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Stem Cells 2004;22:683-695 www.StemCells.com
© 2004 AlphaMed Press

Induction of Tolerance in Quadruple Chimeric Mice

Tian-Xue Fana, Hiroko Hishaa,b,c, Tie-Nan Jina, Kikuya Sugiurae, Muneo Inabaa,b,c, Guo-Xiang Yanga, Qing Lia, Xiao-Li Wanga, Chang-Ye Songa, Yun-Ze Cuia, Qiang Lid, Yuming Zhanga, Xiao-Guang Zhangf, Hong-Xue Fang, Susumu Ikeharaa,b,c

a Department of Pathology,
b Transplantation Center,
c Regeneration Research Center for Intractable Diseases, and
d Department of Neurosurgery, Kansai Medical University, Moriguchi, Osaka, Japan;
e Laboratory of Cell Pathobiology, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, Osaka, Japan;
f Department of Ophthalmology and
g Department of Toxicology, Norman Bethune Medical University, Changchun, China

Key Words. Cord blood cells • Bone marrow transplantation • Reconstitution

Correspondence: Susumu Ikehara, M.D., First Department of Pathology, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi City, Osaka 570-8506, Japan. Telephone: 81-66-993-8283; Fax: 81-66-884-8283; e-mail: ikehara{at}takii.kmu.ac.jp


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human cord blood (CB) contains hematopoietic stem cells and progenitors. Because the major limitation to a widespread use of CB for transplantation lies in its limited volume, it is necessary to combine the CB from several donors. In this study, we show that lethally irradiated mice can be reconstituted with the injection of a mixture of T cell–depleted bone marrow cells (BMCs; total, 3 x 106) obtained from three fully allogeneic mouse strains in two different mouse combinations. A higher survival rate was obtained in the triple injection group than in mice injected with BMCs (1 x106) obtained from a single mouse strain. In the mixed chimeric mice, three kinds of donor-type and recipient-type cells were detected in all the hematopoietic organs 1 month after bone marrow transplantation (BMT). Mixed-lymphocyte reaction showed that the tolerance to both recipient-type and donor-type major histocompatibility complex determinants was induced in the chimeric mice. In the peripheral blood (PB) of these mice, only one type of cells from the three different donor strains became dominant in most chimeric mice and reached a stable level about 4 months after BMT. Polymerase chain reaction analyses, however, revealed that the skins from all the donors were accepted even when no cells with their phenotypes could be detected in the PB. These results suggest that both hemato-lymphoid reconstitution and stable tolerance to not only the recipient strain but also all the donor strains can be achieved in chimeric mice, indicating the possibility of mixed CB transplantation in humans.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Numerous means have been used to conquer the main problems (graft-versus-host disease [GVHD], graft failure, etc.) in allogeneic bone marrow transplantation (BMT) across major histocompatibility complex (MHC) barriers. It has been shown that the transplantation of purified hematopoietic stem cells (HSCs) can reduce the occurrence of GVHD more than can transplantation of whole bone marrow cells (BMCs) [1]. During the past decade, umbilical cord blood (CB), as a source of HSCs, has been used for hematopoietic reconstitution in pediatric patients with both malignant and nonmalignant disorders [2]. Only grade 2 GVHD, which could be controlled with steroid and antithymocyte globulin therapy, developed in patients (body weight <40 kg) transplanted with MHC-mismatched CB cells [3].

In contrast to BMCs, the CB presents multiple advantages. Besides the absence of risk and discomfort to donors, CB offers the clinician a source of HSCs that is rarely contaminated by latent viruses and is readily available [4–6]. In addition, CB has the following advantages: (a) a higher capacity to form colonies in culture, (b) a higher cell-cycle rate, (c) autocrine productions of growth factors, and (d) longer telomeres. Moreover, the relative immaturity of lymphocytes in CB may reduce the risk and severity of GVHD and would allow HLA mismatching between donors and recipients [7–9].

