Resveratrol Enhances NKG2D-Mediated Cytotoxicity Against Leukemia Cells by Upregulating Both NKG2D Receptor on NK Cells As Well As NKG2D Ligands on Target Cells,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3236-3236
Author(s):  
Luis J. Espinoza ◽  
Akiyoshi Takami ◽  
Katsuya Nakata ◽  
Ly Quoc Trung ◽  
Kayoko Yamada ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Specific Inhibitor ◽  
Receptor Expression ◽  
Leukemia Cells ◽  
Target Cells ◽  
Lymphoid Leukemia ◽  
Nkg2d Ligands ◽  
Nkg2d Receptor

Abstract Abstract 3236 NKG2D is a powerful activating receptor expressed by natural killer (NK) cells that promotes cytotoxic lysis of cancer cells expressing NKG2D ligands (NKG2D-Ls). Pharmacological induction of NKG2D-Ls in malignant cells has been an attractive therapeutic approach but has gained poor clinical utility because currently available NKG2D-Ls inducers have been hampered either by their limited efficacy or by their associated toxicity. Resveratrol (RVT), a compound derived from several natural sources, has proved in vivo and in vitro potent anti-tumor effects against various cancers. Extensive research in the last decade has shown that such effects are mediated by targeting various molecules involved in the regulation of proliferation and cell survival and those include, NFκB, STAT3, ATM/ATR and ERK1/2. To date, it is unknown whether RVT has any effect on NKG2D-Ls expression. We report here NKG2D-Ls induction by RVT in a broad range of leukemia cells. RVT upregulated the NKG2D-Ls MICA/B, ULBP1, ULBP2 and ULBP3 in the myeloid leukemia cells OUN-1, NB4, THP-1 and KG1 and upregulated MICA/B, ULBP-1 and ULBP3 ligands in the lymphoid leukemia cells Jurkat and Molt-4. The upregulation of NKG2D-Ls by RVT was associated with increased transcription of each NKG2D-L gene. Ligand upregulation induced by RVT was prevented by cell pretreatment with caffeine, and inhibitor of ATM/ATR, which is the main signal regulator of NKG2D-Ls expression. Leukemia cells treated with RVT were more susceptible to killing by NK cells than untreated cells and the enhanced cytotoxicity of NK cells was blocked by the treatment of NK cells with anti-NKG2D monoclonal antibodies. Interestingly, the same concentration of RVT that effectively induced NKG2D-Ls in tumor cells, consistently upregulated NKG2D receptor expression in primary NK cells from healthy individuals and in the NK cell lines NKL and NK-92 and this effect was also associated with enhanced NKG2D-mediated NK cells cytotoxicity. RVT-induced NKG2D receptor enhancement in NK cells associated with the activation of the MAP kinase ERK1/2 and was prevented by the ERK1/2 specific inhibitor PD98059. Thus, RVT represents the first identified agent capable of activating both arms of the NKG2D axis. Since several clinical trials on RVT are ongoing, these previously unrecognized properties of this non toxic compound have an attractive immunotherapeutic potential. Disclosures: No relevant conflicts of interest to declare.

Fc-Engineered NKG2D-IgG1 Fusion Proteins Target Leukemia Cells for Antibody-Dependent Cellular Cytotoxicity (ADCC) of NK Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1537-1537 ◽  
Author(s):  
Julia Hilpert ◽  
Katrin Baltz-Ghahremanpour ◽  
Benjamin J Schmiedel ◽  
Lothar Kanz ◽  
Gundram Jung ◽  
...  
Keyword(s):  
Nk Cells ◽  
Fusion Proteins ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Target Cells ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity

Abstract Abstract 1537 The capability of anti-tumor antibodies to recruit Fc-receptor (FcR) bearing effector cells like NK cells, a feature considered critical for therapeutic success, can be markedly improved by modifications of the human IgG1 part. At present, Fc-engineered antibodies targeting leukemia cells are yet not available. The various ligands of the NK cell-activating immunoreceptor NKG2D (NKG2DL) are generally absent on healthy cells but upregulated on malignant cells of various origins including leukemia. We aimed to take advantage of the tumor-restricted expression of NKG2DL by using them as target-antigens for Fc-optimized NKG2D-IgG1 fusion proteins targeting leukemia cells for antibody-dependent cellular cytotoxicity (ADCC) and IFN-g production of NK cells. NKG2D-IgG1 fusion proteins with distinct modifications in their Fc portion were generated as previously described (Lazar 2006; Armour 1999). Compared to wildtype NKG2D-Fc (NKG2D-Fc-WT), the mutants (S239D/I332E and E233P/L234V/L235A/DG236/A327G/A330S) displayed highly enhanced (NKG2D-Fc-ADCC) and abrogated (NKG2D-Fc-KO) affinity to the NK cell FcgRIIIa receptor but comparable binding to NKG2DL-expressing target cells. Functional analyses with allogenic NK cells and leukemia cell lines as well as primary leukemic cells of AML and CLL patients revealed that NKG2D-Fc-KO significantly (p<0.05, Mann-Whitney U test) reduced NK cytotoxicity and IFN-g production (about 20% and 30% reduction, respectively), which can be attributed to blockade of NKG2DL-mediated activating signals. Treatment with NKG2D-Fc-WT significantly (p<0.05, Mann-Whitney U test) enhanced NK reactivity (about 20% and 100% increase in cytotoxicity and cytokine production, respectively). The effects observed upon treatment with NKG2D-Fc-ADCC by far exceeded that of NKG2D-Fc-WT resulting in at least doubled NK ADCC and IFN-g production compared to NKG2D-Fc-WT. When applied in combination with Rituximab in analyses with CLL cells, a clear additive effect resulting in a more than four-fold increase of ADCC and FcgRIIIa-induced IFN-g production was observed. The NKG2D-Fc fusion proteins did not induce NK reactivity against healthy blood cells, which is in line with the tumor-restricted expression of NKG2DL. Of note, treatment with NKG2D-Fc-ADCC also significantly (p<0.05, Mann-Whitney U test) enhanced reactivity (up to 70% increase) of NK cells against NKG2DL-positive AML and CLL cells among patient PBMC in an autologous setting. Together, our results demonstrate that Fc-engineered NKG2D-Fc-ADCC fusion proteins can effectively target NKG2DL-expressing leukemia cells for NK anti-tumor reactivity. In line with the hierarchically organized potential of the various activating receptors governing NK reactivity and due to their highly increased affinity to the FcgRIIIa receptor, NKG2D-Fc-ADCC potently enhances NK anti-leukemia reactivity despite the inevitable reduction of activating signals upon binding to NKG2DL. Due to the tumor-restricted expression of NKG2DL, Fc-modified NKG2D-Ig may thus constitute an attractive means for immunotherapy of leukemia. Disclosures: No relevant conflicts of interest to declare.

Expression and Immunomodulatory Function of RANKL in Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 245-245
Author(s):  
Benjamin J Schmiedel ◽  
Tina Baessler ◽  
Miyuki Azuma ◽  
Lothar Kanz ◽  
Helmut R. Salih
Keyword(s):  
Nk Cells ◽  
Cell Function ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Immune Surveillance ◽  
Leukemia Cells ◽  
Target Cells ◽  
Cytotoxic Lymphocytes ◽  
Hematopoietic Malignancies ◽  
Immunomodulatory Function

