scholarly journals NK-cell activation and antibody-dependent cellular cytotoxicity induced by rituximab-coated target cells is inhibited by the C3b component of complement

Blood ◽  
2008 ◽  
Vol 111 (3) ◽  
pp. 1456-1463 ◽  
Author(s):  
Siao-Yi Wang ◽  
Emilian Racila ◽  
Ronald P. Taylor ◽  
George J. Weiner
Keyword(s):  
Cell Activation ◽  
Complement Fixation ◽  
Nk Cell ◽  
Target Cells ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Antitumor Effects ◽  
Surface Enhanced ◽  
The Impact ◽  
The Relationship

Abstract Antibody-dependent cellular cytotoxicity (ADCC) and complement fixation both appear to play a role in mediating antitumor effects of monoclonal antibodies (mAbs), including rituximab. We evaluated the relationship between rituximab-induced complement fixation, natural killer (NK)–cell activation, and NK cell–mediated ADCC. Down-modulation of NK- cell CD16 and NK-cell activation induced by rituximab-coated target cells was blocked by human serum but not heat-inactivated serum. This inhibition was also observed in the absence of viable target cells. C1q and C3 in the serum were required for these inhibitory effects, while C5 was not. An antibody that stabilizes C3b on the target cell surface enhanced the inhibition of NK-cell activation induced by rituximab-coated target cells. Binding of NK cells to rituximab-coated plates through CD16 was inhibited by the fixation of complement. C5-depleted serum blocked NK cell–mediated ADCC. These data suggest that C3b deposition induced by rituximab-coated target cells inhibits the interaction between the rituximab Fc and NK-cell CD16, thereby limiting the ability of rituximab-coated target cells to induce NK activation and ADCC. Further studies are needed to define in more detail the impact of complement fixation on ADCC, and whether mAbs that fail to fix complement will be more effective at mediating ADCC.

The C3b Component of Complement Inhibits NK Cell Activation Induced by Rituximab-Coated Target Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2505-2505 ◽  
Author(s):  
Siao-Yi Wang ◽  
Emilian Racila ◽  
Ronald P. Taylor ◽  
George J. Weiner
Keyword(s):  
Human Serum ◽  
Nk Cells ◽  
Cell Activation ◽  
Complement Fixation ◽  
Nk Cell ◽  
Normal Human Serum ◽  
Heat Inactivation ◽  
Target Cells ◽  
Normal Human ◽  
The Relationship

Abstract Rituximab (RTX) is an accepted therapy for B cell malignancies, but there is still much to learn about the mechanisms responsible for the observed responses and the potential interactions between various mechanisms of action. Some studies suggest that complement fixation followed by lysis through the membrane attack complex contributes to the anti-tumor effects of RTX. Other investigations indicate that antibody dependent cellular cytotoxicity (ADCC) mediated by NK cells is central to the response of therapy. In prior studies, we found that RTX-coated target cells activate NK cells as indicated by NK cell modulation of CD16, upregulation of CD54 and CD69, and production of IFNγ. NK activation induced by RTX-coated target cells was dependent on the affinity of multivalent interactions between Fc γ receptors III (CD16) of the NK cell and Fc regions of cell-bound RTX molecules. We used these in vitro assays to assess the relationship between complement fixation, and the ability of RTX-coated target cells to activate NK cells. Normal human serum inhibited the modulation of NK cell CD16, and also blocked upregulation of CD54, induced by RTX-coated target cells. The ability of serum to inhibit NK activation was dose dependent and was abrogated upon heat inactivation. Serum depleted of C1q or C3 also failed to inhibit NK cell activation. The inhibitory activity of serum depleted of these complement components was restored when purified C1q or C3 were added back respectively. In addition, the level of NK cell inhibition was dependent on the amount of C3b deposited on the target cells. An antibody that stabilizes C3b on the target cell surface (3E7, DiLillo et al., Molec Immunol 2006) further enhanced the inhibition of NK cell activation induced by RTX-coated target cells. One possible explanation for these findings is that complement-mediated lysis destroyed the RTX-coated target cells before they had the opportunity to induce activation of the NK cells. To assess this possibility, lymphoma cells were killed, fixed with formaldehyde, and washed prior to their use as target cells. These RTX-coated and fixed target cells were able to induce modulation of CD16 on the NK cells, which was again inhibited by normal human serum. These findings indicate that the observed inhibition of NK activation by complement is unlikely to be a consequence of complement mediated lysis of the target cells. Instead, these data suggest that C3b deposition on RTX-coated target cells inhibits the interaction between the Fc portions of RTX and CD16 on the NK cells, and so limits the ability of RTX-coated target cells to induce NK activation. These results could have implications in our understanding of the relationship between complement fixation and ADCC, and their relative roles in potentiating destruction of malignant cells in the blood and tissues.

