Ursolic acid promotes robust tolerance to cardiac allografts in mice

Authors

  • Y. Liu,

    1. Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, and Key Laboratory of Ministry of Health and Key Laboratory of Ministry of Education, Wuhan, China
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  • X. Huang,

    1. Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, and Key Laboratory of Ministry of Health and Key Laboratory of Ministry of Education, Wuhan, China
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  • Y. Li,

    1. Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, and Key Laboratory of Ministry of Health and Key Laboratory of Ministry of Education, Wuhan, China
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  • C. Li,

    1. Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, and Key Laboratory of Ministry of Health and Key Laboratory of Ministry of Education, Wuhan, China
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  • X. Hu,

    1. Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, and Key Laboratory of Ministry of Health and Key Laboratory of Ministry of Education, Wuhan, China
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  • C. Xue,

    1. Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, and Key Laboratory of Ministry of Health and Key Laboratory of Ministry of Education, Wuhan, China
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  • F. Meng,

    1. Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, and Key Laboratory of Ministry of Health and Key Laboratory of Ministry of Education, Wuhan, China
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  • P. Zhou

    1. Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, and Key Laboratory of Ministry of Health and Key Laboratory of Ministry of Education, Wuhan, China
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P. Zhou, 1095 Jiefang Road, Wuhan 430030, China.
E-mail: pzhou@tjh.tjmu.edu.cn; pzhou57@hotmail.com

Summary

Nuclear factor (NF)-κB is an important molecule in T cell activation. Our previous work has found that T cell-restricted NF-κB super-repressor (IκBαΔN-Tg) mice, expressing an inhibitor of NF-κB restricted to the T cell compartment, can permanently accept fully allogeneic cardiac grafts and secondary donor skin grafts. In this study, we explore if transient NF-κB inhibition by a small molecular inhibitor could induce permanent graft survival. Ursolic acid, a small molecular compound, dose-dependently inhibited T cell receptor (TCR)-triggered NF-κB nuclear translocation and T cell activation in vitro. In vivo, ursolic acid monotherapy prolonged significantly the survival of cardiac allograft in mice. Assisted with donor-specific transfusion (DST) on day 0, ursolic acid promoted 84·6% of first cardiac grafts to survive for more than 150 days. While the mice with long-term surviving grafts (LTS) did not reject the second donor strain hearts for more than 100 days without any treatment, they all promptly rejected the third-party strain hearts within 14 days. Interestingly, this protocol did not result in an increased proportion of CD4+CD25+forkhead box P3+ regulatory T cells in splenocytes. That adoptive transfer experiments also did not support regulation was the main mechanism in this model. Splenocytes from LTS showed reduced alloreactivity to donor antigen. However, depletion of CD4+CD25+ regulatory T cells did not alter the donor-reactivity of LTS splenocytes. These data suggest that depletion of donor-reactive T cells may play an important role in this protocol.

Introduction

The Holy Grail of transplantation has been to induce antigen-specific tolerance by a short pulse of therapy. This goal has been achieved by various protocols in preclinical models [1–11]. However, there has been no unanimously approved protocol that can induce stable tolerance in the clinic [12]. Thus, it is very important to seek a new protocol that can ultimately induce clinical transplant tolerance. Nuclear factor (NF)-κB is a downstream molecule of T cell receptor (TCR) and co-receptors in T cell activation, and plays a critical role in tolerance induction protocol. In addition, NF-κB is a critical signal molecule following other receptors, such as Toll-like receptors (TLRs) and tumour necrosis factor receptors (TNFRs), which are the most important elements in innate immunity [13]. Thus, inhibiting NF-κB can block both innate and acquired immunity, and may result in more potent immune suppression. Many researchers have proved the importance of NF-κB in immune response. P50-deficient mice have a defect in the T helper type 2 (Th2) response [14]. Survival of cardiac allografts in p50-deficient mice are modestly prolonged [15,16]. Indefinitely prolonged cardiac allograft survival has been demonstrated in the c-Rel-deficient mice because T lymphocytes fail to respond to activation and proliferation signals [16,17]. We and others have shown that the T cell-restricted NF-κB super-repressor (IκBαΔN-Tg) mice, expressing an inhibitor of NF-κB restricted to the T cell compartment, permanently accept cardiac transplants [18,19].

