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The Journal of Immunology, 2006, 177: 863-868.
Copyright © 2006 by The American Association of Immunologists

Targeted Lymphoid Homing of Dendritic Cells Is Required for Prolongation of Allograft Survival1

Kym R. Garrod*, Catherine K. Chang*, Feng-Chun Liu{dagger}, Todd V. Brennan*, Robert D. Foster{dagger} and Sang-Mo Kang2,*

* Transplantation Research Laboratory, Division of Transplantation, Department of Surgery, University of California, San Francisco, CA 94143; and {dagger} Division of Plastic Surgery, Department of Surgery, University of California, San Francisco, CA 94143


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Accumulating evidence that dendritic cells (DC) are important regulators of peripheral immune tolerance has led to the concept that donor-derived DC may be useful for inducing donor-specific transplantation tolerance. Although in vitro studies in this field have been encouraging, in vivo results have been inconsistent. Recent evidence has suggested a critical role of lymphoid organs in tolerance induction. In this study, we use a novel gene transduction technique to show that engineered expression of CCR7 on immature DC can markedly increase DC homing to lymphoid organs, leading to increased interaction with Ag-specific T cells. Moreover, we show that a single infusion of DC coexpressing CCR7 and the immunomodulatory molecule viral IL-10 (vIL-10) markedly prolongs cardiac allograft survival (mean survival time >100 days); importantly, DC expressing either vIL-10 alone or CCR7 alone was not effective. These results demonstrate an important paradigm for immune modulation using DC.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Evidence that steady state dendritic cells (DC)3 are important mediators of peripheral tolerance has led to the hypothesis that exogenous administration of donor-derived DC could be used to induce transplantation tolerance (1). Early studies using immature DC subsets showed promising effects in vitro, yet only modest improvements in allograft survival (1, 2, 3). The majority of subsequent studies have attempted to make DC more tolerogenic via genetic transduction of immunomodulatory genes. Although this approach has been highly effective in vitro, the results in vivo have been inconsistent. For example, donor-derived DC transduced with viral IL-10 (vIL-10), a potent immunomodulatory gene, have been shown in several studies to specifically down-regulate allospecific T cell responses in vitro (4, 5, 6), yet no study has demonstrated prolongation of allograft survival by vIL-10-transduced DC.

Mounting evidence supports the concept that T cell-DC interactions within secondary lymphoid organs are critical for the induction of both immunity and tolerance (7, 8). Notably, immature DC traffic poorly to lymph nodes (LN) (4, 9). We hypothesized that LN trafficking is critical to tolerance induction by DC, and that exogenously generated, immature DC could be targeted to LN via the engineered expression of the chemokine receptor, CCR7. To test this hypothesis, we developed a novel DC gene transduction technique that resolves two confounding factors in the study of genetically modified DC: low transduction efficiency and unintended maturation. Using this technique, we show that transduced CCR7 expression combined with endogenous L-selectin expression results in efficient trafficking of immature DC to the T cell zones of LN and spleen. Moreover, we show that a single pretreatment with donor-derived DC coexpressing an immunomodulatory gene, vIL-10, in conjunction with CCR7 results in striking prolongation of heart allograft survival in otherwise unmanipulated hosts. These results establish an important paradigm for tolerance induction using DC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

Male and female 6- to 12-wk BALB/c, C57BL/6 (CD45.2+), B6.SJL-Ptprca Pep3b/BoyJ (CD45.1+), C3H/HeJ, and CB6F1 (H2d/H2b, (BALB/c x C57BL/6)F1) mice were purchased from the National Cancer Institute. OT-II (OVA323–339 peptide-specific CD4 TCR-transgenic) mice were obtained from A. Weiss (University of California, San Francisco, CA). Mice were used in accordance with protocols approved by the animal care committee (Institutional Animal Care and Use Committee).

Vectors

All transgenes were cloned into a Moloney long terminal repeat-based vector using standard techniques (provided by G. Nolan, Stanford University, Stanford, CA) (10). A Flag-tagged CCR7 cDNA and the control truncated human CD4 (huCD4) cDNA (11) containing a deletion of the intracellular domain were gifts from J. Cyster (University of California, San Francisco, CA). The vIL-10 cDNA was provided by J. Bromberg (Mt. Sinai, NY). CCR7, huCD4, or GFP was placed in the second cistronic position when used in dual expression vectors using the encephalomyocarditis virus internal ribosome entry site element.

DC transduction

Transduction of cycling bone marrow (BM) cells was performed essentially as described by Pear et al. (12). Briefly, 6- to 10-wk-old mice were injected i.v. with 5 mg of 5-fluorouracil. Five days later, BM cells were harvested and cultured in medium containing IL-3, IL-6, and stem cell factor. BM cells underwent two rounds of spin transduction using retroviral supernatants and 8 µg/ml polybrene. Transgene+ cells were selected on a Cytomation cell sorter and cultured in DC differentiation medium (13) (IMDM with 10% FCS (HyClone), L-glutamine, 50 µM beta-ME, 7.5% NIH3T3 supernatant, and 20–30 ng/ml GM-CSF) with the addition of irradiated (3 Gy) syngeneic BM cells (2–5 x 105 transduced cells with 2.5 x 106 irradiated BM cells) in a 60-mm culture plate (Corning Glass). Cells were passaged every 2–3 days into 10-cm plates with 5 x 106 cells and 10 ml of DC medium/plate. DC were generally used 8 days after placement in DC medium.

