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Immunology |
Department of Microbiology and Immunology, Dartmouth Medical School, Lebanon, New Hampshire
Requests for reprints: Charles L. Sentman, Department of Microbiology and Immunology, Dartmouth Medical School, 6W Borwell Building, One Medical Center Drive, Lebanon, NH 03756. Phone: 603-650-8007; Fax: 603-650-6223; E-mail: charles.sentman{at}dartmouth.edu.
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Abstract |
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chain, it was possible to generate a chimeric NKG2D (chNKG2D) receptor that allowed activation of murine T cells on engagement with NKG2D ligand-positive tumor cells leading to antitumor responses in mice. In this study, a human version of the chNKG2D receptor was expressed on primary human T cells, and antitumor responses were determined. Human peripheral blood mononuclear cell–derived T cells were retrovirally transduced with a human chNKG2D receptor gene. These chNKG2D-bearing human T cells responded to NKG2D ligand-positive tumor cells by producing T-helper 1 cytokines, proinflammatory chemokines, and significant cellular cytotoxicity. This response could be blocked by anti-NKG2D antibodies, and it was dependent on NKG2D ligand expression on the target cells but not on expression of MHC molecules. In addition, the activity of chNKG2D-bearing T cells remained unimpaired after exposure to a soluble NKG2D ligand, soluble MICA, at concentrations as high as 1.5 µg/mL. These data indicate the feasibility of using chNKG2D receptors in primary human T cells and suggest that this approach may be a promising means for cancer immunotherapy. (Cancer Res 2006; 66(11): 5927-33) |
Introduction |
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NK cells can use multiple activating receptors to recognize target cells. However, the ligands for many of them remain poorly defined, and some are expressed on normal tissues (1, 2). For cancer immunotherapy, it would be ideal to use those activating NK receptors, where ligand expression is preferentially on tumor cells to minimize the risk of autoimmunity. In humans, NKG2D receptors are expressed on NK cells, NKT cells, 
T cells, and CD8+ 
ß T cells (9). Ligands for human NKG2D include MICA, MICB, UL-16-binding proteins, and Letal (9, 10). NKG2D ligands are primarily expressed on tumor cells but are absent on most normal tissues. In a previous study, we described a novel strategy to redirect murine T cells against tumors through a chimeric NKG2D (chNKG2D) receptor (11). The chNKG2D receptor contains NKG2D fused to a CD3 cytoplasmic domain, which allows direct activation of T cells via the Syk family tyrosine kinase cascade after engagement with NKG2D ligand-positive tumor cells. Besides direct killing of NKG2D ligand-positive tumor cells, the chNKG2D-bearing T cells were able to induce host immunity against related tumors that did not express NKG2D ligands in mice (11).
In this study, we describe a version of the chNKG2D receptor for use in primary human T cells. Our results showed that chNKG2D-modified T cells produced large amounts of T-helper 1 cytokines and lysed target cells in a NKG2D ligand-dependent manner. chNKG2D-mediated responses require the presence of NKG2D ligands but not MHC expression on target cells. In addition, chNKG2D-bearing T cells were resistant to inhibition by high concentrations of soluble MICA (sMICA). These data support the possibility of immunotherapy of human cancers using chNKG2D-modified T cells.
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Materials and Methods |
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Retroviral vector construction. The full-length human NKG2D cDNA was purchased from Open Biosystems (Huntsville, AL). Human CD3 chain and Dap10 cDNAs were cloned by reverse transcription-PCR (RT-PCR) using RNAs from Jurkat and U937 cells, respectively. All PCRs were done using High-Fidelity DNA Polymerase PfuUltra (Stratagene, La Jolla, CA) or Phusion (New England Biolabs, Ipswich, MA). All oligos were synthesized by Integrated DNA Technologies (Coralville, IA). chNKG2D was created by fusing the human CD3 chain cytoplasmic region coding sequence (CD3-CYP) to the full-length gene of human NKG2D. chNKG2D, wild-type NKG2D (wtNKG2D), human Dap10, and MICA genes were cloned into a retroviral vector pFB-Neo (Stratagene). In some cases, a modified vector pFB-IRES-GFP was used to allow coexpression of green fluorescent protein (GFP) with genes of interest (11). A soluble human NKG2D gene was made by fusion of the extracellular portion of human NKG2D (73-216) with the mouse IgG1 hinge-CH2-CH3 portion (243-469). To allow the fusion protein to be secreted, mouse Dap10 signal peptide (1–18) was added to the NH2 terminal of the fusion protein. To make a sMICA plasmid, the DNA fragment coding for extracellular domain of MICA (1-308) was amplified with a histine tag (6 x His) at the COOH terminal. Both NKG2D-mIgG1 and sMICA genes were cloned into pEGFP-C1 vector (BD Clontech, Palo Alto, CA) by replacing the EGFP gene.
