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Stem Cells, Vol. 19, No. 1, 80-87, January 2001
© 2001 AlphaMed Press

Immunogenicity of Ly5 (CD45)-Antigens Hampers Long-Term Engraftment Following Minimal Conditioning in a Murine Bone Marrow Transplantation Model

Ronald van Osa, Tara M. Sheridana, Simon Robinsona, Dainius Drukteinisa, James L.M. Ferrarab, Peter M. Maucha

a Department of Radiation Oncology, Brigham and Women's Hospital and the Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA;
b Combined Bone Marrow Transplantation Program, University of Michigan Cancer Center, Ann Arbor, Michigan, USA

Key Words. Engraftment • Gpi • Ly5 • Immunity

Ronald van Os, Ph.D., Department of Hematology, Leiden University Medical Center Building 1, C2-R, PO Box 9600, 2300 RC Leiden, The Netherlands. Telephone: 31-71-5262271; Fax: 31-71-5266755.


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Various techniques are available for distinguishing donor from host cells evaluating the efficacy of conditioning regimen for experimental bone marrow transplantation (BMT). Techniques include the use of extracellular immunological markers, such as Ly5 (CD45), and intracellular biochemical markers, such as glucose-phosphate-isomerase (Gpi). Because Ly5 is an extracellular protein, the disparity between donor (Ly5.1) and host (Ly5.2) antigens may induce a weak immune response whereas with Gpi, no immune response is expected. This difference may be of particular concern in experimental transplantation approaches that use minimal conditioning such as low-dose total body irradiation (TBI). Such mild conditioning may not induce the immunosuppression required to overcome host rejection of Ly5 disparate cells.

To compare the relative engraftment of Ly5.1 and Gpi-1a donor marrow, B6 (Gpi-1b/Ly5.2) mice were irradiated with low-level TBI (0-6 Gy) and transplanted with several bone marrow (BM) doses (2 x 106-5 x 107 cells). At 8, 26, and 52 weeks post-BMT, the level of donor engraftment was measured using flow cytometry (Ly5) or Gpi-electrophoresis.

Lower engraftment levels were found in mice transplanted with Ly5 congenic BM in groups given low-dose TBI (<=4 Gy) and/or low doses of BM cells (BMC) (2 x 106). However, when higher TBI or BMC doses were used, similar engraftment levels were found, suggesting sufficient immune suppression to allow equal engraftment of both sources of BM.

These data suggest that even a minor phenotypic disparity between donor and host, such as Ly5, may necessitate high-dose TBI to prevent rejection. The combination of low-dose TBI or other nonmyeloablative conditioning strategies with small numbers of BMC may lead to reduced engraftment when extracellular immunological markers such as Ly5 are used for transplantation studies. Therefore, small immunological differences must be considered when using the Ly5 marker for engraftment.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Since 1980, many investigators have relied on in vivo competitive long-term repopulation assays to measure primitive stem cell function [1]. These assays use varying numbers of distinguishable test and control cells transplanted into lethally irradiated recipients to measure the level of long-term hematopoietic chimerism. Following transplantation into minimally conditioned recipients, the use of marked donor cells allows measurement of chimerism [2]. In this case transplanted donor cells compete with endogenous recipient cells. The assays available for detection of donor or host cells in the treated recipient utilize cytogenetic, immunological or biochemical markers.

Cytological examination of metaphases for the presence of the T6 marker was the first method for the identification of donor cells in a transplanted host [3]. Another method investigates chimerism in sex-mismatched bone marrow transplantation (BMT) by detecting the male Y-chromosome after in situ hybridization [4, 5] or after polymerase chain reaction for Y-chromosome-specific DNA sequences on Southern blots [6]. The engraftment of bone marrow (BM) into sex-mismatched hosts can be influenced by an immune reaction against the H-Y antigen; this reaction has been documented to be weakly immunogenic [7].

