The Journal of Immunology, 2004, 172: 1444-1448.
Copyright © 2004 by The American Association of Immunologists
Chimeric Donor Cells Play an Active Role in Both Induction and Maintenance Phases of Transplantation Tolerance Induced by Mixed Chimerism1
Akira Kanamoto and
Takashi Maki2
Transplant Center, Department of Surgery, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215
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Abstract
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Donor hemopoietic cell engraftment is considered to be an indicator of allograft tolerance. We depleted chimerism with cells specifically presensitized to the bone marrow donor to investigate its role in mixed chimera-induced tolerance. Three experimental models were used: model A, B10.A cells presensitized to B6 (a anti-b cells) were injected into (B6 x D2)F1 
B10.A mixed chimeras grafted with DBA/2 skin; model B, anti-B6 presensitized cells prepared in DBA/2 B10.A mixed chimeras, thus unresponsive to DBA/2 (a anti-b/tol-d cells), were injected into (B6 x D2)F1 B10.A mixed chimeras grafted with DBA/2 skin; and model C, (BALB/c x B6)F1 cells presensitized to CBA (d/b anti-k cells) were injected into (B6 x CBA)F1 BALB/c mixed chimeras grafted with B6 skin. Skin was grafted on day 30. Injection of each cell type before skin grafting abolished hemopoietic cell engraftment and prevented allograft acceptance. Injection of presensitized cells after skin grafting resulted in different outcomes depending on the models. In model A, injection of a anti-b cells completely depleted chimerism and caused allograft rejection. In model B, injection of a anti-b/tol-d cells markedly reduced, but did not deplete, peripheral chimerism and maintained skin allograft survival. In model C, d/b anti-k cells reduced chimerism to the background levels but failed to cause graft rejection, probably due to persistence of injected cells which share MHC with skin grafts. Together, the results show that presence of chimeric donor cells is essential in both the induction and maintenance phases of tolerance induced by mixed chimerism.
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Introduction
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Bone marrow transplantation (BMT)3 after myeloablative regimens produces chimerism and tolerance to allografts derived from the bone marrow (BM) donor (1, 2, 3). Engraftment of donor hemopoietic cells is considered to be an indicator for the presence of specific allograft tolerance. However, whether chimeric donor cells play an active role in induction and/or maintenance of tolerance has not been fully elucidated. Sharabi et al. (4) reported that in vivo depletion of donor cells by treatment of mixed chimeric mice with mAb resulted in rejection of donor skin transplanted >4 mo later. Ko et al. (5) selectively depleted graft-derived microchimeric donor leukocytes in a rat model of cyclosporin-induced tolerance to heart allografts. Successful depletion was achieved at days 0 and 18 post-transplantation, but only day 0 depletion prevented tolerance induction. Depletion on day 18 had no effect on graft survival (>200 day graft survival). They concluded that graft-derived microchimerism is essential for induction, but not maintenance, of tolerance. Kawai et al. (6) showed that peripheral chimerism lasting <70 days following BMT appeared both necessary and sufficient for induction of specific tolerance toward a kidney graft placed at the time of BMT. Anderson and Matzinger (7) found that donor T cell microchimerism could result in different outcomes depending on the host’s immunological maturity and the antigenic disparities involved. In immunologically mature hosts, microchimerism resulted in immunity and graft rejection. In immature hosts, it resulted in tolerance to the chimeric T cells but not to graft Ags not expressed by the chimeric cells. We recently demonstrated that, while failure to create stable chimerism is always associated with failure to induce tolerance, allografts can be rejected in the presence of long-term stable non-T cell chimerism (8), suggesting that stable chimerism itself in the absence of donor T cells does not contribute to acceptance of donor skin allografts.
In the present study, we investigated the role of chimerism in the induction and maintenance phases of allograft tolerance induced in a mixed chimerism model. We designed a mixed chimera in which tolerance to an A strain skin allograft was induced in C hosts by whole body irradiation and transplantation of a mixture of (A x B)F1 strain plus C strain BM (A x B C mixed chimeras). We assumed that administration of C cells presensitized to B would eliminate A x B chimeric cells yet spare A grafts from direct destruction. The results reported herein unequivocally show that chimeric donor cells play an active role both in the induction phase and in the maintenance phase of allograft tolerance induced by mixed chimerism.