Although a great deal of data have demonstrated that NF-κB can be a target to induce transplant tolerance in mice, until now there has been no applicable clinical NF-κB inhibitor. Ursolic acid, a pentacyclic triterpenoid, has been shown to potently suppress NF-κB activation in various tumour cell lines [20–23], but it is not clear if ursolic acid can suppress NF-κB activation in normal immune cells such as T cells. Here we report that ursolic acid inhibits T cell activation dose-dependently by suppressing NF-κB nuclear translocation. Treatment of C57BL/6 mice with a brief course of ursolic acid plus donor-specific transfusion (DST) on day 0 permits cardiac allografts to survive indefinitely.

Materials and methods

Mice

Six–8-week-old male BALB/c (H2d), C57BL/6 (B6, H2b), C3H/HEN(C3H, H2k) and B6/RAG−/− (H2b) mice were maintained separately at the Animal Facility of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China, under controlled conditions (specific pathogen-free, 22°C, 55% humidity and 12-h day/night). All experimental procedures on animals used in this study were performed under a protocol approved by the Institutional Animal Care and Use Committee at the Tongji Medical College.

T cell stimulation and activation

The single-cell suspensions from lymph nodes of B6 mice were obtained by grinding and filtration through nylon mesh. T cells were then purified with a Pan T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) (95% purity by CD3+ flow cytometry; eBioscience, San Diego, CA, USA). T cells (2 × 106/ml) were stimulated by plate-bound anti-CD3 (2 µg/ml) and soluble anti-CD28 (1 µg/ml) monoclonal antibodies (mAbs) at 37°C with 5% CO2 in RPMI-1640. Ursolic acid was purchased from Sigma-Aldrich Co. (St Louis, MO, USA; purity ≥90%). For ursolic acid inhibition assay, T cells were preincubated with different concentrations of ursolic acid (5 µM, 10 µM and 25 µM) for 2 h before stimulation with CD3/CD28 mAbs. NF-κBp65 activity was detected using the Transfactor Kit (Clontech, Mountain View, CA, USA). CD25 and CD69 expressed on the T cell surface were determined by flow cytometry. Interleukin (IL)-2 in culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA) using a cytokine assay kit (R&D Systems, Minneapolis, MN, USA).

Heterotopic cardiac transplantation

For the initial transplantation, a mouse abdominal cardiac transplant model was employed. Cardiac allografts were transplanted in the abdominal cavity by anastomosing the aorta and pulmonary artery of the graft end-to-side to the recipient's aorta and vena cava, respectively. For secondary transplantation, a mouse cervical transplant model was employed. In this model, end-to-side anastomoses were performed between aorta and pulmonary artery of the donor heart and the carotid artery and jugular vein of the recipients, respectively. The day of operation was recorded as day 0. Graft beating was monitored by daily palpation. The day of rejection was defined as the last day of a detectable heartbeat in the graft, which was confirmed by direct inspection.

Treatment protocol

Cardiac recipients were left untreated or given either ursolic acid, DST, ursolic acid plus DST or control vehicle (0·5% carboxymethyl cellulose). Ursolic acid (20 mg/kg/day) was administered from days −1 to 14. For DST, 1 × 107 donor strain (BALB/c) splenocytes was administered intravenously just post-transplantation.

Enzyme-linked immunospot (ELISPOT) assay for interferon (IFN)-γ producing cells

Ninety-six-well plates (Millipore, Billerica, MA, USA) were coated with anti-IFN-γ mAbs (eBioscience) and blocked with complete RPMI-1640 medium. Splenocytes were isolated from naive B6 mice, mice that had rejected BALB/c hearts (Rej) and mice with long-term surviving grafts (LTS, >100 days). These cells (1 × 106/well) were co-cultured with BALB/c, C3H or B6 spleen lymphocytes (5 × 105/well) pretreated with mitomycin C. After 48 h, biotinylated detection antibody was added for 2 h. Then, avidin–horseradish peroxidase was added for 45 min. Finally, 3-amino-9-ethylcarbazole (AEC) substrate was added for about 4 min. The number of spots per well was calculated using the ImmunoSpot Analyzer (CTL Analyzers, LLC, Cleveland, USA).

