NK Cells in Hodgkin Lymphoma Are Impaired but Can Be Activated

Abstract Abstract 2182 Introduction: NK cells represent the key component of the innate immune system to recognize and eliminate cancer cells. Defects in NK cell function including impaired cytotoxicity/cytokine secretion, aberrant receptor expression profile, NK cell number and NK cell anergy are reported in non Hodgkin lymphoma and correlate with a bad prognosis. So far, nothing is known about the phenotype of peripheral NK cells and serum levels of ligands for NK cell receptors in Hodgkin Lymphoma (HL) patients. Here, cytotoxicity, expression pattern of activating NK cell receptors and the serum levels of several ligands for the key cytotoxic receptors NKG2D and NKp30 are determined. Methods: The cytotoxicity of NK cells isolated from HL patients was analysed by europium release assay using the HL cell line L428 as target cells. The serum level of the NKp30-ligand BAT3 and ligands for NKG2D (MICA, MICB and ULBP1,2,3) was estimated in sera of 117 HL patients and 40 healthy donors by ELISA. The expression pattern of NKp30, NKp44, NKp46, CD16 and the activation markers CD25, CD69 and CD71 was determined by 4-colour FACS analysis of peripheral blood lymphocytes. Results: The cytotoxicity assays reveal a significantly reduced killing efficacy of NK cells from HL patients against the Hodgkin cell line L428 in comparison to NK cells from healthy donors. Correlating with the impaired NK cell function, we observed that the serum level for BAT3 and MICA was significantly elevated in HL patients, whereas other ligands (MICB and ULBP1,2,3) remained unchanged. NKG2D showed a significantly decreased expression on NK cells of HL patients. No significant difference was observed for all other receptors and activation markers tested. Conclusion: Our results suggest that soluble BAT3 and MICA, ligands for NKp30 and NKG2D, contribute to the NK cell inhibition in HL patients. Since soluble ligands for NK cell receptors are known to inhibit NK cell-cytotoxicity, the release of these ligands might represent an immune escape mechanism of HL tumors to avoid detection and killing by the innate immune system. To overcome NK cell inhibition in HL patients we design, express and purify bispecific proteins (immunoligands) that target NKG2D and a HL-specific tumorantigen. Work to activate HL-derived NK cells with immunoligands ex vivo will be discussed. Disclosures: Engert: Affimed Therapeutics AG: Honoraria, Research Funding.

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Sialic Acids and Their Influence on Human NK Cell Function

Cells ◽

10.3390/cells10020263 ◽

2021 ◽

Vol 10 (2) ◽

pp. 263

Author(s):

Philip Rosenstock ◽

Thomas Kaufmann

Keyword(s):

Nk Cells ◽

Immune Cells ◽

Cell Function ◽

Nk Cell ◽

Sialic Acids ◽

Innate Immune ◽

Target Cells ◽

Carbon Backbone ◽

Nk Cell Receptors ◽

Cell Inhibition

Sialic acids are sugars with a nine-carbon backbone, present on the surface of all cells in humans, including immune cells and their target cells, with various functions. Natural Killer (NK) cells are cells of the innate immune system, capable of killing virus-infected and tumor cells. Sialic acids can influence the interaction of NK cells with potential targets in several ways. Different NK cell receptors can bind sialic acids, leading to NK cell inhibition or activation. Moreover, NK cells have sialic acids on their surface, which can regulate receptor abundance and activity. This review is focused on how sialic acids on NK cells and their target cells are involved in NK cell function.

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Potential Role of Hsp70 and Activated NK Cells for Prediction of Prognosis in Glioblastoma Patients

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.669366 ◽

2021 ◽

Vol 8 ◽

Author(s):

Dominik Lobinger ◽

Jens Gempt ◽

Wolfgang Sievert ◽

Melanie Barz ◽

Sven Schmitt ◽

...