Immunological markers use differences in membrane antigens between donor and host that are then tagged with specific monoclonal antibodies. The difference in major histocompatibility complex (MHC) (H-2) is the most widely used immunological marker in murine BMT research. Both lysis of labeled cells with complement in a microcytotoxicity assay and flow cytometric analysis of antibody binding are used to measure donor chimerism [8-13]. However, H-2 antibody typing of spleen cells is restricted to MHC-mismatched allogeneic BMT where host-versus-graft and graft-versus-host reactions influence BM engraftment. Flow cytometric analysis of other surface markers (Ly-1, Ly-5, and Thy-1) was developed as a method for determination of long-term chimerism in congenic recipients [14, 15]. Ly-1 and Thy-1 markers can only be used for measurement of lymphoid chimerism and the Ly-5 antigen (CD45) is expressed on all BM-derived cells except erythrocytes and erythroblasts [16]. In this study, it was investigated whether the congenic differences in these lymphoid antigens may induce an immune response against donor cells that affect engraftment.

Biochemical markers utilize electrophoretic differences in erythrocyte proteins such as hemoglobin [1, 17-19] or carbonic anhydrase [20] to distinguish donor from host-derived mature RBC. Hemoglobin and carbonic anhydrase are erythrocyte-specific enzymes and can therefore not be used for measuring engraftment along other lineages. Other biochemical markers that can be used to measure engraftment in all cells are the glycolytic enzymes phosphoglycerate kinase [21-23] or glucose-phosphate isomerase (Gpi) [24-27]; these can be used in congenically marked syngeneic as well as allogeneic (MHC-matched and mismatched) BMT. Two murine forms of the above enzymes exist that can be separated using electrophoresis and the relative contribution of donor and host stem cells to hematopoiesis can be determined in all cell lineages.

In this study, two techniques of monitoring chimerism in congenic mice were compared. An immunological method (Ly5) and a biochemical method (Gpi) were used to measure engraftment of congenically marked BM following several doses of BM cells (BMC) and varying doses of total body irradiation (TBI) in order to obtain a range of donor/host chimerism levels to compare the two methods. Furthermore, we investigated whether Ly5 and Gpi congenic cells were able to generate an immune response in vitro following presensitization in vivo.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
Male C57BL/6J (B6)-mice (Ly5b, Gpi-1b) purchased from Jackson Laboratories (Bar Harbor, ME; http://www.jax.org) were used as recipients of congenic BM. Male congenic C57BL/6J-Gpi-1a/Gpi-1a (B6-Gpi-1a) and B6.SJL-Ptprca Pep3b/BoyJ-Ly5a (B6.SJL) mice purchased from Jackson Laboratories, were used as a source of normal untreated marrow in the competitive repopulation assays.

Competitive Repopulation in Vivo
Donors and recipients were between three and five months of age and were sex-matched. Recipient mice were exposed to 137Cs-gamma irradiation (Gamma Cell 40, Atomic Energy of Canada; Ottawa, Canada) at 90-100 cGy/min with single doses up to 6 Gy TBI. Some mice were prepared by a two-week 5-fluorouracil (5-FU) and kit-ligand (KL) protocol as described previously in our laboratory to lead to low levels of engraftment [2]. Tibiae and femora were removed from untreated donor mice and crushed to obtain a BMC suspension in Hank's balanced salt solution. The cell suspension was filtered through a metal sieve and the number of nucleated cells was determined with a hematocytometer. Cell suspensions were kept on ice until use and recipients were injected intravenously with 2 x 106, 107, or 5 x 107 nucleated cells in 0.2-0.5 ml within 3-6 h after the end of the irradiation. Each radiation dose group consisted of four to six mice. Recipients were bled at eight weeks, 26 weeks, and 52 weeks after BMT for Gpi-phenotyping [22] in erythrocytes or Ly5-phenotyping in WBC. All animals were sacrificed at 52 weeks for chimerism determination in blood and BM.

Chimerism Assay (Gpi-Phenotyping)
Erythrocytes were typed for Gpi-1 at different time intervals after transplantation. Twenty µl blood were collected from the tail tip and the percentage of A-type (Gpi-1a) was determined following electrophoresis on cellulose acetate strips as described previously [28]. At 52 weeks after BMT, all surviving mice were sacrificed and BMC suspensions were made to determine the level of donor chimerism in the BM as well as erythrocytes.

