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Dissociation of Hemopoietic Chimerism and Allograft Tolerance After Allogeneic Bone Marrow Transplantation¹

Akihisa Umemura^*, Hirofumi Morita^*, Xian Chang Li, Steven Tahan, Anthony P. Monaco^* and Takashi Maki^2,*

Departments of ^* Surgery and Medicine and Pathology, Transplant Center, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215

	Abstract

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Creation of stable hemopoietic chimerism has been considered to be a prerequisite for allograft tolerance after bone marrow transplantation (BMT). In this study, we demonstrated that allogeneic BMT with bone marrow cells (BMC) prepared from either knockout mice deficient in both CD4 and CD8 T cells or CD3E-transgenic mice lacking both T cells and NK cells maintained a high degree of chimerism, but failed to induce tolerance to donor-specific wild-type skin grafts. Lymphocytes from mice reconstituted with T cell-deficient BMC proliferated when they were injected into irradiated donor strain mice, whereas lymphocytes from mice reconstituted with wild-type BMC were unresponsive to donor alloantigens. Donor-specific allograft tolerance was restored when donor-type T cells were adoptively transferred to recipient mice given T cell-deficient BMC. These results show that donor T cell engraftment is required for induction of allograft tolerance, but not for creation of continuous hemopoietic chimerism after allogeneic BMT, and that a high degree of chimerism is not necessarily associated with specific allograft tolerance.

	Introduction

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Complete depletion of donor T cells from bone marrow cell (BMC)³ preparations is often associated with failure of donor bone marrow engraftment following allogeneic bone marrow transplantation (BMT), suggesting that some T cells may facilitate donor bone marrow engraftment and creation of stable chimerism (1, 2, 3, 4, 5). Moreover, it has been suggested that a correlation exists between high levels of initial donor T cell reconstitution and stable chimerism, which is considered to be a prerequisite for allograft tolerance in the radiation-based mixed allogeneic chimera model (6). The role of chimerism in allograft tolerance induction by nonradiation-based protocols is controversial (7, 8, 9). In this study, we demonstrate that dissociation of hemopoietic chimerism and allograft tolerance, i.e., absence of donor-specific allograft tolerance despite continuous chimerism, occurs after allogeneic BMT with T cell-deficient BMC. Requirement of T cells for achieving donor-specific allograft tolerance was confirmed by restoration of donor-specific allograft tolerance after adoptive transfer of donor T cells to mice given T cell-deficient bone marrow transplants.

	Materials and Methods

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Mice

B10.A (H-2^a) mice (Charles River Laboratories, Kingston, NY) and B10.D2 (H-2^d) mice (The Jackson Laboratory, Bar Harbor, ME) were used as BMT recipients. BMC donors included wild-type (wt) C57BL/6 (wtB6; H-2^b) mice (Taconic Farms, Germantown, NY), B6 mice deficient in CD4 expression (CD4 knockout (KO)) (10) or CD8a expression (CD8 KO) (11), B6129SF₂ (H-2^b/b) mice deficient for both CD4 and CD8a expression (CD4/8 double KO (DKO) mice) (12), and B6CBAF₁ (H-2^b/k) mice carrying a human CD3E transgene (CD3E-tg mice) (13). T cell-deficient mice, their wt controls, and DBA/1 mice were purchased from The Jackson Laboratory. BMT recipients were killed 81–83 days after skin grafting (111–113 days after BMT). All care and handling of animals was conducted in accordance with guidelines provided in the Guide for Care and Use of Laboratory Animals published by the U.S. Department of Health and Human Services.

Preparation of radiation chimeras

Recipient mice were irradiated with a single dose of 6.5 or 7.5 Gy from a ¹³⁷Cs source (Nordion, Ontario, Canada). BMC were harvested from the femurs and humeri of donor mice by flushing with HBSS. Wild-type BMC as well as CD4 KO and CD8 KO BMC were incubated with rat anti-mouse Thy-1.2 mAb (clone 53-2.1; BD PharMingen, San Diego, CA) and immunomagnetic beads conjugated with goat anti-rat IgG (Dynabeads M-450; Dynal, Lake Success, NY) before BMT to prevent graft-vs-host disease. T cell depletion with immunomagnetic bead separation was always >95%. Recipient mice were reconstituted with 25 x 10⁶ allogeneic BMC 4–6 h after irradiation.

