The Importance of CD25 CD4 Regulatory T Cells in Mouse Hepatic Allograft Tolerance

Transcription

1 LIVER TRANSPLANTATION 12: , 2006 ORIGINAL ARTICLE The Importance of CD25 CD4 Regulatory T Cells in Mouse Hepatic Allograft Tolerance Xiaofeng Jiang, 1,2 Miwa Morita, 3 Atsushi Sugioka, 3 Michishige Harada, 1 Satoshi Kojo, 1 Hiroshi Wakao, 1 Hiroshi Watarai, 1 Nobuhiro Ohkohchi, 2 Masaru Taniguchi, 1 and Ken-ichiro Seino 1 1 RIKEN Research Center for Allergy and Immunology, Yokohama City, Kanagawa, Japan, 2 Department of Surgery, Institute of Clinical Medicine, University of Tsukuba, Tsukuba Science City, Ibaraki, Japan, and 3 Department of Surgery, Institute of Clinical Medicine, Fujita Health University, Toyoake, Aichi-Ken., Japan In mouse liver transplantation, tolerance is readily inducible. Recent studies have revealed that CD25 CD4 regulatory T cells play an important role in regulating various immune responses, including transplant tolerance. However, the contribution of these cells to tolerance in mouse liver transplantation has not been elucidated. We showed here that depletion of CD25 CD4 T cells increased proliferative response of CD4 T cells and cytotoxic T lymphocyte induction of CD8 T cells. Depletion of these cells in the recipient but not in the donor before liver transplantation caused rejection. Furthermore, the number of CD25 CD4 population and forkhead/winged helix transcription factor expression in liver mononuclear lymphocytes derived from tolerant mice were higher than those from grafts undergoing rejection. In conclusion, these results indicate that CD25 CD4 regulatory T cells in the recipient but not in the donor of liver transplantation are important for the tolerance induction. Liver Transpl 12: , AASLD. Received October 26, 2005; accepted February 25, Transplantation is now an established therapeutic modality for the correction of a wide range of disease states. The fate of organ transplants between unrelated individuals of the same species is almost always rejection unless the recipient receives immunosuppressive drugs. However, liver transplants are an exception. In an allogeneic mouse liver transplantation model, tolerance is inducible without immunosuppressive measures, even when the transplant combination is fully major histocompatibility mismatched, and wild, not laboratory bred mice, are used. 1 Similarly, human liver allografts are relatively easily accepted, as some cases in which tolerance is induced after programmed weaning of standard immunosuppression have been reported. 2 Therefore, it seems important to understand the mechanisms underlying the immune-tolerogenic properties of liver transplantation to develop a more effective protocol to induce transplant tolerance. Concerning immunological tolerance, there is accumulating evidence that, apart from clonal deletion and anergy, immune-regulatory CD25 CD4 T cells contribute to the transplant tolerance. 3,4 CD25 CD4 T cells were originally identified in mice; subsequently, a comparable population with identical phenotypic and functional properties has been defined also in humans. 5 They have unique immunological characteristics; for example, they show a partially anergic phenotype in that they proliferate poorly upon T cell receptor stimulation in vitro and can potently suppress the activation and proliferation of other CD4 and CD8 T cells. 6-8 They offer protection against autoimmune diseases, 5 inflammatory bowel disease, 9 and graft versus host disease, 10,11 and they can also impede antitumor immunity. 12 However, their contribution to liver transplantation tolerance has not been clarified in detail, although an increased frequency of CD25 CD4 T cells in operationally tolerant patients with liver transplantation was observed. 13,14 Using a mouse liver transplantation model, we report a critical contribution of CD25 CD4 T cells of recipients but not of donors to liver transplant tolerance induction. Recently, it has been shown that a transcrip- Abbreviations: Foxp3, forkhead/winged helix transcription factor; mab, monoclonal antibody; IL, interleukin; TNF, tumor necrosis factor; HGF, hepatocyte growth factor. Address reprint requests to Ken-ichiro Seino, Institute of Medical Science, St. Marianna University School of Medicine, Sugao, Miyame-ku, Kawasaki, Kanagawa , Japan. Telephone: ; FAX: ; seino@rcai.riken.jp DOI /lt Published online in Wiley InterScience ( American Association for the Study of Liver Diseases.

