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Prolongation of skin graft survival by modulation of the alloimmune response with alternatively activated dendritic cells1

Roelen, Dave L.2; Schuurhuis, Danita H.; van den Boogaardt, Daniëlle E. M.; Koekkoek, Karin; van Miert, Paula P. M. C.; van Ŝchip, Jolien J.; Laban, Sandra; Rea, Delphine; Melief, Cees J. M.; Offringa, Rienk; Ossendorp, Ferry; Claas, Frans H. J.

doi: 10.1097/01.TP.0000086340.30817.BA

Background.  Activation of immature dendritic cells (DC) in the presence of the glucocorticoid hormone dexamethasone (DEX) results in alternatively matured DC that present antigen in the absence of a proper co-stimulatory context. This maturation process is irreversible, making these cells an attractive potential tool for the induction of antigen-specific T-cell tolerance in vivo. The authors explored the possibility of using these DC for the induction of transplantation tolerance in a fully allogeneic setting in mice.

Methods.  Immature dendritic cells (D1, an immature splenic DC line derived from B6 mice) were pretreated with DEX for 24 hr, after which lipopolysaccharide or nothing was added to the culture for another 48 hr. These cells were analyzed for their in vitro and in vivo stimulating or tolerizing capacities.

Results.  In line with their phenotype, including decreased interleukin (IL)-12 production, in vitro co-culture of alternatively matured D1 (B6 origin; H-2b) with completely allogeneic T cells of BALB/c origin led to a significant decrease in the alloreactive T-cell response. A single injection of 1×106 alternatively matured H-2b DC into BALB/c mice induced a different alloimmune response compared with mature DC. The responding T cells showed a lower proliferation rate and a lower interferon-γ production, whereas a significantly higher proportion of the cells produced IL-10 as measured ex vivo by enzyme-linked immunospot assay. Furthermore, injection with alternatively matured DC, followed by transplantation of fully mismatched skin grafts (C57BL/6), led to a significantly prolonged survival compared with that of mature DC-pretreated mice or untreated mice. The immunomodulatory effect was antigen specific, as third-party reactive alloresponses were not affected.

Conclusions.  The authors’ data constitute the first direct demonstration that DC alternatively matured in the presence of glucocorticoid hormones can be exploited for the specific suppression of the alloreactive Th1 response, resulting in a delayed skin graft rejection in a complete major histocompatibility complex-incompatible strain combination.

Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands.

2 Address correspondence to: Dave Roelen, Ph.D., Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Albinusdreef 2, 2333 ZA, Leiden, The Netherlands. E-mail:

1 This work was supported by a grant from the Dutch Kidney Foundation, C.00.6011.

Received 14 February 2003.

Revision requested 16 April 2003.

Accepted 3 June 2003.

For patients with end-stage organ failure, organ transplantation often results in an extension and improvement of the quality of life. Because most transplantations concern partially human leukocyte antigen-mismatched donor-recipient combinations, it is necessary to suppress the host immune responses with potent immunosuppressive drugs to prevent rejection. However, long-term immunosuppression increases the risk for life-threatening infections and cancer, and patients receiving these drugs still experience chronic graft rejection. The alternative to immunosuppression would be the induction of donor-specific hypo- or nonresponsiveness. Pretransplant exposure to donor alloantigens seems a useful strategy, although it has already been half a century since Billingham et al. first demonstrated the possibility of transplantation tolerance induction (1).

The fact that dendritic cells (DC) are the most potent inducers of T-cell responses led to high interest for their clinical application to boost immune responses against tumors and viruses (2,3). A role for DC in tolerance induction was first recognized in the context of intrathymic self-tolerance (4,5). Thymic lymphoid-related DC are involved in the deletion of developing T cells with autoreactive potential (6). These DC can also influence mature T cells and therefore may be involved in the maintenance of peripheral tolerance.