Skin grafting

Full-thickness skin grafts were transplanted onto the lateral thoracic area of the recipients using standard techniques 30 days after BMT, as described previously (14).

Flow cytometry

The cells were incubated with an anti-CD16/32 mAb for 10 min to block nonspecific binding of labeled Abs. Splenocytes were stained with the FITC-, PE-, CyChrome-conjugated mAbs directed to H-2K^b, H-2K^k, CD4, CD8a, CD11b (macrophages/monocytes), CD11c (dendritic cells), and CD45R (B cells) (BD PharMingen). FITC-, PE-, CyChrome-conjugated isotype Abs were used as controls. Stained cells were analyzed on a FACScan (BD Biosciences, Mountain View, CA). Donor cell chimerism was determined by flow cytometric analysis of recipient peripheral and/or splenic lymphoid cells with normal donor- and recipient-type cells as positive and negative controls. The percentage of chimeric H-2K^b-positive cells was calculated using the formula: 100 x ((net percentage in the test samples) - (net percentage in the negative control samples))/((net percentage in the positive control samples) - (net percentage in the negative control samples)). Net percentage refers to the percentage obtained after subtraction of staining with the appropriate isotype controls.

In vivo assay for T cell proliferation

Lymphocytes were isolated from spleens and peripheral lymph nodes of B10.A recipients 28 days after BMT (without skin grafting), and labeled with CFSE (Molecular Probes, Eugene, OR), as described previously (15). CFSE-labeled cells (40–60 x 10⁶) were injected through the tail vein into corresponding syngeneic mice (B10.A), wt BMC donor mice (B6 or B6129SF₂), and third-party mice (DBA/1), all of which were lethally irradiated (10 Gy). In the case of lymphocytes prepared from B10.D2 mice transplanted with CD3E-tg BMC, they were injected into lethally irradiated B10.D2 (syngeneic), wtB6CBAF₁ (donor-specific), or DBA/1 (third-party) mice. Lymphoid cells prepared from naive B10.A mice or B10.A mice sensitized to wtB6 by skin grafting were also labeled with CFSE and injected into irradiated B10.A, wtB6, or DBA/1 mice. One to four days after adoptive transfer of CFSE-labeled cells, a single-cell suspension of spleens was prepared and stained with PE-conjugated anti-CD4 mAb and analyzed on a FACScan. Injected CD4⁺ T cells were identified in the CFSE⁺CD4⁺ gate. The frequency of proliferating CD4⁺ T cells was calculated as described previously (16).

Reconstitution with donor-type T cells

B10.A mice were lethally (9.5 Gy) irradiated and reconstituted with 25 x 10⁶ T cell-depleted wtB6129SF₂ BMC. Maturation of donor T cells was usually seen 30 days after reconstitution and stabilized by 60 days (14). Splenic and lymph node lymphocytes harvested 45 days after BMT were enriched for T cells (>85%) by a nylon wool column separation. Flow cytometric analysis showed that >95% of T cells were of donor type. Enriched T cells (15 x 10⁶) were injected i.v. into CD4/8 DKO BMC-reconstituted B10.A mice 21 days after BMT. The presence of injected donor-type T cells was determined by flow cytometry on day 28 after BMT (7 days after T cell transfer). The mice were transplanted with wtB6129SF₂ skin grafts on day 30 after BMT.

	Results

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Effect of radiation dose on induction of chimerism and allograft tolerance

Transplantation of 25 x 10⁶ T cell-depleted wtB6 BMC in sublethally irradiated (7.5 Gy) B10.A mice achieved varying degrees of hemopoietic chimerism on day 28 after BMT (Table I, group a). The majority (11 of 12) of mice showed chimerism of >88% and remained highly chimeric on day 90 in all lymphoid cell types, including CD4⁺ and CD8⁺ T cells, B cells, macrophages/monocytes, and dendritic cells (Table II). These chimeric mice accepted wtB6 skin grafts transplanted on day 30 (Table III, group a). In contrast, B10.A mice irradiated at 6.5 Gy and given 25 x 10⁶ T cell-depleted wtB6 BMC (wtB6/6.5Gy BMT recipients) failed to achieve either chimerism or allograft tolerance. Flow cytometric analyses on day 28 showed a lower degree of chimerism (26%) as compared with wtB6/7.5Gy BMT recipients (87%; Table I, group b). Donor-type skin grafts placed on day 30 were rejected within 15 days (Table III, group b). The degree of chimerism decreased progressively without corresponding to the timing of graft rejection, and by day 90 none of the recipient mice were chimeric. As both CD4⁺ and CD8⁺ donor-type T cells were not detected in these mice on day 28 despite the presence of other donor-type lymphoid cells, we examined whether donor T cell reconstitution is essential in establishment of chimerism and allograft tolerance in this model.