Chimerism Assay (Ly5-Phenotyping)
WBC were isolated from 200 µl whole blood collected from the orbital sinus. Subsequently, RBC were lysed by a 10-15 sec hypotonic shock. WBC were stained with fluorescein isothiocyanate-conjugated anti-CD45 (Ly5). Anti-CD45.1 (Ly5.1) was mixed with phycoerythrin (PE)-conjugated anti-CD4 and CD8; anti-CD45.2 (Ly5.2) with PE-conjugated anti-Gr-1. All antibodies were obtained from PharMingen (San Diego, CA; http://www.pharmingen.com). Samples were analyzed on a FACScan (Becton Dickinson; San Jose, CA; http://www.bd.com) flow cytometer. The percentage of donor engraftment was calculated from the two separate measurements (Ly5.1-positive and Ly5.2-negative) after subtraction of background (donor [B6.SJL] or host [B6]) staining.

Presensitization
Presensitization was performed by intravenous injection of 2 x 107 lethally irradiated (20 Gy) donor spleen cells at one week prior to TBI and BMT. A separate group of identically treated mice were sacrificed on day 0 and the frequency of precursors of proliferating T lymphocytes (pPTL) and precursors of cytotoxic T lymphocytes (pCTL) was determined.

Determination of Splenic pPTL and pCTL Frequencies
To measure immune reactivity the frequencies of pPTL and pCTL were determined. These assays utilize the ability of precusor lymphocytes to expand in culture, enabling the measurement of either proliferation or cytotoxicity of a single clone. Spleen cells from normal B6-mice (unsensitized) or mice presensitized against donor cells seven days earlier were used as a source of responders, and lethally irradiated (20 Gy) B6-Gpi-1a or B6.SJL spleen cells were used as a source of stimulators. Spleens from two or three mice were pooled. The tissue was minced, passed through a hypodermic needle (17G) and filtered through a metal sieve. Spleen cellularity was determined with a hematocytometer. Each well contained a fixed number of stimulator cells (5 x 105/well) and a limiting dilution of responder cells. Eight dilutions in 12 wells per dilution were used. The cells were cultured in RPMI (GIBCO; Gaithersburg, MD) with 5% heat-inactivated fetal bovine serum (GIBCO) and 5 x 10–5 M ß-mercaptoethanol. Six percent conditioned medium (supernatant of rat spleen cell cultures after phorbol-2,3-myrisate acetate and concanavalin A-stimulation as described in detail by Kast et al. [29] was added as a source of cytokines. After six days of culture (37°C, 5% CO2 and humidified atmosphere), 3H-thymidine (TdR) was added and the cells were cultured for another 16-20 h. DNA was collected on a filter using an automatic cell-harvester (TomTec; Hamden, CT). 3H-TdR uptake was determined on the filters from liquid scintillation counting. Precursor frequencies (pPTL) were determined as described [30] from the proportion of negative wells in each dilution. Wells are considered negative when they did not exceed the mean 3H-TdR-uptake + three standard deviations (SD) of the control wells (only stimulators, no responders). pPTL frequencies are expressed relative to unsensitized controls for comparison.

pCTL cultures were set up as described above. On day 7, 10,000 prelabeled target cells were added to measure the development of cytotoxic T-cell clones. Target cells were prepared by culturing donor cells (Ly5.1 or Gpi-1a congenic cells) for three days in the presence of 10 µg/ml lipopolysaccharide. Twenty hours before harvesting, 3H-TdR was added and cells were washed in culture medium and diluted before addition of 50 µl of cells to each well. Then the plates were incubated for 4-5 h at 37°C, 5% CO2 and humidified atmosphere before cells were harvested to determine 3H-TdR uptake from liquid scintillation counting. A well was considered negative when it exceeded the mean 3H-TdR-uptake - three SD of the control wells (only stimulators, no responders). pCTL numbers are expressed per 106 spleen cells for comparison with unsensitized controls.