Transplants

Cardiac allografts were transplanted heterotopically into the abdomen of C57BL/6 (H2b) recipients, as described (14). F1 donor hearts and DC were CB6F1 (H2d/H2b, (BALB/c x C57BL/6)F1). Third-party hearts were from C3H (H2k) mice. Rejection was defined by cessation of cardiac contractions for 2 consecutive days.

Abs and flow cytometry

Fluorochrome-labeled mAbs (I-Ab PE, Vbeta5.1, 5.2 TCR PE, CD11c PE, CD40 PE, CD62L PE, CD80 PE, CD86 PE, IFN-{gamma} allophycocyanin, SAV allophycocyanin, B220 FITC), CD45.2 biotin, Fc block (anti-CD16/CD32), anti-mouse IgD, Mel-14, and isotype controls were purchased from BD Pharmingen. Anti-Flag Ab (Sigma-Aldrich) was used to detect engineered CCR7-Flag-tagged expression. CCL19-Fc was a gift from J. Cyster. CCL19-Fc binding was detected with a biotinylated anti-human IgG (Jackson ImmunoResearch Laboratories), followed by SAV allophycocyanin, as previously described (11).

In vitro proliferation and assays

MLRs. A total of 1 x 105 CD4+ T cells from BALB/c mice was cocultured in a 96-well U-bottom plate with irradiated (3 Gy) DC for 60 h, and pulsed overnight with 1 µCi of [3H]thymidine.

OT-II responses. A total of 2.5 x 104 CD4+ T cells from spleen and lymph nodes of OT-II mice were cocultured in a 96-well U-bottom plate containing irradiated (3 Gy) transduced DC pulsed with 6.7 µg/ml OVA323–339 peptide (AnaSpec). After 48 h, supernatant was harvested for ELISA, and wells were pulsed with 1 µCi of [3H]thymidine for 6–8 h. IL-2 production in vitro was measured by ELISA (BioSource International).

Adoptive transfer experiments

CD4+ OT-II (CD45.2+) donor T cells were labeled with 2 µM CFSE (Molecular Probes) and injected i.v. into C57BL/6.CD45.1+ recipients. After 24 h, animals were challenged with 5 x 105 OVA323–339-pulsed DC given i.v. Spleen and LN were harvested at 72 h and analyzed.

In vivo DC migration

A total of 5 x 106 DC labeled with 5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (Molecular Probes) or CFSE (Molecular Probes) was injected i.v. into syngeneic B6 mice. Spleen and LN from recipients were harvested at 24 h and embedded in OCT compound (Sakura).

Lymph nodes. Frozen sections (6–8 µm) were stained with B220 FITC (BD Pharmingen).

Spleen. Frozen sections (6–8 µm) were fixed in acetone, dried overnight, and stained. Staining with sheep anti-FITC alkaline phosphatase (Roche) was detected using Fast Red (Sigma-Aldrich). B cell and T cell zones were identified by staining with anti-mouse IgD or anti-CD3 (BD Pharmingen), respectively, and detected with a secondary Ab conjugated with HRP (BD Pharmingen). Peroxidase acitivity was developed using 3–3' diaminobenizidine (Sigma-Aldrich). Tissue sections were counterstained in hematoxylin.

To quantify relative DC migration to lymphoid organs, congenic CFSE-labeled DC were adoptively transferred into Ly-5.1+ recipients, as above. Spleen and LN were finely minced, subjected to collagenase digestion (1 mg/ml collagenase II with 40 µg/ml DNase I (Worthington Biochemical)), stained with CD45.2 allophycocyanin, and analyzed by FACS. To examine the effect of L-selectin blockade on migration, DC were incubated with 150 µg of Mel-14 or isotype control for 15 min at 4°C before coinjection.

Statistics

MedCalc Statistics software (Mariakerke) was used. Student’s t test or log rank tests were used, as indicated in the figure legends. Area under the curves for Fig. 1C was calculated using the trapezoidal method.


Figure 1
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  		FIGURE 1. Phenotypic and functional characterization of GFP-transduced DC. A, CD11c expression of transduced cells. B, Two-color FACS for transgene expression (x-axis) and a panel of various markers (y-axis). Representative FACS plot from untreated (Imm, immature; upper panel) and LPS-treated DC (+LPS, lower panel). C, FACS histogram of MHC II (I-Ab) expression. Staining with isotype control Ab superimposed within histograms (thin line). GFP-transduced cells (left and middle panels) compared with nontransduced, bulk BM-derived DC (right panel). Cells analyzed in B and C are from independent experiments. D, MLR. Left panel, Immature (triangles) and LPS-matured (circles) GFP-transduced DC can stimulate allogenic T cells in vitro. *, Denotes p < 0.005 comparing the area under the curves between immature and LPS-matured DC using unpaired Student’s t test. Right panel, Nontransduced DC derived from bulk BM cultures used as stimulators. Untreated (diamonds) and LPS-treated (squares) DC. MLR using transduced and bulk DC were performed simultaneously using identical conditions.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Novel method for gene expression on DC

DC possess danger receptors that sense environmental changes and the presence of microbes (15). Gene transfer directly into DC has been shown to lead to maturation and acquisition of increased immunostimulatory properties (16, 17), potentially antagonizing tolerance induction. We sought to overcome this problem via retroviral transduction of BM precursors prior to their differentiation into DC. After transduction of BM stem cells with a retroviral construct expressing GFP, the transduced cells were purified on a MoFlo cell sorter. The purified cells were then placed into a standard myeloid DC propagation protocol (13) along with the addition of irradiated BM feeder cells.

