EZH2 Mediates Resistance To Apoptosis In Nktl By Activating Nfkb Signaling Through Repression Of TNFAIP3/A20 By H3K27 Trimethylation

Abstract Extranodal nasal-type natural killer/T-cell lymphoma (NKTL) is a rare and aggressive form of lymphoma with poor outcome. A better understanding of the molecular pathophysiology of the disease is needed to development better treatment and improve patient’s outcome. We previously showed that EZH2, a histone methyltransferase, is abnormally over-expressed in NK cell lines as compared to normal NK cells, and may therefore be important for the pathogenesis and treatment of NKTL. 3- Deazaneplanocin A (DZNep) is a drug that can deplete EZH2 levels. Downregulation of EZH2 by DZNep induces apoptosis in a panel of NKTL cell lines but not normal NK cells. We perform gene expression studies to identify possible mechanism by which DZNep induce apoptosis in NKTL. There is significant enrichment of NFKB target genes amongst the genes downregulated after DZNep treatment. In addition, 2 inhibitors of the NFKB pathways, TNFAIP3/A20 and NFKBIA were upregulated. The downregulation of these genes were validated by PCR. We further found that NFKB2 was downregulated after DZNep treatment on western blot suggesting that NFKB signaling was downregulated. Similar results were obtained when we silence EZH2 using siRNA suggesting that much of the effect of DZNep is mediated through downregulation of EZH2. When TNFAIP3/A20 was expressed in untreated NKTL cells, apoptosis was induced. Conversed with TNFAIP3/A20 was knocked down in cells treated by DZNep, DZNep induced apoptosis was reduced. These data suggest in NKTL, EZH2 downregulate TNFAIP3/A20 and activate NFKB leading to resistance to apoptosis. We next investigated if EZH2 directly regulate TNFAIP3/A20 expression at its promoter. Using chromatin immunoprecipitation, we found that there is high occupancy of EZH2 and H3K27me3 at the TNFAIP3/A20 promoter in NKTL. Upon EZH2 knockdown, EZH2 and H3K27me3 is markedly reduced at the promoter and there is concurrent increase in TNFAIP3 expression. Consistent with cell lines data, in clinical samples, there is a strong association between H3K27me3 and nuclear p50 expression on a tissue microarray of NKTL (Chi-square p-value 0.01). We have therefore uncovered a novel mechanism by which EZH2 activates the NFKB pathway through its histone methyltransferase activity on the promoter of TNFAIP3/A20 resulting in resistance of NKTL to apoptosis. Disclosures: No relevant conflicts of interest to declare.

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STAT3 Is Activated By EBV in T or NK Cells Leading to Development of EBV-T/NK-Lymphoproliferative Disorders

Blood ◽

10.1182/blood.v124.21.3026.3026 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3026-3026

Author(s):

Honami Komatsu ◽

Ken-Ichi Imadome ◽

Haruna Shibayama ◽

Tomotaka Yada ◽

Momoko Yamada ◽

...

Keyword(s):