It has been estimated that pluripotent HSCs are contained to a greater extent in CB than in peripheral blood (PB); however, individual samples of umbilical CB are quite variable in both quantity (the volume obtained from a single donor) and quality (the number of colony-forming cells per ml of CB). Another important impediment is the volume of CB. The median number of nucleated cells in CB was 1.1 billion cells per unit [7], which is not enough for BMT in an adult patient. There are some reports that better engraftment can be achieved using a megadose of HSCs, even in MHC-incompatible recipients [10–12]. We have recently demonstrated that donor-specific tolerance could be achieved by the injection of 3 x107 BMCs into mice irradiated with a sublethal dose and without additional immunosuppressants [13].

Ende et al. [8] have shown that the storage of human CB at 4°C in a gas-permeable bag can preserve the capacity of the CB cells for mitosis and cellular expansion.

All of these reasons suggest that a mixture of CB cells obtained from stored samples could be one method of collecting an adequate number of HSCs for HLA-mismatched hematopoietic transplantation. Moreover, HLA-mismatched and unrelated multi-CB cells have already been used for the treatment of advanced solid tumors, and little or no GVHD was observed [14]. These findings suggest that the pooled CB can supply a sufficient quantity or even a megadose of HSCs for an adult patient and can reconstitute recipients.

We investigated whether pooled BMCs obtained from three fully allogeneic mouse strains can reconstitute lethally irradiated recipients for the long-term. An examination using at least three different donor strains is necessary, because a pooling of the CB from at least three different sources is required to obtain sufficient quantities to reconstitute an adult recipient (3 x109 CB cells per adult of 60 kg). In the study reported here, we show that the transplantation of T cell–depleted BMCs (TCD-BMCs) obtained from three different donors can be used to induce persistent tolerance in allogeneic recipients.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Eight-week-old female or male C57BL/6 (H-2b), BALB/c (H-2d), C3H/HeN (H-2k), and DBA/1 (H-2q) mice were purchased from Shizuoka Experimental Animal Laboratory (Hamamatsu, Japan). Before experiments, they were kept in a pathogen-free environment for 1 or 2 weeks and were fed with acidified water. C57BL/6 (B6) mice were used as recipients. A.SW (H-2s) mice were kindly supplied by Dr. Noriyoshi Hashimoto (Kanazawa University). DBA/1,A.SW, BALB/c, and C3H mice were used as donors. Twelve-week-old female B6 mice were also used as recipients when skins of two or three combinations from allogeneic male donors were grafted together with a mixture of BMCs from three different strains.

Preparation of Cell Suspension and BMT
Mice were killed by decapitation. BMCs, taken from the humeri, femurs, and tibias, were flushed out using a 22-gauge needle and kept in phosphate-buffered solution (PBS) containing 2% heat-inactivated fetal calf serum (FCS) on ice. After washing two times, the BMCs were treated together with rat monoclonal antibodies (mAbs)—anti-CD4 (clone: GK 1.5) and CD8 (53-6.72) mAbs (PharMingen, San Diego)—for 30 minutes on ice and washed twice, and then incubated with sheep antirat IgG-conjugated immunobeads (Ibs; Dynabeads M-450, Dynal A.S., Oslo, Norway) at 4°C for 30 minutes with gentle agitation at a 3:1 bead/cell ratio. Ibsrosetted cells were removed using a magnetic particle concentrator. The nonrosetted cells were considered as TCD-BMCs. These TCD-BMCs (1 x106 cells per 0.3 ml) were injected into 9.5-Gy–irradiated (18–20 hours prior) B6 mice intravenously (i.v.) via the tail vein. Another group of recipients were i.v. injected with the mixture of the three kinds of TCD-BMCs, for a total of 3 x106/0.3 ml (combination I: DBA/1, BALB/c, and C3H; combination II:A.SW, BALB/c, and C3H). To compare the same dose of injection, TCD-BMCs (3 x106/0.3 ml) from a single strain were injected into the lethally irradiated B6 mice as controls.

Each group in each combination consisted of more than 10 mice. Each experiment was performed five or more times.