2 HEPATIC ALLOGRAFT TOLERANCE AND REGULATORY T CELLS 1113 Figure 1. Depletion of CD25 CD4 T cells in the recipient but not in the donor before liver transplantation caused hepatic graft rejection. (A) C3H or DBA/2 mice were injected with 0.25 mg of PC61 on day 0. Splenocytes and liver mononuclear cells were obtained from these mice at the indicated time points. Percentage of CD25 CD4 cells was analyzed using a fluorescenceactivated cell sorter (Calibur). Experiments were repeated twice with similar results. (B) Orthotopic liver transplantation was performed using DBA/2 or C3H mice as donors and C3H mice as recipients. In allogeneic transplantation, the DBA/2 donors or C3H recipients were injected once with 0.25 mg of PC61 4 days or 1 day, respectively, before transplantation. As a control, allogeneic transplantation was performed with control antibody treatment to the recipients. Each group includes 5 recipients. Circles represent C3H to C3H; open squares represent DBA/2 to C3H, control Ab to recipients; closed squares represent DBA/2 to C3H, PC61 to recipients; triangles represent DBA/2 to C3H, PC61 to donors. tion factor, forkhead/winged helix transcription factor (Foxp3), is responsible for the development of CD25 CD4 T cell population Induction of Foxp3 expression converts CD25 CD4 T cells toward a regulatory T cell phenotype similar to that of CD25 CD4 T cells. 15 Therefore, the expression level of Foxp3 reflects the regulatory function of the CD25 CD4 T cell population. In our present study, the number of CD25 CD4 T cells and Foxp3 expression were upregulated in the tolerant hepatic allografts but not in the spleens. These results suggest that some liver-specific factor(s) are involved in the increase of CD25 CD4 T cells, which is important for the acceptance of liver allografts. MATERIALS AND METHODS Reagents Fluorescein isothiocyanate or phycoerythrin-conjugated anti-mouse CD4 (L3T4) monoclonal antibody (mab) and CD25 mab (7D4), unconjugated anti-cd16/ CD32 (2.4G2; Fc block), CD3 mab and CD25 mab (PC61) were purchased from BD Biosciences (San Jose, CA). Rat immunoglobulin G mab (control antibody) and recombinant interleukin (IL)-2 tumor necrosis factor (TNF) were purchased from Sigma (St. Louis, MO). Recombinant hepatocyte growth factor (HGF) was purchased from R&D Systems, Inc. (Minneapolis, MN). Anti-CD4 and CD8 microbeads were purchased from Miltenyi Biotec (Auburn, CA). Percoll was purchased from Amersham Bioscience (Uppsala, Sweden). Mouse Liver Transplantation Wild-type C3H/HeJ (C3H, H-2 k ) and DBA/2 (H-2 d ) mice were purchased from Japan Clea (Tokyo, Japan). All mice were bred and maintained in RIKEN and Fujita Health University animal facilities under specific pathogen-free conditions and used in accordance with the guidelines of RIKEN and Fujita Health University. The animal committee of the RIKEN and Fujita Health University approved the experiments. Male mice 8 to 12 weeks old were used in this study. Orthotopic liver transplantation was performed as previously reported, 21 with some modifications (without artery revascularization). C3H and DBA/2 mice were used as donors and C3H mice were used as recipients. Survival of each recipient was determined daily as graft survivals. Anti-mouse CD25 mab (PC61) Treatment For in vivo CD25 CD4 T-cell depletion, mice were injected once with 0.25 mg of PC61. PC61 was injected into donor DBA/2 mice at day 4 before liver transplantation and to recipient C3H mice at day 1 before liver transplantation (Fig. 1A). In another protocol, recipients were injected with 0.25 mg of PC61 after the liver transplantation (Table 1). Analysis of Percentage of CD25 CD4 T Cells Liver mononuclear cells, splenocytes, and blood were obtained from recipients or naïve mice with treatment of PC61. Hepatic lymphocytes were separated from hepatocyte nuclei and other cellular debris by spinning them through a Percoll density gradient. 22 After incubation with 2.4G2 for 10 minutes, they were sequentially stained with fluorescein isothiocyanate -conjugated anti-mouse CD4 mab and phycoerythrin -conjugated anti-mouse CD25 mab (7D4). Then these cells were analyzed using a fluorescence-activated cell sorter (Calibur, Becton Dickinson, San Jose, CA). Histological Examination Livers were removed at indicated time point and fixed in buffered 10% formalin. Five-micrometer paraffin-em-