Keyword(s):

Nk Cells ◽

Tumor Cells ◽

Nk Cell ◽

Human Tumor ◽

Serum Levels ◽

Hsp70 Expression ◽

Cell Receptors ◽

Nk Cell Receptors ◽

Dismal Prognosis ◽

Prediction Of Prognosis

Despite rapid progress in the treatment of many cancers, glioblastoma remains a devastating disease with dismal prognosis. The aim of this study was to identify chaperone- and immune-related biomarkers to improve prediction of outcome in glioblastoma. Depending on its intra- or extracellular localization the major stress-inducible heat shock protein 70 (Hsp70) fulfills different tasks. In the cytosol Hsp70 interferes with pro-apoptotic signaling pathways and thereby protects tumor cells from programmed cell death. Extracellular Hsp70 together with pro-inflammatory cytokines are reported to stimulate the expression of activatory NK cell receptors, recognizing highly aggressive human tumor cells that present Hsp70 on their cell surface. Therefore, intra-, extracellular and membrane-bound Hsp70 levels were assessed in gliomas together with activatory NK cell receptors. All gliomas were found to be membrane Hsp70-positive and high grade gliomas more frequently show an overexpression of Hsp70 in the nucleus and cytosol. Significantly elevated extracellular Hsp70 levels are detected in glioblastomas with large necrotic areas. Overall survival (OS) is more favorable in patients with low Hsp70 serum levels indicating that a high Hsp70 expression is associated with an unfavorable prognosis. The data provide a first hint that elevated frequencies of activated NK cells at diagnosis might be associated with a better clinical outcome.

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Elevated Proportions of Activated Nk Cells at Diagnosis Predict a Favourable Prognosis in Glioblastoma Patients

10.20944/preprints202012.0293.v2 ◽

2021 ◽

Author(s):

Dominik Lobinger ◽

Jens Gempt ◽

Wolfgang Sievert ◽

Melanie Barz ◽

Sven Schmitt ◽

...

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Human Tumor ◽

Serum Levels ◽

Hsp70 Expression ◽

Human Tumor Cells ◽

Cell Receptors ◽

Favorable Prognosis ◽

Nk Cell Receptors ◽

Dismal Prognosis

Despite rapid progress in the treatment of many cancers, glioblastoma remains a devastating disease with dismal prognosis. The aim of this study was to identify immune-related biomarkers that more effectively predict outcome of glioblastoma. Since heat shock protein 70 (Hsp70) and IL-2 are known to increase the expression of activatory NK cell receptors, recognizing aggressive human tumor cells that present Hsp70 on their cell surface, extracellular Hsp70 levels were determined in glioma patients together with activatory NK cell receptors. All gliomas are membrane Hsp70-positive (mHsp70+) and high grade gliomas more frequently show an overexpression of Hsp70 in the nucleus and cytosol. Significantly increased extracellular Hsp70 levels are detected predominantly in glioblastomas with large necrotic areas. Overall survival (OS) is more favorable in patients with low Hsp70 serum levels indicating that a high Hsp70 expression is associated with an unfavorable prognosis. Elevated frequencies of NK cells are associated with a more favorable outcome. Of caution, a glucocorticoid therapy reduces the prevalence of NK cells. In summary, elevated frequencies of Hsp70-reactive NK cells at diagnosis and lower Hsp70 levels predict a more favorable prognosis in glioblastoma patients.

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Low Levels of Circulating CS1, a Newly Identified Multiple Myeloma (MM) Antigen for a Novel Humanized HuLuc63 Monoclonal Antibody, Is Detected in MM Patient Sera and Correlates with Active Disease.

Blood ◽

10.1182/blood.v110.11.1509.1509 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1509-1509 ◽

Cited By ~ 1

Author(s):

Yu-Tzu Tai ◽

Audie G. Rice ◽

Marav Leiba ◽

Xian-Feng Li ◽

Peter Burger ◽

...

Keyword(s):

