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Marked Prolongation of Cardiac Allograft Survival by Dendritic Cells Genetically Engineered with NF-B Oligodeoxyribonucleotide Decoys and Adenoviral Vectors Encoding CTLA4-Ig¹

C. Andrew Bonham^2,*, Lansha Peng^*, Xiaoyan Liang^*, Zongyou Chen^*, Lianfu Wang^*, Linlin Ma^*, Holger Hackstein^*, Paul D. Robbins, Angus W. Thomson^*,, John J. Fung^*, Shiguang Qian^* and Lina Lu^3,*

^* Department of Surgery and Thomas E. Starzl Transplantation Institute, University of Pittsburgh Medical Center, and Department of Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA 15213

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bone marrow-derived dendritic cells (DCs) can be genetically engineered using adenoviral (Ad) vectors to express immunosuppressive molecules that promote T cell unresponsiveness. The success of these DCs for therapy of allograft rejection has been limited in part by the potential of the adenovirus to promote DC maturation and the inherent ability of the DC to undergo maturation following in vivo administration. DC maturation occurs via NF-B-dependent mechanisms, which can be blocked by double-stranded "decoy" oligodeoxyribonucleotides (ODNs) containing binding sites for NF-B. Herein, we describe the combined use of NF-B ODNs and rAd vectors encoding CTLA4-Ig (Ad CTLA4-Ig) to generate stably immature murine myeloid DCs that secrete the potent costimulation blocking agent. These Ad CTLA4-Ig-transduced ODN DCs exhibit markedly impaired allostimulatory ability and promote apoptosis of activated T cells. Furthermore, administration of Ad CTLA4-Ig ODN-treated donor DCs (C57BL10; B10(H-2^b)) before transplant significantly prolongs MHC-mismatched (C3HHeJ; C3H(H-2^k)) vascularized heart allograft survival, with long-term (>100 days) donor-specific graft survival in 40% of recipients. The mechanism(s) responsible for DC tolerogenicity, which may involve activation-induced apoptosis of alloreactive T cells, do not lead to skewing of intragraft Th cytokine responses. Use of NF-B antisense decoys in conjunction with rAd encoding a potent costimulation blocking agent offers promise for therapy of allograft rejection or autoimmune disease with minimization of systemic immunosuppression.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Dendritic cells (DCs)⁴ are uniquely well-equipped professional APC that play crucial roles in innate and acquired immunity (1, 2). The potent immunostimulatory capacity of mature DCs (mDCs) reflects high levels of cell surface MHC class II and costimulatory molecules (CMs) and the production of T cell stimulatory cytokines, in particular bioactive IL-12. Additionally, DCs express ICAMs and chemokine receptors that enable them to migrate efficiently from peripheral sites of Ag uptake to regional lymphoid tissue, where they interact intimately with Ag-specific T cells (3). It has become increasingly apparent that DCs not only can induce immunity, but also play an important role in regulating immune responses (1, 4, 5, 6, 7). Thus, mouse and human DC subsets appear to differentially regulate Th1 and Th2 responses (4, 5), whereas immature DCs (iDCs) can induce T regulatory cells in vitro (5, 8, 9) and promote Ag-specific tolerance in vivo (10). Moreover, while immature myeloid DCs are able to induce alloantigen-specific T cell anergy (11, 12), DCs that express Fas ligand (FasL; CD95 ligand) can promote apoptotic death of alloactivated T cells (13, 14).

The inherent ability of DCs to traffic exquisitely to T cell areas of secondary lymphoid tissues and to regulate immune responses makes them attractive targets for manipulation with genes encoding immunosuppressive molecules. Thus, use of genetically engineered DCs has been proposed for the inhibition of organ allograft rejection and the treatment of various autoimmune diseases (15, 16). DCs engineered to express CTLA4-Ig, viral IL-10, or TGF- all inhibit Ag-specific T cell responses in vitro, and survive longer and in greater numbers in unmodified allogeneic hosts (17, 18, 19). FasL- and CTLA4-Ig-transduced donor DCs prolong murine cardiac and pancreatic islet allograft survival, respectively (20, 21), whereas IL-4-transduced DCs strikingly inhibit collagen-induced arthritis (22, 23). Although this strategy has met with some success in transplantation, indefinite donor-specific graft survival has not been achieved using genetically engineered DCs.

Techniques used to deliver genes to DCs include transfection with lipofectin, electroporation, biolistic (gene gun) delivery, and transduction with viral vectors (17, 20, 21, 24). Of these, transduction with adenoviral (Ad) vectors is the most efficient, with high and sustained levels of transgene expression in the majority of cells exposed to the vector (17, 18). A potential obstacle to the successful use of genetically engineered DCs for therapeutic immunosuppression is their maturation/activation in vivo following interaction with proinflammatory factors that may overcome the desired effect of the transgene product. In addition, rAd can induce DC maturation (25). Both DC maturation and immunostimulatory ability depend on NF-B-dependent gene transcription. Thus, LPS up-regulates CD80, CD86, and inducible NO synthase gene expression in DCs through nuclear translocation of NF-B and subsequently increased NF-B-dependent gene transcription. Similarly, adenovirus activates NF-B and promotes DC maturation (25).