Flow cytometry analysis of regulatory T cells (Treg) in mice spleen

The spleen lymphocytes were prepared as a single-cell suspension. For Treg analysis, fluorescein isothiocyanate (FITC)-conjugated anti-CD4 (RM4-5; eBioscience) and allophycocyanin (APC)-conjugated anti-CD25 (PC61·5, eBioscience) were added to the cell suspension simultaneously and incubated at 4°C for 30 min. After fixation and permeabilization, the cell suspensions were stained with phycoerythrin (PE)-conjugated forkhead box P3 (FoxP3) (FJK-16s; eBioscience). After washing twice, pellets were resuspended in 200 µl of cold staining buffer and analysed by fluorescence activated cell sorter (FACS).

Adoptive transfer protocols

Splenocytes were isolated from naive B6 mice, LTS and B6 Rej mice. Indicated numbers of splenocytes were resuspended in 250 µl of phosphate-buffered saline (PBS) and injected intravenously through the retro-orbital plexus of the mice. There were four protocols for DST: (i) 1 × 107 B6 lymphocytes or 1 × 107 LTS lymphocytes into B6/RAG−/− recipients which had been transplanted 25 days previously with BALB/c cardiac allografts; (ii) 1 × 107 B6 lymphocytes and 1 × 107 LTS lymphocytes into B6/RAG−/− recipients that had been transplanted 25 days previously with BALB/c cardiac allografts; (iii) 1 × 107 Rej lymphocytes into B6/RAG−/− recipients that had been transplanted 25 days previously with BALB/c cardiac allografts; and (iv) 1 × 107 Rej lymphocytes into LTS.

Depletion of CD4+CD25+ Tregs and mixed lymphocyte culture

Splenocytes were isolated from LTS and naive B6 mice. LTS splenocytes were depleted of CD4+CD25+ Tregs using the CD4+CD25+ Regulatory T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). These cells were used as responders. Mitomycin C-treated BALB/c splenocytes were used as stimulator. Equal amounts of responder and stimulator (5 × 105) were cultured for 2 days. Then, IL-2 and IFN-γ in culture supernatants were measured by standard sandwich cytokine ELISA (R&D Systems, Minneapolis, MN, USA). Unstimulated responders were used as control.

Statistical analysis

Statistical analysis of survival curves was performed by the Kaplan–Meier log-rank test. Other comparisons were made using one-way analysis of variance (anova). A value of P less than 0·05 was considered statistically significant.

Results

Ursolic acid suppresses NF-κB nuclear translocation and T cell activation

To examine the effect of ursolic acid on T cell activation in vitro, naive B6 T cells were stimulated with CD3/CD28 mAbs without or with ursolic acid at different concentrations. Ursolic acid suppressed NF-κBp65 nuclear translocation triggered by CD3/CD28 mAbs dose-dependently (Fig. 1a). CD25 and CD69 expression on T cells was up-regulated strongly after CD3/CD28 mAb stimulation for 48 h. In contrast, 25 µM ursolic acid inhibited the up-regulation of these two markers significantly (Fig. 1b). IL-2 secretion was also suppressed dose-dependently by ursolic acid compared to the control group (Fig. 1c).

Figure 1.

Ursolic acid inhibits nuclear factor (NF)-κBp65 nuclear translocation and T cell activation. Isolated B6 T cells were stimulated with CD3/CD28 monoclonal antibodies (mAbs) with or without different concentrations of ursolic acid. (a) The activity of NF-κBp65 was determined using the NF-κB TransFactor kit (Clontech) in the nuclear fraction (representative of three independent experiments; *P < 0·05 compared with B6 T cells; #P < 0·05 compared with B6 T cells + CD3/CD28 mAbs). (b) CD25 and CD69 expression on T cell were analysed by flow cytometry. (c) Interleukin (IL)-2 in culture supernatants were measured by enzyme-linked immunosorbent assay (representative of three independent experiments; *P < 0·05 compared with B6 T cells; #P < 0·05 compared with B6 T cells + CD3/CD28 mAbs). UA: ursolic acid.

