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Stem Cells, Vol. 19, No. 5, 425-435, September 2001
© 2001 AlphaMed Press

A New Method for Tolerance Induction: Busulfan Administration Followed by Intravenous Injection of Neuraminidase-Treated Donor Bone Marrow

Takashi Nagahamaa,b, Kikuya Sugiuraa,c, Shinryu Leea, Haruo Moritab,c, Yasushi Adachia,c, A-Hon Kwonb, Yasuo Kamiyamab,c, Susumu Ikeharaa,b

a The First Department of Pathology, Kansai Medical University;
b The First Department of Surgery, Kansai Medical University;
c Transplantation Center, Kansai Medical University, Osaka, Japan

Key Words. Bone marrow cells • Intrahepatic retention • Donor-specific tolerance • Busulfan • Neuraminidase

Susumu Ikehara, Ph.D., First Department of Pathology, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi City, Osaka 570-8506 Japan. Telephone: 81-6-6993-9429; Fax: 81-6-6994-8283; e-mail: ikehara{at}takii.kmu.ac.jp


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The portal venous (p.v.) administration of foreign cells induces donor-specific tolerance. Recently, we have demonstrated that the p.v. administration of donor cells elicits donor-specific tolerance across major histocompatibility complex barriers. In the present study, utilizing the intrahepatic tolerance-inducing system, we have established a new method for organ transplantation using both busulfan ([Bu] to provide a sufficient "space" for the donor hematopoietic cells to expand in the recipient) and neuraminidase ([Neu] to enhance the trapping of i.v.-injected cells in the liver).

Radiolabeled bone marrow cells (BMCs) were found to exclusively accumulate in the livers of the recipients as a result of the Neu treatment. Furthermore, hematopoietic progenitors (forming hematopoietic foci) in the accumulated BMCs were retained in the recipient livers for at least 18 days.

C57BL/6 (B6) mice that had been transplanted with skins of BALB/c mice immediately after the injection of BALB/c BMCs showed a 90% skin graft survival rate over 400 days as a result of using the combination of injecting 50 mg/kg Bu into the B6 mice and treatment of the BALB/c BMCs with 0.25 U/ml Neu (50 Bu + 0.25 Neu). However, the survival rate significantly decreased when either the Bu or Neu treatment was omitted. In tolerant recipients, microchimerism was observed in the various hematolymphoid organs. T cells collected from the tolerant recipients suppressed proliferative responses to the donor-alloantigens but enhanced the production of Th2 and Th3 cytokines.

These findings suggest that the enhanced retention of donor BMCs in the recipient livers as a result of the Bu and Neu treatments efficiently induces tolerance induction. Therefore, this "single-day protocol" would be of great advantage for human organ transplantation.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Organ transplantation is an effective treatment for various diseases if the graft rejection is persistently controlled. Portal venous (p.v.) administration of foreign cells has been reported to induce donor-specific tolerance across major [1-7], minor [8], and xeno [9] histocompatibility complex barriers. We have previously found that a more significant number of allogeneic bone marrow cells (BMCs) are retained in the livers or spleens of recipients after p.v. injection than after i.v. injection [10], and that the BMCs retained in the recipient livers elicit anergy to recipient CD8+ cytotoxic T-lymphocytes [11].

Recently, we have found that the engraftment of allogeneic BMCs was significantly enhanced by administering BMCs via the portal vein [12]. Furthermore, we have succeeded in inducing persistent donor-specific tolerance across major histocompatibility complex (MHC) barriers utilizing the intrahepatic tolerance-inducing system in mice [13] and pigs [14]; the injection of donor BMCs via the p.v. (on day 0) plus i.v. (on day 5) and the administration of an immunosuppressant (cyclosporin A [Cs A] or FK506) on days 2 and 5 led to a 100% skin graft survival rate for more than 300 days when the skin grafts were carried out 7 days after the p.v. injection. However, the skin graft survival rate significantly decreased (40%) when the grafting was carried out on the day of the p.v. injection (single-day protocol). Moreover, the p.v. injection requires operation and efforts to control bleeding.

