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International Immunology Advance Access originally published online on January 7, 2008
International Immunology 2008 20(2):285-293; doi:10.1093/intimm/dxm142
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Tolerogenic dendritic cells transferring hyporesponsiveness and synergizing T regulatory cells in transplant tolerance

Mu Li1,2,3,*, Xusheng Zhang1,2,3,*, Xiufen Zheng1,2,3, Dameng Lian1,2,3, Zhu-Xu Zhang1,2,3,4, Hongtao Sun1,2,3, Motohiko Suzuki1,2,3, Costin Vladau1,2,3, Xuyan Huang1,2,3, Xiaoping Xia1,2,3, Robert Zhong1,2,3,4,5,6, Bertha Garcia1,2,3,4 and Wei-Ping Min1,2,3,4,5,6

1 Department of Surgery, University of Western Ontario, London, Ontario, Canada
2 Department of Pathology, University of Western Ontario, London, Ontario, Canada
3 Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada
4 Multi-Organ Transplant Program, London Health Sciences Centre, London, Ontario, Canada
5 Transplantation and Regenerative Medicine, Lawson Health Research Institute, London, Ontario, Canada
6 Robarts Research Institute, London, Ontario, Canada

Correspondence to: W.-P. Min; E-mail: mweiping{at}uwo.ca


   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 Funding
 References
 
Dendritic cells are among the most potent antigen-presenting cells and are important in the development of both immunity and tolerance. Tolerogenic dendritic cell (Tol-DC) is a key factor in the induction and maintenance of tolerance during transplantation. However, the precise mechanism and direct evidence of in vivo immune modulation remain unclear. In the present study, we identified critical roles of immune modulation on transplant tolerance through interactions between Tol-DCs and regulatory T (Treg) cells. Tol-DCs remained in an immature state and were insensitive to maturation stimuli. Tol-DCs in tolerant recipients heightened the expression of indoleamine 2,3-dioxygenase (IDO) that induced allogeneic T-cell apoptosis. Adoptive transfer of Tol-DCs isolated from primary tolerant recipients resulted in augmentation of CD4+CD25+CTLA4+ Treg cells and prolonged graft survival in secondary allogeneic heart transplantation and synergized with Treg cells to induce tolerance in secondary recipients. This study indicates that Tol-DC offers two functions during the process of tolerogenesis: suppression of anti-donor T-cell responses through production of IDO and interaction with Treg cells, which provides a framework for future research into tolerance induction.

Keywords: adoptive transfer, dendritic cells, indoleamine 2,3-dioxygenase, T regulatory cells, transplant tolerance


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Funding
 References
 
Dendritic cells (DCs) are the most potent antigen-presenting cells, consequently, and are primarily responsible for initiating protective immune responses (1). On the other hand, DCs also play a critical role in the induction of immune tolerance. It has been clearly demonstrated that the properties of DC are dependent upon their maturation status, phenotype and source of origin and that they can be either immunostimulatory or immunoregulatory (2). Likewise, DCs that inhibit immune responses have been described as being immature (3), having plasmacytoid morphology (4) or being alternatively activated (5). Collectively, suppressive DCs have been termed tolerogenic dendritic cells (Tol-DCs) (2). Donor-specific, allogeneic Tol-DCs can enhance the survival of transplanted grafts (6–8). Due to the lack of one or more activation signals, immature DC can inhibit T-cell responses. This was clearly demonstrated in a study by Thomson et al., in which bone marrow-derived DC progenitors that were deficient in the expression of CD80 and CD86 co-stimulatory molecules were able to induce antigen-specific hyporesponsiveness in vitro (9). Such immune unresponsiveness has been postulated to occur via the mechanisms of apoptosis (10), anergy (11) and default Th2 differentiation (12, 13). More recently, DCs have been found to engage in the active process of tolerance induction via immune modulation and interaction with T cells (14).

