PHENOTYPE, FUNCTION, AND IN VIVO MIGRATION AND SURVIVAL OF... : Transplantation (original) (raw)
*Abbreviations: Ad, adenoviral; APC, antigen-presenting cell; BM, bone marrow; CTL, cytotoxic T lymphocytes; DC(p), dendritic cell (progenitor); ELISA, enzyme-linked immunosorbent assay; FITC, fluorescein isothiocyanate; GM-CSF, granulocyte-macrophage colony-stimulating factor; Ig, immunoglobulin; IL, interleukin; mAb, monoclonal antibody; MHC, major histocompatibility complex; MLR, mixed leukocyte reaction; MOI, multiplicity of infection; MST, median survival time; r, recombinant; TGF-β, transforming growth factor-β.
Bone marrow (BM*)-derived dendritic leukocytes are highly specialized migratory antigen-presenting cells (APC) that are essential for naive T-cell activation and proliferation (1,2). The potent T-cell stimulatory capacity of dendritic cells (DC) is attributed to their constitutive expression both of major histocompatibility complex (MHC) class II antigens and an array of accessory and costimulatory molecules, which includes CD40, CD54, CD58, CD80, and CD86 (1-3). There is also evidence that DC can play a role in the induction and/or maintenance of tolerance (4,5). Thus, DC have been shown to be important both in central tolerance (6-8) and in promoting peripheral or systemic tolerance in various experimental models (9-12). Immature, antigen-processing DC, resembling those present in nonlymphoid tissues and that are deficient in cell-surface expression of costimulatory molecules, can induce alloantigen-specific T-cell anergy in vitro (13) and prolong cell (14) or organ allograft survival (15). These "tolerogenic" DC progenitors (DCp) can be propagated from mouse BM in response to granulocyte macrophage colony-stimulating factor (GM-CSF) plus transforming growth factor-β (TGF-β) (16). In addition to inhibiting the functional maturation of DC generated in vitro, TGF-β has been implicated as a key factor in the regulation of tolerance (17) and the induction of immune privilege (18), including inhibitory effects of donor BM-derived cells (candidate "veto" cells) on antiallograft immunity (19).
Previously, we reported that systemic administration of donor-derived DCp could prolong MHC-mismatched cardiac allograft survival in nonimmunosuppressed recipients, although the grafts were rejected eventually (15). This attrition may be caused by the eventual in vivo maturation of these cells in an allogeneic environment after withdrawal of TGF-β and consequent loss of their tolerogenic properties. One approach to augmenting the tolerogenic potential of donor APC is to genetically engineer these cells to express immunosuppressive molecules. In this study, we used a replication-deficient adenoviral (Ad) vector to transduce donor BM-derived DCp with cDNA encoding TGF-β1. The influence of vector and gene transfer on DC phenotype and function was examined, and the in vivo migration and survival of these cells in unmodified allogeneic recipients was investigated. The findings clearly show that Ad-TGF-β transduction of immature DC antagonizes the stimulatory influence of the viral vector alone on DC function and that, compared with control gene (Ad-LacZ)-transduced DC, enhances and prolongs their survival in unmodified, MHC-mismatched recipients. These observations suggest that overexpression of TGF-β in donor DCp may be useful approach to augmenting their survival, restricting systemic effects of the immunosuppressive transgene product, and promoting potentially tolerogenic donor APC- host T-cell interactions.
MATERIALS AND METHODS
Mice. Male C57BL/10J (B10, H2b, IAb) and C3H/HeJ mice (C3H, H2k, IEk) were purchased from The Jackson Laboratory (Bar Harbor, ME). They were maintained in the specific pathogen-free facility of the University of Pittsburgh Medical Center, provided with Purina rodent chow and tap water ad libitum, and used at 8-12 weeks of age.
