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Blood, 1 August 2008, Vol. 112, No. 3, pp. 699-707. Prepublished online as a Blood First Edition Paper on June 2, 2008; DOI 10.1182/blood-2007-11-122465.
IMMUNOBIOLOGY Fc-dependent expression of CD137 on human NK cells: insights into "agonistic" effects of anti-CD137 monoclonal antibodies1 Department of Otorhinolaryngology-Head and Neck Surgery, University of Maryland, Baltimore; 2 Department of Pathology, University of Maryland School of Medicine, Baltimore; 3 GTC Biotherapeutics, Framingham, MA; 4 Institute of Human Virology, University of Maryland, Baltimore; 5 Department of Dermatology, Johns Hopkins University, Baltimore, MD; 6 Division of Biostatistics, University of Maryland Greenebaum Cancer Center, Baltimore; and 7 Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore
CD137 (4-1BB) is a costimulatory mol-ecule that can be manipulated for the treatment of cancer and autoimmune disease. Although it is known that agonistic antibodies (mAbs) against CD137 enhance the rejection of murine tumors in a natural killer (NK) cell– and T cell–dependent fashion, the mechanism for NK dependence is poorly understood. In this study, we evaluated the ability of 2 different glycoforms of a chimerized antihuman CD137 mAb, an aglycosylated (GA) and a low fucose form (GG), to react with human NK cells. Both mAbs bound similarly to CD137 and partially blocked the interaction between CD137 and CD137 ligand. However, unlike GA mAb, immobilized GG mAb activated NK cells and enhanced CD137 expression. These effects were seemingly dependent on Fc interaction with putative Fc receptors on the NK-cell surface, as only the immobilized Fc-fragment of GG was required for CD137 expression. Furthermore, CD137 expression could be enhanced with antibodies directed against non-CD137 epitopes, and the expression levels directly correlated with patterns of Fc-glycosylation recognized to improve Fc interaction with Fc  receptors. Our data suggest that CD137 can be enhanced on NK cells in an Fc-dependent fashion and that expression correlates with phenotypic and functional parameters of activation.
CD137 (4-1BB), a member of the TNF receptor superfamily, is increasingly recognized as an important target for the treatment of both cancer and autoimmunity.1–5 Specifically, in murine models, it is clear that ligation of CD137 on the surface of activated T cells, through either CD137 ligand (CD137L) or agonistic monoclonal antibodies (mAbs), potentiates the immune response to weakly immunogenic tumors in a natural killer (NK)–dependent fashion.3,6 Unlike other costimulatory-based antitumor immunotherapies (eg, CTLA-4 blockade), CD137 ligation does not induce self-reactivity, but rather has therapeutic benefit in multiple murine models of autoimmune disease such as rheumatoid arthritis,7 systemic lupus erythematosus,8 and inflammatory bowel disease.9 In many studies, functional conclusions regarding CD137 have been based on mAb or fusion protein manipulation of receptor/ligand pathways, with the assumption being that observed effects were secondary to Fab region or ligand interaction with CD137. Importantly, little attention has been paid to the potential link between the Fc region of these molecules and alternate pathways of CD137 regulation through Fc receptors (Fc Rs)
It is now evident that Fc cross-linking of Fc
Based on the therapeutic potential of anti-CD137 mAbs and the recognized importance of Fc-Fc
Surprisingly, we observed that, after IL-2 stimulation and subsequent culture, human NK cells do not express high levels of CD137. However, CD137 is enhanced on IL-2–stimulated human NK cells after culture in the presence of immobilized glycosylated mAbs or papain-cleaved Fc fragments. The ability to enhance CD137 expression is independent of the antigen specificity of the Fab region, and the magnitude of CD137 expression is associated with patterns of glycosylation known to enhance the interaction between Fc and Fc