Skin Graft
Skin grafts were performed on the day of BMT by a slight modification of the technique of Jin et al [15]. Briefly, full-thickness skin grafts (1x1 cm) were harvested from the dorsal wall of the donor mouse strains (male, 8 weeks old) from which hair had been completely removed by depilatory, and the skin was then kept in the dishes with PBS. The recipients (female, 12 weeks old) were anaesthetized using somnopentyl, and the dorsal skin of the left and right parts was gently removed. The prepared donor skins were sutured to the graft beds using 6-0 nylon and then covered with Vaseline gauze and protective tape. Because the middle dorsum is easily affected by the recipient’s movements, and because three different skin grafts on one recipient would decrease their chances of survival, the recipients were mainly transplanted with two combinations from allogeneic donors. In these experiments, 12-week-old mice were used as recipients. This was because they are large enough to take the skin grafts, which the 8-week-old mice are not. Some of the recipients were divided into three groups, and the mice in these groups were engrafted with A.SW + BALB/c, A.SW + C3H/HeN, or BALB/c + C3H/HeN. The engrafted mice were injected with antibiotics for 3 weeks until the special bandage had been opened. From the third week after the transplantation, the grafted skins were checked every day for 2 weeks; thereafter, checks were carried out every 3 days. Some of the mice whose grafted skins had become hairless were chosen for polymerase chain reaction (PCR) analysis.

Analysis of Chimerism
The PB of the recipient mice was collected from the tail vein 4 weeks after BMT. The PB cells were suspended in PBS containing heparin (10 units/ml). Mononuclear cells in the PB (PBMNCs) were isolated by discontinuous density gradient centrifugation using lymphocyte-mammal (1.0875 g/ml; Cedarlane, Ontario, Canada, http://www.cedarlanelabs.com). The PBMNCs were double stained with appropriate fluorescent isothiocyanate (FITC)–conjugated and phycoerythrin (PE)–conjugated anti-H-2 mAbs (mouse antibodies). In some experiments, PBMNCs were triple stained with FITC-conjugated and PE-conjugated anti-H-2 mAbs together with a panel of Red 617-biotinylated anti-Mac-1, Gr-1, B220, CD4, or CD8 mAbs. The stained cells were then quantified with a FACStar (Becton, Dickinson, Franklin Lakes, NJ).

Mitogen Response
Mitogen reactivity was evaluated using spleen cells from the chimeric mice 11 months after BMT. The 200,000 spleen cells collected from chimeric mice and normal control mice were plated in 96-well flat-bottomed plates (3595, Corning Glass Works, Corning, NY) containing 200 µl of RPMI 1640 medium (Nissui Seiyaku Co., Tokyo) supplemented with 2 mM L-glutamine (Wako Pure Chemicals, Tokyo), penicillin (100 units/ml), streptomycin (100 µg/ml), and 5% heat-inactivated FCS. These cells were incubated with either concanavalin A (Con A, 2.5 µg/ml [Calbiochem, San Diego]), phytohemagglutinin P (PHA, x400 [Difco, Detroit, MI]), or lipopolysaccharide (LPS, 25mg/ml [Difco]) for 72 hours at 37°C in a humidified atmosphere of 5% CO2 in air. 3H-thymidine (3H-TdR, 0.5 µCi in 20 µl [New England Nuclear, Cambridge, MA]) was introduced during the last 18 hours of the culture period. Incorporation of 3H-TdR was measured using Matrix 96 (Packard, Meriden, CT).

Mixed Lymphocyte Reaction (MLR)
The spleen cells from the chimeric mice were checked to estimate their reactivity with various donor-type spleen cells. Triplicate cultures were set up in 96-well round-bottomed microtiter plates (25850, Corning) containing responder cells (4 x 105 cells per well) and 15 Gy-irradiated stimulator cells (3 x 105 cells per well), which were suspended in 200 µ1 of the medium mentioned above. These cells were cultured for 5 days. During the last 18 hours of culture, 0.5 µCi of 3H-TdR was added. The uptake of 3H-TdR was counted using Matrix 96.