Nk Cells ◽

Cell Function ◽

Nk Cell ◽

Clinical Investigation ◽

Sandwich Elisa ◽

Serum Levels ◽

Healthy Donors ◽

Physiological Serum ◽

Stage System ◽

Xenograft Mice

Abstract A new humanized anti-CS1 mAb HuLuc63 significantly induces antibody-dependent cellular cytotoxicity (ADCC) against autologous CS1-expressing patient MM cells. Here we examined whether soluble CS1 is detectable in MM patient sera and affects HuLuc63-induced cytotoxicity against MM cells. Using a sandwich ELISA, CS1 was detected in 44% (23/52) of sera of 52 MM patients (1.2 to 35.3 ng/ml). Immunoprecipitation of sera from CS1 ELISA-positive and -negative MM patients using anti-CS1 mAbs (HuLuc63 and ChLuc90) followed by immunoblotting with HuLuc63 and 1G9 recognizing different epitopes of CS1, further confirmed two forms of CS1 only in CS1-positive MM patient sera. Detection of CS1 is associated with MM (p < 0.0001), since it is detected in MM patient sera, but not in MGUS (n=15) or in healthy donors (n=100). In additional sera of 199 MM patients with newly diagnosed MM, 90% (181/199) of MM patients have detectable CS1 (1 to >80 ng/ml). Median CS1 levels for International Stage System (ISS) I (n=100), II (n=53), and III (n=46) patients are 5.87, 9.37, and 8.37 ng/ml, respectively. The correlation between ISS and CS1 is moderate (spearman correlation coefficient = 0.197, p=0.005). Patients with ISS II/III had significantly higher CS1 levels compared with those with ISS I (median 9.0 vs. 5.9 ng/ml, p=0.006), suggesting a correlation of serum CS1 with active MM. Since patients with ISS II and III require therapy, while those with ISS I do not, these results suggest that circulating CS1 may indicate need for therapy and further support clinical investigation of anti-CS1 therapy using HuLuc63 in MM. Importantly, HuLuc63 has demonstrated significant dose-dependant activity, leading to reduced or eliminated human myeloma tumors (MM1S, OPM2, and L363) in xenograft mice models. Anti-MM activity of HuLuc63 in mice could be observed at sustained serum levels of >2 mg/ml, which is well above the levels of circulating CS1 protein observed in MM patients. Thus, it is proposed that serum CS1 will be an unlikely antibody sink in patients treated with optimal doses of HuLuc63. Since NK cells also express CS1, albeit at lower levels than MM cells, we next determined the effects of HuLuc63 on NK cell function. Pretreatment of NK cells with HuLuc63 (0.1 mg/ml) for 3 days did not alter NK-mediated ADCC against CS1-expressing MM cells (MM1S, H929, INA-6) via HuLuc63. Moreover, recombinant CS1-Fc at physiological serum levels (<200 ng/ml) did not significantly inhibit HuLuc63-induced ADCC against MM cells. Inhibition of HuLuc63-induced MM cell lysis by CS1-Fc was observed at higher concentrations (>200 ng/ml), confirming specific HuLuc63-CS1 binding. Together, these studies strongly support continued clinical investigation of HuLuc63 in MM.

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Natural killer cell receptors: new biology and insights into the graft-versus-leukemia effect

Blood ◽

10.1182/blood-2002-02-0350 ◽

2002 ◽

Vol 100 (6) ◽

pp. 1935-1947 ◽

Cited By ~ 352

Author(s):

Sherif S. Farag ◽

Todd A. Fehniger ◽

Loredana Ruggeri ◽

Andrea Velardi ◽

Michael A. Caligiuri

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Killer Cell ◽

Natural Cytotoxicity ◽

Receptor Ligand ◽

Cell Receptors ◽

Nk Cell Receptors ◽

Ligand Interactions ◽

Nk Receptor

AbstractNatural killer (NK) cells have held great promise for the immunotherapy of cancer for more than 3 decades. However, to date only modest clinical success has been achieved manipulating the NK cell compartment in patients with malignant disease. Progress in the field of NK cell receptors has revolutionized our concept of how NK cells selectively recognize and lyse tumor and virally infected cells while sparing normal cells. Major families of cell surface receptors that inhibit and activate NK cells to lyse target cells have been characterized, including killer cell immunoglobulinlike receptors (KIRs), C-type lectins, and natural cytotoxicity receptors (NCRs). Further, identification of NK receptor ligands and their expression on normal and transformed cells completes the information needed to begin development of rational clinical approaches to manipulating receptor/ligand interactions for clinical benefit. Indeed, clinical data suggest that mismatch of NK receptors and ligands during allogeneic bone marrow transplantation may be used to prevent leukemia relapse. Here, we review how NK cell receptors control natural cytotoxicity and novel approaches to manipulating NK receptor-ligand interactions for the potential benefit of patients with cancer.

