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International Immunology Advance Access originally published online on October 26, 2006
International Immunology 2006 18(12):1771-1777; doi:10.1093/intimm/dxl111
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Distinctive role of donor strain immature dendritic cells in the creation of allograft tolerance

Yon Su Kim1, Seung Hee Yang1, Hee Gyung Kang2, Eun Young Seong1, Se Han Lee1, Wenda Gao2, James Kenny2, Xin Xiao Zheng2 and Terry B. Strom2

1 Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea
2 Department of Medicine and Surgery, Division of Immunology and Transplant Research Center, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

Correspondence to: Y. S. Kim; E-mail: yonsukim{at}snu.ac.kr


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Dendritic cells (DCs) are pivotal antigen-presenting cells and serve a unique role in initiating immunity. To test the hypothesis that pre-immunization of recipient with certain DC subsets of donor origin can influence graft outcome, we have studied the effects of immunization with allogeneic CD4+CD8–CD11c+ dendritic cell (CD4+DC) and CD4–CD8+CD11c+ dendritic cell (CD8+DC) on the allograft response. Although both immature CD4+DC and CD8+DC subsets from DBA/2 were able to prime naive allogeneic C57BL/6 (B6) T cells in mixed lymphocyte reaction (MLR), CD8+DC exerted more vigorous alloimmune responses than CD4+DC did. Also, CD4+DC-driven allogeneic T cell response was attenuated more significantly by anti-CD154 mAb than CD8+DC-driven response. Consistent with the MLR results, combined pre-treatment with CD4+DC, but not CD8+DC, plus anti-CD154 mAb produced donor strain-specific long-term graft survival and induced tolerance while treatment with CD8+DC plus anti-CD154 mAb created minimal prolongation of allograft survival in a pancreas islet transplant model (DBA/2->B6). The beneficial effects exerted by CD4+DC and anti-CD154 mAb pre-treatment were correlated with Th1 to Th2 immune deviation and with the amplified donor-specific suppressive capacity by recipient CD4+CD25+ T cells. These findings highlight the capacity of CD4+DC to modulate alloimmune responses, and suggest therapeutic approaches for the induction of donor-specific tolerance.

Keywords: dendritic cells, tolerance, transplantation


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Dendritic cells (DCs), a subgroup of bone marrow-derived antigen-presenting cells (APCs), are unrivalled in their capacity for activating T cells (1). The life history of DCs unfolds in two main developmental stages, termed immature and mature (1). Maturation of DCs radically boosts the immunogenic properties by inducing the stable expression of peptide–MHC complexes, up-regulation of co-stimulatory and adhesion molecules, secretion of chemokines and cytokines and swift migration to T cell zones of regional lymph nodes (2). The improved techniques for the isolation of DCs from a variety of mouse tissues has led to the discovery of a number of DC subsets, defined by their expression of a wide range of cell-surface molecules (1–5). There is considerable heterogeneity within the DC pool. The nature of the activating DC as well as its maturation status can determine the type of response generated. Distinct subsets of DC differ in their lineage affiliation and function as they transmit distinct DC subset-specific T cell polarizing signals (3, 6). CD8+CD11blow or CD8–CD11bhigh phenotypes among CD11c+DCs have been identified and isolated from both mouse lymphoid and non-lymphoid tissues (4, 5), Their relative incidence varies with tissue distribution: CD8–DCs are the predominant subset both in bone marrow and blood (5, 7), whereas CD8+DCs are the principal thymic DCs (8).

In mice, all recognized DC subsets express the CD11c integrin (9), although there is a report regarding CD11c– APCs (10). And again CD11c+DCs can be further delineated by the presence of the CD4 and CD8 molecules noted on splenic DC representing authentic expression of the gene product, that is, CD4+CD8–CD11c+ dendritic cell (CD4+DC) and CD4–CD8+CD11c+ dendritic cell (CD8+DC) (11, 12). In mice, CD8+DCs induce a Th1 response, whereas CD4+DCs induce a Th2 response (13–15). In accordance with this data, CD8+DCs, but not CD4+DCs, robustly express IL-12 (13–16). The distinct patterns of cytokine expression by these DC subsets account, at least in part, for the apparent differences in the ability of DC subsets to foster Th1 or Th2 responses (16), although some studies have challenged this sharp distinction (17). The role of different subsets and maturation status of DC in regulating immune responses against allo-antigen has been explored elsewhere (18–20). In these studies, the precise cell surface phenotype-based classification of DC subset was not provided and DC-recruiting cytokines were used. It is notable that these cytokines may affect the biologic functions of DCs (21, 22).