In the present study, we have modified the former method to induce more potent tolerance and become useful for clinical application as a single-day protocol. The protocol is as follows: A) to enhance the trapping of allogeneic cells into the liver after i.v. injection (instead of p.v. injection), donor BMCs are treated with neuraminidase (Neu), as previously described [15], and B) to provide a sufficient "space" for donor hematopoietic cells to expand in the recipients, the recipients are pretreated with the myeloablative drug busulfan (Bu) 2 days before the injection of Neu-treated BMCs. Skin grafts are carried out on day 0. Subsequently, non-treated donor BMCs are i.v.-injected on day 5, and Cs A is i.p.-injected on days 2 and 5. Using these treatments, we show here that a 90% skin graft survival rate can be obtained for more than 400 days after the transplantation. In addition, we show microchimerism in the various hematolymphoid organs of recipients and increases in Th2 and Th3 cytokine productions.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
C57BL/6 (B6, H-2b), BALB/c (H-2d), and C3H/HeN (C3H, H-2k) mice were purchased from Japan SLC, Inc. (Hamamatsu, Japan), and bred under pathogen-free conditions. Female C57BL/6 mice at the age of 9-11 weeks were used as recipients. Male BALB/c mice at the age of 8-11 weeks were used as donors for BMCs to be injected via the i.v. route. Female BALB/c mice at the age of 8-11 weeks were used as donors for skin grafting.

Preparation of Allogeneic Cells for Injection
BMCs were flushed from the femoral and tibial bones of the male BALB/c mice, and suspended in phosphate-buffered saline (PBS) (pH 7.2). The BMCs were then filtered through a 70-µm nylon mesh (Becton Dickinson Labware; Franklin Lakes, NJ; http://www.bd.com), washed and adjusted to 1 x 108 cells/ml in PBS. The treatment of the BMCs with Neu was carried out as described by Sano et al. [15]. Two ml of the BMC solution (2 x 108 cells) were mixed with various concentrations of Neu (acylneuraminyl hydrolase, derived from Vibrio cholerae; Roche Diagnotics; Mannheim, Germany; http://www.roche.com/diagnostics) in 2 ml physiological saline. The mixture was incubated at 37°C for 30 minutes. After the incubation, the BMCs were washed three times with PBS supplemented with 3% fetal bovine serum ([FBS] Irvine Science; Santa Ana, CA). To estimate the toxicity of Neu, the in vitro hematopoietic activity (the formation of hematopoietic colonies) of the treated BMCs was examined as described previously [16]. There was no effect on the hematopoietic activity of the BMCs when the concentrations of Neu were less than 0.25 U/ml.

Analyses of Fate of Injected Cells
The BMCs of BALB/c mice treated with Neu (0.25 U/ml), and the non-treated BMCs were radiolabeled with 51Cr by incubating 2 x 108 viable cells with 14.8 Mbq Na51CrO4 (NEN Life Science Products; Boston, MA; http://www.nenlifesci.com) at 37°C for 90 minutes. After the incubation, the labeled BMCs (3 x 107 cells in 0.3 ml PBS) were injected via the i.v. or p.v. into B6 mice. The injected mice were sacrificed 15 minutes, 1 hour, 6 hours and 24 hours after the injection. Subsequently, the organs (liver, lungs, spleen, thymus, kidneys, mesenteric lymph nodes [LN], femoral bones, and peripheral blood) were collected from the injected mice, and the distribution of the injected BMCs into the organs was evaluated by counting the radioactivity of 51Cr using a gamma counter.

In the other experiments, BALB/c BMCs treated with or without Neu were injected i.v. or p.v. into B6 mice. Five, 7, 10, 12, 15, and 18 days after the injection, the livers were collected from the recipient B6 mice, and histological examination was carried out to observe the hematopoietic foci, as previously described [10]. Briefly, liver tissue specimens were fixed in 4% buffered formalin, dehydrated, and embedded in paraffin. The sections were stained with hematoxylin and eosin. One hundred randomly chosen "high dry" (400x) microscope fields were examined. Hematopoietic foci were defined as cell clusters consisting of at least 10 cells of erythroid, granulocytic, and unidentifiable large mononuclear cells.