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Materials and Methods
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Mice
All mice except C57BL/6 (B6) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). B6 mice were purchased from Taconic Farms (Germantown, NY). Mice were used at 8–10 wk of age. All care and handling of animals was performed in accordance with guidelines provided in the Guide for the Care and Use of Laboratory Animals published by the U.S. Department of Health and Human Services.
Preparation of mixed chimeras
Recipient mice were irradiated with 1000 rad from a 137Cs source (Nordion, Ottawa, Ontario, Canada). BM was harvested from the femurs and humeri. Irradiated mice were reconstituted with 5 x 106 host BM plus 10 x 106 donor BM. BM was depleted of T cells by anti-Thy 1.2 mAb and magnetic beads (Dynal, Lake Success, NY).
We used two mixed chimerism models. In one model, irradiated B10.A mice (H-2a) were infused with T cell-depleted (C57BL/6 x DBA/2)F1 (B6D2F1, H-2b/d) BM plus B10.A BM (b/d a mixed chimeras) followed by DBA/2 (D2, H-2d) skin grafting. In the second model, irradiated BALB/c (H-2d) mice were infused with a mixture of (B6 x CBA)F1 (H-2b/k) and BALB/c BM (b/k d mixed chimeras) followed by B6 (H-2b) skin grafting.
Skin grafting
Full-thickness skin was transplanted using standard techniques 30 days after BMT as described previously (8, 9). Rejection was defined as complete loss of viable donor epithelium.
Thymectomy
Under anesthesia, a partial sternotomy was made and the thymus was removed by suction. The absence of thymic tissue was confirmed when thymectomized animals were euthanized. Animals with residual thymic tissue were excluded from analysis.
Flow cytometry
An anti-CD16/32 mAb was used as a blocking Ab. PBLs were stained with the FITC-, PE-, Cychrome-conjugated mAb directed to H-2Kb, H-2d, H-2k, and Thy1.2 (BD PharMingen, San Diego, CA). Isotype Abs were used as controls. Stained cells were analyzed on a FACScan (BD Biosciences, Mountain View, CA). The degree of background staining in negative control cells was 1.2 ± 0.1% (range: 0.1–3.2, n = 47).
Presensitized cells for depletion of chimerism
Three types of presensitized cells were prepared. 1) Naive B10.A mice were sensitized to H-2b by B6 skin grafting and injection of 100 x 106 B6 splenocytes after skin graft rejection. Splenocytes were prepared from sensitized mice 7–10 days after immunization and used as "a anti-b presensitized cells" in the b/d a mixed chimera model. 2) After establishment of stable mixed chimerism (40–50 days post-BMT), D2 + B10.A B10.A mixed chimeric mice were sensitized to H-2b as described above. Splenocytes from sensitized chimeric mice were depleted of chimeric D2 cells with the use of anti-H-2Kd mAb and magnetic beads. H-2d-positive cells in the splenocytes were reduced from 63.2 ± 1.7% (n = 23) to 1.7 ± 0.2% after treatment. After removal of H-2d cells, the splenocytes were cytotoxic against H-2b but unresponsive to H-2d in 51Cr-release assays (data not shown). The splenocytes were used as anti-b presensitized cells that were tolerant to D2 (a anti-b/tol-d presensitized cells) in the b/d a mixed chimera model. 3) (BALB/c x B6)F1 (H-2d/b) mice were sensitized to H-2k by CBA (H-2k) skin grafting and immunization with CBA splenocytes. Splenocytes from sensitized mice were used as d*/b anti-k presensitized cells. When injected into b/k d* mixed chimera bearing b skin grafts, these cells were reactive with b/k chimeric cells but unresponsive to b skin grafts. The hosts (d*) were also unresponsive to injected d*/b cells by virtue of b/k chimerism.
Detection of male Ag by PCR
We prepared b/d a mixed chimeras bearing d skin grafts using male B6D2F1 (BM donor), female B10.A (recipients and syngeneic BM donor), and female DBA/2 (skin donor). Presensitized cells were prepared with the use of female mice for hosts as well as skin and splenocyte donors. Thus, following injection of presensitized cells, only chimeric b/d cells expressed male Ags.
