Cross-species transplantation: NK cell number and function are normal in fully xenogeneic chimeras (rat----mouse).

When untreated F344 rat bone marrow is transplanted into B10 mouse recipients conditioned with total body irradiation, stable fully xenogeneic chimerism (rat----mouse) results. Chimeras are specifically tolerant to the donor strain of rat, survival is excellent (greater than 80% at 8 months), and all stem-cell-derived lineages are produced by the rat stem cell. We have previously demonstrated normal function of T-lymphocytes in these chimeras, but have not examined the immune function of natural killer (NK) cells present. Because NK cells play a critical role in immune surveillance, absence of function could result in a serious immunodeficiency state. We present data here to suggest that rat NK cells that have developed in a mouse stromal environment are normal in function as well as number. In all fully xenogeneic chimeras tested from 8 weeks to 8 months following bone marrow transplantation, NK cells were present at a normal level (10% to 16%). NK cells function in these chimeras, as tested by spontaneous lysis of YAC tumor cell targets, was normal or superior to normal F344 rat and B10 mouse NK cells.     

Long-term survival of cardiac xenografts in fully xenogeneic (mouse --> rat) bone marrow chimeras

BACKGROUND: The shortage of human hearts remains a major barrier to the efficacy of heart transplantation for the treatment of end-stage heart disease. One potential solution to the supply problem would be the use of hearts from nonhuman donors (xenografts). We have established a model of mouse to rat xenogeneic bone marrow chimerism, and in this study we have hypothesized that such chimeric rats will accept both donor and recipient specific heart grafts while rejecting third-party mouse and rat grafts. We also investigated humoral responses in naive and chimeric rats with and without donor murine cardiac grafts. METHODS: Recipient Lewis rats (n = 22) were given 1100 cGy lethal total body irradiation and the same day received 300 x 10(6) 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 naive 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 naive 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.     

Kinetics of early T-cell repopulation in fully xenogeneic chimeras (F344 rat----B10 mouse): evidence for rat T-cell maturation in a xenogeneic mouse thymus.

We recently reported the model of fully xenogeneic chimerism achieved by transplantation of rat bone marrow into mouse recipients (F344 rat----B10 mouse), resulting in stable long-term rat lymphoid chimerism. We have now extended this model to examine whether developing precursor rat T cells from rat bone marrow stem cells can undergo normal differentiation in mature lymphocytes under the influence of a xenogeneic mouse thymus. We examined thymic and splenic lymphoid cells from fully xenogeneic chimeras starting 1 week after bone marrow transplantation to characterize early T-cell repopulation and phenotype. Our data suggest that developing rat precursor T cells are able to undergo normal differentiation in the mouse thymus. The first precursor T cells appeared 2 weeks after reconstitution and by week 10 accounted for more than 90% of thymocytes present in the chimeras. In chimeras, developing rat T lymphocytes in the mouse thymus exhibited an immature pattern (Thy 1.1+, alpha beta-TCRdull, CD4+ plus CD8+) when analyzed by flow cytometry. This pattern was similar to a normal rat. In contrast, splenic T-lymphoid cells showed a mature rat phenotype (Thy 1.1-, alpha beta-TCRhi, CD4+ or CD8+), again similar to a normal rat. This development began 2 weeks after bone marrow transplantation, and both thymus and spleen from chimeras exhibited "normal" rat T-cell staining profiles by 10 weeks after reconstitution. Overall, these data indicate that developing rat T cells are capable of undergoing normal maturation in a xenogeneic mouse thymus of tolerant animals.     

Long-term survival of donor-specific pancreatic islet xenografts in fully xenogeneic chimeras (WF rat----B10 mouse).