Treatment of Recipient Mice
As shown in Figure 1Go, upper column, female B6 mice were injected i.p. with various doses of Bu (0, 25, 50, or 100 mg/kg; LD50, 160 mg/kg; Wako Pure Chemical Industries; Osaka, Japan) on day –2. The Neu-treated BMCs of male BALB/c mice (3 x 107 in 0.3 ml PBS) were i.v. injected through the tail vein using a 26-gauge needle on day 0. Cs A (Sandimmun®, Novartis; Basel, Switzerland; http://www.pharma.novartis.com; 10 mg/Kg) was injected i.p. into the recipient mice on days 2 and 5 following the injection of the BMCs. Non-treated BMCs (3 x 107 in 0.3 ml PBS) collected from male BALB/c mice were injected through the tail vein on day 5.



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  		Figure 1. Experimental protocol. The protocol for tolerance induction in the present study is shown in the upper column, and the protocol in the previous study is shown in the lower column. Female C57BL/6 (B6) mice were i.p.-injected with Bu on day –2. The Neu-treated BMCs obtained from male BALB/c mice were i.v.-injected into the B6 recipients on day 0. Subsequently, BALB/c skins were engrafted on the B6 recipients. On day 5, non-treated BMCs of BALB/c mice were i.v.-injected. Cy A (10 mg/kg) was administered i.p. on days 2 and 5. In the previous protocol, non-treated BMCs were injected p.v. into B6 recipients on day 0, and additional i.v. injection of non-treated BMCs was carried out on day 5. Cy A was administered on days 2 and 5.

 
Skin Grafting
The skin grafting was carried out as previously described [13] on the day (but not on day 7) after the injection of donor BMCs (Fig. 1Go). In brief, full-thickness skin grafts (1 cm x 1.5 cm) were harvested from the dorsal wall of the female BALB/c mice from which hair had been completely removed by using a depilatory. Taking care to preserve the panniculus carnosus, full-thickness graft beds were surgically prepared in the lateral thoracic wall of the B6 recipients, which were anesthetized with sodium pentobarbital (25-35 mg/kg). The skins were transplanted to the graft beds on the right thoracic wall using 6-0 nylon; the caudal edges of the grafts were aligned with the rostral edges of the beds [17]. The grafts were covered with a sterile bactericidal dressing and a protective tape.

The first inspection was made 14 days after the skin grafting and followed by daily inspections. Rejection of the skin graft was estimated from the loss of hair, necrotic change and the size of the graft skin. A graft was scored as rejected when it had shrunk to 30% of the initial size or had been covered with a scab over 100% of the original area.

Analyses of Chimerism
Detection of donor (male BALB/c)-derived cells in the female B6 recipients was carried out in a polymerase chain reaction (PCR) using primer sequences contained within the murine Y chromosome, as described [18]: 5'-GCATTTGCCTGTCAGAGAGAG-3' (sense strand), 5'-ACTGCTGCTGCTTTCCAACTA-3' (anti-sense strand) and 411-bp products. More than 300 days after the skin grafting, genomic DNA was isolated from hepatic mononuclear cells (HMNCs), spleen cells, thymocytes, LN cells, BMCs, and peripheral blood of either the skin graft-accepting or skin graft-rejecting mice. The HMNCs were collected as previously described [11]. PCR amplification was performed for 35 cycles in PCR buffer containing dNTP and Taq polymerase (Toyobo; Osaka, Japan; http://www.toyobo.co.jp).

Mixed Lymphocyte Reaction (MLR)
More than 300 days after the skin grafting, CD8+ or CD4+ cells were isolated from the spleen cells of either the skin graft-accepting or -rejecting mice using the magnetic cell separation system (MACS, Miltenyi Biotec GmbH; Bergisch-Gladbach, Germany; http://www.miltenyibiotec.com) and anti-mouse CD8/CD4 monoclonal antibody-conjugated ferritdextran (anti-mouse CD8/CD4 microbeads, Miltenyi Biotec), as previously described [13]. In the MLR, the isolated CD4+ or CD8+ cells (as responders) were incubated for 48 hours with irradiated (15 Gy) spleen cells collected from BALB/c, B6, and C3H mice (3 x 105, as stimulators). The responses were evaluated by measuring the uptake of [3H]-thymidine.