Ursolic acid plus DST on day 0 promotes donor-specific transplant tolerance

Ursolic acid monotherapy (20 mg/kg/day, from days −1 to 14) prolonged the survival of allografts to 36·8 ± 0·6 days [P < 0·05 versus control vehicle (7·8 ± 05 days)]. When DST was given alone to B6 recipients immediately after transplantation with BALB/c hearts, these grafts were promptly rejected at a similar rate to those transplanted into untreated controls (P > 0·05, 8 ± 0·7 days versus 7·4 ± 0·3 days). However, ursolic acid together with DST on day 0 led to 84·6% of grafts surviving for more than 150 days (Fig. 2). To test if donor-specific tolerance was induced, LTS were challenged with a second donor-specific or third-party C3H heart. While these mice did not reject the second BALB/c heart for more than 100 days without any treatment, they all promptly rejected the C3H hearts within 14 days. In accordance with the above results, IFN-γ ELISPOT also showed that LTS splenocytes had an attenuated response to BALB/c antigen in vitro and had the same response to C3H antigen as that in the control group (Fig. 3).

Figure 2.

Ursolic acid plus donor-specific transfusion (DST) on day 0 leads to permanent allograft survival. BALB/c hearts were transplanted into B6 mice. Mice were either left untreated (untreated control) or received 0·5% carboxymethyl cellulose (control vehicle) or received 1 × 107 BALB/c spleen lymphocytes intravenously on day 0 (DST), or received ursolic acid (UA), or received both DST and UA (UA + DST).

Figure 3.

Attenuated donor-specific response of splenocytes from LTS. Splenocytes from BALB/c, B6, LTS or Rej mice were stimulated with mitomycin C-treated BALB/c, C3H or B6 splenocytes. The frequency of interferon-γ producing cells was determined using an enzyme-linked immunospot assay. Quantification is depicted above a representative plate as the mean number of spots ± standard error of the mean (*P < 0·01). LTS: mice with long-term-surviving grafts; Rej: B6 mice that had just rejected BALB/c hearts.

Ursolic acid plus DST on day 0 did not increase the proportion of CD4+CD25+FoxP3+ T cells

To clarify the mechanisms involved in transplant tolerance induced by ursolic acid plus DST on day 0, we detected the frequency of CD4+CD25+FoxP3+ T cells in recipient spleens 7 days post-transplantation. Results showed that neither DST on day 0 alone nor ursolic acid plus DST on day 0 resulted in an increase in CD4+CD25+FoxP3+ T cells (Fig. 4). Furthermore, LTS did not have a higher frequency of CD4+CD25+FoxP3+ T cells (Fig. 4).

Figure 4.

Ursolic acid plus donor-specific transfusion (DST) on day 0 does not increase the frequency of CD4+CD25+forkhead box P3 (FoxP3)+ regulatory T cells (Tregs) in mice spleen. Splenocytes were stained with fluorescein isothiocyanate (FITC)-CD4, allophycocyanin (APC)-CD25 and phycoerythrin (PE)-FoxP3 7 days post-transplantation. CD25+FoxP3+ T cells were analysed within the CD4-positive gate. B/c: BALB/c; CMC: carboxymethyl cellulose; UA: ursolic acid; LTS: mice with long-term-surviving grafts.