When 25 x 10⁶ BMC prepared from CD4 KO or CD8 KO mice were transplanted in sublethally irradiated (7.5 Gy) B10.A mice, a high degree of chimerism (Table I, groups c and d) was maintained for at least 90 days, correlating with donor-specific allograft tolerance (Table III, groups c and d). Loss of chimerism in these mice was always associated with graft rejection, as seen in wtB6/7.5Gy BMT recipients. However, when BMC prepared from CD4/8 DKO mice were transplanted to sublethally irradiated (7.5 Gy) B10.A recipients, the outcome was strikingly different. Five of seven mice promptly rejected wtB6129SF₂ skin allografts even in the presence of a high-degree donor chimerism (Table I, group e, and Table III, group f). The dissociation of hemopoietic chimerism and allograft tolerance was also observed with the use of BMC from CD3E-tg mice, which are deficient in all T cells and NK cells (13) (Table I, group f, and Table III, group h). On day 28 after BMT, the degree of chimerism was moderate, with approximately one-half of B10.D2 (H-2^d) recipients showing <50% chimerism. On day 90, 5 of 11 mice showed a high-degree chimerism (80–95%), while chimerism disappeared in the rest of the mice. Again, donor-type (wtB6CBAF₁) skin grafts were acutely rejected not only in mice that lost chimerism, but in mice that maintained a high degree of chimerism. Transplantation of wt B6129SF₂ or B6CBAF₁ BMC induced both a high degree of chimerism and acceptance of corresponding skin grafts (Table III, groups e and g). Thus, induction of donor-specific allograft tolerance, but not continuous chimerism, required donor T cell reconstitution. Moreover, highly chimeric mice were capable of rejecting donor-specific skin allografts in this model.

Histology of skin grafts

Histology of the rejecting skin grafts in mice given BMC from CD4/8 DKO mice (Fig. 1B) revealed intense cellular infiltration, consisting predominantly of neutrophils, monocytes/macrophages, and rare small lymphocytes forming a bandlike infiltrate in the plane along the dermal-s.c. tissue interface. The epidermis exhibited ischemic changes with areas of ulceration and outward elimination of degenerate reticular dermal collagen into the overlying crust. A similar picture was seen in rejecting skin grafts in mice given CD3E-tg BMC (data not shown). Skin grafts in wtB6/6.5Gy BMT recipients (Fig. 1A) showed more cellular infiltration, with a high number of neutrophils mixed with monocytes/macrophages and rare small lymphocytes. Skin grafts in tolerant mice at 140 days showed no cellular infiltration (Fig. 1C).

View larger version (110K): [in this window] [in a new window]   FIGURE 1. Histology of skin grafts. A, wtB6 skin graft (60% rejected) removed 9 days after grafting in B10.A mice given 6.5 Gy irradiation and wtB6 BMC (H&E; original magnification, x600). B, B6129SF2 skin graft (50% rejected) removed 12 days after grafting in B10.A mice given 7.5 Gy irradiation and CD4/8 DKO BMC (H&E; original magnification, x600). C, B6129SF2 skin graft (100% intact) removed 140 days after grafting in B10.A mice given CD4/8 DKO BMT, and adoptive transfer of donor T cells 21 days after BMT (H&E; original magnification, x200).

To quantitatively analyze the host T cell reactivity to alloantigens after allogeneic BMT, lymphocytes harvested at day 28 were labeled with a fluorochome and CFSE and injected into lethally irradiated (10 Gy) stimulator mice. Because CFSE segregates equally between two daughter cells with each cell division, the CFSE profile of T cells recovered from the stimulator mice correlates with the degree of proliferation by the adoptively transferred host T cells against alloantigens of the stimulator mice (15, 16, 17) (in vivo MLR).