All experimental work related to human materials was approved by the University of Maryland's institutional review board (IRB), under IRB number H-27785. Chimeric monoclonal antibodies Mouse anti–human CD137 mAb (G6) was generated in the laboratory of L. C. A glycosylated chimeric version of G6, hereafter called GG, was developed by replacing the mouse IgG2a Fc region with a human IgG1 Fc region. Likewise, an aglycosylated (GA) chimeric anti-CD137 mAb was created by mutating Asn to glutamine (Gln) at amino acid position 297 in the Fc region. Both GA and GG mAbs were produced in the milk of transgenic goats. Cetuximab (hereafter called CTM) was obtained through the Greenebaum Cancer Center (University of Maryland Medical System, Baltimore). For flow cytometric experiments, all chimeric mAbs were directly labeled to allophycocyanin (APC) through the custom conjugation service of Invitrogen (Invitrogen, Carlsbad, CA). Cells and transfectants Human blood samples were purchased (Biological Specialty Corporation, Colmar, PA) and whole peripheral blood mononuclear cells (PBMCs) were isolated using density-gradient centrifugation. Freshly purified resting NK cells (CD3–CD56+CD16+ as defined by flow cytometry) were obtained by negative magnetic cell sorting using NK-cell isolation beads (Miltenyi Biotec, Auburn, CA) according to the manufacturer's instructions. Chinese hamster ovary (CHO) cells stably transfected with human CD137 receptor and nontransfected wild-type controls were established in the laboratory of L.C. CHO cells were maintained in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Atlanta Biological, Lawrenceville, GA), 1% penicillin/streptomycin (P/S), 1% Glutamax-1 (both Invitrogen), and 1% HEPES (Mediatech, Herndon, VA). K562 cells were cultured in RPMI 1640 supplemented with 10% FBS, 1% P/S, and 1% Glutamax-1. In vitro NK experiments Fresh purified resting NK cells (CD3–CD56+CD16+ as defined by flow cytometry; > 90% purity) were stimulated for 72 hours in RPMI 1640 supplemented with 10% FBS, 1% P/S, 1% HEPES, 1% Glutamax-1, and 200 IU/mL recombinant human IL-2 (rhIL-2, aldesleukin; Chiron, Emeryville, CA). After 72 hours, NK cells were washed 3 times with phosphate-buffered saline and resuspended in RPMI 1640 supplemented with 10% FBS, 1% P/S, 1% HEPES, and 1% Glutamax-1. IL-2–stimulated NK cells were plated at a concentration of 2 x 105 cells/well in a 96-well flat-bottom plate in the presence of either precoated (immobilized) or soluble chimeric mAb or polyclonal human IgG1 (huIgG1; Sigma-Aldrich, St Louis, MO) at a concentration of 20 µg/mL. Control wells were precoated overnight with RPMI 1640 supplemented with 10% FBS to minimize cross-linking of soluble positively charged immunoglobulins present in the culture media. At the indicated time points, NK cells and cell-free supernatants were harvested and analyzed for cell surface expression of defined proteins and secreted cytokines. Flow cytometry NK-cell phenotype was determined by staining with directly conjugated mouse antihuman mAbs against CD3, CD56, CD16, CD69, CD54, and CD137 (BD Biosciences, San Diego, CA). Unless indicated otherwise, CD137 expression was measured using commercial mouse anti–human CD137 mAb (clone 4B4-1). In some experiments, NK cells were stained with annexinV/7-AAD (annexin V apoptosis detection kit I; BD Biosciences) to distinguish live cells from apoptotic and/or necrotic cells. The number of degranulating NK cells was determined by staining with CD107a mAb (BD Biosciences). In brief, NK-cell cultures were incubated for 1 hour at 37°C in 5% CO2 after which brefeldin A and monensin (both BD Biosciences) were added. NK cells were cultured for an additional 5 hours and subsequently stained for CD56 and CD16. Directly conjugated mouse IgGs were used as isotype controls. A minimum of 10 000 events were acquired using a BD LSR II flow cytometer and analyzed with BD FACS DIVA Software (BD Biosciences). Analysis of collected cell-free supernatants
Cell-free culture supernatants were collected at indicated time points during incubation. Concentrations of IFN- Flow cytometry–based competitive binding assays CD137-expressing CHO cells were incubated with different concentrations (0.8 µg/mL-100 µg/mL) of G6, GA, and GG mAb at 4°C for 1 hour. Purified mouse IgG2a (muIgG2a) and huIgG1 (BD Biosciences) were used as control antibodies at a concentration of 100 µg/mL. Next, cells were incubated with 5 µg/mL human CD137L-muCD8 (Ancell, Bayport, MN) for 45 minutes at 4°C. Cells were further stained with PE-conjugated anti-muCD8 mAb (BD Biosciences) and acquired using a BD LSR II flow cytometer. Generation of Fab fragments and Fc fragments Fab and Fc fragments from GG, CTM, and huIgG1 were generated by papain digestion followed by protein A and protein L chromatography using an ImmunoPure Fab Preparation kit (Pierce, Rockford, IL) according to the manufacturer's instructions. Briefly, all mAbs were digested into Fc and Fab fragments using papain. The digests were dialyzed against protein