Analysis for Cytokine Production
The cytokine production by CD4+ cells of recipient mice in the responses to donor (BALB/c) alloantigens was evaluated in reverse transcriptase (RT)-PCR assays. CD4+ cells (5 x 105) were incubated with irradiated (15 Gy) BALB/c spleen cells (5 x 105) in 2 ml of RPMI 1640 medium with 10% FBS and 50 µM 2-mercaptoethanol. After 24 and 48 hours in the culture, the cells were harvested, and the total RNA of the cultured cells was extracted with TRIZOL reagent (GIBCO BRL; Gaithersburg, MD) according to the manufacturer's instructions. The isolated RNA, dissolved in diethylpyrocarbonate-H2O, was then employed in reverse transcription, in which the reactive solution (20 µl) contained 20 units of Moloney murine leukemia virus-RT (Toyobo), 20 units of RNase inhibitor (Toyobo), and 50 pmol of random nonamer (Takara Shuzo Co., Ltd.; Ohtsu, Japan) in an appropriate RT buffer. The RT solution was incubated at 37°C for 10 minutes and 42°C for 30 minutes. The reaction was terminated by heating to 100°C for 5 minutes. Generated cDNAs were amplified in PCR using recombinant Taq DNA polymerase (Toyobo). Primers to detect the cDNA of murine interferon-{gamma} (IFN-{gamma}) and interleukin 10 (IL-10) were purchased from Maxim Biotec, Inc. (San Francisco, CA; http://www.maximbio.com), and for murine transforming growth factor-ß1 (TGF-ß1), purchased from Clonotech Laboratories, Inc. (Palo Alto, CA; http://www.clontech.com). Amplifications (30-35x) were performed according to the manufacturer's instructions associated with the primer sets.

Statistical Analyses
Skin graft survival rates were statistically analyzed by the log rank test. The data on MLR were analyzed by analysis of variance and the Tukey-Kramer multiple comparisons test.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Accumulation and Retention of Neu-Treated BMCs in Recipient Liver
As a first step, we examined whether the Neu treatment indeed increases the affinity of BMCs to the liver, and whether the treated BMCs can proliferate after being trapped in the liver, as seen after p.v. injection [10]. Since we have found that the hematopoietic activity of BMCs is not affected when the concentrations of Neu are less than 0.25 U/ml, we used 0.25 U/ml Neu for the following assay. The Neu-treated or non-treated BMCs of BALB/c mice were radiolabeled with 51Cr and injected via the i.v. or p.v. into B6 mice. Fifteen minutes, 1 hour, 6 hours, and 24 hours after the injection, the liver and other organs were collected from the injected mice, and the accumulation of the injected cells in the organs was evaluated by measuring the radioactivity of 51Cr in the organs. As shown in Figure 2AGo, the largest numbers of BMCs had accumulated in the recipient livers 15 minutes after the p.v. injection, but then gradually decreased. In contrast, significant accumulations of Neu-treated BMCs were seen in the livers 1 hour after the i.v. injection, and reached a plateau through 6 hours to 24 hours. However, the accumulation of Neu-treated BMCs in the lung was the same as that of non-treated BMCs injected via the p.v. or i.v. (Fig. 2BGo). The accumulation of allogeneic cells in the other organs was similar to that in the lung (data not shown). As shown in Figure 3AGo, the BMCs retained in the recipient livers formed hematopoietic foci; the formation of foci peaked 5 and 15 days after the injection. The largest numbers of foci were observed in the livers of recipients that had been injected with Neu-treated BMCs. Not only the number but also the size of foci were larger in the livers injected with the Neu-treated BMCs (Fig. 3BGo). From these data, it is suggested that the treatment of BMCs with Neu enhances the liver-specific accumulation required for the induction of donor-specific tolerance [10].




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  		Figure 2. Distribution of allogeneic BMCs in recipient organs. The Neu-treated BMCs (3 x 107) of BALB/c mice (0.25 U/ml) were radiolabeled with 51Cr and i.v.-injected into B6 mice (). As controls, 51Cr-labeled non-treated BMCs were injected i.v. ({square}) or via the p.v. (). Various organs were extracted from recipient mice 15 minutes, 1 hour, 6 hours, and 24 hours after the treatment. The distribution of the injected BMCs was evaluated by counting the radioavtivity of 51Cr using a gamma counter. The results of the liver (A) and lung (B) are shown.

