Chinese Medical Journal 2004;117(8):1184-1189
Transplantation tolerance mediated by regulatory T cells in mice

FENG Ning-han 冯宁翰,  WU Hong-fei 吴宏飞,  WU Jun 吴 军,  ZHANG Wei 张 炜,  SUI Yuan-geng 眭元庚,  HE Hou-guang 贺厚光,  ZHANG Chun-lei 张春雷,  ZHENG Jun-song 郑峻松

FENG Ning-han 冯宁翰 (Department of Urology, Center of Kidney Transplantation, First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, China)

WU Hong-fei 吴宏飞 (Department of Urology, Center of Kidney Transplantation, First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, China)

WU Jun 吴 军 (Burn Research Institute, Southwest Hospital, Third Military Medical University, Chongqing 400038, China)

ZHANG Wei 张 炜 (Department of Urology, Center of Kidney Transplantation, First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, China)

SUI Yuan-geng 眭元庚 (Department of Urology, Center of Kidney Transplantation, First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, China)

HE Hou-guang 贺厚光 (Department of Urology, Center of Kidney Transplantation, First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, China)

ZHANG Chun-lei 张春雷 (Burn Research Institute, Southwest Hospital, Third Military Medical University, Chongqing 400038, China)

ZHENG Jun-song 郑峻松 (Burn Research Institute, Southwest Hospital, Third Military Medical University, Chongqing 400038, China)

Correspondence to:Zheng Jun-song,Burn Research Institute, Southwest Hospital, Third Military Medical University, Chongqing 400038, China (Tel: 86-23-68752311. Fax:. E-mail:zhengalpha@yahoo.com)
Keywords
T-lymphocytes;immune tolerance;skin transplantation;lymphocyte culture test, mixed
Abstract
Background With potent suppressive effect on responder T cells, CD4+CD25+ regulatory T (Treg) cells have become the focus of attention only recently and they may play an important role in transplantation tolerance. However, the mechanism of action is not clear. This study was designed to assess the possibility of using CD4+CD25+ Treg cells to induce transplantation tolerance and to investigate their mechanism of action.
Methods CD4+CD25+ Treg cells were isolated using magnetic cell separation techniques. Mixed lymphocyte reactions were used to assess the ability of Treg cells to suppress effector T cells. Before skin transplantation, various numbers of CD4+CD25+Treg cells, which have been induced using complex skin antigens from the donor, were injected into the host mice either intraperitoneally [0.5×10(5), 1×10(5), 2×10(5), 3×10(5), 4×10(5), or 5×10(5)] or by injection through the tail vein [5×10(3), 1×10(4), 2×10(4), 5×10(4), 1×10(5), 2×10(5)]. Skin grafts from two different donor types were used to assess whether the induced Treg cells were antigen-specific. The survival time of the allografts were observed. Single photon emission computed tomography was also used to determine the distribution of Treg cells before and after transplantation.
Results Treg cells have suppressive effect on mixed lymphocyte reactions. Grafts survived longer in mice receiving CD4+CD25+ Treg cell injections than in control mice. There was a significant difference between groups receiving intraperitoneal injection of either 2×10(5) or 3×10(5) CD4+CD25+Treg cells and the control group (P<0.05, respectively). Better results were achieved when Treg cells were injected via the tail vein than when injected intraperitoneally. The transplantation tolerance induced by CD4+CD25+ Treg cells was donor-specific. Analysis of the localization of Treg cells revealed that Treg cells mainly migrated from the liver to the allografts and the spleen.
Conclusions CD4+CD25+Treg cells can induce donor-specific transplantation tolerance. Cell-to-cell contact may be the primary mechanism by which Treg cells act on effector T cells.
It is known that transplantation significantly prolongs the lives of patients with end-stage organ function failure. Unfortunately, this procedure is limited by immunological rejection of the grafted organ. Though many new immuno-suppressive drugs have been used in clinical treatment, the immunosuppressants still possess many adverse effects. A more attractive method of inducing graft acceptance is through selectively coaxing the recipient immune response into accepting the transplanted organ, while at the same time maintaining normal immune reactivity against foreign antigens. This approach is called tolerance induction.

