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The Journal of Immunology, 2006, 177: 2175-2185.
Copyright © 2006 by The American Association of Immunologists

Induction of Allospecific Tolerance by Immature Dendritic Cells Genetically Modified to Express Soluble TNF Receptor1

Quanxing Wang2,*, Yushan Liu2,*, Jianli Wang{dagger}, Guoshan Ding*, Weiping Zhang*, Guoyou Chen*, Minghui Zhang*, Shusen Zheng{dagger} and Xuetao Cao3,*

* Institute of Immunology, Second Military Medical University, Shanghai, People’s Republic of China; and {dagger} Institute of Immunology and Center for Organ Transplantation, Zhejiang University, Hangzhou, People’s Republic of China


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The ability of dendritic cells (DC) to initiate immune responses or induce immune tolerance is strictly dependent on their maturation state. TNF-{alpha} plays a pivotal role in the differentiation and maturation of DC. Blockade of TNF-{alpha} action may arrest DC in an immature state, prolonging their window of tolerogenic opportunity. Immature DC (imDC) were transfected with recombinant adenovirus to express soluble TNF-{alpha} receptor type I (sTNFRI), a specific inhibitor of TNF-{alpha}. The capacity of sTNFRI gene-modified imDC (DC-sTNFRI) to induce immune tolerance was analyzed. sTNFRI expression renders imDC resistant to maturation induction and impairs their capacity to migrate or present Ag. This process leads to induction of allogeneic T cell hyporesponsiveness and the generation of IL-10-producing T regulatory-like cells. In vivo pretreatment of transplant recipients with DC-sTNFRI induces long-term survival of cardiac allografts in 50% of cases, and leads to a substantial increase in the generation of microchimerism and T regulatory cell numbers. Thus, blockade of TNF-{alpha} action by sTNFRI genetic modification can inhibit the maturation of DC and potentiate the in vivo capacity of imDC to induce donor-specific immune tolerance and prolong allograft survival.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Induction of alloantigen-specific tolerance is critical for the prolonged survival or permanent acceptance of allografts. Well-characterized mechanisms of peripheral tolerance include induction of programmed cell death in immune cells, development of T cell anergy, and active suppression by regulatory T cells (Treg)4 (1, 2). A promising approach that has emerged in recent years is the use of immature dendritic cells (imDC) to induce peripheral tolerance in allograft recipients (3). Unlike mature DC (mDC), which express high levels of MHC and costimulatory molecules on their surface and induce immune responses (4), imDC deficient in costimulatory molecules or DC in the steady state with the characteristic of regulatory DC have the potential to induce tolerance by inhibiting alloreactive T cell proliferation, inducing Ag-specific T cell anergy or triggering generation of Tregs (5, 6, 7, 8, 9). Systemic administration of donor-derived imDC or manipulation with immunosuppressive cytokines such as IL-10 and TGF-beta can significantly prolong the survival of allografts in non-immunosuppressed recipients (10, 11, 12). A major limitation of this approach is that imDC are likely to encounter inflammatory stimuli at some stage following infusion into recipients, triggering the terminal maturation of DC. Therefore, maintaining imDC in an immature steady state is critical for the induction of long-term immune tolerance in vivo (4).

TNF-{alpha} plays important roles in both DC maturation and migration (13, 14, 15, 16). Exogenous administration of TNF-{alpha} switches DC to a mature immunostimulatory state (16, 17, 18), whereas in the absence of TNF-{alpha}, DC remain in an immature state (13, 18). Produced by activated T cells, macrophages, and DC (19, 20), TNF-{alpha} acts as a potent proinflammatory cytokine involved in the pathogenesis of chronic local and systemic inflammation. Clinical trials of human TNF-{alpha} inhibitors have demonstrated a remarkable efficacy in controlling symptoms of autoimmune diseases. For example, infliximab (an anti-TNF-{alpha} Ab) and etanercept (a soluble TNF-{alpha} receptor (sTNFR)-Fc fusion protein) have been used to treat rheumatoid arthritis (21). Circulating levels of TNF-{alpha} are elevated during allograft rejection and may precede clinical manifestations (22). Prophylactic administration of an anti-TNF-{alpha} Ab has been shown to prolong allograft survival (23).

TNF-{alpha} binds to two independent cell surface receptors, type I (p55) and type II (p75) with TNF receptor I (TNFRI) being the major signal transducer (24, 25). TNFR1 and TNFR2 are also found as soluble receptors (sTNFRs) in the circulation. sTNFRs compete with membrane-bound receptors for available TNF-{alpha}, but cannot induce cell signaling, and thus function as specific inhibitors of TNF activity in target tissues (26, 27). sTNFRI and sTNFRII have both been effectively used to block TNF-{alpha}-mediated immunostimulation in the treatment of immunological disorders such as rheumatoid arthritis and experimental autoimmune encephalomyelitis (28, 29). However, it remains unclear whether blockade of TNF-{alpha} by sTNFRI can inhibit the maturation of DC and potentiate imDC tolerogenicity. In addition, there is no report to date documenting the immunomodulatory effects of immune cells and, in particular, imDC genetically modified to express sTNFRI. To address this, the sTNFRI gene was transfected into imDC by recombinant adenovirus (Ad), and the immunological characteristics of sTNFRI gene-modified imDC (DC-sTNFRI) were analyzed. The results show that DC-sTNFRI promotes tolerance induction and allograft acceptance.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice, cell lines, and reagents