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Soluble and Membrane Bound MICA in Myeloma Do Not Adversely Effect Regulation of Natural Killer Cell Function Via NKG2D..

Blood ◽

10.1182/blood.v104.11.2462.2462 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2462-2462

Author(s):

Jumei Shi ◽

Susann Szmania ◽

Fenghuang Zhan ◽

John Shaughnessy ◽

Bart Barlogie ◽

...

Keyword(s):

Cell Surface ◽

Nk Cells ◽

Cell Function ◽

Nk Cell ◽

Elevated Serum ◽

Serum Levels ◽

Down Regulation ◽

Nkg2d Expression ◽

Mica Gene ◽

Membrane Bound

Abstract The human MHC class I -related chain gene A (MICA), a highly polymorphic MHC-encoded cell-surface glycoprotein, is a stress-induced and transformation related molecule absent from most normal tissues. Expression of MICA has been reported on a variety of mostly epithelial tumors. MICA can activate Natural Killer (NK) cells via interacting with the immunoreceptor NKG2D. Soluble MICA (sMICA), when shedded from tumor by the action of matrix-metalloproteinases (MMPs), can be detected at high levels in the sera of cancer patients. High levels of sMICA can down-regulate NKG2D expression and lead to functional impairment of NK cells thus providing for a mechanism of tumor escape. Gene array expression profiling of bone marrow biopsies revealed high levels of MMP2 and MMP9 in the bone marrow microenvironment, but not in the purified myeloma cells of the same patients. Since we are developing allogeneic NK cell therapy as a novel treatment for high-risk myeloma we decided to study whether the high activity of MMP2 and MMP9 resulted in 1) elevated sMICA levels in myeloma, and 2) affected NKG2D expression by NK cells. We found by ELISA assays that 28% (13/46) of patients with MM at diagnosis contained elevated serum levels of sMICA (median: 265.2 pg/ml; range: 125.9 – 806.5 pg/ml). sMICA levels were low in 16 control healthy donors tested (median: 0.8 pg/ml; range: 0 – 91 pg/ml). Next, we correlated sMICA levels with indicators of tumor burden and prognosticators of outcome in MM. We observed that sMICA levels in the serum of MM patients was not associated with Ras associated oncogene (RAN) expression, presence of abnormal cytogenetics, elevated CRP, elevated b2-microglobulin or elevated serum M-protein levels. There was also no correlation between MICA gene expression in purified MM cells and increased sMICA levels in the serum. We therefore examined several myeloma cell lines and found that high MICA gene expression does not always correlate with MICA expression at the cell surface as detected by flow cytometry. We hypothesize that the lack of correlation between MICA RNA expression and sMICA may be due to variation in translation of the MICA mRNA or failure to transport the MICA protein to the cell surface. Further, we tested the expression of NKG2D on CD3−/CD56+ cells of 5 MM patients with elevated sMICA serum levels and detected no down-regulation of NKG2D on NK cells compared to controls comprising MM patients with normal sMICA levels (n=3) or normal donors (n=5). Recent studies have suggested that tumor-membrane bound MICA may also play a role in the down-regulation of NKG2D on NK cells. We next incubated normal donor NK cells with a) the high membrane bound MICA expressing MM cell line U266 or b) serum from MM patients containing high MICA levels neither of which resulted in down-regulation of NKG2D. We conclude that despite the high expression of MMP2 and MMP9 both soluble or membrane bound MICA do not down-regulate NKG2D on NK cells and hence do not adversely affect NK cell function in MM. One explanation may be that the sMICA levels we found in myeloma patients were in the order of pg/ml, whilst the levels reported in epithelial tumors are 2–3 logs higher.

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Comparative Analysis of NK Receptor and T-Cell Receptor Repertoires in Patients with Chronic Myeloid Leukemia Treated with Different Tyrosine Kinase Inhibitors

Blood ◽

10.1182/blood.v124.21.5508.5508 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5508-5508 ◽

Cited By ~ 1

Author(s):

Gianni Binotto ◽

Luca Frison ◽

Elisa Boscaro ◽

Renato Zambello ◽

Federica Lessi ◽

...