As shown in Fig. 2A, CD4⁺ T cells of naive B10.A mice proliferated at least six times by day 3 after injection in B6 stimulator mice with 9.5% of CFSE-labeled CD4⁺ T cells proliferating. The same T cells also proliferated in response to DBA/1 alloantigen with responder frequency of 11.3%, while they exhibited minimum proliferation in syngeneic B10.A mice (2.8%). More T cells responded to stimulator alloantigens 4 days after adoptive transfer (Fig. 2C). T cells prepared from B10.A mice given 7.5 Gy and wtB6 BMC failed to proliferate in wtB6 hosts (responder frequency of 2% on day 3), suggesting that they were unresponsive to B6 alloantigens. The same T cells proliferated in the third-party DBA/1 hosts as vigorously as naive B10.A T cells (Fig. 2, B and C). Similarly, T cells from B10.A mice reconstituted with CD4 KO BMC or CD8 KO BMC failed to respond to B6 alloantigen, but proliferated in response to DBA/1 alloantigen (data not shown). In contrast, T cells from B10.A mice given CD4/8 DKO BMC or B10.D2 mice given CD3E-tg BMC proliferated in B6129SF₂ or B6CBAF₁ stimulator mice, respectively, with responder frequencies of 10.3 and 10.1%, respectively, suggesting the presence of the host T cell clones reactive to stimulator alloantigens. T cells from wtB6/6.5Gy BMT recipients (B10.A) proliferated more in B6 hosts with greater frequency (12%), although the response was not as vigorous as proliferation by T cells of B10.A mice sensitized to B6 alloantigen (18.2%). The kinetics of in vivo proliferation is summarized in Fig. 2C. Thus, in vivo proliferative response by host T cells strongly correlated with the fate of skin allografts.

View larger version (38K): [in this window] [in a new window]   FIGURE 2. Quantitative analysis of in vivo proliferation by alloreactive T cells (in vivo MLR). CFSE-labeled splenic cells from chimeric mice following allogeneic BMT were injected into irradiated syngeneic controls, corresponding wt BMC donors, and third-party controls (DBA/1). Splenic and lymph node cells from the hosts were collected 1–4 days later and stained with PE-conjugated Ab against CD4. Proliferation was analyzed by flow cytometry. Proliferation frequency of CFSE-labeled CD4+ T cells (percent) is shown in the right histograms (A and B). Data are representative of two to four experiments. A, Day 3 in vivo proliferative responses of naive and sensitized B10.A T cells in syngeneic B10.A and allogeneic wtB6 and DBA/1 hosts. B, Day 3 in vivo proliferative responses of CFSE-labeled CD4+ T cells of chimeric mice in wt BMC donor hosts. C, Changes in precursor frequency of alloreactive CD4+ T cells in in vivo MLR.

To confirm that donor-type T cells are indeed required for induction of allograft tolerance after BMT, we adoptively transferred donor-type T cells to B10.A mice 21 days after CD4/8 DKO BMT. T cells were prepared from the spleens and lymph nodes of lethally irradiated B10.A mice given T cell-depleted wtB6129SF₁ BMT. T cells harvested 45 days after BMT were >95% donor (B6129SF₁) type by flow cytometric analysis and unresponsive to B10.A alloantigen in vivo and in vitro (data not shown). Fig. 3A shows that donor-type T cells were present 7 days after adoptive transfer (28 days after BMT) in mice reconstituted with CD4/8 DKO BMC, whereas no donor-type T cells were detected without adoptive transfer. The mice given donor T cells were transplanted with wtB6129SF₂ skin grafts 30 days after BMT (9 days after adoptive transfer). All skin grafts were accepted for 140 days (Fig. 3B). Flow cytometric analysis on day 102 show a high degree of chimerism (>90%) in all mice (data not shown). Thus, engraftment of donor-type T cells restored allograft tolerance in T cell-deficient BMT recipients by down-regulating the immune response directed against their own alloantigens.

View larger version (36K): [in this window] [in a new window]   FIGURE 3. Effect of adoptive transfer of wtB6129SF2 T cells into B10.A mice given CD4/8 DKO BMT. T cells derived from fully chimeric B10.A mice given wtB6129SF2 BMT (95% B6129SF2 phenotype) were adoptively transferred to CD4/8 DKO BMC-reconstituted B10.A mice 21 days after BMT. A, Flow cytometric analysis of CD4+ T cell chimerism 28 days after wtB6 BMT, CD4/8 DKO BMT, or CD4/8 DKO BMT plus adoptive transfer of donor-type T cells (7 days after T cell transfer). B, Skin graft survival after BMT. After adoptive transfer of donor-type T cells, the mice were transplanted with wtB6129SF2 skin grafts on day 30 after BMT (9 days after T cell transfer) (n = 7). Survival of wtB6129SF2 skin in B10.A mice given CD4/8 DKO BMT is also shown (n = 9).

