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The Journal of Immunology, 2005, 175: 7819-7828.
Copyright © 2005 by The American Association of Immunologists

Silencing Human NKG2D, DAP10, and DAP12 Reduces Cytotoxicity of Activated CD8+ T Cells and NK Cells1

Mobin Karimi*, Thai M. Cao*, Jeanette A. Baker*, Michael R. Verneris{ddagger}, Luis Soares{dagger},§ and Robert S. Negrin2,*

* Division of Blood and Marrow Transplantation and {dagger} Division of Immunology and Rheumatology, Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305; {ddagger} Division of Pediatric Blood and Marrow Diseases, Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455; and § Institute of Biomedical Pharmacology, Curitiba, Brazil


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Human CD8+ T cells activated and expanded by TCR cross-linking and high-dose IL-2 acquire potent cytolytic ability against tumors and are a promising approach for immunotherapy of malignant diseases. We have recently reported that in vitro killing by these activated cells, which share phenotypic and functional characteristics with NK cells, is mediated principally by NKG2D. NKG2D is a surface receptor that is expressed by all NK cells and transmits an activating signal via the DAP10 adaptor molecule. Using stable RNA interference induced by lentiviral transduction, we show that NKG2D is required for cytolysis of tumor cells, including autologous tumor cells from patients with ovarian cancer. We also demonstrated that NKG2D is required for in vivo antitumor activity. Furthermore, both activated and expanded CD8+ T cells and NK cells use DAP10. In addition, direct killing was partially dependent on the DAP12 signaling pathway. This requirement by activated and expanded CD8+ T cells for DAP12, and hence stimulus from a putative DAP12-partnered activating surface receptor, persisted when assayed by anti-NKG2D Ab-mediated redirected cytolysis. These studies demonstrated the importance of NKG2D, DAP10, and DAP12 in human effector cell function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Natural killer cells are frontline guardians in surveillance of pathogens and tumors, and their activation is regulated by the integration of excitatory and inhibitory signals initiated by membrane-bound receptor molecules (1, 2). Inhibitory signals are mediated by monomeric receptors possessing intracytoplasmic immunoreceptor tyrosine-based inhibition motifs that recruit tyrosine protein phosphatases (3). Activating signals are generated from more diverse and complementary groups of membrane molecules that, upon ligand binding, can oligomerize with different cytoplasmic adaptor polypeptides to form distinct signaling complexes. In human NK cells, these types of activating receptors include NKG2D, which associate with the adaptor molecule disulphide adaptor molecule (DAP)3 -10, and the low-affinity IgG FcR CD16, which noncovalently couples with the adaptor molecules CD3{zeta} and FcR{gamma}. CD3{zeta}, FcR{gamma}, and another adaptor molecule found in NK cells, named DAP12 (4, 5), have in common a membrane-bound structure with a cytoplasmic domain bearing one or more ITAM motifs. Human DAP12 is known to mediate activating signals from diverse binding partners that include killer cell Ig-like receptors S such as killer cell Ig-like receptor 2DS2, the activation-induced NKp44 receptor, and HLA-E binding CD94/NKG2C heterodimers (5, 6, 7, 8). DAP12 has been shown to associate with a host of other NK cell surface proteins that have yet to be identified, and it is possible that these interactions may yield additional activating signals (9). CD3{zeta}, FcR{gamma}, and DAP12 intracellular ITAM motifs initiate protein tyrosine kinase-dependent signaling pathways (10), whereas DAP10 contains a YxxM activation motif that triggers the lipid kinase cascade (11, 12, 13).

In human NK cells, DAP10 is the exclusive binding partner and signaling intermediate for NKG2D (14). NKG2D is a potent activating receptor whose ligands include proteins induced by cellular stress and malignant transformation, such as MHC class I-related protein (MICA) and -B and members of the UL16-binding proteins, and is thought to have a particularly important role in antitumor immunity (15).

The expression and function of these activating signaling complexes are not limited to NK cells. DAP12 is expressed in granulocytes, monocytes, as well as CD4+ T cells, and DAP12-deficient mice have been shown to have defects in dendritic cells and NK cells (16, 17, 18, 19). NKG2D has been identified on {gamma}{delta} TCR+ T cells and human CD8+ T cells, where it appears to have costimulatory activity and can be induced on activated mouse CD8+ T cells (20, 21, 22). We have recently reported that NKG2D expression is up-regulated by human CD8+ T cells activated and expanded ex vivo in the presence of IFN-{gamma}, high-dose IL-2, and TCR cross-linking with an anti-CD3 mAb (23). Activated and expanded T cells up-regulate cytotoxic effector molecules, acquire functional and phenotypic properties that resemble NK cells, and develop broad cytotoxicity against a variety of malignant cell targets, including autologous leukemic blasts (24, 25, 26, 27, 28). This acquisition of cytotoxicity, which is MHC unrestricted, coincides with the induction of DAP10 expression, and the majority of killing ability is accounted for by NKG2D signaling.

Activated and expanded CD8+ T cells may have considerable therapeutic utility as an immune-based strategy aimed at eradication or suppression of malignant diseases. These T cells have broad in vitro and in vivo biological activity against inoculated tumors after both syngeneic and allogeneic bone marrow transplantation in rodent models (29, 30). Autologous infusion of cells of this type in patients has been associated with minimal toxicity, can reduce the risk of tumor recurrence in patients with hepatocellular carcinoma after surgical resection, and may induce partial remissions in patients with relapse of lymphoma after bone marrow transplantation (31, 32). In this study, we extended our evaluation of the signaling requirements for induction of cytotoxicity by activated and expanded human CD8+ T cells. We used a combination of in vitro and in vivo assays against tumor cell lines and autologous tumor targets and gene targeting by RNA interference (RNAi). We show that both DAP10 and DAP12, in addition to NKG2D, contribute major activating signals in regulating the cytotoxicity of activated and expanded human CD8+ T cells. We show, furthermore, that each of these molecules, including DAP12, has a nonredundant and partially obligate signaling role in triggering tumor killing.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
mAb reagents and flow cytometry