Male C57BL/6 (H-2b), BALB/C (H-2d), and C3H (H-2k) mice, 8–10 wk of age, were purchased from Shanghai Joint Venture SIPPR BK Experimental Animal. All mice were maintained in a specific pathogen-free environment. ELISA kits for IL-2, IL-4, IL-10, IL-12 (p70), IFN-{gamma}, and sTNFR-p55 were purchased from Endogen and BioSource Europe. FITC- or PE-conjugated mouse mAb to CD4, Iab, CD11c, CD25(7D4), CD40, B220, CD45RB, CD69, CD86, CXCR4, and isotype control mAbs were purchased from BD Pharmingen, and PE-conjugated mouse mAbs to CCR7 were purchased from eBioscience. Microbeads-conjugated mAbs to CD4, CD11c, FITC, and PE, and Lineage Cell Depletion Kit were obtained from Miltenyi Biotec. Fluorescein-conjugated mAbs to IL-10 and TGF-beta and neutralization Abs to IL-10, TGF-beta, and isotype control mAbs were purchased from R&D Systems. The replication-defective recombinant Ad AdsTNFRI encoding sTNFRI was a gift from Dr. P. D. Robbin (Pittsburgh University, Pittsburgh, PA) (28). Generation of recombinant Ad harboring the LacZ reporter gene (Ad-LacZ) has been described previously (30). Ad titers were determined by PFU assay. Anti-CD25 (IL-2R{alpha}) mAb was purified by ammonium sulfate precipitation from PC61 hybridoma (American Type Culture Collection) ascites fluid produced in SCID mice. Rat IgG was used as a control (Sigma-Aldrich).

Genetic modification and phenotypic analysis of DC

Generation of imDC and mDC was performed as described previously (30, 31, 32). imDC were derived from bone marrow (BM) cell culture in the presence of GM-CSF (Sigma-Aldrich) at 2 ng/ml for 6 days and purified to >95% CD11c+ by positive immunomagnetic selection with anti CD11c-conjuagted MicroBeads (Miltenyi Biotec). mDC were generated by stimulating imDC with rTNF-{alpha} (500 U/ml) (Sigma-Aldrich) or LPS (1 µg/ml) (Sigma-Aldrich) for 2 days. imDC were transfected with AdsTNFRI or Ad-LacZ at a multiplicity of infection of 1:100 for 2 h in serum-free RPMI 1640, then washed twice, and cultured in RPMI 1640 supplemented with recombinant mouse GM-CSF. Transfected imDC were designated as DC-sTNFRI or DC-LacZ (imDC genetically modified with LacZ), respectively. Maturation responses were assayed by stimulating imDC, DC-sTNFRI, or DC-LacZ with LPS (1 µg/ml) or TNF-{alpha} (500 U/ml) for 24 h, then examining the expression of Iab, CD40, B7.2, CD25, and CD11c by FACS analysis. LPS-stimulated cell supernatants were collected for IL-12 measurement by ELISA. The phagocytic ability of DC was assessed by uptake of FITC-conjugated OVA at a final concentration of 100 µg/ml in RPMI 1640 containing 10% FCS at 37°C for 4 h, followed by FACS analysis. Cells incubated with OVA-FITC at 4°C were used as a negative control (33).

MLR and assay for allogeneic T cell hyporesponsiveness

CD4+ T cells were purified by autoMACS at a purity of >95% as determined by FACS. For MLR assay, CD4+ T cells (2 x 105/well) from BALB/c mice (H-2d) were cocultured with allogeneic DC-sTNFRI, or irradiated (30 Gy) imDC, DC-LacZ, or mDC, at various ratios, in complete RPMI 1640 for 3 days. T cell proliferation was measured by pulsing with [3H]thymidine (1µCi/well; Amersham Pharmacia Biotech) for the final 18 h of culture, and thymidine incorporation was determined using a liquid scintillation counter (Wallac). MLR supernatants were collected for cytokine detection.

To test T cell hyporesponsiveness, BALB/c splenic T cells (2 x 106/well) were first incubated with irradiated (30 Gy) imDC, DC-LacZ, DC-sTNFRI, or nonfixed DC-sTNFRI from C57BL/6 mice at a fixed stimulator:responder cell ratio (1:10) (primary MLR). After 3 days, residual cells were incubated with FITC-anti-H-2Kb (BD Pharmingen), followed by MACS anti-FITC microbeads to remove C57BL/6 cells. The BALB/c T cells were rested in culture medium for a further 2 days and then restimulated with irradiated (30 Gy) splenocytes from C57BL/6, BALB/c, or C3H mice for 72 h (secondary MLR). T cell proliferation was measured by pulsing with [3H]thymidine.

Analysis of Tregs in vitro and in vivo

CD4+ T cells were cultured with irradiated allogeneic imDC, DC-LacZ, or DC-sTNFRI at a stimulator:responder ratio of 1:10 in MLR for 5 days. The T cells were expanded in the presence of 50 U/ml IL-2 for an additional 10 days. These CD4+ T cells expressed high levels of CD25 and were regarded as Treg-like cells (34, 35). To study the regulatory effects of Treg-like cells, naive CD4+ T cells were cocultured with irradiated allogeneic mDC at a stimulator:responder ratio of 1:10 in the presence of different numbers of Treg-like cells for 5 days. In some experiments, anti-IL-10 and -TGF-beta neutralizing mAbs were added at the beginning of Treg-like cell/mDC/CD4 coculture. T cell proliferation was determined by [3H]thymidine incorporation. In some cases, CD4+CD25+ T cells were isolated by two-step autoMACS selection and used in a coculture system as above described.

Detection of DC chemokine receptor expression and migration

CCR7 and CXCR4 expression was analyzed by cytometry analysis. Chemotaxis analysis was performed in 24-well Transwell cell culture chambers (BD Biosciences) as described previously (36).