Recently, regulatory T (Treg) cells, with their potent suppressive effects on normal responder T cell function, have become the focus of transplant immunology. CD4+CD25+ T cells have been reported as important immunoregulatory cells for T cell homeostasis and the prevention of autoimmunity.
[1-4] It has been suggested that Treg cells play an important role in transplantation tolerance. However, no experiments have examined directly whether or not they can prolong the survival of grafts. In this study, we build on our previous work on allograft transplantation.

METHODS

Animals
Eight to ten week-old inbred Balb/c (H-2d) mice weighing 23-25 g and 8-10 week-old Kunming mice weighing 23-25 g were purchased from the Animal Center of the Third Military Medical University, China. Eight to ten week-old inbred C57BL/6 (H-2b) mice weighing 20-23 g were purchased from the Animal Center of the Chinese Academy of Sciences. The animals were maintained under standard conditions according to the principles of laboratory animal care and our institution’s guidelines for the care and use of laboratory animals.

Reagents
Phycoerythin (PE)-labeled anti-mouse CD4, PE-labeled anti-mouse CD8, PE-labeled anti-mouse CD25, anti-PE microbeads, PE-labeled anti-mouse CTLA4, and PE-labeled anti-mouse CD28 were obtained from Pharmingen, USA. A MiniMACS magnetic cell separator (MACS) and MACS separation column were purchased from Miltenyi, USA. Mouse T cell enrichment columns were obtained from R&D Systems, USA and 99mTc and SnCl2 were purchased from the Department of Nuclear Medicine of Southwest Hospital, China. Millicell-PCF cell culture inserts were obtained from Millipore, USA, and single photon emission computed tomography (SPECT) was purchased from Beckman, USA.

Preparation of CD4+CD25+ Treg cells
CD4+CD25+Treg cells were prepared as previously described.[5] Splenic T cells from Balb/c mice were purified on a T cell enrichment column. Then, CD4+CD25+ Treg cells were isolated using MACS technique, first by depleting the samples of magnetically labeled non-CD4+ T cells. CD4+ T cells labeled with PE-conjugated anti-CD25 were then magnetically labeled with anti-PE microbeads. The magnetically labeled cells were then passed through a column placed in the magnetic field of a MACS separator. The purity of CD4+CD25+cells ranged from 85% to 90% according to flow cytometric analysis.

Mixed lymphocyte reaction (MLR)
The inhibitory effects of various regulatory cells on the proliferation of alloreactive responder cells were measured using MTT assay. Briefly, 5×10(5) splenic lymph cells from Balb/c and C57BL/6 mice were cultured in 96-well plates. Treg cells [5×10(5), 2×10(5), 1×10(5), 5×10(4), or 1×10(4)] were added to each well except for the control well. Triplicate assays were carried out in 96-well flat-bottomed plates in a total volume of 0.2 ml/well culture medium at 37℃ in 5% CO2. On the third day, 10 μl supernatant was removed from each well, and 10 μl MTT was added for an additional incubation of 6 hours. Finally, the absorption value at 595 nm was measured to determine the extent of MLR.

Skin transplantation
C57BL/6 mice and Kunming mice were used as skin graft donors and the Balb/c mice were used as the recipients. Full-thickness skin grafting was performed as previously described.[6] All skin transplantations were performed under pentobarbital anesthesia (100 mg/kg intraperitoneally). Allografts were transplanted onto the back of the recipient mice. The day of transplantation was set as the day 0. On the day 7, the grafts were photographed and graft survival was assessed.

Treg cell injections
Recipient mice were divided into three groups. In group 1, different numbers of Treg cells [0.5×10(5), 1×10(5), 2×10(5), 3×10(5), 4×10(5), 5×10(5), 6 mice per group] were injected intraperitoneally into the recipients before skin transplantation. Recipients who did not receive Treg cells served as the controls. In group 2, different numbers of Treg cells [5×10(3), 1×10(4), 2×10(4), 5×10(4), 1×10(5), 2×10(5)] were injected into recipients through the tail vein. Recipients who did not receive Treg cells served as the controls. In group 3 (6 mice), recipients were injected with 1×10(5) Treg cells through the tail vein, which had been induced using the skin complex antigen from C57BL/6 mice. All injections of Treg cells were performed prior to skins grafting. After the injections, skins from C57BL/6 and Kunming mice were transplanted onto Balb/c mice.