Propagation and purification of BM-derived DCp. BM cells harvested from femurs of normal B10 mice were cultured in 24-well plates (2×106/well) in 2 ml of RPMI-1640 (Life Technologies, Gaithersburg, MD), supplemented with antibiotics and 10% v/v fetal calf serum (referred to subsequently as complete medium), 4 ng/ml recombinant (r) mouse GM-CSF (Schering-Plough, Kenilworth, NJ), and 0.2 ng/ml r human TGF-β1 (R & D Systems Inc., Minneapolis, MN). The culture and selection procedures used to generate and purify these TGF-β-cultured DCp were as described previously in detail (16).
Flow cytometry. Cell surface molecule expression was analyzed extensively by cytofluorography using an EPICS ELITE flow cytometer (Coulter Corporation, Hialeah, FL). Staining with primary hamster or rat monoclonal antibodies (mAbs), including anti-DEC205 and anti-CD11c (directed against mouse DC-restricted antigens), and rat anti-mouse CD40, CD80, or CD86 (PharMingen, San Diego, CA) was followed by fluorescein isothiocyanate (FITC)-conjugated goat anti-hamster or goat anti-rat immunoglobulin (Ig)G2a, as described (20). MHC class II (IAb) staining was performed using biotin-conjugated mouse anti-mouse mAbs, with FITC streptavidin as the secondary reagent (21).
Quantitation of allogeneic T-cell proliferation and the generation of cytotoxic T lymphocytes (CTL). The capacity of graded numbers of γ-irradiated DCp infected with Ad-LacZ, Ad-d1703 ("empty" virus), or Ad- TGF-β1 to induce naive, resting, allogeneic T-cell proliferation, or cytotoxic T-cell activity was assessed in 72-hr, one-way mixed leukocyte reactions (MLR) or in 4- to 5-day CTL 51Cr-release assays, respectively, as described (20).
Ad vector-mediated gene transfer and expression in DCp in vitro. Ad-TGF-β1, an E1-deleted replication-deficient recombinant adenovirus carrying the cDNA of porcine TGF-β with a mutation of cysteine to serine at positions 223 and 225, was constructed (22). This mutation renders the expressed TGF-β1 biologically active (23). Ad-LacZ contains the β-galactosidase gene driven by a mouse cytomegalovirus immediate early promoter and terminated by the SV40 polyadenylation signal inserted into the E1 region of Ad5 using the BHG10 backbone described by Bett et al. (24). A control virus (empty virus) Ad-d1703 was constructed as previously described (24). All Ad vector were grown in 293 cells, banded by two rounds of CsCl density centrifugation, and purified over a Sephadex G-25 M column (Amersham Pharmacia Biotech, Piscataway, NJ). Harvested viral vectors were titered by plaque-forming assay on 293 cells. To assess the ability of the Ad vectors to transfer and express genes in DC in vitro or in vivo, BM-derived DCp were infected on day 5 with Ad-LacZ or Ad-d1703 at various multiplicities of infection (MOI). Twenty-four hours later, LacZ expression was assessed by incubating fixed slides (0.5% glutaraldehyde; 10 min; in 5 mg/ml 5-bromo-4-chloro-3-indolyl D-galactopyranoside; x-gal), 5 mM K3Fe (CN) 5; 5 mM K4 Fe (CN)6, and 1 mM MgCl2 in phosphate-buffered saline for 3 hr in the dark at 37°C. The efficiency of gene transfer was estimated by counting positive (blue) and negative cells.
Quantitation of TGF-β1 gene product. This was performed on cell culture supernatants by enzyme-linked immunosorbent assay (ELISA), using kits provided by R & D Systems (Minneapolis, MN) and following the manufacturer's instructions.
In vivo cell migration. Gene-transduced or control B10 (H2b; IAb) DCp were injected subcutaneously (5×105 cells in 50 µl) into one hind footpad of normal allogeneic recipients (C3H; H2k; IEk). At various times thereafter, groups of five mice were killed, and the draining popliteal lymph node, spleen, and thymus were 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 and then stored at -70°C until used.
Immunohistochemistry. Donor MHC class II+ cells were identified in 5 µm cryostat sections using biotinylated mouse IgG2a antimouse IAb (Pharmingen) in an avidin-biotin-peroxidase complex staining procedure, as described previously (20). Controls included sections of normal donor or recipient strain tissues. The incidence of donor MHC class II+ (IAb+) cells in sections was determined in a "blinded" fashion by ascertaining the mean number of positive cells per 100 high power fields (25).
