell Contact ◽

Ifn Γ

AbstractSeveral recent publications have focused on the newly described interactions between natural-killer (NK) cells and dendritic cells (DCs). Activated NK cells induce DC maturation either directly or in synergy with suboptimal levels of microbial signals. Immature DCs appear susceptible to autologous NK-cell-mediated cytolysis while mature DCs are protected. NK-cell-induced DC activation is dependent on both tumor necrosis factor-α (TNF-α)/interferon-γ (IFN-γ) secretion and a cell-cell contact involving NKp30. In vitro, interleukin-12 (IL-12)/IL-18, IL-15, and IFN-α/β production by activated DCs enhance, in turn, NK-cell IFN-γ production, proliferation, and cytotoxic potential, respectively. In vivo, NK-cell/DC interactions may occur in lymphoid organs as well as in nonlymphoid tissues, and their consequences are multiple. By inducing DC activation, NK-cell activation induced by tumor cells can indirectly promote antitumoral T-cell responses. Reciprocally, DCs activated through Toll-like receptors (TLRs) induce potent NK-cell activation in antiviral responses. Thus, DCs and NK cells are equipped with complementary sets of receptors that allow the recognition of various pathogenic agents, emphasizing the role of NK-cell/DC crosstalk in the coordination of innate and adaptive immune responses.

Download Full-text

Development and Function of NK and T Cells in AML Patients after Allogeneic Stem Cell Transplantation (SCT).

Blood ◽

10.1182/blood.v104.11.3244.3244 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3244-3244

Author(s):

Gabriele Multhoff ◽

Catharina Gross ◽

Anne Dickinson ◽

Ernst Holler

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

Stem Cell ◽

Nk Cells ◽

Nk Cell ◽

Granzyme B ◽

Hla Class I ◽

K562 Cells ◽

Cytolytic Response

Abstract Purpose: Hsp70 was frequently found on the plasma membrane of bone marrow-derived leukemic blasts, but not on normal bone marrow cells. Hsp70 membrane expression could be correlated with protection against therapy-induced apoptosis (Nylandsted et al 2004). In contrast, these tumor cells have been found to be highly sensitive to the cytolytic attack mediated by NK cells. In vitro, Hsp70-activated NK cells efficiently lysed autologous Hsp70 membrane-positive leukemic blasts (Gehrmann et al 2003). Granzyme B release served as a surrogate marker for estimating the cytolytic response of NK cells against Hsp70 membrane-positive tumor target cells (Gross et al 2003). Here, we studied the development of NK and T cells in AML patients (n=6) after allogeneic SCT at different time points (days 14–20, 45, 90, 180, 1 year) after allogeneic stem cell transplantation (SCT). Methods: HLA class I, HLA-E and Hsp70 surface expression was determined on all patient-derived leukemic blasts of the bone marrow by flow cytometry. The amount of NK and T cells was investigated by multicolor flow cytometry using CD3/ CD16 and CD56 and CD94/ CD56 antibody-combinations detecting NK cell specific markers. Effector cell function was tested in a granzyme B ELISPOT assay against patient-derived leukemic blasts and K562 cells. Results: All tested leukemic blasts were positive for HLA class I, HLA-E, and Hsp70. After induction therapy the amount of CD3-negative, CD56/CD94-positive NK cells was 28±16%, that of CD3-positive T cells was 58±3%. On days 14–21 after allogeneic SCT, 58±9% of the donor-derived peripheral blood lymphocytes (PBL) were CD3-negative, CD56/CD94-positive NK cells; the amount of CD3-positive T cells was 26±7.5%. On day 45, the amount of NK cells further increased up to 68±7.9%; that of T cells further decreased down to 16±5.6%. On day 90 and day 180 the amount of NK cells was still 41±10%; that of T cells was 29±12%. Interestingly, high NK cell counts correlated with an increased cytolytic response against leukemic blast and K562 cells. One year after allogeneic SCT, NK (20±1%) and T cell (52±18%) ratios were comparable to that of healthy human individuals. Conclusions: Between days 14 and 180 after allogeneic SCT, the amount of NK cells was significantly elevated if compared to that of T cells. Concomitantly, cytolytic function against leukemic blasts was significantly elevated. Normal levels, in the composition of NK and T cells were reached 1 year after SCT. Project funded by EU-TRANS-EUROPE grant QLK3-CT-2002-01936.

Download Full-text

Enhanced NK Cell Activation, Cytotoxicity and Ex-Vivo Expansion (EvE) of Cryopreserved Cord Blood (CB) Natural Killer (NK) Cells: Potential Role for CB NK Cells in Adoptive Cellular Immunotherapy (ACI).

