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CD45 Congenic Bone Marrow Transplantation: Evidence for T Cell–Mediated Immunity

^a Institute for Cellular Therapeutics, University of Louisville, Louisville, Kentucky, USA;
^b Department of General and Thoracic Surgery, University of Kiel, Kiel, Germany

Key Words. Hematopoietic microenvironment • Niche • Nonmyeloablative conditioning • CD45 • Congenic bone marrow transplantation • Chimerism

Correspondence: Suzanne T. Ildstad, M.D., University of Louisville, 570 South Preston Street, Suite 404, Louisville, Kentucky 40202-1760, USA. Telephone: 502-852-2080; Fax: 502-852-2079; e-mail: stilds01{at}louisville.edu

	ABSTRACT

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CD45 congenic mice have been used to study stem cell engraftment in the absence of alloreactivity. Recently, impaired engraftment was reported in this model and attributed to weak immune reactivity. We have confirmed that there is indeed low-level reactivity mediated by CD8⁺ cells and ß-TCR⁺ T cells. B6 (CD45.2) recipients were conditioned with total body irradiation (TBI) and transplanted with increasing doses of B6 (CD45.1) bone marrow cells (BMCs). Although chimerism was present at 1 month in all recipients, durable engraftment did not occur with <150 cGy of TBI, emphasizing the importance of long-term follow-up in evaluating nonmyeloablative conditioning approaches. A single dose of cyclophosphamide on day 2 also significantly enhanced engraftment. When B6 TCRß^–/–, TCR^–/–, or TCRß^–/–/^–/– (CD45.2) mice were transplanted with CD45.1 bone marrow, significantly enhanced engraftment occurred in recipients lacking ß-TCR⁺ T cells (p < .00005). Similarly, removal of ß-TCR⁺ host T cells in wild-type recipients resulted in enhanced engraftment. Engraftment was also significantly increased in CD8^–/– and CD4^–/–/8^–/– recipients (p < .0005). Taken together, these results demonstrate that ß-TCR⁺ and CD8⁺ T cells play a critical role in regulating engraftment of CD45 congenic marrow and suggest that these cells are the main effector cells in low-level alloreactivity to the CD45 disparity.

	INTRODUCTION

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The morbidity associated with ablative conditioning has prevented the widespread application of bone marrow transplantation (BMT) to induce tolerance and treat a wide variety of autoimmune diseases and hemoglobinopathies. Nonmyeloablative conditioning has significantly reduced the risk-to-benefit ratio of BMT [1–6]. Most nonmyeloablative conditioning approaches use a combination of immunosuppressive agents and low-dose irradiation [3,4], both of which act non-specifically. To further reduce the toxicity of conditioning, the mechanisms regulating engraftment must be understood. Whether the primary role of conditioning is to make space rather than control host-versus-graft alloreactivity or enhance competition remains controversial [7–9].

Resistance to engraftment of transplanted bone marrow is mediated by immune responses. In previous studies, we characterized the cells in the recipient hematopoietic micro-environment that prevent allogeneic marrow engraftment using different T cell–deficient mice as recipients. Major histocompatibility complex (MHC)–disparate allogeneic bone marrow cells (BMCs) engrafted more readily in mice lacking ß-TCR⁺, ß-TCR⁺ plus -TCR⁺, or CD8⁺ cells, but not in mice lacking -TCR⁺ or CD4⁺ cells, suggesting that ß-TCR⁺ and CD8⁺ T cells in the host played critical and nonredundant roles in preventing engraftment of allogeneic bone marrow [10]. Similarly, targeting host T cells with anti-ß-TCR or CD8 decreased the requirement of total body irradiation (TBI) to establish allogeneic chimerism [11–13]. Additionally, conditioning of mice with a single dose of cyclophosphamide (CyP) on day-2 enhanced engraftment, most likely by targeting alloreactive host cells responding to donor alloantigen [3]. Taken together, these data support the fact that the major role of conditioning is to control host-versus-graft reactivity rather than to make space.

