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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
Correspondence:
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.
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ABSTRACT |
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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.
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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.
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RESULTS |
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shows engraftment in BM at 52 weeks after transplantation of 107 cells and Figure 2 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 1). 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 2). 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 3 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 1 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 2).
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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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). 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 4). 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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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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ACKNOWLEDGMENTS |
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REFERENCES |
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