T Cell ◽

Nk Cells ◽

Cell Lines ◽

Nk Cell ◽

Sh2 Domain ◽

Lymphoproliferative Disorders ◽

Clinical Samples ◽

Ebv Infection ◽

Xenograft Models ◽

T Cell Lines

Abstract Introduction Epstein–Barr virus (EBV) genome is positive not only in B-, but also T- or NK-lymphoid neoplasms: extranodal NK/T-cell lymphoma (ENKL), aggressive NK-cell leukemia, and EBV-positive T- or NK-cell lymphoproliferative disorders (EBV-T/NK-LPDs). EBV-T/NK-LPDs are disorders formerly called chronic active EBV infection presenting sustained inflammation, such as infectious mononucleosis-like symptoms, hypersensitivity to mosquito bites, or hydroa vacciniforme-like eruption accompanied by clonal proliferation of EBV-infected T or NK cells. EBV makes the infected B-cells immortal leading to B-cell lymphomas. However, why and how EBV infects T or NK cells and the mechanism of action responsible for the development of EBV-T/NK-neoplasms has not been elucidated yet. STAT3 is a transactivation factor which mediates proliferation and anti-apoptotic signaling. It was reported that a large variety of primary tumor cells as well as tumor-derived cell lines from patients harbored constitutively activated STAT3. In addition, tyrosine 705 (Y705) of STAT3 was constitutively phosphorylated in ENKL cells (Leukemia, 23, p1667, 2009). Objectives We designed this study to investigate STAT3 activation and its contribution to EBV-T/NK-LPDs development. Materials and Methods EBV-positive T-cell lines, SNT8, SNT15, SNT16, and NK-cell lines, SNK1, SNK6, SNK10, were examined. The EBV-negative T-cell lines HPB-ALL, Jurkat, MOLT4 and peripheral blood mononuclear cells (PBMCs) from healthy donors were used as the negative controls. Clinical samples were obtained from EBV-T/NK-LPDs patients who were diagnosed according to the previously described criteria (Blood, 119, p.673, 2012). To detect and isolate EBV-infected cells, T and NK cells were separated using magnetic beads from PBMCs. STAT3 phosphorylation in EBV-T/NK-LPDs cells were examined in clinical samples and xenograft models of EBV-T/NK-LPDs generated by transplantation of PBMCs from the EBV-T/NK-LPDs patients to NOD/Shi-scid/IL-2Rγnull mice. Mutation of STAT3 was examined by direct sequencing. For in vitro EBV infection, EBV was prepared from the culture medium of B95-8 cells and added to MOLT4 cells (Proc Natl Acad Sci, 100, p7836, 2003). Results First, we investigated the activation of STAT3 in EBV-positive T- or NK-cell lines. Phosphorylation of STAT3 on Y705 and serine 727 (S727) was more clearly detected in comparison with that in EBV-negative cell lines by western blotting under their maintenance condition. STAT3 was localized in the nucleus in the EBV-T/NK-cells. These results indicated STAT3 was constitutively activated in EBV-T/NK-cells. We validated the results in EBV-infected T or NK cells derived from 5 EBV-T/NK-LPDs patients (infected cell types: CD4, 1; CD8, 2; and CD56, 2). Phosphorylation of Y705 and S727 of STAT3 was detected in EBV-infected T or NK cells in them. Immunohistological staining also detected the phosphorylation of EBV-positive cells in the tissue of the xenograft models. In these EBV-T/NK-cells, gene mutation was not identified in SH2 domain of STAT3. Next, we examined the direct effect of EBV on STAT3 activation by in virto EBV infection on MOLT4 cells. Immunofluorescence staining detected that STAT3 moved to the nucleus after the infection. Finally, STAT3 specific inhibitor STA-21 suppressed the proliferation of EBV-T/NK-cells. Conclusions STAT3 is activated by EBV leading to growth promoting effects on EBV-T/NK-LPDs. STAT3 can be an attractive molecular target of the treatment for the disorders. Disclosures No relevant conflicts of interest to declare.

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JAK1 and JAK2 Modulate Myeloma Cell Susceptibility to NK Cells Through the Interferon Gamma (IFN-γ) Pathway,

Blood ◽

10.1182/blood.v118.21.3960.3960 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3960-3960

Author(s):

Roberto Bellucci ◽

Allison Martin ◽

Marc Buren ◽

Hong-Nam Nguyen ◽

Davide Bommarito ◽

...

Keyword(s):