Induction of tolerance, a state of antigen-specific non-responsiveness, has been embraced metaphorically as the ‘holy grail’ in the prevention of autoimmune diseases and transplant rejection. Findings suggest that T regulatory (Treg) cells are associated with the establishment of a stable state of tolerance (15, 16). Furthermore, DCs have been linked with the induction of Treg cells. It has been shown by two independent groups, of Groux and Umetsu, that phenotypically mature and immature DCs, which express high levels of IL-10, can stimulate the development of Tr1 or Tr1-like cells, respectively (17, 18). Using an allogeneic heart transplantation model, we previously have demonstrated that stable tolerance is associated with the emergence of Tol-DCs and Treg cells, which interchangeably interact to form a self-maintaining immunoregulatory feedback loop in an in vitro co-culture system (19). However, in vivo evidence of interaction between these two regulatory cells has not been demonstrated, to date.

In our previous study, we showed that both Treg cells and Tol-DCs can be isolated from allograft recipients made tolerant by a short course of treatment with anti-CD45RB mAb and LF15-0915 (LF), an analog of 15-deoxyspergualine (20). Treg cells isolated from an in vivo state of tolerance can guide the in vitro differentiation of DCs into Tol-DCs and in vivo isolated Tol-DCs can stimulate the in vitro production of Treg cells. This suggests that tolerance is associated with a self-maintaining feedback loop between Treg cells and DCs (19). In the present study, we defined critical roles of immune modulation on transplant tolerance via the interaction between Tol-DC and Treg cell. We discovered that the adoptive transfer of Tol-DC isolated from primary tolerant recipients prolonged graft survival in secondary allogeneic heart transplantation. Furthermore, Tol-DCs synergized Treg cells, while inducing transplant tolerance. By adoptively transferring Tol-DCs from primary recipients, a significant augmentation of CD4+CD25+CTLA4+ Treg cells was observed, providing direct evidence of Tol-DC-generating Treg cells in vivo.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 Funding
 References
 
Animals and tolerance induction
C57BL/6 (H-2b) and BALB/c (H-2d) male mice (8–12 weeks old) were purchased from the Jackson Laboratory (Bar Harbour, ME, USA) and used as donors and recipients, respectively. Recipient mice (BALB/c) were divided into groups and treated with a combination protocol: (i) anti-CD45RB mAb 3 mg kg–1 day–1, intravenously (i.v.), days –1 to 7 and (ii) LF 2 mg kg–1 day–1, subcutaneously, days 0–7. Untreated recipients and age-matched BALB/c naive mice were used as rejecting and normal controls.

Heterotopic cardiac transplantation
Treated and untreated BALB/c mice were subjected to allogeneic cardiac transplantation using C57BL/6 donors. Direct abdominal palpation was used to assess graft viability. Heterotopic heart transplantation was performed in accordance with routine procedures in this laboratory (21). The pulsation of heart grafts was monitored daily by two independent observers without prior knowledge of the treatment protocol. The degree of pulsation was scored as follows: A, beating strongly; B, noticeable decline in the intensity of pulsation or C, complete cessation of pulsation. Recipients with grafts surviving >100 days were defined as tolerant and were used for in vitro experiments. Untreated recipients (BALB/c) rejected allografts on day 8 and were used as rejecting controls.

Isolation of splenic DCs and Treg cells
DCs were isolated from the spleens of long-term (>200 days) allograft survivors and of controls by gradient centrifugation over Ficoll-Paque (Amersham Pharmacia Biotech, Uppsala, Sweden) and labeled with anti-mouse CD11c mAb-conjugated super-paramagnetic MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). In order to increase the yield of DC, tolerant recipients and naive mice were administered Flt3-L (Amgen, Thousand Oaks, CA, USA), intra-peritoneally, 10 µg day–1 for 10 days. CD11c+ cells were isolated by passage through a magnetic column.

CD4+CD25+ T cells were isolated from tolerant and control mice using the EasySep System (Stem Cell Technologies, Vancouver, Canada). The experimental procedure utilized was in accordance with the manufacturer's instructions. In brief, CD4+ spleen cells were first isolated, using the negative selection SpinSepTM kit (Stem Cell Technologies). CD4+ cells were then further purified, based upon CD25 expression, using the EasySepTM kit (Stem Cell Technologies). Enriched CD4+ cells were incubated with species-specific Fc-blocking antibody and with PE-conjugated anti-CD25 antibody to obtain CD25+CD4+ T cells.