Determination of Treg cell localization
SPECT was used to determine the distribution of the Treg cells and their mechanism of action. After the skin transplantations, 0.5 ml Treg cells labeled with 99mTc [2×10(6)] total cells) were injected into the recipient via the tail vein. Five hours later, mice were scanned by SPECT to determine radiation levels in the liver, spleen, lungs, and at the site of the skin grafts.

Statistical analysis
All values were expressed as mean±standard deviation (SD). Statistical analysis was conducted using SPSS 10.0 software. Statistical significance of differences among groups was assessed using either one-way ANOVA or the Kaplan-Meier survival analysis. P<0.05 was considered statistically significant.

RESULTS

Treg cell suppression rate of MLR
The suppressive effects on MLR of different numbers of Treg cells are shown in Fig. 1. 5×10(4) Treg cells could significantly suppress MLR, decreasing 77.10% compared with the control group (P<0.01). Suppression is initially enhanced with increases in Treg cell numbers. However, no significant difference was found between groups when the number of Treg cells was greater than 5×10(4).

Rejection inhibition of Treg cells administered intraperitoneally
After Treg cells induced by the donor’s complex antigen were injected into the recipient, the grafted skin survived longer ( Fig. 2 ). The more Treg cells were injected, the longer the grafts survived. However, this dose-dependent effect was limited in scope. Grafts had the longest survival time (17 days) when the recipient received 2×10(5)-3×10(5) Treg cells. Mice recipients died when 4×10(5)-5×10(5) cells were injected. There was a marked difference between groups receiving 2×10(5) or 3×10(5) Treg cells and the control group (P<0.05).

Rejection inhibition of Treg cells administered by tail vein injection
Identical numbers of Treg cells injected through the tail vein produced better results than when administered intraperitoneally ( Fig. 3 ). For example, a tail vein injection of 1×10(5) Treg cells was comparable to an intraperitoneal injection of 3×10(5) cells. Interestingly, the longest survival time was still 17 days, the same maximum survival time for recipients receiving Treg cells by intraperitoneal injection. When the number of Treg cells was raised to 2×10(5), recipient mice died. There was a significant difference between the group receiving 1×10(5) Treg cells and the control group (P<0.05).

Specificity of immune tolerance induced by Treg cells
Immune tolerance induced by Treg cells was found to be donor-specific. After Balb/c mice received Treg cells [1×10(5)] induced by C57BL/6 skin antigens through the tail vein, skin from both Balb/c and Kunming mice was simultaneously transplanted onto the backs of the recipients (Balb/c). The allografted skin from C57BL/6 mice survived 17 days, whereas grafts from Kunming mice survived only 9 days (P<0.05).

Localization of Treg cells in vivo
There was no difference between the distribution of induced and uninduced Treg cells in normal mice that did not receive grafts: liver 40%-50%, spleen 20%-22%, lungs 7%-8%, and other tissues 20%-33% ( Fig. 4 ). After skin transplantation, the distribution of Treg cells changed greatly. Uninduced Treg cells localized mainly at the site of the skin allograft (36.80%) and in the spleen (21.30%), liver (10.70%), and other tissues (31.2%) ( Fig. 5 ). On the other hand, Treg cells induced by donor antigens localized mainly at the site of the skin allograft (45.5%) and in the spleen (38.3%) ( Fig. 6 ). These data imply that induced Treg cells migrate primarily from the liver to the spleen and the allograft.

DISCUSSION

Organ transplantation is the only effective therapy for patients who suffer from certain serious diseases such as uremia, serious burns, end-stage liver function failure, and so on. The long-lasting acceptance of allografts requires life-long administration of immunosuppressive drugs such as cyclosporine A, FK506, and rapamycin. These drugs can prevent allograft loss from rejection.[7-9] However, these drugs lack immunological specificity, and lead to many side-effects including opportunistic infections, malignancies, and other fatal diseases. Thus, it is important to find new strategies to avoid organ rejection. It is know that T lymphocytes play an important role in cell-mediated immunity. The most important host immunological reaction against allo- and xenogenic grafts after transplantation is acute cellular rejection, mediated primarily by T cells.[10] The body contains various kinds of T cells. Selectively inhibiting the activation of donor-specific reactive T cells without influencing the function of other T cell groups is now regarded as an ideal strategy for avoiding rejection and allowing long-term drug free acceptance of allografts.