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.

Different NK cell–activating receptors preferentially recruit Rab27a or Munc13-4 to perforin-containing granules for cytotoxicity

Blood ◽  
2009 ◽  
Vol 114 (19) ◽  
pp. 4117-4127 ◽  
Author(s):  
Stephanie M. Wood ◽  
Marie Meeths ◽  
Samuel C. C. Chiang ◽  
Anne Grete Bechensteen ◽  
Jaap J. Boelens ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Reciprocal Relationship ◽  
Target Cells ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Loss Of Function ◽  
Lytic Granules ◽  
Molecular Events ◽  
Cell Release

Abstract The autosomal recessive immunodeficiencies Griscelli syndrome type 2 (GS2) and familial hemophagocytic lymphohistiocytosis type 3 (FHL3) are associated with loss-of-function mutations in RAB27A (encoding Rab27a) and UNC13D (encoding Munc13-4). Munc13-4 deficiency abrogates NK-cell release of perforin-containing lytic granules induced by signals for natural and antibody-dependent cellular cytotoxicity. We demonstrate here that these signals fail to induce degranulation in resting NK cells from Rab27a-deficient patients. In resting NK cells from healthy subjects, endogenous Rab27a and Munc13-4 do not colocalize extensively with perforin. However, phorbol 12-myristate 13-acetate and ionomycin stimulation or conjugation to susceptible target cells induced myosin-dependent colocalization of Rab27a and Munc13-4 with perforin. Unexpectedly, individual engagement of receptors leukocyte functional antigen-1, NKG2D, or 2B4 induced colocalization of Rab27a, but not Munc13-4, with perforin. Conversely, engagement of antibody-dependent cellular cytotoxicity receptor CD16 induced colocalization of Munc13-4, but not Rab27a, with perforin. Furthermore, colocalization of Munc13-4 with perforin was Rab27a-dependent. In conclusion, Rab27a or Munc13-4 recruitment to lytic granules is preferentially regulated by different receptor signals, demonstrating that individual target cell ligands regulate discrete molecular events for lytic granule maturation. The data suggest Rab27a facilitates degranulation at an early step yet highlight a reciprocal relationship between Munc13-4 and Rab27a for degranulation.

scholarly journals Natural Killer (NK) Cell Education Differentially Influences HIV Antibody-Dependent NK Cell Activation and Antibody-Dependent Cellular Cytotoxicity

2017 ◽  
Vol 8 ◽  
Author(s):  
Nicole F. Bernard ◽  
Zahra Kiani ◽  
Alexandra Tremblay-McLean ◽  
Sanket A. Kant ◽  
Christopher E. Leeks ◽  
...  
Keyword(s):  
Natural Killer ◽  
Cell Activation ◽  
Nk Cell ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity

scholarly journals HIV-1 Env- and Vpu-Specific Antibody-Dependent Cellular Cytotoxicity Responses Associated with Elite Control of HIV

2017 ◽  
Vol 91 (18) ◽  
Author(s):  
Vijaya Madhavi ◽  
Bruce D. Wines ◽  
Janaki Amin ◽  
Sean Emery ◽  
Ester Lopez ◽  
...  
Keyword(s):  
Immune Responses ◽  
Hiv Infection ◽  
Specific Antibody ◽  
Cell Activation ◽  
Nk Cell ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Elite Controllers ◽  
Hiv Replication

