Immunosuppression is essential for successful allogeneic transplantation of the metanephros.

BACKGROUND: Transplanted metanephroi vascularize and develop features of mature kidney. One group reported the intriguing finding that metanephric allografts and congenic, major histocompatibility complex-mismatched grafts developed without rejection in the absence of immunosuppression. Our experiments aim to investigate the hypothesis that metanephroi lack immunogenicity and identify immunosuppressives that do not inhibit development. METHODS: We transplanted syngeneic metanephric grafts, allografts, and class II mismatched transplants to adult rats along with control grafts to nude recipients. glomerular filtration rates (GFRs) were measured where possible and transplants assessed by histology, immunohistochemistry, electron microscopy, and polymerase chain reaction. RESULTS: Allografts underwent reliable growth and vascularization followed by vigorous rejection (n>200). Rejection was conserved across a class II-mismatched strain and when the earliest dissectable metanephric structures were transplanted. Immunosuppressive drugs other than cyclosporine demonstrated no in vivo toxicity to transplants and treatment with FTY720 and tacrolimus could ablate histologic evidence of allograft rejection. Syngeneic transplants exhibited function of up to 8% of a normal GFR. Renal mass reduction and growth factor treatment was associated with higher GFR than controls. The anatomical site of implantation was also linked strongly with achieved function. CONCLUSIONS: Fetal kidney rudiments can provide a source of functioning renal tissue. These results suggest that such structures are no less immunogenic than mature organs, but the observed rejection is controllable.

A patient with Huntington's disease and long-surviving fetal neural transplants that developed mass lesions.

Transplantation of human fetal neural tissue into adult neostriatum is an experimental therapy for Huntington's disease (HD). Here we describe a patient with HD who received ten intrastriatal human fetal neural transplants and, at one site, an autologous sural nerve co-graft. Although initially clinically stable, she developed worsening asymmetric upper motor neuron symptoms in addition to progression of HD, and ultimately died 121 months post transplantation. Eight neural transplants, up to 2.9 cm, and three ependymal cysts, up to 2.0 cm, were identified. The autologous sural nerve co-graft was found adjacent to the largest mass lesion, which, along with the ependymal cyst, exhibited pronounced mass effect on the internal capsules bilaterally. Grafts were composed of neurons and glia embedded in disorganized neuropil; robust Y chromosome labeling was present in a subset of grafts and cysts. The graft-host border was discrete, and there was no evidence of graft rejection or HD pathologic changes within donor neurons. This report, for the first time, highlights the potential for graft overgrowth in a patient receiving fetal neural transplantation.

Immune privilege of allogeneic neuroretinal transplants in the subconjunctival space.

BACKGROUND: The extent of site and tissue-associated immune privilege is of great interest in transplantation experiments involving the CNS. In the present paper we have explored neuroretinal immune privilege by transplantation to a non-immune privileged site. METHODS: Fetal and adult full-thickness rabbit neuroretinal grafts were placed in the subconjunctival space of immunocompetent rabbit hosts. Morphological examination was performed after 2-31 days (fetal grafts, n = 46), and after 8 days (adult grafts, n = 4). RESULTS: Hematoxylin and eosin-stained sections and immunohistochemistry directed against microtubule-associated protein 2 (MAP2) revealed surviving grafts containing retinal neurons in the majority of eyes with fetal grafts. In all specimens, a mild inflammatory reaction was evident as seen with major histocompatibility complex class II (MHC-II) labeling. Short-term grafts survived well and displayed lamination and rosette formation whereas older grafts appeared more disorganized and were more often rejected. Müller cell fibers labeled with glial fibrillary acidic protein (GFAP) were present in grafts from 15 days and onwards. Adult grafts were destroyed after 8 days. CONCLUSIONS: Allogeneic fetal full-thickness neuroretinal transplants can survive for several weeks in a non-immune privileged environment in which adult grafts are rapidly rejected. Fetal grafts gradually shrink, lose their architecture and go through a glial transformation accompanied by low-grade inflammation. The rabbit neuroretina thus appears to enjoy partial immune privilege, the extent of which depends on the development state of the tissue. The characterization of neuroretinal immune privilege will hopefully influence future clinical trials of retinal transplantation.

Donor major histocompatibility complex class I expression determines the outcome of prenatal transplantation.