The characteristics of the transduced DCs are shown in Fig. 1. After several days in culture, typical clusters of DC could be seen. Transduced cells expressed moderate levels of the DC marker CD11c (Fig. 1A), as previously reported for in vitro generated, myeloid DC (13). Greater than 95% of the DC were found to express the transgene. To assess the phenotype of these cells, FACS analysis was performed using typical markers of myeloid DC: MHC II (I-Ab), CD80, CD86, and CD40. This revealed a near homogenous population of relatively immature DC with low levels of MHC II, CD86, CD40, and constitutive expression of CD80 (upper panel, Fig. 1B). Overnight treatment with LPS (lower panel, Fig. 1B) increased the activation markers CD86, CD40, and moderately up-regulated MHC II (Fig. 1, B and C). In comparison, nontransduced DC from bulk BM cultures showed higher baseline levels of MHC II (Fig. 1C, right panel) with several apparent stages of maturity, as previously reported (18).

To confirm that the transduced cells retained functional properties of DC, they were tested for their ability to stimulate alloreactive T cells in vitro. Transduced cells were potent stimulators in an allogeneic MLR (Fig. 1D, left panel). Maturation of transduced DC with LPS resulted in a ~2- to 3-fold increased proliferation of T cells, similar to previous reports using nontransduced DC (19). Transduced DC were not as potent as nontransduced DC derived from bulk BM (Fig. 1D, right panel), consistent with an overall less mature phenotype. Notably, cells can be expanded greater than 1000-fold after transduction, yielding up to 100 x 106 transduced DC per donor mouse. Thus, we have developed a method for producing large numbers of immature DC with nearly uniform expression of a transgene.

Targeted delivery of immature DC to secondary lymphoid organs

Intravenously infused DC are known to migrate poorly to secondary lymphoid organs (4, 9). CCR7 and L-selectin have been identified as two key mediators of blood-borne migration of lymphoid cells to LN (20). CCR7 is not expressed on immature DC, but is rapidly up-regulated on mature DC (20). L-selectin is expressed on DC precursors, blood-borne DC, as well as some subsets of LN DC (21). FACS analysis of transduced, immature DC propagated in our protocol revealed endogenous expression of L-selectin (Fig. 2A). To test whether combined L-selectin and CCR7 expression could improve lymphoid homing, DC were transduced with a murine CCR7 construct carrying an N terminus Flag epitope (Fig. 2B). CCR7-transduced DC were able to bind CCL19 Fc, while control-transduced DC displayed no binding activity (Fig. 2C). Consistent with previous reports (4), transduced CCR7 appeared to be functional on DC, as evidenced by migration in response to CCL19 in vitro (data not shown). Control-transduced and CCR7-transduced DC expressed VLA-4 and LFA-1, as previously reported (13), but did not express CD103.


Figure 2
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  		FIGURE 2. Engineered expression of CCR7 facilitates immature DC migration to T cell zones of secondary lymphoid organs. CCR7-transduced DC were stained with anti-CD62L Abs (A), anti-Flag Abs (B), or CCL19 Fc (C), and analyzed by FACS. D, In vivo migration of 5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine-labeled, transduced DC (red) to lymph nodes after 24 h. B cell zones were identified by staining with B220 FITC (green). Immunofluorescence microscopy, magnification x20. E, In vivo migration of CFSE-labeled, transduced DC to spleen after 24 h. CFSE-labeled DC were identified by staining with sheep anti-FITC alkaline phosphatase and detected using Fast Red (red stain). B cell zones were identified with anti-IgD, or T cell zones were identified by anti-CD3 staining, as indicated (brown staining). Counterstained with hematoxylin. Control DC were transduced with a truncated huCD4 gene. F, FACS analysis of CFSE-labeled DC in spleen after 24 h. Infused DC identified by Ly-5.2 and CFSE staining. G, Relative fold increase in migration to spleen and lymph node of CCR7-transduced DC compared with control huCD4-transduced DC by FACS analysis. *, Denotes p < 0.05 for migration of CCR7 DC vs control DC using unpaired Student’s t test. H, Blockade of LN migration with anti-L-selectin Ab (Mel-14) or isotype control. CFSE-labeled DC expressing CCR7 were preincubated before coinjection with the Abs. Migration to LN was quantitated after 24 h by FACS. *, Denotes p < 0.05 compared with isotype control using unpaired Student’s t test.