Blood ◽

10.1182/blood.v108.11.726.726 ◽

2006 ◽

Vol 108 (11) ◽

pp. 726-726

Author(s):

Janet Ayello ◽

Julia Nemiroff ◽

Prakash Satwani ◽

Carmella van de Ven ◽

Evan Shereck ◽

...

Keyword(s):

Nk Cells ◽

Cell Activation ◽

Nk Cell ◽

Ex Vivo ◽

Granzyme B ◽

Cellular Immunotherapy ◽

Common Mechanism ◽

Short Term ◽

Cd107a Expression ◽

Short Term Culture

Abstract CD56+ NK subsets exhibit differential NK receptors (NKR ) such as NCR profiles including killer-Ig-like receptors (KIR), C-lectin (NKG2) and natural cytoxicity receptors (NCR) involved with tumor target recognition (Farag et al Blood, 2002). NK cell activation and NK mediated cytolysis is induced by several NKRs such as NCR (i.e. NKp44, NKp46) and NKG2 surface receptors like NKG2D (Moretta et al, Curr Opinion in Immunol, 2004). Target cell killing by activated NK cells via the granule-dependent pathway is a common mechanism of NK and CTLs and degranulation is followed by the expression of lysosomal-associated membrane protein-1 [LAMP-1] on the cell surface (Penack et al, Leukemia, 2005). CB is limited by the absence of available donor effector cells (NK, CTL, LAK and NKT cells) for infusion after UCBT (Cairo, et al, Transfusion, 2005). We have demonstrated the ability to EvE CB in short-term culture (48 hrs) with IL-2, IL-7, IL-12 and anti-CD3 (ABCY) cryopreserved, thawed, recryopreserved, rethawed and EvE (CTCTE) CB with significant increase in CD3−/16+/56+ bright/dim subsets expressing KIR3DL1, KIR2DL1/S1, KIR2DL2 and CD94/NKG2a (Ayello/Cairo et al BBMT, 2006). In this study, we compared short-term culture (48 hrs) with prolonged cultures (4 to 10 days) on expansion, expression of NCR, NKG2, KIR and cytolytic ability and mechanisms in CTCTE CB. Rethawed nonadherent CB cells were cultured (2–10 days) in serum-free media alone or with anti-CD3 (50 ng/ml), IL-2 (5 ng/ml), IL-7 (10 ng/ml) and IL-12 (10 ng/ml) [ABCY]. NKR expression (CD94, NKG2D, Nkp44 and KIR2DS4), intracellular perforin, granzyme B activity and LAMP-1 receptor (CD107a) expression were determined by flow cytometry. Cytoxicity was measured by europium release assay and tumor targets used were K562, Daudi, neuroblastoma (SHSY5Y) and AML (Kasumi-1) at a 20:1 E:T ratio. C-lectin activating receptor CD94/NKG2D was increased at day 7 vs 2 following ABCY EvE (41.4±0.43 vs 23.7±2.%, p<0.001). Significant increases were seen in activating KIR2DS4 at day 10 vs 2 in ABCY in both CD3−/16+/56+dim and bright subsets (16.9±0.4 vs 2.1±0.2% and 22.3±0.3 vs 0.9± 0.2%, p<0.001, respectively). In contrast, NCR expression in CD3−/16+/56+dim NKp44 subset was significantly decreased at day 10 vs 2 of EvE CB in ABCY (15.2±0.7 vs 27.2±0.7%, p<0.001). Granzyme B expression was increased from day 2 to 10 (25.8± vs 45.1± 1.7%, p<0.001) yet perforin was decreased in EvE CB in ABCY at day 7 vs 2 (68.3±2.19 vs 84.3±1.3%, p<0.001). CD107a expression was significantly increased at day 7 vs 2 in ABCY EvE CB (12.95±1.47 vs 69.34±2.22%, p<0.001). In addition, significant increases in cytolytic activity was demonstrated at day 7 vs 2 of EvE CB cells in ABCY against tumor targets K562 (71.5±±0.81 vs 53.8±3.9%, p<0.001), Daudi (63.9±0.73 vs 31.8±1.8%, p<0.001), SYSY5Y (76.8±6.5 vs 57.5±3.4%, p<0.05) and Kasumi-1 (56.6.5±0.4 vs 38±1.1%, p<0.001). In summary, CB MNC may be thawed at time of CB transplantation, recryopreserved, rethawed at a later date, EvE and activated for up to 10 days to yield significantly increased cytotolytic activity against NHL, AML and neuroblastoma with increased expression of NK KAR KIR2DS4 and granzyme B, LAMP-1 degranulation (NK activation) but decreased NK C-lection CD94/NKG2D, NCR NKp44 and perforin expression.