The CD45.1/CD45.2 model has been used extensively to characterize engraftment in the absence of alloreactivity [14–19]. Notably, skin grafts from CD45.1 donors are not rejected by CD45.2 recipients [16], and this model was therefore used as a tool to study engraftment in the absence of alloreactivity. However, a recent report has challenged that paradigm and demonstrated that there is a weak immune response to BMC engraftment when the only disparity is between the CD45.1 and CD45.2 antigens. Although host T cells were hypothesized by the authors to be involved in the immunological resistance against the stem cells, this was not conclusively demonstrated [20]. In the present studies, we have confirmed the findings of van Os et al. [20] and have specifically demonstrated that host CD8⁺ and ß-TCR⁺ cells are the effector cells for this reactivity. We first reproduced the work of others using the CD45.1/CD45.2 congenic model. Surprisingly, although high doses of BMCs established chimerism with little or no conditioning and there was a linear relationship between cell dose and level of chimerism, chimerism was durable only in recipients conditioned with 150 cGy TBI. Unconditioned knockout (KO) mice lacking ß-TCR⁺ T cells or CD8⁺ cells engrafted more readily and with significantly higher levels of donor chimerism compared with wild-type controls. In contrast, mice deficient in only CD4⁺ or -TCR⁺ T cells showed no increase in engraftment. Unlike wild-type recipients, the level of donor chimerism increased significantly over time in the mice that engrafted, with the most notable increases occurring in mice lacking ß-TCR⁺ T cells and the CD4^–/–/8^–/– recipients. Because donor cells of all hematopoietic lineages were detected in recipient KO mice, durable chimerism was not merely attributable to homeostatic proliferation of the missing lineages. These results suggest that host CD8⁺ and ß-TCR⁺ cells play a significant role in the regulation of hematopoietic stem cell (HSC) engraftment in this congeneic model and confirm that low-level T cell–mediated reactivity against HSCs is present in this strain combination. As such, caution must be exercised in the interpretation of data using this model as it relates to mechanism of engraftment.

	MATERIALS AND METHODS

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Mice
Three- to five-week-old C57BL/6 (B6: CD45.2) recipient mice and B6 TCRß^–/–, TCR^–/–, TCRß^–/–/^–/–, CD8^–/–, CD4^–/–, and CD4^–/–/8^–/– (all congenic on CD45.2 background), as well as B6.SJL-PtprcaPep3b/BoyJ (B6: CD45.1), donor mice were purchased from Jackson Laboratory (Bar Harbor, ME). Mice were housed in a barrier facility at the Institute for Cellular Therapeutics and cared for according to National Institutes of Health animal care guidelines.

Chimera Preparation
Bone marrow was prepared as previously described [4]. Briefly, bone marrow was harvested and reduced to a single-cell suspension and transplanted with recipients conditioned with either varying doses of TBI or CyP (200 mg/kg, i.p.) as a single dose on day 2.

Flow Cytometric Analysis
Peripheral blood (PB) was collected in heparinized Eppendorf tubes as previously described [3, 10, 21]. Two-color flow cytometric analysis with donor-specific anti-CD45.1 monoclonal antibody (mAb; clone A20, mouse immunoglobulin G2a [IgG2a]) conjugated to phycoerythrin and recipient-specific anti-CD45.2 (clone 104, mouse IgG2a) conjugated to fluorescein-isothiocyanate (FITC) was used to determine the level of donor chimerism. The following FITC-conjugated mAbs were used to confirm the presence of various lineages: anti-CD4 (RM4-5, rat IgG2a), anti-CD8 (53-6.7, rat IgG2a), anti-TCRß chain (H57-597, hamster IgG), anti-B220 (RA3-6B2, rat IgG2a), anti-NK1.1 (PK136, mouse IgG2a), anti-Gr-1 (RB6-8C5, rat IgG2b), and anti-Mac-1 (M1/70, rat IgG2b). All monoclonal antibodies were obtained from Pharmingen (San Diego). An amount of donor cells as low as 0.05% can be reproducibly detected.

mAb Preconditioning
Anti-CD8 or anti-ß-TCR mAb 100 µg was injected intravenously at day 3 before BMT. The antibodies were diluted to a 1-ml volume in phosphate-buffered saline solution (BioWhitaker, Walkersville, MD). Antibodies were titered for optimum dose before use. Adequacy of depletion was confirmed on PB by flow cytometric analysis.