Statistics
To test differences between treatment groups for statistical significance, p-values were calculated with the student's t-test assuming unequal variances of the two variables.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Long-Term Engraftment of Ly5 and Gpi-1-Congenic BM
Mice irradiated with TBI doses up to 6 Gy were transplanted with various doses of BMC. At 8 and 26 weeks post-BMT, engraftment of Ly5-congenic marrow was significantly lower than Gpi-1-congenic marrow (data not shown), and the level of donor chimerism remained stable until 52 weeks. Figure 1Go shows engraftment in BM at 52 weeks after transplantation of 107 cells and Figure 2Go after 2 x 106 cells. Transplantation of 1 x 107 Ly5-congenic cells leads to significantly (p < 0.05) lower engraftment levels than 1 x 107 Gpi-1-congenic marrow cells when transplanted following 0 or 4 Gy (Fig 1Go). After 2 Gy or 4 Gy TBI and 2 x 106 transplanted BMC, engraftment of Ly5-congenic marrow was significantly lower than engraftment of Gpi-1-congenic marrow (Fig 2Go). When TBI doses of 6 Gy were used, comparable levels of engraftment of both BM sources were found when 2 x 106 or 107 cells were transplanted, suggesting that immune suppression was sufficient to allow equal growth of Ly5- and Gpi-1-congenic marrow. Figure 3Go shows engraftment of 107 or 5 x 107 cells without conditioning and with the nonimmunosuppressive two-week 5-FU/KL conditioning protocol. The difference in engraftment at 52 weeks between Ly5- and Gpi-1-congenic marrow is apparent with both stem cell doses with and without conditioning. Only when high numbers of BMC (5 x 107 cells) were transplanted following 5-FU/KL did we find significant engraftment with Ly5 BM; in all other groups Ly5 engraftment was significantly lower than Gpi-1 engraftment (p < 0.05). Table 1Go compares engraftment at 52 weeks in peripheral blood (PB) and BM and shows that the differences in engraftment between Ly5- and Gpi-1-congenic marrow are found in various hematopoietic tissues. Only minor differences were found between engraftment in PB cells and in BM. More extensive analysis of Ly5 engraftment in defined subsets revealed that engraftment of T cells and granulocytes in the BM was usually lower than in their counterparts found in the PB, but different blood cell types within each tissue showed similar engraftment (Table 2Go).



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  		Figure 1. Donor engraftment after transplantation of 107 Ly5- or Gpi-1-congenic BMC. The dependence of donor engraftment on TBI dose is shown ± standard error of the mean for Ly5- and Gpi-1-congenic donor BM for four to five mice per group. Ly5 chimerism was measured in BMC at 52 weeks after transplantation using flow cytometry of unseparated nucleated cells. Gpi-1 chimerism was determined at 52 weeks after transplantation in BMC lysates. For details of chimerism determination see Materials and Methods. *Significantly lower Ly5 engraftment than Gpi-1 engraftment (p < 0.05).

 


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  		Figure 2. Donor engraftment after transplantation of 2 x 106 Ly5- or Gpi-1-congenic BMC. As in Figure 1Go but after transplantation of 2 x 106 donor BMC in four to five mice per group. *Significantly lower Ly5 engraftment than Gpi-1 engraftment (p < 0.05).

 


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  		Figure 3. Donor engraftment in nonimmunosuppressed recipients after transplantation of 107 or 5 x 107 Ly5- or Gpi-1-congenic BMC. The level of donor engraftment ± standard error for Ly5- and Gpi-1-congenic donor BM for four to five mice per group is shown following no conditioning or the nonimmune suppressive 5-FU/KL conditioning protocol. Ly5 chimerism was measured at 52 weeks after transplantation in BMC using flow cytometry of unseparated nucleated cells (shown as open bars). Gpi-1 chimerism was determined at 52 weeks after transplantation in BMC lysates (shown as solid bars). *Significantly lower Ly5 engraftment than Gpi-1 engraftment (p < 0.05).

 

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  		Table 1. Engraftment at 52 weeks in PB and BM; comparison between Ly5- and Gpi-1-congenic BM
 

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  		Table 2. Ly5-engraftment at 52 weeks in PB and BMC, T cells and granulocytes
 
Long-Term Engraftment in Presensitized and Unsensitized Recipients
To test whether the difference in engraftment between Ly5- and Gpi-1-congenic BM can be enhanced by presensitization (preimmunization) with donor cells, we transplanted 107 cells into 2 and 4 Gy-irradiated recipients. When T cells are involved in the rejection of Ly5-congenic cells, presensitization should increase the likelihood of rejection. Figure 4Go shows that following 2 Gy, no effect of presensitization was found. The levels of donor chimerism were too low to show an effect of presensitization (5.8% ± 2.5 in unsensitized mice versus 2.8% ± 1.7 in presensitized mice; p = 0.33). There was a significant difference in engraftment between Ly5 and Gpi-1 marrow as previously shown (p < 0.001). Following 4 Gy TBI, mice that were presensitized against Ly5 showed significantly lower levels of engraftment than unsensitized recipients (p = 0.02). This difference was found in various hematopoietic lineages (data not shown). These results suggest the involvement of T cells in the immunological resistance against Ly5-congenic stem cells.