Production of soluble human NKG2D-mIgG1 protein and sMICA. For production of soluble human NKG2D protein, Bosc23 cells were transiently transfected with the NKG2D-mIgG1 vector by Fugene 6 (Roche, Indianapolis, IN) according to the manufacturer's instructions. Cell culture was done in serum-free medium. Supernatants were collected every 24 hours starting from 2 days after transfection for an additional 5 days. Collected supernatants were then precipitated by 50% saturated ammonium sulfate and resuspended in 5% original volume of PBS. Crude NKG2D-mIgG1 protein [in 1.5 mol/L glycine and 3 mol/L NaCl (pH 8.5)] was subjected to affinity chromatography using a rProtein A FF column (Amersham Biosciences, Piscataway, NJ) according to the manufacturer's instruction. Eluted fractions were then concentrated and desalted using Amicon Ultra columns (50K MWCO, Millipore, Billerica, MA). Purified NKG2D-mIgG1 protein was adjusted in PBS, filtered (0.22 µm), and stored in 4°C in the presence of 0.1% sodium azide. Generation and purification of sMICA were done in a similar way as human NKG2D-mIgG1, except that the collected supernatants (10 mmol/L imidazole was added) were loaded onto HisTrap columns (Amersham Biosciences) according to the manufacturer's instruction. In addition, purified sMICA was kept sterile in 4°C in the absence of 0.1% sodium azide.
Retrovirus production and transduction. Dualtropic packaging cell line PT67 was used to generate retroviruses for transduction of human peripheral blood mononuclear cells (PBMC). Virus-producing PT67 cells were prepared by cross-infection using ecotropic viruses collected from retroviral plasmid-transfected Bosc23 cells as described previously (11, 12). Human PBMCs were isolated from healthy donors by density gradient centrifugation using lymphocyte separation medium (density, 1.077; Mediatech, Herndon, VA) according to the manufacturer's instructions. All procedures were approved by the Internal Review Board at Dartmouth College. PBMCs were then stimulated with phytohemagglutinin (1 µg/mL) for 3 days. Retroviral transduction of PBMCs was done in 12-well plates with collected retroviruses from PT67 in the presence of 50 or 100 IU human interleukin (IL)-2 and 8 µg/mL polybrene. Cells (
0.5 x 106/mL) were spun at 32°C at 1,500 x g for 60 minutes. Viral supernatants were changed to complete RPMI after 8 or 20 hours. Two days after infection, transduced primary T cells (0.5 x 106/mL) were selected in RPMI-10 medium containing G418 (0.5 mg/mL) plus 50 or 100 units/mL recombinant human IL-2 for an additional 3 days. NKG2D ligand MICA-expressing P815 (P815/MICA) cells were established by triple transduction with PT67-derived MICA-expressing vectors followed by G418 (1 mg/mL) selection for 10 days. Both wtNKG2D- and chNKG2D-expressing B3Z cells (B3Z/wtNKG2D and B3Z/chNKG2D) were established using a similar protocol, except that cotransduction of the NKG2D genes with the mouse Dap10 gene was done. NKG2D-positive B3Z cells were then isolated by cell sorting.