Because antigen presentation without co-stimulation can lead to decrease of T-cell function, immature DC may be useful for the induction of transplantation tolerance because these cells show only moderate expression of major histocompatibility complex (MHC) class II molecules and no or low levels of co-stimulatory molecules. This is in contrast to mature myeloid DC with high expression of MHC class II and CD40, CD80, and CD86, which are prerequisites for their high T-cell–stimulating capacity. When properly activated through inflammatory stimuli or CD4+ T helper cells, mature DC present MHC-restricted antigen in a strong co-stimulatory context, resulting in activation of T-cell immunity. In their immature state, however, antigen presentation takes place in the absence of sufficient co-stimulatory signals. There are now multiple indications that immature DC function in the establishment of peripheral tolerance for autoantigens in vivo (4–6). Moreover, the tolerogenic properties of immature DC can be exploited for immune intervention strategies that aim at the induction of antigen-specific tolerance. For instance, bone marrow-derived co-stimulation–deficient DC progenitors were successful in inducing T-cell tolerance in vitro (7) and could even prolong allograft survival in vivo (8). Similar in vitro tolerogenic properties were found for DC that were inhibited in their maturation through treatment with immunoregulatory cytokines (interleukin [IL]-10 or transforming growth factor-β) (9–15), corticosteroids such as dexamethasone (16,17), or the biologically active metabolite of vitamin D3, 1α,25-dihydroxyvitamin D3 (18,19).

An essential risk of using in vitro prepared immature DC for antigen-specific tolerization in vivo is that in the host these DC may be exposed to inflammatory stimuli or CD4+ T helper cells that could trigger DC activation. As a result, such treatments may result in stimulation rather than suppression of the immune response of interest. Interestingly, we have recently demonstrated that in vitro treatment of immature DC with an activating trigger in combination with the glucocorticoid dexamethasone (DEX) results in DC maturation through an alternative maturation pathway, leading to DC capable of inducing Th1-type hyporesponsiveness in vitro (20). An essential aspect of this alternative maturation treatment is that it is irreversible. Consequently, the DC are fixed in their tolerogenic state and cannot be reconverted to immunostimulatory DC after application in vivo. By using an alternative activation of the murine DC cell line D1, we now demonstrate in a mouse transplantation model that such alternatively matured DC can successfully be exploited for the specific suppression of the alloreactive Th1 response.

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Female BALB/c (H-2d), C57BL/6 (B6; H-2b), and CBA/Ca (H-2k) mice were obtained from IFFA Credo (Paris, France). B6.C-H2bm1/ByJ (class I Kb mutant phenotype) were purchased from The Jackson Laboratories (Bar Harbor, ME). Mice were maintained under specific pathogen-free conditions and used at 6 to 10 weeks of age.

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Cell Lines

The D1 cell line, a long-term growth-factor–dependent immature splenic DC line derived from B6 mice, was cultured as described (21). Both floating and adherent D1 cells (detached using 2 mM EDTA) were collected and used.

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Treatment of DC

The D1 cells were pretreated with DEX, 10−6 M for 24 hr, after which lipopolysaccharide (LPS) or nothing was added to the culture for another 48 hr. D1 cells treated with LPS only (for 48 hr) were also used. Studies with lower doses (10−7 M and 10−8 M) of DEX revealed that the immature DC could almost mature normally after incubation with LPS (or FGK) and did not result in alternatively activated DC, whereas treatment of the D1 cells with higher doses of DEX (10−5 M and 10−4 M) resulted in an increase in D1 cell death. For this reason and because our human studies (20) also revealed an optimal dose of 10−6 M, we proceeded with this dose.

Both DEX and LPS (of Escherichia coli serotype 026:B6) were purchased from Sigma-Aldrich (St. Louis, MO). After treatment, supernatants were analyzed for the presence of IL-10 or IL-12.

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Antibodies and Cell Surface Immunofluorescence

The following antibodies (Ab) were purchased from PharMingen (San Diego, CA): fluorescein isothiocyanate-coupled anti-CD86 (B7.2), phycoerythrin (PE)-coupled anti-CD80 (B7.1), PE-coupled anti-CD40, and PE coupled anti–I-Ab/d (M5/114, MHC class II). Staining was carried out at 4°C for 30 min. Stained cells were analyzed using a FACScan flow cytometer equipped with CellQuest software (Becton Dickinson, San Jose, CA).