PURPOSE: The failure of in utero transplantation in immune-competent recipients suggests the existence of a fetal immune barrier. The importance of donor major histocompatibility complex (MHC) class I expression in the induction of prenatal tolerance remains undefined. We hypothesized that donor cell MHC class I expression facilitates engraftment in prenatal allogeneic recipients rather than promoting immune rejection. METHODS: B6.Ly5.2 (class I(+)) or B6.TAP(-/-) (class I(-)) murine fetal liver cells were transplanted into age-matched allogeneic fetal recipients. Survival to weaning and subsequent growth was assessed. Engraftment rates and peripheral blood chimerism levels were measured serially. RESULTS: The presence or absence of class I expression did not affect survival or growth of recipients and no graft-vs-host disease developed. Allogeneic recipients of B6.Ly5.2 cells exhibited significantly higher levels of donor hematopoietic chimerism when compared to recipients of B6.TAP(-/-) cells (27% + 10% vs 11% + 8%; P = .004) that deteriorated further over time. CONCLUSIONS: Donor class I MHC antigen expression is essential for stable long-term engraftment and maintenance of donor-specific tolerance. Further studies are needed to better characterize the role of the fetal innate immune system in prenatal allotransplantation.

Reduced immunogenicity of first-trimester human fetal pancreas.

OBJECTIVE: The use of human fetal pancreatic tissue may provide a potential source of transplantable beta-cells as a therapy for type 1 diabetes. Human fetal pancreas has a remarkable capacity to grow and differentiate in vivo and has been shown to reverse diabetes in rodents. However, it is known that human fetal pancreas obtained from the second trimester of gestation is immunogenic and is rejected after transplantation. Tissue obtained from earlier stages might prove to be immune privileged, as has been shown for other tissues. RESEARCH DESIGN AND METHODS: In this study, we determined the immunogenicity of human fetal pancreatic tissue obtained from the first trimester of gestation in a humanized mouse model. A microarray study of immunoregulatory gene expression in first- and second-trimester human fetal pancreas was also undertaken. RESULTS: The analysis of transplanted human fetal pancreata revealed a significantly decreased immunogenicity of the first-trimester tissue. The first-trimester grafts showed only limited cellular infiltration and contained numerous insulin-positive cells, whereas second-trimester tissue was completely infiltrated and rejected. Furthermore an analysis of immunoregulatory genes expressed in first- and second-trimester human fetal pancreas by microarray demonstrated the upregulation of several key immunoregulatory genes in the second-trimester tissue. This might account for the reduced immunogenicity of the younger tissue. CONCLUSIONS: Our results provide the first indication that the use of first-trimester human fetal pancreas for transplantation might increase the survival of the grafts and might decrease the requirement for immunosuppressive drugs.

Local effects of dexamethasone on immune reaction in neural transplantation.

This study was performed to see whether local injection of dexamethasone may protect the neural grafts from immunological rejection and increase the successive rate of graft. Rats with unilateral 6-hydroxydopamine lesions of the mesostriatal dopamine pathway received fetal ventral mesencephalic (FVM) cells and dexamethasone in two regions of the striatum and showed significant (P<0.001) reduction in rotational asymmetry as compared to the non-immunosuppressed group. A significantly greater number of total TH-ir cells (P<0.001) and fewer number of total GFAP -ir cells (P<0.001) and inflammatory cells were observed in the striatum of animals in immunosuppressed group than those in non-immunosuppressed group. This results indicated that local injection of dexamethasone could not only reduce the immune rejection and increase the survival grafted cell but also avoid the side effects brought by long systemic administer of immunosuppressant.

Association between islet xenograft rejection mediated by activated macrophages and upregulated chemokines.

OBJECTIVE: Our previous study has shown that porcine antigen-primed and CD4+ T cells activated macrophages are capable of the Recognition and rejection of porcine xenografts but not mouse allografts, and therefore suggested the involvement of signaling between the graft and macrophages in this specific graft recognition and destruction. METHODS: NOD-SCID mice were transplanted with fetal pig pancreatic fragment (FPP) before adoptive transfer with exogenous macrophages isolated from rejecting FPP xenografts of BALB/c recipient mice. The exogenous macrophages were tracked by Ly5.1 surface antigen or via CSFE staining. Gene expression of CCR2 and CCR5 and their chemokines in transplanted FPP xenografts was evaluated by real-time PCR. RESULTS: After the adoptive transfer, recently transplanted but not established FPP xenografts were rejected by exogenous activated macrophages. In the meantime, greater level of chemokine gene expression was detected in recently-transplanted compared with the established xenografts. Furthermore, expression of both CCR2 and CCR5 genes was enhanced significantly in activated macrophages when compared with non-activated macrophages. CONCLUSION: Upregulated chemokines were associated with macrophage recruitment and destruction of islet xenografts.