Allo-DTH assay

Allo-DTH assay was performed as reported by Matsue et al. (37) with modifications. BALB/c mice were immunized on the dorsal flank by s.c. inoculation of spleen cells (1 x 107 cells/animal) isolated from allogeneic mice (B6) on day 0 and 12, and challenged on day 7, 14, and 60 at the right hind footpad by injecting the same Ags (1 x 107 cells/animal). Footpad thickness was then measured on day 8, 15, and 61 with a calipers-type engineer’s micrometer by a third experimenter masked to the sample identity. The extent of swelling was calculated as the thickness of the right footpad (receiving B6 spleen cells) minus the baseline thickness of the left footpad (receiving phosphate-buffer saline). imDC, DC-LacZ, or DC-sTNFRI derived from B6 mice were injected i.v. (1 x 106 cells/injection/animal) on day –7, –4, 0, and 6. In some experiments, these animals were killed on day 15. CD4+ T cells isolated from the recipients (BALB/c mice) were labeled with CFSE (5 µM), cocultured with irradiated allogeneic mDC at a ratio of 20:1 for 3 days to analyze their response to alloantigen stimulation.

Heterotopic heart transplantation and skin transplantation

BALB/c mice were infused i.v. with 2 x 106 C57BL/6 donor-derived DC-sTNFRI, DC-LacZ, or imDC 7, 3, and 0 days before transplant. To provide a comparison between the effects of direct TNF-{alpha} blockade by secreted sTNFRI and those of DC-sTNFRI in vivo, 2 x 106 sTNFRI gene-modified L929 cells (L929-sTNFRI, H-2d), which secreted comparable levels of sTNFRI to DC-sTNFRI in vitro, were infused into separate recipients. On day 0, fully allogeneic cervical vascularized heart transplantation was performed from C57BL/6 or C3H (as third-party) donors to size-matched BALB/c recipients as described previously (12). Graft survival was assessed by daily palpation of grafted hearts, with rejection defined as total cessation of cardiac contraction. At indicated intervals, animals from sister groups were sacrificed for immunological analysis. Recipient mice with graft survival >100 days were grafted with allogeneic C57BL/6 and third-party C3H skin as described previously (38).

Statistical analysis

Statistical analysis of graft survival data was performed by lifetable methods, and group comparisons were made using log-rank test statistics.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Resistance of DC-sTNFRI to maturation induction

Our previous studies showed that the efficacy of Ad-mediated transfection of BM-derived DC was >95% (12, 30). First, the levels of sTNFRI in supernatants of gene-modified DC were determined. A high level of sTNFRI (19.3 ng/ml/5 x 105cells) was detected in the supernatant of DC-sTNFRI 12 h posttransfection with Ad-sTNFRI, peaking at 24 h (26.5 ng/ml/5 x 105cell; Fig. 1A), indicating effective gene transfer. Moreover, sTNFRI gene-modified DC cultured in medium containing GM-CSF continued to express a high level of sTNFRI for >14 days. As expected, sTNFRI was not detected in the supernatants of imDC or DC-LacZ.


Figure 1
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  		FIGURE 1. sTNFRI expression in DC-sTNFRI and resistance of DC-sTNFRI to LPS-induced maturation. Day 6 BM-derived imDC were purified and transfected with Ad-sTNFRI or Ad-LacZ at a multiplicity of infection 1:100. A, Supernatants from DC-sTNFRI, DC-LacZ, and imDC cultures were measured for sTNFR-p55 by ELISA. Data are presented as mean sTNFR(p55) concentration (ng/ml/5 x 105cells) ±SD of three independent experiments performed in triplicate. B, DC-sTNFRI, DC-LacZ, or imDC were cultured for 24 h in the absence or presence of LPS (1 µg/ml), or TNF-{alpha} (500 U/ml), then stained with FITC- or PE- anti-Iab, -CD40, -CD86, and -CD25 Abs for FACS analysis. Numbers in each quadrant represent percentage of positive cells. Results shown are representative of three independent experiments. C, Supernatants from B were assayed for IL-12 secretion. Results represent the mean ± SEM of four independent experiments. (*, p < 0.01; brackets indicate groups being compared.) D, Chemokine receptor expression of DC with or without LPS stimulation was detected by FACS analysis. Values showing percentage of positive cells measured over surface are indicated by horizontal bars. Results shown are representative of three independent experiments. E, Chemotaxis assay. LPS (1 µg/ml)-stimulated DC-sTNFRI, DC-LacZ, or imDC were loaded into upper chemotaxis chambers. Cells migrated through the filters into the lower chambers that contained MIP-3beta (200 ng/ml) or medium alone were counted by flow cytometry. Each assay was performed in triplicate. Error bars represent SEM (*, p < 0.05).

 
Next, the effect of sTNFRI gene expression on DC maturation was examined. Like imDC, both DC-sTNFRI and DC-LacZ expressed lower levels of CD40, CD86, and Ia, and a high level of CD11c, but hardly expressed CD25 (Fig. 1B). No significant phenotypic differences were observed among DC-sTNFRI, DC-LacZ, and imDC, demonstrating that sTNFRI gene expression itself does not induce significant phenotypic changes in imDC. Furthermore, the immature phenotype of DC-sTNFRI was little changed by stimulation of LPS or TNF-{alpha}, well-characterized inducers of DC maturation (18, 19). In contrast, imDC and DC-LacZ expressed high levels of MHC class II, CD40, and CD86 upon TNF-{alpha} or LPS stimulation (Fig. 1B). In addition, whereas LPS-stimulated imDC and DC-LacZ secreted high levels of IL-12, DC-sTNFRI expressed low levels of this maturation-associated cytokine (Fig. 1C). Together, these results suggest that sTNFRI gene expression inhibits LPS-mediated maturation of imDC.