 
We next tested whether CCR7 expression would enhance homing to secondary lymphoid organs. DC transduced with either CCR7 or a control gene (huCD4 lacking the intracellular domain) were labeled with intravital dye and injected i.v. into syngeneic mice. Twenty-four hours later, peripheral LN and spleen were assessed for the presence of migrated DC by immunofluorescence microscopy (for LN) or immunohistochemistry (for spleen). Few control-transduced DC could be seen in peripheral LN (Fig. 2D) or spleen (Fig. 2E). In contrast, CCR7-transduced DC were readily detected in both the LN and spleen (Fig. 2, D and E). CCR7-transduced DC were localized to the T cell zones of both the LN and spleen (Fig. 2, D and E), corresponding to known expression patterns of the CCR7 ligands, CCL19 and CCL21 (20, 22). Migrated DC could not be detected beyond 3 days (data not shown), consistent with the life span of most subtypes of endogenous DC (23) as well as previous kinetic studies of injected DC (9).

To rule out the possibility that the observed signal was the result of phagocytosis of infused DC fragments by host cells, we performed FACS analysis after infusion of CFSE-labeled, Ly-5.2 congenic DC. Essentially all of the CFSE-labeled cells were Ly-5.2+ (Fig. 2F), indicating that these were intact DC that had migrated to the LN after infusion. CCR7 expression appeared to increase localization to the spleen and LN ~10- and 40-fold, respectively, compared with controls (Fig. 2G). Migrated, CCR7-transduced DC comprised 0.10 ± 0.03% of the nucleated cells in the spleen. Previous estimates of endogenous DC in the spleen have ranged from 0.5 to 2% of nucleated cells (24). Migrated CCR7-transduced DC comprised 0.020 ± 0.006% of LN cells. This migration appears to be L-selectin dependent, as demonstrated by the ability of the anti-L-selectin Ab MEL-14 to substantially inhibit LN migration compared with an isotype control Ab (Fig. 2H). The effect of MEL-14 suggests that the DC are entering through the high endothelial venule of lymph nodes, rather than through a secondary migration via lymphatics after extravasation into the tissues. Thus, CCR7 transduction can significantly increase the migration of immature DC to secondary lymphoid organs.

vIL-10 transduction of DC

vIL-10 is an IL-10 homologue derived from the EBV with pleiotropic immunomodulatory effects on APCs as well as T and B lymphocytes (25). Several groups have shown that vIL-10-transduced DC could induce allospecific hyporesponsiveness in vitro (5, 6, 26). To test the impact of lymphoid homing on the effects of vIL-10-expressing DC in vivo, we generated DC-expressing vIL-10 with and without concomitant CCR7 expression. Supernatants of vIL-10-transduced DC contained 200–350 ng/ml vIL-10 at the end of the culture period. All transduced DC appeared to be phenotypically comparable, although DC expressing vIL-10 appeared to have slightly lower expression levels of MHC II, CD80, and CD40 than those expressing CCR7 alone (Fig. 3A). This finding is consistent with a previous report showing that vIL-10 helps to maintain DC in an immature state (4). As previously reported by others (5, 6, 27), vIL-10 expression by DC strongly down-regulated T cell proliferation (Fig. 3B) and IL-2 production (Fig. 3C) in vitro.


Figure 3
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  		FIGURE 3. Effect of vIL-10 on DC phenotype and T cell responses in vitro. A, DC transduced with CCR7 alone or coexpressing vIL-10 and CCR7 were analyzed for the expression of MHC II and costimulatory molecules by FACS. MFI, Mean fluorescence intensity. *, Denotes p < 0.05 using unpaired Student’s t test. B, The effect of vIL-10 expression on proliferation by OVA-specific OT-II T cells. A total of 2.5 x 104 OT-II cells incubated with indicated numbers of DC loaded with OVA323–339 peptide. CCR7-transduced DC, {diamondsuit}. vIL-10 + CCR7-transduced DC, {circ}. C, Effect of vIL-10 on IL-2 production in vitro. Supernatants from the proliferation assay in B were pooled and assayed for IL-2 production using ELISA. Results representative of three independent experiments.

 
Effects of lymphoid migration and vIL-10 expression on Ag-specific T cells in vivo

To define the effect of lymphoid migration on T cell responses in vivo, we used the well-characterized OT-II TCR transgenic system (28), which is responsive to an OVA peptide (OVA323–339) presented on I-Ab. A tracer population of OT-II T cells was adoptively transferred into congenic B6 mice. Twenty-four hours later, mice were treated with OVA323–339-pulsed DC expressing either control huCD4 alone, CCR7 alone, or vIL-10 plus CCR7. CCR7-mediated migration of pulsed DC greatly increased the proportion of OT-II cells undergoing division (Fig. 4A), demonstrating the importance of LN trafficking for the engagement of Ag-specific T cells, as well as the ability of the immature DC to engage naive T cells. However, vIL-10 coexpression with CCR7 appeared to suppress the proliferative response of cognate T cells (Fig. 4A) as well as the acquisition of an effector phenotype, as indicated by intracellular IFN-{gamma} staining of OT-II T cells (Fig. 4, B and C). Notably, this pattern of proliferation without the acquisition of effector function has been reported in the induction of tolerance to a model Ag by steady state resident DC in vivo (29).


