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Ann Thorac Surg 2001;72:740-745
© 2001 The Society of Thoracic Surgeons
rat) bone marrow chimeras
a Department of Cardiovascular-Thoracic Surgery, Rush Presbyterian St. Lukes Medical Center, Chicago, Illinois, USA
Accepted for publication May 1, 2001.
Address reprint requests to Dr Mohiuddin, Department of Cardiovascular-Thoracic Surgery, Rush Presbyterian St. Lukes Medical Center, 1653 W Congress Pkway, Chicago, IL 60612
e-mail: mmohiudd{at}rush.edu
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Abstract |
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Methods. Recipient Lewis rats (n = 22) were given 1100 cGy lethal total body irradiation and the same day received 300 x 106 donor B10.BR mouse bone marrow cells intravenously. Peripheral blood of surviving rats (n = 18) was typed at 4 weeks and then monthly thereafter. Donor and recipient specific and third-party heterotopic heart transplantations were performed at 6 to 8 weeks after reconstitution with bone marrow.
Results. Multilineage bone marrow chimerism was produced in all experimental animals with complete replacement of recipient marrow by donor cells. Murine donor and rat recipient strain hearts transplanted in chimeric rats survived indefinitely. Third-party rat and mouse hearts were rejected, though at a slower rate than bone marrow matched naïve controls. High levels of antimouse antibodies were detected in rats with rejected hearts. These antibodies were absent in chimeric animals with long-term surviving heart grafts.
Conclusions. Long-term multilineage bone marrow chimerism can be produced in a mouse 
rat bone marrow transplant model. Long-term survival of donor specific and recipient specific vascularized cardiac grafts can be produced in these chimeric animals. These animals are clinically normal but show signs of subclinical immunosuppression regimen as they reject third-party hearts later than naïve animals. Our results suggest that antibodies also play a significant role in concordant xenograft rejection, and induction of bone marrow chimerism can overcome this barrier.
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Introduction |
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A nonhuman biological source of hearts for transplantation might solve this problem. This possibility has stimulated interest in using organs from animal species for human transplantation. Most investigators have focused on the pig as a likely donor because of its wide potential availability and the likelihood that the economic and ethical issues surrounding its use would be tractable [2–5]. The vigorous rejection response in the pig-to-human species combination [6], however, remains a major problem because it is not controlled by currently available immunosuppressive agents [7, 8].
Both our group and others are attempting to address some of the immunologic obstacles in a rodent model by producing stable xenogeneic bone marrow chimerism as a preparatory regimen to induce tolerance to cardiac xenografts [9–11]. We have previously shown that stable, complete, xenogeneic multilineage, hematopoietic chimerism with complete replacement by donor bone marrow can be induced in a mouse-to-rat model by lethally irradiating the recipient Lewis rats with 1,100 cGy and rescuing with 300 x 106 bone marrow cells from B10.BR mice [9, 12]. The technique of inducing bone marrow chimerism by lethal irradiation is not new. However, our mouse-to-rat model has only been reported once before. Because of the availability of a variety of reagents to study murine cells, this model is advantageous to the study of murine cell development in detail in the rat. In this study we examined the survival of cardiac xenografts in these complete xenogeneic mouse rat bone marrow chimeras to specifically determine if the xenograft tolerance produced by bone marrow chimerism is donor specific or in a generalized immunosuppressed state. We have assessed cardiac xenograft tolerance by examining length of graft survival and also by analysis of an in vitro immune response. Finally, we have studied the role of antibodies in the rejection of concordant cardiac xenografts in this model.
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Material and methods |
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Total body irradiation
Recipient rats were conditioned with a single dose of 1,100 cGy total body irradiation (Nordion Gamma Cell 40, Ontario, Canada) 4 to 6 hours before marrow infusion.
Bone marrow transplantation
Bone marrow was extracted from B10.BR mice and a cell suspension was made as previously described [13]. Briefly, B10.BR mice were euthanized by CO2 inhalation and the long bones (tibia, femur, and humerus) were sterilely harvested. Bone marrow was flushed with Media 199 (Life Technologies, Grand Island, NY) containing 0.2 µL/mL gentamicin (Life Technologies, Grand Island, NY) using a 21-guage needle, and it was filtered through sterile nylon mesh (Tetco, Kansas City, MO). Cells were washed, resuspended, and counted. Cells were diluted in Media 199 to allow the injection of the desired cell number in a 2 mL volume per animal. Bone marrow containing 300 x 106 cells was injected intravenously into each rat 4 to 6 hours after total body irradiation.
Monoclonal antibodies
Antirat RT1Aabl-FITC (B5), antimouse H2Kk-FITC or PE (36–7–5), were purchased from Pharmingen (San Diego, CA).