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Cytokine Analysis

Harvested supernatants were tested for IL-12 p40/p70, IL-10, or interferon (IFN)-γ content using a standard sandwich enzyme-linked immunosorbent assay (ELISA). Coating Ab consisted of rat anti-mouse IL-12 p40/p70 monoclonal Ab (mAb) (clone C15.6, PharMingen), rat anti-mouse IL-10 mAb (clone JES5-2A5, PharMingen), or rat anti-mouse IFN-γ mAb (clone R4-6A2, PharMingen). Detection Ab consisted of biotinylated rat anti-mouse IL-12 p40/p70 mAb (clone C17.8, PharMingen), biotinylated rat anti-mouse IL-10 mAb (clone SXC-1, PharMingen), or biotinylated rat anti-mouse IFN-γ mAb (clone XMG1.2, PharMingen). Streptavidin-horseradish peroxidase and ABTS (Sigma-Aldrich) were used as enzyme and substrate, respectively. OD405 was read by an ELISA reader (Wallac, Turku, Finland).

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Proliferation Assays

To study alloreactivity, splenocytes (0.5 or 1×105 cells/well) of BALB/c mice were co-cultured with irradiated (30 Gy, twofold dilutions from 2×104 cells/well) D1 cells or splenocytes (30 Gy, 1×105 cells/well). Immature DC pretreated with DEX (DEX), DEX-pretreated immature DC subsequently activated with LPS (DEX-LPS), and LPS-matured DC (LPS) were used as antigen-presenting cells in the stimulation assays. The cells were plated in U-bottom 96-well plates (Costar, Cambridge, MA) in Iscove’s modified Dulbecco’s medium (BioWhittaker, Walkersville, MD) containing 8% heat-inactivated fetal calf serum (Greiner, Alphen, The Netherlands), 100 IU/mL penicillin, 2 mM l-glutamine, and 20 μM 2-ME. Supernatant was harvested after 48 hr and stored at −20°C. Wells were pulsed with 1 μCi 3H-thymidine (Amersham International, Amersham, United Kingdom) and the cultures harvested onto glass fiber filters 18 hr later. Proliferation was measured as 3H-thymidine incorporation by liquid scintillation spectroscopy using a betaplate (Wallac).

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In Vivo Treatment with Modulated DC Analyzed by In Vitro Alloreactivity

After washing, 106 D1 cells were injected intravenously or subcutaneously in BALB/c mice in phosphate-buffered saline with 0.5% bovine serum albumin. After 7 days, spleen cells were used for detection of alloreactive cellular responses (proliferation and cytokine analysis) by in vitro stimulation with splenocytes from syngeneic mice (BALB/c), from donor mice (C57BL/6), or from third-party mice (CBA/Ca). The statistical significance of the differences between the several groups was analyzed using the Mann-Whitney test.

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Enzyme-Linked Immunospot Analysis

For enzyme-linked immunospot analysis (ELISPOT), 1×106 splenocytes (from in vivo treated mice) were incubated with 1×106 C57BL/6 splenocytes in 24-well plates (Costar) in Iscove’s modified Dulbecco’s medium containing 8% heat-inactivated fetal calf serum, 100 IU/mL penicillin, 2 mM l-glutamine, and 20 μM 2-ME. The cells were harvested and incubated (at 1 or 2×105 cells/well) with irradiated (3,000 rad) splenocytes (1×105 cells/well) from C57BL/6, medium, or concanavalin A controls during 24 hr in a plate (MAHA S45 10; Millipore, Billerica, MA) that was precoated with 5 μg/mL antibody (IFN-γ, R4-6A2; IL-10, JES5-2A5). Next, the wells were washed and the detection antibody was added at 0.3 μg/mL (IFN-γ, XMG1.2-biotin; IL-10, SXC-1-biotin) and incubated for 2 hr at room temperature. After another washing step, the conjugate (ExtrAvidin alkaline phosphatase, Sigma E2636) was added and incubated for 1 hr at room temperature. After washing, the substrate was added and incubated for 10 min at room temperature, after which the reaction was stopped with tap water. Analysis of spots was performed by using a BioReader 3000 Pro (BioSys, Karben, Germany).

