Stem Cells, Vol. 18, No. 4, 273-280,
July 2000
© 2000 AlphaMed Press
Long-Term (>1 year) Analyses of Chimerism and Tolerance in Mixed Allogeneic Chimeric Mice Using Normal Mouse Combinations
Haruki Hayashia,b,
Junko Tokia,
Lian Zhexionga,
Kikuya Sugiuraa,
Kyoichi Inoueb,
Susumu Ikeharaa
a First Department of Pathology,
b Third Department of Internal Medicine, Kansai Medical University, Osaka, Japan
Key Words. Mixed allogeneic chimeras • Pancreas allografts • Mice • Tolerance
Susumu Ikehara, M.D., 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
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ABSTRACT
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We examined the induction of tolerance using pancreas allografts over the long term (>1 year) in mice for the human application of mixed allogeneic bone marrow transplantation (BMT). T cell-depleted BM cells (BMCs) of C57BL/6 (B6) and C3H/He (C3H) mice were transplanted at various ratios into lethally irradiated B6 mice. The percentages of C3H cells in the chimeric mice gradually decreased, finally declining to only a small percentage, except when the ratio of donor to recipient BMCs was 100:1. However, despite the marked decreases in C3H-type cells, all the pancreas allografts of C3H mice were accepted when more than 1% C3H cells were detected in the peripheral blood. To examine the relationships between percentages of transplanted donor cells and acceptance of pancreas allografts, various percentages of donor and recipient BMCs (5% to 30%) were transplanted. It was found that more than 10% donor cells were necessary for the pancreas allografts to be accepted. In vitro assays for mixed lymphocyte reaction and generation of cytotoxic T-lymphocytes revealed that spleen cells in chimeric mice accepting pancreas allografts are tolerant to both host-type and donor-type major histocompatibility complex (MHC) determinants, but show a vigorous responsiveness to third-party MHC determinants. Since donor-type hemopoietic stem cells (HSCs) were detected in the BM and the liver of the chimeric mice, donor-derived HSCs and donor-derived hematolymphoid cells are responsible for the induction of tolerance. It should be noted that the percentage of donor-type HSCs is higher in the liver (6.2%) than in the BM (0.9%).
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INTRODUCTION
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In humans, organ allografts require the use of immunosuppressants to prevent graft rejection. In mice, we have previously shown that allografts of organs such as the liver and pancreas, in conjunction with allogeneic bone marrow transplantation (BMT) from the same major histocompatibily complex (MHC) donors, can induce permanent tolerance without using immunosuppressants [1, 2]. Ildstad and Sachs have established a system of mixed allogeneic chimerism by carrying out mixed allogeneic BMT and have demonstrated that mixed allogeneic BMT can be used for organ transplantation [3]. Mixed allogeneic chimerism has several advantages over fully allogeneic chimerism. The presence of syngeneic (or autologous) bone marrow cells (BMCs) appears to provide the necessary cells to overcome the impaired immunologic functions and prevent the graft-versus-host-disease (GVHD) observed in fully allogeneic chimeras, while the allogeneic BM elements appear to be responsible for the induction of donor-specific tolerance. Starzl et al. have found in humans that there are some cases in which liver allografts survive without using immunosuppressants, and that, in such cases, a small number of hemopoietic cells derived from the transplanted organs are detected: they described this as microchimerism [4]. In mice, mixed allogeneic chimerism can be achieved by carrying out mixed allogeneic BMT. In this condition, donor-specific tolerance can be induced. However, for human application to organ transplantation, long-term observation using allografts of organs other than the skin is necessary, since we have very recently found that the skin is not rejected after donor-derived Langerhans' cells have been replaced by host-derived Langerhans' cells, even when host-derived hematolymphoid cells become dominant (manuscript in preparation). In addition, we have found that the pancreas is more immunogeneic than the skin (manuscript in preparation), although it has been thought that the skin is the most immunogeneic tissue. Therefore, we examine the induction of tolerance, chimerism, and pancreas allograft acceptance in normal mouse combinations over a long term (>480 days after transplantation). In the present study, we show that mixed allogeneic BMT can be used for organ allografts, although the number of allogeneic hematolymphoid cells gradually decreases.