Characterization of chimeras by flow cytometry
Thirty days after reconstitution with donor marrow peripheral blood lymphocytes of surviving rats were typed using one- or two-color flow cytometry (FACS Calibur, Becton-Dickinson, Mountain View, CA) to determine the relative percentage of peripheral blood lymphocytes of host (RT1A) and donor (H2Kk) origin as described previously [13]. Briefly, peripheral blood from the tail vein was collected into heparinized vials and aliquots of 100 µL were incubated with FITC-labeled antirat-RT1A antibody or PE/FITC-labeled antimouse H2Kk antibody, or both, for 30 minutes at 4°C in the dark. After washing with FACS media (prepared in the laboratory), red blood cells were lysed using ammonium chloride lysing buffer (ACK; prepared in the laboratory), and washed again with FACS media. Cells were fixed in 0.3 mL 2% paraformaldehyde (Tolousimis Research Corp, Rockville, MD) and analyzed using FACS Calibur. Rats were typed monthly until sacrificed at 4 to 6 months.
Measurement of antibody levels and antibody-induced cytotoxic assays
Sera from naïve rats or chimeric rats with or without cardiac grafts were collected. The levels of rat antimouse IgG and IgM in naïve rat or chimeric rat sera were determined by incubating B10.BR mouse bone marrow cells with rat sera, staining with fluorescein-conjugated mouse antirat isotypic-specific secondary antibodies (Pharmingen, San Diego, CA), and analyzing them by using flow cytometry. The complement-dependent cytotoxicity of xeno-reactive antibodies in rats against mouse bone marrow cells was measured by a flow cytometry-based assay using propidium iodide (Sigma, St. Louis, MO) as a marker of cell death.
Heart transplantation
Heterotopic heart transplantation was performed to the inguinal vessels of recipient rats using a technique modified from that described by Ono and Lindsey [14]. Transplantation of the first heart was always performed by end-to-side anastomosis of the donor aorta and pulmonary artery to the right femoral artery and vein, respectively. The second heart graft was either transplanted in the left inguinal region or in the abdomen to the aorta and inferior vena cava. Heart survival was monitored both visually and by palpation, and rejection was confirmed by histology.
Determination proliferation response in mixed lymphocyte reaction
Lymphocytes from chimeric and naïve mice were used as responders. Irradiated lymphocytes from naïve B10.BR mice, naïve Lewis rats, and third party C57BL/6 mice were used as stimulators. In a 96 well plate, 4 x105 stimulator and responder cells were incubated together for 4 days. Tritiated thymidine was added on day 4, the cells were harvested and counts per minutes were determined on a liquid scintillation counter (Beckman Coulter, Schaumburg, IL) after 16 hours of further incubation.
Determination of graft versus host disease
Animals were observed for changes in gross appearance such as wasting, diarrhea, and hair loss. Histologic evidence of graft versus host disease (GVHD), (which includes mononuclear cell infiltration with cells showing shrinkage, nuclear fragmentation, and eosinophilic cytoplasmic degeneration and necrosis), was sought on examination of postmortem tissues.
Statistical analysis
The survival of hearts was analyzed using the Kaplan-Meier method.
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Results |
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Peripheral blood lymphocytes typing of chimeras
Rats surviving 4 weeks (n = 18) were typed for donor chimerism using antirat RT1A and antimouse H2Kk antibodies (Fig 1). All surviving rats showed murine marrow engraftment with more than 99% expression of donor B10.BR mouse cells. Thereafter, these rats were typed monthly using antimouse and antirat Class I monoclonal antibodies until sacrifice at 4 to 6 months. All rats maintained greater than 99% donor chimerism for the length of survival.
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Comment |
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TopAbstractIntroductionMaterial and methodsResultsCommentReferences |
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rat model and demonstrated development of functional mouse T cells in the rat environment without the concomitant presence of rat hematopoietic cells. All chimeric rats engrafted with more than 99% of donor cells and the level of engraftment was stable until the animals were sacrificed 6 months after marrow transplantation. In our earlier studies we have shown that all mouse hematopoietic cell lineages develop normally in the chimeric rat [9].
Our mouse-to-rat model is unique to the study of donor bone marrow elements in detail because multiple murine reagents are available. Furthermore, bone marrow cells from commercially available knockout and transgenic mice can be used to study other details of xeno-chimerism. On the other hand, the large number of mice (4 to 5 mice per rat) required to generate enough marrow cells to produce stable chimerism in one animal makes this model relatively expensive. Heart transplantation is technically easier to perform in this model, and viability of the heart can be monitored visually when it is transplanted in the inguinal region. This model does not mimic a clinically relevant human situation because of the presence of 
-Gal epitopes on both recipient and donor cells and absence of anti--Gal antibodies. However, this model is relevant since once the -Gal barrier is removed, cellular rejection will play an important role in xenograft rejection. Also, in recent experiments, we have observed that bone marrow chimerism can produce B-cell tolerance to -Gal (unpublished data). Finally, this model provides an alternative to the widely used rat-to-mouse model that our results complement.
