Induction of Tolerance in Quadruple Chimeric Mice
http://www.100md.com
《干细胞学杂志》
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@takii.kmu.ac.jp
ABSTRACT
Numerous means have been used to conquer the main problems (graft-versus-host disease , 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) . 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 . 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 .
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 . 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 .
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 , 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 . 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 .
Ende et al. 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 . 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
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. 1). 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. 2). 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.
Figure 1. Experimental protocol for transplantation of TCD-BMCs. Abbreviation: TCD-BMC, T cell–depleted bone marrow cell.
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. 2), 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. 3): 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 3. 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. 4), which was compatible with the highest survival rate in the DBA/1 single-strain group (Fig. 2). 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. 5). In all the recipient mice, recipient-type cells were also detected, together with donor-type cells, indicating that they are quadruple chimeric mice.
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.
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.
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. 6). 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).
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. 7). 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.
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. 8). 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.
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. 9). 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. 10). 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.
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).
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
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
Lie AK, To LB. Peripheral blood stem cells: transplantation and beyond. Oncologist 1997;2:40–49.
Wagner JE. Umbilical cord blood transplantation: overview of the clinical experience. Blood Cells 1994;20:242–244.
Kurtzberg J, Graham M, Casey J et al. The use of umbilical cord blood in mismatched related and unrelated hemopoietic stem cell transplantation. Blood Cells 1994;20: 275–284.
Young JC. Retention of quiescent hematopoietic cells with high proliferative potential during ex vivo stem cell culture. Blood 1996;87:545–556.
Brossard Y,Van Nifterik J, De Lachaux V et al. Collection of placental blood with a view to hemopoietic reconstitution. Nouv Rev Fr Hematol 1990;32:427–429.
Newburger PE, Quesenberry PJ. Umbilical cord blood as a new and promising source of unrelated-donor hematopoietic stem cells for transplantation. Curr Opin Pediatr 1996;8:29–32.
Gluckman E, Rocha V, Chammard AB et al. Outcome of cord-blood transplantation from related and unrelated donors. N Engl J Med 1997;337:373–381.
Ende N, Lu S,Alcid MG et al. Pooled umbilical cord blood as a possible universal donor for marrow reconstitution and use in nuclear accidents. Life Sci 2001;69:1531–1539.
Gabutti V, Timeus F, Ramenghi U et al. Human cord blood progenitors: kinetics, regulation and their use for hemopoietic reconstitution. Bone Marrow Transplant 1993;12(suppl 1):84–86.
Bachar-Lustig E, Reich-Zeliger S, Gur H et al. Bone marrow transplantation across major genetic barriers: the role of megadose stem cells and nonalloreactive donor anti-third party CTLS. Transplant Proc 2001;33:2099–2100.
Reisner Y, Martelli MF. Bone marrow transplantation across HLA barrier by increasing the number of transplanted cells. Immunol Today 1995;16:437–440.
Bachar-Lustig E, Rachamim N, Li HW et al. Megadose of T cell-depleted bone marrow overcomes MHC barriers in sublethally irradiated mice. Nat Med 1995;1:1268–1273.
Jin T, Toki J, Sugiura K et al. A novel strategy for organ allografts using sublethal (7 Gy) irradiation followed by injection of donor bone marrow cells via portal vein. Transplantation 2001;71:1725–1731.
Shen BJ, Hou HS, Zhang HQ et al. Unrelated, HLA-mismatched multiple human umbilical cord blood transfusion in four cases with advanced solid tumors: initial studies. Blood Cells 1994;20:293–295.
Jin T, Sugiura K, Ishikawa J et al. Persistent tolerance induced after portal venous injection of allogeneic cells plus cyclophosphamide treatment. Immunobiology 1999;200: 215–226.
Soderling CC, Song CW, Blazer BR et al. A correlation between conditioning and engraftment in recipients of MHC mismatched T-cell-depleted murine bone marrow transplants. Immunology 1985;135:941–946.
Uharek L, Gassmann W, Glass B et al. Influence of cell dose and graft-versus-host reactivity on rejection rates after allogeneic bone marrow transplantation. Blood 1992;79: 1612–1621.
Kim HS, Meuli-Simmen C, Buncke HJ et al. DNA amplification determines donor-cell fate in cryopreserved skin allografts. J Reconstr Microsurg 1997;13:497–501,
Chen BJ, Cui X, Chao NJ. Addition of a second, different allogeneic graft accelerates white cell and platelet engraftment after T-cell-depleted bone marrow transplantation. Blood 2002;99:2235–2240.