PCR Analyses
Genomic DNA was isolated from the hairless skins of recipients using ISOHAIR (DNA Isolation Kit; Nippon Gene Company Ltd., Tokyo) following the manufacturer’s instructions. PCR amplification was performed for 35 cycles in PCR buffer containing dNTP and Taq polymerase (Perkin Elmer-Cetus, Branchburg, NJ). The following oligonucleotide primers with the sequences 5'-GCATTTGCCTGTCAGAGAGAG-3' (sense strand) and 5'ACTGCTGCTGCTTTCCAACTA-3' (anti-sense strand) were constructed by and purchased from Biologica Co. (Nagoya, Japan). This primer pair was created to amplify a 411-bp region contained within the murine Y chromosome.

Statistics
Statistical differences in survival rates were analyzed by a log-rank test. Each experiment was carried out four or more times. Reproducible results were obtained, and therefore representative data are shown in the figures.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mixture of TCD-BMCs from Different Mouse Strains Increases Survival
In our preliminary experiments, we found that 1 x106 BMCs from allogeneic mice was insufficient to rescue lethally irradiated B6 mice: the survival rates were only 20%–60%. Lethally irradiated B6 mice were therefore divided into four groups: one group was given a mixture (3 x106) of BMCs obtained from three mouse strains, and the other three groups were given a small amount (1 x106 per mouse) of BMCs from only one mouse strain (Fig. 1Go). In combination I (B6 as recipient and BALB/c, C3H, and DBA/1 mice as donors), 80% of the recipients given a mixture of three kinds of TCD-BMCs survived more than 80 days, whereas the survival rate of the single-donor group was less than 60% (Fig. 2Go). Of the three donor mouse strains, it seems that DBA/1 mice have a higher ability to reconstitute lethally irradiated B6 mice than do the other two mouse strains.



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  		Figure 1. Experimental protocol for transplantation of TCD-BMCs. Abbreviation: TCD-BMC, T cell–depleted bone marrow cell.

 


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  		Figure 2. Survival rates after transplantation of TCD-BMCs. In combination I, 80% of triple chimeric mice survived at least 80 days after BMT. No significant difference was observed between triple and single (DBA/1) chimeric mice. These two groups showed a better survival rate than the other two groups that were injected with TCD-BMCs obtained from a single strain (BALB/c or C3H). In combination II, mice injected with a mixture of three kinds of TCD-BMCs showed a significantly higher survival rate than all three groups injected with only 1 x106 from a single mouse strain. Furthermore, at least 90% of recipients survived when injected with 3x106 from a single mouse strain. Statistical analyses were carried out using a log-rank test. Each group consisted of 24 or more mice. Abbreviations: BMT, bone marrow transplant; TCD-BMC, T cell–depleted bone marrow cell.

 
We next replaced DBA/1 mice with another strain, the A.SW (H-2s) mouse. In combination II (Fig. 2Go), 90% of triple chimeric mice (when 3 x106 BMCs per mouse were injected) survived more than 40 days after BMT. In the groups of mice injected with TCD-BMCs (1 x106) obtained from a single mouse strain, 55%, 42%, and 39% survival rates were observed in A.SW, BALB/c, and C3H mice, respectively. Thus, in combination II as well, a higher survival rate was obtained in mice injected with a mixture of TCD-BMCs than in mice injected with BMCs from a single mouse strain. However, 3 x106 of BMCs from a single mouse strain could reconstitute the recipient mice to a slightly greater extent than did 3 x106 BMCs obtained from triple strains. Single-strain donor groups showed 100% (A.SW), 100% (BALB/c), and 90% (C3H) survival rates.