Download Full-text

Tim-3, a Novel Immune Receptor, Is Constitutively Expressed on Human Natural Killer Cells and Functions as An Activating Coreceptor

Blood ◽

10.1182/blood.v116.21.106.106 ◽

2010 ◽

Vol 116 (21) ◽

pp. 106-106

Author(s):

Michelle Gleason ◽

Todd Lenvik ◽

Valarie McCullar ◽

Sarah Cooley ◽

Michael Verneris ◽

...

Keyword(s):

Nk Cells ◽

Board Of Directors ◽

Cell Activation ◽

Low Dose ◽

Cell Function ◽

Nk Cell ◽

Effector Function ◽

Advisory Committees ◽

Immune Receptor ◽

Ifn Γ

Abstract Abstract 106 NK cells are an attractive option for immunotherapy as they do not require pre-sensitization for anti-tumor activity and do not induce graft versus host disease (GvHD) in an allogeneic transplant setting. The potential of NK cells in controlling human hematological malignancies has been increasingly recognized in recent years, as the adoptive transfer of alloreactive NK cells in hematopoietic cell transplantation (HCT) clinical trials have demonstrated therapeutic anti-leukemia effects. NK cell function is regulated by the integration of antagonist signals received from cell surface activating and inhibitory receptors. Tim-3 is a novel immune receptor that is a member of the T cell immunoglobulin and mucin-containing domain (TIM) family of glycoproteins. While its role in T cells and antigen presenting cells has been described, little is known about its function in human NK cells. While Tim-3 is present on a variety of immune cells, resting NK cells constitutively express Tim-3 compared to other lymphocyte populations (NK: 73±3%; NKT: 6±1%; T: 1±1%; n=14) and we hypothesized that Tim-3 may be important in mediating NK cell function. The unique subset of cytokine producing CD56Bright NK cells exhibited significantly lower resting Tim-3 expression compared to CD56Dim NK cells (53±3% vs. 75±3%; p<0.001, n=14). Distinct Tim-3 expression patterns were found on resting CD56Dim NK cells and activation with low dose IL-12 (1ng/mL) and IL-18 (10ng/mL), intended to more closely mimic physiologic conditions, resulted in further differentiation of this unique expression pattern dividing NK cells into 4 distinct populations: Tim-3 was homogeneously up-regulated on all CD56Bright NK cells after activation while CD56Dim NK cells were further stratified into 3 defined populations with Tim-3hi, Tim-3lo and Tim-3neg expression. The only identified ligand of Tim-3 is galectin-9 (Gal-9), a β-galactoside binding lectin, which is expressed on a wide range of healthy and malignant cells. To investigate the potential function of Tim-3, an expression vector containing human Gal-9 was transduced into K562 and Raji cells, both without endogenous Gal-9 expression. Resting NK cytotoxicity (51Cr release) was found to be increased in the presence of Gal-9 compared to the non-Gal-9 expressing targets [E:T=0.7:1, K562 vs. K562-Gal-9: 25±3% vs. 33±3% (n=8, p<0.05); E:T=20:1, Raji vs. Raji-Gal-9: 8±1% vs. 17±2% (n=4, p<0.05)]. Analysis of CD107a degranulation showed that resting Tim-3+ CD56Bright cells were more functional against Gal-9 expressing targets than Tim-3− CD56Bright cells, suggesting that Tim-3 might also play a role in IFN-γ production. To further investigate this, resting NK cells were activated with low-dose IL-12/IL-18 overnight and IFN-γ levels were measured in response to soluble rhGal-9 (0, 2.5, 5, 10 and 20nM). Exposure to soluble rhGal-9 alone without IL-12/IL-18 did not induce IFN-γ production. For both the CD56Bright and CD56Dim IL-12/IL-18 activated NK populations, only Tim-3+ NK cells displayed a dose dependent increase in IFN-γ production upon exposure to soluble rhGal-9 compared to Tim-3− NK cells. To understand the relevance of the distinct Tim-3 populations circulating in resting blood, CD56Bright, CD56Dim/Tim-3hi, CD56Dim/Tim-3lo and CD56Dim/Tim-3neg populations were sorted, cultured overnight in IL-12/IL-18 and exposed to soluble rhGal-9. Results showed the Tim-3 expressing populations contain the predominant IFN-γ producing cells that were responsive to rhGal-9 (results for the sorted CD56Dim/Tim-3lo population shown in the figure below). This increase in IFN-γ production within the Tim-3 expressing NK cell populations was abrogated by the addition of β-lactose, a β-galactoside that binds and blocks Gal-9 activity. Lastly, Western blot and immunohistochemistry analysis of human primary acute leukemia blasts revealed high Gal-9 expression. As the presence of ligands for NK cell activating receptors on tumors provide an important prerequisite for NK cell activation and effector function, we show a novel functional role for the receptor Tim-3 in human NK cell biology in the presence of its ligand Gal-9. We, therefore, propose a model where constitutively expressed Tim-3 is up-regulated by NK cell activation and effector function is enhanced by Tim-3/Gal-9 interaction, which may potentiate the elimination of Gal-9 positive tumors by NK cells. Disclosures: Niki: GalPharma: Membership on an entity's Board of Directors or advisory committees. Hirashima:GalPharma: Membership on an entity's Board of Directors or advisory committees.