We have demonstrated previously that double-stranded oligodeoxyribonucleotides (ODNs) containing binding sites for NF-B specifically inhibit NF-B-dependent gene transcription in DCs, suppress IL-4-induced DC maturation, and promote the ability of immature donor DCs to prolong organ allograft survival (26). In this study, we report that LPS- or rAd-induced DC maturation is prevented by these NF-B ODN "decoys," and that the maturation of DCs induced by allogeneic T cells is inhibited. We have combined NF-B ODNs and rAd encoding the costimulation-blocking molecule CTLA4-Ig (Ad CTLA4-Ig) to generate tolerogenic DCs (Ad CTLA4-Ig/NF-B ODN DCs). Thus, exposure to NF-B ODNs prevents rAd-induced DC maturation, without interfering with CTLA4-Ig transgene expression and protein secretion. Moreover, a single i.v. injection of donor-derived rAd CTLA4-Ig/NF-B ODN DCs before transplant induces long-term (>100 days) MHC-mismatched vascularized cardiac allograft survival in a high proportion of recipients and without systemic levels of the gene product. These tolerogenic effects correlate with the capacity of the rAd CTLA4-Ig/NF-B ODN DCs to enhance apoptosis of activated T cells in vitro and with decreased expression of proinflammatory cytokines (both Th1 and Th2) within the grafts. These novel findings demonstrate the potential of NF-B ODN decoys and transgenic costimulation-blocking molecules to render DCs capable of promoting long-term organ transplant survival.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals

C57BL/10J (B10; H-2^b), C3H/HeJ (C3H; H-2^k) and BALB/c (H-2^d) mice, 10–12 wk old, were obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained in a specific pathogen-free facility at the University of Pittsburgh Medical Center (Pittsburgh, PA). Animals were provided with Purina rodent chow (Ralston Purina, St. Louis, MO) and tap water ad libitum.

Propagation and purification of bone marrow (BM)-derived DCs

BM cells harvested from femurs of B10 mice were cultured in 24-well plates (2 x 10⁶/well) in 2 ml RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with antibiotics and 10% v/v FCS (referred to subsequently as complete medium), and 4 ng/ml mouse GM-CSF ± 1000 U/ml rIL-4 (Schering-Plough, Kenilworth, NJ). Thus, two populations of predominantly iDCs (GM-CSF-stimulated) or mDCs (GM-CSF + IL-4-stimulated) were obtained. The selection and purification procedures were similar to those reported initially by Inaba et al. (27) with minor modifications as described (28). In certain experiments, the cells were cultured with 10 µg/ml LPS (Escherichia coli serotype O₂6:B6; Sigma-Aldrich, St. Louis, MO) for 18 h to induce NF-B nuclear translocation and DC maturation, then subjected to further analysis.

ODN decoys

Double-stranded NF-B ODN decoys were generated using equimolar amounts of single-stranded sense and antisense phosphorothioate-modified oligonucleotides containing two NF-B binding sites (sense sequence 5'-AGGGACTTTCCGCTGGGGACTTTCC-3'; NF-B binding sites bold and underlined) as described (26, 29). Sense and antisense strands were mixed in the presence of 150 mM NaCl, heated to 100°C, and allowed to cool to room temperature to obtain dsDNA. A total of 10 µM NF-B ODNs were added at the initiation of DC cultures. Appropriate double-stranded control ODNs were used to confirm the absence of nonspecific effects (26).

Construction of rAd vectors

Ad vectors (E1, E3) were constructed through Cre-lox recombination using the pAd-lox shuttle vector and 5 helper virus DNA in the Ad packaging cell line CRE-8 (30). To construct Ad vectors encoding the reporter gene enhanced green fluorescent protein (EGFP; Ad-EGFP), a SnaBI-HpaI fragment containing part of the CMV promoter, the EGFP_N1 cDNA, and part of the SV40 poly(A) derived from pEGFP_N1 (Clontech Laboratories, Palo Alto, CA) was inserted into the shuttle vector pAdlox. To construct Ad CTLA4-Ig, a HindIII-EcoRI fragment containing the mouse CTLA4 mouse IgG3 cDNA (kindly provided by Dr. A Shaked, Department of Surgery, University of Pennsylvania, Philadelphia, PA) was inserted in the shuttle vector. The rAd were generated by cotransfection of SfiI-digested pAdlox-EGFP_N1 or pAdlox-mouse CTLA4 mouse IgG3 and 5 helper virus DNA into the CRE-8 cell line, propagated, and purified as described (30).

rAd vector-mediated gene transfer and expression in DCs

All vectors were propagated in CRE-8 cells, purified by two rounds of CsCl density centrifugation, dialyzed, and stored at -70°C in 3% sucrose. Viral titers were determined by plaque-forming assay using 293 cells. All vector preparations were free of replication-competent virus. DCs were infected after 5 days in culture with an empty Ad vector (Ad-), Ad-EGFP, or Ad CTLA4-Ig for 2 h at 50 multiplicity of infection (MOIs). Gene expression was assessed 2 days after transduction. EGFP expression was determined by flow cytometry and by direct visualization of cells using a fluorescence microscope.

Flow cytometry

Expression of DC surface Ags was analyzed by cytofluorography, using an EPICS ELITE flow cytometer (Coulter, Hialeah, FL). Cells were stained with primary hamster or rat mAbs directed against CD11c, CD40, CD80, or CD86 (BD PharMingen, San Diego, CA), followed by FITC- or PE-conjugated goat anti-hamster or goat anti-rat IgG2a, as described (11). MHC class I and II Ags were detected with FITC-conjugated mAbs directed against H-2^b and I-A^b, respectively.