	Discussion

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Although B10.A mice reconstituted with CD4/8 DKO BMT acutely rejected wt (B6129SF₂) skin allografts, adoptive transfer of donor wt T cells in these mice led to acceptance of skin grafts, suggesting that donor T cells were essential in achieving allograft tolerance. Adoptively transferred T cells were derived from lethally irradiated hosts (B10.A) given T cell-depleted wt (B6129SF₂) BMT. Thus, the T cells were mostly of B6129SF₂ origin, but incapable of causing graft-vs-host response upon transfer to B10.A hosts. These donor T cells contained immunoregulatory T cells that were capable of inhibiting the host T cell response directed against their own (donor) alloantigens. Results of skin graft survival suggest that these immunoregulatory T cells differentiate from wt, CD4 KO, or CD8 KO BMC, but not from CD4/8 DKO or CD3E-tg BMC. As maturation of CD8⁺ or CD4⁺ T cells is unaffected in CD4 or CD8 KO mice, respectively, it is possible that both CD4^-CD8⁺ and CD4⁺CD8^- T cells are capable of exerting immunoregulatory activity. Alternatively, as two mice given CD4/8 DKO BMT accepted wt skin grafts, CD4^-8^- -TCR-positive cells derived from CD4/8 DKO BMC (23) may function as immunoregulatory cells. Complete lack of allograft tolerance in mice reconstituted with CD3E-tg BMC is probably attributable to more complete loss of T cells through deficiency in early T cell differentiation by CD3- gene disruption (13). It is not known whether the immunoregulatory T cells identified in the present study are the same as previously described tolerance-inducing veto cells or bone marrow facilitator cells (5, 24, 25, 26, 27, 28, 29, 30).

We have previously reported that rejection of donor-type skin allografts in the presence of a high degree of hemopoietic chimerism occurs following allogeneic BMT with MHC class II Ag-deficient BMC (14). Skin allografts showed delayed-type hypersensitivity-like histology (A. Umemura, unpublished observation). Taken together, the present results suggest that chimerism and allograft tolerance are probably two interrelated, but distinct events. They are interrelated because allogeneic BMT that achieves thymic clonal deletion by donor class II Ag-bearing cells (31, 32) and peripheral immunoregulation by donor T cells, and/or full depletion or inactivation of host peripheral T cells by the strong immunoablative regimens induces tolerance to donor alloantigen, leading to both durable chimerism and allograft tolerance. They are also distinct because donor skin graft rejection does not lead to rejection of chimerism as long as the clonal size of donor-reactive T cells remains too small to generate direct cytotoxicity against donor lymphoid cells. It is possible that chimerism could be lost at a later time after the end of the observation period (110 days after BMT or 80 days after skin grafting) in mice given CD4/8 DKO or CD3E-tg BMT. As skin graft rejection took place 8–28 days (mostly within 14 days) after transplantation, and a high degree (average 90%) of chimerism continued for at least 30 days or more in these mice, these results would still argue for the dissociation of chimerism and allograft tolerance in these mice.

Thus, in the allogeneic BMT model, presence of chimerism may not necessarily guarantee allograft tolerance, particularly when residual host T cells are not completely inactivated or deleted. On the other hand, absence or loss of chimerism after BMT is caused by a large residual host T cell clone and possibly newly developing host T cells, and is usually associated with absence/loss of tolerance.

	Footnotes

 
¹ This study was supported by National Institutes of Health Grant RO1 AI14551. A.U. and H.M. were supported by a fellowship from the Mitsui Memorial Hospital Fund.

² Address correspondence and reprint requests to Dr. Takashi Maki, Research North, Beth Israel Deaconess Medical Center, P.O. Box 15707, Boston, MA 02215. E-mail address: tmaki{at}caregroup.harvard.edu

³ Abbreviations used in this paper: BMC, bone marrow cell; BMT, bone marrow transplantation; KO, knockout; DKO, double KO; tg, transgenic; wt, wild type.

Received for publication April 26, 2001. Accepted for publication July 6, 2001.

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