We recently reported that reconstitution of lethally irradiated B10 mouse recipients with 40 x 10(6) untreated WF rat bone marrow cells resulted in stable fully xenogeneic chimerism (WF rat----B10 mouse). In these animals, the tolerance induced for skin xenografts was highly MHC specific in that donor-specific WF rat skin grafts were significantly prolonged while MHC-disparate third-party xenografts were rapidly rejected (median survival time [MST] = 9 days). We have now examined whether islet cell xenografts placed under the renal capsule of chimeras rendered diabetic with streptozotocin would be accepted and remain functional to maintain euglycemia. Animals were prepared, typed for chimerism at 6 weeks, and diabetes induced with streptozotocin. Donor-specific WF (Rt1Au) islet cell xenografts were significantly prolonged (MST greater than 180 days) in WF----B10 chimeras, while MHC-disparate third-party F344 rat (Rt1A1) grafts were rejected with a time course similar to unmanipulated B10 mice (MST = 8 days). The transplanted donor-specific islet cells were functional to maintain euglycemia, since removal of the grafts at from 100 to 180 days in selected individual chimeras uniformly resulted in return of the diabetic state. These data suggest that donor-specific islet cell xenografts are accepted and remain functional in mice rendered tolerant to rat xenoantigens following bone marrow transplantation.     

Establishment of fully xenogeneic (mouse-->rat) bone marrow chimeras: evidence for normal development and clonal deletion of mouse T cells

BACKGROUND: Xenotransplantation is a potential solution to the critical shortage of transplantable organs. However, conventional immunosuppressive agents do not control the vigorous cellular and humoral rejection across species disparities. The induction of donor specific tolerance via bone marrow chimerism may be a method to avoid xenograft rejection. In xenogeneic chimeras, T cell repertoire selection plays an important role in the induction of tolerance. Until now a model of mouse-->rat multilineage chimerism has not been reported. This study reports the establishment of fully xenogeneic mouse-->rat multilineage chimeras and evaluates the role of T cell development and repertoire selection in tolerance induction in a xenogeneic environment. METHODS: Recipient rats were irradiated at a dose of total body irradiation ranging between 800-1100 cGy and injected with 120-300x10(6) donor mouse bone marrow cells. Chimeras were typed for engraftment at 4 weeks and then monthly thereafter. T cell repertoire was evaluated in chimeras using two-color flow cytometry and monoclonal antibodies directed against the variable portion of the beta chain of the T cell receptor. RESULTS: Fully xenogeneic multilineage bone marrow chimerism was produced in a mouse-->rat model by using ablative radiation and a high dose of donor cells. Mouse T cells develop in a phenotypically normal fashion in chimeric rats and the host rat is capable of deleting T cells that are reactive to the donor mouse strain. CONCLUSION: Long-term multilineage bone marrow chimerism can be produced in a mouse-->rat bone marrow transplant model. Mouse T cells develop in a phenotypically normal fashion and negative selection of specific T cell receptor-Vbeta occurs in a xenogeneic environment in a predictable fashion paralleling that for syngeneic or allogeneic transplantation.     

Cross-species bone marrow transplantation: evidence for recognition of skin-specific antigens across a species barrier (rat----mouse).

We have developed a model to study cross-species bone marrow transplantation and the associated donor-specific transplantation tolerance induced using fully xenogeneic chimeras. Reconstitution of lethally irradiated B10 mice with untreated F344 rat bone marrow cells results in fully xenogeneic chimerism (F344 rat----B10 mouse). Survival of recipients is excellent (greater than 80% at 100 days) and stable rat lymphoid and multilineage chimerism are present throughout the life of the chimeras. Recipients are specifically tolerant to donor-type skin xenografts yet are competent to reject major histocompatibility complex (MHC)--disparate third party strain rat xenografts. Although prolonged, donor-specific skin xenografts underwent chronic rejection which had its onset at approximately 40 days following skin graft placement. We have now examined these chimeras by serial flow cytometry typing to determine whether this is due to skin-specific antigens expressed on skin, but not on the bone marrow elements to which the chimeras were rendered tolerant. In all animals examined, lymphopoietic chimerism persisted unchanged even after the onset of inflammation in the grafts, suggesting the presence of skin specific antigens. This model may provide a method to study tissue and organ specific antigens recognized across a species barrier.     