EMSA

EMSA were performed using commercially available kits (Promega, Madison, WI). Briefly, nuclear proteins were isolated (31) from 2 x 10⁶ cultured DCs manipulated as described earlier in the presence or absence of NF-B ODNs. An NF-B oligonucleotide supplied with the kit was used as a probe (sense sequence: 5'-AGTTGAGGGGACTTTCCCAGGC-3'). The probe was end-labeled with [-³³P]ATP (NEN, Boston, MA). Excess unlabeled probe was added as competitor for the radiolabeled probe in the binding reaction. The specificity of the NF-B band was further determined by supershifting of complex (p50-SS) using specific anti-NF-B p50 Ab (Santa Cruz Biotechnology, Santa Cruz, CA). Nuclear proteins were incubated with labeled probe and the mobility shift was detected by running the mixture on a 4% acrylamide gel. Shifted bands were visualized by autoradiography.

Mixed leukocyte reaction

To determine the Ag-presenting capacity of DCs in vitro, one-way MLRs were performed with gamma-irradiated (20 Gy) DCs derived from B10 BM as stimulators and nylon wool-purified C3H splenic T cells (2 x 10⁵) as responders. Cultures were established in triplicate in 96-well, round-bottom microculture plates (200 µl/well) and maintained in complete medium for 4 days in 5% CO₂ in air at 37°C. [³H]TdR (1 µCi/well) was added for the final 18 h of culture. Cells were harvested onto glass fiber disks using an automated system, and incorporation of [³H]TdR into DNA was assessed by liquid scintillation counting. Results are expressed as mean cpm ± 1 SD.

CTLA4-Ig assays

M38 cells expressing CD80 were used as indicator cells as described (17). Supernatants from Ad CTLA4-Ig-transduced or control DCs were added to CD80⁺ or CD80^-M38 cells, followed by staining with a secondary FITC-conjugated mAb against mouse IgG and flow cytometric analysis. Serum samples were assayed for CTLA4 by ELISA (detection limit 200 pg/ml) using goat anti-mouse CTLA4 (R&D Systems, Minneapolis, MN) and biotin-conjugated rabbit anti-goat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) with mouse CTLA4/Fc (R&D Systems) as a standard.

T cell activation

Nylon wool-purified C3H splenic T cells were cultured (2 x 10⁶/ml) in 24-well plates with Con A (2.5 µg/ml) in complete medium in 5% CO₂ in air at 37°C. After 3 days, dead cells were removed by Ficoll gradient centrifugation, and Con A blasts used to stimulate DCs. Alternatively, C3H T cells were stimulated by gamma-irradiated (20 Gy) mature allogeneic DCs derived from B10 BM (stimulator:responder ratio = 1:10) for 3 days before isolation and addition to DC cultures.

In vivo DC migration

B10 DCs were injected s.c. (5 x 10⁵ in 50 µl) into one hind footpad of normal C3H allogeneic recipients. At various times thereafter, groups of three mice were killed and the spleens removed, embedded in Tissue-Tek (OCT compound; Miles, Elkart, IN) and frozen at -70°C. Cryostat sections (5 µm) were air dried at room temperature overnight, then stored at -70°C until further processing. Donor MHC class II⁺ (I-A^b+) cells were identified in cryostat sections of lymphoid tissue using biotinylated mouse IgG2a anti-mouse I-A^b (BD PharMingen) in an avidin-biotin-alkaline phosphatase complex (ABC) staining procedure, as described (32). Controls included sections of normal donor or recipient strain tissues. The incidence of donor MHC class II⁺ cells in sections was determined by the mean number of positive cells per 100 high power fields.

RNase protection assay

Total RNA was extracted by the guanidinium isothiocyanate phenol chloroform method using TR1 reagent (Sigma-Aldrich) as described (33). The purity of RNA was determined from the A_260/280 absorbance ratio. Cytokine transcript levels were determined using the Ribonuclease Protection Assay kit (RiboQuant, San Diego, CA). Briefly, probes were synthesized by T7 RNA polymerase with incorporation of [³²P]-UTP. A total of 5 µg of total RNA extracted from DCs was hybridized overnight with synthesized probes (sp. act.: 800 Ci/mM) at 56°C, followed by treatment with RNase A (80 µg/ml) and T1 (250 U/ml) for 45 min at 30°C. The murine L32 and GADPH riboprobes were used as controls. Protected fragments were submitted to electrophoresis through a 7.0 M urea/5% polyacrylamide gel, then exposed to Kodak X-omat film (Kodak, Rochester, NY) for 72 h.

Detection of apoptosis

T cells were stained with PE-conjugated anti-CD3 mAb and DNA strand breaks identified by TUNEL as described (34). Following surface CD3 staining, cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 and 0.1% sodium citrate. TUNEL reaction mixture of the Cell Death Detection kit (Roche Diagnostics, Indianapolis, IN) was then added according to the manufacturer’s instructions. Cells incubated with label solution in the absence of terminal transferase were used as negative controls. Quantitative analysis was performed by flow cytometry, with 5000 events acquired from each sample.

Heart transplantation

Fully allogeneic intraabdominal vascularized heart transplantation was performed from normal B10 or BALB/c (third party) donors to size-matched C3H recipients as described (12). Surgical procedures were performed under methoxyflurane (Medical Development, Springvale, Australia) inhalation anesthesia. Graft survival was assessed by daily transabdominal palpation. Rejection was defined as total cessation of cardiac contraction, and confirmed by histological examination. To assess the effect of donor-derived DCs on allograft survival, animals received 2 x 10⁶ cells i.v., 7 days before heart transplantation in the absence of immunosuppression.