We observed no evidence of GVHD in chimeric animals. Other investigators who have reported GVHD have either used considerably higher numbers of bone marrow cells or a different immunosuppressive regimen [17]. We have seen GVHD when a marrow dose of more than 400 x 106 was used. It is our understanding that the incidence of GVHD is directly related to the numbers of mature T cells in the bone marrow inoculum, and our presumption is that the T cell number increases with increasing numbers of marrow cells.
In these experiments we have shown that long-term cardiac xenograft survival can be achieved in chimeric rats. All donor-specific mouse or rat recipient identical cardiac grafts survived indefinitely in mouse-rat chimeras. Indefinite survival of recipient specific Lewis heart grafts was also expected because the T cells developing in the rat environment should recognize both the recipient Lewis rat and B10.BR mouse tissues as self. Chimeric rats rejected third-party mouse and rat hearts, although in some instances at a delayed rate. Two rats failed to reject either third-party mouse or rat hearts. These findings suggest that some chimeric rats were not immune-competent, although they were free of clinical disease.
Our data also showed that lymphocytes from chimeric rats were hyporesponsive to both donor B10.BR mouse and recipient Lewis rat’s irradiated lymphocytes in vitro in a mixed lymphocyte reaction. A normal proliferative response was seen to be irradiated lymphocytes from third-party mouse or rat strains. In mixed lymphocyte reaction, we observed a difference in response between B10.BR and B6 mice. In our experience we always see a higher response with B10.BR mice than B6 mice. We do not have data to explain this difference. The response of irradiated B6 plus chimera lymphocytes was higher than the response of irradiated B6 plus naïve Lewis because the latter response is from Lewis rat lymphocytes to B6 cells, whereas the former is from the B10.BR lymphocytes that reconstitute the Lewis rat chimera.
Although the chimeric mice were significantly immunosuppressed, these results show that they maintained a normal response to lymphocytes from third-party mice. Also, all third-party heart grafts were ultimately rejected. Therefore, the long-term survival of donor- and recipient-specific cardiac grafts cannot be attributed to immunosuppression regimen alone. Previously, we have shown that mouse lymphocytes migrate to the rat thymus where they pass through normal developmental stages, including deletion of self-reactive T cells. During this process rats maintain the ability to react to foreign antigens, including third-party antigens. It is reasonable to conclude that the T cells reactive to third-party lymphocytes are not deleted because they are not exposed to these antigens during development, and this plays a significant role in rejecting third-party grafts.
Previously it has been shown that preformed antibodies are responsible for the resistance to bone marrow engraftment in a rat mouse xenograft model [18]. In these experiments, we have shown that xeno-reactive antibody levels are strongly associated with xenograft rejection in a mouse rat concordant heart transplant model and that these antibodies are cytotoxic to donor cells. Our data suggest that xenogeneic chimerism also induces humoral tolerance to xenografts because these antibody levels were low in chimeric rats, even after exposure to donor heart grafts.
In summary, we have produced xenogeneic mouse-to-rat complete bone marrow chimeras that exhibit durable engraftment and excellent survival. Our studies provide evidence that long-term cardiac xenograft survival can be achieved in chimeric animals that are apparently tolerant to organs from isogeneic donors to the marrow donors. Furthermore, the bone marrow grafts are tolerant of the recipient because GVHD was not seen. Although some chimeric animals are without clinical disease or other manifestations of immune suppression, they may not be fully immune-competent as illustrated by delayed rejection of third-party donor heart grafts. However, this was observed in a minority of chimeras.
Although the regimen of lethal irradiation and complete bone marrow replacement successfully demonstrates production of long-term survival of vascularized organ xenografts, it is not likely to be feasible in a clinical situation. Lethal total body irradiation is indicated for treatment of certain life-threatening malignancies. Its numerous toxicities preclude its use in other conditions. We are now attempting to modify our model to produce mixed xenogeneic chimerism without the requirement for lethal irradiation or long-term immunosuppressive agents. Such a regimen might be appropriate for use in patients with end-stage cardiac disease, and it may solve the problem of donor organ shortage and eliminate the complications presently associated with long-term nonspecific immunosuppression regimen.
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References |
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TopAbstractIntroductionMaterial and methodsResultsCommentReferences |
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rat) bone marrow chimeras: evidence for normal development and clonal deletion of mouse T cells. Transplantation 2000;69:731-736.[Medline]
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  Y. L. Colson, H. Xu, Y. Huang, and S. T. Ildstad Mixed Xenogeneic Chimerism Induces Donor-Specific Humoral and Cellular Immune Tolerance for Cardiac Xenografts J. Immunol., November 1, 2004; 173(9): 5827 - 5834. [Abstract] [Full Text] [PDF]
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