 



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  		Figure 3. Formation of hematopoietic foci in recipient liver. Five, 7, 10, 12, 15 and 18 days after the injection of Neu-treated or non-treated BMCs (3 x 107) of BALB/c mice, the livers were removed from the B6 recipients, and the hepatic hematopoietic foci were examined. (A) Hepatic hematopoietic focus numbers of 100 hpf (x 400). Closed circles () represent the number of foci in the recipients injected i.v. with Neu-treated BMCs. Open squares ({square}) represent the number of foci in the recipients injected p.v. with non-treated BMCs, and open circles () represent the number of foci in the recipients injected i.v. with non-treated BMCs. (B) Light microscopical findings of hematopoietic foci after the i.v. injection of Neu-treated allogeneic BMCs (x 400).

 
Myeloablation
The numbers of peripheral blood cells decreased to 25%-50% by 7-10 days after the injection of 25-50 mg/kg Bu. The numbers then gradually increased but were still 50%-75% 20 days after the injection (data not shown). The treatment with 50 mg/Kg Bu exerted a larger (but not significant) decrease in comparison with 25 mg/Kg Bu (data not shown).

Skin Graft Survival Rates
When recipient mice (n = 20) were conditioned with: A) 50 mg/Kg Bu (on day –2); B) the injection of donor BMCs treated with 0.25 U/ml Neu (on day 0); C) the Cs A treatment (on days 2 and 5), and D) the injection of non-treated BMCs (on day 5), a 100% skin graft survival rate (for 130 days), a 95% skin graft survival rate (for 250 days), and a 90% skin graft survival rate (for more than 400 days) were obtained (Fig. 4Go). The survival rates decreased as the dose of Bu was reduced to 25 mg/Kg (67% survival for >400 days) and further decreased when recipient mice were not treated with Bu (54% survival for >400 days). However, in the recipients treated with the same dose of Bu, the injection of 0.25 U/ml Neu-treated BMCs enhanced skin graft survival rates (in the 50 mg/Kg Bu-treated recipients, p < 0.01), as shown in Figure 4Go. Furthermore, in the recipients that were not treated with Bu, the injection of Neu-treated BMCs was more effective in the graft survival rate (54%) than p.v. injection (40%) in the single-day protocol. Recipients that had received 100 mg/Kg Bu and non-treated BMCs showed a 100% skin graft survival rate, but 80% of the recipients died within 30 days due to the cytotoxicity of Bu. All of the recipients that had not been treated with Bu but injected with non-treated BMCs (n = 10) rejected the skin grafts within 60 days (Fig. 4Go). As shown in Figure 5Go, luxurious hair growth was observed, with the growth being counterpoint to the normal hair growth and usually of different color to the normal hair of the recipient strains.



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  		Figure 4. Skin graft survival rates in recipients after various treatments. B6 mice were i.p.-injected with Bu (0-50 mg/kg) on day –2. The Neu-treated (0 or 0.25 U/ml) BMCs (3 x 107) from BALB/c mice were i.v.-injected into the B6 recipients on day 0. Subsequently, BALB/c skins were engrafted on the B6 recipients. On day 5, non-treated BMCs (3 x 107) of BALB/c mice were i.v.-injected. Cy A (10 mg/kg) was administered i.p. on days 2 and 5. Closed symbols ({blacksquare}, , {blacktriangleup}) indicate skin graft survival rates in the recipients injected with the Neu (0.25 U/ml)-treated BMCs, and open symbols ({square}, , {Delta}) indicate skin graft survival rates in the recipients injected with non-treated BMCs. Squares indicate the skin graft survival rates in the recipients treated with 50 mg/kg Bu; circles, 25 mg/kg Bu; triangles, 0 mg/kg Bu. Non-treated BMCs were p.v.-injected into B6 recipients on day 0, and additional i.v. injection of non-treated BMCs was carried out on day 5. Cy A was administered on days 2 and 5 ({blacklozenge}). * = p < 0.01 compared with open squares ({square}), open circles (), open triangles ({Delta}) and closed diamonds ({blacklozenge}) by the log rank test. ** = p < 0.05 compared with closed triangles ({blacktriangleup}). *** = p < 0.05 compared with open triangles ({Delta}) and closed diamonds ({blacklozenge}).