ABSTRACT Studying HIV-infected individuals who control HIV replication (elite controllers [ECs]) enables exploration of effective anti-HIV immunity. HIV Env-specific and non-Env-specific antibody-dependent cellular cytotoxicity (ADCC) may contribute to protection from progressive HIV infection, but the evidence is limited. We recruited 22 ECs and matched them with 44 viremic subjects. HIV Env- and Vpu-specific ADCC responses in sera were studied using a novel enzyme-linked immunosorbent assay (ELISA)-based dimeric recombinant soluble FcγRIIIa (rsFcγRIIIa)-binding assay, surface plasmon resonance, antibody-dependent natural killer (NK) cell activation assays, and ADCC-mediated killing assays. ECs had higher levels of HIV Env-specific antibodies capable of binding FcγRIIIa, activating NK cells, and mediating granzyme B activity (all P < 0.01) than viremic subjects. ECs also had higher levels of antibodies against a C-terminal 13-mer Vpu peptide capable of mediating FcγRIIIa binding and NK cell activation than viremic subjects (both P < 0.05). Our data associate Env-specific and Vpu epitope-specific ADCC in effective immune responses against HIV among ECs. Our findings have implications for understanding the role of ADCC in HIV control. IMPORTANCE Understanding immune responses associated with elite control of HIV may aid the development of immunotherapeutic and vaccine strategies for controlling HIV infection. Env is a major HIV protein target of functional antibody responses that are heightened in ECs. Interestingly, EC antibodies also target Vpu, an accessory protein crucial to HIV, which degrades CD4 and antagonizes tetherin. Antibodies specific to Vpu are a common feature of the immune response of ECs that may prove to be of functional importance to the design of improved ADCC-based immunotherapy and preventative HIV vaccines.

scholarly journals CD4- and Time-Dependent Susceptibility of HIV-1-Infected Cells to Antibody-Dependent Cellular Cytotoxicity

2019 ◽  
Vol 93 (10) ◽  
Author(s):  
Wen Shi Lee ◽  
Jérémie Prévost ◽  
Jonathan Richard ◽  
Reneé M. van der Sluis ◽  
Sharon R. Lewin ◽  
...  
Keyword(s):  
Late Stage ◽  
De Novo ◽  
Nk Cell ◽  
Early Stage ◽  
Time Dependent ◽  
Target Cells ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Infected Cells ◽  
Hiv 1

ABSTRACTHIV-1-specific antibody-dependent cellular cytotoxicity (ADCC) antibodies within HIV-1-positive (HIV-1+) individuals predominantly target CD4-induced (CD4i) epitopes on HIV-1 envelope glycoprotein (Env). These CD4i epitopes are usually concealed on the surface of infected cells due to CD4 downregulation by the HIV-1 accessory proteins Nef and Vpu. We hypothesized that early-stage infected cells in the process of downregulating CD4 could be more susceptible to ADCC than late-stage infected cells that have fully downregulated CD4. There was significantly higher binding of antibodies within plasma from HIV-1-infected individuals to early-stage infected cells expressing intermediate levels of CD4 (CD4-intermediate cells) than in late-stage infected cells expressing low levels of CD4 (CD4-low cells). However, we noted that HIV-1-uninfected bystander cells and HIV-1-infected cells, at various stages of downregulating CD4, were all susceptible to NK cell-mediated ADCC. Importantly, we observed that the cytolysis of bystander cells and early infected cells in this culture system was driven by sensitization of target cells by inoculum-derived HIV-1 Env or virions. This phenomenon provided Env to target cells prior tode novoEnv expression, resulting in artifactual ADCC measurements. Future studies should take into consideration the inherent caveats ofin vitroinfection systems and develop improved models to address the potential role for ADCC against cells with nascent HIV-1 infection.IMPORTANCEAn increasing body of evidence suggests that ADCC contributes to protection against HIV-1 acquisition and slower HIV-1 disease progression. Targeting cells early during the infection cycle would be most effective in limiting virus production and spread. We hypothesized that there could be a time-dependent susceptibility of HIV-1-infected cells to ADCC in regard to CD4 expression. We observed NK cell-mediated ADCC of HIV-1-infected cells at multiple stages of CD4 downregulation. Importantly, ADCC of early infected cells appeared to be driven by a previously unappreciated problem of soluble Env and virions from the viral inoculum sensitizing uninfected cells to ADCC prior tode novoEnv expression. These results have implications for studies examining ADCC against cells with nascent HIV-1 infection.