Statistical analysis. Significances of differences between means were determined using the two-tailed Student's t test.
RESULTS
Surface immunophenotype of BM-derived cells (DCp) propagated in GM-CSF plus TGF-β1. Nonadherent cells were propagated from normal B10 mouse BM in GM-CSF and TGF-β1 to enrich for DCp as described under Materials and Methods and harvested at 5 days. They were stained for surface expression of lineage-restricted and other antigens using an extensive panel of mAbs, then analyzed by flow cytometry. As described previously (16), the cells were negative for lymphoid (including natural killer cell) markers but expressed the mouse DC-restricted antigens CD11c and DEC205 and MHC class II (IAb). They exhibited only low levels of CD40, CD80, and CD86 (Fig. 1) and moderate to high levels of F4/80, CD11b, and CD32 (FcγRII; data not shown). After withdrawal of TGF-β1 after 5 days in culture and maintenance of the cells for a further 48 hr in GM-CSF plus interleukin (IL)-4, up-regulated expression of cell surface DEC 205, CD40, CD80, and CD86 was observed, with concomitant down-regulation of macrophage-associated markers (F4/80, Cd11b, and CD32). In addition, there was a striking increase in allostimulatory activity of these cells, as demonstrated in primary MLR (data not shown). These findings are consistent with the TGF-β-cultured cells being DCp or immature DC (16,26), capable of differentiation into potent allostimulatory DC after withdrawal of the TGF-β and stimulation with GM-CSF plus IL-4.
Cell surface expression of costimulatory molecules and MHC antigens determined by flow cytometric analysis on B10 mouse BM-derived DCp propagated in GM-CSF plus TGF-β1 for 5 days, as described under Materials and Methods, then transduced with adenoviral vectors encoding LacZ (control gene) or TGF-β1. Staining was performed 48 hr after gene transduction. Open profiles denote isotype controls. The results are representative of five separate experiments.
Adenoviral transduction of DCp. To evaluate the ability of Ad vectors to transfer and express genes in DCp propagated in GM-CSF plus TGF-β1, the cells were infected with Ad-LacZ, and the incidence of cells expressing β-gal activity was determined 24 hr after infection. Naive and Ad-d1703 (empty virus)-infected cells were negative. Expression of β-gal was readily detected, however, in the majority (80%) of DCp infected with Ad-LacZ at MOI of 50-100, confirming that successful gene transfer and expression could be accomplished in these cells using an Ad vector. Concomitant studies, using other MOI, demonstrated 40% of cells expressing LacZ at a MOI of 20 and 10-15% at a MOI of 10. All subsequent studies were performed at a MOI of 50.
Detection of transgene product (TGF-β1) in cell culture supernatants. To determine whether transgene product could be detected in the culture supernatant of BM-derived DCp transduced with the TGF-β1 gene, supernatants were harvested from control (Ad-LacZ, Ad-d1703) and Ad-TGF-β-transduced cells 24 hr after exposure to virus at 50 MOI. By using an ELISA technique, accumulation of 10.8±1.8 (n=3) ng TGF-β1/ml/106 DCp/24 hr was detected in Ad-TGF-β-transduced cells, whereas TGF-β1 was not detected in control (Ad-LacZ or Ad-d1703) culture supernatants.
Cell surface phenotype and allostimulatory activity of TGF-β1-transduced DCp. Flow cytometric analysis was used to determine the influence of Ad-mediated gene expression in DCp on key cell surface molecules that play a role in T-cell allostimulatory activity. Analyses were performed 48 hr after gene transduction. Transduction with Ad-LacZ or Ad-TGF-β1 at 50 MOI did not significantly affect the expression of the DC-restricted antigens CD11c or DEC 205 or MHC class I or II. The intensity of expression of the costimulatory molecules CD40 and CD86, however, was marginally but reproducibly increased (Fig. 1). The stimulatory activity of the nontransduced and transduced DCp for naive, allogeneic splenic T cells was poor (compared with mature DC) although superior to that of bulk spleen cells over the range of stimulator cell numbers tested (Fig. 2). DCp transduced with Ad-LacZ showed enhanced allostimulatory activity, whereas this effect was suppressed in DCp transduced with Ad-TGF-β1 at 50 MOI that showed similar activity to nontransduced cells. This indicated that TGF-β transduction negated the modest enhancing effect of the Ad vector on DCp allostimulatory function. This effect was also observed with respect to CTL induction (Fig. 3). Transduction with the control Ad-LacZ gene increased the capacity of DCp to induce CTL, an effect that was prevented by the TGF-β transgene.