DC migratory capacity is closely related to DC maturation. To analyze the migratory capacity of DC-sTNFRI, the expression of the maturation up-regulated chemokine receptors CXCR4 and CCR7 were measured using FACS analysis. Like imDC, both DC-sTNFRI and DC-LacZ expressed low levels of CXCR4 and CCR7. After LPS stimulation, both imDC and DC-LacZ expressed high levels of CXCR4 and CCR7, whereas DC-sTNFRI displayed a modest increase in CCR7 expression, but retained low levels of CXCR4, suggesting that CCR7 and CXCR4 expression on DC-sTNFRI were inhibited after LPS stimulation (Fig. 1D). The chemotactic responses of DC-sTNFRI to MIP-3beta were then evaluated. Upon LPS stimulation, both imDC and DC-LacZ exhibited marked migratory responses to MIP-3beta, but DC-sTNFRI exhibited comparatively weak MIP-3beta-induced migration (Fig. 1E). These results further indicate that sTNFRI gene expression inhibits DC maturation, in this instance interfering with up-regulation of DC chemokine receptors.

Impaired Ag-presenting capacity of DC-sTNFRI

To examine DC phagocytic ability, purified imDC, DC-LacZ, or DC-sTNFRI were incubated with FITC-conjugated OVA and analyzed by FACS. Both imDC and DC-sTNFRI displayed high phagocytic ability (Fig. 2A); however, the phagocytic ability of imDC or DC-LacZ decreased after LPS stimulation. In contrast, DC-sTNFRI exhibited high phagocytic ability like imDC after LPS stimulation. The results imply that DC-sTNFRI restricts LPS-induced maturation.


Figure 2
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  		FIGURE 2. Phagocytic ability and Ag-presenting capacity of DC-sTNFRI. A, The phagocytic ability of imDC, DC-LacZ, and DC-sTNFRI was tested by measuring OVA-FITC phagocytosis using FACS. Filled histograms represent imDC, DC-LacZ, and DC-sTNFRI or LPS-treated DCs (lower line) cultured with 100 µg/ml OVA-FITC at 37°C. Shown are geometric mean fluorescence values. Open histogram represents cells cultured with 100 µg/ml OVA-FITC at 4°C for 2 h. Data are representative of three independent experiments. B, CD4+ T cells (2 x 105/well) from BALB/c mice (H-2d) were cocultured for 3 days with different numbers of irradiated allogeneic mDC, imDC, DC-LacZ, and DC-sTNFRI (H-2b). Results represent the mean ± SEM of three independent experiments (*, p < 0.01). C, Expression of CD25 and CD69 on CD4+ T cells stimulated by mDC, imDC, DC-LacZ, or DC-sTNFRI in 3-day allo-MLR were tested by FACS. One representative experiment of three is shown.

 
The allostimulatory activities of mDC, imDC, DC-LacZ, and DC-sTNFRI in allogeneic MLR were compared. mDC, imDC, DC-LacZ, and DC-sTNFRI were cocultured with allogeneic CD4+ T cells from BALB/c mice for 3 days. DCs demonstrated different levels of capacity to induce T cell proliferation, with mDC having the highest and DC-sTNFRI the lowest capacity. mDC potently provoked the proliferation of T cells. Compared with mDC, imDC was less able to stimulate the proliferation of CD4+ T cells in 3-day allogeneic MLR (p < 0.05), whereas DC-sTNFRI did not significantly promote T cell proliferation (Fig. 2B). The activation of CD4+ T cells after coculture with DC-sTNFRI in MLR was analyzed (Fig. 2C). DC-sTNFRI also stimulated relatively lower expression of activation marker CD25 and CD69 in T cells when compared with imDC. These results suggest that DC-sTNFRI resists maturation and exhibit a compromised allostimulatory activity in vitro.

Induction of allogeneic T cell hyporesponsiveness and IL-10-producing T regulatory-like cells in vitro by DC-sTNFRI

The ability of DC-sTNFRI to induce T cell hyporesponsiveness to alloantigenic restimulation was investigated. Although CD4+ T cells from BALB/c mice primed with imDC or DC-LacZ in primary MLR could expand significantly in response to stimulation with C3H splenocytes (third-party), their response to restimulation with C57BL/6 splenocytes in secondary MLR was weak (Fig. 3A). However, this T cell-hyporesponsiveness could be partially reversed by adding exogenous IL-2 (Fig. 3B). In contrast, the responses of T cells that were exposed to DC-sTNFRI in primary MLR and to C57BL/6 splenocytes in secondary MLR were significantly weaker than those of imDC or DC-Lac-Z-primed T cells, although the T cells did retain the capacity to proliferate in response to third-party C3H splenocytes (Fig. 3C). Moreover, this DC-sTNFRI-induced alloantigen -specific T cell hyporesponsiveness could not be reversed by adding exogenous IL-2 (Fig. 3B). These results indicate that DC-sTNFRI is able to induce donor-specific T cell hyporesponsiveness in vitro.