Genomic DNA was extracted from blood, spleen, liver, thymus, and BM using the QIAamp Tissue kit from Qiagen (Chatsworth, CA). The reactions were completed in a total volume of 50 µl of reaction buffer (200 mM Tris-HCl, pH 8.4, 500 mM KCL, 50 mM MgCl2) containing 30 ng of template DNA, 0.2 mM each of 10 mM dNTP mixture (Invitrogen Carlsbad, CA), 1.0 U Taq polymerase (Invitrogen Carlsbad, CA), and 0.2 mM each of Y chromosome-specific primers under the following conditions in a thermocycler (PerkinElmer, Branchburg, NJ): 10 min denaturation at 95°C followed by 38 cycles amplification, each containing 1 min denaturation at 95°C, 1 min annealing at 60°C, 1 min polymerization at 72°C, followed by a 10-min amplification step at 72°C (holding temperature 4°C). The sequence of the Y chromosome-specific primers was: 5'-CTAAGCCATGTACCACCT and 5'-CTCTCTCTTTATCAATTTTTTCT (10). For analysis, the probes were run in a 1.5% agarose gel and detected by ethidium bromide staining. The Y chromosome-specific primers amplified the positive signal of male donors with a minimal detection level of 1:105 (male to female cells).
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Results
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Injection of a anti-b presensitized cells depletes chimerism and causes graft rejection in b/d a mixed chimeras bearing d skin
In B6D2 + B10.A B10.A (b/d a) mixed chimeras (n = 6), the levels of peripheral chimerism gradually increased from 69.4 ± 4.5% (range: 55.9–81.3%) on day 28 (2 days before skin grafting) to 91.2 ± 1.7% (range: 84.2–96.1%) on day 150 post-MT. All D2 skin allografts were intact 120 days after transplantation.
To deplete chimeric donor cells without directly damaging skin grafts, we prepared a B10.A splenocyte sensitized to B6 (a anti-b presensitized cells). The a anti-b presensitized cells lysed H-2b targets (EL4 lymphoma) but not H-2d target (P815 mastocytoma) in standard 4-h 51Cr-release assays (not shown). We injected these a anti-b presensitized cells (20 x 106) into b/d a mixed chimeric mice either before or at various time points after d skin grafting (Table I). When the cells were injected before skin grafting, the process of BM engraftment was abrogated, and, as a result, establishment of chimerism was prevented. Skin graft rejection followed in 4–5 days. Both prevention of peripheral chimerism and skin graft rejection were also induced when the presensitized cells were injected 14 days before skin grafting (14 days post-BMT) (not shown). When the a anti-b presensitized cells were injected into b/d a mixed chimeric mice after skin grafts were accepted (30–141 days after grafting), the peripheral chimerism was reduced to the background levels (<1%) in 
40 days followed by skin graft rejection in 3–20 days. The effect of the a anti-b presensitized cells was specific for b-bearing cells because injection of the a anti-b cells into D2 + B10.A B10.A (d a) mixed chimeras bearing D2 (d) skin allografts failed to cause significant reduction of peripheral chimerism or skin allograft rejection (data not shown).
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Table I. Chimerism and skin graft survival after injection of a anti-b presensitized cells into b/d mixed chimeras bearing d skin grafts
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To determine whether chimeric cells were completely depleted or remained at very low levels, we created b/d a mixed chimeras in which only b/d chimeric cells expressed male Ags. After injection of a anti-b presensitized cells prepared in female mice caused marked reduction of peripheral chimerism (Fig. 1, Ia), mice were euthanized and various tissues were analyzed for the presence of a Y chromosome Ag. PCR analysis showed the absence of Y Ag, indicating that injection of the a anti-b presensitized cells into b/d a chimeras resulted in complete elimination of chimeric cells (Fig. 1, Ib).
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FIGURE 1. State of chimerism 120–200 days after injection of a anti-b cells (I) and a anti-b/tol-d cells (II) into b/d a mixed chimeras. Ia and IIa, Flow cytometric analyses. Ib and IIb, PCR analyses of the PBLs. P, Positive control (male naive B10.A); N, negative control (female naive B10.A); 1–4 and 5–6, individual mice given a anti-b or a anti-b/tol-d cells, respectively. The same results were obtained with PCR analyses of other tissues.