Nk Cells ◽

Cell Lines ◽

Nk Cell ◽

Conflicts Of Interest ◽

Specific Antigen ◽

Innate Immune ◽

Target Cells ◽

Jak Inhibitor ◽

Stat Proteins ◽

Ifn Γ

Abstract Abstract 3960 Multiple myeloma (MM) is a B cell neoplasm characterized by clonal expansion of malignant plasma cells in the bone marrow. Despite the use of new drugs such as lenalidomide and bortezomib, MM remains an incurable disease. Successful treatment of MM with allogeneic stem cell transplantation suggests that MM is susceptible to immunologic approaches. NK cells are the primary effectors of the innate immune response against infectious pathogens and malignant transformation. Unlike T and B cells, NK cells do not recognize antigens in the context of classical major histocompatibility complex (MHC) but lyse target cells without specific antigen recognition. Nevertheless, MM cells have developed mechanisms to evade innate immune surveillance and the molecular basis for target resistance to NK cell-mediated lysis is not well understood. To identify novel pathways that modulate MM cell resistance to the immune system, we previously developed a genetic screen to detect cell-cell interactions using a large lentiviral shRNA library containing a total of 6,144 shRNAs targeting more than 1,000 human genes. Using this approach we found that silencing JAK1 and JAK2 results in significantly increased MM cell susceptibility to NK cell lysis. This effect was not noted when JAK3 and TYK2 were targeted. JAK1, JAK2 JAK3 and TYK2 are members of a family of tyrosine kinases that are constitutively associated with many membrane cytokine receptors. After activation, JAK proteins regulate phosphorylation/activation of STAT proteins, which subsequently initiate gene transcription. To understand JAK1 and JAK2 involvement in MM resistance to NK cells, we undertook a series of experiments to analyze the JAK signaling pathway in MM cells. We first analyzed the activation status of STAT proteins in a series of MM cell lines (IM-9, KM12BM, RPMI 8226, U266) in which JAK1 and JAK2 expression was reduced by specific shRNAs. Constitutive activation of STAT proteins was not affected by JAK1 or JAK2 gene silencing suggesting that these kinases were not activated in the absence of cytokine receptor-mediated signaling. Since JAK1 and JAK2 are associated with the IFN-γ receptor and we previously showed that JAK1 and JAK2 silencing induces increased secretion of IFN-γ from NK cells, we pre incubated MM cell lines with NK activated supernatant or recombinant IFN-γ and tested them for STAT activation. 15 min incubation was sufficient to initiate phosphorylation of STAT1 but no other STATs were activated. Silencing of JAK1 or JAK2 with specific shRNAs prevented STAT1 activation. To validate this finding, we tested primary MM cells treated with different concentrations of Jak inhibitor 1 (0 nM, 10 nM, 30 nM and 40 nM). These cells had a similar STAT profile at their basal level when compared with the previously tested MM cell lines. Pre-incubation with NK activated supernatant or IFN-γ also induced rapid activation of STAT1, which was completely inhibited when cells were pre-treated with Jak inhibitor 1. Treatment of MM cells with 10, 30 and 40 nM of Jak inhibitor enhanced killing by NK cells by 46.6%, 51% and 53%, compared to untreated cells (p=0.0036, p=0.0011 and p=0.0010 respectively). These findings demonstrate that IFN-γ signals rapidly enhance resistance of MM cells to NK cells but inhibition of this pathway at the level of JAK1 and JAK2 reverses this effect and induces susceptibility to NK cell mediated lysis. Disclosures: No relevant conflicts of interest to declare.

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A Novel Method to Study the in Vivo Trafficking and Homing of Adoptively Transferred NK Cells in Rhesus Macaques and Humans

Blood ◽

10.1182/blood.v124.21.659.659 ◽

2014 ◽

Vol 124 (21) ◽

pp. 659-659 ◽

Cited By ~ 3

Author(s):

Jan Davidson-Moncada ◽

Noriko Sato ◽

Robert F Hoyt ◽

Robert N Reger ◽

Marvin Thomas ◽

...

Keyword(s):