Fc-Engineered RANK-Fc Fusion Proteins for Neutralization of Soluble RANKL and Induction of Antibody-Dependent Cellular Cytotoxicity (ADCC) Against Multiple Myeloma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3039-3039
Author(s):  
Benjamin J Schmiedel ◽  
Carolin Scheible ◽  
Tina Baessler ◽  
Constantin M Wende ◽  
Stefan Wirths ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Nk Cells ◽  
Fusion Proteins ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Soluble Form ◽  
Target Cells ◽  
Published Data ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity

Abstract Abstract 3039 Bone resorption is commonly associated with aging, but also with certain cancers. Recent studies identified Receptor Activator of NF-κB (RANK) ligand (RANKL) and its receptors RANK and osteoprotegerin as key regulators of bone remodelling. Multiple myeloma (MM) disrupts the balance within this molecule system towards osteoclastogenesis and bone destruction. Neutralization of RANKL by the monoclonal antibody Denosumab (AMG162) is presently being evaluated for treatment of both non-malignant and malignant osteolysis. We found, in line with previously published data, that primary MM cells (9 of 10) express substantial levels of RANKL at the cell surface and that MM cells directly release RANKL in soluble form (sRANKL). Next we evaluated the possibility to combine neutralization of sRANKL with targeting of MM cells for antibody-dependent cellular cytotoxicity (ADCC) of NK cells utilizing RANK-Ig fusion proteins with modified Fc portions. Compared to wildtype RANK-Fc, our mutants (S239D/I332E and E233P/L234V/L235A/DG236/A327G/A330S) displayed highly enhanced (RANK-Fc-ADCC) and abrogated (RANK-Fc-KO) affinity, respectively, to the NK cell FcγRIIIa, but comparable capacity to neutralize RANKL in binding competition and osteoclast formation assays. Analyses with RANKL transfectants and RANKL-negative controls confirmed the high and lacking potential of the RANK-Fc-ADCC and the RANK-Fc-KO to induce NK ADCC, respectively, and ascertained that the RANK-Fc-ADCC specifically induced NK cell lysis of RANKL-expressing but not RANKL-negative target cells. Most notably, in cultures of NK cells with RANKL-expressing primary MM cells RANK-Fc-ADCC potently enhanced NK cell degranulation, cytokine release and MM cells lysis due to enhanced NK reactivity. Thus, our Fc-engineered RANK-Fc-ADCC fusion protein may both neutralize detrimental effects of sRANKL and enhance NK anti-tumor reactivity by targeting RANKL-expressing malignant cells thereby constituting an attractive immunotherapeutic means for treatment of MM. Disclosures: No relevant conflicts of interest to declare.

scholarly journals GA101 induces NK-cell activation and antibody-dependent cellular cytotoxicity more effectively than rituximab when complement is present

2013 ◽  
Vol 54 (11) ◽  
pp. 2500-2505 ◽  
Author(s):  
Delila J. Kern ◽  
Britnie R. James ◽  
Sue Blackwell ◽  
Christian Gassner ◽  
Christian Klein ◽  
...  
Keyword(s):  
Cell Activation ◽  
Nk Cell ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity

scholarly journals IL-15 enhanced antibody-dependent cellular cytotoxicity mediated by NK cells and macrophages