3 1114 JIANG ET AL. TABLE 1. Survival of Liver Transplant Recipients with Delayed CD4 CD25 Cell Depletion Recipient Number Time of PC61 Injection (days after liver transplantation) Survival (days) 1 29, , 36 80* 3 20, 29, 39 79* 4 21, 30, 40 80* 5 30, 37, 55, 60, NOTE: DBA/2 mice were used as donors and C3H mice as recipients. Recipients were injected with 0.25 mg PC61 at indicated time points after the liver transplantation. Survival of each recipient was determined daily as graft survivals. *Recipients were sacrificed at indicated time points, and liver mononuclear cells, splenocytes, and blood lymphocytes were obtained to examine the percentage of CD25 CD4 T cells or Foxp3 expression. bedded sections were cut and stained with hematoxylin & eosin. Mixed Lymphocyte Reaction and Cytotoxic T-lymphocyte Assay Twenty-five days after the liver transplantation, splenocytes were obtained from allogeneic recipients with treatment of PC61 or control antibody. CD4 and CD8 T cells were purified with a magnetic-activated cell sorter (Miltenyi Biotec, Bergisch-Gladbach, Germany) according to the manufacturer s protocol. The resulting purity was 98% in all experiments /well CD4 T cells were cocultured with 35 Gy-irradiated, T-cell-depleted (using a magnetic-activated cell sorter, CD90 negative fraction) splenocytes from DBA/2 or C3H mice at an indicated number of stimulator cells for 4 days, and proliferative responses were measured by [ 3 H] thymidine incorporation of the last 8-hour culture. CD8 T cells were cocultured with 35 Gy-irradiated T-cell-depleted DBA/2 or C3H splenocytes at 1:2 cell number ratio in the presence of IL-2 (50 U/mL). Five days later, live cells were collected and cytotoxic activity against P815 cells (H-2 d, /well) at indicated effector-to-target ratios was assessed by a standard 51 Cr release assay. Real-Time Reverse Transcriptase Polymerase Chain Reaction Analysis At an indicated time point after liver transplantation, grafts, naïve livers, and spleens were harvested, and mononuclear lymphocytes were purified. Total RNA was extracted from the lymphocytes using RNeasy Micro kit or RNeasy Protect Mini kit (QIAGEN GmbH, Hilden, Germany). After complementary DNA synthesis, quantity of messenger RNA for Foxp3 expression was examined by the ABI PRISM 7000 (Applied Biosystems, Foster City, CA), normalized with hypoxanthine phosphoribosyltransferase (HPRT) expressions. The HPRT-normalized value from liver lymphocytes, CD25 CD4 T cells, or naïve DBA/2 splenocytes gene expressions was designated as the calibrator. Final relative quantity of messenger RNA in each sample was expressed relative to the calibrator. Proliferation of CD25 CD4 T Cells CD25 CD4 T cells were sorted from untreated C3H splenocytes using a fluorescence-activated cell sorter (Vantage, Becton Dickinson). The purity of the cells was 99%. The CD25 CD4 T cells ( /well) were cultured with anti-cd3 mab (10 g/ml). IL-2 (100 U/mL), TNF- (100 ng/ml), or HGF (100 ng/ml) was added to the culture. For the last 8 hours of the 3-day culture, poliferative responses were measured by [ 3 H] thymidine incorporation. Statistical Analysis Comparisons were performed using the Mann-Whitney U test. A value of P 0.05 was considered significant. RESULTS CD25 CD4 T Cells in the Recipient but not in the Donor are Important for the Tolerance Induction of Hepatic Allograft We first investigated the effect of the in vivo administration of anti-cd25 mab (PC61) on CD25 CD4 T cell population in spleens and livers of C3H or DBA/2 mice. A single injection of PC61 temporarily eliminated splenic and hepatic CD25 CD4 T cells of not only C3H but also DBA/2 mice (Fig. 1A). After an injection of 0.25 mg PC61, CD25 CD4 T cells in C3H mice reduced maximally to 0% almost on day 1 and continued for 27 days. They fully recovered by day 80. In DBA/2 mice, CD25 CD4 T cells reduced maximally on day 1 and continued for 4 days. They began to recover on day 9 and fully recovered by day 28 in spleens and by day 45 in livers (Fig. 1A). Therefore, for subsequent analyses, we used a single injection of 0.25 mg PC61 for donor DBA/2 mice on day 4 before the transplantation and for recipient C3H mice at day 1 before the transplantation. Next we performed liver transplantation experiments. C3H recipients treated with a control antibody receiving DBA/2 hepatic allografts survived over 70 days as in the syngeneic group (Fig. 1B), confirming that the mouse liver transplantation is a spontaneous tolerance-inducing model. However, the allograft acceptance did not occur, and 80% of recipients died within 40 days of when PC61 was given to the liver transplant recipients (P 0.05 compared with the control antibody treatment in DBA/2 to C3H combination; Fig. 1B). This decreased survival was accompanied by a visible increase in cellular infiltration, periportal inflammation, atrophy of hepatocytes and necrosis (Fig. 2A), when compared with allografts from recipients treated with a control antibody, which showed well-preserved hepatic