Nk Cells ◽

Adoptive Transfer ◽

Nk Cell ◽

Conflicts Of Interest ◽

Raji Cell ◽

K562 Cells ◽

Ct Imaging ◽

Pet Ct ◽

Hematological Cancers

Abstract Adoptive transfer of allogeneic or autologous natural killer (NK) cells is now being developed for therapy of both hematological and solid malignancies. The efficacy of NK immunotherapy to mediate anti-tumor effects will ultimately be dependent on their ability to traffic and home to the tumor microenvironment. Recent data suggest expanded NK cells are ineffective at homing to the bone marrow (BM) and lymph nodes (LN) where hematological malignancies reside. A variety of techniques to maintain and/or enforce expression of homing receptors in NK cells are now being explored in preclinical models to improve their localization to the BM and LN. Historically, xenogeneic human into mouse or mouse into mouse models have been utilized for preclinical development of adoptive NK transfer. These experiments often use fluorescent dye-labeled NK cells and require repeated invasive biopsies, which can be confounded by sampling error, or the requirement for post mortem analysis. Here we present a method to track in real time and in vivo adoptively infused zirconium-89 (89Zr) labelled NK cells by PET imaging. A rhesus macaque (RM) model was used for these preclinical experiments as RM and human NK cells have similar expansion kinetics, and have greater similarity than mice in their phenotype, function, and homing receptors and ligands. PBMCs collected from the PB of 13 RMs were enriched for NK cells by CD3+ T-cell depletion and were then expanded for 14 days by culturing with irradiated human EBV-LCL cells in X-VIVO 20 media containing 10% human AB serum and 500 IU/μl of human IL-2. RM NK cells expanded a mean 145±41 fold and contained >99% pure CD3- and CD56+ cells. The phenotype and tumor cytotoxicity of RM NK cells were similar to NK cells expanded from humans (n=3) using similar expansion cultures; at a 10:1 E:T ratio, 67% and 73% of K562 cells were lysed by RM and human NK cell respectively. To label NK cells, 89Zr was conjugated to oxine, which readily permeabilized the cellular membrane and was retained in the cells. Expanded NK cells from both humans and RM showed no changes in CD16 or CD56 expression for up to 6 days following radiolabeling. Human and RM NK cell viability 0 to 24 hours following radiolabelling was 60-100% then declined to 20-30% after 6 days. 89Zr retention by both human and RM NK cells was 75-80% in the first 24 hours of culture but gradually declined with time, decreasing to 20-30% after 7 days of culture. Culturing radiolabeled human NK cells for 24-36 hours with different cellular populations including Ramos and Raji cell lines and normal human PBMCs revealed no significant transfer of radioactivity (max 2% above baseline), establishing that 89Zr was not transferred from labeled to unlabeled cells. Oxine labeling did not alter the cytotoxicity of human or RM NK cells vs K562 cells compared to unlabeled controls. 89Zr-oxine labeling of expanded RM NK cells is currently being used to quantify NK cell trafficking and survival following adoptive transfer in autologous macaques. In these experiments, RM recipients of adoptively infused 89Zr labeled NK cells receive concurrent deferoxamine to chelate and then enhance renal excretion of any free 89Zr that is released from dead cells. In the experiments shown below, 13 x 107 autologous ex vivo expanded 89Zr-labeled RM NK cells were injected IV into a 5.7 kg RM and tracked by sequential PET/CT imaging for 7 days. Up to 1-hour post infusion, most NK cell activity was restricted to the lungs. By 4 hours, NK cells began to traffic from the lungs to the liver and spleen. By 2 days, NK cells were no longer detectable in the lungs and resided largely in the liver and spleen, where they remained for the remainder of the 7 day imaging period. During the entire observation period, little to no NK cell radioactivity was detected in the LN or BM. In conclusion, 89Zr oxine labelling of NK cells followed by PET/CT imaging represents a powerful tool to track the in vivo fate of adoptively transferred NK cells. The RM model presented here provides a method to evaluate and optimize various strategies aimed at altering the phenotype of NK cells, with the goal of improving their homing to the BM and LN where hematological cancers reside. These preclinical in vitro and in vivo data suggest this technology could be safely extended to humans and could be applied to other cellular populations besides NK cells. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Feasibility of Targeting Traf2-and-Nck-Interacting Kinase in Synovial Sarcoma

Cancers ◽

10.3390/cancers12051258 ◽

2020 ◽

Vol 12 (5) ◽

pp. 1258

Author(s):

Tetsuya Sekita ◽

Tesshi Yamada ◽

Eisuke Kobayashi ◽

Akihiko Yoshida ◽

Toru Hirozane ◽

...

Keyword(s):

Wnt Signaling ◽

Synovial Sarcoma ◽

Cell Lines ◽

Target Genes ◽

Xenograft Model ◽

Clinical Samples ◽

Sarcoma Cell ◽

Molecular Therapeutics ◽

Mouse Xenograft Model ◽

Sarcoma Cells

Background: The treatment of patients with metastatic synovial sarcoma is still challenging, and the development of new molecular therapeutics is desirable. Dysregulation of Wnt signaling has been implicated in synovial sarcoma. Traf2-and-Nck-interacting kinase (TNIK) is an essential transcriptional co-regulator of Wnt target genes. We examined the efficacy of a small interfering RNA (siRNA) to TNIK and a small-molecule TNIK inhibitor, NCB-0846, for synovial sarcoma. Methods: The expression of TNIK was determined in 20 clinical samples of synovial sarcoma. The efficacy of NCB-0846 was evaluated in four synovial sarcoma cell lines and a mouse xenograft model. Results: We found that synovial sarcoma cell lines with Wnt activation were highly dependent upon the expression of TNIK for proliferation and survival. NCB-0846 induced apoptotic cell death in synovial sarcoma cells through blocking of Wnt target genes including MYC, and oral administration of NCB-846 induced regression of xenografts established by inoculation of synovial sarcoma cells. Discussion: It has become evident that activation of Wnt signaling is causatively involved in the pathogenesis of synovial sarcoma, but no molecular therapeutics targeting the pathway have been approved. This study revealed for the first time the therapeutic potential of TNIK inhibition in synovial sarcoma.