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MATERIALS AND METHODS
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Mice
Female C57BL/6J (B6: H-2b), C3H/HeN (C3H: H-2k), and BALB/c (H-2d) mice (five to eight weeks old) were purchased from SLC (Shizuoka, Japan), and raised at the Kansai Medical Animal Care Center.
Mixed Allogeneic BMT ([B6 + C3H]
B6)
Mixed allogeneic chimeric mice were prepared as previously described [3]. Briefly, inbred B6 female recipient mice were lethally irradiated with 10 Gy from a 137Cs source (Gammacell 40 Exactor, Nordion International Inc.; Kanta, Ontario; http://www.MDSNordion.com). The BM was flushed from the femurs of the B6 and C3H donor mice with RPMI 1640 using a 23-gauge needle. The BMCs were gently resuspended with a 21-gauge needle, and the suspension was filtered through a nylon mesh. The BMCs were then washed at 1,500 rpm for 5 min, resuspended in RPMI, and counted. T cells in the BMCs were depleted using anti-Thy1.2 monoclonal antibody (mAb) (F7D5, Olac; Bicester, England) at 4°C for 30 min. They were then washed and resuspended in guinea pig complement at a 1/16 dilution in RPMI (1 x 107 cells/ml in diluted guinea pig complement) at 37°C for 40 min. The BMCs were then washed twice and resuspended in RPMI at an appropriate concentration for injection of 0.5 ml of final volume per mouse. The recipient mice were reconstituted within 12 to 24 h after irradiation with 1 x 107 BMCs mixed at various ratios (B6: C3H = 2:1, 1:1, 1:3, 1:10, or 1:100, and B6 or C3H only). In some experiments, the recipient mice were reconstituted in the ratios of 5% (18:1), 10% (9:1), 20% (4:1), and 30% (7:3), respectively.
All recipients were evaluated for the presence of clinical GVHD as manifested by weight loss, alopecia, ruffled fur, diarrhea and a decreased level of activity associated with a "hunched over" appearance. In addition, histological evidence for GVHD was evaluated using samples of the skin, liver, intestine and hematolymphoid organs (the spleen, lymph nodes, thymus and BM).
Transplantation of Fetal Pancreas Tissue
The procedure was as described previously [5]. Briefly, recipients were anesthetized with an i.p. injection of somnopentyl (0.1mg/g body weight). Light ether anesthesia was used, if necessary, during the operation. A vertical incision was made in the lumbar region, and the underlying kidney gently pulled out of the abdomen.
A longitudinal incision was made in the renal capsule. The edge of the incised capsule was lifted up with fine forceps, and the fetal pancreas grafts placed under the capsule, and pushed away from the incision. The fetal pancreas was used because it contains more islets, but less exocrine glands, than the adult pancreas. The kidney was replaced within the peritoneal cavity and abdominal muscles, and the skin incision closed with silk sutures. It required about 10 min for a single engraftment.
Cell Preparation
Peripheral blood (PB) was collected into heparinized plastic vials from the orbital cavity. After mixing, the suspension was layered over 1.5 ml of room temperature lymphocyte separation medium (Lympholyte-M; Cedarlane; Hornby, Ontario; http://www.cedarlanelabs.com) and centrifuged at 3,000 rpm for 30 min at 23°C. The lymphocyte layer was aspirated from the saline-Lympholyte-M interface and washed with medium.
BMCs were collected from the femurs of recipient mice, as previously described [5]. The spleen and lymph nodes were gently teased on a fine steel mesh, and cell suspensions washed twice in RPMI-1640 medium (Nissui; Tokyo, Japan; http://www.nissui.co.jp/top.html), and finally suspended in medium containing 10% fetal calf serum (FCS) (HyClone Laboratories; Logan, UT; http://www.hyclone.com).