Adoptive transfer of Tol-DCs and Treg cells
In total, 2 x 106 CD11c+ DCs were administered i.v. into naive recipient BALB/c mice 3 days prior to and 1 day after heart transplantation. All recipients received a low dose (2 Gy, 7 days before transplantation) of whole-body irradiation. No immunosuppressive drugs were given to the recipients. In some experiments, a sub-therapeutic dose of Treg cells (105 cells, i.v., day +1) was co-administered with Tol-DCs (2 x 106, days –3 and +1).

Mixed leukocyte reaction
Splenic DCs were isolated from tolerant or rejecting recipients (BALB/c) and were used as stimulators. Total T cells (105 per well) from C57BL/6 mice were purified by means of the T cell purification kit (Stem Cell Technologies) and added to the DC cultures, with the final mixed leukocyte reaction (MLR) taking place in 200 µl of complete RPMI 1640 (Life Technologies). Cells were cultured at 37°C in a humidified atmosphere of 5% CO2 for 3 days and pulsed with 1 µCi of [3H]thymidine (Amersham Pharmacia Biotech) for the last 16 h of culture. Cells were harvested onto glass fiber filters and incorporated radioactivity was quantified using a Wallac Betaplate liquid scintillation counter. Results were expressed as mean counts per minute of triplicate cultures ± SEM.

To determine the ability of Treg cells to inhibit an MLR, CD4+CD25+ and CD4+CD25– T cells isolated from tolerant or control BALB/c mice were added to an MLR, using purified normal BALB/c total T cells as responders (105 per well) and irradiated (25 Gy) C57BL/6 spleen cells as stimulators (105 per well). The experimental procedure of incubating and harvesting cells was the same as described above.

Flow cytometry
Phenotypic analysis of isolated or cultured DCs was performed on a FACScan (Becton Dickinson, San Jose, CA, USA). All antibodies were purchased from BD PharMingen (San Diego, CA, USA), unless otherwise indicated. For T cells, we used FITC-, PE- or CyChrome-conjugated anti-mouse CD4, CD152 (CTLA4) and CD25 (eBioscience, San Diego, CA, USA). CD152 expression was assessed by intracellular staining, using a cell permeabilization kit (Caltag Laboratories, Burlingame, CA, USA). For characterization of DCs, we used FITC- or Cy5-conjugated anti-mouse I-Ad, CD11c, CD40 and CD86 and CD8{alpha} mAb. T-cell and DC subsets were analyzed by two- or three-color sorting with various combinations of mAbs. All flow cytometric analyses were performed using appropriate isotype controls (Cedarlane Laboratories, Hornby, Ontario, Canada).

Western blot
Splenic CD11c+ DCs from control and tolerant mice were lysed in 100 µl lysis buffer (PBS, pH7.4, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS and proteinase inhibitor cocktail tablet). Total protein was assayed using protein assay reagents (Bio-Rad, Hercules, CA, USA). Lysates were separated on 10% SDS–PAGE. Indoleamine 2,3-dioxygenase (IDO) protein was detected using mouse anti-IDO antibody 1:250 (Chemicon, Temecula, CA, USA) and goat anti-mouse IgG–HRP (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), using standard blotting and detection techniques.

Apoptosis assay
After co-culture of control DCs or Tol-DCs with allogeneic T cells for 24 h, apoptosis of T cells was measured using the Annexin-V (Bio-Rad)-binding assay, and the cells were then analyzed on a FACScan.

Real-time PCR
Quantitative PCR was performed on an ABI 7900 PCR Instrument (PerkinElmer, Wellesley, MA, USA) using Universal SYBR Green PCR Master Mix (PerkinElmer). Primers used in this study were as follows: IDO, sense 5'-CGGACTGAGAGGACACAGGTTAC-3' and anti-sense 5'-ACACATACGCCATGGTGATGTAC-3'; FoxP3, sense 5'-CAGCTGCCTACAGTGCCCCTAG-3' and anti-sense 5'-CATTTGCCAGCAGTGGGTAG-3' and GAPDH, sense 5'-TGATGACATCAAGAAGGTGGTGAA-3' and anti-sense 5'-TCCTTGGAGGCCATGTAGGCCAT-3'. A relative quantitative assay was adopted. Mouse GAPDH mRNA was used for normalization to ensure equal amounts of starting RNA. Each sample was tested in triplicate with samples obtained from at least three independent experiments.

