Rapamycin combined with allogenic immature dendritic cells selectively expands CD4+CD25+Foxp3+regulatory T cells in rats

Guo-Ying Wang, Qi Zhang, Yang Yang, Wen-Jie Chen, Wei Liu, Nan Jiang and Gui-Hua Chen

Guangzhou, China

Rapamycin combined with allogenic immature dendritic cells selectively expands CD4+CD25+Foxp3+regulatory T cells in rats

Guo-Ying Wang, Qi Zhang, Yang Yang, Wen-Jie Chen, Wei Liu, Nan Jiang and Gui-Hua Chen

Guangzhou, China

BACKGROUND:Dendritic cells (DCs) can initiate the expansion of regulatory T cells (Tregs), which play an indispensable role in inducing transplantation tolerance. Some studies have investigated the effect of the immunosuppressant rapamycin (Rapa) on Tregsin vitro. However, thein vivoeffect of Rapa combined with immature DCs (iDCs) on Tregs is unknown. This study was undertaken to determine whether allogenic iDCs combined with a short course of Rapa have the ability to selectively expand the CD4+CD25+Foxp3+Tregs in a rat model.

METHODS:Brown Norway rats were injected intravenously with 2×106Lewis iDCs followed by 1 mg/kg per day Rapa intraperitoneally for 7 consecutive days. On day 8, the levels of CD4+CD25+Foxp3+Treg cells in peripheral blood and spleen cells were analyzed by flow cytometry. IL-2, IL-4, TGF-β1, and IFN-γlevels in serum were assessed by ELISA. The experimental animals were divided into four groups: control, Rapa-treated, iDC-treated, and combination-treated.

RESULTS:CD4+CD25+Foxp3+Tregs comprised 7%-8% of CD4+T cells in control rats. Rapa combined with iDCs enhanced this percentage in the peripheral blood and spleen. However, the levels of Tregs did not significantly change after treatment with Rapa or iDCs alone. The levels of CD4+CD25-Foxp3+T cells and CD4+CD25+Foxp3-T cells in CD4+T cells did not significantly change in the combined group. The TGF-β1 level in serum from the combined group increased significantly compared with the other groups.

CONCLUSIONS:A significantly higher percentage of CD4+CD25+Foxp3+Tregs was found in rats treated with allogenic iDCs and a short course of Rapa, along with an increase in the TGF-β1 level in serum. This improved protocol may be a promising therapeutic strategy to increase Tregs, which are beneficial to the induction of peritransplant tolerance.

(Hepatobiliary Pancreat Dis Int 2012;11:203-208)

dendritic cells; regulatory T cells; rapamycin

Introduction

Dendritic cells (DCs), professional antigenpresenting cells, play a pivotal role in the induction and regulation of immune reactivity. The tolerogenic properties of DCs may invariably be linked to stimulating the development of regulatory T cells (Tregs), which are emerging as key players in the downstream effects of tolerogenic DCs.[1]The hallmark of Tregs is the expression of transcriptional repressor forkhead winged helix protein-3 (Foxp3), which is highly expressed by Tregs and is associated with the suppressive effects and phenotype.[2,3]CD4+CD25+Foxp3+Tregs play an important role in immune tolerance to selfantigens and allografts by their ability to suppress the proliferation of conventional effector cells.[4,5]Immature or tolerogenic DCs are important in the normal control and induction of Treg function and expansionin vitroandin vivo.[6]

It is important to minimize immunosuppression and to develop alternative therapeutic strategies to induce and maintain immune tolerance in organ transplantation. One promising way is the induction of the alloantigenspecific immune regulation and maintenance of transplant tolerance by Tregs, in particular the CD4+CD25+Foxp3+Tregs. Rapamycin (Rapa), a macrolide antibiotic with potent immunosuppressive activity, has been introduced in recent years as an anti-rejectiontherapy in organ transplantation. Rapa significantly promotes the expansion and function of murine and human Tregsin vitro.[7,8]Rapa favors Treg survival and function and, by suppressing effector T cells, shifts the balance from immunity towards tolerance.[8-10]Furthermore, Rapa affects endocytosis, antigen presentation, phenotypic maturation and apoptosis of DCs and impairs the ability of DCs to induce allogeneic T cell proliferation while expanding naturally existing Tregs.[11-16]Our previous studies have shown that Rapa inhibits the allostimulatory activity of both immature and mature DCs.[17,18]However, thein vivoeffect of Rapa combined with allogenic immature DCs (iDCs) on the level of the CD4+CD25+Foxp3+Treg cell population is still unknown. Based on previous studies of their respective positive effects of Rapa and iDCs on Tregs, we hypothesized thatin vivoadministration of donor bone marrow-derived iDCs followed by a short course of Rapa could synergize for selective expansion of the CD4+CD25+Foxp3+Tregs. The aim of the present study was to test this hypothesis in an allogenic rat model.