Use of T-cell antibodies for donor dosaging in a canine model of in utero hematopoietic stem cell transplantation.

AIM: Microchimerism following canine in utero hematopoietic stem cell transplantation (IUHSCT) development of T-cell dosing regimens. OBJECTIVE: To investigate the use of anti-T-cell antibodies for cell dosing of the donor graft in a canine model of IUHSCT. STUDY DESIGN: Canine IUHSCT was performed by ultrasound-guided intraperitoneal injection in days 35-38 of fetal canines with CD34(+) cells at doses of 4.5 x 10(8) to 1.3 x 10(9) cells/kg and T cells (CD3(+) CD5(+)) at doses of 8 x 10(6) to 8.8 x 10(8) cells/kg. Postnatal studies included tissue histology and polymerase chain reaction-based chimerism analysis. RESULTS: Term survival was 86-100%. Microchimerism (0-2%) was detected in five of eight recipients in multiple tissues. Histopathology revealed no evidence of graft-versus-host disease (GVHD). CONCLUSION: Canine IUHSCT is a useful model to investigate the role of donor T cells in engraftment and GVHD. IUHSCT at early gestational ages with high doses of donor T cells in the graft yields microchimerism in multiple tissues without GVHD.

Porcine fetal ventral mesencephalic cells are targets for primed xenoreactive human T cells.

Xenotransplantation of porcine fetal ventral mesencephalic (pfVM) cells to overcome the dopamine shortage in the striatum of patients with Parkinson's disease seems a viable alternative to allotransplantion of human fetal donor tissue, especially because the latter is complicated by both practical and ethical issues. There is, however, little known about the xenospecific immune responses involved in such an intracerebral xenotransplantation. The aim of our study was to investigate whether (1) naive human peripheral blood mononuclear cells (PMBC) display cytotoxicity against pfVM cells of E28 pig fetuses, and (2) priming of human PBMC by xenogeneic antigen presenting cells (APC) modulates pfVM-directed cellular cytotoxicity. For this purpose fresh PMBC from nine individual donors were primed by incubation with either irradiated pfVM cells or porcine spleen cells (PSC) as APC in the presence of IL-2 for 1 week before assessing cytotoxicity in a 51Cr release assay. Also, direct NK reactivity and antibody-dependent cellular cytotoxicity (ADCC) of fresh PMBC against pfVM cells was assessed. No direct cytotoxicity of naive cells (either NK reactivity or ADCC) against pfVM cells could be determined. Only PMBC primed with PSC were capable of lysing pfVM cells. PBMC primed with pfVM cells did not show cytolytic capacity towards pfVM. Interestingly, large differences in xenospecific T-cell responses exist between individual donor PBMC. Thus, human T cells are capable of killing pfVM cells in a xenoreactive response, but only after priming by donor APC. The large interindividual differences between human donors in their xenoreactive response may influence patient selection for xenotransplantation and chances of graft survival for individual patients.

Allogeneic acute rejection on fetal small-bowel graft: role of gangliosides.

In previous work, it was shown that gangliosides (Gang) have an inhibitory effect on lymphocyte proliferation as well as on delayed-type hypersensitivity response and mixed lymphocyte reaction. Therefore, we decided to examine the effect of gangliosides in acute allorejection after fetal intestinal transplantation. We used two female C57BL/6 mice on pregnancy day 19 as a source of fetal intestine. All animals were anesthetized with ketamine (70 mg/kg) and xylazine (10 mg/kg), intramuscularly. We harvested intestinal segments of 1 cm to transplant into BALB/c and C57BL/6 mice (male, weighing around 20 g) used as recipients. They were divided into groups of six animals each: isogeneic and allogeneic without treatment, or treated with tacrolimus 1 mg/kg/day, or gangliosides 3 and 9 mg/kg/day, during 7 days posttransplantation, intramuscularly. On postoperative day 7, intestinal grafts were collected and fixed in 10% formalin solution. Using an anesthetic overdose as euthanasia, we removed the intestinal grafts. Tissue samples were stained with hematoxylin-eosin for histological analysis regarding grafts development (D) and rejection (R) aspects. Data were analyzed by the Kruskal-Wallis test, considering P < or = 0.05 as significant. In the isogeneic and tacrolimus groups, we observed a very good degree of development (D = 9 +/- 0.5; D = 9 +/- 0.4, respectively), but a severe degree of rejection (R = 15 +/- 1.3) and a low degree of development (D = 1 +/- 0.8) in animals without treatment. The ganglioside groups showed D = 5 +/- 1.6 and R = 13 +/- 3.3, and D = 7 +/- 2.9 and R = 9 +/- 1.9, for the 3-mg and 9-mg groups, respectively. There was a statistically significant difference between the ganglioside groups and allogeneic groups without treatment. Based on the above data, we conclude that avascular fetal intestine transplantation is a good experimental model for studying immunological events, and that gangliosides only partially modulate the allorejection response, allowing intestinal development, mainly at the highest ganglioside dose. Maybe immunomodulation would be better observed by using isolated types of gangliosides or association with other immunosuppressive drugs.

Murine fetal small-intestine grafts: morphometric and immunohistochemical evaluation.

We investigated histopathological changes following murine fetal intestinal transplantation. Fetal intestine, obtained from a pregnant C57BL/6 mouse, was transplanted into BALB/c and C57Bl/6 mice. Recipients were divided into three groups: isogeneic, and allogeneic treated with 3 mg/kg/day gangliosides (Allo-a) or 9 mg/kg/day (Allo-b). One week after transplant, all grafts showed good viability, confirmed by cellular mitosis in the mucosa and a well-defined propria muscular layer. Isogeneic grafts showed a thicker muscular layer than in the Allo-a (P = 0.02) and Allo-b (P = 0.004) groups. There was no difference in number of mitotic cells among groups. Goblet cells were significantly reduced in allografts treated with 3 mg gangliosides (P = 0.013) or 9 mg gangliosides (P = 0.002) compared to isografts. Villi height was similar in all studied groups. There was no difference in positivity of the enteric nervous system among groups. Atrophy was more common in the allogeneic groups, suggesting that isografts had better development than allografts treated with gangliosides. (

Beta1 integrin as a xenoantigen in fetal porcine mesencephalic cells transplanted into the rat brain.

Xenografts of fetal porcine mesencephalic cells implanted into the rat striatum are generally rejected within several weeks. The fetal donor mesencephalon predominantly consists of neurons, but also contains microglial and endothelial cells, which are more immunogenic. In the present work, we investigated the occurrence of donor endothelial cells in grafts of porcine mesencephalic cells implanted into the rat striatum. Pig endothelial cells were monitored by immunochemical methods, using a monoclonal antibody (mAb) that recognizes a peptidic epitope of the porcine beta1 integrin, and isolectin IB4, for the staining of the Galalpha1,3Gal epitope. The analysis also involved the detection of the pig hyaluronate receptor CD44, and the cell adhesion molecule CD31. The anti-beta1 integrin mAb revealed endothelial-like cells in grafts of porcine mesencephalic cells as soon as 1 week after implantation. A similar staining pattern was obtained with the IB4 lectin. Unlike aortic endothelial cells, these pig brain-derived endothelial-like cells were not recognized by the anti-CD44 antibody. They also failed to express the CD31 adhesion molecule, a fact which suggests that they remained poorly mature, even in grafts maintained during 45 days in immunosuppressed rats. Interestingly, a strong expression of beta1 integrin immunoreactivity was noticed in a large proportion (80%) of the cells freshly dissociated from the fetal pig mesencephalic tissue. The immunoreactivity decreased progressively after transplantation of the cells into the rat brain. This observation suggests that dissociated neuroblasts are capable of a temporary expression of beta1 integrin. This molecule is known to participate in the process of cell sorting and migration in the developing brain. Hence, its expression could be the hallmark of a rescue mechanism triggered by the disruption of the cell/matrix interactions during the dissociation of the fetal mesencephalon. This disruption might account for part of the dramatic cell death process that occurs during the manipulation of the donor tissue.

Autoamplification of NFATc1 expression determines its essential role in bone homeostasis.

NFATc1 and NFATc2 are functionally redundant in the immune system, but it was suggested that NFATc1 is required exclusively for differentiation of osteoclasts in the skeletal system. Here we provide genetic evidence that NFATc1 is essential for osteoclast differentiation in vivo by adoptive transfer of NFATc1(-/-) hematopoietic stem cells to osteoclast-deficient Fos(-/-) mice, and by Fos(-/-) blastocyst complementation, thus avoiding the embryonic lethality of NFATc1(-/-) mice. However, in vitro osteoclastogenesis in NFATc1-deficient cells was rescued by ectopic expression of NFATc2. The discrepancy between the in vivo essential role of NFATc1 and the in vitro effect of NFATc2 was attributed to selective autoregulation of the NFATc1 gene by NFAT through its promoter region. This suggested that an epigenetic mechanism contributes to the essential function of NFATc1 in cell lineage commitment. Thus, this study establishes that NFATc1 represents a potential therapeutic target for bone disease and reveals a mechanism that underlies the essential role of NFATc1 in bone homeostasis.

Transgenic expression of CTLA4-Ig by fetal pig neurons for xenotransplantation.

The transplantation of fetal porcine neurons is a potential therapeutic strategy for the treatment of human neurodegenerative disorders. A major obstacle to xenotransplantation, however, is the immune-mediated rejection that is resistant to conventional immunosuppression. To determine whether genetically modified donor pig neurons could be used to deliver immunosuppressive proteins locally in the brain, transgenic pigs were developed that express the human T cell inhibitory molecule hCTLA4-Ig under the control of the neuron-specific enolase promoter. Expression was found in various areas of the brain of transgenic pigs, including the mesencephalon, hippocampus and cortex. Neurons from 28-day old embryos secreted hCTLA4-Ig in vitro and this resulted in a 50% reduction of the proliferative response of human T lymphocytes in xenogenic proliferation assays. Transgenic embryonic neurons also secreted hCTLA4-Ig and had developed normally in vivo several weeks after transplantation into the striatum of immunosuppressed rats that were used here to study the engraftment in the absence of immunity. In conclusion, these data show that neurons from our transgenic pigs express hCTLA4-Ig in situ and support the use of this material in future pre-clinical trials in neuron xenotransplantation.

Embryonic stem cell immunogenicity increases upon differentiation after transplantation into ischemic myocardium.

BACKGROUND: We investigated whether differentiation of embryonic stem cells (ESCs) in ischemic myocardium enhances their immunogenicity, thereby increasing their chance for rejection. METHODS AND RESULTS: In one series, 129/SvJ-derived mouse ESCs (ES-D3 line) were transplanted by direct myocardial injection (1 x 10(6) cells) into murine hearts of both allogeneic (BALB/c, n=20) and syngeneic (129/SvJ, n=12) recipients after left anterior artery ligation. Hearts were procured at 1, 2, 4, and 8 weeks after ESC transplantation and analyzed by immunohistochemistry to assess immune cell infiltration (CD3, CD4, CD8, B220, CD11c, Mac-1, and Gr-1) and ESC differentiation (hematoxylin and eosin). In a second series (allogeneic n=5, sham n=3), ESC transplantation was performed similarly; however after 2 weeks, left anterior descending artery-ligated and ESC-injected hearts were heterotopically transplanted into naive BALB/c recipients. After an additional 2 weeks, donor hearts were procured and analyzed by immunohistochemistry. In the first series, the size of all ESC grafts remained stable and there was no evidence of ESC differentiation 2 weeks after transplantation; however, after 4 weeks, both allogeneic and syngeneic ESC grafts showed the presence of teratoma. By 8 weeks, surviving ESCs could be detected in the syngeneic but not in the allogeneic group. Mild inflammatory cellular infiltrates were found in allogeneic recipients at 1 and 2 weeks after transplantation, progressing into vigorous infiltration at 4 and 8 weeks. The second series demonstrated similar vigorous infiltration of immune cells as early as 2 weeks after heterotopic transplantation. CONCLUSIONS: In vivo differentiated ESCs elicit an accelerated immune response as compared with undifferentiated ESCs. These data imply that clinical transplantation of allogeneic ESCs or ESC derivatives for treatment of cardiac failure might require immunosuppressive therapy.

Immunogenicity of adult mesenchymal stem cells: lessons from the fetal allograft.

Herein we review recent data that support host tolerance of allogeneic adult mesenchymal stem cells (MSC). Evidence is emerging that donor MSC deploy a very powerful array of mechanisms that allow escape from host allogeneic responses. These mechanisms include limited expression of alloantigen by the stem cell and cell contact-dependent and -independent mechanisms. MSC modulate host dendritic cell and T cell function, promoting induction of suppressor or regulatory T cells. These effects are complemented by the induction of divisional arrest anergy in T cells and by stem cell production of soluble immunomodulatory factors, including interleukin-10, transforming growth factor-beta, prostaglandin E2, and hepatocyte growth factor. In addition, MSC express the enzyme indoleamine 2,3-dioxygenase, which creates a tryptophan-depleted milieu that promotes immunosuppression. We propose that these observations show striking similarity to emerging data on the maternal acceptance of the fetal allograft. This comparison suggests new approaches to determine the contribution of different mechanisms to the successful use of MSC in regenerative medicine.

Regulatory T cells in the establishment and maintenance of self-tolerance: role of the thymic epithelium.

The thymus constitutes the microenvironment for T lymphocyte differentiation and acquisition of self-tolerance. Aiming to specify the contributions of the two essential parts of the thymus, namely hemopoietic and epithelial, we have devised experimental models in birds and mice. Chimeric thymuses, xenogeneic in birds and allogeneic in mice, were constructed early in development. In both models we could demonstrate a critical role of the epithelial component of the thymic stroma in induction and maintenance of self-tolerance. These experiments showed that suppression mechanisms are also implicated in these events, strongly suggesting the existence of regulatory T cells in both models. Before these experiments the control of self-tolerance was usually attributed to suppressive cells. However, as the cell phenotypes were not identified, the role of these cells was disregarded. Numerous studies since our investigations argue in favour of regulatory mechanisms. The work we initiated several years ago represents a contribution to our understanding of the two linked and opposite aspects of immune-responded control, namely self-tolerance and autoimmunity.

Insulin-like growth factor-1 controls type 2 T cell-independent B cell response.

The IGF-1 receptor (IGF-1R) is expressed on T and B lymphocytes, and the expression of the insulin- and IGF-1-signaling machinery undergoes defined changes throughout lineage differentiation, offering a putative role for IGF-1 in the regulation of immune responses. To study the role of the IGF-1R in lymphocyte differentiation and function in vivo, we have reconstituted immunodeficient RAG2-deficient mice with IGF-1R(-/-) fetal liver cells. Despite the absence of IGF-1Rs, the development and ex vivo activation of B and T lymphocytes were unaltered in these chimeric mice. By contrast, the humoral immune response to the T cell-independent type 2 Ag 4-hydroxy-3-nitrophenyl acetyl-Ficoll was significantly reduced in mice reconstituted with IGF-1R-deficient fetal liver cells, whereas responses to the T cell-dependent Ag 4-hydroxy-3-nitrophenyl acetyl-chicken globulin were normal. Moreover, in an in vitro model of T cell-independent type 2 responses, IGF-1 promoted Ig production potently upon polyvalent membrane-IgD cross-linking. These data indicate that functional IGF-1R signaling is required for T cell-independent B cell responses in vivo, defining a novel regulatory mechanism for the immune response against bacterial polysaccharides.

Induction of transplantation tolerance in humans using fetal cell transplants.

When engrafted with donor stem cells and lymphoid cells, patients develop transplantation tolerance to donor antigens. We analyzed the mechanism of tolerance induction in immunoincompetent recipients whose immunity has been reconstituted by transplantation of mismatched stem cells. Seven infants or human fetuses received fetal liver transplants as a treatment for severe combined immunodeficiency disease. After reconstitution of immunity by lymphocytes developed from donor stem cells, T-cell clones were produced and analyzed. Because donors and recipients were HLA mismatched, it was easy to demonstrate the donor origin of the T-cell clones. These clones were shown to have developed tolerance to histocompatibility antigens of the stem cell donor via a process of clonal deletion (probably as a result of contact with donor-derived macrophages and dendritic cells). They were also tolerant to histocompatibility antigens of the host but through a different mechanism: many clones recognized these antigens but had no detrimental effect on the target cells exhibiting host antigens, either in vitro or in vivo. Clonal anergy was therefore the cause of this tolerance to host determinants, resulting in a lack of graft-versus-host disease and of autoimmunity. The contact between developing T cells of donor origin and host epithelial cells within the host thymus may explain this colonal anergy. It should be noted that all patients had high serum levels of interleukin-10, which might have contributed to the persistent engraftment and tolerance.