Abstract Abstract 245 The TNF family member RANKL and its receptors RANK and osteoprotegerin (OPG) are key regulators of bone remodelling, but have also been shown to influence progression of malignancies like breast cancer (Tan et al., Nature 2011), myeloma (Sordillo et al., Cancer 2003) and CLL (Secchiero et al. J Cell Physiol. 2006). NK cells are cytotoxic lymphocytes that play an important role in tumor immune surveillance especially of hematopoietic malignancies. Their reactivity is influenced by a variety of activating and inhibitory molecules expressed by their target cells including several members of the TNF family. Recently, we reported that RANK, upon interaction with RANKL which can be expressed by malignant hematopoietic cells, mediates signals that impair NK reactivity (Schmiedel et al., Blood 2010 116,21:893–893). Here we extended these analyses and comprehensively studied the expression and immunomodulatory function of RANKL in leukemia. Analysis of primary leukemia cells revealed substantial RANKL surface expression in a high proportion of the investigated cases (AML, 47 of 65 (72%); ALL, 16 of 21 (76%); CML, 6 of 10 (60%); CLL, all 54 (100%)). Signaling via surface-expressed RANKL into the malignant cells mediated the release of cytokines like TNF, IL-6, IL-8 and IL-10 which have been shown to act as autocrine and paracrine growth and survival factors in leukemia. Moreover, the factors released upon RANKL signaling upregulated RANK expression on NK cells. In line, NK cells from leukemia patients (n=75) displayed significantly (p<0.001, Mann-Whitney U-test) higher RANK expression compared to healthy controls (n=30) confirming our notion that RANK-RANKL interaction may contribute to leukemia pathophysiology. We further found that RANK-RANKL interaction, beyond directly inhibiting NK cell function via RANK, may contribute to evasion of leukemia cells from NK immunosurveillance by creating an NK inhibitory cytokine milieu. This was revealed by impaired cytotoxicity and degranulation in response to leukemia targets following exposure of the NK cells to the factors released upon RANKL signaling by leukemia cells. Notably, the RANKL-mediated cytokine release of leukemia cells could be disrupted by the clinically approved RANKL antibody Denosumab/AMG162. Thus, RANKL signaling may trigger a “vicious cycle” comprising of release of immunosuppressive cytokines and also upregulation of RANK on NK cells. The latter both directly inhibits NK reactivity and may result in augmented RANKL signaling into leukemia cells. Our data suggest that therapeutic modulation of the RANK/RANKL system e.g. with Denosumab/AMG162, which is approved for treatment of osteolysis, may be a promising strategy to reinforce NK reactivity against hematopoietic malignancies. Disclosures: No relevant conflicts of interest to declare.

Enhanced Elimination of 6-Mercaptopurine or Methotrexate-Treated Acute Lymphoblastic Leukemia Cells by Natural Killer Lymphocytes In Vitro: A Potential Mechanism of Action of Maintenance Chemotherapy

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2128-2128 ◽  
Author(s):  
Abdual H. Siddiqui ◽  
Mohammad Bhuiyan ◽  
Akila Muthukumar ◽  
Steven Buck ◽  
Yaddanapudi Ravindranath ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Drug Exposure ◽  
Nk Cell ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cells ◽  
Target Cells ◽  
Lymphoma Cells ◽  
Nkg2d Ligands ◽  
Reh Cells

Abstract Abstract 2128 Background: Maintenance chemotherapy (MC) is an important component of childhood B-precursor acute lymphoblastic leukemia (ALL) therapy; however, it is not necessary in the treatment of mature B cell neoplasms. The operational mechanisms of MC are not understood. Improvement in immunologic function including near normal levels of natural killer (NK) lymphocytes was reported during ALL MC. We hypothesize that in addition to their direct cytotoxicity, MC drugs alter surviving lymphoblasts, rendering them susceptible to innate immune response, likely through cell mediated cytotoxicity via stress proteins such as NKG2D ligands, co-stimulatory or adhesion molecules. Objective: The effect of 6-mercaptopurine (6MP) or methotrexate (MTX) treatment of B-precursor and mature B leukemia/lymphoma cells in their elimination by NK lymphocytes was investigated in this study. Design and Methods: Allogeneic NK cell-mediated elimination of REH (TEL/AML-positive B-precursor ALL) and Raji (mature B cell lymphoma) cells treated with standard MC drugs was studied. High dose cytarabine (Ara-C) and MTX are used during the consolidation chemotherapy; therefore, Ara-C and MTX-resistant REH and Raji cell sub-lines were established by exposing wild type cells to increasing concentrations of drugs over several months. Natural killer cells from 17 healthy volunteers were separated using the MACS NK cell isolation kit. After purity evaluation, NK cells were incubated with interleukin-15 overnight. Leukemia cells were incubated in minimally toxic (20% cytotoxicity) concentrations of 6MP and MTX. The leukemia/lymphoma cells were then co-incubated with NK cells at different ratios. The NK cell-mediated leukemia/lymphoma cell cytotoxicity was measured by flow cytometric cell-mediated cytotoxicity assay, marking effector cells with lineage-specific monoclonal antibodies and staining target cells with propidium iodide and annexin-V and using microspheres for quantification of viable and apoptotic cells. The level of resistance of the respective cell sub-lines was evaluated using MTT assay. We also investigated whether NK cell exposure to same concentrations of MC drugs before co-incubation alters cytotoxicity. Surface expression of NKG2D ligands, ULBP 1, 2 and 3, MICA and MICB was studied by flow cytometry. Results: 6-mercaptopurine treatment of REH cells and MTX treatment of Raji cells resulted in enhanced NK cell-mediated elimination when compared to untreated cells by 25% and 20%, respectively. The results were similar when NK cells were exposed to the same concentrations of MC drugs before co-incubation, indicating lack of negative effect of the drug exposure in NK cells’ ability to kill. Similar experiments were conducted on resistant cells, in order to make the target cells more comparable to the residual lymphoblasts during MC. Most interestingly, the REH cells, but not the Raji cells, resistant to Ara-C and MTX showed about 14% and 4% enhancement of NK cell-mediated killing, respectively, after being exposed to the minimally toxic concentrations of MC drugs. This indicates that resistant B precursor ALL cells can be eliminated by NK cells upon MC drug exposure, but not mature B lymphoblasts, in this experimental setting. No increase in the expression of NKG2D ligands on drug treated ALL cells was observed. Conclusion: These findings suggest that enhanced susceptibility of drug-exposed leukemia cells to innate immune response may be an operational mechanism of MC. This mechanism may involve pathways other than NKG2D. Disclosures: No relevant conflicts of interest to declare.