RT-PCR. Total RNAs from vector-, wtNKG2D-, and chNKG2D-transduced primary human T cells were extracted using the RNeasy Mini kit (Qiagen, Valencia, CA). Synthesis of first strands of cDNAs using random hexamer primers (Fermentas, Hanover, MD) was done according to the manufacturer's instructions. The resulting cDNA, corresponding to 33 ng of total RNA, was subjected to PCR amplification in a total volume of 20 µL buffer, including 0.5 µmol/L of each primer, 0.2 mmol/L of each deoxynucleotide triphosphate, and 1 unit Taq DNA polymerase (New England Biolabs). The primers used for amplification of the human ß-actin sequence were 5'-GATCATTGCTCCTCCTGAGC-3' (ß-actin sense) and 5'-CGTCATACTCCTGCTTGCTG-3' (ß-actin antisense). The primers used for amplification of the wtNKG2D sequence were 5'-CACAGCTGGGAGATGAGTGA-3' (wtNKG2D sense) and 5'-TCGAGGCATAGAGTGCACAG-3' (wtNKG2D antisense). The primers used for amplification of the chNKG2D sequence were 5'-AGGGCCAGAACCAGCTCTAT-3' (chNKG2D sense) and 5'-AGAAGGCTGGCATTTTGAGA-3' (chNKG2D antisense). The PCR conditions were as follows: 95°C for 5 minutes followed by 30 cycles of 95°C for 30 seconds (denaturation), 57°C for 45 seconds (annealing), and 72°C for 1 minute (extension), with 5-minute incubation at 72°C at the end. The PCR products were run on agarose gels and visualized by staining with ethidium bromide.
Flow cytometry and magnetic cell sorting. For fluorescence-activated cell sorting analysis of NKG2D ligand expression, human tumor cells were stained with human NKG2D-mIgG1 fusion protein followed by staining with PE-labeled rat anti-mouse IgG1 (A85-1, BD PharMingen, San Diego, CA). MICA expression on P815 cells as determined by the human NKG2D/Fc chimera (R&D Systems, Minneapolis, MN) followed by staining with FITC-goat F(ab')2 anti-human IgG (Caltag, Burlingame, CA). Purified anti-NKG2D antibody (1D11, mouse IgG1) was obtained from BD PharMingen. APC-anti-CD3 (S4.1) and PE-anti-NKG2D (1D11) were obtained from Caltag. FITC-anti-CD8 (RPA-T8), anti-CD4 (OKT4), and all isotype controls were obtained from eBiosciences (San Diego, CA). CD8+ T cells were purified by a magnetic cell sorting kit (Miltenyi Biotec, Inc., Auburn, CA) according to the manufacturer's instruction and expanded for 2 more days without G418 before use as effector cells. Cell fluorescence was monitored using a FACSCalibur cytometer (Becton Dickinson, San Jose, CA). Cell sorting was done on a FACSAria sorter (Becton Dickinson).
Cytokine production by gene-modified T cells. For tumor cells grown in suspension, coculture with gene-modified primary human T cells (105) was done in round-bottom 96-well plates at a ratio of 1:1, whereas adherent tumor cells (2.5 x 104) were cocultured with T cells in flat-bottom plates. Tumor cells were irradiated (120 Gy) before use. Cell-free supernatants were collected after 24 and 72 hours. Twenty-four-hour supernatants were assayed for IFN- by ELISA using DuoSet ELISA kits (R&D Systems). Seventy-two-hour supernatants were used for detection of other cytokines using Bio-Plex kits (Bio-Rad, Hercules, CA) based on the manufacturer's protocol. Bio-Plex analysis was done by the Immune Monitoring Laboratory of the Norris Cotton Cancer Center (Lebanon, NH).
Cytotoxicity assay. Lysis of target cells was determined by a 4-hour 51Cr release assay as described previously (11). To block NKG2D receptors, T cells were preincubated for an hour with the anti-NKG2D antibody (clone: 1D11, 20 µg/mL, sodium azide-free) before addition to the target cells. Inhibition experiments using sMICA (0-15 µg/mL) were done in a similar way except that the ratio of effector to target (E:T) was fixed at 10:1.
ß-red staining. The LacZ activity in B3Z cells was determined by using a BetaRed ß-galactosidase assay kit (EMD Biosciences, San Diego, CA) according to the manufacturer's instruction.
Statistical analysis. Differences between groups were analyzed using the Student's t test. Ps < 0.05 were considered significant.