Mixed TCD-BMCs Have Ability to Generate Multilineage Cells
All surviving mice were analyzed for their chimerism 4 weeks after BMT using fluorescence-activated cell sorting (FACS). PBMNCs were stained with anti-H-2 antibodies (Abs). When BMCs from only one mouse stain were injected, the percentages of donor cells were 7.2% (BALB/c-type), 13.2% (C3H-type), and 43.0% (DBA/1-type) in combination I. These percentages in each single-donor group were higher than those in each cell type in the triple-donor group (average: 3.9%, 2.2%, and 34.3%, respectively). The chimeric mice showed three kinds of reconstitution states (Fig. 3Go): type A shows all donor-type BMCs, type B shows two kinds of donor-type BMCs, and type C shows one kind of donor-type BMCs. Types B and C are further divided into three subgroups, respectively, as shown in Figure 3Go. Type A mice were 18% of all the triple chimeric mice, type B mice were 23%, and type C mice were 59% in combination I. In type A, three kinds of donor-type cells were detected in the PB, bone marrow, and other hematopoietic organs, whereas type C mice showed mostly DBA/1 phenotype (H-2q) 8 weeks after BMT (Fig. 4Go), which was compatible with the highest survival rate in the DBA/1 single-strain group (Fig. 2Go). In combination II, as well as in combination I, the percentages of donor-type cells were higher in the single-donor groups (BALB/c, C3H, and A.SW alone) than those in the triple-donor group. The recipient mice given BMCs from three strains also showed three types: A, B, and C. The type A, B, and C mice were 50%, 20%, and 30% in all the triple chimeric mice respectively (Fig. 5Go). In all the recipient mice, recipient-type cells were also detected, together with donor-type cells, indicating that they are quadruple chimeric mice.




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  		Figure 3. Representative H-2 staining patterns in mononuclear cells in the peripheral blood of chimeric mice in combination I (4 weeks after bone marrow transplantation). Chimeric mice are divided into three groups (A, B, and C) according to reconstitution states of donor phenotypes. Type B is further divided into three subgroups: Type B-1 has H-2b, H-2d, and H-2k phenotypes; type B-2 has H-2b, H-2k, and H-2q phenotypes; and type B-3 has H-2b, H-2d, and H-2q phenotypes. Type C also shows three reconstitution states: Type C-1 has H-2b and H-2k phenotypes, type C-2 has H-2b and H-2q phenotypes, and type C-3 has H-2b and H-2d phenotypes. Abbreviation: BMC, bone marrow cell.

 


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  		Figure 4. H-2 typing of various tissues from chimeric mice in combination I (8 weeks after bone marrow transplantation). The mice in combination I were sacrificed, and mononuclear cells from the bone marrow, spleen, and thymus were stained with fluorescent isothiocyanate–conjugated and phycoerythrin–conjugated anti-H-2 mouse antibodies and analyzed using a fluorescence-activated cell sorter scan. Abbreviations: BMC, bone marrow cell; PBC, peripheral blood cell.

 



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  		Figure 5. Representative H-2 staining patterns in mononuclear cells in the peripheral blood of chimeric mice in combination II (4 weeks after bone marrow transplantation). Chimeric mice in combination II are also divided into seven groups according to reconstitution states of donor phenotypes: type A, type B-1, type B-2, type B-3, type C-1, type C-2, and type C-3. Abbreviation: BMC, bone marrow cell.

 
In type A of combinations I and II, three kinds of donor strains could be detected in the PB 1 month after BMT. However, 4 months after BMT, the reconstituting situation changed: One kind of donor-type cells became dominant, while the other kinds of cells, including recipient-type cells, decreased gradually (Fig. 6Go). Seven or 9 months after BMT, this tendency became even more evident. Some of the combination I chimeric mice were sacrificed 5 months after BMT, and the BMCs, spleen cells, and thymus cells were then analyzed. Similar patterns to those seen in the PB were detected (data not shown).



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  		Figure 6. Predominant expansion of single phenotype cells in peripheral blood of quadruple chimeric mice. Mononuclear cells in the peripheral blood (PBMNCs) of chimeric mice in combination I were collected and stained with fluorescent isothiocyanate (FITC)–conjugated and phycoerythrin (PE)–conjugated anti-H-2 mouse antibodies (mAbs) 1, 2, and 7 months after bone marrow transplantation (BMT). PBMNCs of chimeric mice in combination II were collected and stained with FITC-conjugated and PE-conjugated anti-H-2 mAbs 1, 4, and 9 months after BMT.

 
Various Immunological Functions Are Restored in Quadruple Chimeric Mice
Spleen cells from the mice of types A and C (combination I) responded well to PHA, Con A, and LPS when mitogenic stimulation assays were carried out 11 months after BMT (Fig. 7Go). There was no obvious difference in mitogen response between both chimeric mice. The responsiveness of chimeric mice was higher or similar to that of normal C3H and DBA/1 mice, although it was lower than that of normal BALB/c and B6 mice in PHA or LPS stimulation.