RANKL Expressed by Acute Myeloid Leukemia Cells Impairs NK Cell-Mediated Immune Surveillance

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2164-2164
Author(s):  
Benjamin J Schmiedel ◽  
Constantin M Wende ◽  
Tina Baessler ◽  
Carolin Scheible ◽  
Stefan Wirths ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Nk Cells ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Immune Surveillance ◽  
Surface Protein ◽  
Leukemia Cells ◽  
Acute Myeloid Leukemia Cells ◽  
Acute Myeloid

Abstract Abstract 2164 NK cells play an important role in tumor immunosurveillance, especially of leukemia. Their reactivity is governed by various activating and inhibitory molecules expressed by their targets including multiple members of the TNF family. The TNF family member Receptor Activator of NF-κB ligand (RANKL) and its receptors RANK and osteoprotegerin (OPG) are key regulators of bone remodelling, but recently have also been shown to influence progression of hematopoetic malignancies. Here we studied the yet unkown role of the RANK/RANKL molecule system in NK cells and their reactivity against acute myeloid leukemia (AML). Primary leukemia cells from AML patients were found to substantially express RANKL mRNA and surface protein in 75% of the investigated cases (n=40). Reverse signaling via surface-expressed RANKL into AML blasts induced the release of soluble factors including the immunoregulatory cytokines TNF and IL-10, which impaired NK cell anti-tumor reactivity. Moreover, we observed upregulation of RANK on NK cells among PBMC of healthy donors upon exposure to IL-10. This was not caused by direct effects on NK cells, but was rather due to yet unidentified factors released by monocytes among the PBMC upon IL-10 exposure and could be prevented by the activating cytokine IL-2. Furthermore, functional experiments with NK cells and RANKL transfectants or RANKL-negative controls revealed that forward signaling into RANK-expressing NK cells by tumor-expressed RANKL also directly impaired NK cytotoxicity and IFN-γ production. In line, blocking RANK-RANKL interaction using anti-RANKL antibodies or RANK-Fc fusion protein increased cytotoxicity and cytokine production of allogenic NK cells in cultures with RANKL-positive primary AML cells. Our data indicate that RANKL expression enables immune evasion of leukemia cells both by directly inhibiting reactivity of RANK-expressing NK cells and by orchestrating a reciprocal interplay between AML cells, monocytes and NK cells resulting in an immunosuppressive cytokine milieu. Thus, therapeutic modulation of the RANK/RANKL system, e.g. with Denosumab/AMG162, which is presently being evaluated for treatment of both non-malignant and malignant osteolysis, holds promise to reinforce NK reactivity against hematopoietic malignancies. Disclosures: No relevant conflicts of interest to declare.