Download Full-text

AFM13 Is the Most Advanced Bispecific NK-Cell Engaging Antibody in Clinical Development Substantially Enhancing NK-Cell Effector Function and Proliferation

Blood ◽

10.1182/blood.v128.22.1764.1764 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1764-1764 ◽

Cited By ~ 1

Author(s):

Jens Pahl ◽

Uwe Reusch ◽

Thorsten Gantke ◽

Anne Kerber ◽

Joachim Koch ◽

...

Keyword(s):

Nk Cells ◽

Cell Activation ◽

Nk Cell ◽

Target Cells ◽

Cell Cytotoxicity ◽

Cell Receptors ◽

Cell Responses ◽

Nk Cell Receptors ◽

Ifn Γ ◽

Nk Cell Cytotoxicity

Abstract Introduction: AFM13 is an NK-cell engaging CD30/CD16A bispecific tetravalent TandAb antibody currently in phase 2 clinical development in Hodgkin lymphoma (HL) and other CD30+ malignancies. It engages NK-cells through CD16A with high affinity and specificity and confers significantly stronger NK-cell activation compared to other therapeutic antibodies. We have previously shown synergistic efficacy when NK-cell activation by AFM13 is combined with check-point modulation such as anti-PD-1 treatment, which is known to unleash T cell and NK-cell activity. The goal of this study was to identify further candidates for combination treatments and biomarkers that potentially indicate NK-cell responses to AFM13 treatment. Methods: AFM13-mediated NK-cell cytotoxicity and IFN-γ production after 4-hour interaction with HL cell lines was measured by 51Cr release assays and flow cytometry, respectively. Expression of NK-cell receptors, NK-cell proliferation (CFSE dilution) and expansion (absolute cell counts) was analyzed by flow cytometry. Results: The interaction of NK-cells with AFM13-coated tumor cells up-regulated the expression of NK-cell receptors such as CD25, CD69, CD137/4-1BB as well as molecules that may serve as NK-cell check-points when compared with the unrelated NK-cell binding TandAb AFM12 that does not bind to target cells. Importantly, CD16A engagement by AFM13 enhanced the proliferation and expansion potential of NK-cells when subsequently incubated with IL-15 or with particularly low doses of IL-2. NK-cell cytotoxicity and IFN-γ production was substantially increased towards CD30+ tumor cells in the presence of AFM13. Even target cells resistant to naïve and IL-2/IL-15-activated NK-cells were susceptible to AFM13-induced NK-cell cytotoxicity. AFM13 concentrations of as low as 10-2 µg/mL resulted in maximal activity while AFM13 was significantly more potent than native anti-CD30 IgG1 antibody. NK-cell activation by IL-2 or IL-15 had a synergistic effect on AFM13-mediated cytotoxicity. Conclusion: AFM13 specifically enhances the cytotoxic, proliferative and cytokine-producing potential of NK-cells. Our data indicate that the distinctive modulation of NK-cell receptors can be utilized to monitor NK-cell responses during AFM13 therapy and provides candidates for therapeutic combination strategies. Moreover, the combination with low doses of IL-2 or with IL-15 may expand the quantity of tumor-reactive NK-cells after AFM13 treatment and promote NK-cell functionality in the tumor microenvironment in cancer patients. Disclosures Reusch: Affimed: Employment, Patents & Royalties: Patents. Gantke:Affimed GmbH: Employment. Kerber:Affimed: Employment. Koch:Affimed: Employment. Treder:Affimed: Employment. Cerwenka:Affimed: Research Funding.

Download Full-text

Natural killer (NK) cell activation, cytokine production, and cytotoxicity in human PBMC/myeloma cell co-culture exposed to elotuzumab (Elo) alone or in combination with lenalidomide (Len).