The mAbs used in this study were the following: CD3 (UCHT1), CD8 (RPA-T8), CD16 (3G8), CD56 (NCAM16-2), and isotype control (IgG) were purchased from BD Biosciences as either FITC or PE conjugates. Purified NKG2D were purchased from R&D Systems, and PE NKG2D-PE and NKG2D-allophycoyanin were purchased from Beckman Coulter. Labeled cells were analyzed on either a FACScan or an LSR-II instrument (BD Biosciences) maintained by the Stanford Shared FACS Facility (Stanford University). For micromagnetic cell separation, human CD8 (BW135/80) MicroBeads, human CD16 MicroBeads (130-045-701), or human HEA MicroBeads (130-061-101) were used for positive selection using an AutoMACS device according to the vendor’s instructions (Miltenyi Biotec). Where stated, additional cell isolation by FACS sorting was performed with a MoFlo instrument (DakoCytomation).

Isolation and generation of ex vivo expanded and activated CD8+ T cells

CD8+ T cells were activated and expanded as previously described (23). Briefly, mononuclear cells were isolated from healthy donors by Ficoll-Hypaque density centrifugation, washed three times with PBS, and separated by positive selection with CD8 MicroBeads. Cells were then labeled and further purified by FACS sorting for the CD3+CD8+ population. The final product was resuspended at 2 x 106 cells/ml in complete RPMI 1640 (cRPMI) medium supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µg/ml 2-ME at 37°C in 5% CO2. On day 0, cells were activated with human IFN-{gamma} (1000 U/ml; Genentech). On day 1, cells were stimulated with OKT-3 (25 ng/ml; OrthoBioTech) and human rIL-2 (300 U/ml; Chiron). On day 4, cells were divided into flasks with new medium and not restimulated with OKT-3. Every 3–5 days thereafter, fresh medium was added supplemented with IL-2 (300 U/ml) to maintain a cell density of 1.5–2 x 106/ml for a total of 14–28 days. Bulk populations of cells have been referred to as cytokine-induced killer cells. Twenty-four to 48 h before use, cells were labeled and further purified by FACS sorting for the CD3+CD8+ population. For some experiments CD8+ T cells were also activated and expanded from patients with ovarian cancer according to protocols approved by the Stanford University administration panel on human subjects.

Isolation of NK cells

Mononuclear cells from healthy donors freshly isolated with Ficoll-Hypaque were purified for NK cells by a two-step procedure. Cells were first positively selected with CD16 MicroBeads, followed by additional purification with FACS sorting for the CD3–D56+ cell population. NK cell purity was typically >98% as determined by reanalysis of an aliquot of separated cells. During lentiviral infection for RNAi, isolated NK cells were maintained in DMEM/Ham’s F-12 medium (2/1 mixture) for 7 days supplemented with 10% human type AB serum, 600 U/ml IL-2, 100 U/ml penicillin, 100 µg/ml streptomycin, 50 µM ME, 10 ng/ml ethanolamine, 10 µg/ml ascorbate, and 50 ng/ml selenium.

NKG2D, DAP10, and DAP12 RNAi

Nucleotide sequences for short hairpin RNA (shRNA) are summarized in Table I. A previously reported RNAi sequence specific for murine CD8{alpha} (mCD8) was used as a nonspecific control (33). Oligonucleotides were designed that incorporated these sequences within a short hairpin structure, using the stem loop sequence 5'-TCAAGAGA-3', which were then cloned between MluI and ClaI sites downstream of an H1 promoter in the plasmid pLVTHM as previously described (34). Plasmid pLVTHM, derived from pSUPERn which contains a GFP expression cassette upstream of the H1 promoter (35), was a gift from D. Trono (University of Geneva, Geneva, Switzerland). For some experiments we replaced the GFP gene on pLVTHM with a DsRed reporter gene via PmeI and SpeI linkers. 293 cells were transfected using the calcium phosphate method with 10 µg pLVThM lentivirus, 3.5 µg of vesicular stomatitis virus G plasmid, and 6.5 µg of CMV{Delta}R8.74 according to standard protocols (36). After 16 h, the medium was changed, and recombinant lentivirus vectors were harvested 24–48 h later. CD8+ T cell or NK cell infection was performed three times at 24-h intervals. For each infection, cells were plated in 48-well plates at 1 x 105 cells/well and infected in the presence of protamine and hexadimethrin. Spin infection was performed at 1200 rpm for 90 min at 37°C. Four days after the first infection, transduced cells were isolated by FACS sorting for GFP+ cells to >99% purity.


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  		Table I. shRNA sequences for human NKG2D, DAP10, and DAP12 RNAIa

 
Cell lines and autologous tumor cell isolation

RPMI 1640, a human myeloma cell line cultured in cRPMI, and P815, a murine mastocytoma cell line cultured in complete DMEM, were both purchased from American Type Culture Collection. UCI101 (a gift from S. Y. Liao, University of California, Irvine, CA) is a human ovarian carcinoma cell line cultured in complete IDMEM. Luciferase expressing UCI101 (UCI101-luc) was generated according to a modified protocol that we have previously described (37). In brief, retrovirus generated in Phoenix A producer cells transfected with the pMSCVpuroBA-L2G plasmid (a gift from Dr. C. Contag, Stanford University, Stanford, CA) containing a firefly luciferase insert cloned into the SalI and NotI sites, was used to infect UCI101 cells plated in the presence of polybrene and protamine.

For preparation of tumor targets used for evaluation of autologous activated and expanded CD8+ T cell cytolysis, fresh tumor was collected from six ovarian cancer patients at the time of surgical resection and digested and homogenized to single-cell suspensions using a metal cell strainer. The tumor cells were incubated on a shaker at 1200 rpm for 30 min at 37°C with collagenase 2 (10 µg/ml) or collagenase D (10 µg/ml) in cRPMI. The cells were then selected in T25 flasks and subsequently positively enriched using HEA MicroBeads to >80% purity. At the time of surgery, PBMC were concurrently obtained from each patient and used for the generation of autologous activated and expanded CD8+ T cells as noted above. The protocol for collection of ovarian and peripheral blood cells was approved by the institutional review board at Stanford University.