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Pharmacologic inhibition of lysine-specific demethylase 1 as a therapeutic and immune-sensitization strategy in pediatric high-grade glioma

Neuro-Oncology ◽

10.1093/neuonc/noaa058 ◽

2020 ◽

Vol 22 (9) ◽

pp. 1302-1314 ◽

Cited By ~ 5

Author(s):

Cavan P Bailey ◽

Mary Figueroa ◽

Achintyan Gangadharan ◽

Yanwen Yang ◽

Megan M Romero ◽

...

Keyword(s):

Nk Cells ◽

Cell Lines ◽

Nk Cell ◽

Gene Signature ◽

Immune Gene ◽

High Grade ◽

Cell Cytotoxicity ◽

Lysine Specific Demethylase

Abstract Background Diffuse midline gliomas (DMG), including brainstem diffuse intrinsic pontine glioma (DIPG), are incurable pediatric high-grade gliomas (pHGG). Mutations in the H3 histone tail (H3.1/3.3-K27M) are a feature of DIPG, rendering them therapeutically sensitive to small-molecule inhibition of chromatin modifiers. Pharmacological inhibition of lysine-specific demethylase 1 (LSD1) is clinically relevant but has not been carefully investigated in pHGG or DIPG. Methods Patient-derived DIPG cell lines, orthotopic mouse models, and pHGG datasets were used to evaluate effects of LSD1 inhibitors on cytotoxicity and immune gene expression. Immune cell cytotoxicity was assessed in DIPG cells pretreated with LSD1 inhibitors, and informatics platforms were used to determine immune infiltration of pHGG. Results Selective cytotoxicity and an immunogenic gene signature were established in DIPG cell lines using clinically relevant LSD1 inhibitors. Pediatric HGG patient sequencing data demonstrated survival benefit of this LSD1-dependent gene signature. Pretreatment of DIPG with these inhibitors increased lysis by natural killer (NK) cells. Catalytic LSD1 inhibitors induced tumor regression and augmented NK cell infusion in vivo to reduce tumor burden. CIBERSORT analysis of patient data confirmed NK infiltration is beneficial to patient survival, while CD8 T cells are negatively prognostic. Catalytic LSD1 inhibitors are nonperturbing to NK cells, while scaffolding LSD1 inhibitors are toxic to NK cells and do not induce the gene signature in DIPG cells. Conclusions LSD1 inhibition using catalytic inhibitors is selectively cytotoxic and promotes an immune gene signature that increases NK cell killing in vitro and in vivo, representing a therapeutic opportunity for pHGG. Key Points 1. LSD1 inhibition using several clinically relevant compounds is selectively cytotoxic in DIPG and shows in vivo efficacy as a single agent. 2. An LSD1-controlled gene signature predicts survival in pHGG patients and is seen in neural tissue from LSD1 inhibitor–treated mice. 3. LSD1 inhibition enhances NK cell cytotoxicity against DIPG in vivo and in vitro with correlative genetic biomarkers.

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Lenalidomide Strongly Enhances Natural Killer (NK) Cell Mediated Antibody-Dependent Cellular Cytotoxicity (ADCC) of Rituximab Treated Non-Hodkin’s Lymphoma Cell Lines In Vitro.

Blood ◽

10.1182/blood.v108.11.3714.3714 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3714-3714 ◽

Cited By ~ 2

Author(s):

Lei Wu ◽

Peter Schafer ◽

George Muller ◽

David Stirling ◽

J. Blake Bartlett

Keyword(s):