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In Vivo Treatment with Modulated DC Analyzed by Skin Transplantation

After washing, 106 D1 cells were injected intravenously or subcutaneously into BALB/c mice in phosphate-buffered saline with 0.5% bovine serum albumin. After 7 days, mice underwent transplantation on the tail with skin grafts derived from the tail from donor mice.

The skin grafts were protected with a 4.5-cm-long glass pipe, which was kept on the tail for 7 days. Besides this protection, little irritation (and therefore inflammation) was observed because of the fact that the mice were kept on an individual basis in cages with a high-tech artificial bedding (Omega-Dri) instead of normal sawdust. Graft survival was followed by daily visual inspection. Scoring was performed by comparison with syngeneic grafts and was based on redness, crust-forming, and the presence of hairs. The grafts were scored as rejected when they were fully necrotic or fallen off. Statistical analysis was performed using the log-rank test.

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Characteristics of Alternatively Activated Dendritic Cells (Phenotype and Cytokine Production)

A typical fluorescence-activated cell sorter profile of the immature DC cell line D1 and the influence of DEX treatment and LPS triggering on these DC can be seen in Figure 1(A). DC matured with LPS showed significant increase of CD86, CD40, and MHC class II when compared with immature DC, whereas DC pretreated with DEX and subsequently matured with LPS (DEX-LPS) did not show increase of CD86 and only marginal increase of CD40 and in general a lowered expression of MHC class II. We investigated whether DEX affected the production of the proinflammatory cytokine IL-12. As shown in Figure 1(B), LPS triggering of immature DC strongly induced IL-12 (p40/p70) secretion. Combined treatment with DEX and LPS resulted in a strongly reduced (sevenfold) IL-12 production compared with LPS treatment alone, whereas DEX treatment only also resulted in a dramatically reduced IL-12 production. We explored the possibility that DEX-pretreated mature DC produced IL-10 on the basis of our previous findings with human DC (20). However, IL-10 production by these DEX-pretreated mature DC according to the followed protocol was below the detection level of the ELISA used.

Figure 1

Figure 1

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Impaired Stimulating Capacity of Alternatively Activated DC

The reduced IL-12 production by DEX-treated LPS-triggered DC (DEX-LPS) prompted us to assess the T-cell stimulatory capacity of these DC. As shown in Figure 2(A), proliferation of BALB/c (H-2d) splenocytes in a primary mixed leukocyte reaction response to stimulation with DEX-LPS DC (H-2b) was strongly reduced (and similar to the allogeneic response to untreated immature D1 cells). Similar striking differences of the allogeneic (major and minor histocompatibility antigens mismatched) response were observed when IFN-γ production of the BALB/c splenocytes in response to the various DC used as stimulator cells was measured (Fig. 2B). Besides DEX-LPS DC, the DEX-treated immature DC (DEX) also induced strongly reduced alloreactive responses as measured by IFN-γ production. Therefore, these results show that mature DC pretreated with DEX have an impaired stimulating capacity.

Figure 2

Figure 2

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In Vivo Reactivity Induced by Classic versus “Alternatively Activated” DC

To study the modulation of allospecific immunity of these DC in vivo, these in vitro pretreated cells were injected by means of two different routes, intravenously or subcutaneously. Spleen cells were harvested at different times after injection and restimulated with allogeneic splenocytes in vitro. Spleen cells from mice injected intravenously with mature DC (LPS) exhibited a high proliferative allogeneic response, which was higher than that of untreated control mice or of mice treated with immature DC, DEX-treated immature DC (DEX), or DEX-LPS DC (Fig. 3A). This difference could not be observed for the response against third-party splenocytes. Analysis of the production of IFN-γ by the splenocytes of mice injected with the different DC revealed that mice injected intravenously with the DEX DC showed IFN-γ production similar to the mice injected with DEX-LPS DC. This response was slightly higher compared with untreated control mice but significantly lower compared with mice injected with untreated immature DC or with LPS DC (Fig. 3B) (P =0.0286). The IFN-γ production in response to third-party splenocytes was low. The proliferative responses of spleen cells from mice injected subcutaneously with DEX-LPS DC exhibited a low proliferative allogeneic response, which was similar to that of untreated controls and to that of DEX DC (Fig. 4A). The allogeneic IFN-γ response after the DEX-LPS DC treatment was slightly higher or comparable to that of untreated controls but significantly reduced when compared with mature DC treatment, whereas the DEX DC induced a response similar to mice injected with untreated immature DC (Fig. 4B) (DEX-LPS DC vs. immature DC or vs. LPS DC, P =0.0286). The number of IFN-γ–producing cells as measured by ELISPOT analysis was four times lower after treatment with DEX-LPS DC than after treatment with the DEX DC but comparable to that of untreated controls (data not shown). When the splenocytes were in vitro stimulated with C57BL/6 alloantigens for 6 days and restimulated with concanavalin A or C57BL/6 splenocytes, the ELISPOT analysis showed an increase in the number of IL-10–producing cells when compared with untreated or with LPS DC-treated mice (Fig. 4C).