51Cr release cytotoxicity assay

Tumor targets were labeled with 51Cr (DuPont-NEN) by incubating 1 x 106 cells in 300 µCi (11.1 MBq) of 51Cr for at 37°C for 2 h in 5% CO2. The labeled cells were washed three times with PBS, resuspended in cRPMI, and plated in 96-well plates at a concentration of 1 x 104 cells/ml in triplicate. Effector cells were added at specified ratios (10:1 or 40:1) and incubated at 37°C for 4 h in 5% CO2. At the completion of each assay, supernatant was collected and counted using a gamma counter (Cobra/AII; Packard). The percent-specific 51Cr lysis was calculated with the following equation: percent-specific lysis = 100 x (test release) – (spontaneous release)/(maximal release) – (spontaneous release).

Redirected cytotoxicity assay

For redirected killing assay, 1 x 106 P815 cells were labeled with 51Cr as described above. Excess 51Cr was removed by washing, and the cells were resuspended in 500 µl of medium. Anti-NKG2D (5 µg) was added and incubated for 30 min. The P815 target cells were then used as a target in the 51Cr release assay as described.

Cytokine production

Activated and expanded CD8+ T cells at 21 days of culture were removed, washed, and incubated with 20 ng/ml PMA (Sigma-Aldrich) and 1 µM ionomycin or medium alone in a total volume of 200 µl/well in a 96-well plate and cultured for 48 h. The supernatant was harvested and assayed for cytokine production by ELISA for IL-2 and IFN-{gamma} (eBioscience) according to the vendor’s instructions.

Northern blotting

RNA was prepared with the RNeasy kit (Qiagen) according to the vendor’s instructions, and RNA blotting was performed according to standard protocols (38). Three {alpha}-32P-labeled probes were constructed with the following PCR primers: NKG2D, 5'-CTGGGAGATGAGTGAATTTCATA-3' and 5'-GACTTCACCAGTTTAAGTAAATC-3' (417-bp fragment); DAP10, 5'-CATCTGGGTTCACATCCTCTT-3' and 5'-CAGAAGTCAAAGGTCCAAGC-3' (306-bp fragment); and DAP12, 5'-CCGCAAAGACCTGTACGCCA-3' and 5'-TGGACTTGGGAGAGGACTGG-3' (650-pb fragment). PCR products were cloned by TA cloning into pCR2.1 (Invitrogen Life Technologies), digested with EcoRI, separated by 1.5% agarose gel electrophoresis, and purified with the QiaQick gel extraction kit (Qiagen).

Immunoprecipitations and Western blotting

CD8+ T cells or NK cells were lysed using freshly prepared lysis buffer (10 mM Tris (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, complete Protease Inhibitor Cocktail (Roche), and 500 µM PMSF) and clarification at 12,000 x g for 10 min at 4°C. Seventy micrograms of each cell lysate was separated on an 18% denaturing polyacrylamide gel and transferred to polyvinylidene difluoride membranes for immunoblot analysis. Membranes were blocked with 5% nonfat milk and probed with monoclonal anti-NKG2D (M585, mouse IgG) or polyclonal DAP10 (C-20, goat IgG; Santa Cruz Biotechnology), followed by incubation with a species-specific, secondary, HRP-conjugated Ab (Santa Cruz Biotechnology). Blots were developed using ECL reagent (Amersham Biosciences).

For immunoprecipitation, clarified lysate was labeled with 1 µg of anti-DAP12 polyclonal rat IgG Ab (FL-113; Santa Cruz Biotechnology) for 1 h at 4°C, followed by cross-linking to protein G-agarose beads (Pierce) pre-equilibrated with lysis buffer, for 1 h at 4°C on a rotator. After washing, immunoprecipitated lysate was resuspended in Laemmli buffer with 5% 2-ME and boiled for 5 min before separation and immunoblotting as described above.

SCID/Hu tumor model and bioluminescent imaging

C.B-17 SCID mice, 8–12 wk old, were obtained from the colony at the Stanford University Research Animal Facility and irradiated with a single 2-Gy fraction using a Phillips Unit Irradiator (250 kV, 15 mA) at a dose rate of 100 rad/min. After irradiation, mice were maintained on antibiotic water containing trimethoprin/sulfmethoxole. Recipient mice received 1 x 106 luciferase-expressing UCI101-luc cells (1 ml/injection) via i.p. injection. Intraperitoneal injection with UCI101-luc into immune-deficient mice establishes tumors with peritoneal implants within 1 wk and eventually massive ascites and death (39). On days 6 and 12 after tumor inoculation, each mouse was given an injection with 40 x 106 activated and expanded CD8+ T cells. Mice were evaluated every second day from the time of tumor injection for 30 days by bioluminescence imaging with the IVIS 200 Imaging System (Xenogen) as previously described (37).