Statistical analysis
Data are expressed as means ± SEMs. Statistical comparisons between groups were performed using Student's t-tests. Graft survival was compared among experimental groups using the log-rank test. MLR data were analyzed using one-way analysis of variance, followed by the Newman–Keuls test. Differences with P values <0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Funding
 References
 
DCs in tolerant recipients are mature resistant
To characterize the role of Tol-DCs and Treg cells in transplant tolerance, we have established a murine model of tolerance, using heart transplantation of C57BL/6 (H-2b) donors into BALB/c recipients (H-2d) after a short course of treatment with anti-CD45RB mAb and LF (20). We have found that Treg cells isolated from an in vivo state of tolerance can guide the in vitro differentiation of DCs into Tol-DCs and that in vivo isolated Tol-DCs can stimulate the in vitro production of Treg cells. This suggests that tolerance is associated with a self-maintaining feedback loop between Treg cells and DCs, after treatment with anti-CD45RB mAb and LF (19). In this model system, the allogeneic grafts in the recipients survived >200 days and readily accepted donor strain, but not third-party strain skin grafts (20). These long-term survivors of allografts were defined as primary tolerant recipients and used for testing immune modulation in this study. We first examined DCs' maturation in tolerant recipients after treatment with a short course of anti-CD45RB and LF. The splenic DCs were isolated from tolerant recipients, in which allografts had survived >100 days. The phenotypical changes of DCs were determined by flow cytometry. As shown in Fig. 1(A), a significantly greater proportion of DCs in tolerant recipients, versus non-treated/rejecting mice, did display an immature phenotype with low expressions of MHC II, CD40 and CD86.


Figure 1
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  		Fig. 1. Tol-DCs in tolerant recipients are maturation resistant. (A) Phenotypic analysis of maturation of Tol-DC. Splenic CD11c+ DCs were isolated from allogeneic (C57BL/6 to BALB/c) heart transplant recipients that were made tolerant through a short-course treatment with anti-CD45RB and LF. Control DCs were isolated from non-treated rejecting recipients. DCs were stained with indicated mAbs (solid lines) and isotype controls (doted lines) and analyzed on flow cytometry. (B) Tol-DCs isolated from tolerant recipients are insensitive to maturation stimulation. DCs were isolated from spleens of tolerant recipients by CD11c+ MACS beads. DCs were cultured in complete RPMI containing 10 ng ml–1 granulocyte macrophage colony-stimulating factor and IL-4, in the absence (non-activated) or in the presence (activated) of LPS (10 ng ml–1) and TNF{alpha} (10 ng ml–1). After 24 h incubation, DCs were stained with FITC-labeled anti-I-Ad, CD40 and CD86 mAbs, followed by flow cytometry analysis. The graph represents pooled data from three experiments. (C) MLR. BALB/c DCs were isolated and activated as in (B) and were used as stimulants at the indicated cell numbers. C57BL/6 T cells (105 cells per well) were used as responders. MLR was performed in a 3-day culture and pulsed with 1 µCi of [3H]thymidine for the last 16 h of culture. Data represent mean ± SEM and are representative of three experiments (*P < 0.01 by one-way analysis of variance and Newman–Keuls test).

 
It has been reported that immature, donor-derived DCs are capable of initiating antigen-specific tolerance (22). Additionally, in vitro-generated maturation-resistant immature DCs induced tolerance in a cardiac transplantation model (23). Accordingly, we next examined whether Tol-DCs are maturation resistant. DCs isolated from tolerant recipients were cultured with DC maturation stimuli [tumor necrosis factor (TNF) {alpha} and LPS] for 24 h. The expression of MHC II and co-stimulatory molecules (CD40 and CD86) on the DCs from tolerant recipients expressed lower levels than the control DCs from naive mice (Fig. 1B, non-activated group). After activation with TNF{alpha} and LPS, the expressions of MHC and co-stimulatory molecules were significantly up-regulated in control DCs. However, the levels of these molecules on DCs from tolerant mice retained compatibility with unstimulated immature DC phenotypes (Fig. 1B, activated group), indicating that DCs in tolerant recipients are maturation resistant. Functionally, DCs isolated from tolerant recipients, whether non-activated or activated in vitro, failed to stimulate an allogeneic T-cell response (Fig. 1C); conversely, control DCs vigorously stimulated an allogeneic T-cell response after activation. These data suggest that maturation-resistant Tol-DCs may play an important role in the maintenance of tolerance in transplantation since allografts, as strong allo-antigens, could stimulate mature DCs, resulting in graft rejection.