 


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  		Figure 5. A representative BALB/c skin graft on a B6 recipient mouse 300 days after skin grafting. Female B6 mice were i.p.-injected with Bu (50 mg/kg) on day –2. The Neu-treated (0.25 U/ml) BMCs (3 x 107) obtained from male BALB/c mice were i.v.-injected into the B6 recipients on day 0. Subsequently, BALB/c skins were engrafted on the B6 recipients. On day 5, non-treated BMCs (3 x 107) of BALB/c mice were i.v.-injected. Cy A (10 mg/kg ) was administered i.p. on days 2 and 5.

 
The skin graft survival rates also decreased as the concentrations of Neu were reduced from 0.25 U/ml to 0.125 U/ml (67% survival for >360 days), and the skin graft survival rates further decreased when recipient mice were injected with non-treated BMCs (50% survival for >400 days) (Fig. 6Go). The treatment of BMCs with 0.5 U/ml Neu, although not showing any cytotoxicity during the treatment, significantly impaired the in vitro hematopoiesis, and resulted in a significantly lower graft survival (33% survival for >400 days) (Fig. 6Go). These results suggest that both Bu and Neu treatments are critical for inducing donor-specific tolerance to obtain a high skin graft survival rate in the single-day protocol. As shown in Figure 7Go, either Cs A treatment or the additional BMC injection is also necessary in order to induce potent tolerance.



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  		Figure 6. Effect of Neu treatment on skin graft survival rates. B6 mice were i.p. administered with Bu (50 mg/kg) on day –2. On day 0, BALB/c BMCs treated with various concentrations of Neu (0.5 U/ml, n = 6; 0.25 U/ml, n = 20; 0.125 U/ml, n = 12; 0 U/ml, n = 20) were injected. Subsequently, BALB/c skins were engrafted on the B6 recipients. On day 5, non-treated BMCs (3 x 107) of BALB/c mice were i.v. injected. Cy A (10 mg/kg) was administered i.p. on days 2 and 5. * = p < 0.01 compared with i.v. injection of non-treated BMCs and 0.5 U/ml Neu-treated BMCs by the log rank test.

 


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  		Figure 7. Effect of Cs A and additional injection of non-treated BMCs. B6 mice were administered i.p. with Bu (50 mg/kg) on day –2. The Neu-treated (0.25 U/ml) BALB/c BMCs (3 x 107) were injected i.v. into the B6 recipients on day 0. Subsequently, BALB/c skins were engrafted on the B6 recipients. Recipient mice were administered with Cs A (10 mg/kg) on days 2 and 5, and injected with non-treated BALB/c BMCs (3 x 107) on day 5 (Group I: n = 20). Recipient mice were administered with Cs A, but not injected with BMCs on day 5 (Group II: n = 10). Recipient mice were not administered Cs A or BMCs (Group III: n = 10). * = p < 0.01 compared with Group III by the log rank test.

 
Maintenance of Chimerism
Since we injected BMCs of male BALB/c mice into female B6 mice to determine whether the injected BMCs were maintained in the recipients, cells with the Y chromosome in various organs were examined in PCR analyses. As shown in Figure 8Go, donor cells were maintained for more than 300 days in the liver, spleen, thymus, LNs, and BM of the skin graft-accepting recipients, whereas no donor cells were detected in the organs of skin graft-rejecting recipients.



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  		Figure 8. Detection of donor-derived cells in recipient mice by PCR. More than 300 days after the skin grafts, genomic DNA was isolated from the indicated organs of the female recipient mice that had received the BMCs from male allogeneic donors, and a 411-bp region contained within the murine Y chromosome was detected in PCR. Lanes 1, 3, 5, 7, and 9 = genomic DNA from BALB/c skin-accepting B6 recipients. Lanes 2, 4, 6, 8, and 10 = genomic DNA from BALB/c skin-rejecting B6 recipients. Genomic DNA from male (lane 11) and female (lane 12) mice served as positive and negative controls. Experiments were performed three times, and reproducible results were obtained. Representative data are therefore shown.