Statistics

Differences in the mean observations between experimental groups were determined with Student’s t test. Statistical significance was conferred at the p < 0.05 level.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
RNAi with NKG2D-specific shRNA suppresses cytotoxicity of activated and expanded CD8+ T cells

We have recently demonstrated that human CD8+ T cells activated and expanded during culture in the presence of IFN-{gamma}, IL-2, and OKT3 lose cytolytic potential when NKG2D is silenced by transient transfection with a small interfering RNA (siRNA) duplex oligonucleotide (23). To complement these studies and to develop a model for in vivo analysis of NKG2D function, we sought to establish stable NKG2D knockdown by RNAi in activated CD8+ T cells via transduction of shRNA with the pLVTHM lentiviral vector. NKG2D-sh6, which incorporates nucleotide sequences targeting a portion of the NKG2D gene transcript previously shown functional for silencing NKG2D by siRNA transfection, was evaluated along with five other NKG2D-specific shRNA. NKG2D-sh6 was again found to most effectively silence NKG2D gene expression in CD8+ T cells activated and expanded after 14–28 days in culture (Fig. 1, A and B). FACS analyses of transduced cells, identified by positive GFP reporter gene expression, demonstrate that infection with empty pLVTHM vector does not affect NKG2D surface levels (Fig. 1, C and D). At the same time, NKG2D RNAi with NKG2D-sh6 is specific insofar as it has no effect on other surface receptors, including CD3, CD8, CD16, and CD56. Activated and expanded CD8+ T cells can lyse RPMI tumor targets (Fig. 1E) and retain this ability when RNAi is attempted with NKG2D-specific shRNA that do not effectively silence NKG2D. Cytolysis is similarly potent after RNAi with mCD8-sh, an shRNA specific for murine CD8{alpha} (33), and after transduction by empty pLVTHM vector without RNAi. Cytolysis is suppressed to a level comparable with background cytolysis of P815 targets, however, if NKG2D RNAi is induced by NKG2D-sh6 (p < 0.0001). P815 is a murine mastocytoma cell line known to be resistant to killing by activated and expanded human CD8+ T cells. Thus, we confirmed our prior observations that NKG2D is required for in vitro tumor lysis by activated CD8+ T cells.



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  		FIGURE 1. NKG2D RNAi with shRNA by lentiviral transduction in activated and expanded human CD8+ T cells. Human CD8+ T cells activated and expanded after 14–28 days of culture in the presence of IFN-{gamma}, IL-2, and OKT3 were infected with pLVTHM lentivirus expressing one of six shRNA-targeting NKG2D. As shown by RNA (A) and Western blotting (B) of transduced cells, NKG2D silencing is most effective with NKG2D-sh6, which targets a sequence (nt 1468–1486) in the 3' untranslated region of the NKG2D transcript. C and D, FACS analysis of transduced cells. NKG2D RNAi with NKG2D-sh6 induces complete silencing of NKG2D while preserving surface expression of CD3, CD8, CD16, and CD56. E, Cytolysis of 51Cr-labeled RPMI human plasmacytoma cell line by activated and expanded CD8+ T cells is suppressed >90% by NKG2D RNAi with NKG2D-sh6 (p < 0.0001), whereas RNAi with nonspecific mCD8 shRNA (mCD8-sh) and transduction with empty pLVTHM (No RNAi) had no effect. Cytolysis of murine P815 mastocytoma was not observed. Shown are representative results from one experiment repeated three times.

 
NKG2D is required for cytolysis of autologous tumor by activated and expanded CD8+ T cells

We and others have shown that human plasmacytoma cell lines, such as U266, and plasmacytoma cells from patients with multiple myeloma express MICA and ULBP3, and that cytotoxicity by activated CD8+ T cells and NK cells can correlate with the expression of these known NKG2D ligands (23, 40). We sought to extend these observations by examining requirements for NKG2D signaling in killing of autologous tumor by CD8+ T cells activated and expanded from human patients undergoing cancer therapy. A novel NKG2D ligand, termed Letal, has recently been found to be expressed by ovarian carcinoma patients (41). Patients with ovarian cancer undergoing surgical resection of their tumor were selected as sources of tumor targets for in vitro cytolysis studies. A PBMC sample was simultaneously obtained at the time of tumor collection for activation and expansion of CD8+ T cells. After expansion, NKG2D expression in CD8+ T cells from these patients can be functionally silenced using NKG2D-sh6 and pLVTHM lentiviral transduction (Fig. 2, A and B). In 51Cr release assays, we then observed that activated CD8+ T cells can effectively lyse labeled tumor cell targets isolated from the same patient when transduced without RNAi or with nonspecific mCD8-sh (Fig. 2C). In contrast, cytolysis of each ovarian tumor target paired with patient activated CD8+ T cells was strongly suppressed when NKG2D was silenced by NKG2D-sh6 (p < 0.0001).



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  		FIGURE 2. NKG2D RNAi suppresses cytolysis of autologous ovarian tumor by activated and expanded CD8+ T cells. CD8+ T cells activated and expanded from patients were evaluated for cytolysis of autologous ovarian tumors isolated at the time of surgical resection. RNA (A) and Western blots (B) from a representative patient show that RNAi with NKG2D-sh6 by pLVTHM lentivirus effectively silences NKG2D expression. C, Cytolysis of 51Cr-labeled ovarian tumor targets by paired patient activated and expanded CD8+ T cells (E:T cell ratio, 10:1 or 40:1) is significantly reduced by NKG2D RNAi (p < 0.0001) compared with nonspecific RNAi and mock pLVTHM lentiviral transduction. Shown are representative results from one experiment repeated four times with other ovarian cancer autologous T cells paired samples. ELISA for IFN-{gamma} (D) by activated CD8+ T cells showing that production of this cytokine is not influenced by NKG2D silencing and does not significantly increase with stimulation by PMA and ionomycin.

 
NK cells can kill tumor targets through a combination of cell-mediated and humoral cytotoxicity mechanisms. We previously showed that activated human CD8+ T cells require cell-cell contact and rely on granule release to effect cytolysis (42, 43). We evaluated whether cytokine production may contribute to killing by activated CD8+ T cells by ELISA for IL-2 and IFN-{gamma}(Fig. 2D). After 21 days of activation and expansion in culture, these cells constitutively produce IFN-{gamma}. Stimulation with PMA and ionomycin failed to augment levels of IFN-{gamma}, and production of these cytokines was not influenced by NKG2D silencing. Similar amounts were obtained for IL-2. These results suggest that the production of cytotoxic cytokines does not contribute to killing by activated and expanded CD8+ T cells.