Pseudoexpression of Glucocorticoid-Induced TNF-Related (GITR) Ligand Upon Coating of Cancer Cells by Platelets Impairs NK Cell Anti-Tumor Reactivity

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3193-3193 ◽  
Author(s):  
Theresa Placke ◽  
Lothar Kanz ◽  
Helmut R. Salih ◽  
Hans-Georg Kopp
Keyword(s):  
Nk Cells ◽  
Tumor Cells ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Immune Surveillance ◽  
Substantial Reduction ◽  
Cell Activity ◽  
Target Cells ◽  
Tumor Immune Escape ◽  
Tumor Spread

Abstract Abstract 3193 NK cells as part of the innate immune system substantially contribute to cancer immune surveillance. They prevent tumor progression and metastasis due to their ability to mediate cellular cytotoxicity and to produce cytokines like IFN-γ, which, among others, stimulates subsequent adaptive immune responses. NK reactivity results from an integrative response emerging upon recognition of multiple ligands for activating and inhibitory NK cell receptors including various members of the TNFR family. Apart from the direct interaction with their target cells, NK cell activity is further influenced by the reciprocal interplay with various other hematopoietic cells like e.g. dendritic cells. Metastatic tumor spread in experimental animals is dramatically reduced in thrombopenic mice. Additional depletion of NK cells reverses this effect, indicating that platelets may impair NK anti-tumor reactivity. However, the underlying mechanisms have not been fully elucidated, especially in humans. Recently, we demonstrated that NK anti-tumor immunity is impaired by platelet-derived TGF-β, which is released upon interaction of platelets with tumor cells (Kopp et al., Cancer Res. 2009). Here we report that the ligand for the TNFR family member GITR (GITRL) is upregulated on megakaryocytes during maturation resulting in substantial GITRL expression by platelets. Since we recently identified GITR as inhibitory NK receptor involved in tumor immune escape (e.g., Baltz et al., Blood 2008, Baessler et al., Cancer Res. 2009) we investigated how platelet-derived GITRL influences platelet function and NK immune surveillance. Signaling via GITRL into platelets upon interaction with NK-expressed GITR or recombinant GITR-Ig fusion protein did not alter platelet activation as revealed by analysis of the activation marker CD62P and release of TGF-β. Interestingly, we found that GITRL-negative tumor cells rapidly get coated by platelets, which confers a seemingly GITRL-positive phenotype. “GITRL pseudoexpression” on tumor cells caused a substantial reduction of NK cell cytotoxicity and cytokine production. This reduced NK reactivity was not due to induction of apoptosis via GITR and could be restored by addition of a blocking GITR antibody. Thus, coating of tumor cells by platelets inhibits NK reactivity, which is in part mediated by platelet-derived GITRL. Our data provide a functional basis for the previously observed finding that platelets increase metastasis i.e. by enabling evasion of tumor cells from NK-mediated immune surveillance. Disclosures: No relevant conflicts of interest to declare.

Engineering Anti-AML Antibodies for Improved NK Cell ADCC

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3629-3629
Author(s):  
Vladimir Senyukov ◽  
William Kelton ◽  
Nishant Mehta ◽  
George Georgiou ◽  
Dean Lee
Keyword(s):  
Cytotoxic Activity ◽  
Nk Cells ◽  
Adoptive Immunotherapy ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Cell Culture Supernatant ◽  
Current Therapy ◽  
Target Cells ◽  
Wild Type ◽  
Related Donor