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We also investigated the involvement of host thymus in skin graft rejection caused by injection of the presensitized cells. A group of b/d a mixed chimeras bearing d skin grafts underwent thymectomy 60 days after BMT (30 days after skin grafting). We injected a anti-b presensitized cells into both thymectomized mice (21 days after thymectomy) and nonthymectomized mice (51 days after skin grafting). Injection of the presensitized cells abolished peripheral chimerism within 35 days in both thymectomized and nonthymectomized mice (Fig. 2A). However, skin allograft rejection in thymectomized mice was markedly delayed while the skin allograft was rejected within 30 days after cell injection in nonthymectomized mice (p = 0.012 by Mantel’s log-rank test) (Fig. 2B). Both chimerism and skin allografts remained intact after thymectomy without administration of the presensitized cells (not shown).
Injection of a anti-b presensitized cells that are unresponsive to skin donor Ag diminishes chimerism but fails to cause skin graft rejection in b/d a mixed chimeras
To further eliminate the possibility that the injected presensitized cells directly damaged skin grafts, we injected a splenocytes that were sensitized to the b but tolerant to d (a anti-b/tol-d presensitized cells) into b/d a mixed chimeras. These a anti-b/tol-d cells were highly cytotoxic to H-2b targets after 3 days of in vitro sensitization to H-2b stimulators, but failed to generate anti-H-2d cytotoxicity after in vitro sensitization with H-2d stimulators (not shown). We injected the cells into b/d a mixed chimeric mice before or after d skin grafting. Injection of the cells 9 days before skin grafting induced rapid reduction of chimerism and skin graft rejection (Table II). However, injection of the cells 30 days after skin grafting failed to cause skin graft rejection despite the same degrees of reduction of the peripheral chimerism as those induced by a anti-b cells. In some mice, injection of 30 x 106 (n = 2) or 20 x 106 cells (n = 2) on day 30 had little effect on the degree of chimerism while second injection of 50 x 106 or 30 x 106 cells, respectively, was necessary to significantly reduce chimerism. In these mice, skin grafts remained intact for >150 days. When the a anti-b/tol-d cells prepared in female mice were injected into female b/d a mixed chimeric mice bearing male b/d chimeric cells, peripheral chimerism determined by flow cytometric analyses was markedly reduced (Fig. 1, IIa). However, PCR analysis of various tissues revealed the presence of the Y chromosome-specific Ag, suggesting the persistence of low level chimerism (Fig. 1, IIb).
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Table II. Chimerism and skin graft survival after injection of a anti-b cells that were tolerant to d (aanti-b/tol-d cells) into b/d a mixed chimeras bearing d skin grafts
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Injection of d*/b anti-k presensitized cells diminishes chimerism but fails to cause skin graft rejection in b/k d mixed chimeras bearing b skin
We injected (BALB/c x B6)F splenocytes prepared from mice sensitized to CBA (d*/b anti-k presensitized cells) into (B6 x CBA)F1 + BALB/c BALB/c (b/k d*) mixed chimeras bearing B6 (b) skin grafts at various time points (Table III). The d*/b anti-k cells were able to deplete b/k chimeric cells, but were unresponsive to b (skin grafts) and d* (hosts), while the d* mixed chimeric hosts were unresponsive to the injected d*/b cells because of b/k chimerism. Injection of the cells 9 days before skin grafting caused reduction of chimerism to the background levels and skin graft rejection. Significant numbers of H-2Kb-positive cells persisted. Injection of the presensitized cells 30 or 70 days after skin grafting again resulted in marked reduction of H-2Kk-positive cells but persistence of H-2Kb-positive cells. Skin allografts remained intact for up to 150 days in all mice. H-2Kb-positive cells were doubly positive for H-2Kd (Fig. 3), indicating that these cells represented injected d*/b anti-k presensitized cells, not the host (d*) or chimeric (b/k) cells.