Evidence for nonimmune mechanisms in the loss of hematopoietic chimerism in rat→mouse mixed xenogeneic chimeras

Abstract: We have recently described a relatively nontoxic, nonmyeloablative conditioning regimen allowing engraftment of T cell-depleted (TCD) rat bone marrow in mice, leading to a state of mixed xenogeneic chimerism and donor-specific skin graft tolerance, apparently by a deletional mechanism. The conditioning regimen involves depletion of host T and NK cells with mAbs, followed by administration of 7 Gy thymic irradiation (TI) and 3 Gy whole body irradiation (WBI) prior to transplantation of TCD F344 rat bone marrow cells (BMC). Although the percentage of rat cells gradually declines over time post-BMT in these animals, they demonstrate prolonged survival of donor-specific rat skin grafts and an absence of anti-rat antibody responses long after this decline is underway. We therefore hypothesized that the loss of rat hematopoietic repopulation may not be due to a loss of tolerance at the T cell or B cell level. We have now evaluated the effect of a repeat, late, donor marrow infusion on chimerism and tolerance. Treatment with 3 Gy WBI followed by TCD rat marrow infusion at 22 weeks following the original BMT led to a marked increase in rat cell repopulation of both myeloid and lymphoid lineages and prolonged the period of complete central and peripheral deletion of Vp5⁺ and Vβ11⁺ host T cells, which recognize endogenous (presumably mouse) superantigens presented by rat hematopoietic cells. Furthermore, the second marrow infusion did not induce a cytotoxic antibody response to rat marrow. Since these results suggested that the decline in rat chimerism in our model was not associated with a loss of T cell or B cell tolerance to the donor, we evaluated the possibility that a failure of NK cell tolerance led to the decline by comparing chimerism in animals receiving chronically NK cell-depleting mAb and controls. Chronic NK depletion did not markedly enhance the level of rat repopulation in any lineage. Together, our results are most consistent with the interpretation that the gradual decline in rat repopulation in our chimeras reflects a competitive advantage of host hematopoietic cells over xenogeneic cells rather than immune-mediated rejection, possibly due to species selectivity of cytokines and adhesion molecule interactions of hematopoietic progenitors and the marrow microenvironment. This host hematopoietic advantage might be counteracted by repeat donor-specific marrow infusions.     

Mixed xenogeneic chimeras (rat + mouse to mouse). Evidence of rat stem cell engraftment, strain-specific transplantation tolerance, and skin-specific antigens.

We report the induction of stable and reliably detectable mixed xenogeneic chimerism through the coadministration of a mixture of untreated rat bone marrow plus T cell-depleted mouse bone marrow into B10 recipients conditioned with total body irradiation (TCD B10 mouse + untreated F344 rat----B10 mouse). Recipients repopulated as true mixed lymphopoietic chimeras, with from 1-21.6% rat-derived lymphoid cells in peripheral blood and splenic lymphoid tissue. Production of rat platelets was also demonstrated. Rat platelet and lymphoid chimerism was reliably detectable in chimeras from 1 to 7 months following reconstitution, suggesting engraftment of the rat bone marrow stem cell. Production of each stem cell-derived lineage appeared to be under independent regulation since a significantly greater proportion of platelets were rat-derived (24-81%) than were lymphocytes (1-21.6% rat), while erythrocytes were preferentially syngeneic (less than 2% rat). The tolerance induced by this model was highly donor strain-specific: donor-specific rat and mouse skin grafts were accepted while MHC-disparate third-party mouse (C3H; H-2k) and rat (Wistar Furth; Rt1Au) skin grafts were promptly rejected. Although specifically prolonged xenogeneic donor rat skin grafts underwent a slow chronic rejection, and some totally disappeared. In spite of this, chimeras retained their lymphoid chimerism, suggesting the presence of skin-specific antigens. This model for mixed xenogeneic chimerism with reliably detectable rat lymphoid cells may provide a model to study the existence of tissue-specific antigens across a species barrier, as well as mechanisms responsible for the induction and maintenance of this strain-specific transplantation tolerance.     