Abstract Abstract 3629 Acute myeloid leukemia (AML) is an aggressive malignancy for which current therapy fails to provide durable remission in approximately half of cases. Natural killer (NK) cells, as a key component of innate immunity, have recently shown clinical potential for adoptive immunotherapy against AML, particular when the donor and recipient are KIR mismatched. In addition to patients who do not have a suitable related donor, approximately 30% of patients bear all three families of KIR ligands and therefor cannot benefit from KIR mismatch. Thus, finding a related donor with predicted KIR mismatch is a major obstacle for adoptive NK cell immunotherapy. The majority of peripheral blood NK cells express CD16a (FcγRIIIa), which is the most potent receptor among the activating receptors that NK cells posses. NK cells express CD16a in association with disulflde-linked homo- or hetero-dimers of FcRγ or CD3ζ. Clustering of CD16a initiated by binding to the Fc-portion of IgG1 or IgG3 that opsonize target cells induces signals strong enough to overcome KIR inhibition. Thus, combining NK cell adoptive immunotherapy with Abs against tumor antigens could help overcome the limitations of KIR mismatching. Indeed, many promising anticancer Abs have failed in clinical trials because of insufficient efficacy, which, at least in part, may result from low affinity CD16a binding. Indeed, it was shown that the affinity between Fc and FcγRs correlates with cytotoxicity in cell-based assays and that the Abs with optimized FcγR affinity induced strong cytotoxicity against targeted tumor cells. CD33 is expressed on the blast cells of most cases of AML and represents a suitable antigen for antibody-based therapies. Lintuzumab, an unconjugated, humanized anti-CD33 mAb (HuM195), failed to improve patient outcomes in two randomized trials when combined with conventional chemotherapy. Gemtuzumab ozogamicin, an anti-CD33 mAb conjugated to the calicheamicin, in combination with chemotherapy, improved survival in a subset of AML patients, but has been withdrawn from US market by safety concerns. We optimized the FcγR affinity of HuM195 mAb (mNuM195) by cloning into pMaz-IgH Herceptin recipient vector containing S239D, A330L, I332E mutations that, as previously shown, leads to significant improvement of IgG1 binding to CD16a. To generate control wild type variant (wHuM195) we cloned the variable domains of HuM195 into pMaz-IgH Herceptin. Plasmids were transfected into HEK293F, and Abs were purified from cell culture supernatant with protein A resin, eluted with glycine HCL, and then the samples were buffer exchanged into PBS pH 7.4 for long-term storage. This S239D-A330L-I332E triple mutation in Fc portion of IgG1 did not affect antigen-biding affinity for CD33 target protein but showed more than 14-fold higher binding to CD16a than the wild type variant. The mHuM195 Abs increased cytotoxic activity of expanded human NK cells in Calcein AM-release assay when used in concentration as low as 0.01 μg/ml to pretreat murine thymoma EL-4 cells gene-modified to express human CD33 (ADCC, Mean±SD: 38.7±2.25% vs 11.7±3.49% for optimized vs wild type HuM195, and 5±3.15% without Abs, E:T ratio 2:1). We obtained the similar results when using K562 as targets, which naturally express CD33. K562 cells pretreated with mHuM195 Abs induced degranulation in 34±5.25% of NK cells where wHuM195 did so only in 17±4.6% of NK cells. Thus, optimization of HuM195 Ab to improve CD16a affinity results in dramatic increases NK cell cytotoxic activity. Disclosures: No relevant conflicts of interest to declare.

Chemokine Receptor Expression, Migration, and Survival of NK Cells Expanded with Mbil-15 or Mbil-21

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4683-4683
Author(s):  
Dean Lee ◽  
Maureen Aliru ◽  
Cecele J. Denman ◽  
Srinivas S. Somanchi
Keyword(s):  
Nk Cells ◽  
Chemokine Receptors ◽  
Conflicts Of Interest ◽  
Specific Antigen ◽  
Xenograft Model ◽  
Receptor Expression ◽  
Target Cells ◽  
Expression Levels ◽  
Tissue Migration