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Table III. Chimerism and graft survival after injection of d/b anti-k presensitized cells into b/k d mixed chimeras bearing b skin grafts
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FIGURE 3. Flow cytometric analysis of b/k d mixed chimeras 120 days after injection of d/b anti-k cells.
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Discussion
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Injection of cells that were cytolytic to chimeric cells but not to skin grafts into mixed chimeric hosts, i.e., injection of a anti-b cells or a anti-b/tol-d into b/d a mixed chimeras bearing d skin and injection of d*/b anti-k presensitized cells into b/k d* mixed chimeras bearing b skin, before skin grafting abolished engraftment of BM and prevented acceptance of the BM-MHC matched skin allografts. These results are in agreement with previous reports by others (11) and us (8) that robust chimerism is essential for induction of tolerance in a mixed radiation chimera or allogeneic radiation chimera model. For example, Taniguchi et al. (11) reported that mice with >30% chimerism could accept skin grafts whereas mice with <10% chimerism showed prolonged but not permanent graft survival. Incomplete depletion of residual host T cells associated with <10% chimerism was considered responsible for failure of allograft tolerance induction. A study by Sharabi et al. (4) also showed that depletion of chimerism before skin grafting led to failure of allograft tolerance induction. We recently observed that administration of anti-H-2KbDb mAb in B6 x D2 B10.A mixed chimeras starting 16–9 days before skin grafting caused rapid reduction of chimerism and rejection of D2 skin grafts even in the presence of a significant degree of chimerism (10%) which persisted up to 150 days (A. Kanamoto, unpublished data).
Injection of the presensitized cells into mixed chimeras after establishment of tolerance to skin allografts led to different results depending on the type of presensitized cells used as summarized in Table IV. When a anti-b presensitized cells were injected into b/d a mixed chimeras bearing d skin grafts, peripheral chimeric cells were completely eliminated as confirmed by PCR analysis. Effective elimination of chimeric cells may be attributable to the continuous presence of injected cells and/or expansion of anti-b cells in vivo by their restimulation by the b/d chimeric cells. It is unlikely that injected cells directly mediated rejection of tolerant grafts. Reduction of chimerism to the background levels (<1%) always preceded skin allograft rejection by several days. Injection of a anti-b presensitized cells into d a mixed chimeras bearing d skin grafts failed to abolish peripheral chimerism or cause graft rejection. Host thymectomy before cell injection had no effect on depletion of chimerism by presensitized cells, but prevented rejection of tolerant skin grafts, indicating that the host thymus, most likely host thymus-derived T cells, is required for rapid rejection of skin grafts. Khan et al. (12) have demonstrated that tolerance can be broken when nontolerant T cells emerge from the thymus after intentional depletion of donor Ag or after exogenous administration of nontolerant T cells.
When the a anti-b/tol-d cells were injected into b/d a mixed chimeras bearing d skin grafts, peripheral chimerism determined by flow cytometry was reduced to the same background levels as induced by the a anti-b cells, but skin grafts remained intact. PCR analysis revealed that low level chimerism persisted in these mice for up to 200 days. The reasons why tolerant presensitized cells were unable to completely eliminate chimeric cells are not immediately known. As the a anti-b/tol-d cells were prepared in d a mixed chimeras, it is possible that small numbers of chimeric D2 (d) cells were present in the injected cells despite their careful removal before injection. Once injected into b/d a mixed chimeras, these residual d cells might survive and possibly function as immunoregulatory cells preventing complete elimination of b/d chimeric cells and rejection of tolerant d skin.
When the d*/b anti-k cells were injected into b/k d* mixed chimeras bearing b skin grafts, b/k chimeric cells were reduced to the background levels but skin grafts remained intact. It is possible that the background levels of b/k chimerims represented low but true chimerism as in the case in which a anti-b/tol-d cells were injected into b/d a mixed chimeras. Alternatively, as injected d*/b cells persisted, albeit in a small number, it is conceivable that these cells served as protectors of b skin allografts in place of abolished b/k chimeric cells.