Nk Cells ◽

Tumor Cells ◽

Cell Lines ◽

Nk Cell ◽

Dependent Manner ◽

Activation Markers ◽

Early Results ◽

Ifn Γ ◽

Dose Dependent

Abstract Lenalidomide (Revlimid® is approved for the treatment of transfusion-dependent patients with anemia due to low- or intermediate-1-risk MDS associated with a del 5q cytogenetic abnormality with or without additional cytogenetic abnormalities, and in combination with dexamethasone is for the treatment of multiple myeloma patients who have received at least one prior therapy. Encouraging early results suggest a potential for clinical efficacy in B cell non-Hodgkin’s lymphoma (NHL). Potential mechanisms of action include anti-angiogenic, anti-proliferative and immunomodulatory activities. Lenalidomide has been shown to enhance Th1-type cytokines and T cell and NK cell activation markers in patients with advanced cancers. Furthermore, lenalidomide has been shown to enhance rituximab-mediated protection in a SCID mouse lymphoma model in vivo. We have utilized an in vitro ADCC system to assess the ability of lenalidomide to directly enhance human NK cell function in response to therapeutic antibodies, such as rituximab (chimeric anti-CD20 mAb). Isolated NK cells produced little or no IFN-γ in response to IgG and/or IL-2 or IL-12. However, pre-treatment of NK cells with lenalidomide greatly enhanced IFN-γ production by NK cells in a dose-dependent manner. In a functional ADCC assay, NHL cell lines (Namalwa, Farage & Raji) were pre-coated with rituximab and exposed to NK cells pre-treated with lenalidomide in the presence of either exogenous IL-2 or IL-12. After 4 hours in culture the viability of the tumor cells was assessed. Lenalidomide consistently and synergistically increased the killing of tumor cells in a dose-dependent manner and up to >4-fold compared to rituximab alone. Rituximab alone had only a small effect in this model and there was no killing of cells in the absence of rituximab. The presence of either exogenous IL-2 or IL-12 was required to see enhanced killing by lenalidomide. In cancer patients lenalidomide has been shown to increase serum IL-12 levels and is also known to induce IL-2 production by T cells in vitro. Potential mechanisms for enhanced ADCC include increased signaling through NK FCγ receptors and/or IL-2 or IL-12 receptors. However, we found that these receptors are unaffected by lenalidomide, although downstream effects on NK signaling pathways are likely and are being actively investigated. In conclusion, we have shown that lenalidomide strongly enhances the ability of rituximab to induce ADCC mediated killing of NHL cells in vitro. This provides a strong rationale for combination of these drugs in patients with NHL and CLL.

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High Anti-Tumor Activity of Natural Killer Cells after Non-Myeloablative Conditioning and Allogeneic Stem Cell Transplantation.

Blood ◽

10.1182/blood.v108.11.5146.5146 ◽

2006 ◽

Vol 108 (11) ◽

pp. 5146-5146

Author(s):

Lars Fischer ◽

Olaf Penack ◽

Andrea Stroux ◽

Chiara Gentilini ◽

Eckhard Thiel ◽

...

Keyword(s):

Stem Cell ◽

Nk Cells ◽

Cell Lines ◽

Nk Cell ◽

Normal Values ◽

Leukemia Cell ◽

Myeloablative Conditioning ◽

Conventional Dose ◽

Leukemia Cell Lines ◽

Anti Tumor Activity

Abstract Background: It is hypothesized that enhanced graft-versus-leukemia activity of natural killer (NK) cells contributes to the clinical efficacy of non-myeloablative allogeneic hemotopoietic stem cell transplantation (HSCT). We compared NK cell cytotoxic activity after transplantation with full and reduced dose regimens with normal values obtained from healthy volunteers. Methods&Material: Using a flow cytometric assay that detects CD107a expression after coincubation with leukemia cell lines as a marker for NK cell degranulation we prospectively quantified and characterized NK cells mediating anti-tumor activity in patients after HSCT. Mononuclear cells (MNC) were isolated from peripheral blood of 17 healthy individuals and 31 patients transplanted with G-CSF mobilized peripheral blood stem cells at day +30. MNC were incubated with leukemia cell lines HL60 and K562 (effector/target ratio 1:1) and expression of CD107a was measured after 3 hours. The absolute number of degranulating (CD107a+) NK cells was calculated. Results: Thirty one patients (five with ALL, four with NHL, one with Aplastic Anemia and 21 with AML) were enrolled, of whome 22 were transplanted from a matched related donor, seven from a matched unrelated donor, and two from a haploidentical donor. Nine patients had been transplanted after conditioning with 2 Gy TBI only, whereas 22 patients had received various conventional dose regimens. NK cell counts at day +30 after HSCT with conventional conditioning were comparable to normal values, but percentage and number of degranulating cells were significantly reduced (2.4% and 7/μl vs. 6.2% and 18/μl; p<0.0001 and p=0.0003). In contrast, in patients after 2 Gy TBI the percentage of degranulating NK cells was comparable to healthy donors (7.6%; p=0.9), and interestingly, absolute numbers of all and of degranulating NK cells (41/μl; p=0.004) were higher than normal values and consequently higher than in patients with conventional dose conditioning. Conclusions: Using this new method we were able to exactly quantify tumor-reactive NK cells after HSCT in a feasible way. We found a high cytotoxic activity of NK cells towards leukemia cell lines in patients undergoing non-myeloablative conditioning with 2Gy TBI. Further studies to determine the clinical impact of these findings are needed.