Abstract Abstract 4683 Natural killer (NK) cells can kill malignant or virus-infected cells through the interaction of activating and inhibitory receptors without needing specific antigen recognition of target cells, and therefor have broad therapeutic applications for treatment of human malignancies. However, due to their limited life-span in vivo and poor expansion in vitro, production of sufficient numbers of NK cells for effective adoptive immunotherapy poses an obstacle. Genetically engineered artificial antigen presenting cells (aAPCs) consisting of K562 modified 4-1BBL and membrane bound IL-15 or IL-21 have been reported for their ability to support ex vivo NK cell proliferation. aAPCs with mbIL-21 were shown to promote increased proliferation of NK cells with shorter telomeres, but differences in in vivo survival or tumor or tissue migration have not been assessed. Tumor and/or tissue migration is primarily mediated by the expression of chemokine receptors. Using aAPCs bearing mbIL15 or mbIL21, we expanded NK cells for 3 weeks and assessed their expression of chemokine receptors, organ migration, and in vivo survival in a xenograft model. Propagated NK cells showed relatively similar levels of low to modest expression of CCR2, CCR7, CXCR4 and CXCR5, and high expression levels of CXCR3. Mean CCR5 expression levels were similar on cells that were positive, but CCR5 was expressed on a higher percentage of NK cells expanded with mbIL-15 than those expanded with mbIL-21. In contrast, about 20% of mbIL-21 expanded NK cells expressed CX3CR1 expression whereas mbIL-15 NK cells showed almost no expression of this receptor. Results from ongoing migration and survival experiments will also be presented. Disclosures: No relevant conflicts of interest to declare.

scholarly journals HIV-1 Vpr up-regulates expression of ligands for the activating NKG2D receptor and promotes NK cell–mediated killing

Blood ◽  
2010 ◽  
Vol 115 (7) ◽  
pp. 1354-1363 ◽  
Author(s):  
Jonathan Richard ◽  
Sardar Sindhu ◽  
Tram N. Q. Pham ◽  
Jean-Philippe Belzile ◽  
Éric A. Cohen
Keyword(s):  
Dna Damage ◽  
Cell Surface ◽  
Nk Cell ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Target Cells ◽  
Nkg2d Ligands ◽  
Infected Cells ◽  
Nkg2d Receptor ◽  
Hiv 1

AbstractHIV up-regulates cell-surface expression of specific ligands for the activating NKG2D receptor, including ULBP-1, -2, and -3, but not MICA or MICB, in infected cells both in vitro and in vivo. However, the viral factor(s) involved in NKG2D ligand expression still remains undefined. HIV-1 Vpr activates the DNA damage/stress-sensing ATR kinase and promotes G2 cell-cycle arrest, conditions known to up-regulate NKG2D ligands. We report here that HIV-1 selectively induces cell-surface expression of ULBP-2 in primary CD4+ T lymphocytes by a process that is Vpr dependent. Importantly, Vpr enhanced the susceptibility of HIV-1–infected cells to NK cell–mediated killing. Strikingly, Vpr alone was sufficient to up-regulate expression of all NKG2D ligands and thus promoted efficient NKG2D-dependent NK cell–mediated killing. Delivery of virion-associated Vpr via defective HIV-1 particles induced analogous biologic effects in noninfected target cells, suggesting that Vpr may act similarly beyond infected cells. All these activities relied on Vpr ability to activate the ATR-mediated DNA damage/stress checkpoint. Overall, these results indicate that Vpr is a key determinant responsible for HIV-1–induced up-regulation of NKG2D ligands and further suggest an immunomodulatory role for Vpr that may not only contribute to HIV-1–induced CD4+ T-lymphocyte depletion but may also take part in HIV-1–induced NK-cell dysfunction.

scholarly journals The Traffic of the NKG2D/Dap10 Receptor Complex during Natural Killer (NK) Cell Activation

2009 ◽  
Vol 284 (24) ◽  
pp. 16463-16472 ◽  
Author(s):  
Pedro Roda-Navarro ◽  
Hugh T. Reyburn
Keyword(s):  
Cell Surface ◽  
Nk Cells ◽  
Cell Activation ◽  
Cell Function ◽  
Nk Cell ◽  
Receptor Expression ◽  
Cell Surface Expression ◽  
Target Cells ◽  
Receptor Complex ◽  
Secretory Lysosomes