Figure 4
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  		FIGURE 4. vIL-10 suppresses Ag-specific T cell-mediated immune responses in vivo. A, In vivo proliferative responses. Congenic mice were loaded with OT-II CD4+ CFSE-labeled T cells, and challenged 24 h later by i.v. injection of OVA323–339-pulsed DC. OT-II T cell expansion was determined by CFSE dilution, and the number in the upper right corner represents the stimulation index (total number of divided cells/total number of undivided cells). The No DC group received only an injection of saline. B, Induction of IFN-{gamma}-producing OT-II T cells in LN and spleen, as detected by intracellular cytokine staining after challenge with OVA-pulsed DC or saline, as in A. Representative data from one of three independent experiments. C, Relative induction of IFN-{gamma}-producing OT-II cells. Values expressed relative to induction by CCR7-transduced DC. *, Denotes p < 0.05 compared with CCR7 using unpaired Student’s t test. Data pooled from three independent experiments.

 
Targeted migration of immature DC to LN is critical for prolonging heart allograft survival

Although DC expressing vIL-10 have been reported to down-regulate alloresponses in vitro, they have not been shown to prolong allograft survival (5, 27). We hypothesized that this discrepancy between in vitro and in vivo results could be due to failed LN migration after i.v. injection. To test this hypothesis, we investigated the ability of DC expressing vIL-10 alone, CCR7 alone, or both vIL-10 and CCR7 to prolong heart allograft survival.

Transduced DC were tested for their ability to prolong allograft survival using an F1->parent (BALB/c x B6 (CB6F1)->B6) combination because of the ability of F1 DC to engage both the direct and indirect pathways of allopresentation. Simultaneous targeting of both the direct and indirect pathways has previously been shown to be critical to graft prolongation using immunomodulatory DC (30). Normal B6 mice were treated with a single i.v. injection of 0.5 x 106 F1 DC 1 wk before heart transplantation. No other immunosuppressants or immunomodulatory agents were used.

Immature, transduced DC expressing either vIL-10 alone or CCR7 alone were unable to prolong heart allograft survival (Fig. 5A). Strikingly, injection of F1 DC expressing both vIL-10 and CCR7 resulted in indefinite survival of F1 heart allografts in the majority of recipients (Fig. 5B). Thus, both an immunomodulatory and a lymph node-targeting gene are required for graft prolongation. The graft prolongation was allospecific, as third-party (C3H) graft survival was unaffected (Fig. 5B). The effect of vIL-10 on graft prolongation is likely to be related to local rather than systemic effects, evidenced by the need for lymphoid migration, as well as the inability to detect vIL-10 in the serum after DC infusion by ELISA. Syngeneic DC (B6) expressing both vIL-10 and CCR7 failed to prolong F1 heart survival in B6 recipients (mean survival time (MST) = 7.5 ± 0.6 days; n = 4), ruling out a nonspecific effect of the vIL-10-expressing DC. Notably, histologic examination of cardiac allografts after 100 days revealed a lymphocytic infiltrate characteristic of chronic rejection, and secondary F1 skin transplants after 100 days were rejected with normal kinetics (MST = 10 days; n = 3), indicating an incomplete induction of tolerance. Thus, targeted lymphoid migration of immunomodulatory DC expressing vIL-10 appears to be critical to preventing acute rejection and prolonging graft survival.


Figure 5
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  		FIGURE 5. Lymphoid targeting is required for prolongation of cardiac allograft survival by vIL-10-expressing DC. Kaplan-Meier survival analysis of F1 heart grafts in parental (B6) recipients receiving 0.5 x 106 F1 DC 7 days before transplant (A). Third-party graft depicted is C3H hearts after treatment with vIL-10 + CCR7 F1 DC. Cardiac allograft survival was significantly different (p < 0.005) only for the vIL-10 + CCR7 DC group (log rank test) (B).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Previous studies using genetically modified DC have generally reported transduction efficiencies of <50–60%, reflecting the difficulty of directly transducing DC. In this study, we report the development of a highly efficient methodology for the genetic manipulation of DC, resulting in nearly uniform expression of the transgene. An important advantage of this method is that it allows for the production of transduced DC with an immature phenotype. Notably, the presence of untransduced, immunostimulatory DC is likely to be antagonistic in tolerance studies. Indeed, in our system, the deliberate admixture of untransduced DC to simulate a transduction efficiency of 60% abrogated graft prolongation (MST = 11.5 ± 3.1 days; n = 4). Another important advantage of our methodology is the ability to generate large numbers of transduced DC from BM precursors. Thus, it will be a useful tool for studying DC biology, as well as for the genetic engineering of DC for manipulating immunogenicity. Although many obstacles remain before genetically modified DC can be applied in humans, our transduction strategy should be readily translatable to the clinical arena.

Our results clearly show that an immunomodulatory gene and lymphoid targeting of DC are critical to prolonging allograft survival in an otherwise normal host, defining an important concept for the design of DC tolerance induction strategies. Our findings add further support to the emerging paradigm that the lymphoid organs are critical for the priming of alloimmune responses (31), as well as to the development of tolerance (7, 32). These findings provide an impetus to re-examine prior studies using genetically or pharmacologically modified DC in the context of lymphoid homing, as an opportunity to improve in vivo efficacy.