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Results |
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chain to the NH2 terminal of the human NKG2D receptor. NKG2D is a type II protein, in which the NH2 terminal is located intracellularly, whereas the CD3 chain is a type I protein, with the COOH terminal in the cytoplasm (9, 11). On expression of chNKG2D, the orientation of the CD3 portion is reversed inside the cells. The extracellular and transmembrane domains are derived from NKG2D. The structures of the chNKG2D and wtNKG2D receptors used are diagramed in Fig. 1
. To determine whether the chNKG2D receptor could be expressed in a similar manner as wtNKG2D, we cotransfected these genes with an adaptor protein gene (Dap10) into Bosc23 cells. wtNKG2D requires Dap10 to be expressed at the cell surface. A bicistronic vector with GFP gene downstream of internal ribosome entry site (IRES) was used to identify those cells that were transfected. NKG2D surface expression was analyzed by flow cytometry in the GFP+ cell population. As expected, Bosc23 cells did not express either NKG2D or Dap10, and transfection with only one of the two components did not cause surface expression of NKG2D (Fig. 2A
). However, cotransfection of a NKG2D gene along with an adaptor protein gene led to significant membrane expression of NKG2D. Surface expression of NKG2D was comparable after transfection with chNKG2D to those where the wtNKG2D gene was used. Thus, like wtNKG2D, human chNKG2D needs to be associated with an adaptor protein Dap10 for surface expression. chNKG2D and wtNKG2D expression in primary human T cells was initially determined by RT-PCR 7 days after retroviral transduction. As shown in Fig. 2B, wtNKG2D was expressed in vector-, wtNKG2D-, and chNKG2D-transduced human T cells, whereas chNKG2D expression was only found in chNKG2D-transduced T cells. The intensity of the wtNKG2D signal is greater in chNKG2D-transduced T cells because both wtNKG2D and chNKG2D can be templates for the wtNKG2D primers. This result indicated that chNKG2D could be expressed in primary T cells. Flow cytometry was then done on primary human T cells. Seven days after retroviral transduction, only a small percentage of CD4+ T cells (data not shown) were positive for NKG2D, whereas majority of CD8+ T cells expressed NKG2D (Fig. 2C). Compared with vector-transduced T cells, wtNKG2D and chNKG2D-modified CD8+ T cells had higher NKG2D surface expression (>100-400% increase in geometric mean fluorescent intensity), supporting that the exogenously transduced chNKG2D gene can be expressed on the surface of T cells.
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after coculture with NKG2D ligand-positive cells but not with ligand-negative P815 cells, indicating that these chNKG2D-modified T cells could functionally recognize NKG2D ligand-bearing tumor cells. In contrast, vector or wtNKG2D-modified T cells did not show any significant response to the stimulation by NKG2D ligand-positive cells. All T cells produce IFN- in the presence of RPMI8866 cells, although chNKG2D-bearing T cells produced 13-fold more IFN- than vector- or wtNKG2D-modified T cells. In addition, on chNKG2D engagement, chNKG2D-modified T cells also released significant amounts of proinflammatory chemokines [CCL3 (Fig. 3A
) and CCL5 (Fig. 3B)] as well as other T-helper 1–type cytokines, granulocyte macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor- (TNF-; Fig. 3C and D), but not T-helper 2 cytokine IL-10 (Supplementary Fig. S2). These data indicate that a chNKG2D-NKG2D ligand interaction induces human T cells to produce T-helper 1–type cytokines.