Keyword(s):

T Cell ◽

Nk Cells ◽

T Cell Receptor ◽

Kinase Inhibitors ◽

Nk Cell ◽

Cell Receptor ◽

Cell Receptors ◽

Receptor Repertoire ◽

Nk Cell Receptors ◽

T Cell Receptor Repertoire

Abstract Introduction: Given the critical role of BCR–ABL kinase activity in chronic myeloid leukemia (CML), tyrosine kinase inhibitors (TKIs) are currently considered the cornerstone of CML treatment. Previous studies have suggested that TKIs may influence anti-tumor immunity through off-target modulation of different immune effectors. Natural killer (NK) cells, as well as T cells in the context of adaptive immunity, are a key component of the innate immune system, providing first-line defense against virally infected cells and tumors. The activity of NK cells is modulated by a finely-tuned balance between signals received from inhibitory and activating cell surface receptors. Aims: We sought to evaluate the impact of first and second generation TKIs on modulating different NK cell receptors patterns; secondly we studied the effect of a TKIs driven NK subpopulations selection on treatment response. Finally, we analyzed the T cells Vβ-TCR repertoire to identify any restrictions. Materials and Methods: Peripheral blood samples from 25, 9 and 8 chronic phase CML patients treated with imatinib frontline, nilotinib and dasatinib as first or second line therapy, respectively, were collected. Patients characteristics are described below (see table 1). After separation of mononuclear cells (PBMC), the expression of several NK cell receptors (Killer Immunoglobulin-like Receptors, KIR: p70, p140, p58/p50; Killer Lectin-like Receptors, KLR: CD94, NKG2A, NKG2C/A, NKG2D; Natural Cytotoxicity Receptors, NCR: NKp30, NKp44, NKp46, NKp80; Co-receptors: 2B4; LIR1/ILT2, GPR56) and Vβ TCR-repertoire were analized by flow cytometry analysis. Treatment response was assessed with standardized real quantitative polymerase chain reaction and cytogenetics according to ELN recommendations. Results: The leukocyte count was not statistically different between groups (WBC = 5.5 x 109 / L vs. 6.8 x 109 / L vs. 5.6 x 109 / L, p = 0.09, respectively); also lymphocytes, considered either in percentage or absolute number, were comparable (32% vs 26% vs 35%, p = 0.08), as well as the percentage and absolute number of NK cells (20%; 0.37 x 109 / L vs. 15%; 0.26 x 109 / L vs. 24%; 0.54 x 109 / L (p = 0.17, p = 0.10).The analysis of NK receptors expression showed that patients treated with Imatinib exhibited a preferential selection of NK cells subpopulations harboring activating receptors (NKp30, NKp46, NKp80 and NKG2D), while in Dasatinib treated patients an increased expression of KIR (KIR2DL1) receptors was observed (figure 1). Interestingly, these effects were documented also in the absence of lymphocytosis. 44.4% (4 of 9 patients) of patients treated with nilotinib showed preferential expression of Vβ chains, compared with 87.5% of patients treated with dasatinib; no TCR-repertoire restriction was documented in the sole TKI primary resistant patient. 8 out of 17 patients showed a preferential expression of more than oneVβ chain (figure 2). No specific NK receptors profiles were found to be associated with different degrees of treatment response. Conclusions: These preliminary data suggest the existence of a different NKRs and T cell receptor repertoire modulation, mediated by Tyrosine-Kinase Inhibitors. Since no significant correlation between response and specific NK receptor profiles has been demonstrated, TKIs immunomodulatory effect seems secondary compared to direct inhibition of BCR-ABL kinase. However, it's conceivable that NK and T cells subpopulations selection, induced by TKIs, may become relevant in the immunological control of leukemic disease at the time of drug discontinuation. These observations are currently being investigated on a larger series of patients. Figure 1 NK cell receptors differentially expressed between imatinib, nilotinib and dasatinib treated patients. Figure 1. NK cell receptors differentially expressed between imatinib, nilotinib and dasatinib treated patients. Figure 2 Figure 2. Figure 3 T cell receptor repertoire in nilotinib (A) and dasatinib (B) treated CML patients Figure 3. T cell receptor repertoire in nilotinib (A) and dasatinib (B) treated CML patients Disclosures No relevant conflicts of interest to declare.

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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.

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