Tolerance in LTS is not dominant

Ursolic acid plus DST on day 0 did not increase the proportion of CD4+CD25+FoxP3+ T cells in recipient splenocytes. To understand the situation further, adoptive transfer experiments were carried out. B6RAG−/− mice pretransplanted with BALB/c hearts for 25 days transferred with 1 × 107 LTS splenocytes did not reject BALB/c hearts (Fig. 5a). However, the BALB/c hearts were rejected promptly in B6RAG−/− mice transferred with either 1 × 107 naive B6 splenocytes alone or 1 × 107 naive B6 splenocytes and 1 × 107 LTS B6 splenocytes (Fig. 5a). To exclude the possibility that Tregs existed outside the spleen, splenocytes from B6 mice which had just rejected BALB/c hearts were transferred into LTS, and also into B6RAG−/− mice as controls. Results indicated that both groups rejected cardiac grafts within 14 days (Fig. 5b).

Figure 5.

Tolerance in LTS is not dominant. (a) BALB/c hearts were transplanted into B6/RAG−/− mice. Twenty-five days post-transplant, B6/RAG−/− mice received an intravenous injection with 1 × 107 LTS splenocytes, or 1 × 107 naive B6 splenocytes, or 1 × 107 naive B6 splenocytes and 1 × 107 LTS splenocytes. (b) 1 × 107 Rej splenocytes were transferred into LTS or B6/RAG−/− mice (transplanted with BALB/c hearts 25 days previously). LTS: mice with long-term-surviving grafts; Rej: B6 mice that had just rejected BALB/c hearts.

Depletion of CD4+CD25+ Tregs did not alter donor-reactivity of LTS splenocytes

To investigate if donor-reactive T cells were depleted in LTS, CD4+CD25+ Tregs were removed from LTS splenocytes. Results showed that LTS splenocytes being depleted of CD4+CD25+ Tregs produced the same levels of IL-2 and IFN-γ as that of LTS splenocytes in response to mitomycin C-treated donor splenocytes. However, both of them secreted lower levels of IL-2 and IFN-γ compared to naive B6 splenocytes (Fig. 6).

Figure 6.

Depletion of CD4+CD25+ regulatory T cells (Tregs) did not alter donor-reactivity of LTS splenocytes. Splenocytes were isolated from BALB/c, LTS and naive B6 mice. BALB/c splenocytes treated by mitomycin C were used as stimulator. Naive B6 splenocytes, LTS splenocytes and LTS splenocytes, depleted of CD4+CD25+ Tregs, were used as responders; 5 × 105 stimulators were left unstimulated or stimulated by 5 × 105 responders for 2 days. Interleukin-2 and interferon-γ in culture supernatants were measured by standard sandwich cytokine enzyme-linked immunosorbent assay (representative of three independent experiments; *P < 0·01 compared with stimulated B6).

Discussion

The present study showed two principal findings. (i) ursolic acid inhibits NF-κB translocation and T cell activation triggered by TCR ligation; and (ii) ursolic acid plus DST on day 0 induces robust donor-specific tolerance.

NF-κB is an important downstream molecule of TCR, co-receptors and TLR. It is critical in both acquired and innate immunity [24]. Our previous work found that IκBαΔN-Tg mice, expressing an inhibitor of NF-κB restricted to the T cell compartment, can permanently accept fully allogeneic (H-2d) cardiac grafts and secondary donor skin grafts [19]. It suggests that impaired NF-κB activation can lead to long-term graft survival without any other treatment. This was supported by other research using c-Rel-deficient mice [17], in which long-term allograft survival was also achieved. These phenomena indicate that NF-κB could be a potential target for inducing transplant tolerance. However, permanently impaired NF-κB activation must affect anti-infection immunity, anti-tumour immunity and other physiological functions mediated by NF-κB. It may be not beneficial to use long-term NF-κB inhibition in clinical transplantation, so we question if transient NF-κB inhibition by small molecular inhibitors can lead to long-term graft survival.