A or protein L binding solutions and subsequently passed through protein A or protein L columns, and the flow through, containing Fab or the Fc fragments, respectively, was collected. Purity, determined by both immunoblotting and ELISA, was greater than 75% and 95% for the Fc and Fab fractions, respectively. Compositional monosaccharide analysis of IgG1-Fc Purified Fc fragments (100-200 µg) were diluted to 200 µL using deionized water and mixed with an equal volume of 4N TFA (trifluoroacetic acid; Sigma-Aldrich). Samples were hydrolyzed at 100°C for 5 hours. After lyophilization, the residues were reconstituted with water prior to high-performance anion-exchange chromatography (HPAEC) analysis. The chromatography was performed on a Dionex DX600 chromatography system (Dionex, Sunnyvale, CA) equipped with an electrochemical detector (ED50). Monosaccharides were separated on a CarboPac PA10 (Dionex) column (4 x 250 mm) with an isocratic 18 mM NaOH as the eluent. Monosaccharides were detected by the pulsed amperometric electrochemical detector with the following waveforms (potentials and durations): E1 = +0.05 V (T1 = 0 to 0.4 seconds), E2 = +0.75 V (T2 = 0.41 to 0.6 seconds), and E3 = –0.15 V (T3 = 0.61 to 1 seconds). Typically, 10 µL of sample containing 0.1 to 1 nmol of monosaccharides was injected. For quantification, the peaks corresponding to the monosaccharides were integrated and peak areas were normalized to molar quantity by comparison with standard solution of monosaccharide standards. Cytotoxicity assays NK cytotoxicity was measured by 4-hour chromium-51 (51Cr)–release assays. K562 tumor targets were labeled with 150 µCi (5.55 MBq) 51Cr (Na251CrO4; Perkin Elmer, Shelton, CT) for 1 hour at 37°C in 5% CO2. After incubation, cells were washed 2 times and incubated for an additional 30 minutes to reduce spontaneous release. Finally, tumor targets were plated at 5000 targets/well in triplicate wells in a 96-well V-bottom plate. Effector cells consisted of CD3–CD56+ NK cells that were IL-2 stimulated for 72 hours and subsequently cultured for 24 hours in the presence of immobilized GA or GG mAb. NK effectors (100 µL) were added at indicated effector-target ratios to give a final volume of 200 µL per well. After 4 hours of incubation, 100 µL supernatant was harvested and mixed with 100 µL scintillation cocktail (Perkin Elmer) in a 96-well sample plate (Perkin Elmer). Its radioactive content was measured using a gamma-counter and the percentage of specific lysis was calculated using the formula: 100 x (experimental release – spontaneous release)/(maximum release – spontaneous release). Statistical analysis A Q-Q plot was used to assess whether normality could be assumed for the flow cytometric parameters. Statistical significance, as measured by a 2-sided paired Student t test (or the nonparametric sign test if the normality was not plausible) was calculated using Excel v2003 (Microsoft, Redmond, WA) and Splus (Insightful, Seattle, WA), based on the number of experiments as indicated in the figure legends. NK-cell cultures with immobilized GA mAb were used as control cultures to calculate statistical differences. Differences were considered to be significant at P value less than .05.
Immobilized glycosylated mAbs enhance CD137 expression on human NK cells Several groups, including ours, have demonstrated in murine models that the antitumor activity of anti-CD137 agonistic mAbs is NK dependent.2,3,6 Based on the crucial role of NK cells in antitumor activity and the recognized expression of CD137 on IL-2–stimulated murine NK cells, we initially hypothesized that human NK cells cultured in IL-2 would express high levels of CD137 and that chimeric anti-CD137 mAbs would react with CD137 expressed on IL-2–stimulated human NK cells, inducing degranulation and cytokine release. Interestingly, we found that after IL-2 stimulation and subsequent culture in RPMI 1640 supplemented with FBS, human NK cells did not demonstrate high levels of CD137 expression (data not shown). In contrast, IL-2–stimulated NK cells cultured in the presence of immobilized glycosylated mAbs showed high levels of CD137 expression (Figure 1). Expression of CD137 was transient and was not observed to a measurable extent after 72 hours of culture (data not shown). Of note, NK cells from one individual evidenced relatively high levels of CD137 after IL-2 stimulation. This exception may reflect ongoing infection/inflammation in this donor or individual variation in susceptibility for IL-2.