4 HEPATIC ALLOGRAFT TOLERANCE AND REGULATORY T CELLS 1115 Figure 2. Histological analysis of DBA/2 hepatic allografts in C3H recipients. C3H recipients were treated with PC61 (A) or control antibody (B) at 1 day before transplantation. Hepatic allografts were removed on days after transplantation and stained by hematoxylin & eosin staining. Representative data of more than 3 grafts in each group. morphology with sparse mononuclear cell infiltrates (Fig. 2B). However, depletion of CD25 CD4 T cells in DBA/2 donors had no effect on the survival time (Fig. 1B). These results indicate that tolerance induction on this system is dependent on CD25 CD4 T cells in the recipients but not in the donors. Depletion of CD25 CD4 T cells Increased Proliferative Response of CD4 T Cells and Cytotoxic T Lymphocyte Induction of CD8 T Cells Next, we analyzed the effect of CD25 CD4 T cells on proliferation of CD4 T cells and cytotoxic activity of CD8 T cells against allogenic stimulation. We found that depletion of CD25 CD4 T cells in C3H recipients resulted in enhancement of both the proliferative response of CD4 T cells and the cytotoxicity of CD8 T cells against donor-type alloantigen (Fig. 3A,3B). When syngenic cells were used as the stimulators, no significant proliferation and cytotoxicity were observed, indicating that these T-cell responses were alloantigen specific. Therefore, CD25 CD4 T cells suppress the function of alloreactive CD4 and CD8 T cells in the recipients. CD25 CD4 T Cells are Concentrated in the Grafts We further examined the population of CD25 CD4 T cells in the hepatic grafts. The accepted liver allografts with control antibody treatment contained increased percentage of CD25 CD4 T cells compared with rejected allografts and syngeneic grafts (Fig. 4A). This increase of CD25 CD4 T cells lasted at least for 70 days after the transplantation (data not shown). The number of mononuclear cells in the liver allografts was not significantly changed (data not shown); therefore, the absolute number of CD25 CD4 T cells increased. We also examined expression of Foxp3, a specific transcription factor for regulatory CD25 CD4 T cells Figure 3. Depletion of CD25 CD4 T cells increased proliferative response of CD4 T cells and cytotoxic T lymphocyte induction of CD8 T cells against alloantigen. Twenty-five days after liver transplantation, splenocytes were obtained from allogeneic recipients that received the treatment with PC61 (represented by circles) or control antibody (represented by squares). CD4 and CD8 T cells were purified from splenocytes with a magnetic-activated cell sorter. (A) /well CD4 T cells were cocultured with 35 Gyirradiated T-cell-depleted splenocytes from DBA/2 or C3H mice at the indicated number of stimulator cells, and proliferative responses were measured by [ 3 H] thymidine incorporation of the last 8-hour culture. When C3H-derived cells were used as the stimulator, the CD4 T-cell responses in either group were less than at each stimulator number. (B) CD8 T cells were cocultured with 35 Gy-irradiated T-cell-depleted DBA/2 or C3H splenocytes at 1:2 of cell number ratio. Five days later, live cells were collected and cytotoxic activity against /well P815 cells (H-2 d ) in vitro was assessed by a standard 51 Cr release assay. When C3H-derived cells were used as the stimulator of exvivo stimulation, the cytotoxicity of CD8 T cells in either group was less than 8.8% even at 40 of effector to target ratio. The data are the mean of triplicates and are representative of two independent experiments with similar results. MLR, mixed lymphocyte reaction; CTL, cytotoxic T lymphocyte; cpm, counts per minute; E/T, effector to target. Results showed that it was upregulated in the mononuclear cells of accepted liver allografts (Fig. 4B), thereby confirming the concentration of CD25 CD4 regulatory T cells in the liver allografts. On the other hand, no increase of the percentage of CD25 CD4 T cells and no upregulation of FoxP3 expression were observed in recipients spleens (Fig. 4A,4C), suggesting that the increase of CD25 CD4 T cells occurred only in the transplanted livers. In an attempt to elucidate the mechanism of CD25 CD4 T-cell expansion only in the accepted liver allograft, we next examined a possible effect of TNF- and HGF, known to be involved in liver regeneration, on the expansion of CD25 CD4 T cells. By using a proliferation assay of CD25 CD4 T cells in an antigenpresenting cell-free system, we found that TNF- but not HGF reversed the anergic state of CD25 CD4 T cells, although the efficacy was one-tenth that of IL-2 (Fig. 4D). No synergistic effect between TNF- and HGF was observed (data not shown).