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Role of CD137/4-1BB and Its Ligand in the NK Cell Mediated Immune Surveillance of Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v110.11.880.880 ◽

2007 ◽

Vol 110 (11) ◽

pp. 880-880

Author(s):

Tina Baessler ◽

Matthias Krusch ◽

Katrin M. Baltz ◽

Benjamin J. Schmiedel ◽

Helga M. Schmetzer ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Nk Cells ◽

Cell Lines ◽

Myeloid Leukemia ◽

Nk Cell ◽

Immune Surveillance ◽

Cell Reactivity ◽

Ifn Γ ◽

Tnfr Superfamily ◽

Acute Myeloid

Abstract NK cells play an important role in the reciprocal interaction of tumor cells with the immune system and participate in the surveillance and eradication of hematological malignancies including acute myeloid leukemia (AML). NK cell reactivity is governed by a balance of activating and inhibitory receptors including various members of the TNF receptor (TNFR) superfamily. The TNFR superfamily member CD137/4-1BB has been shown to stimulate proliferation and IFN-γ production, but not cytotoxicity of NK cells in mice. Surprisingly, yet nothing is known regarding the consequences of CD137-CD137 ligand (CD137L) interaction for NK cell reactivity in humans. In this study we demonstrate that CD56dimCD16+ but not CD56brightCD16− NK cells express CD137 upon stimulation with the activating cytokines IL-2 and IL-15 with peak expression between 48 and 60h. Furthermore, we found that 5 of 7 investigated AML cell lines and 16 of 51 (33%) primary AML cells of patients expressed substantial CD137L levels, while no CD137L expression was detected on CD34+ cells of healthy donors (n=5). CD137L expression was not restricted to a specific French-American-British (FAB) subtype, but was significantly (p<0.05, one-way ANOVA) associated with monocytic (FAB M4, M5) differentiation. In addition, no association with a particular cytogenetic abnormality or with expression of MHC class I was observed. Reverse signaling via CD137L into AML cells (n=10) significantly induced the release of the immunoregulatory cytokines IL-10 and TNF (both p<0.05, Mann-Whitney U-test). Surprisingly and in contrast to available data regarding the function of murine CD137, we found that in humans blocking CD137-CD137L interaction caused a significant increase in NK cell cytotoxicity and IFN-γ production about 50% (both p<0.05, Mann-Whitney U-test) in coculture assays with CD137L-expressing patient AML cells and AML cell lines. The inhibitory effect of CD137 on NK cell reactivity was further confirmed in cocultures of NK cells with CD137L-transfectants and by triggering CD137 with an agonistic monoclonal antibody. This indicates that CD137 mediates opposite effects in murine compared to human NK cells. Furthermore we conclude that CD137L expression substantially influences tumor immunoediting by AML cells and diminishes NK cell reactivity against AML.

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Aberrant Overexpressions of MicroRNA-21 and MicroRNA-155 Activate AKT Signaling Via Downregulation of Tumor Suppressors in NK-Cell Lymphoma/Leukemia.

Blood ◽

10.1182/blood.v114.22.1917.1917 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1917-1917

Author(s):

Hiroyuki Tagawa ◽

Yasuo Yamanaka ◽

Atsushi Watanabe ◽

Naoto Takahashi ◽

Ken-ichi Sawada

Keyword(s):