Figure 3

Figure 3

Figure 4

Figure 4

The third-party reactivity was not altered in the DC-treated mice compared with untreated mice, indicating that the treatment with alternatively activated D1 (H-2b) was specific for the H-2b alloantigens. These experiments demonstrate that DEX-LPS DC induce an alloimmune response which, on the basis of the in vitro parameters tested, showed both quantitative and qualitative differences compared with the alloimmune response found after injection with mature DC.

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Prolonged Skin Allograft Survival after Injection with Alternatively Activated DC

Subsequently, we analyzed the in vivo “modulatory” potential of the DEX-LPS DC in a fully allogeneic skin graft model. BALB/c mice were injected subcutaneously with LPS DC or DEX-LPS DC or left untreated. One week after treatment, these mice underwent transplantation with a skin graft derived from the tail of a donor C57BL/6 mouse. The skins derived from C57BL/6 mice were rejected by the mice injected with LPS DC, with a median survival time (MST) of 14 days, which is not significantly different from the survival in untreated mice (MST, 16 days) (Fig. 5A). However, when mice underwent transplantation after injection with DEX-LPS DC, a significantly prolonged allograft survival was found (MST, 34 days; P =0.039). A similar significant prolongation was observed in two other independent experiments using BALB/c mice as the responding strain (P =0.023 and P =0.009) and in another study using BM1 mice as the responder strain (P =0.008, data not shown).

Figure 5

Figure 5

The prolonged skin graft survival after treatment with alternatively activated D1 (H-2b) was specific for the H-2b alloantigens, as mice injected with DEX-LPS DC rejected skin grafts from DBA/1 mice (H-2q) in the same time (MST, 14 days) (Fig. 5B) as control mice (MST, 14 days; untreated or LPS DC-treated mice P =0.9 or P =0.92, respectively). These results show that the DEX-LPS DC are capable of inducing a specific prolongation of complete MHC-incompatible skin allograft survival.

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The present study in the mouse extends our in vitro observation with human DC to a clinically relevant in vivo setting (20). Delivery of a maturation signal (by LPS) after DEX pretreatment leads to an alternatively activated DC with in vitro and in vivo tolerogenic properties.

Usually, DC maturation is associated with changes in the phenotype and function of DC, including increase of MHC antigens, co-stimulatory molecules, and adhesion molecules; expression of chemokine receptors; and migration to the regional lymph nodes, where DC interact with recirculating T cells and initiate T-cell immunity (2,12,21–23). Glucocorticoids such as DEX can decrease DC function in vitro and in vivo, with concomitant reduction of surface expression of CD86 and reduction of T-cell proliferation (24). DEX influences the DC phenotype only when DC are pretreated by DEX before maturation, as was shown by several studies (16,17,20,25), and not when stimulation occurs before DEX treatment (26,27).

The present study shows that DEX affects the LPS-triggered maturation of mouse immature DC not only by preventing the increase of co-stimulatory and MHC molecules but also by blocking the IL-12 secretion of these cells. In contrast to human DC, murine alternatively activated DC do not produce detectable amounts of IL-10. Nevertheless, these DC are still able to decrease alloreactive T cells in vitro and to induce prolonged allograft survival (in vivo). Other immunoregulatory cytokines produced by these DC or the lack or decreased production of IL-12 may be critical factors for this immunomodulatory effect (26,27). Furthermore, we have recently observed increased IL-10 levels of DEX-LPS human DC when these DC are analyzed at an earlier time point after LPS addition (6 or 12 hr instead of 48 hr; D.L.R., unpublished data, 2003). Whether the same holds true for murine DC is currently under investigation.