Figure 3
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  		FIGURE 3. Induction of allogeneic T cell hyporesponsiveness by DC-sTNFRI. BALB/c splenic CD4+ T cells (2 x 106/well) were incubated at a fixed stimulator:responder cell ratio (1:10) with irradiated allogeneic imDC, DC-LacZ, or DC-sTNFRI (H-2b) in primary MLR. After 3 days, DCs were removed by autoMACS selection of H-2Kb+ cells. The CD4+ T cells were rested in culture medium for a further 2 days, and then restimulated with irradiated C57BL/6 (H-2b, allogeneic) splenocytes (A), or plus 20 U/ml IL-2 (B), or C3H (H-2k, third-party) splenocytes (C) for 72 h (secondary MLR) at different stimulator:responder cell ratio. Cell proliferation was determined by [3H]thymidine uptake. Results represent the mean ± SEM of three independent experiments (*, p < 0.05), D and E, Cytokine production in allogeneic MLR. CD4+ T cells (2 x 105/well) from BALB/c mice were cocultured with irradiated allogeneic DC-sTNFRI, DC-LacZ, or imDC from C57BL/6 mice at different ratios. After culture for 5 days, the supernatants were assayed for production of IL-2, IFN-{gamma} (D), IL-4, and IL-10 (E) by ELISA. Data represent the mean ± SD of three independent experiments performed in triplicate.

 
The cytokine profiles of CD4+ T cells were analyzed after stimulation with DC in allo-MLR supernatants. High levels of IFN-{gamma} and IL-2 were detected in the supernatants of allo-MLR stimulated by mDC, whereas the level of IL-10 was very low (Fig. 3, D and E). In contrast, supernatants of allo-MLR stimulated with imDC or DC-LacZ contained high levels of IL-10 but low levels of IFN-{gamma}. Importantly, the highest levels of IL-10 were found in allo-MLR cultures stimulated with DC-sTNFRI (p < 0.05). However, there were no marked changes in the production of IL-4 in allo-MLR stimulated with imDC or DC-sTNFRI. These results indicate that both imDC and DC-sTNFRI possess the ability to polarize CD4+ T cells to the Th2 cell subset and can induce the generation of IL-10-producing T cells.

The IL-10-producing Treg-like cells induced by DC-sTNFRI were characterized further. CD4+ T cells (H-2d) were cultured with irradiated allogeneic imDC or DC-sTNFRI (H-2b) in MLR. Five days later, cells were expanded in the presence of 50 U/ml IL-2 for an additional 10 days. Intracellular cytokine staining indicated that CD4+ T cells cocultured with DC-sTNFRI expressed higher levels of IL-10 and TGF-beta than T cells cocultured with imDC or mDC after 5 days in MLR (Fig. 4A). Furthermore, these cells became CD25+, CD45RBlow, and CD69low following expansion in a low concentration of IL-2 (Fig. 4B). High levels of IL-10 and TGF-beta were also found in the supernatant of DC-sTNFRI and CD4+ T cells coculture (Fig. 4C). Therefore, these T cells are regarded as Treg-like cells. Interestingly, Treg-like cells induced by imDC could significantly inhibit the proliferation of syngeneic CD4+ T cells in response to allogeneic Ag (B6-derived mDC). Treg-like cells induced by DC-sTNFRI displayed the most potent inhibitory activity (Fig. 4D). However, Treg-like cells induced by either imDC or by DC-sTNFRI could not inhibit the proliferation of syngeneic CD4+ T cells in response to third-party allogeneic Ag, indicating that the suppressive effect of Treg-like cells is allospecific (Fig. 4C). Moreover, an Ab-blocking assay identified that TGF-beta was involved in the inhibitory function of Treg-like cells induced by DC-sTNFRI (Fig. 4E). These results demonstrate the capacity of DC-sTNFRI in the induction of Treg-like cells.


Figure 4
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  		FIGURE 4. Induction of IL-10-producing T regulatory-like cells in vitro by DC-sTNFRI. A, Intracellular cytokine staining of CD4+ T cells in allo-MLR. CD4+ T cells were stimulated with allogeneic DC-sTNFRI or irradiated imDC and DC-sTNFRI at a ratio of 10:1 for 5 days and stained for IL-10 and TGF-beta. One representative experiment of three is shown. B, After culture for 5 days, Treg-like cells in allogeneic MLR (DC:T cell ratio of 1:10) described above were expanded in the presence of 50 U/ml IL-2 for a further 10 days, and the phenotype of the cells was analyzed. Numbers in each quadrant represent percentage of positive cells. C, The supernatants were assayed for production of IL-10 and TGF-beta by ELISA. Data represent the mean ± SD of three independent experiments performed in triplicate (p < 0.01). D, Isolated Treg-like cells were cocultured with different numbers of syngeneic CD4+ T cells, in the presence of irradiated allogeneic mDC (from C57BL/6 mice or C3H mice), at a stimulator:responder ratio of 1:10. Results represent the mean ± SEM of three independent experiments (*, p < 0.05). E, The inhibitory function of Treg-like cells was determined after blocking of IL-10 and TGF-beta. Anti-IL-10 or anti-TGF-beta-neutralizing mAbs were added at the beginning of Treg-like cell/mDC/CD4 coculture. On day 5, CD4+ T cells proliferation was determined by [3H]thymidine incorporation. Results represent the mean ± SEM of three independent experiments (*, p < 0.05).