Although a certain subset of DCs from recipients may prolong allograft survival by the reinforcement of regulatory T cell capacity (23), the differential effect of donor-derived DC subsets has not been investigated thoroughly. Insofar as pre-transplant donor-specific transfusion can aid tolerance (24, 25), we have revisited the potential therapeutic use of donor strain DCs for pre-transplant immunization. In this study, we have analyzed the cell surface phenotype of the DC populations employed and we have gathered DCs without use of DC-recruiting cytokines. We test the hypothesis that immunization of distinct DC subsets of donor strain leads to the development of quantitatively and qualitatively differential effects on the manifestations of allograft responses to fully MHC-mismatched tissues in this study. Also, we examined the potential for synergistically promoting transplant tolerance by immunization with donor DCs plus imposition of CD40/CD154 pathway blockade (26–28).


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Animals
C57BL/6 (H-2b) and DBA/2 (H-2d) mice, aged 6–8 weeks, were purchased from Taconic Farms (Germantown, NY, USA). C3H (H-2k) mice were purchased from the Jackson Laboratories (Bar Harbor, ME, USA). All the animals were housed in a pathogen-free animal facility at the Beth Israel Deaconess Medical Center Research North and Clinical Research Institute Seoul National University Hospital. The Internal Review Board of our institutions approved the research protocol used for this study.

Antibodies
Following antibodies conjugated with FITC, PE, Cychrome or APC were purchased from BD PharMingen (San Jose, CA, USA); anti-CD4 mAb, anti-CD8 mAb, anti-CD11c mAb, anti-CD40 mAb, anti-CD80 mAb, anti-CD86 mAb, anti-CD45RA, anti-IFN-{gamma} mAb and anti-IL-10 mAb. Anti-CD154 mAb (MR1) was purchased from Taconic Biotechnology (Germantown, NY, USA) and Bio Express (West Lebanon, NH, USA).

Isolation of DC
The procedure was performed as described recently (11, 29). Briefly, spleen fragments were digested for 20 min at 37°C CO2 incubator with collagenase D (Roche Applied Science, Indianapolis, IN, USA), and then treated for 5 min with EDTA to disrupt T cell–DC complexes. All subsequent procedures were performed at 4°C in a divalent metal-free HBSS. Splenocytes were re-suspended with 10 ml of media, and 4 ml of OptiPrep (Sigma–Aldrich, St Louis, MO, USA) was mixed with cell suspension. Cell suspension/OptiPrep mixture was overlaid with 7.5 ml of 1.078 g ml–1 lymphocyte-specific density layer. This layer was overlaid with 20 ml of 1.068 g ml–1 solution and 0.5 ml of HBSS. The mixture was centrifuged at 600 x g for 25 min at room temperature. A brake was not applied during deceleration. Light-density cells (the first 18–20 ml, with care not to collect any of the WBC band observed at the interface of the 1.078 and 1.068 g ml–1 layers) were collected. At this stage, the splenic DCs were usually 20–25% pure. This preparation was then used for presorting when required or directly for immunofluorescent labeling before analysis.

Flow cytometric analysis and sorting of DC
Analyses were performed on FACSort instrument (BD Biosciences, San Jose, CA, USA). When large DC numbers were needed, the MoFlo High-Performance Cell Sorter (DakoCytomation, Carpinteria, CA, USA) was used. DCs were sorted either into CD4+DC or CD8+DC. After sorting, the DC subsets were ~90% pure.