The present study demonstrates that chimerism plays an active role not only in the induction phase but also in the maintenance phase of allograft tolerance induced by mixed chimerism. In the induction phase, macrochimerism (
10%) is required for the induction of allograft tolerance. Prevention of allogeneic BM engraftment was always associated with inability to accept BM-MHC bearing skin allografts. In the maintenance phase, complete depletion of chimeric donor cells led to rejection of existing tolerant grafts, probably due to loss of immunoregulation by chimeric cells and activation of host antigraft immunity. In contrast, persistence of peripheral chimerism, even at the low levels detectable only by PCR, or continuous survival of injected cells that expressed graft MHC, was sufficient to maintain allograft tolerance. Failure by Schlitt and colleagues (5) to elicit rejection of allografts by mAb administered in the maintenance phase of cyclosporin-induced tolerance was explained by lack of involvement by the graft-derived chimeric cells in the maintenance of tolerance, implicating the involvement of tolerance mechanism(s) other than that mediated by chimerism. Alternatively, in view of our present results, their results could suggest that persistent microchimerism which they detected at 200 days by PCR is responsible for maintenance of tolerance. Thus, in their model, chimerism was indeed essential in both the induction and maintenance phase of allograft tolerance.
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Footnotes
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1 This work was supported National Institutes of Health Grant AI14551. A.K. was a recipient of a fellowship from the Mitsui Memorial Hospital Fund. 
2 Address correspondence and reprint requests to Dr. Takashi Maki, Transplant Center, Department of Surgery, Beth Israel Deaconess Medical Center, Research North, 330 Brookline Avenue, Boston, MA 02215. E-mail address: tmaki{at}bidmc.harvard.edu
3 Abbreviations used in this paper: BMT, bone marrow transplantation; BM, bone marrow; MT, marrow transplantation.
Received for publication August 4, 2003.
Accepted for publication November 17, 2003.
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References
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- Colson, Y. L., H. Li, S. S. Boggs, K. D. Patrene, P. C. Johnson, S. T. Ildstad. 1996. Durable mixed allogeneic chimerism and tolerance by a non-lethal radiation based cytoreductive approach. J. Immunol. 157:2820.[Abstract]
- Tomita, Y., D. H. Sachs, A. Khan, M. Sykes. 1996. Additional monoclonal antibody (mAb) injections can replace thymic irradiation to allow induction of mixed chimerism and tolerance in mice receiving bone marrow transplantation after conditioning with anti-T cell mAbs and 3-Gy whole body irradiation. Transplantation 61:469.[Medline]
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- Ko, S., A. Deiwick, M. D. Jager, A. Kinkel, F. Rohde, R. Fischer, T. Y. Tsui, K. L. Rittmann, K. Wonigeit, H. J. Schlitt. 1999. The functional relevance of passenger leukocytes and microchimerism for heart allograft acceptance in the rat. Nat. Med. 5:1292.[Medline]
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- Anderson, C. C., P. Matzinger. 2001. Immunity or tolerance: opposite outcomes of microchimerism from skin grafts. Nat. Med. 7:80.[Medline]
- Umemura, A, H. Morita, X. C. Li, S. Tahan, A. P. Monaco, T. Maki. 2001. Dissociation of hematopoietic chimerism and allograft tolerance after allogeneic bone marrow transplantation. J. Immunol. 167:3043.[Abstract/Free Full Text]
- Umemura, A., A. P. Monaco, T. Maki. 2000. Donor MHC class II antigen is essential for induction of transplantation tolerance by bone marrow cells. J. Immunol. 164:4452.[Abstract/Free Full Text]
- Peters, S. O., K. Bauermeister, J. P. Simon, B. Branke, T. Wagner. 2002. Quantitative polymerase chain reaction-based assay with fluorogenic Y-chromosome specific probes to measure bone marrow chimerism in mice. J. Immunol. Methods. 260:109.[Medline]
- Taniguchi, H., M. Abe, T. Shirai, K. Fukao, H. Nakauchi. 1995. Reconstitution ratio is critical for alloreactive T cell deletion and skin graft survival in mixed bone marrow chimeras. J. Immunol. 155:5631.[Abstract]
- Khan, A., Y. Tomita, M. Sykes. 1996. Thymic dependence of loss of tolerance in mixed allogeneic bone marrow chimerism after depletion of donor antigen. Transplantation 62:380.[Medline]
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Abstract
Introduction