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  		Figure 7. Mitogen responsiveness of spleen cells from chimeric mice (types A and C) in combination I. Eleven months after bone marrow transplantation, the spleen cells of types A and C mice were collected and cultured in the presence of PHA, Con A, or LPS for 72 hours. Proliferation was assessed by 3H-TdR incorporation for 18 hours before harvesting. Each column represents mean ± standard deviation of triple cultures. Abbreviations: Con A, concanavalin A; 3H-TdR, 3H-thymidine; LPS, lipopolysaccharide; PHA, phytohemagglutinin P.

 
The plaque-forming cell (PFC) assay also showed that spleen cells from the chimeric mice produced anti-sheep red blood cell Abs, as did the normal age-matched recipients (data not shown).

Quadruple Chimeric Mice Show Tolerance to Both Donor-Type and Recipient-Type MHC Determinants
Spleen cells from quadruple chimeric mice of types A and C in combination I were analyzed in MLR 4 months after BMT (Fig. 8Go). Spleen cells from type A mice showed no proliferative response to BALB/c, C3H, or DBA/1 stimulator (donor-type), because there was no significant difference in the values on the stimulation index between donor-type and recipient-type stimulators. Surprisingly, spleen cells from type C mice also showed a low degree of proliferative response to BALB/c and C3H stimulator cells, but nearly no proliferative response to the DBA/1 stimulator cells, although the type C mice used in this reaction mainly showed the DBA/1 phenotype in the PB. In contrast, spleen cells did respond to the third-party (A.SW) cells.



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  		Figure 8. No responsiveness of spleen cells from chimeric mice (types A and C) in combination I to donor-type spleen cells in a mixed-lymphocyte reaction. Four months after bone marrow transplantation, the spleen cells of types A and C mice were collected and cultured in the presence of irradiated syngenic or allogenic spleen cells. These mice show tolerance to all donor stimulators. Each column represents the mean ± standard deviation of triple cultures. Abbreviation: 3H-TdR, 3H-thymidine.

 
As the skin is more immunogenic than other organs, we performed skin grafts to examine whether donor-type skins would be accepted in the quadruple chimeric mice. Skins from three donor strains were transplanted onto the chimeric mice of combination II on the day of BMT. Because the mice were too small to be transplanted with three skin grafts, most mice were transplanted with two kinds of graft. As expected, thus-treated mice accepted both grafts, whether or not donor phenotypes were detected in PBCs at 4 months after BMT (Fig. 9Go). A higher tendency to become hairless tissue was observed in the mice grafted with three kinds of skin than in those that received two kinds. PCR was performed on some of the engrafted mice (female) in order to detect donor (male)-derived cells (Fig. 10Go). Although the grafted skin had changed to hairless tissue, and not all donor phenotypes were detected in the chimeric mouse PBCs (H-2s in chimeric mouse 1, and both H-2d and H-2k in chimeric mouse 2), all the phenotypes of grafted skins were detected in all the skin samples from the graft.



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  		Figure 9. Acceptance of donor-type skins in chimeric mice (type A) in combination II. The B6 mouse (12 weeks old) was injected with a mixture of T cell–depleted bone marrow cells (total 3 x106) of three different mouse strains and engrafted with C3H and BALB/c skins on the same day. Skin grafts were performed in a total of 30 chimeric mice, and a representative recipient mouse is shown (4 months after skin grafts).