Hepatic mononuclear cells (MNCs) were obtained as follows: the liver was perfused in situ via the portal vein with 10 ml of Dulbecco's phosphate-buffered saline (PBS) and 10 ml of prewarmed (38°C) PBS(-) containing 150 U/ml Type IV collagenase (Sigma Chemical Co.; St. Louis, MO; http://www.sigma-aldrich.com). The liver was removed and cut into small pieces. The tissue was transferred into a 50-ml tube, dispersed by pipetting, and added to 40 ml of PBS containing 2% FCS. The cell suspension was centrifuged at 35x g for 1 min at 4°C to remove tissue debris and parenchymal cells. The hepatic MNCs in the supernatant were washed three times at 250x g for 5 min. The hepatic MNCs in the pellet of the last centrifugation were suspended in 2 ml of 31.5% Percoll solution (Pharmacia; Uppsala, Sweden; http://www.pnu.com), layered onto 2 ml of 70% Percoll solution in a 15-ml tube, and covered with 2 ml of PBS. After the centrifugation at 450x g for 20 min, the hepatic MNCs in the lower interface were removed and washed twice. The recovery for hepatic MNCs was about 31%, and the contamination of PB MNCs was less than 1%.
Flow Cytometry
Fluorescein isothiocyanate (FITC)-coupled anti-H-2Kk (030-39F) and H-2Kb (030-11F) mAbs from Meiji Institute of Health Service (Odawara, Japan) and phycoerythrin (PE)-coupled anti-H-2Kb mAb from PharMingen (San Diego, CA; http://www.pharmingen.com) were used for determining the percentage of cells bearing MHC class I (H-2Kb and H-2Kk) surface markers in the PB lymphocytes, BM, spleen, lymph nodes, and liver. PE-coupled anti-CD4, CD8, B220, Mac-1, Gr-1, and CD71 mAbs from PharMingen were used for characterizing the donor-derived BM and liver MNCs. mAbs against erythroid lineage cells (TER119) were kindly donated by T. Kina (Chest Disease Institute; Kyoto University, Kyoto, Japan; http://www.Kyoto-u.ac.jp). The cells were suspended in PBS containing 2% FCS plus sodium azide, then incubated on ice with the appropriate mAbs for 30 min and analyzed by flow cytometry on FACScan (Becton Dickinson & Co.; Mountain View, CA; http://www.bd.com) equipped with logarithmic scales.
Mixed Lymphocyte Reaction (MLR)
Triplicate cultures from four chimeric mice and four control mice were set up in round-bottom 96-well microwell trays (Corning Inc.; Corning, NY; http://www.corning.com). Each well contained 2 x 105 responder cells and 105 stimulator cells in a total of 0.2 ml of RPMI 1640 medium supplemented with 2mM L-glutamine, penicillin (100 units/ml), streptomycin (100 mg/ml) (Sigma-Aldrich; St. Louis, MO), 10% heat-inactivated FCS, and 50 mM 2-mercaptoethanol (2-ME; Wako; Osaka, Japan). Stimulator cells were irradiated with 20 Gy. The cultures were incubated for 96 h in a humidified 5% CO2 atmosphere. [3H] thymidine (0.5 mCi) was introduced during the last 4 h of the culture period. [3H] incorporated into trichloroacetic acid-insoluble materials was measured using a liquid scintillation counter.
Generation of Cytotoxic T-Lymphocytes (CTLs)
Responder cells (7.5 x 106) and stimulater cells (2.5 x 106) were cocultured in RPMI 1640 medium containing 10% heat-inactivated FCS, supplemented with 50 mM 2-ME. Cultures were incubated for five days at 37°C in a 5% CO2 atmosphere. Cells of the cell lines (P815 [H-2d], EL-4 [H-2b] and X5563 [H-2k]) were used as target cells. These cells were labeled by incubation for 1 h at 37°C with 100 mCi of Na251CrO4 (NEN Life Science Products Inc.; Boston, MA; http://w.nenlifesci.com). After washing three times, labeled cells (5 x 104) were mixed with effector cells in 100 ml of RPMI 1640 medium in round-bottom microwells and incubated at 37°C in 5% CO2 for 4 h. The Titertek supernatant system was used for determination of released radioactivity of 51Cr.
Percent-specific lysis was calculated as ([experimental release-spontaneous release]/[maximal release-spontaneous release]) x 100. In the analyses, spleen cells were pooled from mice, and the analyses were performed in triplicate.
Histology
Recipient mice were sacrificed each month after engraftment. The grafts were easily identified as a rounded white swelling on the surface of the kidney. The acceptance and growth of the grafts was assessed using a dissecting microscope, and the kidneys from individual mice were fixed in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin for the histological examination.