Murphy WJ, Kumar V, Cope JC et al. An absence of T cells in murine bone marrow allograft leads to an increased susceptibility to rejection by natural killer cells and T cells. J Immunol 1990;114:3305–3311.
Asiedu C, Meng Y, Wang W et al. Immunoregulatory role of CD8alpha in the veto effect. Transplantation 1999;67: 372–380.
Schwaighofer H, Kernan NA, O’Reilly RJ et al. Serum levels of cytokines and secondary messages after T-cell-depleted and non-T-cell-depleted bone marrow transplantation: influence of conditioning and hematopoietic reconstitution. Transplantation 1996;62:947–953.
Weissman IL. Stem cells: units of development, units of regeneration, and units in evolution. Cell 2000;100: 157–168.
Watt FM, Hogan BL. Out of Eden: stem cells and their niches. Science 2000;287:1427–1430.
Ikehara S. Successful allogeneic bone marrow transplantation: crucial roles of stromal cells in prevention of graft rejection.Acta Haematol 2001;105:172–178.
Hisha H, Nishino T, Kawamura M et al. Successful bone marrow transplantation by bone grafts in chimeric-resistant combination. Exp Hematol 1995;23:347–352.
Sugiura K, Hisha H, Ishikawa J et al. Major histocompatibility complex restriction between hematopoietic stem cells and stromal cells in vitro. STEM CELLS 2001;19:46–58.
Kushida T, Inaba M, Hisha H et al. Intra-bone marrow injection of allogeneic bone marrow cells: a powerful new strategy for treatment of intractable autoimmune diseases in MRL/lpr mice. Blood 2001;97:3292–3299.
Bryder D, Ramsfjell V, Dybedal I et al. Self-renewal of multipotent long-term repopulating hematopoietic stem cells is negatively regulated by Fas and tumor necrosis factor receptor activation. J Exp Med 2001;194:941–952.
Thomson AW, Lu L. Dendritic cells as regulators of immune reactivity: implication for transplantation. Transplant 1999;68:1–8.(Tian-Xue Fana, Hiroko His)
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@takii.kmu.ac.jp
ABSTRACT
Numerous means have been used to conquer the main problems (graft-versus-host disease , 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) . 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 . 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 .
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 . 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 .
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 , 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 . 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 .
Ende et al. 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 . 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
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. 1). 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. 2). 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.
Figure 1. Experimental protocol for transplantation of TCD-BMCs. Abbreviation: TCD-BMC, T cell–depleted bone marrow cell.
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. 2), 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. 3): 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 3. 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. 4), which was compatible with the highest survival rate in the DBA/1 single-strain group (Fig. 2). 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. 5). In all the recipient mice, recipient-type cells were also detected, together with donor-type cells, indicating that they are quadruple chimeric mice.
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.
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.
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. 6). 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).
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. 7). 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.
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. 8). 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.
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. 9). 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. 10). 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.
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).
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
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
Lie AK, To LB. Peripheral blood stem cells: transplantation and beyond. Oncologist 1997;2:40–49.
Wagner JE. Umbilical cord blood transplantation: overview of the clinical experience. Blood Cells 1994;20:242–244.
Kurtzberg J, Graham M, Casey J et al. The use of umbilical cord blood in mismatched related and unrelated hemopoietic stem cell transplantation. Blood Cells 1994;20: 275–284.
Young JC. Retention of quiescent hematopoietic cells with high proliferative potential during ex vivo stem cell culture. Blood 1996;87:545–556.
Brossard Y,Van Nifterik J, De Lachaux V et al. Collection of placental blood with a view to hemopoietic reconstitution. Nouv Rev Fr Hematol 1990;32:427–429.
Newburger PE, Quesenberry PJ. Umbilical cord blood as a new and promising source of unrelated-donor hematopoietic stem cells for transplantation. Curr Opin Pediatr 1996;8:29–32.
Gluckman E, Rocha V, Chammard AB et al. Outcome of cord-blood transplantation from related and unrelated donors. N Engl J Med 1997;337:373–381.
Ende N, Lu S,Alcid MG et al. Pooled umbilical cord blood as a possible universal donor for marrow reconstitution and use in nuclear accidents. Life Sci 2001;69:1531–1539.