DAP10 RNAi in activated and expanded human CD8+ T cells suppresses in vitro cytolysis

NKG2D is a type II homodimeric, C-type, lectin-like transmembrane molecule with a short cytoplasmic tail and by itself lacks intrinsic signaling capabilities (44). Instead, NKG2D forms a stable complex with the predominantly intracellular DAP10 adaptor protein to deliver activating signals via recruitment of the p85 subunit of the PI3K. We therefore used RNAi to evaluate the role of DAP10 signaling in activated and expanded CD8+ T cells. We first determined that DAP10 is expressed by activated T cells and the CD8+ T cell subset (Fig. 3A). We then generated three DAP10-specific shRNA and found that DAP10-sh3, which targets exon 4 of the DAP10 mRNA near the translation stop codon, most effectively silenced DAP10 gene expression when transduced with lentivirus (Fig. 3, B and C). Activated CD8+ T cells subjected to DAP10 RNAi using the DAP10-sh3 lost ~75% of cytolytic activity (p = 0.0003) compared with transduction using ineffective DAP10 shRNA, nonspecific mCD8-sh, and empty pLVTHM vector.



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  		FIGURE 3. DAP10 RNAi in activated and expanded CD8+ T cells. A, RNA blot showing that DAP10 is constitutively expressed by freshly isolated human NK cells and all activated and expanded T cells including the CD8+ subset. By RNA (B) and Western blotting (C), DAP10 silencing by three DAP10 shRNA in activated CD8+ T cells by pLVTHM lentivirus was most effective with DAP10-sh3. DAP10-sh3 (nt 343–361) targets exon 4 of the DAP10 transcript, nine nucleotides upstream of the translation stop codon. D, Cytolysis of 51Cr-labeled RPMI 1640 targets by activated and expanded CD8+ T cells is suppressed by ~75% (p = 0.003) after DAP10 RNAi with DAP10-sh3. Shown are representative results from one experiment repeated three times.

 
DAP12 silencing also suppresses cytolysis by activated and expanded CD8+ T cells

DAP10 gene knockdown suppressed cytolysis by activated and expanded CD8+ T cells less potently than did silencing of NGK2D, suggesting that additional signaling pathways contribute to maximum cytolysis triggered by NKG2D-mediated activation. No association between NKG2D and DAP12, another intracytoplasmic activating adaptor protein, has been observed in humans (9, 14). DAP12 is a promiscuous binding partner, however, and thus may contribute signals important for cytolysis in activated CD8+ T cells through pairing with other activating surface receptors. Similar to DAP10, we found levels of DAP12 expression in activated and expanded CD8+ T cells comparable to those observed in freshly isolated NK cells (Fig. 4A). Of four DAP12-specific shRNA evaluated, we determined that DAP12-sh3 induced the most suppression of DAP12 gene expression in activated CD8+ T cells (Fig. 4B). The DAP12-sh3 nucleotide sequence is complementary to an entirely translated portion of exon 4 of the DAP12-coding sequence. In accordance with our speculation, we observed a partial and significant reduction in cytotoxicity by activated and expanded CD8+ T cells when DAP12 was silenced by RNAi using DAP12-sh3 (p = 0.002; Fig. 4C). Although less profound than requirements for NKG2D- and DAP10-mediated signaling, these results suggest that activated human CD8+ T cells also rely on DAP12-dependent signaling pathways for optimal tumor cytolysis.



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  		FIGURE 4. DAP12 RNAi in activated and expanded CD8+ T cells. A, RNA blot showing that DAP12 is expressed by activated and expanded human T cells at a level comparable to that in freshly isolated NK cells. B, RNA blot; DAP12 silencing using four DAP12 shRNA in activated CD8+ T cells by pLVTHM lentivirus was functionally achieved with DAP12-sh3. DAP12-sh3 (nt 310–328) targets exon 4, an entirely translated portion of the DAP12 coding sequence. C, Cytolysis of RPMI 1640 targets in a 51Cr release assay by activated and expanded CD8+ T cells is partially suppressed by DAP12 RNAi with DAP12-sh3 (p < 0.002) and to a lesser extent by DAP12-sh4 at the higher E:T cell ratio of 40:1 compared with negative mCD8-sh and empty pLVTHM vector controls. Shown are representative results from one experiment repeated three times.

 
Double-knockdown of DAP10 and DAP12 in activated CD8+ T cells

The proximal signaling pathway for DAP10 includes Grb-2, phospholipase C-2, SLP-76, and PI3K (45). For DAP12, initial signaling involves activation of Src protein kinases, which phosphorylate the ITAM on tandem tyrosine residues, followed by second-line recruitment of Syk family protein kinases. It is thought that these disparate signals then culminate in a common pathway leading to activation of Rac, MAPK kinase, and ERK and, ultimately, cytolysis. To gain insight into whether the integration of DAP10 and DAP12 signaling contributes to induction of optimal tumor killing, we generated activated CD8+ T cells silenced for both DAP10 and DAP12. To isolate double transductants for in vitro cytolysis assays, we replaced the GFP reporter gene on the pLVTHM lentiviral vector with DsRed for transduction of DAP10-sh3. Thus, DAP10-sh3-transduced cells can be FACS sorted based on DsRed fluorescence, as opposed to GFP fluorescence for successful DAP12-sh3 transduction, and fluorescence of both DsRed and GFP in DAP10-sh3 and DAP12-sh3 double transductants (Fig. 5, A–C). NKG2D expression was preserved even with simultaneous silencing of DAP10 and DAP12 after activation in 14–28 days of culture, as determined by FACS. Suppression of in vitro cytolysis by DAP10 and DAP12 double knockdown was significant, with a >80% reduction (p < 0.0001) in cytotoxicity compared with negative controls (Fig. 5D), reflecting more potent suppression than silencing of DAP10 or DAP12 alone.