CCR7 transduction improved homing to both the spleen and LN, consistent with patterns of CCR7-ligand expression. The relative importance of splenic vs LN homing of DC in prolonging cardiac allograft survival is not known. Although splenic migration of donor DC from the cardiac allograft is likely to be the dominant pathway (31, 33), it is clear that LN priming in the absence of a spleen is sufficient for robust cardiac allograft rejection (34). Thus, it is likely that targeting of both spleen and LN is important for preventing acute rejection.

Although the model immunomodulatory gene used for this study, vIL-10, dramatically prolonged cardiac allograft survival, it did not result in robust tolerance, as evidenced by the appearance of chronic rejection and the failure to prevent skin allograft rejection. This pattern of incomplete tolerance induction has been reported in a number of models, including anti-CD4 Ab treatment (35) and costimulation blockade (36). The differential susceptibility of heart and skin transplants is a well-established phenomenon that is not yet fully understood (37). Skin transplants can be rejected by CD8+ T cells alone (38, 39), CD4+ T cells alone (39), as well as CD4+ TCR transgenic T cells (40). In contrast, heart transplants are not rejected by CD8+ T cells alone (38, 39, 41) nor by CD4+ TCR transgenic T cells (40). Although some have speculated that the skin is more immunogenic due to the presence of skin-resident DC (Langerhans cells), other data have implicated graft size as an important variable (42). Moreover, it is clear that a small number of alloreactive T cells can reject a skin graft, while larger numbers are required for heart transplant rejection (37). Likewise, a pathway that is not able to cause acute rejection can mediate chronic rejection of heart allografts (42). In this context, our results suggest an incomplete modulation of alloreactivity with the approach used in this study. An additional possibility is that regulatory T cells are induced by vIL-10-expressing DC, which may then act in a site-specific manner (43) and would not necessarily protect against a subsequent skin transplant at a distant site. The results of our study provide a useful platform for further improvement of DC-based therapy via mechanistic studies as well as the exploration of alternate immunomodulatory genes, optimization of administration schemes, and evaluation of synergy with other immunomodulatory agents.


   Acknowledgments
 
We thank Jeffrey Bluestone and Abul Abbas for helpful comments, discussion, and critical review of the manuscript. We thank Cliff McArthur and Richard M. Locksley for assistance with cell sorting, as well as Tracy Hayden for expert technical assistance.


   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 by grants from the American Society of Transplant Surgeons (to C.K.C., T.V.B., and S.-M.K.) and the National Institutes of Health (DK61970; to S.-M.K.). Back

2 Address correspondence and reprint requests to Dr. Sang-Mo Kang, University of California, 513 Parnassus Avenue, Box 0780, San Francisco, CA 94143-0780. E-mail address: kangs{at}surgery.ucsf.edu Back

3 Abbreviations used in this paper: DC, dendritic cell; vIL-10, viral IL-10; BM, bone marrow; huCD4, human CD4; LN, lymph node; MST, mean survival time. Back