StatisticalAnalysis
Data are presented as average ± standard deviation (SD). The Wilcoxon test (one-tailed t-test: two samples assuming unequal variances) was used to evaluate statistical differences. The difference between groups was considered to be significant if p < .05.

	RESULTS

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Level of Engraftment of CD45.1 BMCs Is Directly Correlated with Cell Dose and Amount of TBI Administered
To establish our model, increasing numbers of congenic (CD45.1) B6 BMCs were transplanted into unconditioned B6 (CD45.2) mice. A strict linear relationship between cell dose and percentage of donor chimerism was observed (Fig. 1). The level of chimerism was 0.4 ± 0.1% at 1 month after transplantation for animals receiving 15 x 10⁶ BMCs. When the cell dose was increased to 50 x 10⁶ (3.3-fold), the chimerism increased 3.3-fold. Similarly, when recipients were transplanted with 75 x 10⁶ cells, a fivefold increase in level of donor chimerism occurred, compared with the level for 15 x 10⁶ cells. The correlation factor between 15 x 10⁶ and 100 x 10⁶ cells was 6.7. The expected level of chimerism with a linear correlation between cell dose and level of engraftment would be 2.7% when 100 x 10⁶ cells are transplanted, and the observed level was 2.6 ± 0.2%. When the cell dose was increased twofold, the level of chimerism doubled. This pattern for engraftment differs from that for nonmyeloablatively conditioned recipients of MHC-disparate bone marrow, in which an abrupt transition to 100% engraftment at significantly higher doses of TBI is observed [3,4].

View larger version (9K): [in this window] [in a new window]   Figure 1. Cell dose and level of chimerism are directly correlated in the absence of conditioning. Unconditioned B6 recipients (CD45.2) were transplanted with increasing doses of congenic B6 (CD45.1) bone marrow cells. Engraftment was assessed by flow cytometric analyses of peripheral blood lymphocytes. Data represent average percent donor chimerism ± standard deviation from 6 to 12 recipients in each group 1 month after transplantation.

To evaluate the influence of low-dose TBI on engraftment, B6 (CD45.2) mice were conditioned with increasing amounts of TBI (50, 100, and 150 cGy) and transplanted with 5, 10, or 15 x 10⁶ BMCs from B6 (CD45.1) donors. The percent donor engraftment was directly correlated with cell dose for a given dose of TBI, ranging from 0.4 ± 0.1% to 7.6 ± 1.5% when 5 x 10⁶ cells with 50 cGy TBI versus 15 x 10⁶ cells with 150 cGy TBI were infused. The degree of conditioning even more significantly influenced the level of chimerism for each given cell dose (Fig. 2). However, sustained long-term engraftment in 100% of recipients occurred in only those recipients given 150 cGy TBI and 15 x 10⁶ BMCs. Only some animals from each of the other groups maintained their chimerism 8 months (Table 1).

View larger version (12K): [in this window] [in a new window]   Figure 2. Low-dose TBI and cell number are directly correlated with engraftment. B6 (CD45.2) recipients were conditioned with increasing amounts of TBI (50, 100, or 150 cGy) and transplanted with 5, 10, or 15 x 106 bone marrow cells from CD45.1 donors. Data represent average donor chimerism ± standard deviation of six recipients in each group 1 month after bone marrow transplantation from three experiments. Abbreviation: TBI, total body irradiation.