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55 kDa in a SDS-PAGE gel, consistent with previous findings (17). To confirm that the recombinant sMICA was functional, we tested whether plate-bound sMICA could activate B3Z cells that expressed chNKG2D. Because B3Z cells contain the IL-2 promoter-controlled LacZ gene as a reporter, after activation, B3Z cells will express LacZ (ß-galactosidase). The ability of immobilized sMICA to stimulate chNKG2D-expressing B3Z cells was evaluated by measurement of the A570 nm values after addition of a ß-red substrate. As shown in Fig. 5B, incubation of chNKG2D-bearing B3Z cells, but not wtNKG2D-bearing B3Z cells with immobilized sMICA, led to cell activation in a dose-dependent manner, indicating that the recombinant sMICA is functional. Next, we determined the extent to which the sMICA affects the activity of chNKG2D-bearing primary human T cells. There was no significant drop in the cytotoxicity of chNKG2D-bearing T cells against K562 and RPMI8866 cells when sMICA was added at a concentration of as high as 1.5 µg/mL (Fig. 5C and D). Reduced cytotoxicity (44.2%) against RPMI8866 was observed after addition of a high concentration of sMICA (15 µg/mL). However, the cytotoxicity against K562 remained unimpaired in the presence of sMICA at this concentration. These sMICA concentrations are much higher than those reported in sera from cancer patients (0.2-10 ng/mL). These data suggest that sMICA in cancer patients may not inhibit the function of chNKG2D-modified human T cells in vivo.
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Discussion |
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and ß chains has been shown to have an effect in T-cell–mediated killing of tumor cells both in vitro and in vivo (18–20). Down-regulation of HLA molecules on tumors, which is a common way for tumor cells to evade T-cell recognition, will render this strategy less effective (18). To overcome the dependence on the "MHC restriction" by TCR, chimeric antigen receptors (CAR) are designed to recognize intact membrane proteins. Most of CARs are "T bodies," which consist of a single-chain antibody (extracellular domain) that can bind a membrane antigen on the tumor cells, fused to a cytoplasmic signaling domain, such as the CD3 chain and Fc receptor chain (18–21). However, for a given tumor antigen, either high-affinity HLA-restricted TCRs or T bodies have to be identified and tested, which is not an easy task in many cases. In addition, the potential immunogenicity of the extracellular domain of T bodies may pose a problem for repeated infusion of T body–modified T cells (18–20).
Here, we reported that chNKG2D-modified human T cells were able to respond to NKG2D ligand-positive tumor cells in vitro, showing this as a possibility for immunotherapy of human cancers. One of the major advantages of this strategy is that chNKG2D-modified T cells can be used for immunotherapy of multiple types of malignant tumors, as long as tumors express NKG2D ligands. In this sense, chNKG2D receptor is a useful addition to the "CAR family". Many human cancers, such as colon cancer, leukemia, lymphoma, myeloma, cervical cancer, ovarian cancer, prostate cancer, and melanoma, have been found to express NKG2D ligands (9, 22–24). The ability of chNKG2D-bearing T cells to kill the pancreatic tumor cell line Panc-1 is especially interesting. Pancreatic cancer is the fourth leading cause of cancer death in the United States, accounting for 30,000 death yearly (25). Therefore, infusion of chNKG2D-bearing T cells may be a potential treatment modality for this type of cancer. Unlike T bodies, chNKG2D has the same extracellular domain as wtNKG2D; thus, it would not be expected to induce an immune response against it.
After engagement with NKG2D ligand-positive tumor cells, chNKG2D-bearing human T cells produced significant amounts of IFN-, GM-CSF, and TNF-, which were similar to what has been observed from activated human tumor-specific CTLs (26, 27). IFN- has been shown to activate antigen-presenting cells (such as dendritic cells and macrophages) to promote antigen presentation to CTLs (28). GM-CSF alone or in combination with TNF- plays important roles in generation of mature dendritic cells as well as in tumor antigen presentation (29, 30). Phase I clinical trials using GM-CSF-modified tumor vaccines showed therapeutic effects in some cancer patients (31, 32). Therefore, interaction between chNKG2D-bearing T cells and NKG2D ligand-positive tumor cells may not only allow these T cells to kill tumor cells but also promote tumor antigen presentation due to local production of GM-CSF and IFN-. We observed differences in the amounts of cytokines produced when chimeric receptor-bearing T cells were cultured with different tumor cells. This may be due to the expression of different ligands for NKG2D on the tumor cell lines or different amounts of cytokine receptors expressed on the various tumor cell lines. Production of proinflammatory chemokines CCL3 (MIP-1) and CCL5 (RANTES) has also been shown beneficial to antitumor immunity (33, 34). Taken together, production of T-helper 1 cytokines and proinflammatory chemokines by chNKG2D-bearing T cells is likely to promote host antitumor immunity.