DCs in tolerant recipients express high levels of IDO
We further compared subtypes of Tol-DCs versus control DCs that were generated from non-treated rejecting recipients. The lymphoid subset of DC (CD11c+CD8{alpha}+) was significantly increased in tolerant recipients (Fig. 2A). IDO is a rate-limiting enzyme in the catabolism of tryptophan, an essential amino acid for T-cell survival. Recent studies have revealed that IDO is expressed on CD11c+CD8{alpha}+ DCs in mice and on CD123+ DCs in humans (24, 25). Since CD11c+CD8{alpha}+ DCs are increased in tolerant recipients, it is crucial to understand whether IDO expression on DCs is involved in transplant tolerance. As shown in Fig. 2, we have found that up-regulated expression of IDO exists in DCs isolated from tolerant recipients, as determined by western blot (Fig. 2B) and by real-time PCR (Fig. 2C), in comparison with DCs isolated from rejecting mice.


Figure 2
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  		Fig. 2. Up-regulated expression of IDO in tolerant recipients. Splenic DCs were isolated from tolerant allografted recipients or from non-treated control mice, as described in Fig. 1. (A) Subtype composition of Tol-DCs. DCs were stained with anti-mouse CD11c (FITC conjugated) and CD8{alpha} (Cy5 conjugated) mAbs and analyzed on flow cytometry, as described above. (B) IDO expression on DCs isolated from tolerant recipients or from non-treated control mice was assessed by western blot. (C) IDO expression on DCs isolated from tolerant recipients or from non-treated control mice was assessed by real-time PCR. (D) Tol-DCs induce apoptosis of allogeneic T cells. In total, 104 DCs (BALB/c) from recipient mice were incubated with C57BL/6 T cells (105 per well). After 48 h of co-culture, apoptosis was assessed by Annexin-V-binding assay. Data represent mean ± SEM and are representative of three experiments.

 
To explore the function of IDO in DCs, we examined apoptosis in allogeneic T cells. After co-culture with Tol-DCs obtained from long-term survivors of tolerant allograft recipients, not control DCs, a remarkable increase in apoptosis (Annexin-V+) was noted in allogeneic T cells in vitro (Fig. 2D).

Interplay between Tol-DCs and Treg in vivo
Even though we previously had demonstrated that a ‘regulatory feedback loop’ exists between Tol-DCs and Treg cells in an in vitro co-culture system in tolerant mice, after treatment with anti-CD45RB and LF (19), the in vivo interaction between these two types of regulatory cells had not yet been established. Hence, as a next step, we examined whether Tol-DCs could augment Treg cells in vivo, besides directly suppressing T-cell responses through IDO production. In order to perform the adoptive transfer of tolerance, Tol-DCs were isolated from primary tolerant recipients of allogeneic heart transplants (C57BL/6 -> BALB/c) and subsequently injected into naive BALB/c mice (hereafter referred to as secondary recipients), followed by an allogeneic heart transplant, using the identical (C57BL/6 -> BALB/c) strain combination. In agreement with in vitro experimental data (19), the CD4+CD25+ Treg cell subset in secondary recipients significantly increased in number after an adoptive transfer of Tol-DCs from primary recipients (Fig. 3A). These CD4+CD25+ T cells expressed high levels of CTLA4 (Fig. 3A). Furthermore, these CD4+CD25+CTLA4+ T cells expressed Foxp3, a regulatory T-cell hallmark (Fig. 3B). To verify the inhibitory function of these Tol-DC-induced Treg cells, we isolated the CD4+CD25+ Treg cells from the spleens of secondary recipients and performed a titration into an ongoing MLR. Addition of the CD4+CD25+ Treg cells from the secondary tolerant recipients resulted in significant inhibition of the MLR (Fig. 3C).