 
Induction of T cell hyporesponsiveness in vivo by DC-sTNFRI

The ability of DC-sTNFRI to induce T cell hyporesponsiveness in vivo was investigated by allo-DTH assay. BALB/c mice immunized with B6 spleen cells exhibited marked DTH responses upon challenge (Fig. 5A). This DTH response was inhibited significantly by repeated i.v. injections of DC-sTNFRI. In three independent experiments, DC-sTNFRI induced 80–95% inhibition of mouse footpad swelling, whereas imDC or DC-LacZ injected in the same protocol caused partial inhibition. Moreover, DC-sTNFRI suppressed DTH responses of BALB/c mice to B6 spleen cells after the first rechallenge on day 7, the second rechallenge on day 14, and even after the third rechallenge on day 60. To evaluate the level of alloresponsiveness, CD4+ T cells isolated from the recipients (BALB/c mice) were labeled with CFSE (5 µM) and cocultured with irradiated allogeneic mDC for 3 days to analyze their response to alloantigen stimulation. CD4+ T cells from DC-sTNFRI-treated recipients also exhibited lower responsiveness to alloantigen stimulation than those from imDC- or DC-LacZ-treated recipients (Fig. 5B). These results suggest that i.v. infusion of DC-sTNFRI potently inhibits allo-DTH and can induce T cell hyporesponsiveness in vivo.


Figure 5
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  		FIGURE 5. Induction of T cell hyporesponsiveness in vivo by DC-sTNFRI. A, BALB/c mice (10 mice/experiment) received i.v. injection of imDC, DC-LacZ, o r DC-sTNFRI from B6 mice (1 x 106 cells/injection) on day –7, –4, 0, and 6, and were immunized on the dorsal flank by s.c. inoculation of allogeneic spleen cells (1 x 107 cells/animal) on day 0, and then rechallenged on days 7, 14, and 60 at the right hind footpad by injecting the same Ags (1 x 107 cells/animal). Footpad thickness was measured on day 8 (left panels), day 15 (middle panels), and day 61 (right panels) with a calipers-type engineer’s micrometer by a third experimenter masked to the sample identity. Data are representative of three independent experiments, showing the individual data points and the means ± SD of footpad swelling (*, p < 0.05). B, In some experiments, these animals were killed on day 15. CD4+ T cells from the recipients were labeled with CFSE (5 µM), cocultured with irradiated allogeneic mDC at a ratio of 20:1 for 3 days to analyze cell division using FACS. Data are representative of three independent experiments. C, BALB/c recipients were i.v. infused with 2 x 106 C57BL/6 donor-derived imDC, DC-LacZ, DC-sTNFRI, or L929-sTNFR on days 7, 4, and 0 before heart transplantation. The data are representative of three independent experiments with similar results, showing the survival curves plotted by the Kaplan-Meier method.

 
Induction of long-term cardiac allograft survival by DC-sTNFRI pretreatment

To determine the tolerogenic properties of DC-sTNFRI in vivo, BALB/c recipients were i.v. infused with C57BL/6-derived imDC, DC-LacZ, or DC-sTNFRI at day 7, 3, and 0 before cervical heterotopic heart transplantation. To determine the effect of sTNFRI expression alone on allograft survival, Ad-sTNFRI-transfected L929 cells expressing comparable levels of sTNFRI (data not shown) were used as a control. Survival of cardiac grafts in recipients infused with imDC or DC-LacZ was significantly prolonged (p < 0.01), with median graft survival time (MST) extended from 12 days to 22 or 21 days (Fig. 5C). Recipients infused with imDC acutely rejected third-party allogeneic cardiac grafts from C3H mice (MST = 12 days). Recipients infused with sTNFRI-expressing L929 cells also experienced an extension in cardiac graft survival (MST = 33 days; p < 0.01). However, none of the recipients in these groups demonstrated long-term survival of cardiac grafts, indicating that although pretreatment of recipients with imDC or the use of L929 cells to deliver sTNFRI can prolong cardiac allograft survival, they cannot induce permanent acceptance.

Cardiac allograft survival in recipients infused with DC-sTNFRI was significantly improved over that observed in all the other groups (p < 0.01), with an extension in MST from 12 to 87 days and 50% of cardiac grafts surviving long-term (>100 days). To investigate whether the immune tolerance induced by DC-sTNFRI is alloantigen-specific, third-party skin transplantation was performed in the surviving recipients with long-term cardiac graft. All of these recipients rejected the skin from C3H third-party mice within 8 days, whereas they accepted the skin from B6 mice >60 days into the observation period. These results indicate that infusion of DC-sTNFRI into recipients can effectively induce alloantigen-specific tolerance, significantly increasing allograft survival over that produced by infusion of unmodified imDC or sTNFRI-secreting non-DC.

Increased generation of CD4+CD25+ Tregs and microchimerism in recipients pretreated with donor DC-sTNFRI

imDC may control peripheral tolerance by inducing the differentiation of Tregs (1, 7, 35). DC-sTNFRI was examined for its potential to induce the generation of Tregs in vivo. A significant increase in the percentage of CD4+CD25+ splenic T cells in DC-sTNFRI-pretreated recipients was found (Fig. 6A). An increase was also detected in CD4+CD25+ T cells in the spleens of imDC- and DC-LacZ-pretreated recipients, compared with those in untreated recipients or naive mice. Furthermore, CD4+CD25+ T cells from both imDC- and DC-sTNFRI-pretreated mice suppressed alloreactive CD4+ T cell proliferation. This effect was more pronounced in CD4+CD25+ T cells isolated fromDC-sTNFRI-treated mice. Moreover, this potent suppressive effect of CD4+CD25+ T cells on the proliferation of alloreactive T cells was sustained, and even enhanced, in DC-sTNFRI-pretreated recipients with allografts that had survived 60 days (Fig. 6B).