Treg cells were discoveryed after the treatment of auto-immune diseases and malignancies.[2,11,12] Because of their potent immunosuppressive function on antigen-specific reactive T cells, particular attention has been geared to this cell type. In humans, it has been confirmed by many research groups that CD4+CD25+ peripheral blood T cells and thymocytes have suppressive activity in vitro,[13] and these Treg cells have been defined anergic.[14]

We built on past research by exploring the application of Treg cells after skin transplantation, as skin graft acceptance is an important marker of tolerance. Allogenic skin grafts have more difficulty than other organs surviving in the host because of the dendritic and lymphocytic cells in the skin. Thus, skin transplantation models have been used as the most reliable marker of immune tolerance.

In the case of clinical kidney transplantations, it has been shown that allografts survive longer if immunosuppressive drugs are administered after the recovery of renal function rather than taken during the operation.[7,15] So we attempted to inject Treg cells intraperitoneally to assess the effect on allograft survival time of delayed administration of Treg cells. The results show that more cells are required to obtain the same effect when Treg cells are injected intraperitoneally rather than directly into the tail vein. One possible explanation is that Treg cells suppress effector T cells primarily by cell-to-cell contact, and more time is required for intraperitoneal Treg cells to enter the blood and make contact with effector T cells. Because fewer Treg cells can contact effector T cells, more Treg cells need to be injected if they are administered intraperitoneally.

In our experiment, we found that Treg cells only induce immune tolerance to the skin of the donor and that the host continues to reject allografts from third parties. This demonstrates that the suppressive effect of Treg cells on effector T cells is antigen-specific. On the other hand, our data also show that Treg cells induced by the donor phenotype can only suppress effector T cells with the same phenotype as the inducer. Results from MLR also support these results. All of these results are in accordance with Oluwole’s conclusions. Oluwole and his group[16] found that adoptive transfer of enriched CD4+CD25+ host thymic T cells combined with in vivo P5 primed syngeneic peripheral T cells led to permanent graft survival. However, this technique cannot prolong the survival of third-party allografts. Guo[17] and Wu[18] also came to a similar discovery.

Although it is now clear that Treg cells can suppress effector T cells and that they play a very important role in T cell homeostasis,[2,4] the mechanisms underlying the suppression of immunity remain ill-defined and hotly contested. Some have postulated that the suppressive effects of Treg cells requires T cell receptor engagement and cell-to-cell contact with the target cells.[19] Our experiments ex vivo confirmed that Treg cells act on the effector T cells primarily by cell-to-cell contact (data not shown). It has been postulated that the primary inhibition of T cell activation is mediated by naturally occurring CD25+ Treg cells, which is strictly cell-to-cell contact-dependent and results in local suppression.[20] Others believe that cell-to-cell contact is inevitably the first step in the mechanism of action of Treg cells.[21]

The location of Treg cells according to our data also supports this conclusion. Before skin transplantation, Treg cells are located primarily in the liver, spleen, and lungs, while after transplantation, they are found primarily at the site of the allografts and in the spleen. Treg cells presumably act on effector T cells as early as possible, implying that Treg cells act on effector T cells by cell-to-cell contact. It would also be useful for us to clarify the mechanism of Treg cell circulation. Treg cells could act on effector T cells immediately leading to longer allograft survival, if it was possible to guide the Treg cells directly to the allograft after transplantation. We plan to investigate this possibility.Treg cells have been the focus of recent immunological studies because of their important roles in maintaining immunological homeostasis. However, much knowledge remains to be clarified concerning these cells. With a growing understanding of Treg cells, the day long-term, drug-free acceptance of allografts is approaching.