Treatment protocol
Sorted immature CD4+DCs or CD8+DCs from DBA/2 (H-2d) were injected into C57BL/6 via lateral tail vein (5 x 105 DCs per mouse). Anti-CD154 mAb (0.25 mg) was given intra-peritoneally three times at days 0, 2 and 4 (anti-CD154 mAb alone group) or immediately following the injection of DCs (CD4+DC + anti-CD154 mAb or CD8+DC + anti-CD154 mAb). Four weeks after treatment, these pre-treated C57BL/6 mice were used for in vitro and in vivo experiments. Blocking anti-IL-10R mAb (1B1.2, kindly provided by K. J. Wood, Oxford, UK) was injected in some mice using a regimen described previously (30).

DC-allogeneic T cell mixed lymphocyte reaction (MLR)
Mixed Lymphocytes Reactions (MLRs) were performed to determine the stimulatory capacity of the CD4+DC and CD8+DC subsets. Splenic DCs were sorted from DBA/2 (H-2d) as either CD4+DCs or CD8+DCs. Various numbers of DC were cultured with 2 x 105 T cells purified from C57BL/6 mice using T cell purification column (R&D Systems Inc., Minneapolis, MN, USA) in 200 µl complete medium in U-bottom plates. After 3 days of culture, cells were pulsed with [3H]thymidine ([3H]TdR) (1 µCi in 25 µl) for 16 h. Cells were harvested using a multi-well harvester, and [3H]TdR incorporation was determined in a liquid scintillation counter. Results are expressed as the mean counts per minute ± SD from triplicate cultures. In the experiments that were designed to examine the potency of regulatory T cell, sorted CD4+CD25+ T cells from the pre-treated or control mice were co-mixed with syngeneic naive T effector cells in MLR (1:1 ratio), and T cell proliferation was analyzed via [3H]TdR incorporation.

Islet transplantation
Allogeneic DBA/2 islet cell grafts were transplanted into 8- to 10-week old C57BL/6 recipient mice rendered diabetic by a single intra-peritoneal injection of streptozotocin (250 mg kg–1; Sigma–Aldrich). Islets were isolated from donor DBA/2 pancreata through collagenase digestion (Sigma–Aldrich) and centrifuged on a discontinuous Ficoll (Sigma–Aldrich) gradient. The crude islet isolates containing islets, vascular tissue, ductal fragments and lymphocytes were divided into aliquots of 300 islets and were transplanted under the renal capsule of B6 recipients. Initial allograft function was verified by sequential blood glucose measurements with levels under 200 mg dl–1 on days 2–3 after transplantation, and graft rejection was defined as a rise in blood glucose levels exceeding 300 mg dl–1 following a period of primary graft function. To evaluate the strain specificity, C3H (H-2k) mice were used as third party islet donors to DBA/2 DC pre-treated B6 mice or to mice that have long-term primary graft function.

Enzyme-linked immunosorbent analysis
The technique for enzyme-linked immunosorbent (ELISPOT) analysis has been described previously (31, 32). Briefly, immunospot plates (Cellular Technology, Cleveland, OH, USA) were coated with 4 µg ml–1 rat anti-mouse IFN-{gamma} capture mAb (R4-6A2) or IL-4 capture mAb (11B11) in sterile PBS overnight. The plates were then blocked for 1 h with sterile PBS containing 1% BSA (fraction V) and washed three times with sterile PBS. Purified T cells (3 x 105 in 200 µl of media) were then placed in each well in the presence of 1 x 106 irradiated (2500 rad) syngeneic or allogeneic splenocytes and cultured for 24 h (IFN-{gamma}) or 48 h (IL-4) at 37°C in 5% CO2. After washing with PBS, followed by PBS containing 0.05% Tween (PBST), 2 µg ml–1 biotinylated rat anti-mouse IFN-{gamma} detection mAb (XMG1.2) or anti-IL-4 detection mAb (BVD6-24G2) was added overnight. The plates were then washed four times in PBST, followed by 2-h incubation with streptavidin–HRP (Dako, Carpenteria, CA, USA) diluted at 1/2000 in PBS/1% BSA. All mAbs were purchased from BD PharMingen (San Diego, CA, USA). After washing three times with PBST followed by PBS, the plates were developed using 800 µl 3-amino-9-ethylcarbazole (Sigma–Aldrich; 10 mg dissolved in 1 ml N,N-dimethylformamide) mixed in 24 ml 0.1 M sodium acetate (pH 5.0) plus 12 µl H2O2. The resulting spots were counted on a computer-assisted ELISPOT image analyzer (T Spot Image Analyzer; Cellular Technology).