The data shown in the figures and tables are representative data since reproducible results were obtained.
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RESULTS
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Analyses of Chimerism
Chimerism was analyzed by flow cytometry using FITC-conjugated anti-class I (H-2b and H-2k) mAbs. As shown in Figure 1
, mixed chimerism was observed in almost all chimeric mice except the mice reconstituted at a host:donor ratio of 1:100. Donor-type T cells, B cells and macrophages were detected (data not shown). Since there was no significant difference between the spleen, lymph nodes and PB, the degree of chimerism was shown in the PB. As shown in Figure 1, donor-type cells (H-2Kk) gradually decreased in all mice except for the mice reconstituted with a ratio of 1:100. However, it should be noted that donor-type cells did not completely disappear. Even at 15 months, 38.0%, 12.0%, 8.2% and 6.2% of donor-type cells were detected in mice reconstituted with the ratios of 1:10, 1:3, 1:1 and 2:1, respectively. Although observations were continued for 16 months, no symptoms of GVHD were clinically or histologically observed in the mixed allogeneic chimeric mice. Complete replacement with donor-type cells was observed in mice reconstituted at a ratio of 1:100.
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Figure 1. Percentages of donor (H-2Kk)-derived cells in the PB of mixed chimeras by two-color FACS analyses. B6 hosts were lethally irradiated (10 Gy) and then reconstituted with a mixture (total 1 x107) of T cell-depleted syngeneic and allogeneic BMCs with various proportions, as described in Materials and Methods. PB was collected from the mice in each group every month after treatment. The groups of 1:100, 1:10, 1:3, 1:1 and 2:1 consisted of 10, 15, 15, 13, and 7 mice, respectively. Statistical analyses were performed by Mann-Whitney U-test: p < 0.005, 1:10 versus 1:100 and 1:3.
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Graft Acceptance
The pancreas tissues transplanted under the capsules of the kidneys in the recipient mice were histologically examined at various time points. As shown in Figure 2, the grafts were accepted by the recipient mice with established mixed chimerism, but rejected in mice without mixed chimerism. Even in mice with a markedly decreased percentage of donor-type cells, no destruction of grafted pancreatic tissue was observed when more than 1% of donor cells were detected in the PB. Immunohistochemical studies revealed the presence of insulin-producing cells (brown) in the engrafted islets (Fig. 2 left).
Induction of Tolerance
The next step was to examine whether donor-specific tolerance is induced in mixed allogeneic chimeric mice using MLR and CTL assays.
MLR was performed to examine the induction of tolerance. Representative data are shown in Figure 3. Twelve months after transplantation, 97.13%, 45.8% and 14.3% of donor cells were detected in mice reconstituted with the ratios of 1:100, 1:10, and 1:3, respectively. All these chimeric mice showed low responses to both donor- and host-type stimulators, although they showed significantly high responses to the third-party (BALB/c) cells (Fig. 3). Moreover, when 3.5% and 3.1% of donor cells were detected 12 months after transplantation in mice reconstituted at ratios of 1:1 and 2:1, these chimeric mice showed significantly high responses to the third-party (BALB/c) cells, whereas they showed low responses to both donor- and host-type stimulators (Fig. 3).
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Figure 3. MLR of spleen cells in B6, C3H, BALB/c, and ([B6 + C3H]B6) chimeric mice one year after treatment. More than three experiments were carried out, and reproducible results were obtained. Representative data are therefore shown. Chimeric mice respond only to third-party (BALB/c) stimulator. Asterisks represent p values of responses to donor-type stimulators versus third party by t-test. *p < 0.0001, **p < 0.005.
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Similar results were obtained in CTL assays (Table 1). The spleen cells of the chimeric mice showed cytotoxic activity to third-party (H-2d) cells, but not to host-type or donor-type cells.