Received for publication February 17, 2006. Accepted for publication May 3, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Coates, P. T., A. W. Thomson. 2002. Dendritic cells, tolerance induction and transplant outcome. Am. J. Transplant. 2: 299-307. [Medline]
  2. Fu, F., Y. Li, S. Qian, L. Lu, F. D. Chambers, T. E. Starzl, J. J. Fung, A. W. Thomson. 1997. Costimulatory molecule-deficient dendritic cell progenitors induce T cell hyporesponsiveness in vitro and prolong the survival of vascularized cardiac allografts. Transplant. Proc. 29: 1310[Medline]
  3. Rastellini, C., L. Lu, C. Ricordi, T. E. Starzl, A. S. Rao, A. W. Thomson. 1995. Granulocyte/macrophage colony-stimulating factor-stimulated hepatic dendritic cell progenitors prolong pancreatic islet allograft survival. Transplantation 60: 1366-1370. [Medline]
  4. Takayama, T., A. E. Morelli, N. Onai, M. Hirao, K. Matsushima, H. Tahara, A. W. Thomson. 2001. Mammalian and viral IL-10 enhance C-C chemokine receptor 5 but down-regulate C-C chemokine receptor 7 expression by myeloid dendritic cells: impact on chemotactic responses and in vivo homing ability. J. Immunol. 166: 7136-7143. [Abstract/Free Full Text]
  5. Buonocore, S., S. Van Meirvenne, F. X. Demoor, F. Paulart, K. Thielemans, M. Goldman, V. Flamand. 2002. Dendritic cells transduced with viral interleukin 10 or Fas ligand: no evidence for induction of allotolerance in vivo. Transplantation 73: S27-S30. [Medline]
  6. Lu, L., W. C. Lee, T. Takayama, S. Qian, A. Gambotto, P. D. Robbins, A. W. Thomson. 1999. Genetic engineering of dendritic cells to express immunosuppressive molecules (viral IL-10, TGF-beta, and CTLA4Ig). J. Leukocyte Biol. 66: 293-296. [Abstract]
  7. Bai, Y., J. Liu, Y. Wang, S. Honig, L. Qin, P. Boros, J. S. Bromberg. 2002. L-selectin-dependent lymphoid occupancy is required to induce alloantigen-specific tolerance. J. Immunol. 168: 1579-1589. [Abstract/Free Full Text]
  8. Lakkis, F. G., A. Arakelov, B. T. Konieczny, Y. Inoue. 2000. Immunologic ‘ignorance’ of vascularized organ transplants in the absence of secondary lymphoid tissue. Nat. Med. 6: 686-688. [Medline]
  9. Lappin, M. B., J. M. Weiss, V. Delattre, B. Mai, H. Dittmar, C. Maier, K. Manke, S. Grabbe, S. Martin, J. C. Simon. 1999. Analysis of mouse dendritic cell migration in vivo upon subcutaneous and intravenous injection. Immunology 98: 181-188. [Medline]
  10. Kinsella, T. M., G. P. Nolan. 1996. Episomal vectors rapidly and stably produce high-titer recombinant retrovirus. Hum. Gene Ther. 7: 1405-1413. [Medline]
  11. Reif, K., E. H. Ekland, L. Ohl, H. Nakano, M. Lipp, R. Forster, J. G. Cyster. 2002. Balanced responsiveness to chemoattractants from adjacent zones determines B-cell position. Nature 416: 94-99. [Medline]
  12. Pear, W. S., J. P. Miller, L. Xu, J. C. Pui, B. Soffer, R. C. Quackenbush, A. M. Pendergast, R. Bronson, J. C. Aster, M. L. Scott, D. Baltimore. 1998. Efficient and rapid induction of a chronic myelogenous leukemia-like myeloproliferative disease in mice receiving P210 bcr/abl-transduced bone marrow. Blood 92: 3780-3792. [Abstract/Free Full Text]
  13. Winzler, C., P. Rovere, M. Rescigno, F. Granucci, G. Penna, L. Adorini, V. S. Zimmermann, J. Davoust, P. Ricciardi-Castagnoli. 1997. Maturation stages of mouse dendritic cells in growth factor-dependent long-term cultures. J. Exp. Med. 185: 317-328. [Abstract/Free Full Text]
  14. Corry, R. J., H. J. Winn, P. S. Russell. 1973. Heart transplantation in congenic strains of mice. Transplant. Proc. 5: 733-735. [Medline]
  15. Gallucci, S., P. Matzinger. 2001. Danger signals: SOS to the immune system. Curr. Opin. Immunol. 13: 114-119. [Medline]
  16. Morelli, A. E., A. T. Larregina, R. W. Ganster, A. F. Zahorchak, J. M. Plowey, T. Takayama, A. J. Logar, P. D. Robbins, L. D. Falo, A. W. Thomson. 2000. Recombinant adenovirus induces maturation of dendritic cells via an NF-{kappa}B-dependent pathway. J. Virol. 74: 9617-9628. [Abstract/Free Full Text]
  17. Salmon, P., J. F. Arrighi, V. Piguet, B. Chapuis, R. H. Zubler, D. Trono, V. Kindler. 2001. Transduction of CD34+ cells with lentiviral vectors enables the production of large quantities of transgene-expressing immature and mature dendritic cells. J. Gene Med. 3: 311-320. [Medline]
  18. Lutz, M. B., N. Kukutsch, A. L. Ogilvie, S. Rossner, F. Koch, N. Romani, G. Schuler. 1999. An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J. Immunol. Methods 223: 77-92. [Medline]
  19. Hertz, C. J., S. M. Kiertscher, P. J. Godowski, D. A. Bouis, M. V. Norgard, M. D. Roth, R. L. Modlin. 2001. Microbial lipopeptides stimulate dendritic cell maturation via Toll-like receptor 2. J. Immunol. 166: 2444-2450. [Abstract/Free Full Text]
  20. Cyster, J. G.. 2000. Leukocyte migration: scent of the T zone. Curr. Biol. 10: R30-R33. [Medline]
  21. Nakano, H., M. Yanagita, M. D. Gunn. 2001. CD11c+B220+Gr-1+ cells in mouse lymph nodes and spleen display characteristics of plasmacytoid dendritic cells. J. Exp. Med. 194: 1171-1178. [Abstract/Free Full Text]
  22. Moser, B., P. Loetscher. 2001. Lymphocyte traffic control by chemokines. Nat. Immunol. 2: 123-128. [Medline]