Figure 3
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  		Fig. 3. Expansion of Treg cell populations after Tol-DCs transfer. (A) Flow cytometric analysis. Tol-DCs (2 x 106) from primary BALB/c tolerant recipients were transferred to syngeneic naive secondary recipients, followed by an allogeneic (C57BL/6 to BALB/c) heart transplant. Tol-DCs (upper panels) and control DCs (lower panels) transferred to secondary recipients were analyzed for frequency of CD4+CD25+ Treg cells and CD4+CTLA4+ Treg cells. (B) Tol-DC-induced CD4+CD25+ T cells express Foxp3. RNA from the T cells described above (A) was extracted by means of the Trizol method. Quantitative real-time PCR was performed to assess the expression of FoxP3 and GAPDH, using primers described in Methods. Data are representative of three independent experiments. (C) The inhibitory function of Treg cells isolated from the secondary recipients. The CD4+CD25+ or CD4+CD25– cells were purified from tolerant mice (BALB/c) and added to an ongoing MLR (BALB/c versus C57BL/6), as described in Methods. Data represent mean ± SEM and are representative of five experiments (*P < 0.01 by unpaired Student's t-test).

 
Adoptive transfer of Tol-DCs prolongs graft survival
These newly discovered tolerogenic properties of suppression of T-cell responses by IDO production and induction of Treg cell in vivo prompted us to ask whether Tol-DCs could serve as regulatory cells in vivo, and therefore be able to transfer tolerogenic properties from primary tolerant recipients to naive recipients. In the absence of any immunosuppressive treatment of secondary recipients, adoptive transfer of Tol-DCs, which were isolated from primary tolerant recipients of allogeneic heart transplants, significantly prolonged allograft survival to a median survival time (MST) of 39.4 days (Table 1). In comparison, secondary recipients that received DCs from naive mice survived to an MST of 13 days (P < 0.05). Hence, our data suggest that Tol-DCs can transfer tolerogenic properties to second recipients, thereby leading to prolongation of graft survival.


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  		Table 1. Survival of heart allografts after DC transfera

 
Tol-DCs synergize Treg cells in tolerance induction
As we previously demonstrated, synergistic transplant tolerance can be achieved by simultaneously targeting DCs and T cells in an allogeneic heart transplantation model, in which targeting both cells resulted in an induction of tolerance, while targeting DCs or T cells alone failed to achieve tolerance (20). Whether this synergistic effect also occurs during adoptive transfer remains unclear. The data above indicated that the transfer of Tol-DCs could prolong graft survival, but could not induce tolerance. In order to test if in vivo synergistic interaction between Tol-DCs and Treg cells results in induction of tolerance, we co-administrated Tol-DCs and Treg cells (CD4+CD25+) isolated from primary tolerant allograft recipients. While the sub-therapeutic dose of Treg cells alone failed to transfer tolerance (P > 0.05), the combination of Tol-DCs and Treg cells significantly (P < 0.01) enhanced the tolerance achieved by means of adoptive transfer from tolerant primary recipients to naive secondary recipients. More than 75% of the secondary recipients achieved tolerance, experiencing allograft survival of >100 days (Fig. 4), suggesting that the synergy between Treg cells and Tol-DCs may, in fact, enhance the efficacy of tolerance.


Figure 4
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  		Fig. 4. Co-adoptive transfer of Tol-DCs and Treg cell synergizes tolerance induction. Tol-DCs (2 x 106 cells, i.v., day –3 and day +1) isolated from primary tolerant recipients were co-administered to 2 Gy irradiated syngeneic naive secondary recipients with a sub-therapeutic dose (105, i.v., day +1) of CD4+CD25+ Treg cells, followed by allogeneic (C57BL/6 to BALB/c, n = 6) heart transplantation. Control DCs and control total T cells are isolated from non-transplanted mice.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Funding
 References
 
It has been established that the control of immune activation by Tol-DCs is mediated via the induction of T-cell apoptosis (26), anergy (27) and Treg cell differentiation (28). Classically, the lack of co-stimulatory molecules on Tol-DCs (9, 29), the expression of IL-10 (17, 18) and the presence of inhibitory molecules such as PD-1L (30) have been identified as mediating factors in the immune suppression. We have demonstrated that splenic DCs isolated from tolerant recipients possess the following tolerogenic properties in vitro: immature phenotypes (19), decreased allostimulatory capacity (19, 20), impaired co-stimulatory function (19, 20), polarized Th2 differentiation (31) and an ability to generate Treg cells (19). In this study, we further demonstrated that Tol-DCs are insensitive to maturation stimuli and express high levels of IDO (Fig. 2). Furthermore, we demonstrated a novel phenomenon by which the adoptive transfer of Tol-DCs isolated from primary tolerant recipients prolongs graft survival in secondary allogeneic heart transplanted mice and synergizes Treg cells in tolerance induction in secondary recipients.