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Table 1. Cytotoxic T-lymphocytes from spleen cells of chimeric mice are tolerant to both host-type and donor-type MHC determinants
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Critical Doses of Donor Cells for Allograft Acceptance
To determine the critical doses of initially transplanted donor cells for allograft acceptance, we next carried out BMT using various ratios of donor and recipient BM cells; the ratios of donor BM were adjusted to 5%, 10%, 20%, or 30%. In addition, pancreatic tissue transplantation was performed at the same time. As shown in Table 2, the grafts were accepted by the recipient mice reconstituted with 10%, 20%, or 30% donor BM cells, but not 5% donor BM cells. Mixed chimerism was observed in recipient mice reconstituted with 10%, 20% or 30% donor BM. The induction of tolerance in these mice was examined by MLR. As shown in Figure 4, chimeric mice reconstituted with 5% donor BM cells, in which the grafts had been rejected, showed high responses to C3H mouse MHC determinants, while chimeric mice reconstituted with 10%, 20%, and 30% donor BM cells, in which the grafts were accepted, showed low responses to C3H mouse MHC determinants. From these results, it can be concluded that the presence of chimerism in the PB is useful for evaluating the induction of tolerance; it seems that more than 10% donor BM cells are initially necessary to prevent the rejection of donor pancreas tissue.
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Table 2. Relationship between acceptance of donor pancreatic grafts and percentages of initially transplanted donor cells in chimeric micea
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Figure 4. Relationship between percentages of donor cells and responsiveness in MLR six months after treatment. The spleen cells of mixed chimeric mice with 5% of donor cells show a high responsiveness to donor (C3H)-type MHC determinants, although the spleen cells of mixed chimeric mice with more than an initial 10% of donor cells show low responsiveness to not only host-type but also donor-type MHC determinants. Asterisks represent p values of responses to donor-type stimulators versus third-party by t-test: *p < 0.001.
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Allogeneic Hemopoietic Stem Cells (HSCs) in the BM and Liver
Since donor-type T cells, B cells, and macrophages are observed in the BM and liver of the recipient mice for an extended period after transplantation, it is conceivable that donor-type HSCs are present in the recipient mice. We therefore analyzed whether donor-type HSCs are present in the BM and liver using a fluorescence-activated cell sorter (FACS), as previously described [6]. As shown in Figure 5, Lin–/CD71–/H-2high cells, which are pluripotent HSCs, as previously described [6-8], were observed in the BM and liver; it should be noted that the percentage of donor HSCs is higher in the liver (6.2%) than in BM (0.0%).
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Figure 5. The characterization of the donor-derived BM and liver MNCs by flow cytometry three months after treatment. BM and liver MNCs of chimeric mice and B/6 mice (control) were stained with H-2Kk, H-2Kb, CD71 mAbs, and lineage markers (CD4, CD8, B220, Mac-1, TER119, Gr-1). The populations of lineages CD71– and H-2Khigh are HSC-enriched. Chimeric mice (four mice) were analyzed, and similar results were obtained. Therefore, representative data are shown in this figure.
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DISCUSSION
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A number of approaches to achieve allogeneic chimerism with lethal or nonlethal conditions have been reported in rodent models [3, 9-11]. Mixed allogeneic chimerism has several advantages over fully allogeneic chimerism. The presence of syngeneic (or autologous) BMCs appears to provide the necessary cells to overcome the impaired immunologic functions and prevent the GVHD observed in fully allogeneic chimeras [12], while the allogeneic BM elements appear responsible for the induction of donor-specific tolerance. In mixed chimeras, primary antibody responses to T cell-dependent antigens are completely restored, since T cells can cooperate with B cells and antigen-presenting cells. Therefore, mixed allogeneic chimerism may be a useful approach to induce tolerance for solid organ and cellular grafts.