Statistical analyses

Statistical analysis was performed using the Mann-Whitney U test. Graft survival between groups of transplanted animals was compared using the log-rank test for comparison of survival curves. A value of p < 0.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
NF-B ODNs inhibit LPS-induced DC maturation

Maturation of DCs, including that induced by LPS stimulation or Ad infection is dependent on nuclear translocation of NF-B (25). To test their capacity to inhibit DC maturation, ODNs with binding sites specific for NF-B (NF-B ODNs) were added at the initiation of cultures of GM-CSF-stimulated B10 BM-derived DCs. After 5 days, the DCs were exposed to LPS to maximally activate NF-B nuclear translocation. Surface expression of CD40, CD80, CD86, and MHC class I and II was determined by flow cytometry. Whereas untreated DCs remained relatively immature with low surface MHC class II and CM expression, DCs exposed to LPS showed increased levels of MHC class II, CD80, and CD86 (Fig. 1). NF-B ODNs prevented this LPS-mediated maturation, and maintained the cells in the immature state, with low levels of surface CM expression.

View larger version (30K): [in this window] [in a new window]   FIGURE 1. NF-B ODNs inhibit LPS-induced CM expression by murine B10 (H-2b) DCs. iDCs cultured for 5 days in GM-CSF were exposed to LPS for 18 h. Cell surface molecule expression was determined by flow cytometric analysis. LPS induced maturation of the DCs, as evidenced by increased expression of CD40, CD80, CD86, and MHC class II (I-Ab) molecules. Addition of NF-B ODNs at the initiation of DC cultures prevented LPS-induced up-regulation of these molecules. Data are from a single experiment and the results are representative of three performed.

NF-B ODNs inhibit rAd-induced DC maturation and function

To ascertain whether NF-B ODNs might similarly prevent the up-regulation of CM expression in response to rAd infection, DCs cultured for 5 days in the presence or absence of ODNs were transfected with rAd- at 50 MOI. Twenty-four hours later, surface expression of CM was determined by flow cytometry. As shown in Fig. 2A, iDCs cultured in the absence of NF-B ODNs exhibited marked up-regulation of surface CD86 expression after exposure to Ad-. In contrast, iDCs maintained in NF-B ODNs did not exhibit this Ad-mediated effect, and maintained very low levels of CM expression. The influence of NF-B ODNs and rAd infection on DC allostimulatory capacity was assessed in primary MLR. Allogeneic naive C3H splenic T cells (H-2^k) were stimulated with iDCs propagated from B10 (H-2^b) BM and treated with NF-B ODNs and/or Ad-. As expected, the observed levels of CM expression (Fig. 2A) correlated with the T cell allostimulatory capacity of the DCs (Fig. 2B). Notably, Ad- DCs induced strong T cell proliferation, consistent with activation/maturation of the DCs induced by the viral vector, whereas uninfected iDCs were poor stimulators in MLR. This activity was further reduced by exposure of the DCs to NF-B ODNs that abrogated the effect of Ad- infection, as demonstrated by minimal DC allostimulatory capacity. To verify that NF-B ODNs inhibited rAd-induced DC maturation by binding specifically to NF-B, EMSA was performed on nuclear extracts obtained from the various DC cultures. As shown in Fig. 3, NF-B binding activity was detected in nuclear extracts of mDCs (GM-CSF + IL-4-stimulated) and Ad--infected iDCs. Addition of excess unlabeled consensus NF-B probe to the binding reaction resulted in the disappearance of the binding band. The position of the p50 Ab supershifted complex (p50-SS) indicated that p50 is the main member of NF-B family in nuclear protein extracted from DCs. By contrast, exposure to NF-B ODNs completely inhibited NF-B binding, despite rAd infection.

View larger version (17K): [in this window] [in a new window]   FIGURE 2. NF-B ODNs inhibit rAd-induced CM expression and allostimulatory capacity of DCs. A, CD86 expression on DCs cultured in GM-CSF alone (immature), GM-CSF + IL-4 (mature), or NF-B ODN-treated iDCs was assessed before and after transduction with Ad- as described in Materials and Methods. Ad infection up-regulated CD86 expression on both iDCs and mDCs. In contrast, NF-B ODNs prevented Ad-induced up-regulation of CD86 expression. Data are from one experiment and the results are representative of two performed. B, Purified allogeneic splenic T lymphocytes (2 x 105) from C3H (H-2k) mice were stimulated by various numbers of gamma-irradiated (20 Gy) Ad--transfected or nontransfected, untreated or NF-B-treated DCs propagated from B10 (H-2b) BM. [3H]TdR uptake was assessed in 4-day MLR. Results are expressed as mean cpm ± 1 SD. Ad- iDCs stimulated enhanced T cell proliferation compared with nontransfected cells, consistent with activation/maturation of DCs induced by the vector. Treatment of DCs with NF-B ODNs abrogated the stimulatory effect of Ad- transduction. Nontransfected, NF-B ODN-treated DCs exhibited the lowest levels of allostimulatory activity. Data are means ± 1 SD from triplicate cultures and the results are representative of those obtained from three separate experiments.

View larger version (42K): [in this window] [in a new window]   FIGURE 3. NF-B ODNs specifically inhibit NF-B nuclear translocation induced by rAd transfection in iDCs. B10 BM-derived DCs were propagated in the presence or absence of NF-B ODNs. After 5 days of culture, iDCs were infected with Ad- for 2 h. Two days following transfection, nuclear extracts were obtained from the DCs. NF-B-specific binding activity was determined by EMSA and supershift assay using anti-NF-B p50 mAb. Left to right, free probe in the absence of nuclear protein; addition of excess unlabeled probe to the nuclear protein as competitor for the radiolabeled probe; addition of anti- NF-B p50 Ab for supershift complex (p50-SS); NF-B binding band in nuclear protein extracted from mDCs, Ad- iDCs, and Ad- NF-B ODNs iDCs. Ad transfection induced nuclear translocation of NF-B similar to that induced in mDCs. NF-B ODNs blocked nuclear translocation of NF-B. Data are representative of two experiments performed.