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  		Figure 4. Donor engraftment in presensitized recipients after transplantation of 107 Ly5- or Gpi-1-congenic BMC. The effect of presensitization (at one week before TBI and BMT) with donor cells on donor engraftment is shown ± standard error for Ly5- and Gpi-1-congenic donor BM (6-12 mice per group). Recipients were given 2 Gy (left panel) or 4 Gy (right panel) TBI prior to transplantation. Ly5 chimerism was measured at 26 weeks after transplantation in BMC using flow cytometry of unseparated nucleated cells (shown as open bars). Gpi-1 chimerism was determined at 26 weeks after transplantation in BMC lysates (shown as solid bars).

 
pPTL and pCTL Frequencies in Presensitized and Unsensitized Recipients
In vitro determination of pPTL frequencies demonstrated no increase in reactivity against Gpi-1 upon presensitization, whereas reactivity against Ly5 increased 2.9-fold, but this did not reach statistical significance (Table 3Go). To exclude the influence of hematopoietic proliferation on splenic pPTL frequency measurements, we also studied pCTL frequencies in presensitized and unsensitized mice. The baseline pCTL frequency was fourfold higher against Ly5-congenic cells than Gpi-1-congenic cells, but the relative increase in frequencies upon presensitization was the same for Gpi-1- and Ly5-congenic cells (Table 4Go). These results suggest that T cell reactivity against Ly5-congenic cells is low, but can be slightly increased by preimmunizing the recipients one week prior to transplantation.


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  		Table 3. Measurements of pPTL frequencies against Ly5- and Gpi-1-congenic cells in presensitized and unsensitized mice
 

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  		Table 4. Measurements of pCTL frequencies against Ly5- and Gpi-1 congenic cells in presensitized and unsensitized mice
 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Data presented in this paper suggest that transplantation of Ly5-congenic marrow leads to lower levels of engraftment than Gpi-1-congenic marrow; this is probably caused by immune-mediated processes. The data demonstrate that after low-dose TBI and after low doses of BMC, engraftment was significantly lower in mice transplanted with Ly5-congenic marrow than in mice given Gpi-1-congenic marrow. When TBI doses of 6 Gy were used, comparable levels of engraftment of both marrow sources were found to suggest no significant residual antidonor immunity to cause a difference in engraftment. No host treatment or preparation with a two-week course of 5-FU and KL resulted in the largest differences because these protocols are associated with the least immune suppression. The use of antigenic differences between donor and host has allowed rapid flow cytometric determination of donor chimerism. Our results suggest that following minimal conditioning such as with low-dose TBI or 5-FU and KL, cell surface antigenic differences may influence engraftment. Biochemical markers, such as Gpi-1, that do not result in expression of cell-surface antigens, appear to allow engraftment without immunological resistance even with minimal conditioning.