We observed a higher ratio of the CD11c+CD8{alpha}+ subset of DC in tolerant recipients, highlighting the possibility that increased CD11c+CD8{alpha}+ DC might be associated with the treatment of tolerance protocol involving LF and anti-CD45RB. However, whether Tol-DCs are differentiated from immature DCs or are converted from mature DCs during LF and anti-CD45RB treatment remains unclear. In order to determine the extent to which the Tol-DCs from tolerant recipients were capable of immune modulation, we transferred in vivo extracted Tol-DCs into naive ‘secondary’ recipients. These recipients experienced enhanced survival of their allografts, suggesting that immune modulation was enacted. According to our feedback inhibitory model (19), Tol-DCs should synergize with Treg cells in inducing and maintaining the feedback loop between Tol-DCs and Treg cells in vivo. Our data have shown that transferring both Treg cells and Tol-DCs from the tolerant primary recipient into the naive secondary recipient leads to synergy in tolerance induction (Fig. 4). Thus, the transfer of both cells involved in the immune regulatory loop can lead to reconstitution of the feedback loop in naive recipients. Supporting evidence for synergy between Tol-DCs and Treg cells can be seen in other studies, in which the generation of Tol-DCs by anti-CD40L antibodies has been observed to synergize with the administration of the T-cell-targeting antibody, anti-CD45RB, so as to enhance the survival of allogeneic allografts (32).

IDO is a rate-limiting enzyme involved in the catabolism of tryptophan, an essential amino acid that is crucial for T-cell survival. Pioneering work by Munn et al. identified an essential role for IDO in fetal tolerance. More recent studies have revealed that IDO is expressed on CD11c+CD8{alpha}+ DCs in mice and on CD123+ DCs in humans (24, 25). In this study, heightened expression of IDO was found in Tol-DCs, but not in control DCs from graft-rejecting recipients (Fig. 2). It is vital to understand whether IDO expression in DCs is involved in transplant tolerance. To explore the function of IDO up-regulation on DCs in transplant tolerance, we examined apoptosis in allogeneic T cells in vitro. After co-culture with ex vivo-isolated Tol-DCs obtained from long-term allograft survivors, a remarkable increase in apoptosis was noted in allogeneic T cells in vitro (Fig. 2D).

CTLA4 has been found to play a pivotal role in the function of Treg cells in vivo (33). Blocking of CTLA4 function leads to the abolishment of Treg cell function in vivo (33). Further supporting this notion, our data indicate that Tol-DCs can effectively generate CTLA4+ Treg cells in vivo (Fig. 3). On the other hand, recently it has been demonstrated that CTLA4 in Treg cells can stimulate IDO expression via the ligation of B7 molecules on DCs (34). Since adoptive transfer of Tol-DCs results in an in vivo augmentation of Treg cells with dramatically increased expression of CTLA4 (Fig. 3A), Treg cells might, in turn, up-regulate IDO in DCs. In support of this notion, we observed a significant increase in the expression of IDO on the DCs of Tol-DC-transferred secondary recipients, as compared with the DCs in control DC-transferred recipients (data not shown). Therefore, we postulate that Treg cells not only directly inhibits alloreactive T cells, but also may induce recipient DCs to produce IDO via CTLA4 up-regulation, which indirectly deletes alloreactive T cells by apoptosis. Supporting this concept, we have found that sub-therapeutic Treg cell fails to induce tolerance alone, but dramatically enhances tolerance induction by Tol-DC co-transfer (Fig. 4). It has been extensively reported that IFN{gamma} is another important factor triggering IDO expression, besides CTLA4 engagement. However, Tol-DC self-expressed low levels of IFN{gamma} in our previous publications (19, 20). Tol-DCs might obtain IFN{gamma} from other T cells, particularly from Treg cells that express IFN{gamma} at an early stage of activation (35).