The ability to generate functional activity by engagement of chNKG2D receptor suggests that this receptor leads to full activation of T cells, which is predicted due to the presence of CD3 signaling domain. Some studies have shown that inclusion of CD28 signaling domain into CARs could enhance the signal initiated by CD3 because CD28 provides a costimulation signal (18, 19). Similar to CD28, Dap10 transduces a costimulatory signal via a phosphatidylinositol 3-kinase pathway (9). In T cells, NKG2D associates with Dap10. Therefore, engagement of chNKG2D may activate T cells using both a primary signal through CD3 and a costimulatory signal through Dap10. However, further studies will be needed to clarify the details of chNKG2D-mediated signaling pathways.
It has been shown that human tumor cells produce soluble forms of NKG2D ligands and may lead to evasion from NK and T-cell surveillance (16, 17, 35); therefore, it was important to determine whether soluble ligands altered the function of chNKG2D-bearing T cells. Using a cytotoxicity assay, we have shown that chNKG2D-modified T cells remained functionally intact in the presence of sMICA at a level >1.5 µg/mL, which is a higher level (150- to 7,500-fold more) than those that found in sera of cancer patients (15, 36). However, it is possible that the levels of soluble NKG2D ligands at local tumor areas are different from that in sera. The mechanism for sMICA inhibition of cytotoxicity may be blocking of chNKG2D receptors or down-modulation of receptor expression. The cytotoxicity against RPMI8866 cells is more sensitive to sMICA as well as anti-NKG2D blocking antibody than that against K562 cells. The reason may be that RPMI8866 cells express lower level of NKG2D ligands.
There are two major safety concerns related to the use of chNKG2D. First, genetic modification of T cells may cause transformation of T cells due to preferable integration near transcriptional units, although the occurrence is rare (18, 19, 37). Use of nonviral vectors, such as transposon-based integration vector, may reduce the chance of transformation (38). Second, the risk of autoimmunity following infusion of autologous tumor-reactive T cells exists because NKG2D ligands have been found to be expressed by some normal tissues besides tumors, such as gut epithelial cells (9). One promising strategy to control the potential transformation and autoimmunity elicited by transferred T cells is coexpression of a "suicidal gene" along with the chNKG2D gene. Several suicidal genes, including a thymidine kinase (TK) gene from human herpes simplex virus and a Fas-based "artificial suicide gene," have been evaluated in terms of the ability to eliminate T cells (18, 19). A recent study showed that a modified human caspase-9-based molecular switch in combination with a small-molecule pharmaceutical drug (at concentration of 10 nmol/L) could induce apoptosis in 99% of transduced T cells both in vitro and in vivo (39). Engineering a suitable "suicide gene" into the vector along with the chNKG2D gene will enable the elimination of transferred T cells to prevent potential autoimmunity or T-cell transformation.
In summary, the present study suggests a new strategy for cancer immunotherapy using chNKG2D-modified human T cells, which can kill multiple types of human cancer cells (NKG2D ligand-positive) and produce T-helper 1 cytokines and proinflammatory chemokines simultaneously in vitro.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Gary A. Ward (Englert Cell Analysis Laboratory, Norris Cotton Cancer Center) for cell sorting and the immune monitoring laboratory (Norris Cotton Cancer Center) for assistance in luminex analysis.
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Footnotes |
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Received 1/12/06. Revised 3/ 5/06. Accepted 3/17/06.