 


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  		Figure 10. Detection of donor-derived cells in grafted skin of chimeric mice (type A) in combination II by PCR. Eight months after bone marrow transplantation plus skin graft (male skins ->female recipient), some of the grafted skins had become hairless. The grafted skins were removed, and PCR amplification was performed for 35 cycles. The primer pair was prepared to amplify a 411-bp region containing the murine Y chromosome. Genomic DNA from male and female mice was also examined as positive and negative controls. PB was also collected from the recipient mice when their skins were taken, and the PB cells were stained with fluorescent isothiocyanate–conjugated and phycoerythrin-conjugated anti-H-2 mouse antibody. Phenotype of H-2s was not detected in chimeric mouse 1, and phenotypes of H-2d and H-2k were not detected in chimeric mouse 2. However, all the donor phenotypes (Y chromosome) were detected in all the hairless tissues of grafted skins. Abbreviations: PB, peripheral blood; PBC, peripheral blood cell; PCR, polymerase chain reaction.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the study reported here, we demonstrated that the transplantation of mixed TCD-BMCs can rescue lethally irradiated mice more effectively than can that of a small number of BMCs from only one allogeneic mouse strain (Fig. 2Go). In combination II, the difference was significant (p < .025). Although no significant difference was observed in combination I, the triple chimeric mice did show a higher survival rate than the DBA/1 mice. It was previously reported that in either allogeneic or xenogeneic BMT in lethally irradiated rodents better-quality engraftment and longer-term survival could be achieved using a megadose of BMCs [16, 17]. Bachar-Lustig et al. [10] also showed that a megadose of TCD-BMCs or HSCs could overcome the barriers of MHC in sublethally irradiated mice, and that stable chimerism was maintained in various hematopoietic organs 8 months after BMT. In our experiments, the survival rate of the recipients of mixed TCD-BMCs (3 x106) was similar to that of the recipients of TCD-BMCs (3 x106) obtained from a single mouse strain. When a total of 1 x106 mixed TCD-BMCs were grafted, all the recipients died within 50 days (data not shown). Neither acute nor chronic GVHD was observed in any of the recipient mice. As we expected, reconstitution patterns in the PB were similar to those in other hematopoietic organs (Fig. 4Go). The mitogen-induced proliferative response (Fig. 7Go) and anti-SRBC PFC response (data not shown), which reflect immunological functions, were restored in the quadruple chimeric mice. MLRs (Fig. 8Go), and skin grafts (Figs. 9Go and 10Go) also showed that tolerance was induced to all the kinds of donors.

Some of the skin grafts became hairless due to friction. Therefore, we performed PCR analyses to preclude the possibility of scar formation due to rejection, since PCR analysis has been used to detect traces of donor-derived cells in grafted skins. Kim et al. [18] made a rejection model of skin allografts and reported that skin samples from graft sites where donor skins had been rejected and then replaced by recipient scar tissue did not contain any donor-type cells, even if sensitive PCR was performed. As shown in Figure 10Go, the hairless skins observed in the quadruple chimeric mice were not derived from recipient cells but from donor cells, since the donor Y chromosome was detected.

In the present experiments, we used 12-week-old mice as recipients when skins of donor strains were grafted together with a mixture of BMCs from three different strains. This is because their bodies are large enough for skin grafts, which is not the case with 8-week-old mice. There was no difference in survival and reconstitution rates between 12-week-old and 8-week-old mice (data not shown). Very recently, we have observed that complete reconstitution and tolerance to donor-type cells were induced in aged mice (more than 15 weeks) when donor bones (to recruit stromal cells) were also grafted with BMCs (data not shown). Therefore, if donor stromal cells were to be cografted in the present experimental system, similar success with reconstitution might be achieved even in older mice with much less or no function of the thymus. Indeed, recently, BMT (particularly minitransplantation) is being carried out in humans, even in aged patients (more than 60 years old).

A similar BMT experiment using mixed TCD-BMCs has recently been reported by Chen et al. [19]. They injected mixed BMCs (total, 5 x106) from two different donors into lethally irradiated recipients, with the result that white blood cell and platelet counts were doubled on days 10 to 14 after BMT, in contrast to mice injected with 2.5 x106 BMCs from a single donor. Mixed chimerism was stable more than 200 days after BMT, and normal immune response against ovalbumin challenge was observed in these mixed chimeric mice. These results are consistent with ours. However, experiments with mice that are more than triple chimeric are required in preclinical trials for CB transplantation, since CB cells from two different donors are insufficient for an adult patient, as mentioned in the introduction.