The addition of DEX to immature DC results in a decreased proliferative response and a decrease in IFN-γ production by BALB/c splenocytes stimulated by these DC, which confirms findings of others (16). In addition, we demonstrate that in vivo treatment with DEX-pretreated mature DC decreased the allogeneic Th1 response as shown by a reduced IFN-γ production in vitro and a reduction in number of IFN-γ–producing effector cells when the response was compared with mice pretreated with mature DC. This was the case after subcutaneous or intravenous injection of the DEX-pretreated DC, but even more so after in vivo treatment with the alternatively activated (DEX-LPS) DC. Furthermore, the induction of IL-10–producing cells by the alternatively activated DC-treated mice suggests that a different population of T cells is triggered by this pretreatment. We have tested the production of IL-4, and the production of this cytokine was comparable in mice pretreated with DEX-LPS DC or LPS DC. The number of IL-4– and IL-5–producing cells was increased in the case of DEX-LPS DC when compared with LPS DC, but only marginally. However, the presence of these Th2 cells could have contributed to the observed rejection and had a counteracting effect on the beneficial effect of alternatively activated DC. Multiple injection of alternatively activated DC or prolonged time between injection and analysis did not seem to improve the results, as measured by ex vivo analysis of the Th1 response. We have noticed that a small population of the alternatively activated DC have a high expression of MHC class II, which might be “normally” matured DC with the capacity to induce an Th1 activation, thereby counteracting the beneficial effect of the alternatively activated DC.

Pretreatment of recipients with these DC leads to a significantly prolonged skin graft survival. This prolonged survival was observed in a fully MHC- and minor transplantation-mismatched combination. In future experiments, we will optimize the protocol in donor-recipient combinations with smaller histocompatibility differences and explore whether this pretreatment allows a reduction of the immunosuppressive drug dose after transplantation. Our finding that IL-10–producing cells are found after in vivo treatment with alternatively activated allogeneic DC is in agreement with the observation that DEX-pretreated LPS matured DC were also able to induce ovalbumin-specific “regulatory” T cells producing IL-10 (and no IFN-γ or IL-4) in T-cell receptor–ovalbumin transgenic mice (16). Also, human immature DC have been used for the induction of regulatory T cells. After repetitive stimulation by these DC, T cells are not able to proliferate anymore. This effect cannot be restored by restimulation with mature DC. Actually, these T cells are able to inhibit the responses of alloreactive Th1 cells to mature DC (28). For this inhibitory effect, these nonproliferating, IL-10–producing CD4+ T cells need cell-cell contact. The relevance of such regulatory T cells in humans, being CD4+ and CD25+, was reported in a series of simultaneously published articles (reviewed in Shevach 29). Currently, we explore whether the alternatively activated DC exert their suppression of alloreactive responses by directly anergizing alloreactive T cells or through the induction of regulatory T cells.

Alternatively, treatment of immature DC with 1α,25-dihydroxyvitamin D3 followed by LPS-induced maturation leads also to DC capable of inducing T-cell hyporesponsiveness in vitro (18–20). In a murine system, the combination of this treatment with mycophenolate mofetil led to tolerance with concomitant presence of CD4+CD25+ regulatory T cells (30). In a similar way, the generation of DC with low doses of granulocyte-macrophage colony-stimulating factor resulted in maturation-resistant immature DC that induced T-cell unresponsiveness in vitro and in vivo (as measured by prolonged cardiac allograft survival) (31).

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Our studies confirm and extend the practical use of alternatively activated DC for modulation of the alloimmune response and show that these can induce a prolonged skin graft survival even in a complete MHC-incompatible donor-recipient combination. Further studies should reveal whether the application of immature in vitro manipulated DC may become a suitable tool for the induction of transplantation tolerance in the clinical setting.

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