Statistical analysis
Data were compared by Mann–Whitney U-test, {chi}2 test or the log-rank test where appropriate.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Freshly isolated DCs manifest an immature phenotype but progress into mature state after stimulation
The surface antigenic phenotypes of splenic DC subsets were analyzed via immunofluorescent staining using an informative panel of mAbs. After OptiPrep density gradient DC enrichment, CD4+DCs outnumbered CD8+DCs (Fig. 1). Both DC subsets expressed similar levels of class II MHC, CD40, CD80 (B7.1) and CD86 (B7.2), although CD45RA expression was more prominent in CD8+DC (Fig. 1). After overnight culture in media containing granulocyte macrophage colony-stimulating factor (GM-CSF) (20 ng ml–1) and LPS (40 ng ml–1), the magnitude of up-regulation in above molecules was similar in both DC subsets (data not shown).


Figure 1
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  		Fig. 1 Isolation of CD11c+DCs from spleen that display properties of DCs. (a) Low-density cells isolated over OptiPrep were labeled with antibodies to CD11c, CD4 and CD8. (b) Splenic CD11c+DCs, identified by presence of CD4 or CD8, analyzed for expression of presenting molecules and co-stimulatory molecules by flow cytometry; overlays respresent isotype-matched controls.

 
DC subsets possess differential capacities on alloimmune responses
To evaluate the allostimulatory capacity of CD4+DCs and CD8+DCs, naive T cells from C57BL/6 were stimulated by irradiated DBA/2 CD4+DCs or CD8+DCs in the MLR. Interestingly, T cell proliferation in 72-h culture was significantly greater after stimulation with CD8+DCs than with CD4+DCs at various DC:T cell ratios (Fig. 2, P < 0.05). DC-driven T cell allostimulation was inhibited with the addition of anti-CD154 mAb (2 µg ml–1) into the culture media, and the inhibitory effect of anti-CD154 mAb was more profound in CD4+DC than in CD8+DC-induced T cell proliferation (Fig. 2, Table 1, P < 0.05).


Figure 2
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  		Fig. 2 DC subsets possess differential allostimulatory capacities. T cells (from B6) proliferated more vigorously when they were stimulated with CD8+DCs (from DBA/2) than with CD4+DCs (from DBA/2) on various DC:T cell ratio. The blocking of CD40/CD154 pathway profoundly inhibited the alloreactive T cell responses. (*P < 0.05 when compared between CD8+DC and CD4+DC as stimulator, or between CD8+DC + anti-CD154 mAb (MR1) and CD4+DC + MR1, Mann–Whitney U-test).

 

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  		Table 1 Percent decrement of alloreactivity with the addition of anti-CD154 mAb

 
Pre-treatment with donor strain CD4+DCs, but not CD8+DCs, plus imposition of CD40/CD154 blockade promotes transplant tolerance
To test the effect of pre-immunization of donor strain DC subsets upon the in vivo allograft response, fully mismatched DBA/2 (H-2d) islet allografts were transplanted into B6 (H-2b) mice. As compared with untreated controls, pre-treatment of B6 hosts with either DBA/2 CD4+DCs or CD8+DCs did not produce prolonged islet allograft survival (mean survival time, MST; 14 and 13.5 days, respectively) as compared with untreated control (MST; 14 days). In contrast and consistent with findings in the in vitro T cell MLR model, combined pre-treatment with donor origin CD4+DCs and anti-CD154 mAb induced indefinite allograft survival and donor-specific tolerance (Fig. 3). Indeed, all B6 islet allograft recipients (n = 6) pre-treated with DBA/2 CD4+DCs plus anti-CD154 mAb showed permanent engraftment of DBA/2 islets (Fig. 3). Surgical removal of the left kidney bearing the islet allograft was performed after 120 days of engraftment. In the absence of further immunosuppressive therapy, a second DBA/2 islet allograft was successfully engrafted without evidence of rejection. In contrast, islet transplants from third party (C3H; H-2k) were acutely rejected (Fig. 3). The effect of pre-treatment with donor strain CD8+DCs differed from that noted with CD4+DCs. Combined pre-treatment with donor origin CD8+DCs plus anti-CD154 mAb produced very modest prolongation of allograft survival (MST; 21.5 days). B6 mice pre-treated with DBA/2 CD4+DC plus anti-CD154 mAb did not experience prolonged engraftment of C3H (H-2k) islets as compared with untreated control hosts (MST; 18 days, n = 4). Monotherapy with anti-CD154 mAb pre-treatment a month before transplantation did not enable permanent graft survival (MST; 20 days, n = 5).