After depleting mature T cells from the donor BMCs, only fewer than 0.5% of CD4+ or CD8+ cells could be detected (data not shown). It has been reported that the T-cell depletion from BMCs causes an increased incidence of allograft failure [20]. Furthermore, recent studies have shown that CD8+ allo-BMCs contribute to the deletion of responding T cells in the recipients via apoptosis (veto effect) [21]. However, it has been shown that, compared with non-TCD-BMT, TCD-BMT induces significant alterations in serum cytokines. These cytokines are considered to contain factors that prevent GVHD [22]. It is reasonable, therefore, to assume that improved survival rates could be achieved if mixed BMCs (without the mature T cells) from several donors were engrafted in HLA-mismatched recipients.

In our study, the quadruple chimeric mice (type A) contained cells of all three donor types and recipient types 1 month after BMT, but those of only one phenotype were predominant 4 months later (Fig. 6Go). The cells of this predominant phenotype continued to increase in number until 7 or 9 months after BMT, whereas the percentages of the other donor-type and recipient-type cells decreased gradually. The survival, proliferation, and differentiation of donor HSCs in the recipients are essential factors for reconstitution. For successful engraftment, the recipient marrow space or niche for grafted HSCs is created by the engraftment facilitation procedure [23, 24]. We have shown that the stromal cells can protect the stem cells from attack by effector cells and that the HSCs can proliferate by direct interaction with MHC-compatible stromal cells rather than MHC-incompatible stromal cells [25, 26]. Using B10 congenic mouse strains, we previously found that the MHC preference is restricted by MHC class Ia molecules [27].

Based on these findings, we recently established a new strategy for allogeneic BMT: intra–bone marrow injection of allogeneic bone marrow cells (IBM-BMT). IBM-BMT can successfully treat autoimmune diseases in chimeric-resistant MRL/lpr mice [28]. The allogeneic mouse strains used in the present experiments show different alleles from the recipients at class Ia, class II, and class III but not at class Ib (Table 1Go). DBA/1 and A.SW have the same class Ib alleles with recipient B6. This might be one of the reasons that BMCs from DBA/1 or A.SW mice showed higher reconstitution ability than did BALB/c and C3H mice in combinations I and II. There is also the possibility that the donor MHC antigens have a role in inducing tolerance in such an artificial case (pooled BMCs into one recipient). It is conceivable that donor stromal cells contained in the TCD-BMCs were also grafted together with the donor P-HSCs into the lethally irradiated recipients and that they migrated into the recipient’s bone marrow and spleen. In these organs, there is likely competition not only between donor and recipient stromal cells but also between MHC-matched and MHC-mismatched P-HSCs. There are other possibilities modifying the result of BMT, such as cytokines produced by donor- or recipient-origin cells and the cross-reactivity among these cytokines that inhibit or promote the growth of primitive hematopoietic progenitors [29]. It has been shown that dendritic cells can engulf and process apoptotic cells and then tolerize recipient T cells, followed by induction of alloantigen-specific hyporesponsiveness in vitro and in vivo [30]. Further studies are necessary to elucidate the mechanisms that underlie tolerance induction in quadruple chimeras.


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  		Table 1. Compatible alleles at H-2 loci
 
In conclusion, mixed CB transplantation would be applicable to humans, since, in mice, hematolymphoid reconstitution was achieved and stable tolerance to all the donor mouse strains could be maintained in the recipient.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We thank Mr. Hilary Eastwick-Field and Ms. Keiko Ando for manuscript preparation. This work was supported by a grant from the Haiteku Research Center of the Ministry of Education; a grant from the Millennium program of the Ministry of Education, Culture, Sports, Science and Technology; a grant from the "Science Frontier" program of the Ministry of Education, Culture, Sports, Science and Technology; a grant-in-aid for scientific research (B) 11470062 and a grant-in-aid for scientific research on priority areas (A) 10181225 and (A) 11162221; and a grant from Japan Immunoresearch Laboratories Co., Ltd (JIMRO).

Tian-Xue Fan and Hiroko Hisah contributed equally to this work.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received April 7, 2003; accepted for publication April 21, 2004.




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