  23. Kamath, A. T., J. Pooley, M. A. O’Keeffe, D. Vremec, Y. Zhan, A. M. Lew, A. D’Amico, L. Wu, D. F. Tough, K. Shortman. 2000. The development, maturation, and turnover rate of mouse spleen dendritic cell populations. J. Immunol. 165: 6762-6770. [Abstract/Free Full Text]
  24. Crowley, M. T., K. Inaba, M. D. Witmer-Pack, S. Gezelter, R. M. Steinman. 1990. Use of the fluorescence activated cell sorter to enrich dendritic cells from mouse spleen. J. Immunol. Methods 133: 55-66. [Medline]
  25. Ding, Y., L. Qin, S. V. Kotenko, S. Pestka, J. S. Bromberg. 2000. A single amino acid determines the immunostimulatory activity of interleukin 10. J. Exp. Med. 191: 213-224. [Abstract/Free Full Text]
  26. Takayama, T., H. Tahara, A. W. Thomson. 2001. Retrovirally transduced myeloid dendritic cells expressing mammalian or viral IL-10 differentially affect cell-mediated immunity and growth of transplantable tumors. Transplant. Proc. 33: 590[Medline]
  27. Takayama, T., Y. Nishioka, L. Lu, M. T. Lotze, H. Tahara, A. W. Thomson. 1998. Retroviral delivery of viral interleukin-10 into myeloid dendritic cells markedly inhibits their allostimulatory activity and promotes the induction of T-cell hyporesponsiveness. Transplantation 66: 1567-1574. [Medline]
  28. Barnden, M. J., J. Allison, W. R. Heath, F. R. Carbone. 1998. Defective TCR expression in transgenic mice constructed using cDNA-based {alpha}- and beta-chain genes under the control of heterologous regulatory elements. Immunol. Cell Biol. 76: 34-40. [Medline]
  29. Hawiger, D., K. Inaba, Y. Dorsett, M. Guo, K. Mahnke, M. Rivera, J. V. Ravetch, R. M. Steinman, M. C. Nussenzweig. 2001. Dendritic cells induce peripheral T cell unresponsiveness under steady state conditions in vivo. J. Exp. Med. 194: 769-780. [Abstract/Free Full Text]
  30. Mirenda, V., I. Berton, J. Read, T. Cook, J. Smith, A. Dorling, R. I. Lechler. 2004. Modified dendritic cells coexpressing self and allogeneic major histocompatibility complex molecules: an efficient way to induce indirect pathway regulation. J. Am. Soc. Nephrol. 15: 987-997. [Abstract/Free Full Text]
  31. Lakkis, F. G., A. Arakelov, B. T. Konieczny, Y. Inoue. 2000. Immunologic ‘ignorance’ of vascularized organ transplants in the absence of secondary lymphoid tissue. Nat. Med. 6: 686-688. [Medline]
  32. Ochando, J. C., A. C. Yopp, Y. Yang, A. Garin, Y. Li, P. Boros, J. Llodra, Y. Ding, S. A. Lira, N. R. Krieger, J. S. Bromberg. 2005. Lymph node occupancy is required for the peripheral development of alloantigen-specific Foxp3+ regulatory T cells. J. Immunol. 174: 6993-7005. [Abstract/Free Full Text]
  33. Larsen, C. P., P. J. Morris, J. M. Austyn. 1990. Migration of dendritic leukocytes from cardiac allografts into host spleens: a novel pathway for initiation of rejection. J. Exp. Med. 171: 307-314. [Abstract/Free Full Text]
  34. Beckmann, J. H., S. Yan, H. Luhrs, B. Heid, S. Skubich, R. Forster, M. W. Hoffmann. 2004. Prolongation of allograft survival in ccr7-deficient mice. Transplantation 77: 1809-1814. [Medline]
  35. Pearson, T. C., C. R. Darby, A. R. Bushell, L. J. West, P. J. Morris, K. J. Wood. 1993. The assessment of transplantation tolerance induced by anti-CD4 monoclonal antibody in the murine model. Transplantation 55: 361-367. [Medline]
  36. Nathan, M. J., D. Yin, E. J. Eichwald, D. K. Bishop. 2002. The immunobiology of inductive anti-CD40L therapy in transplantation: allograft acceptance is not dependent upon the deletion of graft-reactive T cells. Am. J. Transplant. 2: 323-332. [Medline]
  37. Jones, N. D., S. E. Turvey, A. Van Maurik, M. Hara, C. I. Kingsley, C. H. Smith, A. L. Mellor, P. J. Morris, K. J. Wood. 2001. Differential susceptibility of heart, skin, and islet allografts to T cell-mediated rejection. J. Immunol. 166: 2824-2830. [Abstract/Free Full Text]
  38. Krieger, N. R., D. P. Yin, C. G. Fathman. 1996. CD4+ but not CD8+ cells are essential for allorejection. J. Exp. Med. 184: 2013-2018. [Abstract/Free Full Text]
  39. Youssef, A. R., C. Otley, P. W. Mathieson, R. M. Smith. 2004. Role of CD4+ and CD8+ T cells in murine skin and heart allograft rejection across different antigenic disparities. Transplant Immunol. 13: 297-304. [Medline]
  40. Sayegh, M. H., Z. Wu, W. W. Hancock, P. B. Langmuir, M. Mata, S. Sandner, K. Kishimoto, M. Sho, E. Palmer, R. N. Mitchell, L. A. Turka. 2003. Allograft rejection in a new allospecific CD4+ TCR transgenic mouse. Am. J. Transplant. 3: 381-389. [Medline]
  41. Pietra, B. A., A. Wiseman, A. Bolwerk, M. Rizeq, R. G. Gill. 2000. CD4 T cell-mediated cardiac allograft rejection requires donor but not host MHC class II. J. Clin. Invest. 106: 1003-1010. [Medline]
  42. He, C., S. Schenk, Q. Zhang, A. Valujskikh, J. Bayer, R. L. Fairchild, P. S. Heeger. 2004. Effects of T cell frequency and graft size on transplant outcome in mice. J. Immunol. 172: 240-247. [Abstract/Free Full Text]
  43. Graca, L., S. P. Cobbold, H. Waldmann. 2002. Identification of regulatory T cells in tolerated allografts. J. Exp. Med. 195: 1641-1646. [Abstract/Free Full Text]




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