View larger version (13K): [in this window] [in a new window]   Figure 3. The effect of CyP on engraftment in congenic BMT. Recipient CD45.2 mice were conditioned with 0, 100, or 200 cGy TBI and transplanted with 15 x 106 CD45.1 bone marrow cells. Recipients were treated with CyP 2 days after BMT. Controls were conditioned with TBI but did not receive CyP. Data represent average percent donor chimerism ± standard deviation of five to six recipients in each group 1 month after BMT from two experiments. Abbreviations: BMT, bone marrow transplantation; CyP, cyclophosphamide; TBI, total body irradiation.

Host ß-TCR⁺T Cells Play an Important Role in Alloreactivity to CD45

View larger version (19K): [in this window] [in a new window]   Figure 4. Congenic engraftment in B6 TCRß–/–, TCR–/–, or TCRß–/–/–/– mice. Unconditioned TCRß–/–, TCR–/–, and TCRß–/–/–/– (CD45.2) mice were transplanted with 15 x 106 CD45.1 bone marrow cells. (A): Level of donor chimerism at 1 month after transplantation. (B): Kinetics of donor chimerism at up to 8 months. Data represent average percent donor chimerism ± standard deviation from six to eight recipients in each group from two experiments. Abbreviation: BMT, bone marrow transplantation.

View larger version (17K): [in this window] [in a new window]   Figure 5. CD45.1 BMC engraftment in CD45.2 CD4, CD8, or CD4–/–/8–/– mice. Unconditioned CD8–/–, CD4–/–, or CD4–/–/8–/–mice were transplanted with 15 x 106 CD45.1 BMCs. The level of chimerism was assessed by flow cytometric analysis. (A): Level of donor chimerism at 1 month. (B): Kinetics of syngeneic donor chimerism up to 10 months after transplant. Data represent average percent donor chimerism ± standard deviation from five to eight recipients in each group in three experiments. Abbreviations: BMC, bone marrow cell; BMT, bone marrow transplantation.

View larger version (13K): [in this window] [in a new window]   Figure 6. Phenotypic analysis of the T-cell populations in the BM microenvironment in wild-type B6 (CD45.2) mice. BM and PB were collected from 6- to 8-week-old unmanipulated wild-type B6 mice. The phenotypic analysis of the T-cell populations was performed by flow cytometry. The percentage of each T-cell population was analyzed from the lymphocyte gate of BM or PB. Data shown are average percent of T-cell subpopulations ± standard deviation of four individual wild-type B6 mice. Abbreviations: BM, bone marrow; PB, peripheral blood.

Kinetics of Chimerism in CD8^–/–, CD4^–/–/8^–/–, TCRß^–/–, andTCRß^–/–/^–/– Recipients

Multilineage Chimerism Occurs in Engrafted Mice
To evaluate whether the increase in donor chimerism was attributable to homeostatic normalization, multilineage typing was performed on PB in TCRß^–/– (n = 3), TCRß^–/–/^–/– (n = 3), CD8^–/– (n = 5), and CD4^–/–/8^–/– recipients (n = 5) 3–4 months after reconstitution. Donor-derived B cells, T cells, natural killer (NK) cells, macrophages, and granulocytes were present in all animals, making homeostatic expansion an unlikely explanation (Table 2).

Anti-ß-TCR mAb Pretreatment Enhances Engraftment

View larger version (16K): [in this window] [in a new window]   Figure 7. B6 mice (CD45.2) were preconditioned with anti-CD8 or anti-ß-TCR monoclonal antibody in vivo and varying doses of TBI followed by infusion of CD45.1 bone marrow cells on day 0 relative to TBI. Engraftment was assessed at 28 days. Data represent five to six animals from two experiments. Abbreviation: TBI, total body irradiation.