5 1116 JIANG ET AL. 1). With this delayed PC61 treatment, complete disappearance of CD25 CD4 T cells was observed by fluorescence-activated cell sorter analysis (Fig. 5A). Similarly, the Foxp3 level in liver mononuclear cells was also decreased to the extent seen in PC61-treated DBA/2 mice (Fig. 5B). However, these protocols could not shorten the survival of hepatic allografts (Table 1), indicating that CD25 CD4 T cells are not essential for the maintenance of tolerance. Figure 4. CD25 CD4 T cells are concentrated in the allografts. (A) The liver mononuclear cells and splenocytes were obtained from the indicated recipients 25 days after the transplantation. The percentage of CD25 CD4 T cells was analyzed using a fluorescence-activated cell sorter (Calibur). Foxp3 messenger RNA levels in the liver (B) mononuclear cells and (C) splenocytes were examined with real-time reverse transcriptase polymerase chain reaction. (D) TNF- relieved CD25 CD4 T cells from their anergic state /well of CD25 CD4 T cells sorted from untreated C3H spleens were cultured with anti-cd3 mab in the presence of IL-2 (100 U/mL), TNF- (100 ng/ml), or HGF (100 ng/ml). Poliferative responses were measured by [ 3 H] thymidine incorporation for the last 8 hours of a 3-day culture. All the experiments were repeated 3-5 times with similar results. MNC, mononuclear cell; cpm, counts per minute. CD25 CD4 Regulatory T Cells are not Essential for the Maintenance of Tolerance Finally, we depleted CD25 CD4 T cells in the recipients with a delayed timing after tolerance was established. Recipients were injected with 0.25 mg of PC61 at indicated time point after liver transplantation (Table DISCUSSION The potential immunoregulatory properties and the profound anergic phenotype of CD25 CD4 T cells make them a very desirable subpopulation for participation in liver transplantation tolerance. Hence, we expect that the depletion of CD25 CD4 T cells will affect mouse liver transplantation tolerance. We found that the spontaneous allograft acceptance did not occur when PC61 was given to the liver transplant recipients but not to the donors (Fig. 1B, Fig. 2). As for the ineffectiveness of donor treatment with PC61 on graft survival, we have examined the presence of DBA/2-derived cells in the C3H recipients with a fluorescence-activated cell sorter and reverse transcriptase polymerase chain reaction by detecting H-2 d -positive cells after the donor treatment. However, H-2 d -positive cells (including CD25 CD4 cells) have never been detected in the peripheral blood, spleen, or liver of C3H recipients from 7 to 52 days after transplantation (data not shown). Therefore, it seems unlikely that the ineffectiveness is due to an insufficient deletion of CD25 CD4 cells in DBA/2 donors. When CD25 CD4 T cells were depleted after tolerance was established, the depletion did not influence the tolerant status (Table 1), indicating that CD25 CD4 T cells are required for the induction but not maintenance of tolerance against hepatic allografts. The results of this study strongly suggested that a mechanism other than CD25 CD4 regulatory T cells might be involved in the maintenance of the spontaneous tolerance to liver allografts. Further studies are needed to clarify this mechanism. Given that there are now a few published examples where CD4 CD25 T cells would seem to suppress the activation of other CD4 and CD8 T cells, 6-8 we next wished to determine the effect of these cells on responses of CD4 and CD8 T cells. We found that elimination of CD25 CD4 T cells in C3H recipients resulted in the enhancement of both proliferative response of CD4 T cells and cytotoxicity of CD8 T cells against donor-type alloantigen (Fig. 3). Therefore, CD25 CD4 T cells suppress the function of alloreactive CD4 and CD8 T cells in the recipients, which may, at least in part, contribute to tolerance induction. We found that the number of CD25 CD4 T cells and Foxp3 expression in the accepted liver allografts increased (Fig. 4). However, it was unclear how they were expanded in allogeneic livers. IL-2 has been indicated as a CD25 CD4 T-cell-growth-promoting factor, 23 but our data show that IL-2 messenger RNA was not up-