CD137 was enhanced only on IL-2–stimulated NK cells in cultures with immobilized GG (range: 22%-61%; P = .007) and not with GA (range: 1%-14%). Cultures of IL-2–stimulated NK cells in the presence of nonimmobilized (soluble) mAbs failed to induce CD137 expression. CD137 was also up-regulated on IL-2–stimulated NK cells cultured with immobilized polyclonal huIgG1 (range: 3%-73%; P = .023) and, to a lesser extent, with immobilized anti-EGFR mAb CTM (range: 3%-10%). The Fab region of GA and GG did not appear to augment CD137 expression, as the levels of CD137 in cultures with immobilized huIgG1 plus immobilized GA (range: 14%-25%) were lower than in cultures with immobilized huIgG1 or immobilized GA alone (range: 3%-73% and 1%-14%, respectively).
In addition, CD137 expression was inversely associated with the presence of Fc To exclude other possible sources of NK-cell activation, we indirectly tested for the presence of LPS and evaluated the binding affinity of individual mAb preparations. The up-regulation of CD137 on the NK-cell surface was likely not induced by LPS contamination, because boiling the GG mAb preparation eliminated its ability to up-regulate CD137 (data not shown). Furthermore, observed differences in CD137 expression were also not likely to be secondary to variable levels of immobilized immunoglobulins because similar levels of immobilized GG, GA, and huIgG1 were present on the surface of precoated wells (data not shown). In addition, 10-fold increases in GG concentration did not further enhance CD137 expression on IL-2–stimulated NK cells. However, CD137 expression levels were slightly enhanced with a 10-fold increase in immobilized CTM concentration (10 µg/mL to 100 µg/mL) and CD137 started to reach expression levels as observed in cultures with 10 µg/mL GG (data not shown). These experiments support the conclusion that CD137 expression on NK cells is Fc dependent and that different Fc regions have variable ability to induce CD137 on human NK cells. CD137 expression on NK cells is induced in an Fc-dependent fashion
The fact that immobilized glycosylated mAbs, but not soluble glycosylated mAbs or aglycoslyated mAbs, induce high levels of CD137 expression on IL-2–stimulated NK cells strongly supports the idea that CD137 expression on the NK-cell surface is mediated through Fc-Fc As expected, NK cells cultured with immobilized GG/Fc fragments induced CD137 expression at levels similar to those observed with immobilized GG mAb (range: 27%-61% and 22%-61%, respectively). In contrast, NK cells cultured with immobilized GA mAb or GG/Fab fragments induced only small amounts of CD137 expression (range: 1%-14% and 6%-13%, respectively), as shown in Figure 2Ai, and this difference was determined to be statistically significant (P = .001). Interestingly, the total number of live NK cells (gated as 7-AAD–/annexinV– cells) decreased in the culture with immobilized GG mAb versus the culture with purified GG/Fc fragments, as indicated by the increased ratio of total number of live cells versus total number of acquired cells, 1:6.7 and 1:3.7, respectively (Figure 2Aii).
CD137 was detected not only by commercial mouse anti-CD137 mAb but also by both chimeric anti-CD137 mAbs, demonstrating that GG and GA cross-react with CD137 induced on IL-2–stimulated NK cells. These data have importance for future clinical translational studies (Figure 2B). Importantly, the purity of the Fab fragments was 95% and of the Fc fragments, 75%. However, given that the Fab fragments did not induce high levels of CD137, it is unlikely that our results are secondary to contamination. Therefore, these data provide convincing evidence that CD137 is induced on IL-2–stimulated NK cells in an Fc-dependent fashion. Chimeric GA and GG mAbs have similar Fab regions despite differences in Fc