Figure 3
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  		Fig. 3 (a) Pre-treatment of DC subsets provoked differential effects on islet allograft survival. Pre-treatment with donor strain CD4+DCs + anti-CD154 mAb (MR1) induced indefinite allograft survival but CD8+DCs + MR1 pre-treatment did not demonstrate synergistic effect (P < 0.01, log-rank test). Also, B6 mice pre-treated with CD4+DC (from DBA/2) + MR1 did not show any survival benefits on third party (C3H; H-2k) islet allograft. (b). Induction of donor-specific allograft tolerance by CD4+DCs + MR1 pre-treatment. Surgical removal of the left kidney bearing the islet allograft was performed after 120 days of transplantation. The second DBA/2 islet allograft was successfully engrafted without rejection in the absence of further immunosuppressive therapy, while islets from third party (C3H; H-2k) were rejected promptly.

 
Pre-treatment with CD4+DC and co-stimulation blockade induces an allograft response that manifests Th1 to Th2 type immune deviation and potent donor-directed suppressive effects by CD4+CD25+ regulatory T cells
To characterize the allograft response mounted by T cells in hosts primed by pre-treatment of donor CD4+DC or CD8+DC subsets, the frequency of alloreactive IL-4 and IFN-{gamma}-producing splenocytes was analyzed 4 weeks after DC pre-treatment. As compared with B6 T cells from hosts pre-treated with donor (DBA/2) CD8+DCs and anti-CD154 mAb, T cells from B6 mice that were pre-treated with CD4+DCs (DBA/2) and anti-CD154 mAb in vivo and challenged with irradiated DBA/2 splenocytes, exhibited an increased frequency of IL-4-producing T cells and decreased frequency of IFN-{gamma}-producing T cells by ELISPOT analysis (Fig. 4). These data are consistent with previous reports that presentation of conventional antigens by immature CD4+DCs may promote Th2 responses (13–15). Moreover, blockade of IL-10-mediated regulatory effects by anti-IL-10R mAbs did not preclude the prolongation of islet allograft survival resulting from combined pre-treatment with CD4+DC and anti-CD154 mAb. Indeed, islet allograft survived >120 days when the recipients were pre-treated with CD4+DC, anti-CD154 mAb and anti-IL-10R mAb (n = 4, DBA/2 islets into B6). Next, we tested the hypothesis that pre-treatment of allograft recipients with the donor CD4+DCs subset plus anti-CD154 mAb would more powerfully strengthen the potency of donor-directed CD4+CD25+ regulatory T cells than pre-treatment with donor CD8+DCs. CD4+CD25+ T cells from CD4+DC plus anti-CD154 mAb pre-treated mice showed more profound suppressive capacity on naive T effector cell proliferation against donor cells or CD8+DC plus anti-CD154 mAb pre-treated mice than CD4+CD25+ T cells from naive mice or from anti-CD154 mAb pre-treated mice. Also, CD4+DC plus anti-CD154 mAb pre-treatment imparted more suppressive capacities to CD4+CD25+ T cells than CD8+LCD plus anti-CD154 mAb pre-treatment when they were added to naive T effector cells (Fig. 5, P < 0.05).