6 HEPATIC ALLOGRAFT TOLERANCE AND REGULATORY T CELLS 1117 Figure 5. Depletion of CD4 CD25 T cells by delayed PC61 treatment. (A) Fluorescence-activated cell sorter analysis. Recipients with delayed PC61 treatment were sacrificed as indicated in Table 1. Liver mononuclear cells, splenocytes, and peripheral blood lymphocytes were examined for their percentage of CD25 CD4 T-cell population. Similar results were obtained from 3 recipients. Data of naïve DBA/2 liver mononuclear cells are shown as a positive control. (B) Foxp3 messenger RNA levels in liver mononuclear cells. Foxp3 expression in liver mononuclear cells derived from different mice was examined by real-time reverse transcriptase polymerase chain reaction. Mean expression of Foxp3 messenger RNA level in the tolerant recipient without PC61 treatment (black bar) is expressed as 1. Gray bar represents tolerant recipient with PC61 treatment. Lined bar represents DBA/2 with PC61 treatment. regulated in liver lymphocytes or CD25 CD4 T cells (data not shown). It was also shown that mature dendritic cells were able to trigger the significant proliferation of these cells. 24 However, we observed that dendritic cells from DBA/2 liver were immature and could not promote the expansion of these cells (data not shown). Accepted liver allografts appear to regenerate after initial attack from the immune system, in which some cytokines such as TNF- or HGF should be involved. 25 For that reason, we tested whether these liverregeneration-related cytokines can affect CD25 CD4 T-cell proliferation in antigen-presenting cell-free systems in vitro, and we found that TNF- revived CD25 CD4 T cells from their anergic state, although their efficacy was one-tenth that of IL-2 (Fig. 4D). Therefore, TNF- produced from regenerating liver may contribute to the increase of CD25 CD4 T cells in liver allografts to some extent. However, other liver-related factor(s) may be also involved, and this possibility is now under investigation. In summary, we have demonstrated for the first time that CD25 CD4 T cells in recipients play an important role in tolerance induction to hepatic allografts. We also provide direct evidence that regulatory CD25 CD4 T cells are accumulated in hepatic allografts. Interestingly, delayed depletion of CD25 CD4 T cells did not affect the course of tolerance, suggesting that an immune-regulatory mechanism other than CD25 CD4 regulatory T cell is elicited in the liver transplant recipients soon after the induction phase of tolerance. These observations might contribute to elucidation of the mechanisms of tolerogenic features of clinical liver transplantation and development of a novel strategy for tolerance induction to transplanted allografts. ACKNOWLEDGEMENT The authors thank Hanae Fujimoto for her excellent technical assistance. REFERENCES 1. Sugioka A, Morita M, Fujita J, Hasumi A, Shiroishi T. Graft acceptance and tolerance induction in mouse liver transplantation using wild mice. Transplant Proc 2001;33: Takatsuki M, Uemoto S, Inomata Y, Egawa H, Kiuchi T, Fujita S, et al. Weaning of immunosuppression in living donor liver transplant recipients. Transplantation 2001; 72: Karim M, Bushell AR, Wood KJ. Regulatory T cells in transplantation. Curr Opin Immunol 2002;14: Waldmann H, Graca L, Cobbold S, Adams E, Tone