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  		FIGURE 5. Double transduction with DAP10-sh3 and DAP12-sh3 for RNAi. A, The GFP cassette in pLVTHM was replaced by a DsRed reporter gene for transduction of DAP10-sh3 to confirm by double DAP10 and DAP12 knockdown FACS analysis in activated T cells as shown by GFP and DsRed fluorescence, also indicating no reduction in NKG2D expression. B, RNAi of DAP10 and DAP12 is specifically mediated by, respectively, DAP10-sh3 and DAP12-sh3, as shown in this RNA blot of activated and expanded CD8+ T cells. C, Western blot for DAP10 and DAP12 after transduction for DAP10-sh3 and DAP12-sh3. D, Suppression of 51Cr-labeled RPMI 1640 cytolysis by activated and expanded T cells in the presence of simultaneous DAP10 and DAP12 RNAi (p < 0.001) compared with mCD8-sh and empty pLVTHM vector negative controls as shown. Shown are representative results from one experiment repeated three times.

 
Human NKG2D has been shown to assemble as a hexameric complex consisting of the NKG2D homodimer interacting with four DAP10 molecules via a three-helix interface in the transmembrane domains (46). In DAP10-deficient mice, NKG2D surface assembly and signaling can be delivered through noncovalent binding with DAP12 via expression of an alternative splice variant (22). Because no human NKG2D splice variant has been identified to date, we were surprised to find that NKG2D surface expression is maintained in activated CD8+ T cells despite DAP10 silencing. It is possible that human NKG2D isoforms may exist at low levels or only in certain conditions or cell types, however, and thus we examined whether NKG2D can interact with DAP12 in activated and expanded CD8+ T cells. We first confirmed by Western blotting that DAP10-sh3 effectively silences DAP10 expression in both CD8+ T cells and NK cells (Fig. 6, A and B). Activated CD8+ T cells differ from NK cells, however, in that we observed that NKG2D can be coimmunoprecipitated with DAP12 regardless of whether DAP10 is silenced, which is lost when DAP12 RNAi is induced (Fig. 6, C–E). These results indicate that human NKG2D expression can be accounted for by pairing with DAP12 when DAP10 is not present in activated and expanded CD8+ T cells.



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  		FIGURE 6. NKG2D pairs with DAP12 in activated CD8+ T cells. Western blot of whole cell lysates from activated and expanded CD8+ T cells (A) or purified NK cells (B) confirming silencing of DAP10 by DAP10-sh3, whereas NKG2D protein expression is preserved. C, Receptor complexes were immunoprecipitated with a polyclonal anti-DAP12 Ab and blotted for NKG2D, showing pairing of DAP12 with NKG2D regardless of DAP10 silencing in activated and expanded CD8+ T cells but not in purified NK cells (D). E, Binding of NKG2D and DAP12 in activated CD8+ T cells was confirmed by immunoprecipitation with anti-NKG2D, followed by blotting for DAP12.

 
NKG2D, DAP10, and DAP12 RNAi in human NK cells

Using the NKG2D-sh6, DAP10-sh3, and DAP12-sh3 constructs, we examined the roles of these signaling molecules in primary NK cells isolated from normal human donors. These three shRNA and the pLVTHM lentivirus were able to elicit high level functional silencing of NKG2D, DAP10, and DAP12 gene expression in NK cells (Fig. 7, A–C). NK cell killing of RPMI 1640 target cells was not compromised by lentiviral transduction, but was potently suppressed by NKG2D silencing, which was not seen with RNAi by control shRNA (Fig. 7D). This finding is in agreement with observations by others who have established the general dependency by NK cells on NKG2D for in vitro killing of NKG2D ligand-bearing tumor targets (15). Because NKG2D-activating signals appear to be exclusively transduced by DAP10 in human NK cells (9), we were not surprised to find a similar degree of NK cell cytolysis suppression with DAP10 gene silencing (Fig. 7E). Unexpectedly, however, we observed that DAP12 RNAi also substantially suppressed NK cell killing compared with control lentiviral transductions.



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  		FIGURE 7. NKG2D, DAP10, and DAP12 RNAi in human NK cells. CD3–CD56+ NK cells freshly isolated from normal human donors were evaluated for cytolysis after RNAi for NKG2D, DAP10, and DAP12. RNA (A) and Western blots (B) demonstrate that functional silencing of NKG2D can be achieved in fresh polyclonal human NK cells using NKG2D-sh6 and pLVTHM lentiviral vector. Similar DAP10 and DAP12 RNAi was observed by RNA blot (C) after transduction with DAP10-sh3 and DAP12-sh3. Cytolysis of 51Cr-labeled RPMI 1640 targets by fresh NK cells is suppressed with NKG2D RNAi (D; p = 0.002) and to a similar extent with either DAP10 (p = 0.005) or DAP12 (p = 0.009) RNAi (E). Shown are representative results from one experiment repeated three times.

 
NKG2D, DAP10, and DAP12 RNAi in redirected cytolysis of NK cells and activated CD8+ T cells

Elegant studies conducted by Rosen et al. (14) provided compelling evidence suggesting that DAP10 is entirely sufficient, and, hence, DAP12 dispensable, for NKG2D function in human NK cells insofar as direct killing of MICA-transduced BaF/3 cell targets. The surface density of NKG2D ligands where natively expressed by tumor cell lines can be at low levels, however, which may account for conflicting observations of only partial dependency on NKG2D for NK tumor cytolysis in these different experimental circumstances (23, 47). We tested whether a strong NKG2D stimulus, serving as the dominant activating signal, can overcome suppression of killing due to DAP12 knockdown by performing redirected cytolysis of Fc{gamma}R-bearing P815 cell targets preincubated with an agonistic anti-NKG2D mAb. Vigorous Ab-mediated redirected killing was observed by freshly isolated human NK cells when transduced with empty pLVTHM vector or with control mCD8-sh RNAi, but not after transduction with NKG2D-sh6 or DAP10-sh3 (Fig. 8A). In contrast to cytolysis of RPMI targets, we observed little suppression of NK cell-redirected killing with DAP12-sh3-mediated RNAi. Suppression of redirected cytolysis by activated and expanded CD8+ T cell persisted, however, when DAP12 was silenced by RNAi (Fig. 8B).