Figure 4
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  		Fig. 4 Pre-treatment with CD4+DC and co-stimulation blockade induced Th2-dominant T cell responses. T cells from B6 mice that were pre-treated with CD4+DCs (DBA/2) and anti-CD154 mAb (MR1) showed increased frequencies of IL-4-producing T cells and decreased frequencies of IFN-{gamma}-producing T cells when encountered DCs from same donors. But CD8+DC plus MR1 treatment did not show the significant deviation of Th1 to Th2 cytokine profile. (*P < 0.05, **P > 0.05, Mann–Whitney U-test).

 

Figure 5
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  		Fig. 5 Pre-treatment with CD4+DC and co-stimulation blocking enhanced suppressive effects of regulatory T cells. Although CD4+CD25+ T cells from either CD4+DC plus anti-CD154 mAb (MR1) or CD8+DC plus MR1 pre-treated mice showed suppressive effects on naive T effector cell proliferation against allo-antigen, CD4+DC plus MR1 pre-treatment granted more profound suppressive activities to CD4+CD25+ T cells than CD8+DC plus MR1 pre-treatment when they were added to naive T effector cells. (*P < 0.05, Mann–Whitney U-test).

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
In the current study, we have examined the effect of pre-immunization with donor strain DC subsets in the allograft response. An improved range of techniques for the isolation of DCs from a variety of mouse tissues has led to the discovery of a number of DC subsets, defined by their expression of distinctive sets of cell-surface markers. Because DCs are rare in secondary lymphoid organs, DC mobilizing factors such as progenipoietin-1, Flt-3 ligand and GM-CSF are often used to mobilize a greater quantity of DCs (21, 33). Nonetheless the use of Flt-3 ligand, for example, produces biased expansion of CD8-bearing DC, and cytokine treatment can alter biologic functions of DCs (22). Because DC-recruiting agents may lead to the qualitative and quantitative changes in DC subsets, we did not use DC-recruiting agents throughout the experiments described herein.

Both immature CD4+DCs and CD8+DCs stimulate allogeneic T cells in the MLR albeit CD8+DCs more vigorously. T cell proliferation was consistently higher after stimulation by CD8+DCs compared with that by CD4+DC subset (Fig. 2). These results are compatible with the notion that CD8+DCs produces high levels of IL-12p70 as reported by some (34–36) but not all investigators (18). Consistent with the in vitro experiments showing that allogeneic cells of either DC subset is stimulatory in the MLR, pre-immunization with either donor strain CD4+DCs or CD8+DCs does not enable prolonged engraftment of islet allograft (Fig. 3). As a cautionary note, DC subtype-related heterogeneity and plasticity may be dependent on the site of DC development (37). In previous work, the failure of donor strain DC infusions to achieve beneficial effects on allograft survival was ascribed to the apparent in vivo maturation of donor DCs into immunostimulatory APCs (38). This proposal is supported by a recent study that injection of maturation-resistant immature myeloid DCs induced permanent allograft acceptance (19). Hence, maintenance of immature phenotype of DC in vivo may be a critical factor for the induction of donor-specific tolerance (27, 28). Because the CD40 to CD154 interaction is a key event for the maturation and activation of immature DC (39), we tested the hypothesis that pre-immunization with donor strain DCs plus adjunctive CD40/CD154 blockade might promote tolerance induction. Anti-CD154 mAb blunts MLR responses to allogeneic CD4+DCs more powerfully than the response to allogeneic CD8+DCs (Fig. 2). As shown in Fig. 3, adjunctive treatment with anti-CD154 mAb at the time of immature CD4+DC, but not CD8+DC, administration can induce donor-specific tolerance. Again the effects noted in vitro were qualitatively similar to those noted in vivo. Pre-treatment with donor strain CD4+DCs, but not CD8+DCs, plus anti-CD154 created donor-specific tolerance. The therapeutic effects of anti-CD154 monotherapy or anti-CD154 plus donor CD8+DCs were unimpressive.