Exposure to NF-B ODNs does not interfere with transgenic expression of EGFP or CTLA4-Ig

We next examined whether exposure to NF-B ODNs would permit transgenic expression of a reporter gene (EGFP) delivered to the DC by rAd. Because the CMV promoter was used to drive gene expression, it was expected that the transgene would still be expressed, despite the presence of NF-B ODNs. As shown in Fig. 4A, most (>75%) of the DCs transduced with Ad-EGFP (Ad-EGFP DCs) fluoresced within 2 days of gene transfer. Importantly, NF-B ODN treatment did not interfere with reporter transgene expression, as evidenced by >85% of Ad-EGFP-transduced, NF-B ODN-treated DCs exhibiting EGFP expression. Similarly, rAd CTLA4-Ig-transduced, NF-B ODN-treated DCs secreted similar amounts of transgenic CTLA4-Ig when compared with Ad CTLA4-Ig DCs that had not been exposed to NF-B ODN (Fig. 4B).

View larger version (44K): [in this window] [in a new window]   FIGURE 4. NF-B ODNs do not inhibit expression of transgenic EGFP and CTLA4-Ig in DCs. A, mDCs and iDCs were transduced with rAd-EGFP at 50 MOI, as described in Materials and Methods. Before transduction, selected cultures were exposed to NF-B ODNs. Transgene expression was assessed 2 days after transduction by fluorescent microscopy, and quantified by flow cytometric analysis of EGFP-expressing cells. Left panels, Typical DC cultures with loosely adherent cells in clusters. Right panels, Green fluorescent cells (EGFP+) in the same fields. Similar numbers of DCs express EGFP after transduction. This was confirmed by flow cytometric analysis, as shown in the far right column. NF-B ODNs did not interfere with EGFP transgene expression. Magnification, x400. B, iDCs were propagated for 5 days in GM-CSF ± NF-B ODNs as described in Materials and Methods. Ad CTLA4-Ig transduction was performed, supernatants collected 2 days later, and assessed for CTLA4-Ig using a CD80+ (M38) indicator cell line and flow cytometric analysis. Exposure of DCs to NF-B ODNs did not prevent CTLA4-Ig secretion by the DCs. Data are from a single experiment representative of three performed.

NF-B ODNs do not completely inhibit DC CM expression following interaction with allogeneic T cells

The potent ability of NF-B ODNs to prevent LPS- and rAd-induced DC maturation raised the question of whether further modification of the cells would be necessary to maximize their potential to subvert T cell responses. To address this issue, B10 NF-B ODN-treated DCs were cocultured with naive allogeneic C3H T cells for 48–72 h at a DC:T cell ratio of 1:4. CD40, CD80, and CD86 expression by gated CD11c⁺ cells was then analyzed by flow cytometry. There was a modest but progressive increase in CD80 and CD86 expression by NF-B ODN DCs over time (Fig. 5). This finding suggested that additional NF-B-independent signaling pathways existed whereby allogeneic T cells could induce CM expression on DCs and thus enhance their stimulatory potential. It was concluded that expression of an immunosuppressive transgene product by NF-B ODN DCs might further impair their potential to promote T cell-mediated responses and enhance their tolerogenicity.

View larger version (21K): [in this window] [in a new window]   FIGURE 5. CM expression is up-regulated on NF-B ODN DCs following interaction with allogeneic T cells. B10 NF-B ODN-treated iDCs and C3H T cells were cocultured for 48–72 h at a DC:T cell ratio of 1:4. CD40, CD80, and CD86 expression on CD11c+ cells were analyzed by flow cytometry. NF-B ODN iDCs exhibited very low CM expression. After culture with allogeneic T cells, the NF-B ODN DCs exhibited modest increases in CD80 and CD86 expression. Results are from one experiment representative of two performed.

Treatment of DCs with NF-B ODNs combined with rAd CTLA4-Ig is more effective than either agent alone in reducing T cell allostimulatory ability

Both NF-B ODN DCs and rAd CTLA4-Ig-transduced DCs exhibit markedly impaired ability to stimulate naive allogeneic T cells in vitro (17, 26), but their capacity to suppress alloimmune reactivity and allograft rejection in unmodified hosts is limited. In an effort to improve their immunosuppressive efficacy and tolerogenic potential, B10 BM-derived DCs were exposed to NF-B ODNs before transduction with Ad CTLA4-Ig, then evaluated as stimulators of naive C3H T cells in MLR. Both Ad- NF-B ODN DCs and Ad CTLA4-Ig DCs exhibited reduced allostimulatory capacity compared with untreated control DCs (Fig. 6). T cell proliferative responses were also profoundly reduced following exposure to DCs treated with both Ad CTLA4-Ig and NF-B ODNs, indicating that the two treatments were not antagonistic and together achieved maximal inhibition of T cell allostimulatory capacity.

View larger version (20K): [in this window] [in a new window]   FIGURE 6. Ad CTLA4-Ig NF-B ODN iDCs exhibit impaired allostimulatory capacity. Splenic T cells from C3H mice (H-2k) (2 x 105) were stimulated in vitro by gamma-irradiated (20 Gy) DCs propagated from B10 BM (H-2b) donors and treated with NF-B ODNs and/or Ad CTLA4-Ig, as described in Materials and Methods. [3H]TdR uptake was assessed in 4-day MLR. Results are expressed as mean cpm. Ad -transfected mDC-stimulated vigorous T cell proliferation, consistent with activation/maturation of DCs induced by the Ad vector. This effect was inhibited by CTLA4-Ig gene transfer. Stably immature (NF-B ODN-treated) iDC Ad transduced to express CTLA4-Ig showed the lowest levels of allostimulatory activity. Results are means ± 1 SD from a single experiment representative of four performed.