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  		FIGURE 8. NKG2D, DAP10, and DAP12 RNAi in redirected cytolysis. P815, which is an FcR-bearing murine plasmacytoma cell line, was used as a target in 51Cr release assays after incubation with anti-NKG2D (10 µg/ml) mAbs. A, Freshly isolated NK cells effectively kill 51Cr-labeled anti-NKG2D-coated P815 targets regardless of whether DAP12 is silenced by RNAi with DAP12-sh3 transduction, but not after either NKG2D or DAP10 RNAi transduction. B, Activated and expanded CD8+ T cells cytolysis is suppressed after NKG2D or DAP10 RNAi transduction; however, in contrast to NK cells, it remains suppressed in the presence of DAP12 RNAi. Neither NK cells nor activated CD8+ T cells kill P815 targets or those preincubated with anti-NKG2D. Shown are representative results from one experiment repeated three times.

 
NKG2D RNAi in an SCID/Hu tumor model

Using NKG2D-sh6 and the lentiviral vector to induce stable gene silencing, we evaluated the role of NKG2D signaling in an in vivo tumor model. We generated a luciferase-expressing human ovarian carcinoma cell line, UCI101-luc, and inoculated these cells in C.B-17 SCID mice, followed by i.v. injections of activated and expanded human CD8+ T cells on days 6 and 12 after tumor seeding. Tracking tumor growth by bioluminescence imaging, we observed that mice treated with expanded and activated CD8+ T cells transduced with NKG2D-sh6 had markedly faster tumor progression than animals treated with CD8+ T cells not containing the NKG2D-sh6 siRNA (Fig. 9). These results indicate that suppression of NKG2D expression significantly impacts the in vivo biological capability of expanded CD8+ T cells in controlling tumor growth and progression.



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  		FIGURE 9. NKG2D RNAi suppresses the antitumor activity of activated and expanded CD8+ T cells in an SCID/hu model. The luciferase-expressing ovarian carcinoma cell line UCI101-luc (1 x 106) was implanted in C.B-17 SCID mice and followed for 30 days by bioluminescence imaging. On days 6 and 12 after tumor inoculation, mice received injections with 40 x 106 activated and expanded human CD8+ T cells transduced with either NKG2D-sh6 or pLVTHM empty vector (No RNAi). A, Bioluminescence imaging on day 30 in representative mice from one experiment. Little tumor was established after i.p. seeding if mice were subsequently given injections with activated CD8+ T cells not subjected to NKG2D RNAi (upper right panel). In contrast, i.p. UCI101-luc leads to bulky tumor on all peritoneal surfaces and massive ascites in mice given NKG2D-silenced activated CD8+ T cells (upper left panel). B, Quantification of tumor signal after injection of expanded CD8+ T cells treated with NKG2D-sh6 siRNA {blacksquare}, vector; {triangleup}, without RNAi (n = 25 mice).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this report we dissect the molecular signaling events leading to cytolysis of tumor cells by activated and expanded human CD8+ T cells and NK cells. We developed shRNA specific for human NKG2D, DAP10, and DAP12 for lentiviral transduction that were effective for stable gene expression silencing. Using these shRNA in RNAi experiments, we confirmed our previous observation that activated and expanded human CD8+ T cells depend on NKG2D for in vitro killing and demonstrated that NKG2D is also required for in vivo antitumor activity. This dependency on NKG2D and consequently DAP10 signaling, although obligate, is not sufficient for optimal cytolysis, because partial suppression of killing solely by DAP12 RNAi suggests a nonredundant role for activation initiated by the DAP12 pathway.

Although selective and robust inhibition of protein function by RNAi has lead to its increasing exploitation as a tool for scientific investigation, some limitations of the technique merit emphasis with regard to the current study. Foremost among these concerns is the confounding effect of transcript variation, mainly alternative splice variants, on the effectiveness of RNAi with the shRNA that we used. Numerous RNA splice variants of human NKG2D, DAP10, and DAP12 have been described (12, 48, 49), and a query of the public nucleotide sequence databases revealed many more that have not been fully characterized. We have not excluded the possibility that alternative expression of these variants between NK cells vs activated and expanded CD8+ T cells or from one normal donor compared with another may influence the extent of gene knockdown with our RNAi. We have otherwise controlled for the mode of RNAi delivery and the physiologic consequences of RNAi induction, including those involved in viral host defense (50), with a nonspecific shRNA approximating the thermodynamic profile expected of an active siRNA.

The regulation of NK cell activation has been an area of intensive investigation, and our results highlight the complexity of the mechanisms that underlie regulation of tumor cytolysis. Using RNAi to target signaling molecules involved in NK cell stimulation, we observed subtle differences in signaling requirements for direct killing of NKG2D ligand-bearing tumor as opposed to anti-NKG2D mediated indirect killing of FcR-bearing tumor targets by freshly isolated polyclonal and unstimulated NK cells. In both instances, cytolysis is NKG2D and DAP10 dependent, because knockdown of either gene by RNAi suppressed killing. We found that in vitro lysis of RPMI targets was also substantially suppressed by DAP12 RNAi, however, indicating that signals mediated by other activating NK receptors are additionally required for the greatest killing. This finding is consistent with reports by others who observed that lysis of tumor cells naturally expressing NKG2D ligands is only partially inhibited by blocking with NKG2D-specific Abs even at saturating doses (47, 51). Conversely, some target tumor cells that lack expression of NKG2D ligands can be sensitive to NK cell-mediated cytotoxicity, again suggesting alternative activating receptors (51). This contrasts with indirect killing of anti-NKG2D-coated P815 targets, where we found that lysis by NK cells was efficient despite DAP12 silencing. This may be a reflection of conditions where selective NKG2D signaling is the predominant stimulus for activation, akin to enhancement of cytotoxicity by tumor cell lines transfected with NKG2D ligands that are otherwise resistant to NK cell killing (14, 20, 52). Whether there is a threshold of NKG2D signaling amplitude that must be exceeded to effect cytolysis is not known, and this may be influenced by differences in the surface density of NKG2D ligands, as previously suggested (47). Alternatively, NKG2D binds with its various ligands at markedly different binding affinities (44); thus, heterogeneous tumor cell expression of NKG2D ligands may influence signaling.