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treatment of K562 cells cannot be explained by augmented major histocompatibility complex class I expression. Immunology 1994;83:75–80.[Medline] chain. J Exp Med 1993;178:361–6. T cells of MICA and MICB. Proc Natl Acad Sci U S A 1999;96:6879–84., and IFN- by human tumor-infiltrating lymphocytes after autologous tumor stimulation. J Immunol 1991;146:3674–81.[Abstract]. Curr Opin Immunol 1999;11:76–81.[CrossRef][Medline] cooperate in the generation of dendritic Langerhans cells. Nature 1992;360:258–61.[CrossRef][Medline], and ultraviolet radiation. J Leukoc Biol 1992;52:209–17.[Abstract], MIP-1ß, RANTES, and ATAC/lymphotactin function together with IFN- as type 1 cytokines. Proc Natl Acad Sci U S A 2002;99:6181–6.This article has been cited by other articles:
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  A. Barber, A. Rynda, and C. L. Sentman Chimeric NKG2D Expressing T Cells Eliminate Immunosuppression and Activate Immunity within the Ovarian Tumor Microenvironment J. Immunol., December 1, 2009; 183(11): 6939 - 6947. [Abstract] [Full Text] [PDF]
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A. Barber and C. L. Sentman Chimeric NKG2D T Cells Require Both T Cell- and Host-Derived Cytokine Secretion and Perforin Expression to Increase Tumor Antigen Presentation and Systemic Immunity J. Immunol., August 15, 2009; 183(4): 2365 - 2372. [Abstract] [Full Text] [PDF]
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 P. S. Woll, B. Grzywacz, X. Tian, R. K. Marcus, D. A. Knorr, M. R. Verneris, and D. S. Kaufman Human embryonic stem cells differentiate into a homogeneous population of natural killer cells with potent in vivo antitumor activity Blood, June 11, 2009; 113(24): 6094 - 6101. [Abstract] [Full Text] [PDF]
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 B. M. Carreno, J. R. Garbow, G. R. Kolar, E. N. Jackson, J. A. Engelbach, M. Becker-Hapak, L. N. Carayannopoulos, D. Piwnica-Worms, and G. P. Linette Immunodeficient Mouse Strains Display Marked Variability in Growth of Human Melanoma Lung Metastases Clin. Cancer Res., May 15, 2009; 15(10): 3277 - 3286. [Abstract] [Full Text] [PDF]
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A. Barber, T. Zhang, and C. L. Sentman Immunotherapy with Chimeric NKG2D Receptors Leads to Long-Term Tumor-Free Survival and Development of Host Antitumor Immunity in Murine Ovarian Cancer J. Immunol., January 1, 2008; 180(1): 72 - 78. [Abstract] [Full Text] [PDF]
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 T. Zhang, A. Barber, and C. L. Sentman Chimeric NKG2D Modified T Cells Inhibit Systemic T-Cell Lymphoma Growth in a Manner Involving Multiple Cytokines and Cytotoxic Pathways Cancer Res., November 15, 2007; 67(22): 11029 - 11036. [Abstract] [Full Text] [PDF]
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A. Barber, T. Zhang, L. R. DeMars, J. Conejo-Garcia, K. F. Roby, and C. L. Sentman Chimeric NKG2D Receptor-Bearing T Cells as Immunotherapy for Ovarian Cancer Cancer Res., May 15, 2007; 67(10): 5003 - 5008. [Abstract] [Full Text] [PDF]
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), wtNKG2D (
), or chNKG2D (
) were used as effector cells in a 51Cr release assay. The ratios of effector to targets (E:T ratios) were 25:1, 5:1, and 1:1. chNKG2D-transduced T cells showed significantly higher (P < 0.05) cytotoxicity against NKG2D ligand-positive tumors (P815/MICA, T47D, MCF-7, Panc-1, and A375) but not ligand-negative P815 cells than vector- or wtNKG2D-transduced T cells at all ratios. B, for NKG2D blocking experiments, wtNKG2D-bearing (circle) or chNKG2D-bearing (triangle) CD8+ T cells were incubated with saturating amounts of anti-NKG2D (1D11, filled symbols) or with control Ig (open symbols) before exposure to tumor cells. Blocking antibody 1D11 significantly reduced the cytotoxicity of chNKG2D-transduced T cells against K562 and RPMI8866 cells (P < 0.05) at all ratios compared with control antibody. Points, mean of two to three experiments done in triplicate; bars, SD.
) were preincubated with varying concentrations of sMICA and then tested in a 51Cr assay with RPMI8866 (C) or K562 (D) at an E:T ratio of 10:1. Points, mean of two experiments done in triplicate; bars, SD. *, P < 0.05, significant difference between the treated samples (in the presence of 15,000 ng/mL sMICA) and the untreated control.