Immunobiology of xenogeneic cornea grafts in mouse eyes. I. Fate of xenogeneic cornea tissue grafts implanted in anterior chamber of mouse eyes

BACKGROUND: The cornea is an immune privileged tissue that, when grafted orthotopically, forms the anterior surface of the immune privileged anterior chamber. We have recently reported that allogeneic cornea fragments implanted in the anterior chamber of mouse eyes resist immune rejection, although such graft fragments are rejected outside the eye. We wished to determine the extent to which xenogeneic cornea fragments placed in the eyes of normal mice are vulnerable to immune rejection. METHODS: Guinea pig corneas, deprived surgically of epithelium, were cut into fragments and inserted into the anterior chamber of eyes of BALB/c and severe combined immune deficient (SCID) mice, adjacent to the central cornea of the recipient. The fate of the grafts was assessed clinically by biomicroscopy and histologically for 8 weeks postimplantation. RESULTS: The majority of guinea pig cornea fragments devoid of epithelium came to rest with the raw stroma adjacent to recipient endothelium. These fragments remained clear for the 8-week observation interval in both BALB/c and severe combined immune deficient mice. Clear grafts displayed viable guinea pig keratocytes and endothelial cell layers for 4 weeks. The endothelium was then replaced by murine cells by 8 weeks. A minority of guinea pig cornea fragments were oriented with donor endothelium adjacent to recipient endothelium. Although these grafts in severe combined immune deficient eyes eventually acquired an endothelial layer that faces the anterior chamber and remained clear, similar fragments in BALB/c eyes became opaque, failed to acquire a proper lining of endothelium that faces the anterior chamber, and incited an inflammatory reaction in adjacent recipient cornea. CONCLUSIONS: Immune privilege is afforded to xenografts of guinea pig cornea placed as stromal: endothelial cell fragments in the anterior chamber of mouse eyes, but only if the surface of the fragments that faces the anterior chamber is promptly covered with corneal endothelium. The possible roles of corneal endothelium in promoting immune privilege of corneal xenografts are discussed.     

Immunobiology of xenogeneic cornea grafts in mouse eyes. II. Immunogenicity of xenogeneic cornea tissue grafts implanted in anterior chamber of mouse eyes

BACKGROUND: Xenogeneic corneal fragments (guinea pig) are highly resistant to immune rejection in the anterior chamber of mouse eyes. Because guinea pigs and mice are discordant (i.e., mouse serum normally contains guinea pig reactive xenoantibodies), we wished to determine the extent to which xenogeneic corneal fragments placed intraocularly in normal and specifically sensitized mice activated xenoreactive T and B cells. METHODS: Guinea pig corneas, deprived surgically of epithelium, were cut into fragments and inserted into the anterior chamber of eyes of BALB/c mice, adjacent to the central cornea of the recipient. Antibody (immunoglobulin [Ig]M, IgG) and delayed type hypersensitivity (DTH) immune responses of recipient mice to guinea pig xenoantigens were assessed. The fate of xenogeneic cornea implants was assessed in mice immunized systemically to guinea pig antigens. RESULTS: Guinea pig spleen cells and corneal fragments implanted s.c. induced within 2 weeks of immunization both DTH and IgG antibodies to guinea pig xenoantigens. By contrast, xenogeneic corneal fragments implanted in the anterior chamber of mouse eyes evoked no change in recipient humoral immune status and induced mild guinea pig-specific DTH only after 5 weeks. Presensitization of mice to guinea pig xenoantigens failed to increase the proportion of grafts that were regarded as rejected, but the onset of rejection in failed grafts occurred earlier than in unsensitized recipients. Active systemic immunization of mice bearing intracameral guinea pig corneal fragments failed to curtail the grafts' survival. Guinea pig corneal fragments implanted in the anterior chamber of normal mice failed to induce anterior chamber-associated immune deviation. CONCLUSIONS: Xenogeneic corneal fragments implanted in the anterior chamber of eyes of normal mice display strikingly reduced immunogenicity, and an inability to induce anterior chamber-associated immune deviation. These properties are discussed in terms of the vulnerability of guinea pig corneal tissue to immune rejection within the eye.