Gabutti V, Timeus F, Ramenghi U et al. Human cord blood progenitors: kinetics, regulation and their use for hemopoietic reconstitution. Bone Marrow Transplant 1993;12(suppl 1):84–86.
Bachar-Lustig E, Reich-Zeliger S, Gur H et al. Bone marrow transplantation across major genetic barriers: the role of megadose stem cells and nonalloreactive donor anti-third party CTLS. Transplant Proc 2001;33:2099–2100.
Reisner Y, Martelli MF. Bone marrow transplantation across HLA barrier by increasing the number of transplanted cells. Immunol Today 1995;16:437–440.
Bachar-Lustig E, Rachamim N, Li HW et al. Megadose of T cell-depleted bone marrow overcomes MHC barriers in sublethally irradiated mice. Nat Med 1995;1:1268–1273.
Jin T, Toki J, Sugiura K et al. A novel strategy for organ allografts using sublethal (7 Gy) irradiation followed by injection of donor bone marrow cells via portal vein. Transplantation 2001;71:1725–1731.
Shen BJ, Hou HS, Zhang HQ et al. Unrelated, HLA-mismatched multiple human umbilical cord blood transfusion in four cases with advanced solid tumors: initial studies. Blood Cells 1994;20:293–295.
Jin T, Sugiura K, Ishikawa J et al. Persistent tolerance induced after portal venous injection of allogeneic cells plus cyclophosphamide treatment. Immunobiology 1999;200: 215–226.
Soderling CC, Song CW, Blazer BR et al. A correlation between conditioning and engraftment in recipients of MHC mismatched T-cell-depleted murine bone marrow transplants. Immunology 1985;135:941–946.
Uharek L, Gassmann W, Glass B et al. Influence of cell dose and graft-versus-host reactivity on rejection rates after allogeneic bone marrow transplantation. Blood 1992;79: 1612–1621.
Kim HS, Meuli-Simmen C, Buncke HJ et al. DNA amplification determines donor-cell fate in cryopreserved skin allografts. J Reconstr Microsurg 1997;13:497–501,
Chen BJ, Cui X, Chao NJ. Addition of a second, different allogeneic graft accelerates white cell and platelet engraftment after T-cell-depleted bone marrow transplantation. Blood 2002;99:2235–2240.
Murphy WJ, Kumar V, Cope JC et al. An absence of T cells in murine bone marrow allograft leads to an increased susceptibility to rejection by natural killer cells and T cells. J Immunol 1990;114:3305–3311.
Asiedu C, Meng Y, Wang W et al. Immunoregulatory role of CD8alpha in the veto effect. Transplantation 1999;67: 372–380.
Schwaighofer H, Kernan NA, O’Reilly RJ et al. Serum levels of cytokines and secondary messages after T-cell-depleted and non-T-cell-depleted bone marrow transplantation: influence of conditioning and hematopoietic reconstitution. Transplantation 1996;62:947–953.
Weissman IL. Stem cells: units of development, units of regeneration, and units in evolution. Cell 2000;100: 157–168.
Watt FM, Hogan BL. Out of Eden: stem cells and their niches. Science 2000;287:1427–1430.
Ikehara S. Successful allogeneic bone marrow transplantation: crucial roles of stromal cells in prevention of graft rejection.Acta Haematol 2001;105:172–178.
Hisha H, Nishino T, Kawamura M et al. Successful bone marrow transplantation by bone grafts in chimeric-resistant combination. Exp Hematol 1995;23:347–352.
Sugiura K, Hisha H, Ishikawa J et al. Major histocompatibility complex restriction between hematopoietic stem cells and stromal cells in vitro. STEM CELLS 2001;19:46–58.
Kushida T, Inaba M, Hisha H et al. Intra-bone marrow injection of allogeneic bone marrow cells: a powerful new strategy for treatment of intractable autoimmune diseases in MRL/lpr mice. Blood 2001;97:3292–3299.
Bryder D, Ramsfjell V, Dybedal I et al. Self-renewal of multipotent long-term repopulating hematopoietic stem cells is negatively regulated by Fas and tumor necrosis factor receptor activation. J Exp Med 2001;194:941–952.
Thomson AW, Lu L. Dendritic cells as regulators of immune reactivity: implication for transplantation. Transplant 1999;68:1–8.(Tian-Xue Fana, Hiroko His)