The transfer of tolerance from a tolerant animal to a naive one classically is believed to be a T-cell-dependent process. This is due to the fact that T cells can expand clonally and influence other T cells; whereas other cell types, such as DCs, are terminally differentiated cells. In contrast, some studies have shown that antigen-specific tolerance can also be induced upon the adoptive transfer of antigen-pulsed, IL-10-expressing Tol-DCs (17, 18). In line with these latter studies, we demonstrated that DCs from a tolerant recipient can act as a bridge between animals to transfer the state of immunological unresponsiveness to a naive host. Assuming that these immunomodulatory DCs are not self-replicating, it may be possible that these DCs from the tolerant recipients actually ‘educate’ the naive T cells in the secondary recipient to tolerate the graft. Support for the concept that DCs may act as a bridge between T cells and Treg cells has been established: a CD40L+ T cell ‘licenses’ DCs to activate other T cells and induces cross-presentation (36). However, there has been little suggestion that such ‘licensing’ of DCs occurs in the inhibitory sense. Supporting evidence for this inhibitory licensing comes from experiments by Matzinger et al., which demonstrated, using an in vivo oral tolerance system, that immunoinhibitory T cells actually use DCs as a bridge to communicate immunomodulation to other naive T cells (37). Although we (20) and others (38, 39) previously have shown that Tol-DCs can enhance Treg cell numbers and activity, this was not demonstrated in vivo. Here, we observed an expansion of Treg cells with the phenotype CD4+CD25+ in secondary naive recipients that received in vivo generated Tol-DCs from primary recipients (Figs 2 and 3). Such an ability of Tol-DCs to generate Treg cells provides support for an active interaction between these cell types in vivo, consistent with the ‘feedback loop’ concept we proposed in one of our previous publications (19). Our previous study indicated that Tol-DCs expand Treg cells in vitro and help to induce tolerance (19). Others also have reported that Tol-DCs particularly might stimulate Treg cells, not normal CD4+ T cells, since they have low expression levels of MHC and CD86 (39). Studies also suggest that the engagement of Toll-like receptors (40, 41) and glucocorticoid-induced tumor necrosis factor receptor molecules (42, 43) on Treg cells can induce Treg cell activation and proliferation. In this study, we demonstrated the in vivo regulatory capacity of Tol-DCs on Treg cells in an adoptive transfer experiment. Adoptive transfer of the Tol-DCs significantly prolonged allograft survival (Table 1) and generated Treg cells in vivo (data not shown). These results suggest that tolerogenic properties through Tol-DCs are associated with Treg cell expansion in vivo, which in turn prolongs allograft survival.

It is generally agreed that the transport of antigen from an infection site or transplanted organ to a draining lymph node by DCs is a crucial component in initial adoptive immune responses. We do not have direct evidence regarding whether adoptive transfer Tol-DCs actually migrate to lymphoid organs. However, we identified an increase of Treg cells in the spleen and lymph nodes in Tol-DC-treated mice, which suggests that Tol-DCs might interact with Treg cells in the lymphoid organs.

In summary, we describe here a novel phenomenon, whereby Tol-DCs adoptively transfer tolerance to syngeneic naive recipients, providing the first direct evidence of in vivo immunomodulation by Tol-DCs in transplantation. The Tol-DC-mediated tolerance occurs by means of two distinct pathways: one through the active generation of Treg cells and the other through passive induction of apoptosis of reactive T cells via IDO. Coincidently, Treg cells may, in turn, enhance the tolerogenic effects of Tol-DCs by up-regulating IDO expression on DCs. In conclusion, the interaction between Tol-DCs and Treg cells plays a key role in the maintenance of transplant tolerance.


   Funding
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
References
 
Heart and Stroke Foundation of Canada; Roche Organ Transplantation Research Foundation; Multi-Organ Transplant Program, London Health Sciences Centre.


   Acknowledgements
 
We thank Dr. Gill Strejan for his critical comments and suggestions.


   Abbreviations
 
DC, dendritic cell
IDO, indoleamine 2,3-dioxygenase
i.v., intravenously
LF, LF15-0195
MLR, mixed leukocyte reaction
MST, median survival time
TNF, tumor necrosis factor
Tol-DC, tolerogenic dendritic cell
Treg, regulatory T

   Notes
 
* These authors contributed equally to this study. Back

Transmitting editor: K. Inaba

Received 13 November 2006, accepted 6 December 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
 References
 

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