Ildstad and Sachs were the first to establish a system of mixed allogeneic chimerism which can be used for organ transplantation [3]. Using skin allografts, they have shown that persistent tolerance can be induced until 380 days by carrying out mixed allogeneic BMT. In the present study using pancreas allografts, we have shown that persistent tolerance can be maintained for more than one year (480 days) after mixed allogeneic BMT, although the percentages of donor-type (C3H) cells gradually decrease (Fig. 1
). This decrease in the percentages of donor (C3H)-type cells can be explained by MHC restriction between HSCs and stromal cells; in the ([B6 + C3H]B6) chimeric mice, B6 HSCs should show a better proliferative response than C3H HSCs, as we have previously shown [13]. It should be noted that all the pancreas allografts are accepted when more than 1% of allogeneic cells are detected in the PB of the recipients. To further examine the relationship between percentages of initially transplanted donor cells and acceptance of pancreatic allografts, we carried out mixed allogeneic BMT using donor BM of decreased percentages (5% to 30%). Table 2 shows that more than 10% of donor cells are necessary to prevent graft rejection. It has been reported that recipient mice with >30% chimerism accept skin allografts in an MHC class II-disparate combination, although recipients with <10% chimerism show prolonged skin graft survival but finally reject skins [14]. It is conceivable that the difference between the data of Taniguchi et al. and ours is due to the grafts of different organs (the skin and pancreas) and different mouse combinations (only class II-disparate combination in the former and both class I- and class II-disparate combinations in the latter).
In the present study, we have shown that pancreas allografts are accepted by the recipient mice with establishment of mixed chimerism in the PB despite marked decreases in donor-type cells (Figs. 1 and 2; Table 2). MLR (Fig. 3) and CTL (Table 1) assays indicate the induction of systemic tolerance in these mice. Mechanisms underlying tolerance induction include clonal deletion [15, 16], anergy [17], and suppression [18, 19]. In the present study, we have demonstrated the presence of donor-derived allogeneic HSCs in the BM and liver (Fig. 4). Since we have previously found that donor HSCs trapped in the liver induce clonal anergy in the recipient CD8+ CTLs [20], it is certain that clonal anergy is involved in tolerance induction in this mixed allogeneic chimerism. Although we have not examined the clonal deletion mechanism in this system, it is also conceivable that clonal deletion, not only in the thymus but also in the periphery, is involved in this system, since Zavazava et al. have recently demonstrated that soluble MHC class I molecules induce apoptosis in alloreactive CTLs [21]. Suppressor mechanisms appear to be involved in the establishment of tolerance induction even in mixed allogeneic BMT. Suppressor cells include suppressor T cells (CD8+ cells), natural killer cells, and natural suppressor (NS) cells; we have previously found that NS cells belong to HSCs in the cycling phase in the BM [22].
The skin and pancreas are candidates to examine functional tolerance in vivo, since they are highly antigeneic and very sensitive to rejection [7]. Starzl et al. have found in humans that there are some cases in which liver allografts survive without using immunosuppressants, and that, in such cases, a small number of donor-derived hemopoietic cells are detected. We have very recently established the method for organ allografts by injecting allogeneic hemopoietic cells (including HSCs) from the portal vein; the recipient mice show microchimerism [17], as shown in the present study. It should be noted that the percentage of donor-type HSCs is higher in the liver (6.2%) than in the BM (0.9%) (Fig. 5). We have previously found that HSCs trapped in the liver induce anergy of recipient CTL2 [20]. We are in the process of analyzing the exact mechanisms underlying tolerance induction in microchimerism.
In summary, we report here that long-term pancreatic allograft survival over a one-year period can be reliably achieved in MHC-disparate allogeneic donor and recipient combinations, although donor-type cells gradually decrease. The tolerance was highly MHC-specific, as evidenced by MLR and CTL assays.
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ACKNOWLEDGMENTS
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We thank Ms. Keiko Ando for preparing the manuscript.
This work was supported by a grant from the Japanese Ministry of Health and Welfare, the Ministry of Education, Science and Culture, Japan, and the Private School Promotion Foundation, Japan.
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REFERENCES
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Sykes M, Eisenthal A, Sachs DH. Mechanism of protection from graft-vs-host disease in murine mixed allogeneic chimeras. I. Development of a null cell population suppressive of cell-mediated lympholysis responses and derived from the syngeneic bone marrow component. J Immunol 1988;140:2903-2911.[Abstract]
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Sykes M, Sachs DH. Mechanisms of suppression in mixed allogeneic chimeras. Transplantation 1988;46(suppl 2):135-142.
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Sugiura K, Kato K, Hashimoto F et al. Induction of donor-specific T cell anergy by portal venous injection of allogeneic cells. Immunobiol 1997;197:460-477.[Medline]
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Received May 12, 2000;
accepted for publication May 23, 2000.
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Abstract
Introduction