The mechanism by which provision of anti-CD154 plus CD4+DCs, but not CD8+DCs, create tolerance was probed. As previously noted, CD8+DCs more robustly expressed IL-12 than CD4+DCs. Hence, we tested the hypothesis that in anti-CD154-treated recipients, the polarization of alloreactive T cells either into Th1 or Th2 type T cells would be strongly influenced by the phenotype of DCs used in the pre-immunization protocol. To address this point, we investigated effector T cell differentiation into either Th1 or Th2 phenotype using ELISPOT analysis. ELISPOT is a sensitive method for the detection and quantification of antigen-specific responses of activated T cells (40) and is based on the detection of cytokines secreted by a single cell within a polyclonal population. As previously noted, pre-treatment with CD4+DCs, but not CD8+DCs, from donor strain plus CD40/CD154 pathway blockade induced donor-specific allograft tolerance. Although the precise role of Th1 or Th2 response in the allograft response remains somewhat controversial, a Th2 response is linked to, although not necessarily causal for, prolonged allograft survival (41, 42). In fact, Th1 to Th2 immunodeviation (Figs 3 and 4) was more evident in the anti-CD154 mAb plus CD4+DC as compared with the anti-CD154 mAb plus CD8+DC treatment group. By comparison to the anti-CD154 mAb monotherapy or anti-CD154 mAb plus CD8+DC treatment groups, an increased frequency of IL-4-producing T cells and a decreased frequency of IFN-{gamma}-producing T cells was noted in the anti-CD154 mAb plus CD4+DC pre-treatment group (Fig. 4). Hence, priming the recipients with donor strain with immature CD4+DCs in the presence of anti-CD154 mAb has the pivotal role in alloimmune responses by tipping the milieu toward a Th2-dominant response. Interestingly, the administration of an IL-10R-blocking mAb did not abrogate the prolongation of islet allograft survival achieved by combined treatment of CD4+DC and anti-CD154 mAb in our model. Moreover, allogeneic CD4+DCs plus anti-CD154 mAb imparted more profound suppressive activities to CD4+CD25+ T cells against donor allogeneic antigens than combined CD8+DCs plus anti-CD154 mAb treatment (Fig. 5). As shown in Fig. 5, regulatory T cells harvested from the mice that were pre-treated with donor strain CD4+DCs and anti-CD154 mAb showed profound suppression on the proliferation of naive effector T cells (CD4+CD25–) stimulated by donor strain APCs compared with the pre-treatment using CD8+DCs and anti-CD154 mAb. Selective depletion of donor-reactive cytopathic T cells while sparing or enhancing the immunosuppressive regulatory T cells is one of the most critical ways to create transplantation tolerance (43). Although little is known about the mechanisms that control the regulatory CD4+CD25+ T cells, the interactions between immature DCs and regulatory T cells, at least in some models, are indispensable for the initiation of proper regulatory responses where CD40 and CD154 interaction is the main abrogating factor (44). In the other direction, certain type of DCs, especially plasmacytoid DCs, from recipients induced the generation of regulatory T cells and mediated graft tolerance (23). Taken together, it is tempting to speculate that reinforcement of DC subtypes consisted of donor-derived DC subset and recipient-resident DC subset may provide a dependable therapeutic modality for achieving the graft tolerance, though a more sophisticated understanding should be provided.


   Acknowledgements
 
This work was supported in part by a grant from MarineBio21, Ministry of Maritime Affairs and Fisheries, Korea (Y.S.K.) and from the National Institute of Allergy and Infectious Diseases and Juvenile Diabetes Foundation (T.B.S.).


   Abbreviations
 
APC, antigen-presenting cell
CD4+DC, CD4+CD8–CD11c+ dendritic cell
CD8+DC, CD4–CD8+CD11c+ dendritic cell
DC, dendritic cell
ELISPOT, enzyme-linked immunosorbent
GM-CSF, granulocyte macrophage colony-stimulating factor
[3H]TdR, [3H]thymidine
MLR, mixed lymphocyte reaction
MST, mean survival time
PBST, PBS containing 0.05% Tween

   Notes
 
Transmitting editor: K. Inaba

Received 12 April 2006, accepted 22 September 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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