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.8087 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. 8087-8087 ◽

Cited By ~ 2

Author(s):

Balaji Balasa ◽

Rui Yun ◽

Nicole Belmar ◽

Gary Starling ◽

Audie Rice

Keyword(s):

Nk Cells ◽

Cell Activation ◽

Nk Cell ◽

Myeloma Cell ◽

Cell Killing ◽

Cell Counting ◽

Target Cells ◽

Human Pbmc ◽

Myeloma Cells ◽

Ifn Γ

8087 Background: Elo is a monoclonal IgG1 antibody targeting CS1, a cell surface glycoprotein highly expressed on >95% of myeloma cells. In preclinical models Elo exerts anti-myeloma activity via NK cell-mediated antibody-dependent cellular cytotoxicity. Len is an immunomodulatory agent that may activate NK cells. The combination of Elo + Len synergistically enhanced anti-tumor activity in myeloma xenograft models. We investigated the mechanism of enhancing NK cell activation and myeloma cell killing with Elo + Len. Methods: Human PBMC/OPM-2 co-cultures were treated for 24-72h with Elo, Len, or Elo + Len. Activation markers and adhesion receptors were evaluated by flow cytometry. Cytokines were measured by Luminex and ELISpot assays. Cytotoxicity was assessed by cell counting. Results: Elo + Len increased IFN-γ secretion significantly more than Elo or Len alone. IFN-γ elevates ICAM-1 expression, and ICAM-1 surface expression on OPM-2 target cells increased synergistically with Elo + Len. Elo, Elo + Len but not Len increased expression of CD25 (IL-2Rα) on NK cells. Len increased the levels of IL-2, but those were decreased in the presence of Elo due to increased consumption by CD25 expressing NK cells. Blocking uptake of IL-2 with anti-CD25 resulted in higher IL-2 levels than with Len. ELISpot assays confirmed that Elo + Len significantly increased the number of IL-2-producing cell colonies compared with Elo or Len. Elo induced NK dependent myeloma cell killing, and the effect was significantly higher with Elo + Len. Conclusions: Elo alone activated NK cells and mediated the killing of myeloma cells in PBMC/OPM-2 co-cultures. Elo + Len synergistically enhanced myeloma cell killing and increased expression/production of IFN-γ, ICAM-1, IL-2, and CD25. [Table: see text]

Download Full-text

Activation of Human NK Cells by Staphylococci and Lactobacilli Requires Cell Contact-Dependent Costimulation by Autologous Monocytes

Clinical and Vaccine Immunology ◽

10.1128/cdli.9.3.649-657.2002 ◽

2002 ◽

Vol 9 (3) ◽

pp. 649-657 ◽

Cited By ~ 17

Author(s):

D. Haller ◽

P. Serrant ◽

D. Granato ◽

E. J. Schiffrin ◽

S. Blum

Keyword(s):

Nk Cells ◽

Cell Activation ◽

Mononuclear Cells ◽

Nk Cell ◽

Interleukin 12 ◽

Accessory Cell ◽

Cell Contact ◽

Gram Positive ◽

Gram Positive Bacteria ◽

Ifn Γ

ABSTRACT NK cells are instrumental in innate immune responses, in particular for the early production of gamma interferon (IFN-γ) and other cytokines necessary to control certain bacterial, parasitic, and viral infections. NK cell-mediated effector functions are controlled by a fine balance between distinct receptors mediating activating and inhibitory signals; however, little is known about activating receptors on NK cells and their corresponding ligands. Several studies have shown that commensal lactobacilli isolated from the human gastrointestinal tract activate human mononuclear cells and are potent inducers of IFN-γ and monocyte-derived interleukin 12 (IL-12). NK cell activation was shown for Lactobacillus johnsonii La1. In this study the cellular mechanisms of in vitro NK cell activation by gram-positive bacteria were analyzed. Staphylococcus aureus- and L. johnsonii La1-mediated activation of CD3− CD16+ CD56+ human peripheral blood NK cells, including expression of the activation antigen CD69 and secretion of IFN-γ, required cell contact-dependent costimulation by autologous monocytes. S. aureus- and L. johnsonii-preactivated monocytes retained their capacity to induce NK cell activation. In contrast, cytokine-primed monocytes completely failed to induce NK cell activation unless bacteria were present. This suggests that phagocytosis of bacteria provided additional coactivation signals on accessory cells that may differ from those induced by tumor necrosis factor and IFN-γ. Blocking of costimulatory molecules by B7.1, B7.2, and IL-12 but not CD14 monoclonal antibodies inhibited S. aureus- and L. johnsonii-induced effector function of NK cells. Our data suggest an important role for accessory cell-derived signals in the process of NK cell activation by gram-positive bacteria.

Download Full-text