Combined treatment of DCs with Ad CTLA4-Ig and NF-B ODN DCs promotes apoptosis of activated T cells

We have shown previously that the markedly reduced T cell stimulatory capacity of DCs exposed to CTLA4-Ig protein in MLR is associated with enhanced apoptotic death of the alloactivated T cells (14, 35). To determine the extent of apoptosis induced by Ad CTLA4-Ig/NF-B ODN DCs in activated T cells, Con A-activated, alloactivated, or naive T cells were cultured for 18 h in the presence of either Ad CTLA4-Ig/NF-B ODN DCs, Ad- DCs, Ad- NF-B ODN DCs, or Ad CTLA4-Ig DCs at a DC:T cell ratio of 1:20. Apoptotic death of T cells was determined by two-color staining of CD3⁺ and TUNEL⁺ cells as described (36). Mature control DCs (Ad- DCs) appeared to protect alloactivated naive T cells from apoptosis (Fig. 7). By contrast, significant levels of apoptosis (15–30%) were observed in Con A blasts. Ad CTLA4-Ig DCs, Ad- NF-B ODN DCs, and Ad CTLA4-Ig/NF-B ODN DCs all promoted apoptosis of both Con A-activated and alloactivated T cells. The greatest degree of apoptosis was noted in Con A-activated T cells exposed to Ad CTLA4-Ig/NF-B ODN DCs, suggesting that concomitant inhibition of both CM expression (by NF-B ODNs) and functional blockade of CM (by transgenic CTLA4-Ig) was more effective than either treatment alone in promoting activated T cell apoptosis.

View larger version (28K): [in this window] [in a new window]   FIGURE 7. Apoptosis of activated T cells is promoted by Ad CTLA4-Ig-transduced DCs. Nylon wool-purified allogeneic (C3H) splenic T cells (2 x 105) were activated by Con A (2.5 µg/ml) or by incubation with B10 mDC stimulators (stimulator:responder = 1:20) for 3 days. Naive T cells served as controls. Following activation, T cells were exposed to Ad- DCs, Ad CTLA4-Ig DCs, Ad- NF-B ODN DCs, or Ad CTLA4-Ig/NF-B ODN DCs for 18 h at a DC:T cell ratio of 1:1. Apoptosis was determined by TUNEL staining and quantified by flow cytometry. mDCs expressing Ad- rescued activated T cells from apoptosis, as evidenced by low levels of TUNEL+ T cells, particularly alloactivated T cells. In contrast, expression of CTLA4-Ig was associated with an increase in the levels of apoptosis detected in alloactivated T cells. NF-B ODN DCs similarly promoted apoptosis in activated T cells, and to a lesser degree in naive T cells. Ad CTLA4-Ig/NF-B ODN DCs displayed the most potent apoptosis inducing capacity, as demonstrated by the high levels of apoptosis in both activated and naive T cells after exposure to these DCs. Results are from a single experiment and are representative of data obtained from three experiments.

DCs exposed to NF-B ODNs and rAd retain in vivo migratory capacity

The ability of DCs to direct immune responses is dependent in part on their capacity to migrate from peripheral sites to secondary lymphoid tissues. Therefore, the effect of NF-B ODNs and rAd transduction on DC migration was examined. A total of 5 x 10⁵ B10 BM-derived Ad- NF-B ODN DCs or Ad CTLA4-Ig-transduced NF-B ODN DCs were administered s.c. into the hind footpad of naive allogeneic (C3H) mice. Spleens were removed at various times and examined by immunohistochemistry for the presence of I-A^b+ cells. Greater numbers of I-A^b+ cells were visible in T cell areas of spleens of animals injected with rAd CTLA4-Ig-transduced NF-B ODN DCs compared with those given rAd- NF-B ODN DCs (Fig. 8), indicating that the dual treatment promoted the homing ability of the DCs and/or their survival in secondary lymphoid tissue.

View larger version (169K): [in this window] [in a new window]   FIGURE 8. Migratory capacity of Ad CTLA4-Ig/NF-B ODN DCs in vivo. A total of 5 x 105 B10 BM-derived Ad CTLA4-Ig/NF-B ODN iDCs were administered s.c. into the hind footpad of untreated allogeneic C3H recipients. Spleens were removed 1 day later and examined by immunohistochemistry for the presence of I-Ab-expressing cells. I-Ab+ cells (brown) are clearly visible. Counterstained with hematoxylin. Magnification, x1000.