M, Tone Y. Regulatory T cells and organ transplantation. Semin Immunol 2004;16: Sakaguchi S. Naturally arising CD4 regulatory t cells for immunologic self-tolerance and negative control of immune responses. Annu Rev Immunol 2004;22: Takahashi T, Kuniyasu Y, Toda M, Sakaguchi N, Itoh M, Iwata M, et al. Immunologic self-tolerance maintained by CD25 CD4 naturally anergic and suppressive T cells: induction of autoimmune disease by breaking their anergic/suppressive state. Int Immunol 1998;10: Thornton AM, Shevach EM. CD4 CD25 immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J Exp Med 1998;188: Piccirillo CA, Shevach EM. Cutting edge: control of CD8 T cell activation by CD4 CD25 immunoregulatory cells. J Immunol 2001;167: Asseman C, Mauze S, Leach MW, Coffman RL, Powrie F. An essential role for interleukin 10 in the function of regulatory T cells that inhibit intestinal inflammation. J Exp Med 1999;190: Hoffmann P, Ermann J, Edinger M, Fathman CG, Strober S. Donor-type CD4 CD25 regulatory T cells suppress lethal acute graft-versus-host disease after allogeneic bone marrow transplantation. J Exp Med 2002;196: Edinger M, Hoffmann P, Ermann J, Drago K, Fathman CG, Strober S, Negrin RS. CD4 CD25 regulatory T cells preserve graft-versus-tumor activity while inhibiting graftversus-host disease after bone marrow transplantation. Nat Med 2003;9: Onizuka S, Tawara I, Shimizu J, Sakaguchi S, Fujita T,

7 1118 JIANG ET AL. Nakayama E. Tumor rejection by in vivo administration of anti-cd25 (IL-2Ra) monoclonal antibody. Cancer Res 1999;59: Li Y, Koshiba T, Yoshizawa A, Yonekawa Y, Masuda K, Ito A, et al. Analyses of peripheral blood mononuclear cells in operational tolerance after pediatric living donor liver transplantation. Am J Transplant 2004;4: Yoshizawa A, Ito A, Li Y, Koshiba T, Sakaguchi S, Wood KJ, Tanaka K. The roles of CD25 CD4 regulatory T cells in operational tolerance after living donor liver transplantation. Transplant Proc 2005;37: Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science 2003;299: Khattri R, Cox T, Yasayko SA, Ramsdell F. An essential role for Scurfin in CD4 CD25 T regulatory cells. Nat Immunol 2003;4: Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4 CD25 regulatory T cells. Nat Immunol 2003;4: Walker MR, Kasprowicz DJ, Gersuk VH, Benard A, Van Landeghen M, Buckner JH, Ziegler SF. Induction of FoxP3 and acquisition of T regulatory activity by stimulated human CD4 CD25 T cells. J Clin Invest 2003;112: Chen W, Jin W, Hardegen N, Lei KJ, Li L, Marinos N, et al. Conversion of peripheral CD4 CD25 naive T cells to CD4 CD25 regulatory T cells by TGF-b induction of transcription factor Foxp3. J Exp Med 2003;198: Karube K, Ohshima K, Tsuchiya T, Yamaguchi T, Kawano R, Suzumiya J, et al. Expression of FoxP3, a key molecule in CD4CD25 regulatory T cells, in adult T-cell leukaemia/ lymphoma cells. Br J Haematol 2004;126: Sugioka A, Morita M, Esaki T, Hasumi A, Kurosawa Y. Evaluation of microchimerism after orthotopic liver transplantation between allogeneic mice. Transplant Proc 1997;29: Harada M, Seino K, Wakao H, Sakata S, Ishizuka Y, Ito T, et al. Down-regulation of the invariant Va14 antigen receptor in NKT cells upon activation. Int Immunol 2004;16: Sakaguchi S, Sakaguchi N, Shimizu J, Yamazaki S, Sakihama T, Itoh M, et al. Immunologic tolerance maintained by CD25 CD4 regulatory T cells: their common role in controlling autoimmunity, tumor immunity, and transplantation tolerance. Immunol Rev 2001;182: Yamazaki S, Iyoda T, Tarbell K, Olson K, Velinzon K, Inaba K, Steinman RM. Direct expansion of functional CD25 CD4 regulatory T cells by antigen-processing dendritic cells. J Exp Med 2003;198: Taub R. Liver regeneration: from myth to mechanism. Nat Rev Mol Cell Biol 2004;5: