Suzanne T. Ildstad

Cell migration, chimerism, and graft acceptance

The chimeric nature of the transplanted liver was first shown in our long-surviving human recipients of orthotopic hepatic allografts in 1969.1 When liver grafts were obtained from cadaveric donors of the opposite sex, karyotyping studies showed that hepatocytes and endothelium of major blood vessels retained their donor specificity, whereas the entire macrophage system, including Kuppfer cells, was replaced with recipient cells.2 Where donor cells that had left the liver had gone was unknown, but their continued presence was confirmed by the acquisition and maintenance in recipient blood of new donor-specific immunoglobulin (Gm) types1,3 and red-blood-cell alloantibodies, if donors with ABO non-identity were used.4 Davies et al5 attributed the secretion of new soluble HLA class I antigens of donor type to transplanted hepatocytes. However, these HLA molecules come from bone-marrow-derived macrophages and/or dendritic cells,6 and probably have the same origin from migrated donor cells as the additional Gm types and red-cell antibodies.

Systemic chimerism in human female recipients of male livers

We have previously reported data from clinical and laboratory animal observations which suggest that organ tolerance after transplantation depends on a state of balanced lymphodendritic cell chimerism between the host and donor graft. We have sought further evidence to support this hypothesis by investigating HLA-mismatched liver allograft recipients. 9 of 9 female recipients of livers from male donors had chimerism in their allografts and extrahepatic tissues, according to in-situ hybridisation and molecular techniques 10 to 19 years posttransplantation. In 8 women with good graft function, evidence of the Y chromosome was found in the blood (6/8), skin (8/8), and lymph nodes (7/8). A ninth patient whose transplant failed after 12 years from recurrent chronic viral hepatitis had chimerism in her lymph nodes, skin, jejunum, and aorta at the time of retransplantation. Although cell migration is thought to take place after all types of transplantation, the large population of migratory cells in, and the extent of their seeding from, hepatic grafts may explain the privileged tolerogenicity of the liver compared with other organs.

Chimerism and donor-specific nonreactivity 27 to 29 years after kidney allotransplantation

Chimerism was demonstrated with immunocytochemical and/or polymerase chain reaction techniques in kidney allografts and in the native skin, lymph nodes, or blood of 5 of 5 patients who received continuously functioning renal transplants from 1 or 2 haplotype HLA mismatched consanguineous donors (4 parents, 1 aunt) 27–29 years ago. In the 4 cases where the kidney donor still was alive to provide stimulator lymphocytes for testing, these provoked no (n=2) or modest (n=2) MLR in contrast to vigorous MLR to third party lymphocytes. In all 4 cases, the donor cells failed to generate in vitro cytotoxic effector cells (cell-mediated lymphocytotoxicity). These findings are in accord with the hypothesis that cell migration, repopulation, and chimerism are seminal events that define graft acceptance and ultimately can lead to acquired donor-specific nonresponsiveness (tolerance).

Chimerism after liver transplantation for type IV glycogen storage disease and type 1 Gaucher's disease.

BACKGROUND Liver transplantation for type IV glycogen storage disease (branching-enzyme deficiency) results in the resorption of extrahepatic deposits of amylopectin, but the mechanism of resorption is not known. METHODS We studied two patients with type IV glycogen storage disease 37 and 91 months after liver transplantation and a third patient with lysosomal glucocerebrosidase deficiency (type 1 Gauchers disease), in whom tissue glucocerebroside deposition had decreased 26 months after liver replacement, to determine whether the migration of cells from the allograft (microchimerism) could explain the improved metabolism of enzyme-deficient tissues in the recipient. Samples of blood and biopsy specimens of the skin, lymph nodes, heart, bone marrow, or intestine were examined immunocytochemically with the use of donor-specific monoclonal anti-HLA antibodies and the polymerase chain reaction, with preliminary amplification specific to donor alleles of the gene for the beta chain of HLA-DR molecules, followed by hybridization with allele-specific oligonucleotide probes. RESULTS Histopathological examination revealed that the cardiac deposits of amylopectin in the patients with glycogen storage disease and the lymph-node deposits of glucocerebroside in the patient with Gauchers disease were dramatically reduced after transplantation. Immunocytochemical analysis showed cells containing the HLA phenotypes of the donor in the heart and skin of the patients with glycogen storage disease and in the lymph nodes, but not the skin, of the patient with Gauchers disease. Polymerase-chain-reaction analysis demonstrated donor HLA-DR DNA in the heart of both patients with glycogen storage disease, in the skin of one of them, and in the skin, intestine, blood, and bone marrow of the patient with Gauchers disease. CONCLUSIONS Systemic microchimerism occurs after liver allotransplantation and can ameliorate pancellular enzyme deficiencies.

Orthotopic liver transplantation in the mouse.

Heart and kidney transplantation in the mouse has been well established (1, 2), but orthotopic liver transplantation has not been reported. Although the mouse is one-tenth the size of the rat, there are many advantages to using the mouse for immunologic research. The mouse genome has been more thoroughly characterized than the rat or any other species of mammal. The mouse H-2 system also bears a striking resemblance to the human HLA system. In addition, there are numerous genetically defined inbred strains and wealth of monoclonal antibodies that are commercially available for mouse investigations. A key development in rat orthotopic liver transplantation (3) was the introduction by Zimmerman et al. (4) and Kamada and Caine (5), of the cuff technique instead of suture for some of the venous vascular anastomoses. This method shortened the clamping time of the portal vein and increased survival. We have applied this principle to mouse orthotopic liver transplantation. In our pilot studies, more than 40 syngeneic mouse liver replacements were performed before 6 long-term survivors were obtained. The experience reported here is with the next 48 attempts, in which the surgical success rate was 83%. Male inbred syngeneic mice 10–12 weeks old (25–32 g), from Jackson Laboratory, Bar Harbor, ME, were used as size-matched donors and recipients under methoxyflurane (2.2-dichloro-1,1-difluoroethyl methyl ether) anesthesia. The strains used were B6AF1 (27 pairs), C57BL/6 (11 pairs), and BALB/c (10 pairs). Clean but not sterile operative technique was used, and all procedures were performed under the operating microscope with 4–6.4× magnification.

Mixed xenogeneic chimerism (mouse + rat → mouse) to induce donor-specific tolerance to sequential or simultaneous islet xenografts

We previously reported that donor-specific rat islet xenografts were accepted by fully xenogeneic (rat-->mouse) chimeras when the islets were transplanted at least six weeks following reconstitution. The purpose of the present study was to examine whether a similar outcome would occur for mixed xenogeneic (mouse+rat-->mouse) chimeras if the islets were placed coincident with the time of bone marrow infusion. As with fully xenogeneic chimeras (rat-->mouse), synchronous donor-specific F344 rat (Rt1A1) islet xenografts were significantly prolonged (MST > 139 days) in mixed xenogeneic (mouse+rat-->mouse) chimeras, while MHC-disparate third-party WF rat (Rt1Au) grafts were rejected (MST = 21.2 days). The transplanted donor-specific islets were functional to maintain euglycemia and they were regulated in function to respond to a glucose challenge. For potential clinical application, it would be of obvious benefit if the islet xenografts could be placed at the time of bone marrow transplantation. We therefore performed similar studies using donor islets administered simultaneously with bone marrow. Donor-specific islet xenografts were permanently accepted by all mouse recipients (n = 5). When MHC-disparate third-party rat islets were transplanted, only 3 of 8 islet xenografts were rejected; the other 5 remained functional from 77 to 90 days posttransplantation. Although prolonged and functional, the MHC-disparate islets that were accepted exhibited histologic evidence of fibrosis and rejection, while those matched to the donor did not. These data therefore suggest that donor-specific islet xenografts are permanently accepted if placed simultaneously or sequentially following mixed xenogeneic bone marrow reconstitution.

Donor dendritic cell repopulation in recipients after rat-to-mouse bone-marrow transplantation

Sir,—Bone-marrow-derived dendritic cells are powerful antigen-presenting cells, which are 100-times more effective than macrophages in activating T lymphocytes in mixed-lymphocyte culture1 and in intact animals.2 These cells are pivotal in the generation of the T-cell repertoire, including provision of the appropriate ligand for negative selection of potentially autoreactive T lymphocytes.3 We now describe the tissue distribution of donor dendritic accessory cells in fully xenogenic (F344 rat → B10 mouse) radiation bone-marrow chimeras that were permanently tolerant to donor-specific xenoantigens, yet fully reactive to third-party mouse and rat lymphoid cells.4,5 Laboratory animals were typed for chimerism by flow cytometry, and 3 per group were killed for a complete tissue survey at 1,2,3,4,6, and 8 weeks and at 8 months after reconstitution. Immunohistochemical staining for rat-derived dendritic cells (class II bright) was with the monoclonal antibody L-21-6 directed at the invariant chain of the rat class II molecule without cross-reactivity in mouse cells (provided by Dr Yuichi Iwaki, University of Pittsburgh). In the first 2–3 weeks after reconstitution, the structure of the class-II-positive rat cells migrating into the tissue of the mice appeared more rounded than stellate and were thought to be an immature, less differentiated form of dendritic cell. By the end of one month and invariably thereafter, the rat dendritic cells were found in all of the mouse tissues examined: thymic cortex and medulla (figure, top), spleen, other lymphoid structures, liver, fat, peripheral nerve roots, brain (cerebellum, cerebrum), trachea, oesophagus, pancreas, lungs, bronchi, heart, and skin (figure, bottom). Their morphology, cell density, and distribution in the chimeras resembled that of normal rat tissues examined as controls. Figure 1 Mouse thymus (top) and skin (bottom) six months after rat-to-mouse bone-marrow transplantation The extent as well as the normal pattern of “homing” of the rat bone-marrow-derived dendritic cells in a xenogeneic mouse environment, suggests the presence of a highly conserved receptor-ligand mechanism. We are currently investigating the possible role of these ubiquitous cells in the induction and maintenance of specific transplantation tolerance across both allogeneic and xenogeneic histocompatibility barriers.

Evidence for early Th 2 T cell predominance in xenoreactivity.

Two distinct subsets of CD4+ Th lymphocytes have been characterized by their cytokine profiles: Th 1 (TH1) and Th 2 (TH2). While TH1 cells predominate in cell-mediated responses, TH2 cells support the humoral response. We have examined the mRNA cytokine profile of normal mouse lymphocytes in response to alloantigen versus xenoantigen (rat) in MLC, and present evidence to suggest that early in proliferative responses, alloreactivity is dominated primarily by TH1-type lymphocytes, while xenoreactivity is predominantly TH2. Normal mouse lymphocyte-responding cells were cultured in a one-way MLR with either allo or xeno antigen and examined for production of mRNA for cytokines characteristically produced by TH1 (IL-2, IFN-γ) or TH2 (IL-4, IL-10) cells. Semiquantitative reverse transcription-polymerase chain reaction analysis was performed for mouse IL-2, IL-4, IL-10, and IFN-γ mRNA. In the mouse anti-rat xeno response, mRNA for TH2 gene products were upregulated, with greater levels of IL-4 and IL-10 at 24 and 48 hr when compared with controls. In contrast, upregulation of mRNA for TH1 gene products occurred in the mouse anti-mouse allo response, with higher levels of IL-2 and IFN-γ at 24 and 48 hr. In the anti-xeno response, upregulation of all 4 cytokines occurred by day 4 and peak levels of mRNA for all cytokines examined were 2—3 times that seen for the peak anti-allogeneic response. These data suggest that early xenorecognition may differ from allorecognition by differential activation of the TH2 subset. A better understanding of the balance between Th subset function and cytokine profile in allo and xeno reactivity may allow a more targeted and specific approach to control the early events in xenograft rejection.

A minimal conditioning approach to achieve stable multilineage mouse plus rat chimerism

Transplantation of untreated rat bone marrow into lethally irradiated (950 cGy) mouse recipients results in durable xenogeneic (rat-->mouse) chimerism and confers donor-specific transplantation tolerance for subsequent xenografts. The purpose of the present study was to characterize the minimal dose of total body irradiation (TBI) which would allow engraftment of rat bone marrow in mouse recipients. We report here that durable and stable lymphohaematopoietic cross-species chimerism can be achieved using a less than totally ablative radiation-based conditioning approach. The percentage of B10 mouse recipients which engrafted with rat bone marrow cells correlated with the dose of TBI. Engraftment of rat bone marrow stem cells occurred in all animals receiving 750 cGy prior to bone marrow transplantation, while no engraftment was detected at doses less than 650 cGy. Although most of the recipients were repopulated with mixed mouse and rat multilineage chimerism, some exhibited a predominance of rat cells. Although mixed xenogeneic rat/mouse chimeras prepared by lethal TBI produced only mouse derived RBC (red blood cells), chimeras prepared by sublethal conditioning produced both rat and mouse RBC. Only animals with detectable chimerism exhibited specific functional transplantation tolerance to donor xenoantigens, as assessed in vitro by mixed lymphocyte reaction assay. This model may offer an in vivo approach to study the role of species-specific growth factors in stem cell biology as well as the mechanisms for the induction of tolerance across species barriers.

FK506 inhibits the differentiation of developing thymocytes but not negative selection of T cell receptor Vβ5+ and Vβ;11+ T lymphocytes in vivo

Abstract To examine the influence of FK506 on lymphocyte development, we employed a syngeneic bone marrow transplantation model using MHC-disparate B10 (H-2 b , I-A b ) and B10.BR (H-2 k , I-A k , I-E k ) mice. B10 mice, which do not express class II I-E, do not delete any known T cell receptor (TCR)-Vβ;, while B10.BR mice (MHC class II I-E k , I-A k ) delete Vβ;5 + and Vβ;11 + TCR. Continuous daily treatment of syngeneically reconstituted B10 mice with FK506 delayed the development of thymocytes from the CD4 + CD8 + to CD4 + CD8 − stage, while no effect was observed at the earlier CD4 − CD8 − to CD4 + CD8 + stage. At the same time, there was a significant reduction in TCR high thymocytes compared with untreated, syngeneically reconstituted controls. These results suggest that FK506 treatment interfered with thymic positive selection. We also examined whether FK506 treatment would influence negative selection. Levels of expression of Vβ5 + and Vβ11 + T cells in FK506-treated B10.BR → B10.BR recipients were similar to those observed in unmanipulated, syngeneically reconstituted B10.BR → B10.BR controls. This was not due to the inhibition of clonal proliferation by FK506, since 35 days after drug withdrawal complete recovery of the peripheral Thy1.2 + population was observed, while the percentages of Vβ5 + and Vβ11 + Thy1.2 + T cells were maintained at values similar to controls. Surprisingly, clonal proliferation stimulated by monoclonal antibody against Vβ5 and Vβ11 TCRs was observed in CsA-treated, syngeneically reconstituted B10.BR mice but not in FK506-treated mice, suggesting that CsA may be more likely to induce autoreactivity. Differences in thymic architecture between FK506- and CsA-treated animals further suggested that the drugs may differ in their effects on T cell development in vivo .