The specific immunological processes resulting in a decrease in engraftment of Ly5-congenic BM remain uncertain. We tested in vitro immune reactivity but the assays were not sensitive enough to detect significant differences in reactivity against Gpi-1- or Ly5-congenic spleen cells. Also, presensitized recipients were tested for T cell involvement in the rejection of congenic marrow. We found a modest decrease in Ly5 engraftment when presensitized recipients were compared with unsensitized recipients irradiated with 4 Gy prior to BMT. Following 2 Gy TBI, no effect of presensitization could be detected, probably because the level of engraftment was very low in both presensitized and unsensitized recipients. In a previous study, the same presensitization greatly enhanced rejection by the host of MHC-matched allogeneic BM [31], suggesting that a much greater genetic difference increases the effect of presensitization. In this study, in vitro determination of the frequency of pPTL reactive against MHC-compatible allogeneic cells was increased considerably upon presensitization [31] whereas in this study, immune reactivity against Ly5-congenic spleen cells showed only a small increase in pPTL and pCTL frequencies following presensitization. Although our results do not determine the exact mechanisms responsible for rejection of Ly5-disparate BMC, T cell-mediated immunity seems to be involved; other immune effector cells such as natural killer cells may be involved as well but were not tested. Recent studies have indicated that anti-Ly5-reactive T cells can be generated that are specific for the polymorphic region of the Ly5 molecule [32]. Ly5-congenic mice could be efficiently immunized using Freund's adjuvant and two immunizations, and T cells isolated from their spleens were grown for several passages in vitro to enrich for Ly5-reactive cells. These T cells were able to induce graft-versus-host disease when administered to mixed Ly5 chimeras leading predominantly to lung injury [32, 33]. In addition, presensitization against a single antigen may require multiple immunizations as was shown for the H-3 and H-4 antigen [34]. These studies show that in vivo generation of a T cell response against a single antigen such as Ly5 is possible, but in our experiments the frequency of reactive cells remained low after one or three immunizations.

Recently, there has been renewed interest in developing a conditioning regimen with minimal toxicity to the host that allows stable mixed chimerism. It has been shown that without conditioning, engraftment could be achieved when high numbers of BMC are transplanted. When very high BMC doses are used, up to 50% donor cells can be detected in unirradiated syngeneic recipients [26, 35-38]. However, the use of low-dose TBI (1 Gy) enables more efficient engraftment of equivalent numbers of BMC than no conditioning [39]. The relationship between radiation dose and BMC dose has previously been studied more extensively. In syngeneic BMT, a reduction in radiation dose can usually be compensated by an increase in the number of transplanted cells [van Os and Down, unpublished results], but in allogeneic BMT there is an additional requirement for immune suppression causing very steep radiation dose-response curves for allogeneic engraftment [27]. However, in an allogeneic setting, immune suppression may be achieved by thymic radiation or the administration of antibodies against immune effector cells or costimulatory molecules [40]. Thus, the method to determine donor chimerism should be evaluated on the possibility of immune reactivity between donor and host cells that may affect the results. In our previously published experiments [2], for instance, exploring an alternative conditioning regimen not including radiation (e.g., 5-FU in combination with KL), the conclusion would have been different when Ly5-congenic BM was used instead of Gpi-1-congenic marrow. Using Gpi-1-congenic marrow is not always feasible, especially when engraftment among various cell lineages needs to be monitored, but one should be aware of possible immunological barriers between other congenic strains. Therefore, extra immune supression should be applied to allow growth of donor cells when immune reactivity against donor cells is expected.

Engraftment following experimental BMT relies on the donor and host combination chosen, especially when minimal conditioning is applied. Development of mouse models to study the ability of a nonmyeloablative regimen to induce stable chimerism will benefit from dissecting between the effects on transplant immunity and the effects on host stem cells.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The authors wish to thank Dr. Julian Down (Biotransplant Inc.; Charlestown, MA) for helpful discussions and John Delmonte (Department of Pediatric Oncology, Dana Farber Cancer Institute; Boston, MA) for assistance with flow cytometry. This research was supported by the NIH RO1 Grant-CA 10941-28 and NIH P50 Grant-HL54785-01.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Harrison DE. Competitive repopulation: a new assay for long-term stem cell functional capacity. Blood 1980;55:77-81.[Abstract/Free Full Text]

  2. van Os R, Dawes D, Mislow JM et al. Host conditioning with 5-fluorouracil and kit-ligand to provide for long-term bone marrow engraftment. Blood 1997;89:2376-2383.[Abstract/Free Full Text]

  3. Ford CE, Hamerton JL, Barnes DWH et al. Cytological identification of radiation chimeras. Nature 1956;177:452-454.[CrossRef][Medline]

  4. van der Sluijs J, van den Bos C, Baert M et al. Loss of long-term repopulating ability in long-term bone marrow culture. Leukemia 1993;7:725-732.[Medline]