 
Inhibition of Proliferative Responses and Increases in Th2 and Th3 Cytokine Production by Tolerant T Cells
More than 300 days after skin grafting, CD4+ or CD8+ cells isolated from either skin graft-accepting or -rejecting recipients were examined for the induction of donor-specific tolerance in MLRs. As shown in Figure 9Go, the responses of both CD4+ and CD8+ cells from the skin graft-accepting recipients were significantly lower in MLRs against donor (BALB/c) alloantigens than those from skin graft-rejecting recipients or non-treated B6 mice. The responses of CD8+ cells were inhibited more significantly than those of CD4+ cells. In contrast, the responses to the third-party (C3H) alloantigens were similarly high in the three groups.



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  		Figure 9. MLR. More than 300 days after skin grafting, CD4+ or CD8+ cells were isolated from spleen cells of the skin graft-accepting recipients, skin graft-rejecting recipients, and non-treated B6 mice. Donor-specific tolerance was evaluated in MLR. Closed circles () represent the responses of T cells from the skin graft-accepting recipients (n = 4). Open squares ({square}) represent the responses of T cells from the skin-rejecting recipients (n = 4). Open circles () represent the responses of T cells from non-treated B6 mice (n = 4). All syngeneic MLRs (background levels) were less than 500 cpm.

 
As shown in Figure 10Go, in the responses to donor alloantigens, CD4+ cells isolated from skin graft-accepting recipients showed a decreased production of IFN-{gamma} by Th1 but increased production of both IL-10 (by Th2) and TGF-ß1 (by Th3).



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  		Figure 10. Cytokine production. More than 300 days after skin grafting, CD4+ cells isolated from recipients were incubated with irradiated BALB/c spleen cells. Twenty-four and 48 hours after culture, the cells were harvested. The expression of IFN-{gamma} , IL-10, and TGF-ß1 was examined in PCR. Lanes 1 and 2 = BALB/c skin graft-accepting B6 mice. Lanes 3 and 4 = BALB/c skin graft-rejecting B6 mice. Lanes 5 and 6 = non-treated B6 mice.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Since it has been shown that the injection of antigens via the p.v. results in specific and systemic immune tolerance [19, 20], preimmunization via the p.v. has been utilized for inducing donor-specific tolerance across MHC barriers and preventing the rejection of allografts such as the heart [3, 4], kidney [6] and pancreas [5]. Although, in these studies, the survival rate of the allografts was indeed prolonged, all except for the liver allograft [7], which itself can induce donor-specific tolerance [21], were finally rejected. To apply the p.v. injection to clinical transplantation, we have recently established a method for inducing persistent tolerance across MHC barriers using the p.v. injection [13]. In this method, in addition to the p.v. injection of hematopoietic cells, we carried out the following treatments: A) hematopoietic cells (BMCs) were additionally injected 5 days after the p.v. injection to supplement the donor cells in the recipient liver, since we have previously found that donor-specific tolerance is significantly prolonged by the supplementation of BMCs [10, 11], and B) Cs A (10 mg/Kg) was administered 2 and 5 days after the p.v. injection to inhibit the generation of T cells, which cause rejection.

In many clinical cases (particularly transplantation from brain-dead donors), tolerance induction and organ transplantation should be carried out on the same day (a single-day protocol). Although a 100% skin graft survival rate was previously obtained when the skin grafts were carried out 7 days after the p.v. injection, the long-term survival rate significantly decreased (40%) when the grafting was carried out immediately after the p.v. injection. These findings suggest that the additional procedures are required for the single-day protocol to be effective.