Methods

Animals

Eight-to-ten week-old Lewis (LEW, RT1l, 200-250 g) and Brown Norway (BN, RT1a, 200-250 g) rats were purchased from Vital River Laboratory Animal Technology Co., Ltd., Beijing, China. Procedures involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with the national and international laws and policies.

Generation of bone marrow-derived DCs

The generation of bone marrow-derived DCs has been described previously.[17,18]Briefly, bone marrow cells removed from LEW rat femurs, tibias, and humeri were depleted of red blood cells with hypotonic buffered tris-ammonium chloride (0.83%, pH 7.21). The cells were washed twice in complete medium consisting of RPMI 1640 (Gibco Labs, Grand Island, NY) supplemented with 10% (v/v) heat-inactivated fetal calf serum, 100 U/mL penicillin, 100 µg/mL streptomycin, 2 mmol/L L-glutamine, and 10 mmol/L HEPES buffer (Gibco). Two million of the resulting cells were then cultured in 1 mL complete medium containing 20 ng/mL recombinant rat granulocyte-macrophage colony-stimulating factor (rrGM-CSF, Peprotech, Rocky Hill, New Jersey, USA) and 10 ng/mL recombinant rat interleukin 4 (rrIL-4, Peprotech) in one well of a 24-well plate. On day 2, nonadherent cells were removed; cultures were fed every second day by exchanging half of the medium for fresh GM-CSF- and IL-4-containing medium. On day 6, cells were harvested. The DC preparations used in this study contained 83%-90% OX62+cells that expressed low levels of CD80, CD86 and MHC class II molecules.[17,18]

In vivoexperimental protocol

The animals were divided into four groups (six per group): control, Rapa-treated, iDC-treated, and combination-treated. The combination-treated BN rats received intravenously via the lateral tail vein, 2×106LEW iDCs (day 1) combined with a subtherapeutic regimen of intraperitoneal Rapa (Wyeth-Ayerst, Princeton, NJ) 1 mg/kg per day for 7 consecutive days (days 1 to 7). Rapa was dissolved in 51% wt/vol polyethylene glycol 300, 2.5% wt/vol polysorbate 80, and 10% vol/vol ethanol. Additional groups received either iDCs alone (iDC-treated group), Rapa alone (Rapa-treated group), or saline alone (control group). On day 8, the rats were sacrificed by cervical dislocation and blood samples were taken. The serum was then collected and stored at -20 ℃until analysis.

T lymphocyte preparation and flow cytometric analysis

On day 8, BN rat peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient (Sigma, St. Louis, MO). The spleen was harvested. Single cell suspensions were prepared by grinding the tissues with the plunger of a 5-mL disposable syringe and then suspended in RPMI1640 medium. Splenocytes were treated with hypotonic buffered tris-ammonium chloride (0.83%, pH 7.21) to remove red blood cells. Splenic T cells were enriched using nylon-wool columns. The PBMCs and splenic T cells (106) were triple-stained with FITC-anti-CD4 (OX35; eBioscience, San Diego, CA), APC-anti-CD25 (OX39; eBioscience) and PE-anti-Foxp3 (FJK-16s; eBioscience), according to the manufacturer's protocol for intracellular staining. The levels of CD4+CD25+Foxp3+Tregs in PBMCs and splenic cells were analyzed by flow cytometry (FACSCalibur with CellQuest software; BD PharMingen, CA).

ELISA assay

Measurements of serum IL-2, IL-4, TGF-β1, and IFN-γ levels were performed by ELISA according to the manufacturer's protocols (KeyGen, Nanjing, China).

Statistical analysis

All values are expressed as mean±standard deviation. Statistical analysis was conducted using SPSS 13.0 software. Statistical differences between the groups weredetermined by one-way ANOVA with least-significant difference multiple comparisons. APvalue <0.05 was considered to be statistically significant.

Results

CD4+CD25+Foxp3+Tregs in BN rats treated with allogenic iDCs and Rapa

Rapa combined with donor bone marrow-derived iDCs enhanced the percentages of CD4+CD25+T cells in CD4+T cells in the combination-treated group compared with the control group (14.97±0.99% and 17.96±0.86% in peripheral blood, and 12.94±0.59% and 14.68±0.66% in the spleens of control and combinationtreated rats, respectively) (Table and Fig. 1). The levels of CD4+CD25+T cells in the Rapa alone and iDCs alone groups had no significant change.