Ad CTLA4-Ig/NF-B ODN DCs promote long-term donor-specific cardiac allograft survival

To ascertain their influence on vascularized organ allograft survival, 2 x 10⁶ unmodified immature or mature B10 DCs, or either NF-B ODN-treated, rAd-transduced DCs or B10 DCs subjected to both treatments, were injected i.v. into fully allogeneic C3H recipients. Seven days later, the mice received B10 or BALB/c (third party) cardiac transplants. mDCs accelerated graft rejection (median graft survival time (MST) 5 days) (Table I). Immature donor DCs modestly but significantly prolonged graft survival compared with untreated controls (MST 13 vs 10 days). Ad CTLA4-Ig DCs did not significantly affect graft survival when compared with untreated controls, suggesting that expression of transgenic CTLA4-Ig alone was insufficient to inhibit alloimmune responses in vivo. In contrast, NF-B ODN DCs exerted a marked effect, and prolonged MST to 24 days. Notably, NF-B ODN treatment before Ad transduction maintained the tolerogenic potential of the DCs. Thus, Ad- NF-B ODN DCs prolonged allograft survival to 23 days. However, the most profound effects were achieved with Ad CTLA4-Ig/NF-B ODN DCs. The combined treatment appeared to be synergistic, as Ad CTLA4-Ig/NF-B ODN DCs extended MST to 71 days. The effect was donor-specific as third party hearts (BALB/c) were rejected acutely. Moreover, 40% of the animals exhibited indefinite (>100 days) graft survival, and were functionally tolerant as evidenced by acceptance of donor-specific (B10) but not third party (BALB/c) skin grafts placed 100 days post heart transplant that were rejected acutely. The effects were achieved in the absence of circulating CTLA4 in serum samples obtained at various times after DC infusion and organ transplant.

Ad CTLA4-Ig/NF-B ODN DCs suppress Th1 and Th2 cytokine gene expression within cardiac allografts

There have been reports that CTLA4-Ig administration may promote allograft survival by selective induction of Th2 cell responses (37). Moreover, DCs rendered tolerogenic in vitro by blocking surface CM as a result of CTLA4-Ig gene delivery appear to promote immune deviation (38). To address the influence of rAd CTLA4-Ig/NF-B ODN DCs on host Th cytokine expression, cardiac allografts were obtained from mice given the transduced DCs or control DCs 7 days before heart transplantation. Grafts were isolated 7 days posttransplant, and subjected to RNase protection assay to determine expression of message for Th1 (IFN-; IL-2) and Th2 (IL-10) cytokines. IL-2, IFN-, and IL-10 gene transcripts were readily detected in grafts of untreated animals (Fig. 9). Although there was evidence that ODN-treated DCs promoted Th skewing toward Th2, no unequivocal evidence of immune deviation was obtained from analysis of hearts from Ad CTLA4-Ig/NF-B ODN DC-treated recipients.

View larger version (19K): [in this window] [in a new window]   FIGURE 9. Cytokine gene expression in cardiac allografts of C3H mice treated with various donor DC populations (treatment groups: A, PBS; B, GM-CSF iDCs; C, NF-B ODN iDCs; D, Ad- NF-B ODN iDCs; E, Ad CTLA4-Ig/NF-B ODN iDCs. Heart allografts from animals treated with DCs 1 wk before transplantation were isolated 7 days posttransplant, RNA extracted, and analyzed by RNase protection assay to determine cytokine gene expression. IL-2, IL-10, and IFN- mRNA were detected in grafts of untreated animals, consistent with immune activation. Injection of iDCs partially down-regulated IFN- and IL-10 expression, but had little effect on IL-2 mRNA levels. NF-B ODN iDCs appeared to skew cytokine expression toward Th2 (IL-10), whereas Ad CTLA4-Ig/NF-B ODN iDCs, or Ad- NF-B ODN DCs showed no evidence of promoting immune deviation. Data are from one experiment with three animals per group and are representative of results from two separate experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

An attractive conceptual approach to enhancing the tolerogenic potential of DCs is their genetic modification to express immunosuppressive molecules, such as viral IL-10, TGF-, FasL, or CTLA4-Ig, and their use to subvert alloantigen-specific T cell responses (17, 18, 19, 24). In principle, transgenic expression of immunomodulatory molecules by donor DC trafficking to the precise microenvironment where Ag presentation and allospecific T cell responses are instigated minimizes systemic delivery of the immunosuppressive gene product with diminution of potential undesired side effects. To date, strategies using genetically engineered DCs alone in experimental organ transplantation have failed to induce tolerance. This may be due to a number of factors, several of which have been addressed in the present study. In the setting of transplantation, proinflammatory cytokines and other factors capable of promoting DC maturation abound within recipient tissues. Thus, late maturation and inherent T cell stimulatory potential of genetically engineered DCs may overcome the effects of localized immunosuppressive transgene expression. However, our data indicate that preconditioning donor DCs ex vivo with ODN to block NF-B nuclear translocation is sufficient to stably suppress up-regulation of CM expression in response to potent activating stimuli, such as LPS, or Ad infection. In previous work, we have shown that this effect is NF-B-specific (26). This is particularly relevant to the current model, in which DC maturation induced by rAd is abrogated by NF-B ODNs without inhibitory effects on transgene expression. Thus, CTLA4-Ig production by dual-engineered DCs is not inhibited by our approach to suppressing the DC maturation-inducing effect of Ad transduction.

The ability of NF-B ODNs to prevent up-regulation of CM expression by DCs is potent, but it affects only those stimuli that use signal transduction pathways involving NF-B. The promoter regions for the genes encoding CD80 and CD86 contain sites to which several other transcription factors may bind. For example, the 5'-flanking region of the CD86 gene contains functional IFN- activation site elements to which promoters such as Stat-1 bind (42). AP-1-binding sites have been identified, and gene expression is promoted in response to IL-3 or oncogenes (43, 44, 45). Thus, while NF-B ODNs block CM expression by DCs in response to a number of stimuli including CD40 ligation, other molecules that signal through NF-B-independent pathways may induce CM expression on ODN DCs. Activated T cells are a potential source of such molecules, which may include IFN- or adhesion molecules. As we have demonstrated, activated T cells can induce CD80 and CD86 expression in NF-B ODN-treated DCs. By transducing ODN DCs with rAd CTLA4-Ig, the effect of CM up-regulation is abrogated.