Allostimulatory activity for resting naive C3H splenic T cells of graded doses of γ-irradiated DCp propagated from B10 BM and transduced with Ad vectors. Mixed leukocyte cultures were set up and maintained for 72 hr, as described under Materials and Methods. The comparatively weak stimulatory activity of DCp grown in GM-CSF plus TGF-β1 (compared with mature DC) was increased by transduction with Ad-LacZ (50 MOI) before the start of the cultures. This effect of the viral vector was suppressed in Ad-TGF-β1-transduced DCp. The stimulatory activity of fresh bulk B10 and C3H (syngeneic control) spleen cells is also shown. Results are means±1 standard deviation and are representative of three separate experiments.
Cytotoxic activity of C3H splenic T cells stimulated for 5 days with either unmodified B10 DCp or DCp transduced with Ad-LacZ (control gene) or Ad-TGF-β1 (DCp: T-cell ratio=1:10) against target cells (EL4) expressing donor (B10) alloantigen (H2Kb). A 4-hr 51Cr-release assay was used to determine cytotoxicity. The activity of C3H T cells stimulated with bulk B10 spleen cells (1:1 ratio) is also shown. Results were calculated as described under Materials and Methods and are means±1 standard deviation. They are representative of three separate experiments.
In vivo migration and survival of Ad-TGF-β-transduced DCp in allogeneic recipients. To examine the in vivo migration and survival of the Ad vector-transduced DCp, 5×105 Ad-LacZ- or Ad-TGF-β-transduced B10 cells were injected subcutaneously into normal naive allogeneic (C3H) recipients. At various times after injection, donor-derived cells (β-gal+ or IAb+) were detected in recipient lymphoid tissues by enzyme- or immunohistochemistry, respectively (Fig. 4). β-gal+ or IAb+ (donor) cells were readily observed beneath the capsule of draining popliteal lymph nodes 24 hr after injection of the gene-transduced DCp. IAb+ donor-derived cells were reduced significantly in number in animals given Ad-LacZ-transduced DCp compared with mice injected with equivalent numbers of nontransduced or TGF-β-transduced DCp (Fig. 5A). IAb+ cells in lymph nodes fell rapidly between 24 and 48 hr after injection. In the spleen, higher numbers of IAb+ cells were detected per unit area compared with lymph nodes; numbers of donor cells were maximal at 24-48 hr (Fig. 5B) and then declined during the subsequent 2-week period after injection. Ad-LacZ transduction of DCp reduced the number of IAb+ cells detected in spleens compared with animals given control (nontransduced) DCp. On the other hand, Ad-TGF-β transduction significantly increased IAb+ cells compared with both nontransduced and, especially, with Ad-LacZ-transduced cells (6- to 7-fold increase) on day 14 postinjection. These findings are consistent with the enhanced T-cell proliferative and cytotoxic responses generated by Ad LacZ (control viral vector)-transduced allogeneic DCp in vitro and with prevention of this effect by the TGF-β transgene, which appears able to enhance the survival of these donor-derived cells compared with nontransduced controls.