NKG2D is an important activating receptor for triggering the NK cell cytotoxic activity, although chronic engagement of specific ligands by NKG2D is also known to provoke decreased cell surface expression of the receptor and compromised NK cell function. We have studied the dynamics of surface NKG2D expression and how exposure to the specific ligand major histocompatibility complex class I chain-related molecule B (MICB) affects receptor traffic and fate. While in the NKL cell line and “resting” NK cells NKG2D was found principally at the cell surface, in activated primary NK cells an intracellular pool of receptor could also be found recycling to the plasma membrane. Exposure of NK cells to targets expressing MICB resulted in degradation of ∼50% of total NKG2D protein and lysosomal degradation of the DAP10 adaptor molecule. Consistent with these observations, confocal microscopy experiments demonstrated that DAP10 trafficked to secretory lysosomes in both transfected NKL cells and in activated primary NK cells upon interaction with MICB-expressing target cells. Interestingly, polarization to the synapse of secretory lysosomes containing DAP10 was also observed. The implications of the intracellular traffic of the NKG2D/DAP10 receptor complex for NK cell activation are discussed. We propose that the rapid degradation of NKG2D/DAP10 observed coincident with recruitment of the receptor to the cytotoxic immune synapse may explain the loss of NKG2D receptor expression after chronic exposure to NKG2D ligands.

Natural Killer Cells with NKDG2D Cytotoxicity but NKp30-Dull Phenotype Predominate in Myelodysplastic Syndrome (MDS).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3436-3436
Author(s):  
Fanqi Bai ◽  
Jeffrey S. Painter ◽  
Cantor Alan ◽  
Zou JianXiang ◽  
Sheng Wei ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Cell Line ◽  
Cell Lines ◽  
Nk Cell ◽  
B Cell Lymphoma ◽  
Target Cells ◽  
Nkg2d Ligands ◽  
Human Stress ◽  
Normal Controls

Abstract Natural Killer (NK) function in patients with MDS as measured by non-MHC-restricted cytotoxicity and activation-dependent cell cytotoxicity (ADCC) are reduced in patients with MDS, however, the mechanisms of the functional impairment are not known. Tumor cytolysis occurs through orchestrated control by inhibitory NK receptors (NKRs) and activating NKRs, which control signaling events that lead to polarized movement of perforin-containing granules toward the NK-tumor contact area. We found that NK cells from 23 out of 35 patients with MDS (66%) displayed reduced lysis of K562 tumor cells compared to age-matched normal controls (p&lt;0.01). To better characterize this defect, we evaluated patient NK function against differential tumor targets including the MDS1 cell line established from an MDS patients. We found that MDS1 incited non-MHC-restricted lysis. Unactivated PBMCs, unactivated NK cells, NK cell lines (NK92 and NKL) but not purified unactivated T cells from normal donors killed MDS1 in 4-hr 51Cr-release assays. Normal NK cells and NK cell lines were also found to rapidly redistrubute perforin granules after exposure to MDS1suggesting that a perforin-dependent lytic pathway was activated. We then performed simultaneous cytolytic assays with K562, MDS1, and the 721.221 B cell lymphoma cell line as target cells. We found that NK cells from MDS patients had greater lytic activity against MDS1 (average 24% vs. average 8% at 50:1 Effector:Target ratio, respectively, p&lt;0.01) Antibody-blocking experiments demonstrated that the NKL cell line and PBMCs from 8 out of 10 MDS patients predominantly used the NKG2D activating receptor to kill MDS1. Consistent with this finding, we showed that MDS1 cells express the major human stress-inducible endogenous proteins MICA and MICB, which are NKG2D ligands. In contast, lysis by NK92 cells and normal PBMCs was not appreciably reduced by NKG2D blocking antibodies suggesting that other unidentified NKR(s) also mediate lysis. To identify the NKRs expressed in MDS patients, we performed immunophenotyping for both the activating NKRs and inhibitory NKRs compared to age-matched normal controls. We found that two activating receptors, NKp30 and CD244 (2B4), were significantly reduced on NK cells from all MDS patients regardless of their ability to lyse NK targets. Inhibitory NKR expression and function were normal. Interestingly, NKG2D expression correlated with reduced cytolytic function. Similar to studies on normal NK cells with low NKp30 and NKp46 (NCRdull) phenotypes, these results suggest that low NKp30 expression leads to predominant NKG2D utilization for tumor cell lysis, which is reduced in MDS patients with defective NK function. Our findings provide critical information about potential importance for immunosurviellance through NKG2D-NKG2D ligands.

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