Figure 6
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  		FIGURE 6. CD4+CD25+ Tregs and microchimerism generation in recipients pretreated with DC-sTNFRI. BALB/c recipient mice were i.v. infused with C57BL/6 donor-derived imDC, DC-LacZ, DC-sTNFRI, or L929-sTNFRI before heart transplantation. A, CD4+ T cells from untreated, DC-LacZ-, L929-sTNFRI-, or DC-sTNFRI-pretreated recipients or DC-sTNFRI-pretreated recipients (21 days after transplantation) were stained with FITC-anti-CD25 and analyzed by FACS. Data are presented as mean ± SD of three independent experiments performed (*, p < 0.05). B, CD4+ T cells from naive BALB/c mice were cocultured with irradiated allogeneic mDC (H-2b) at a DC:T cell ratio of 1:10, and CD4+CD25+ T cells, isolated from imDC- or DC-sTNFRI-treated BALB/c recipients with surviving allografts at 21 days or >60 days, were added to the cultures at a T cell:Treg ratio of 1:1. T cell proliferation was determined by [3H]thymidine incorporation. Results represent the mean ± SEM of four independent experiments (*, p = 0.046; +, p = 0.0095). C, BALB/c recipients were i.v. infused with C57BL/6 imDC or DC-sTNFRI before heart transplantation. Seven days after heart transplant, recipients with surviving allografts were administered 1 mg of PC61 (anti-CD25, i.p., two injections, 3 days apart) or isotype IgG as control. The percentage of CD25+ T cells in spleen cells was determined by FACS. Results are representative of three independent experiments with similar results. D, BALB/c recipients, as described above, with surviving allografts were administered 1 mg of PC61 (anti-CD25, i.p., two injections, 3 days apart) to deplete CD25-positive cells at day 7 and 60. The data are representative of three independent experiments, showing the survival curves plotted by the Kaplan-Meier method. E, Isotype IgG was injected as control. F, Spleens from BALB/c recipients infused i.v. with C57BL/6-derived imDC, DC-LacZ, or DC-sTNFRI before transplant were harvested on day 14, 21, 28, or 42 following heterotopic heart allograft. Splenic DC were stained with FITC-labeled anti-Iab Ab and PE-labeled anti-Iad Ab for FACS analysis. Results shown are representative of three independent experiments.

 
To further test the possible involvement of CD4+CD25+ T cells in tolerance maintenance induced by pretreatment with imDC or DC-sTNFRI, CD25+ T cells were depleted by injecting recipients with anti-CD25 Ab. Following injection of anti-CD25 Ab, CD4+CD25+ T cells were reduced significantly in the recipients (Fig. 6C), and allografts in these imDC-treated mice were rejected within 22 days (MST = 17 days), a significant reduction in survival time compared with that of imDC-pretreated recipients (Fig. 6D). Injection of isotype Ab into imDC-treated recipients did not reduce CD4+CD25+ T cell numbers, and there was no significant difference in allograft survival compared with that following anti-CD25 administration (MST = 21 days).

In DC-sTNFRI-treated recipients, the survival of allografts was markedly reduced following depletion of CD25+ T cells, with MST reduced from 87 to 32 days in day 7-injected recipients, and no observable long-term allograft survival (Fig. 6E). Phenotypic analysis revealed a reduction in the proportion of CD4+CD25+ T cells following anti-CD25 Ab treatment in recipients pretreated with imDC or DC-sTNFRI (Fig. 6C). To further investigate CD4+CD25+ T cell involvement in tolerance maintenance, anti-CD25Ab were injected into DC-sTNFRI-pretreated recipients with allografts that had survived >60 days. Allografts were rejected as early as 5 days after injection, and all allografts were rejected within 17 days of anti-CD25 treatment (Fig. 6E). Rejection was accompanied by a marked reduction in CD4+CD25+ T cell numbers (data not shown). These data demonstrate that pretreatment of transplant recipients with DC-sTNFRI can significantly promote the generation of Tregs, which exert suppressive effects on alloreactive T cells and contribute to tolerance maintenance in vivo.

To determine the fate of donor DC (H-2b) in recipients (H-2d), MHC class II-positive donor cells (Iab+ cells) were analyzed in immune organs of recipients at different times following DC transfusion. Iab+-donor cells could be detected in spleens of all recipients transfused with DC-sTNFRI, DC-LacZ, or imDC 14 days after allograft transplantation (Fig. 6F). These cells were substantially reduced in recipients infused with imDC or DC-LacZ at day 21 and completely disappeared at day 28. In sharp contrast, Iab+ cells were readily detectable in the spleens of recipients infused with DC-sTNFRI even 42 days later. These results indicate that DC-sTNFRI survive in recipients for an extended period, leading to enhanced microchimerism in the recipients.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
This study examined the capacity of genetically modified imDC to express sTNFRI to induce donor-specific immune tolerance. The data demonstrate that sTNFRI genetic modification can inhibit the maturation of DC, promote the production of IL-10-producing Treg-like cells, and potentiate the ability of imDC to induce donor alloantigen-specific T cell hyporesponsiveness both in vitro and in vivo. Importantly, in vivo pretreatment of recipients with imDC genetically modified to express sTNFRI induces long-term survival of 50% of cardiac allografts and leads to a substantial increase in generation of microchimerism and Tregs.

Several approaches have been used to enhance the tolerogenic properties of imDC by maintaining imDC in the more steady immature state. This study shows that imDC-expressing sTNFRI are resistant to maturation induced by TNF-{alpha} or LPS stimulation, retaining low levels of MHC class II and costimulatory markers typical of imDC. In contrast to the decreased phagocytic ability of imDC after LPS stimulation, these DC-sTNFRI retained high phagocytic ability and low Ag-presenting capacity. The allostimulatory activity of DC-sTNFRI was significantly impaired in allogeneic MLR. Interestingly, nonirradiated DC-sTNFRI also resist maturation under the help of CD4+ T cells in MLR (data not shown), which may be due to the involvement of autocrine TNF-{alpha} in T cell activation (39). Furthermore, DC-sTNFRI inhibit LPS-stimulated up-regulation of CXCR4 and CCR7 and migration to MIP-3beta, which may result in low frequency DC migration to lymph nodes following infusion into recipients and may contribute active tolerance induction (40, 41).