  5. Ploemacher RE, van der Loo JCM, Van Beurden CAJ et al. Wheat germ agglutinin affinity of murine hemopoietic stem cell subpopulations is an inverse function of their long-term repopulating ability in vitro and in vivo. Leukemia 1993;7:120-130.[Medline]

  6. Hampson IN, Spooncer E, Dexter TM. Evaluation of a mouse Y chromosome probe for assessing marrow transplantation. Exp Hematol 1989;17:313-315.[Medline]

  7. Simpson E. The role of H-Y as a minor transplantation antigen. Immunol Today 1982;3:97-106.

  8. Soderling CC, Song CW, Blazar BR et al. A correlation between conditioning and engraftment in recipients of MHC- mismatched T cell-depleted murine bone marrow transplants. J Immunol 1985;135:941-946.[Abstract]

  9. Blazar BR, Widmer MB, Soderling CC et al. Enhanced survival but reduced engraftment in murine recipients of recombinant granulocyte/macrophage colony-stimulating factor following transplantation of T-cell-depleted histoincompatible bone marrow. Blood 1988;72:1148-1154.[Abstract/Free Full Text]

  10. Sykes M, Chester CH, Sundt TM et al. Effects of T cell depletion in radiation bone marrow chimeras. III. Characterization of allogeneic bone marrow cell populations that increase allogeneic chimerism independently of graft-vs-host disease in mixed marrow recipients. J Immunol 1989;143:3503-3511.[Abstract]

  11. Lapidot T, Singer T, Reisner Y. Transient engraftment of T cell depleted allogeneic bone marrow in mice improves survival rate following lethal irradiation. Bone Marrow Transplant 1988;3:157-164.[Medline]

  12. Lapidot T, Terenzi A, Singer T et al. Enhancement by dimethyl myleran of donor type chimerism in murine recipients of bone marrow allografts. Blood 1989;73:2025 -2032.[Abstract/Free Full Text]

  13. Salomon O, Lapidot T, Terenzi A et al. Induction of donor-type chimerism in murine recipients of bone marrow allografts by different radiation regimens currently used in treatment of leukemia patients. Blood 1990;76:1872-1888.[Abstract/Free Full Text]

  14. Spangrude GJ, Heimfeld S, Weissman IL. Purification and characterization of mouse hematopoietic stem cells. Science 1988;241:58-62.[Abstract/Free Full Text]

  15. Li CL, Johnson GR. Rhodamine123 reveals heterogeneity within murine Lin–, Sca-1+ hemopoietic stem cells. J Exp Med 1992;175:1443-1447.[Abstract/Free Full Text]

  16. Spangrude GJ, Johnson GR. Resting and activated subsets of mouse multipotent hematopoietic stem cells. Proc Natl Acad Sci USA 1990;87:7433-7437.[Abstract/Free Full Text]

  17. Ferrara JLM, Lipton J, Hellman S et al. Engraftment following T-cell-depleted bone marrow transplantation. I. The role of major and minor histocompatibility barriers. Transplantation 1987;43:461-467.[Medline]

  18. Mauch P, Down J, Warhol M et al. Recipient preparation for bone marrow transplantation. I. Efficacy of total-body irradiation and busulfan. Transplantation 1988;46:205-209.[Medline]

  19. Down J, Tarbell N, Thames H et al. Syngeneic and allogeneic bone marrow engraftment after total body irradiation: dependence on dose, dose-rate, and fractionation. Blood 1991;77:661-669.[Abstract/Free Full Text]

  20. Ferrara JLM, Mauch P, McIntyre J et al. Engraftment following T-cell-depleted bone marrow transplantation. II. Stability of mixed chimerism in semiallogeneic recipients after total-body irradiation. Transplantation 1987;44:495-499.[Medline]

  21. Brecher G, Neben S, Yee M et al. Pluripotent stem cells with normal or reduced self renewal survive lethal irradiation. Exp Hematol 1988;16:627-630.[Medline]

  22. Ansell J, Micklem H. Genetic markers for following cell populations. In: Weir DM, Herzenberg LA, Blackwell CC, eds. Handbook of Experimental Immunology. Blackwell Publishers, Edinburgh. 4th Edition. 1986;4:56.1-56.18