View larger version (36K): [in this window] [in a new window]   Figure 8. Host CD8dim lymphocytes were not removed by host anti-CD8 treatment in vivo. B6 mice were preconditioned with anti-CD8 mAb in vivo. To document depletion, peripheral blood was obtained 3 days after mAb treatment from treated mice and stained with FITC-conjugated anti-CD8 (53–6.7). Staining was also performed with secondary mAb of mouse anti-rat immunoglobulin G2a–FITC to assure that cells were depleted or coated with mAb. The bright CD8+ population was depleted, but there was a CD8dim population comprising 2% of the lymphoid gate that could not be removed by increasing amounts of mAb (A). These CD8dim cells were then stained for ß-TCR, CD4, B220, NK1.1, CD11b, or CD11c expression and analyzed by flow cytometry (B). Profile is representative of three experiments.Abbreviations: FITC, fluorescein-isothiocyanate; FSC, forward scatter; mAb, monoclonal antibody.

CD4^–/–/8^–/– Mice Do Not Produce NK Cells
NK cells have been demonstrated to contribute to BMC rejection [22]. We therefore examined the various strains for the production of NK cells with the rationale that this may be underlying the difference in chimerism between the strain combinations. PB lymphocyte from the CD8^–/–, TCRß^–/–, TCRß^–/–/^–/–, and CD4^–/–/8^–/– mice was analyzed for production of NK cells. Interestingly, CD4^–/–/8^–/– mice do not produce NK cells, whereas the others do at levels similar to wild-type controls (Fig. 9). Therefore, it is possible that the absence of NK cells in these mice may explain the enhanced advantage of wild-type donor marrow.

View larger version (13K): [in this window] [in a new window]   Figure 9. Peripheral blood lymphocytes from CD4–/–/8–/–, CD8–/–, TCRß–/–, and TCRß–/–/–/– mice were analyzed for the presence of NK cells by flow cytometry (mean ± standard deviation from two experiments with three to six animals per group). Abbreviation: NK, natural killer.

	DISCUSSION

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Host T cells clearly influence engraftment of MHC-disparate allogeneic BMCs. We previously reported that 700 cGy of TBI is required for durable engraftment of MHC-disparate allogeneic marrow [3]. This TBI dose can be reduced to 500 cGy with the addition of 200 mg/kg of intraperitoneal CyP 2 days after BMT [3]. In this TBI/CyP-based conditioning approach, the addition of host preconditioning with anti-CD8 mAb further reduced the minimum TBI dose to 300 cGy [12], suggesting that host CD8⁺ cells play an important role in alloresistance. Host ß-TCR⁺ T and T cells play a nonredundant role in allogeneic marrow rejection, because mice deficient in production of both cell types engraft without conditioning [10]. Taken together, these data demonstrate that a major role for conditioning in MHC-disparate transplants is to control host-versus-graft alloreactivity.

In the present study, we found that specific cell subsets in the recipient microenvironment indeed influenced HSC engraftment in CD45 congenic recipients. Notably, mice lacking ß-TCR⁺ T cells exhibited enhanced engraftment. Removal of ß-TCR⁺ T cells by mAb pretreatment of wild-type recipients resulted in a similar enhancement of engraftment. In contrast with what we observed for MHC-disparate allogeneic HSC engraftment [10], -TCR⁺ T cells did not contribute to the reactivity to CD45.1 BMCs. These data suggest a specific role for host ß-TCR⁺, but not -TCR⁺ T cells, in regulating CD45 marrow engraftment. As shown in MHC-disparate recipients, the administration of CyP on day 2 also enhanced engraftment in the CD45 congenic model, further supporting an immune-mediated mechanism. Similarly, mice lacking CD8 cells exhibited enhanced engraftment, whereas those recipients lacking CD4 cells did not. Moreover, mice lacking both CD4⁺ and CD8⁺ cells developed significantly higher levels of donor chimerism compared with mice lacking CD8⁺ cells alone. These results demonstrate that a subset of CD8⁺ cells plays a critical role in regulating engraftment of CD45 congenic bone marrow and that their function is enhanced by CD4⁺ cells. In light of the fact that CD8⁺ cell function is enhanced by CD4⁺ cells, one could hypothesize a functional regulatory cellular mechanism for engraftment, because CD4⁺ T cells function as helper T cells to initiate function mediated by cytotoxic CD8⁺ T cells [24]. Alternatively, the fact that CD4^–/–/8^–/– mice lack NK cells in their marrow and that TCRß^–/–/^–/– mice produce a normal or increased number of NK1.1 and 5E6⁺ cells but lack T/NK and CD8⁺ NK cells whereas CD8^–/– mice lack only CD8⁺ NK cells may mechanistically point to a role for NK cells in contributing to the low level of reactivity.