It has been shown that a state called "microchimerism" (the existence of donor-derived cells in the blood and tissues of recipients) plays a significant role in the stable long-term survival of grafts [22-24]. To efficiently establish microchimerism, the creation of a sufficient "space" for donor-derived hematopoietic cells seems to be important [25]. The myeloablative reagent Bu has been reported to provide such a "space" (but not immune suppression) and to enhance the engraftment of donor-derived hematopoietic stem cells in the recipients [26-30]. Therefore, we treated recipients with Bu 2 days before the BMC injection, as per the previous reports [27-30]; 35 mg/kg Bu is cleared from the blood within 24 hours after i.p. injection. As shown in Figures 4 and 6GoGo, the long-term survival rates of skin grafts significantly increased as a result of the pretreatment of recipients with Bu. Furthermore, as shown in Figure 8Go, donor-derived cells were indeed detected in various hematolymphoid organs of the tolerant recipients more than 300 days after the BMC injection. From these findings, it is suggested that the Bu treatment creates a sufficient "space" for donor hematopoietic cells and enhances microchimerism, resulting in persistent tolerance.

We have previously found that donor hematopoietic cells trapped in the liver play a crucial role in inducing tolerance [10, 11]. However, since BMCs have no receptors for hepatic tissue, the injected donor BMCs, even if injected via the p.v., gradually flow from the liver. Furthermore, in transplantation of other than abdominal organs, additional surgical intervention is required for the p.v. injection. To address these problems, we employed the i.v. injection of Neu-treated BMCs, since it has been reported that Neu-treated hematopoietic cells lose terminal sialic acid residues of ascialoglycoprotein on their surface and exhibit an enhanced affinity for the liver [31-34]. Moreover, it has been reported that the i.v. injection of Neu-treated lymphocytes abrogates the delayed-type hypersensitivity responses to donor alloantigens [15]. As shown in Figure 2Go, the trapping of the Neu-treated BMCs in the liver was low just after the i.v. injection but then increased to significantly higher levels than after p.v. injection. The higher levels were maintained for at least 18 days. The Neu treatment of BMCs significantly increased the skin graft survival rates (Fig. 6Go) and improved the graft survival rates over those resulting from the p.v. injection of non-treated BMCs. From these findings, it is therefore suggested that donor BMCs are more efficiently trapped in the recipient liver, which results in inducing potent tolerance by the Neu treatment.

In MLRs, both CD4+ and CD8+ cells collected from the skin graft-accepting B6 recipients showed significantly low responses to BALB/c alloantigens. These results may be explained by the finding that both activated CD4+ [35] and CD8+ [36] cells are selectively retained in the liver and undergo apoptosis. In addition, we have previously found that anergy of CD8+ cells against donor alloantigens is efficiently induced after the p.v. injection of donor BMCs, since donor hematopoietic stem cells express significant amounts of MHC class I molecules but not costimulatory molecules [11, 13].

It has been reported that the responses of T cells to donor alloantigens deviate to a Th2 pattern in the portal vein tolerance [37-39]. However, it has recently been shown that the abrogation of Th1 activity causes a failure to induce the tolerance [40, 41]. In the present study, the decrease in the production of Th1 cytokines and increase in the production of Th2 and Th3 cytokines were indeed observed in the donor-specific responses of CD4+ cells from the tolerant recipients. However, the Th1 activity was not completely abrogated in the donor-specific responses of the tolerant Th cells, and Th3 activity was also observed in the responses of the non-tolerant Th cells. Therefore, the local proportion or time lag in activation among the three Th populations may relate to the maintenance of the tolerance.

In conclusion, using both Bu and Neu treatments, we have succeeded in inducing long-term tolerance and preventing the rejection of skin allografts by the single-day protocol. To ascertain the applicability of this method to humans, we are now performing the transplantation of vascularized organs in primates.


   ACKNOWLEDGMENT
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The authors thank Ms. Y. Tokuyama, Ms. M. Shinkawa, and Ms. S. Miura for their expert technical assistance, and Mr. H. Eastwick-Field and Ms. K. Ando for their help in the preparation of the manuscript.

This work was supported by a grant from "Haiteku Research Center" of the Ministry of Education, grant-in-aid for scientific research (B) 11470062, grants-in-aid for scientific research on priority areas (A) 10181225 and (A) 11162221, and also a grant from Japan Immunoresearch Laboratories Co., Ltd. (JIMRO).


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received March 5, 2001; accepted for publication March 7, 2001.




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