2018 ◽  
Vol 115 (46) ◽  
pp. E10915-E10924 ◽  
Author(s):  
Meili Zhang ◽  
Bernard Wen ◽  
Olga M. Anton ◽  
Zhengsheng Yao ◽  
Sigrid Dubois ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Activation ◽  
Nk Cell ◽  
Xenograft Model ◽  
Fold Increase ◽  
Antitumor Action ◽  
Host Immune System ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Syngeneic Mouse Model

The goal of cancer immunotherapy is to stimulate the host immune system to attack malignant cells. Antibody-dependent cellular cytotoxicity (ADCC) is a pivotal mechanism of antitumor action of clinically employed antitumor antibodies. IL-15 administered to patients with metastatic malignancy by continuous i.v. infusion at 2 μg/kg/d for 10 days was associated with a 38-fold increase in the number and activation status of circulating natural killer (NK) cells and activation of macrophages which together are ADCC effectors. We investigated combination therapy of IL-15 with rituximab in a syngeneic mouse model of lymphoma transfected with human CD20 and with alemtuzumab (Campath-1H) in a xenograft model of human adult T cell leukemia (ATL). IL-15 greatly enhanced the therapeutic efficacy of both rituximab and alemtuzumab in tumor models. The additivity/synergy was shown to be associated with augmented ADCC. Both NK cells and macrophages were critical elements in the chain of interacting effectors involved in optimal therapeutic responses mediated by rituximab with IL-15. We provide evidence supporting the hypothesis that NK cells interact with macrophages to augment the NK-cell activation and expression of FcγRIV and the capacity of these cells to become effectors of ADCC. The present study supports clinical trials of IL-15 combined with tumor-directed monoclonal antibodies.

scholarly journals Enhancement of Antibody-Dependent Cellular Cytotoxicity of Neonatal Cells by Interleukin-2 (IL-2) and IL-12

1998 ◽  
Vol 5 (1) ◽  
pp. 98-104 ◽  
Author(s):  
Quoc H. Nguyen ◽  
Robert L. Roberts ◽  
Bonnie J. Ank ◽  
Syh-Jae Lin ◽  
Casey K. Lau ◽  
...  
Keyword(s):  
Cord Blood ◽  
Blood Cells ◽  
Nk Cell ◽  
Interleukin 2 ◽  
Cell Activity ◽  
Target Cells ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Cord Blood Cells ◽  
Adult Cells

ABSTRACT Newborn infants are more susceptible to infections due in part to deficiencies in the cytotoxic functions of their lymphocytes. We investigated the ability of interleukin-2 (IL-2) and IL-12 to enhance the cytotoxicity of neonatal (cord blood) and adult mononuclear cells (MNCs) in both natural killer (NK) cell and antibody-dependent cellular cytotoxicity (ADCC) assays. The cytotoxic activity of cord blood MNCs was less than 50% that of adult MNCs in most assays prior to exposure to cytokines. Incubation with IL-2 (100 U/ml) or IL-12 (1 ng/ml) for 18 h increased the NK cell activity (using K562 target cells) of both cord blood and adult MNCs, and the combination of IL-2 and IL-12 increased cord blood cytotoxicity threefold, making the cytotoxicity of cord blood cells equivalent to that of adult cells treated with the same cytokines. In ADCC assays with chicken erythrocyte targets, the combination of IL-2 and IL-12 increased the cytotoxicities of both cord blood and adult MNCs, with greater enhancement again seen with cord blood cells. In assays with NK cell-resistant CEM cells coated with human immunodeficiency virus (HIV) gp120 antigen in the presence of hyperimmune anti-HIV immunoglobulin, ADCC of cord blood MNCs was about 50% that of adult MNCs; ADCC of cord blood MNCs increased two- to threefold with the addition of IL-2 and IL-12, whereas ADCC of adult MNCs did not increase. Incubation of cord blood cells, but not adult cells, with IL-2 or IL-12 for 1 week increased the percentage of CD16+/CD56+ cells two- to fivefold and enhanced ADCC activity. Thus, IL-2 and IL-12 greatly enhance both the NK cell and ADCC activities of neonatal MNCs and increase the number of NK cells in longer-term culture.

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