To further evaluate the potential role of the Fab fragments of GA and GG in inducing CD137 on the NK-cell surface, we compared their ability to bind 4-1BB transfected CHO cells and to block natural 4-1BB-4-1BBL interactions. Both mAbs showed similar binding to CHO/CD137 cells and failed to bind wild-type (CHO/WT) controls (Figure 3A). To determine whether the parent mAb (mouse antihuman CD137) G6 and both chimeric mAbs GA and GG recognized the same CD137 epitope, CHO/CD137 cells were incubated with increasing concentrations of purified G6, GA, or GG mAb, respectively (Figure 3B). All mAbs partially blocked binding of G6-APC in a dose-dependent fashion, suggesting that chimerization did not alter epitope affinity. Finally, to further define the functional characteristics of these mAbs, the ability of G6, GA, and GG to block the interaction of the CD137 receptor with its natural ligand, CD137L, was determined. CHO/CD137 cells, incubated with increasing concentrations of purified G6, GA, or GG, blocked the binding of human CD137L/mouse CD8 fusion protein in a dose-dependent fashion (Figure 3C). These data indicate that both chimeric mAbs bind similarly to CD137 and partially block CD137 receptor-ligand interactions in a dose-dependent fashion, suggesting equal specificity of their Fab region for the CD137 receptor. Thus, the fundamental difference between GA and GG mAbs is the status of N-glycosylation of the mAb Fc region. The fact that the Fab fragments of these antibodies are identical suggests that differences in CD137 expression levels in NK-cell cultures with immobilized GG and GA mAb are secondary to the variable ability of the Fc regions of GG and GA to interact with putative Fc
CD137 expression levels on NK cells are associated with defined patterns of Fc glycosylation
Human IgG1 mAbs carry a conserved N-glycan at Asn297 of the Fc region. Fc-Fc To quantitate the monosaccharide composition, we performed HPAEC profile analysis of hydrolysates of the different mAbs studied. To exclude the interference of potential glycosylation at other sites, we purified respective IgG1-Fc fragments from all mAbs and analyzed the monosaccharide composition of the Fc N-glycan. Treatment of the mAb Fc fragments with trifluoroacetic acid resulted in complete hydrolysis of N-glycan into the compositional monosaccharides, where N-acetylglucosamine (GlcNAc) was released as glucosamine (GlcN). The HPAEC profiles of the hydrolysates of all IgG1-Fc fragments are demonstrated in Figure 4.
As expected, the hydrolysis of GA did not release typical monosaccharides (eg, mannose [Man], GlcN, and galactose [Gal]) of the N-glycan, confirming that no N-glycan was attached to this antibody. In contrast, GG, CTM, and huIgG1 revealed typical monosaccharide patterns compared with a standard monosaccharide profile. For quantification, the peaks corresponding to the different monosaccharides were integrated and peak areas were normalized to molar quantity by comparison with a standard glycopeptide (GP-2) isolated from hen's egg yolk that contains a full-size biantennary complex–type N-glycan with the following molar ratios of monosaccharides: Man/Gal/GlcN = 3:2:4. Based on these calculations, the molar ratios of the monosaccharide composition of all IgG1-Fc fragments are summarized in Table 1. GG showed much less fucosylated N-glycan than CTM and huIgG1. Interestingly, CTM showed a higher content of GlcNAc (Man/GlcN = 3:5) than GG and huIgG1 (Man/GlcN = 3:4), suggesting that this mAb may have an additional bisecting GlcNAc. These observations suggest that the low fucose content on GG might contribute to its enhanced ability to induce CD137 on human NK cells.
CD137 expression on human NK cells is associated with phenotypic markers of activation and the release of proinflammatory cytokines To begin to understand the function of CD137 on human NK cells, we sought to associate CD137 expression with cellular activation, as characterized by NK-cell surface expression of CD69 and CD54 and proinflammatory cytokine secretion. CD137-expressing NK- cell cultures were associated with increased levels of CD69 and CD54bright expression, with the highest levels of CD69 expression in cultures with huIgG1 and GG mAb (range: 32%-62% and 36%-86%, respectively; P = .036 for GG). CD54bright expression varied from 4% to 17% expression in cultures with huIgG1 (P = .019) to 10% to 24% expression in cultures with GG mAb (P = .004), as indicated in Figure 5A,B.