The percentage of CD4+CD25+Foxp3+Treg cells in CD4+CD25+T cells significantly increased after Rapa and allogenic iDC treatment (46.49±0.79% and 51.94± 1.34% in peripheral blood, and 58.91±1.97% and 65.22±3.57% in the spleens of control and combination-treated rats, respectively). Within the CD4+T cell population, the combination group maintained a higher incidence of CD4+CD25+Foxp3+Tregs (6.96±0.62% and 9.31± 0.54% in peripheral blood, and 7.46±0.63% and 9.38± 0.89% in the spleens of control and combination-treated rats, respectively). But the levels of CD4+CD25+Foxp3+T cells had no significant change after Rapa alone or iDCs alone (Table and Fig. 1).

Table. Percentages of CD4+CD25+Foxp3+Tregs in CD4+T cells in the peripheral blood and spleen (mean±SD)

Fig. 1. Rapamycin treatment combined with donor iDCs enhanced the percentage of CD4+CD25+Foxp3+T cells in the peripheral blood and spleen of allogenic rats. Data shown were gated on CD4+T cells. Numbers in the dot plot indicate the percentage of CD4+CD25+(A) or CD4+CD25+Foxp3+T cells (B) in CD4+T cells (n=6 per group).

Cytokine levels in serum in different groups

The ELISA results showed that the IL-2, IL-4, and IFN-γ levels in serum did not change in any groups (Fig. 2). However, the TGF-β1 level increased significantly in the combination-treated group despite the similar levels in the Rapa alone and iDCs alone groups (Fig. 2). These results clearly showed that donor iDCs combined with a short course of Rapa stimulated TGF-β1 productionin vivo.

CD4+CD25-Foxp3+and CD4+CD25+Foxp3-T cells from PBMCs and spleen

Fig. 2. Cytokine levels in serum from different groups. TGF-β1 level increased significantly in rats treated with donor iDCs in combination with a short course of Rapa.

Fig. 3. Percentages of CD25-T cells in CD4+Foxp3+T cells in the peripheral blood and spleen were lower in the combination-treated group than in control rats. (A) Cell numbers of CD4+CD25-Foxp3+T cells in 1×104CD4+PBMCs or splenic T cells. (B) Cell numbers of CD4+CD25+Foxp3-T cells in 1×104CD4+PBMCs or splenic T cells. (C) Percentages of CD25-T cells in CD4+Foxp3+PBMCs or splenic T cells after treatment. *:P<0.05, vs control group. The data are representative of 3 separate experiments with similar results (n=6 per group).

We evaluated the effect of Rapa treatment combined with allogenic iDCs on the percentage of CD4+CD25-Foxp3+and CD4+CD25+Foxp3-T cell subsets. This treatment protocol did not alter the levels of CD25-Foxp3+and CD25+Foxp3-T cells in CD4+T cells obtained from PBMCs and the spleen (Fig. 3A and B). In addition, the percentages of CD25-T cells in CD4+Foxp3+T cells in the peripheral blood and spleens of combination-treated rats were significantly lower than those of control rats, especially in spleen T cells (Fig. 3C).

Discussion

Tregs play a key role in maintaining peripheral immune tolerance and regulating graft rejection. iDCs are able to induce the development of naturally-occurring CD4+CD25+Tregs produced in the thymus, and inducible Tregs derived from peripheral CD4+CD25-T cells.[6,19]Consistent with the ability of iDCs to generate Tregsin vitro, thein vivoadministration of iDCs is effective in promoting Treg responses and graft survival in rodent models.[20,21]Immature DCs in the steady state are essential for the induction and maintenance of peripheral tolerance, but they inevitably become mature under inflammatory conditions such as organ transplantation.

There have been many reports regarding the effects of Rapa on the proliferation and suppressive capacity of Tregs.[7-10,22-24]The current study focused on whether or not donor iDCs combined with a short course of Rapa had the ability to expand the CD4+CD25+Foxp3+Tregsin vivo. We found that only about 45% of CD4+CD25+T cells expressed Foxp3+in peripheral blood and about 60% in the spleen. These results are in accord with other reports.[25,26]Although all CD4+CD25+T cells, including both Foxp3+and Foxp3-T cells, significantly increased in the peripheral blood and spleen after Rapa and allogenic iDC treatment, actually Foxp3+but not Foxp3-T cells expanded in the CD4+CD25+population. In addition, administration of Rapa alone for 7 consecutive days or a single injection of iDCs did not change the level of Tregs. These findings are different from some previous studies showing that Rapa or iDC treatment can induce Treg cell expansion.[6-10]This discrepancy might be explained by differences in the time course and cell numbers. However, our results clearly showed that the combination of donor iDCs with a short course of Rapa increased the Foxp3+Treg levelin vivo.