Although the CD45 congenic model has been historically believed to be nonantigenic, supported by lack of skin graft rejection [16], and has been extensively used as a model to study HSC engraftment in the absence of alloreactivity [17,18], our present data confirm and extend a recent report to the contrary [8]. The report by van Os et al. [8] hypothesized but did not conclusively demonstrate that immune-mediated processes were the underlying explanation for the impaired engraftment observed in the CD45 isoform congenic model [8]. The enhanced engraftment we observed when ß-TCR⁺ T cells were depleted would support a low level of T cell–mediated reactivity in this model, as would the enhanced engraftment after CyP on day 2. Although these data do not negate previous reports using this model, they do suggest a cautionary note that this variable be included in interpretation of the data. The fact that CD45.2 mice do not reject skin grafts from CD45.1 congenic donors [16] leads one to hypothesize that the low level of reactivity is directed at non-MHC antigens unique to BMCs.

Conditioning has also been shown to influence engraftment in congenic recipients [17]. Low-dose TBI significantly reduces the number of BMCs required for engraftment compared with unconditioned recipients [25–27]. However, much less conditioning is required for engraftment of syngeneic marrow compared with allogeneic marrow. There is a linear relationship between the proportion of syngeneic recipients engrafting and TBI dose [28], whereas a more abrupt transition occurs in allogeneic recipients at 550 cGy TBI. Down et al. [28] showed that at least 250 cGy of irradiation was required to achieve engraftment of 10 x 10⁶ syngeneic BMCs. Using a CD45 congenic model, Tomita et al. [17] confirmed this observation, demonstrating that myelosuppressive conditioning is required to achieve engraftment of HSCs when 15 x 10⁶ T cell–depleted BMCs are administered. In our present studies using the CD45 congenic model, although recipients conditioned with low-dose TBI initially exhibited chimerism, durable multilineage chimerism occurred only in recipients conditioned with 150 cGy of TBI. The difference in conditioning requirements between the CD45 congenic and MHC-disparate transplants suggests that there may be at least two functional categories of cells that exist in the host hematopoietic microenvironment: cells that mediate host-versus-graft alloreactivity and cells that occupy or compete for niches. The kinetic for graft rejection differs significantly between MHC disparate versus the CD45 congenic transplants, because chimerism in the latter group was present at 2 months and then tapered more slowly, supporting the fact that there is only a weak response to the CD45 isoform. Notably, our data strongly suggest that long-term follow-up is essential in demonstrating success or failure in developing nonmyeloablative conditioning approaches in closely matched recipients.

The concept that myeloablation is a prerequisite to HSC engraftment has been challenged by studies showing high rates of engraftment in unconditioned syngeneic recipients of large numbers of BMCs [25, 27, 29, 30]. Our own data reproduced that observation. However, outcomes assessment from previous studies used single-lineage typing and relatively short-term follow-up, ranging historically from 1 week to 3 months [9, 17, 29, 31, 32]. Our present data indicate that long-term engraftment was strikingly different in unconditioned versus minimally conditioned recipients. Although significant chimerism was present at 1 month in unconditioned recipients, we found that 150 cGy of TBI was necessary to obtain reliable, durable multilineage engraftment. The fact that very low levels of irradiation provide a significant competitive advantage for donor HSC engraftment and also significantly reduce the number of donor cells required to establish engraftment could indirectly support the concept of competition for niches [25,27]. Alternatively, the low level of T cell–mediated alloreactivity in the CD45 congenic BMT model could be the mechanism underlying the late graft loss, a mechanism we favor in light of the data in the T cell–deficient and anti-ß-TCR mAb-treated recipients. Nevertheless, our data suggest that long-term follow-up is critical in evaluating the success or failure of non-myeloablative conditioning approaches.