Natural Killer ◽

Cell Lines ◽

Antisense Oligonucleotides ◽

Nk Cell ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

Function Analysis ◽

Genetic Alterations ◽

Ebv Infection ◽

Endogenous Expression

Abstract Abstract 1917 Poster Board I-940 Background: Natural Killer (NK) cell lymphomas/leukemias are characterized groups of highly aggressive lymphoid malignancies, which are comprised of “extranodal NK/T cell lymphoma, nasal type” and “aggressive NK-cell leukemia”. Notably, these two subtypes show many similarities in their morphologic features, immmunophenotypes and genotypes, and are invariably associated with Epstein-Barr virus (EBV), which suggests they may share the same genetic alterations. The gene(s) responsible for natural killer (NK)-cell lymphoma/leukemia have not been identified. Materials and Methods: In the present study, we used Northern and quantitative PCR analyses to screen for and quantitatively assess miRNA expression in NK-cell lymphomas/leukemias using 66 probe sets of miRNAs. For function analysis, we conducted antisense oligonucleotides and lentivirus transfection assays using NK-cell lymphoma cell lines. Results: We found that in NK-cell lymphoma lines (n=10) and specimens of primary lymphoma (n=10), levels of miR-21 and miR-155 expression were inversely related, and were significantly higher than those seen in normal natural killer (CD3-CD56+) cells (n=8). To determine the functions of these microRNAs in lymphomagenesis, we examined the effects of antisense oligonucleotides targeting miR-21 (ASO-21) and/or miR-155 (ASO-155) in NK-cell lymphoma lines overexpressing one or both of these miRNAs. Conversely, cells showing little endogenous expression of miR-21 or miR-155 were transduced using lentiviral vectors, leading to their overexpression. Reducing expression of miR-21 or miR-155 led to upregulation of phosphatase and tensin homologue (PTEN), programmed cell death 4 (PDCD4), or Src homology-2 domain-containing inositol 5-phosphatase 1 (SHIP1). ASO-21- and ASO-155-treated cell lines all showed downregulation of phosphorylated AKTser473 (pAKT). Moreover, transduction with either miR-21 or miR-155 led to downregulation of PTEN and PDCD4, or SHIP1 with upregulation of pAKT. Conclusions: Because recent reports suggest that EBV infection can lead directly to upregulation of miR-21 and miR-155, aberrant overexpressions of these miRNAs via EBV infection could be the first-hit genetic alterations during NK-cell lymphomagenesis. Collectively, these results provide important new insight into the pathogenesis of NK-cell lymphoma/leukemia and suggest targeting miR-21 and/or miR-155 may represent a useful approach to treating NK-cell lymphoma/leukemia. Disclosures: No relevant conflicts of interest to declare.

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HLA Influence on NK Cell Expansion

Blood ◽

10.1182/blood.v112.11.4924.4924 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4924-4924

Author(s):

Jennifer Schellekens ◽

Anna Stserbakova ◽

Madis Tõns ◽

Hele Everaus ◽

Marcel GJ Tilanus ◽

...

Keyword(s):

Nk Cells ◽

Cell Lines ◽

Cell Expansion ◽

Nk Cell ◽

Killer Cell ◽

Hla Class I ◽

Class I ◽

Haematopoietic Stem Cell ◽

Specific Priming

Abstract Natural Killer (NK) cells are effector cells in the innate immune system. The anti-leukaemic capacities of NK cells in haematopoietic stem cell transplantation make these cells a potential treatment modality to improve clinical outcome. Immunotherapy with NK cells requires transfusion of large quantities, which obviates the need for an in vitro culture system for NK cells. The killer cell immunoglobulin-like receptors (KIR) on NK cells recognise defined groups of HLA class I alleles. To elucidate the influence of these interactions on proliferation, the peripheral blood mononuclear cells (PBMCs) of 29 patients and donors were cultured in CellGro SCGM with IL-2 and OKT3 antibody to expand the NK cell fraction. The killer cell immunoglobulin-like receptor (KIR) and HLA repertoire were determined by sequence specific priming and sequence based typing respectively. The percentage of NK cell expansion from the total PBMC fraction varied between 5.4% and 71.6%. A significantly better NK cell expansion was observed for individuals homozygous for HLA-C epitope group 2 (p<0.05). For evaluation of cytolytic competence of the cultured NK cells, specific killing of an HLA class I expression deficient LCL 721.221 cell line and three 721.221 cell lines transfected with different HLA-C alleles was determined. A significantly better NK cell-induced specific cytotoxicity was observed towards the untransfected 721.221 cells compared to the HLA-C transfected 721.221 cells. No significant differences were observed between killing of the three HLA-C transfected 721.221 cell lines. We have shown that cytolytic capacities of the cultured NK cells are maintained and in vitro expansion of NK cells is dependant on the presence of HLA-C alleles.

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