  23. Barker JE, Braun J, McFarland-Starr E. Erythrocyte replacement precedes leukocyte replacement during repopulation of W/Wv mice with limiting dilutions of +/+ donor marrow cells. Proc Natl Acad Sci USA 1988;85:7332-7335.[Abstract/Free Full Text]

  24. Harrison D, Astle C, Lerner C. Number and continuous proliferative pattern of transplanted primitive immunohematopoietic stem cells. Proc Natl Acad Sci USA 1988;85:822-826.[Abstract/Free Full Text]

  25. Francescutti LH, Gambel P, Wegmann TG. Characterization of hemopoietic stem cell chimerism in antibody-facilitated bone marrow chimeras. Transplantation 1985;40:7-11.[Medline]

  26. Voralia M, Semeluk A, Wegmann T. Facilitaton of syngeneic stem cell engraftment by anti-class monoclonal antibody pre-treatment of unirradiated recipients. Transplantation 1987;44:487-494.[Medline]

  27. van Os R, Konings AW, Down JD. Radiation dose as a factor in host preparation for bone marrow transplantation across different genetic barriers. Int J Radiat Biol 1992;61:501-510.[Medline]

  28. Ploemacher RE, van Os RP, Van Beurden CAJ et al. Murine hematopoietic stem cells with long-term engraftment and marrow repopulating ability are less radiosensitive to gamma radiation than are spleen colony forming cells. Int J Radiat Biol 1992;61:489-499.[Medline]

  29. Kast WM, de Waal LP, Melief CJ. Thymus dictates major histocompatibility complex (MHC) specificity and immune response gene phenotype of class II MHC-restricted T cells but not of class I MHC-restricted T cells. J Exp Med 1984;160:1752-1766.[Abstract/Free Full Text]

  30. Fazekas de St. Groth S. The evaluation of limiting dilution assays. J Immunol Methods 1982;49:R11-R23.[CrossRef][Medline]

  31. van Os R, de Witte T, Dillingh JH et al. Increased rejection of murine allogeneic bone marrow in presensitized recipients. Leukemia 1997;11:1045-1048.[CrossRef][Medline]

  32. Chen W, Chatta GS, Rubin WD et al. T cells specific for a polymorphic segment of CD45 induce graft-versus-host disease with predominant pulmonary vasculitis. J Immunol 1998;161:909-918.[Abstract/Free Full Text]

  33. Clark JG, Madtes DK, Hackman RC et al. Lung injury induced by alloreactive Th1 cells is characterized by host-derived mononuclear cell inflammation and activation of alveolar macrophages. J Immunol 1998;161:1913-1920.[Abstract/Free Full Text]

  34. Blazar BR, Roopenian DC, Taylor PA et al. Lack of GVHD across classical, single minor histocompatibility (miH) locus barriers in mice. Transplantation 1996;61:619-624.[CrossRef][Medline]

  35. Brecher G, Ansell J, Micklem H et al. Special proliferative sites are not needed for seeding and proliferation of transfused bone marrow cells in normal syngeneic mice. Proc Natl Acad Sci USA 1982;79:5085-5087.[Abstract/Free Full Text]

  36. Saxe DF, Boggs SS, Boggs DR. Transplantation of chromosomally marked syngeneic marrow cells into mice not subjected to hematopoietic stem cell depletion. Exp Hematol 1984;12:277-283.[Medline]

  37. Wu DD, Keating A. Hematopoietic stem cells engraft in untreated transplant recipients. Exp Hematol 1993;21:251-256.[Medline]

  38. Stewart FM, Crittenden RB, Lowry P et al. Long-term engraftment of normal and post-5-fluorouracil murine marrow into normal nonmyeloablated mice. Blood 1993;81:2566-2571.[Abstract/Free Full Text]

  39. Stewart FM, Zhong S, Wuu J et al. Lymphohematopoietic engraftment in minimally myeloablated hosts. Blood 1998;91:3681-3687.[Abstract/Free Full Text]

  40. Wekerle T, Sachs DH, Sykes M. Mixed chimerism for the induction of tolerance: potential applicability in clinical composite tissue grafting. Transplant Proc 1998;30:2708-2710.[CrossRef][Medline]

Received November 1, 2000; accepted for publication November 3, 2000.




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