Ad-TGF-β1 transduction of DCp does not affect their in vivo migratory ability and enhances their survival in unmodified allogeneic recipients. Bone marrow-derived DCp (B10) were transduced with either Ad-LacZ (control) or Ad-TGF-β1 and then injected subcutaneously (2×105) into one hind footpad of normal C3H recipients. One to 7 days later, donor-derived cells were detected in lymphoid tissue by enzyme histochemistry (x-gal staining) or by immunohistochemical staining for donor MHC class II (IAb), as described under Materials and Methods. (A), Ad-LacZ-transduced DCp stained 24 hr after infection for β-galactosidase activity to illustrate transduction efficiency (>80% cells transduced at 50 MOI); (B), x-gal, and (C), IAb staining of donor-derived cells (Ad-LacZ-transduced DCp) in draining popliteal lymph node 24 hr after cell injection; (D)-(F) detection of donor-derived (IAb+) cells in spleen, 7 days after subcutaneous footpad injection of (D), control nontransduced DCp, (E) Ad-LacZ-transduced DCp, and (F), TGF-β1-transduced DCp. Magnifications: (A), ×400; (B), ×400; (C-F), ×200.
Ad-TGF-β1 transduction of DCp enhances their survival in unmodified allogeneic recipients. Bone marrow-derived DCp (B10) was transduced with either Ad-LacZ (control) or Ad-TGF-β1 and then were injected subcutaneously (2×105) into one hind footpad of normal allogeneic (C3H) recipients. The numbers of donor-derived DC in (A) popliteal lymph nodes on day 1 or (B) in spleens on day 7 were determined by immunohistochemical staining for donor MHC class II (IAb), as described under Materials and Methods. Results are means±1 standard deviation obtained from three mice in each experimental group.
DISCUSSION
This study evaluates the use of an adenoviral gene transfer vector to deliver a potent immunoregulatory cytokine (TGF-β1) into immature DC (DCp) in vitro. It also examines the impact of genetic modification on the phenotype and allostimulatory activity of these APC and on their in vivo migration/survival after transplantation into unmodified allogeneic hosts. The data show that an Ad vector encoding TGF-β1 can effectively transfer and express the transgene in BM-derived DCp propagated in GM-CSF plus TGF-β1, a milieu that both we (16) and others (26) have shown arrests DC maturation and promotes the growth of immature, costimulatory molecule-deficient DCp that have tolerogenic properties (16). Transfer of the gene encoding TGF-β1 did not significantly alter the expression of MHC or costimulatory molecules on the cell surface but marginally reduced the modest allostimulatory activity of these cells, which was enhanced by the Ad vector alone (Ad-LacZ-transduced controls). In addition, the pattern of in vivo migratory activity of transplanted TGF-β1-transduced DCp in allogeneic hosts appeared to be unaffected. Notably, the number of these cells in recipient secondary lymphoid tissue (spleen) was markedly enhanced compared with control gene-transduced DCp. Moreover, these TGF-β1-transduced APC persisted with little diminution in numbers during the 2-week period after their injection.
These observations reveal the potential of genetically modified donor DCp to express an immunosuppressive transgene product previously linked with immunologic privilege (18,27) and tolerance induction (17,19). They also show the increased survival of these cells in nonimmunosuppressed, fully allogeneic recipients. This strategy, of genetic manipulation of donor APC with preexisting tolerogeneic potential to express an immunosuppressive molecule(s), may provide an effective novel approach for the therapy of cell or organ allograft rejection. In addition to TGF-β1, candidate molecules may include those already known to subvert DC-allogeneic-T-cell interaction, including IL-10 (28,29), the chimeric fusion protein cytotoxic T lymphocyte antigen 4 (CTLA4)-Ig (30), Fas ligand (L; CD95L; 30,31), nitric oxide (32), and potentially, cytokine antagonists or allogeneic MHC class I peptides.
Various approaches to gene therapy of allograft rejection and autoimmunity are presently under examination. These include donor MHC gene transfer to recipient thymus (33) or BM (34,35). Thus, retroviral constructs have been used for ex vivo transfer of MHC class I genes into recipient hematopoietic cells to confer specific hyporesponsiveness to skin allografts. Alternatively, transfection of donor organs with cDNA encoding immunosuppressive proteins (e.g., vIL-10, TGF-β, Fas L, or CTLA4-Ig; 36-39) has been used to significantly prolong graft survival. Genetic modification of autoreactive T cells to express IL-10 (40,41) or local delivery of IL-4 by retrovirus-transduced T cells (42) can ameliorate experimental autoimmune disease in mice. Transfer of a variety of genes, including the IL-1 receptor (R) antagonist (43), Fas L (44), soluble(s) IL-1R, or sTNFαR gene (45) to the site of inflammation ameliorates experimental antigen-induced arthritis.