Ursolic acid, a small molecule that can suppress NF-κB activation in various tumour cells [23], inhibits p65 nuclear translocation dose-dependently following TCR ligation. In this study, we found that T cell activation is also suppressed dose-dependently by ursolic acid, as shown by IL-2 secretion and cell surface expression of CD25 and CD69. Therefore, we assessed its effect on rejection in mice cardiac transplantation. In accordance with its effect in vitro, ursolic acid alone delayed the rejection of cardiac allograft significantly. However, permanent graft survival cannot be achieved by transient ursolic acid monotherapy. We hypothesize that if assisted with other management, a brief course of ursolic acid administration could promote permanent cardiac survival. Pretransplant DST has proved to be an effective method to induce transplant tolerance [3,25–27]. Combined DST and CD154 mAbs induce all kinds of transplant tolerance [10,25,28]. Because NF-κB is a key molecule downstream of CD154 and other receptors, we speculate that inhibition of NF-κB by ursolic acid, assisted with DST, could also induce transplant tolerance.

Because DST cannot be given before transplantation in many clinical transplant situations, especially when using organs from deceased donors, we chose DST on day 0 in this study. Interestingly, when DST alone was given just after transplantation, it had no effect on cardiac allograft survival. However, when DST was administered on day 0 plus ursolic acid from days −1 to 14 simultaneously, 84·6% of allografts survived for more than 150 days. To test if donor-specific tolerance was induced, secondary cardiac transplants from donor strain BALB/c or third-party C3H were transplanted. In accordance with our expectation, the second BALB/c cardiac allograft survived for more than 100 days without any other treatment, but the second C3H cardiac allograft was rejected at the same rate as that in untreated C57BL/6 recipients. In vitro, IFN-γ ELISPOT also showed that LTS had a reduced response to donor-specific antigen but not to the third-party antigen. All these results indicate that donor-specific tolerance is induced by ursolic acid plus DST on day 0. To explore the mechanisms underlying donor-specific tolerance in this model, we checked CD4+CD25+FoxP3+ Tregs in recipient spleens. Neither DST alone on day 0, nor ursolic acid plus DST on day 0, increased the proportion of CD4+CD25+FoxP3+ Tregs 7 days post-transplantation. Furthermore, LTS also showed no higher frequency of CD4+CD25+FoxP3+ Tregs. Although the proportion of CD4+CD25+FoxP3+ Tregs was not increased, it is possible that other types of regulatory elements might exist. Thus, adoptive transfer was employed. In contrast to wild-type splenocytes, splenocytes from LTS were not capable of rejecting BALB/c hearts in B6/RAG−/− mice. More importantly, when added at a 1:1 ratio, splenocytes from LTS could not suppress the rejection of BALB/c hearts on B6/RAG−/− mice mediated by wild-type splenocytes. This suggests that splenocytes from LTS had not enough regulatory capacity to control the alloresponse of conventional wild-type splenocytes, but the above results cannot exclude the possibility that Tregs resided outside the spleen. Thus, the splenocytes from mice which has just rejected BALB/c hearts were transferred into LTS and B6/RAG−/− mice with BALB/c hearts. Surprisingly, LTS showed the same rate of rejecting BALB/c hearts as did the B6/RAG−/− mice. All the above indicate that regulation is not the main mechanism in the induction and maintenance of transplant tolerance induced by ursolic acid plus DST on day 0.

As revealed by ELISPOT, LTS splenocytes did show strongly reduced donor alloreactivity. Thus, we speculate deletion of alloreactive T cells may be the critical mechanism in this model. Therefore, we depleted CD4+CD25+ Tregs from LTS splenocytes. As we expected, CD25- LTS splenocytes showed the same ability to produce IL-2 and IFN-γ as LTS splenocytes in response to donor antigens. Furthermore, both of them secreted lower levels of IL-2 and IFN-γ compared with naive B6 splenocytes. All these results indicated that deletion of donor-alloreactive T cells was the main mechanism involved in this model. However, this needs further investigation by TCR transgenic mice to provide direct evidence.

In conclusion, our results show that protocol-treated mice involving a brief course of ursolic acid can achieve donor-specific transplantation tolerance related to the depletion of donor-alloreactive T cells. This protocol, perhaps with some modification, should be tested on non-human primates to yield a clinically applicable procedure for the induction of tolerance in solid organ transplant.

Acknowledgements

This research was supported by National Natural Science Foundation of China Grant: 30772041

Disclosure

None.

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