Because cytokines play a pivotal role in the regulation of Treg cell differentiation and proliferationin vivo, we subsequently assessed the serum IL-2, IL-4, TGF-β1, and IFN-γ levels. We found that the serum IL-2, IL-4, and IFN-γ levels did not change in any groups. However, despite unchanged levels of TGF-β1 in the Rapa alone and iDCs alone groups, the TGF-β1 level increased significantly in rats treated with donor iDCs in combination with a short course of Rapa. A recent study has shown that Rapa strongly stimulates TGF-β production by the splenic lymphocytes of BALB/c mice treated with a single dose of Rapa 24 hours after injection of splenic leukocytes (1×106) prepared from C57BL/6 mice.[27]TGF-β has potent anti-proliferative effects on CD4+T cells due to its ability to inhibit IL-2 production. Furthermore, TGF-β can induce the expression of Foxp3 and Treg generation.[28]However, in our study, the TGF-β1 level did not change in rats after Rapa administration for 7 consecutive days. We found a large dose difference between our protocol (1 mg/kg) and the above study (20 mg/kg).[27]It is reasonable that the effect of donor iDCs and a short course of Rapa on Treg proliferation may be mediated at least in part by TGF-β production.

Considering the conflicting results of whether Foxp3 is induced in peripheral CD4+CD25-T cells and they become CD4+Foxp3+Tregs,[29,30]it is still not clear that Rapa treatment combined with allogenic iDCs can induce conventional CD4+CD25-T cells to express both CD25 and Foxp3 simultaneouslyin vivo. Thein vivoimmune environment is so complicated that, in our protocol, we do not know whether the increased CD4+CD25+Foxp3+Tregs naturally occur in the thymus or they are induced from naїve CD4+CD25-or CD4+CD25+T cells in the periphery. However, our experiments showed that the levels of conventional nonregulatory CD4+CD25+Foxp3-T cells did not change significantly in the peripheral blood and spleen of rats treated with Rapa and allogenic iDCs. Taken together with the literature, we suggest that our protocol most probably converted conventional CD4+CD25-Foxp3-T cells to induced CD4+CD25+Foxp3+Treg cells or at least increased CD4+CD25+Foxp3+Treg expansion. The beneficial effects of our protocol can be summarized as follows: 1)In vivoadministration of Rapa suppresses immune responses while significantly promoting the expansion and function of Tregs.[8-10,24]2) A short course of Rapa can minimize the risks of undesirable maturation of donor iDCsin vivo. 3) Rapa not only has the ability to inhibit DC maturation and to impair the ability of DCs to induce allogeneic T cell proliferation while expanding naturally existing Tregs,[11-13]but also inhibits the allostimulatory activity of mature DCs.[17,18]4) Increased TGF-β levels in serum play an important role in the induction of Foxp3 expression.[28]In summary, Rapa promotes the proliferation and expansion of CD4+CD25+Foxp3+regulatory T cells via both direct and indirect pathways (by allogeneic immature or tolerogenic DCs).

In conclusion, significantly higher percentages of CD4+CD25+Foxp3+Treg cells were found in recipient rats treated with donor bone marrow-derived iDCs and a short course of Rapa, along with an increase in the TGF-β1 level in serum. This improved administration protocol may be a promising treatment option for the induction of peritransplant tolerance. A further study on the biological function of slightly increased regulatory T cells will be helpful for developing a method of inducing immune tolerance to a graft in a recipient.

Contributors:WGY proposed the study and wrote the first draft. ZQ analyzed all the data. All authors contributed to the design and interpretation of the study and to further drafts. CGH is the guarantor.

Funding:This work was supported by grants from the Major State Basic Research Development Program of China (973 Program) (2009CB522404), the National Natural Science Foundation of China (30801112, 30972915, and 81000190), and the National Twelfth Five-Year Science and Technology Plan Major Projects of China (2012ZX10002-017).

Ethical approval:All experimental protocol were approved by the Ethics Committees of Sun Yat-Sen University.

Competing interest:No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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February 21, 2011

Accepted after revision July 26, 2011

Author Affiliations: Liver Transplantation Center, Third Affiliated Hospital, Transplantation Research Institute, Sun Yat-Sen University, Organ Transplantation Research Center of Guangdong Province, Guangzhou 510630, China (Wang GY, Zhang Q, Yang Y, Chen WJ, Liu W, Jiang N and Chen GH)

Gui-Hua Chen, MD, Liver Transplantation Center, Third Affiliated Hospital, Transplantation Research Institute, Sun Yat-Sen University, Organ Transplantation Research Center of Guangdong Province, Guangzhou 510630, China (Tel: 86-20-85252276; Fax: 86-20-85252276; Email: chgh1955@263.net)

© 2012, Hepatobiliary Pancreat Dis Int. All rights reserved.

10.1016/S1499-3872(12)60149-0