Immature DC in peripheral sites capture and process antigens and have the capacity to migrate to lymphoid organs, home to T-dependent areas, and secrete cytokines (e.g., IL-12) to initiate immune responses (1-3). In addition to activating lymphocytes, they also have the ability to tolerize T cells to self-antigens, both in the thymus and periphery (4,5). The potential of DC and their progenitors to determine the balance between tolerance and immunity makes them powerful tools for therapeutic manipulation of undesired immune responses and attractive targets for genetic manipulation to improve allograft survival. By using a replication-deficient Ad viral vector to modify donor DCp in vitro, there is much less administration of potentially immunogeneic viral proteins, in contrast to direct in vivo gene delivery to organ allografts. Thus, this approach is likely to minimize the production of neutralizing antiviral antibodies, which preclude repeat administration of viral vectors (46,47). Of the gene transfer technologies available, gene delivery to DC by replication-defective Ad vectors is by far the most efficient. Thus, DC have been engineered to express IL-12 or tumor antigens for experimental cancer therapy (48-50) or viral antigens for the treatment of infectious disease, such as human immunodeficiency virus infection. To date, however, there have been no published reports of the properties of DC genetically engineered to express an immunosuppressive molecule. Recently, we have observed that retroviral delivery of vIL-10 to murine myeloid DC can strikingly inhibit their allostimulatory activity and confer the potential to induce allogeneic T-cell hyporesponsiveness (51).
There are several reasons why, in this study, the TGF-β1 gene was transduced to DCp deficient in costimulatory molecules and with potential tolerogenic activity. TGF-β is a potent immunosuppressive cytokine (52), shown to affect the differentiation and proliferation of hematopoietic progenitors, including the differentiation of granulocytes and macrophages induced by GM-CSF (53,54). Moreover, TGF-β blocks the maturation of GM-CSF-stimulated mouse BM-derived DC (16,26) and may maintain epidermal Langerhans cells at an immature stage in vivo (55). TGF-β has been strongly implicated as a key microenvironmental factor in tolerance induction. It exerts a variety of inhibitory effects on cells of the immune system, including macrophage deactivation, inhibition of T- and B-cell proliferation (52), and suppression of alloantigen presentation by cultured APC, including DC (56,57). In vivo, TGF-β prolongs rodent cardiac allograft survival (58) and has been implicated, acting on APC, in the generation of immune deviation and immunologic privilege (18,27). In addition and of special relevance to the present study is evidence that TGF-β may be responsible for veto cell-mediated induction of tolerance by allogeneic BM cells (17), suggested to be DCp (19).
In summary, we show that primary cultures of murine myeloid DCp can be adenovirally transduced to express the immunosuppressive cytokine TGF-β1. The transduced DCp generated by this method exhibit a similar cell surface phenotype to nontransduced DC, with minimal capacity to induce allogeneic T-cell proliferative or CTL responses. The enhancement of T-cell stimulatory activity associated with the Ad vector is prevented by expression of the TGF-β transgene. TGF-β1-transduced DC shows increased survival and persistence after transplantation into normal allogeneic hosts, compared with both nontransduced and control gene (Ad-LacZ)-transduced DCp. The findings are consistent with the view that genetic engineering of donor DCp to express an immunosuppressive molecule(s) may be a promising novel approach to therapy of cell or organ transplantation and possibly of other immune-mediated disorders.
Acknowledgments. The authors thank Jennifer Little for skillful assistance with cell culture, Alison Logar for flow cytometric analyses, Christy Bruton for enzyme immunoassays, and Shelly L. Conklin for secretarial support.
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**Section Description
The 17th Annual Meeting of the American Society of Transplant Physicians, May 9-13, 1998, Chicago, Illinois
© Williams & Wilkins 1998. All Rights Reserved.