Because activated human NK cells are known to secrete proinflammatory cytokines, we evaluated the supernatants of NK- cell cultures for IFN- and TNF- . Levels of TNF- and IFN- in cultures containing immobilized GG were significantly increased (P = .047 and P = .048, respectively) in comparison with cultures with immobilized huIgG1, CTM, or soluble mAb (Figure 5C,D). The levels of IFN- and TNF- declined over time—remaining detectable only in wells with immobilized GG after 72 hours (data not shown). In conclusion, these data suggest that Fc-dependent CD137 expression is associated with phenotypic markers of activation and proinflammatory cytokine release. Degranulation precedes CD137 expression on human NK cells and results in less efficient lysis of NK-sensitive tumor targets by CD137-expressing human NK cells
Although Fc-Fc
The critical findings of these studies are that (1) immobilized Fc enhances the expression of CD137 on IL-2–stimulated NK cells, (2) the levels of CD137 expression are dependent on patterns of Fc glycosylation known to impact Fc interactions with Fc RIII, and (3) the ability to induce CD137 expression is independent of the antigen specificity of the Fab region. Importantly, our experiments do not specifically address the therapeutic utility of targeting the CD137 pathway on CD137-expressing NK cells, which is perhaps best studied using CD137L. Our findings shed new light onto a secondary function of mAbs against costimulatory molecules and demand caution when describing mAbs as being "agonistic" if their function in in vivo and in vitro studies is assessed with intact Fc regions. Knowledge of the functional import of CD137 on human NK cells is primarily derived from murine studies. It is now clear that IL-2–stimulated murine NK cells express CD137 in vivo and that, in some but not all cases, these cells are required for the antitumor effects of agonistic mAbs against CD137.2,3,6 The function of CD137/CD137L interactions on murine NK cells is not completely understood, yet it appears that CD137 ligation enhances NK-cell proliferation and cytokine secretion, and may provide a helper function in the induction of cytotoxic T lymphocytes (CTLs), thereby bridging the innate and adaptive immune system in mice.20,21
Correlate studies suggest that human NK cells play a pivotal role in the regulation of both innate and adaptive immune responses, yet limited information is available regarding CD137 expression and its role in modulating these interactions.22 For example, it has been demonstrated that low levels of CD137 (
Our experiments using various chimeric IgG1 antibodies with different Fc regions support the novel concept that interactions between Fc and putative Fc
Consistent with the idea that Fc-Fc The higher GlcNAc contents in CTM (Man/GlcN = 3:5) in comparison with GG and huIgG1 (Man/GlcN = 3:4) implicates the presence of a bisecting GlcNAc in the N-glycan of this mAb. It is reported that the addition of bisecting GlcNAc enhances ADCC much less efficiently than the presence or absence of fucose.27 Our observation that, regardless of the higher GlcNAc content, CTM is less efficient in inducing CD137, phenotypic markers of activation, and proinflammatory cytokines is in agreement with these previously published reports.
Previous studies have demonstrated that Fc-Fc Interestingly, the number of live NK cells was substantially decreased in cultures with GG. This observation is likely secondary to the specificity of GG for CD137 and 2 potential mechanisms may be involved. First, GG interaction with CD137 may induce ADCC among CD137-expressing NK cells and/or, second, GG interactions may either block or deliver a signal through CD137. These mechanisms are not mutually exclusive, and both could potentially play a role.
In addition to displaying phenotypic markers characteristic of NK activation, cultures with high levels of CD137 expression demonstrated the proinflammatory cytokines IFN-
In summary, our findings that immobilized Fc induces CD137 expression on IL-2–stimulated NK cells are pivotal to the interpretation of studies using anti-CD137 mAbs with an Fc region capable of binding Fc
Contribution: W.L., X.Z., A.W., E.B., and Y.W. performed experiments and analyzed data; C.J.V. designed and performed experiments, analyzed data, and wrote the paper; L.C. provided transfectants and the mouse antihuman anti-CD137 monoclonal antibody; D.G.S. provided the chimeric anti-CD137 monoclonal antibodies; G.T. performed statistical analysis; L.-X.W., K.T, D.H.S., D.M., and S.E.S. designed experiments, and reviewed data and the paper. Conflict-of-interest disclosure: S.E.S. and L.C. receive royalties from GTC Biotherapeutics through the Mayo Clinic College of Medicine for licensure of intellectual property related to CD137. S.E.S. is a cofounder and major stockholder in Gliknik, a biotechnology company. D.G.S. is employed by and owns stock in GTC Biotherapeutics. All other authors declare no competing financial interests. Correspondence: Scott E. Strome, Department of Otorhinolaryngology-Head and Neck Surgery, University of Maryland, 16 South Eutaw Street, Suite 500, Baltimore, MD 21201-168; e-mail: sstrome{at}smail.umaryland.edu.
This study was partially supported by National Institutes of Health (Bethesda, MD) grant 5R44CA107608-04 and by an Small Business Innovation Research grant from the National Cancer Institute, Bethesda, MD, on which S.E.S. and L.C. are subcontractors.
Submitted November 6, 2007; accepted May 12, 2008.
Prepublished online as Blood First Edition Paper, June 2, 2008
DOI: 10.1182/blood-2007-11-122465
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734.
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Abstract
Introduction
) or immobilized GA mAb (
) and subsequently used in a 4-hour 51Cr-release assay to determine lytic activity against K562 target cells. Non–CD137-expressing NK cells (
2.02%) can be induced on NKB1+ cells when PBMCs are exposed to doxorubicin, bleomycin, or mitomycin, although it remains uncertain whether the small NKB1+CD137+ cell population depicted in this study represents T cells, NK cells, NKT cells, or 
T cells.