It is of note that the CD45 congenic wild-type bone marrow exhibited a highly significant competitive advantage when transplanted into recipients lacking ß-TCR⁺ cells and CD8⁺ cells, as reflected by a significant increase in donor chimerism over time, especially for TCRß^–/–, TCRß^–/–/^–/–, and CD4^–/–/8^–/– recipients. Although CD4^–/– mice did not engraft as readily as the CD8^–/– recipients, the CD4^–/–/8^–/–recipients exhibited significantly higher levels of donor chimerism. This is only partially explained by normalization of cell numbers by homeostatic expansion, because the recipients have all hematopoietic lineages represented in the PB. Additionally, the TCRß^–/– recipients continue to have low levels of T cells in the periphery, even when the percentage donor chimerism is increasing (2– 4 months). The competitive repopulation, as reflected by a progressive increase in donor chimerism, was not observed in other congenic models using wild-type recipients [17]. Moreover, the enhanced engraftment observed after anti-ß-TCR pretreatment of normal recipients supports a role for conventional T cells. Taken together, these data suggest that host CD8⁺ and ß-TCR⁺ T cells play an important role in regulating engraftment in the recipient’s hematopoietic microenvironment even with only minor antigen disparities. In the absence of ß-TCR⁺ and CD8⁺ cells, the donor-derived marrow has a competitive advantage, perhaps via low-level graft-versus-host reactivity. It is possible that the enhanced engraftment observed in the CD4^–/–/CD8^–/– recipients is related to the fact that regulatory NK cells are not present. However, this cannot explain the influence of ß-TCR⁺ cells, because TCRß^–/– mice produce NK1.1⁺ cells in normal numbers [10]. An alternative explanation is that cells producing various engraftment-enhancing cytokines produced by wild-type mice have not developed in these genetically deleted recipients.

In conclusion, we show strong evidence that a relatively weak but definite T cell–mediated immune response to the transplantation of CD45 congenic BMCs is present that differs from the more aggressive response to MHC-disparate allogenic BMC in kinetics of graft rejection and effector cells. Notably, although CD45.2⁺ mice do not reject skin grafts from CD45.1 donors, they do mount an immune-mediated attack on BMCs. These data are important to the study of nonmyeloablative models to establish donor chimerism defining the relative contribution of alloreactivity versus HSC environment and competition for niches. A definition of the mechanism of engraftment of syngeneic versus congenic versus MHC-disparate BMCs is critical to identifying methods to establish mixed chimerism nonmyeloablatively. The fact that near-linear correlations exist between cell dose, irradiation dose, and engraftment in closely matched transplants such as the CD45 congenics will allow one to design individual therapeutic strategies of conditioning to achieve the level of chimerism required to treat a specific disease. We demonstrate for the first time that the absence of CD8⁺ or ß-TCR⁺ cells in the recipient enhanced acceptance of CD45 marrow grafts, strongly pointing to an immune-mediated mechanism influencing engraftment in this model. The combined knowledge of what contributes to the maintenance of niches and what is required to establish durable, sustained hematopoiesis will allow a focused strategy to target and manipulate those cellular populations in the host in vivo to enable engraftment in a highly specific and benign fashion.

	ACKNOWLEDGMENTS

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The authors thank Dr. Francine Rezzoug, Dr. Lee Grimes, and Dr. Isabelle Fugier for review of the manuscript and helpful comments; Carolyn DeLautre and Kim Nichols for manuscript preparation; and the staff of the animal facility for outstanding animal care. This research was supported in part by NIH DK52294 and R01 HL63442-01A2, The Commonwealth of Kentucky Research Challenge Trust Fund, The Jewish Hospital Foundation, and the University of Louisville Hospital.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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