DC-sTNFRI induce allospecific tolerance through cytokine production. It is well known that DC influence Th cell differentiation by producing cytokines (42, 43). Immature or tolerogenic DCs can drive the differentiation of CD4+ type 1 T-regulatory cells (Tr1) (34, 35, 44). mDC-induced T cell expansion and Th1 polarization produced in this study agreed with previous findings (45). In contrast, stimulation of allogeneic CD4+ T cells with imDC resulted in high levels of IL-10 but low levels of IFN-{gamma} and IL-2 production in allogeneic MLR. Interestingly, coculture of CD4+ T with DC-sTNFRI in allogeneic MLR resulted not only in much higher levels of IL-10 and TGF-beta production, but also in the generation of an IL-10-producing T regulatory-like cell subset. These IL-10-producing Treg-like cells induced by DC-sTNFRI could significantly inhibit the proliferation of syngeneic CD4+ T cells in response to allogeneic Ag. Although IL-10 plays a key role in the development of IL-10-producing T cells (1, 46), and other studies report that IL-10 is crucial in T regulatory cell-mediated immune tolerance in vivo (47, 48, 49), the current data show that the anti-TGF-beta Ab can reverse the inhibitory function of Treg-like cells. Consistent with other studies, although the resulting Treg-like cells produce high levels of IL-10, their high levels of expression of TGF-beta, CTLA-4, the transcription factor Foxp3, and cell contact-dependent mechanisms may lead to potent inhibitory effects on T cells (42, 49).

Two lines of in vivo evidence further suggest that DC-sTNFRI controls peripheral tolerance by inducing the production of T-regulatory cells. First, treatment of DC-sTNFRI dramatically enhanced the appearance of CD4+CD25+ T cells in recipient mice. Second, anti-CD25 Ab-mediated in vivo depletion of Tregs significantly reduced allograft survival in recipients pretreated with DC-sTNFRI. Moreover, in DC-sTNFRI-pretreated recipients with allografts that had survived >60 days, administration of anti-CD25 Ab provoked particularly rapid allograft rejection. TNF-{alpha} has been shown to impact T-regulatory cell generation. Administration of TNF-{alpha} to neonatal NOD mice decreases the number of thymic CD4+CD25+ T cells, whereas anti-TNF-{alpha} Ab has the reverse effect (50, 51). There are reports documenting that mDC, not imDC, express CD25 (52, 53). Although there is the possibility that anti-CD25 Ab may influence the results by targeting DC in the recipients, CD25 expression on imDC or DC-sTNFRI administered to the recipients was not detected.

The fate of donor imDC in recipients is pivotal to the maintenance of tolerance. The observation of multilineage microchimerism in long-surviving recipients of organ allografts indicates that chimerism is an essential prerequisite for tolerance induction (54, 55). Although passenger leukocytes were reported in the observation of chimerism in long-surviving recipients, donor hemopoietic cells and DC are also important in generation of microchimerism (56). Hence, the preservation of imDC tolerogenic potential and efforts to manipulate microchimerism using immunosuppressive therapy are promising approaches. In our experiments, Iab+ donor cells could be detected in the spleens of all recipients (H-2d) transfused with DC-sTNFRI or imDC 14 days after transplantation. Iab+ cells dwindled over time in imDC-infused recipients, disappearing within 35 days. However, in recipients infused with DC-sTNFRI, these cells remained detectable in spleens up to 49 days later. These results suggest that DC-sTNFRI with immature steady state (57) may persist in recipients for a certain period and contribute to microchimerism formation in recipients, which may be a possible mechanism for donor-specific tolerance.

In conclusion, sTNFRI genetic modification effectively confers imDC with resistance to maturation induction and allows them to induce allogeneic T cell hyporesponsiveness and the generation of IL-10-producing T-regulatory-like cells in vitro. In vivo pretreatment of recipients with DC-sTNFRI induces long-term survival of 50% of cardiac allografts and leads to a substantial increase in microchimerism and T-regulatory cell numbers. Thus, infusion of DC-sTNFRI into recipients appears to be an effective approach for prolonging allograft survival.


   Acknowledgments
 
We thank Dr. Jane Rayner and Prof. You-Wen He (Department of Immunology, Duke University Medical Center, Durham, NC) for critically reading this manuscript.


   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 National Key Basic Research Program of China (2003CB515503, 2001CB510002), the Ministry of Education of China (0109), and the National Natural Science Foundation of China (30170896, 30121002). Back

2 Q.W. and Y.L. contributed equally to this study. Back

3 Address correspondence and reprint requests Dr. Xuetao Cao, Institute of Immunology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, People’s Republic of China. E-mail address: caoxt{at}public3.sta.net.cn Back

4 Abbreviations used in this paper: Treg, regulatory T cell; DC, dendritic cell; imDC, immature DC; mDC, mature DC; sTNFR, soluble TNF-{alpha} receptor; sTNFRI, sTNFR type I; Ad, adenovirus; DC-sTNFRI, sTNFRI gene-modified imDC; BM, bone marrow; DC-LacZ, imDC genetically modified with LacZ; MST, median graft survival time. Back

Received for publication September 12, 2005. Accepted for publication May 25, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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