We unexpectedly observed that NKG2D expression is maintained in activated and expanded CD8+ T cells despite DAP10 silencing and showed that binding with DAP12 may account for this phenomenon. The receptor specificity of DAP10 and DAP12 resides entirely in their transmembrane domains (9, 14, 46). It is possible that there are structural variants in the transmembrane domain of either NKG2D or the adaptor proteins permissive for pairing of NKG2D with DAP12 in activated CD8+ T cells, although evidence supporting this has not been found in studies of polyclonal NK cells and IL-2-activated blood mononuclear cells (53). This consideration is further complicated by our observation that NKG2D is expressed by activated CD8+ T cells even when both DAP10 and DAP12 are simultaneously silenced. Similarities can be drawn to observations made in Jurkat cell lines, which do not express DAP12 and express DAP10 only at low levels (5, 12), whereby NKG2D expression can be induced by transfection with an NKG2D expression vector alone (20). Although binding of NKG2D with adaptor proteins other than DAP10 and DAP12 has not been reported, we can speculate on novel associations between NKG2D and yet to be identified receptor complex subunits. Double knockdown of DAP10 and DAP12 abolishes much of the killing by activated and expanded CD8+ T cells, however, implying that these novel NKG2D receptor complexes are unlikely to be important in signaling for activation of cytolysis.

Unlike NK cells, activated and expanded CD8+ T cells require simultaneous stimulation mediated by DAP10 as well as DAP12 pathways for all aspects of in vitro killing evaluated in this study. From previous studies we had characterized cytolysis by activated and expanded T cells as being MHC unrestricted and TCR independent, requiring cell-cell contact, and relying on an intact granule release pathway; more recently, we identified NKG2D as the critical activating intermediary (23, 29, 42, 43). Our finding that killing by activated and expanded CD8+ T cells is sensitive to DAP10 silencing by RNAi is predictable given the exquisite relationship between cytolysis and Ab-mediated NKG2D blockade that we had observed from previous studies. That cytolysis can also be inhibited by DAP12 RNAi was unexpected and raises questions about whether there are key DAP12-binding receptor partners expressed by activated and expanded CD8+ T cells influencing cytolysis. Of clinical relevance, DAP12 loss-of-function mutations have been identified (49, 54), and it would be important to understand whether CD8+ T cells activated and expanded from such individuals would be impaired with regard to tumor killing.

Many emerging lines of evidence now point toward remarkably convergent evolution between NK cells and T cells, and our results in this study lend additional support to this idea. Although NK cells used a strategy of balancing selection to diversify its repertoire of recognition receptors as opposed to T cells, which acquired diversity through germline recombination of the TCR, both center on interaction with ligands bearing the general structure of MHC molecules for activation (1, 55). NK cells constitutively express granzyme, perforin, and IFN-{gamma}, whereas cytotoxic T cells must undergo proliferation and de novo transcription of these gene products to become competent effector cells, yet both ultimately use the same defense mechanisms to defend against invading pathogens or tumor (56). It is interesting to speculate about whether physiologic conditions mimicking our in vivo activation and expansion protocol exist and how these could be beneficial for effective immune function, or why a T cell would maintain, but keep dormant, pathways for activation of cytolysis shared with NK cells and, moreover, use it preferentially over its native TCR. Our results in this study clearly show that activated and expanded CD8+ T cells rely on NKG2D and DAP10 signaling to acquire cytolysis of tumor targets, but we have yet to understand the role of DAP12 in the activation cascade. We have also not clarified the distinction between cytolysis as opposed to proliferation and cytokine secretion and the dependency on each of these proximal signaling pathways, although our results suggest that cytokine production does not contribute to killing in activated CD8+ T cells. We have observed that activated and expanded murine CD8+ T cells efficiently eradicate tumor, but do not cause graft-vs-host disease (30), and it is possible that subtle differences in these activation pathways, compared with those used by unmanipulated alloreactive T cells, may be crucial for avoidance of deleterious immune effects.

Ultimately, we aim to test the antitumor activity of activated and expanded CD8+ T cells in rationally designed clinical studies involving patients with malignant diseases. We provide additional evidence that CD8+ T cells activated and expanded from normal donors have in vivo tumor activity using an SCID/hu model. We furthermore show that these activated cells can be generated from patients with ovarian cancer, which gain the ability to lyse autologous tumor targets isolated from the same patient. Findings from this study provide us with a better understanding of the broad antitumor potential of these cells, enhance our understanding of regulatory mechanisms supporting cytolysis, and will aid in the implementation of activated and expanded CD8+ T cells as a potentially efficacious form of cellular immunotherapy.


   Acknowledgments
 
We thank Dr. John Chan for assistance with acquisition of PBMC and tumor samples from ovarian cancer patients, and the patients themselves for participating in our study. We also thank Dr. Tim Doyle for providing us with the pMSCVpuroBA-L2G retroviral vector, and Dr. Christopher H. Contag for his thoughtful advice. Many thanks to Dr. Leon Su for help with vector cloning and discussion of experimental results. We thank Dr. Ruby Wong for assistance with statistical analyses.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.

1 This work was supported in part by Grants KO8HL04505, P01CA049605, and R01CA080006 from the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Robert S. Negrin, Division of Blood and Marrow Transplantation, Stanford University, CCSR Room 2205, 269 West Campus Drive, Stanford, CA 94305-5170. E-mail: negrs{at}stanford.edu Back

3 Abbreviations used in this paper: DAP, disulphide adaptor molecule; MICA, MHC class I-related protein; RNAi, RNA interference; cRPMI, complete RPMI 1640; m, murine; shRNA, short hairpin RNA; siRNA, small interfering RNA. Back

Received for publication May 18, 2005. Accepted for publication September 21, 2005.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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