Suboptimal inhibition of immune reactivity by genetically engineered DCs in vivo may also be ascribed to inadequate numbers of injected DCs, or to failure of these cells to persist for sufficient time in recipient tissues to maintain inhibition of immune responses. Terminal DC maturation is associated with shortened survival, due in part to increased Fas expression on the DCs (46). By preventing DC maturation, NF-B ODNs may extend their effective in vivo lifespan. As shown in the present study, the duration of survival of Ad CTLA4-Ig-/NF-B ODN DCs in vivo appears to be enhanced in host lymphoid tissue after their injection. This suggests that improved delivery to and survival of immunoregulatory DCs within T cell areas of lymphoid tissue may enhance their tolerogenicity. Escalation of DC dose and frequency of administration, and alternative methods of gene delivery are being investigated in an effort to augment delivery of genetically engineered donor DCs to recipient lymphoid tissue.

Indirect presentation of donor alloantigen by host APC may play a role in failure of donor DC therapies to sustain indefinite graft survival. However, after homing to recipient lymphoid tissue, release of the immunosuppressive transgene product CTLA4-Ig by engineered DCs in the local microenvironment is likely also to impair recipient APC function, and thus inhibit the indirect pathway of Ag presentation, at least temporarily. Indeed, others have shown that transient inhibition of host DC maturation in situ by systemic immunosuppressive drug administration promotes induction of organ transplant tolerance (47).

The mechanism(s) whereby Ad CTLA4-Ig/NF-B ODN donor DCs prolong cardiac allograft survival is at present unclear. We found in vitro evidence that the stably immature (NF-B ODN-treated) Ad CTLA4-Ig-transduced DCs could enhance apoptotic death of both naive and activated T cells. These findings recapitulate our earlier observation that the inhibitory effect of soluble CTLA4-Ig on the allostimulatory activity of myeloid DCs was associated with marked dose-related enhancement of the apoptotic death of the responder T cell population (14). Apoptotic death of alloreactive T cells appears to be an important mechanism underlying the induction of organ transplant tolerance (48, 49), whether tolerance is achieved in the absence of or in response to immunosuppressive therapy. Our recent findings further suggest that blockade of costimulation provided by donor-derived DCs (by administration of CTLA4-Ig or neutralizing anti-IL-12 mAb) markedly extends apoptotic death of alloreactive T cells in host lymphoid tissue and promotes organ graft survival (36, 50). Thus, stably immature donor DCs expressing ectopic CTLA4-Ig may promote graft survival by promoting deletion of alloreactive T cells. Pretreatment of graft recipients (in the present study, 7 days before transplantation) may be necessary for effective elimination of donor-reactive T cell clones. Measuring the frequency of alloreactive T lymphocytes within both host lymphoid tissue and graft-infiltrating cell populations will define the extent of donor-reactive T cell elimination in the prolongation of allograft survival by Ad CTLA4-Ig/NF-B ODN DCs.

In summary, we confirm that murine BM-derived myeloid DCs can be genetically engineered with Ad vectors to express a potent costimulation-blocking agent. We also show for the first time that DC maturation in response to Ad infection can be effectively suppressed by DC pretreatment with NF-B ODNs. CTLA4-Ig transgene expression is not impaired by the ODNs, and appears sufficient to overcome functional CM expression by DCs in response to other NF-B-independent signals. This novel dual-engineering strategy allows generation of stably iDCs with impaired T cell allostimulatory capacity and tolerogenic potential. Furthermore, Ad CTLA4-Ig ODN DCs significantly prolong cardiac allograft survival in the absence of systemic levels of the gene product, leading to donor-specific, long-term (>100 days) transplant survival in a high proportion of recipients. The mechanism(s) responsible may involve enhanced apoptotic death of alloreactive T cells. No evidence was found of skewing of the immune response to a Th2 phenotype. Conceivably, repeated administration of Ad CTLA4-Ig/NF-B ODN DCs may significantly increase the number of tolerant recipients. These findings add credence to the view that genetic engineering of DCs may be a promising modality for suppression of undesired immune reactivity in transplantation or other immune-mediated disorders.

	Acknowledgments

 
We thank Alison Logar for expert assistance with flow cytometry, Jo Harnaha for tissue culture, Dr. Adrian E. Morelli for graft histology, Dr. Katsuhiko Kaneko for CTLA4 assays, and Shelly L. Shaplye for skilled assistance with manuscript preparation.

	Footnotes

 
¹ This work was supported in part by Roche Organ Transplantation Research Foundation (to L.L.), grants from Pittsburgh Foundation (to C.A.B.), and National Institutes of Health Grant AI41011 (to A.W.T.).

² Current address: Department of Surgery, Stanford University School of Medicine, Stanford, CA 94304.

³ Address correspondence and reprint requests to Dr. Lina Lu, Thomas E. Starzl Transplantation Institute, University of Pittsburgh Medical Center, Biomedical Science Tower, W1544, 200 Lothrop Street, Pittsburgh, PA 15213. E-mail address: lul{at}msx.upmc.edu

⁴ Abbreviations used in this paper: DC, dendritic cell; Ad, adenoviral; Ad-, empty Ad vector; Ad CTLA4-Ig, Ad vector encoding CTLA4-Ig; BM, bone marrow; CM, costimulatory molecule; EGFP, enhanced green fluorescent protein; ODN, oligodeoxyribonucleotide; FasL, Fas ligand; MOI, multiplicity of infection; MST, median graft survival time; mDC, mature DC; iDC, immature DC.

Received for publication April 26, 2002. Accepted for publication July 18, 2002.

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