REFERENCES

1.Waldmann H, Cobbold S. Regulating the immune response to transplants: a role for CD4+ regulatory cells? Rev Immunity 2001;14:399-406.
2.Shevach EM. Regulatory T cells in autoimmmunity. Annu Rev Immunol 2000;18:423-449.
3.Takahashi T, Kuniyasu Y, Toda M, et al. Immunologic selftolerance maintained by CD25+CD4+ naturally anergic and suppressive T cells: induction of autoimmune disease by breaking their anergic/suppressive state. Int Immunol 1998;10:1969-1980.
4.Tamatani T, Tezuka K, Hanzawa-Higuchi N. AILIM/ICOS: a novel lymphocyte adhesion molecule. Int Immunol 2000;12:51-55.
5.Zheng JS, Wu J, Yi SX, et al. The mechanisms of CD4+CD25+Treg cels acting on effector T cells. Acta Acad Med Milit Tertiae (Chin) 2003;25:1881-1884.
6.Baharav E, Lider O, Margalit M, et al. A modified technique for experimental skin grafting. J Immunol Methods 1986;90:143-144.
7.Li B, Hartono C, Ding R, et al. Noninvasive diagnosis of renal-allograft rejection by measurement of messenger RNA for perforin and granzyme B in urine. N Engl J Med 2001;344:947-954.
8.Starzl TE, Demetris AJ, Trucco M, et al. Cell migration and chimerism after whole-organ transplantation: the basis of graft acceptance. Hepatology 1993;17:1127-1152.
9.Devlin J, Doherty D, Thomson L, et al. Defining the outcome of immunosuppression withdrawal after liver transplantation. Hepatology 1998;27:926-933.
10.Zhang WH, Cai SR, Wang JP, et al. The effect of FasL expression on pancreatic islet allografts. Chin Med J 2002;115:1006-1009.
11.Sakaguchi S, Sakaguchi N, Asano M, et al. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25): breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 1995;155:1151-1164.
12.Suri-Payer E, Amar AZ, Thornton AM, et al. CD4+CD25+ T cells inhibit both the induction and effector function of autoreactive T cells and represent a unique lineage of immunoregulatory cells. J Immunol 1998;160:1212-1218.
13.Stephens LA, Mottet C, Mason D, et al. Human CD4+CD25+ thymocytes and peripheral T cells have immune suppressive activity in vitro. Eur J Immunol 2001;31:1247-1254.
14.Taams LS, Smith J, Rustin MH, et al. Human anergic/suppressive CD4+CD25+ T cells: a highly differentiated and apoptosis-prone population. Eur J Immunol 2001;31:1122-1131.
15.Soulillou JP. Immune monitoring for rejection of kidney transplants. N Engl J Med 2001;344:1006-1007.
16.Oluwole S F, Oluwole O, DePaz HA, et al. CD4+CD25+ regulatory T cells mediate acquired transpl tolerance. Transpl Immunol 2003;11:287-293.
17.Guo L, Fujino M, Kimura H, et al. Simultaneous blockade of co-stimulatory signals, CD28 and ICOS, induced a stable tolerance in rat heart transplantation. Transpl Immunol 2003;12:41-48.
18.Wu A, Yamada K, Ierino F L, et al. Regulatory mechanism of peripheral tolerance: in vitro evidence for dominant suppression of host responses during the maintenance phase of tolerance to renal allografts in miniature swine. Transpl Immunol 2003;11:367-374.
19.Chen WJ, Wahl SM. TGF-β1: the missing link in CD4+CD25+ regulatory T cell-mediated immunosuppression. Cytokine Growth Factor Rev 2003;14:85-89.
20.Taams L, Vukmanovic-Stejic M, Salmon M, et al. Immune regulation by CD4+CD25+ regulatory T cells: implications for transplantation tolerance. Transpl Immunol 2003;11:277-285.
21.Jonuleit H, Schmitt E, Kakirman H, et al. Infectious tolerance: human CD25+ regulatory T cells convey suppressor activity to conventional CD4+ T helper cells. J Exp Med 2002;196:255-260.

  1. National Natural Science Foundation of China,No.30200264;Key Project of the National Natural Science Foundation of China,No.39993430-2;