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.

Thymic selection of the human T cell receptor V beta repertoire in SCID-hu mice.

Implantation of pieces of human fetal liver and thymus into SCID mice results in the development of a human thymus-like organ, in which sustained lymphopoiesis is reproducibly observed. In this model, T cell development can be experimentally manipulated. To study the influence of thymic selection on the development of the human T cell repertoire, the T cell receptor (TCR) V beta gene repertoire of double-positive (CD4+CD8+) and single-positive (CD4+CD8- and CD4-CD8+) T cells was analyzed in the SCID-hu thymus using a multiprobe ribonuclease protection assay. TCR diversity in double-positive SCID-hu thymocytes was found to be comparable with that present in the thymus of the fetal liver donor, did not change with time, and was independent of the origin of the thymus donor. Thymic selection in SCID-hu thymus induces changes in V beta usage by the single-positive CD4+ or CD8+ T cells comparable with those previously reported for single-positive cells present in a normal human thymus. Finally, significant differences were observed in the V beta usage by CD4 or CD8 single-positive T cells that matured from genetically identical stem cells in different thymic environments. Collectively, these data suggest: first, that the generation of TCR diversity at the double-positive stage is determined by the genotype of the stem cells; and second, that polymorphic determinants expressed by thymic epithelium measurably influence the V beta repertoire of mature single-positive T cells.

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.

Chimerism and tolerance to host and donor in severe combined immunodeficiencies transplanted with fetal liver stem cells.

We have studied the peripheral T cell repertoire of two patients with severe combined immunodeficiency who were successfully treated with human histocompatibility leukocyte antigen (HLA)-mismatched fetal liver stem cell transplantation. The patients presented a split chimerism. T cells were of donor origin, whereas the B cells/monocytes were of the host phenotype. Interestingly, the natural killer (NK) cells in one patient were donor derived and in the other patient of host origin. The NK cells were functional but did not have antihost or donor reactivity. Despite the HLA mismatch between donor and host cells, complete tolerance was achieved in vivo, and a specific unresponsiveness of peripheral blood mononuclear cells from both patients toward the host cells was demonstrated in vitro. Nevertheless, we could isolate T cell receptor (TCR)alpha beta, CD4+ or CD8+, T cell clones specifically reacting with HLA class I and II molecules of the host. The CD4+ host-reactive T cell clones from both patients produced interleukins 2 and 5, interferon-gamma, granulocyte/macrophage colony-stimulating factor but are specifically defective in interleukin 4 production. The frequencies of CD8+ host-reactive T cells were high, and were in the same range as those observed for CD8+ alloreactive T cells. In contrast, no donor-reactive CD8+ T cells or host or donor-reactive TCR gamma delta + T cells were detected. These data indicate that, after fetal stem cell transplantation, donor-reactive, but not host-reactive cells, are deleted from the T cell repertoire. Therefore, a peripheral mechanism of suppression or clonal anergy, rather than clonal deletion, is involved in maintaining in vivo tolerance toward the host.

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.

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.

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.

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.

A distinct Th1 immune response precedes the described Th2 response in islet xenograft rejection.

Previous studies using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) have demonstrated that islet xenograft rejection in mice is dominated by Th2-associated cytokines, i.e., interleukin (IL)-4 and IL-10. However, immunohistochemical stainings show that the morphological pattern in this model is more reminiscent of a delayed-type hypersensitivity (DTH) reaction, which is associated with a Th1 response. This study was designed to resolve the mechanisms of acute cellular xenograft rejection in rats transplanted with fetal porcine islet-like cell clusters (ICCs). Real-time quantitative RT-PCR was used to quantify the mRNA expression of cytokines in the grafts and lymph nodes, and the findings were related to the immunopathology of the rejecting grafts. By day 1, mRNA expression levels of IL-1 beta, IL-2, IL-12p40, interferon-gamma, and tumor necrosis factor-alpha were already induced in the lymph nodes. From days 3 to 12, an increasing amount of activated macrophages was seen in the grafts, whereas T- and NK-cells were fewer and mainly accumulated in the periphery of the grafts. Most of the ICCs were rejected by day 5. Transcripts of Th1-associated cytokines were dominant in both regional lymph nodes and in the grafts, with peak levels on days 3 and 5, respectively. The mRNA expression of IL-4 was increased on day 12, and it correlated with the infiltration of eosinophils and an increased level of xenoreactive IgG. The data presented indicate that an islet xenograft triggers a sequential activation of 1) a Th1-associated response characterized by graft destruction in a DTH-like reaction and then 2) a subsequent Th2-associated response characterized by increased levels of xenoreactive antibodies.

A critical role for human CD4+ T-cells in rejection of porcine islet cell xenografts.

T-cell-mediated rejection is likely to present a significant barrier to porcine islet xenotransplantation. Little is known, however, about human anti-porcine islet rejection because no suitable model exists to study this process. To address this problem, we have developed an immunodeficient mouse model to study rejection of fetal porcine islet cell clusters (ICCs) by human lymphocytes. Transplantation of porcine ICCs into hyperglycemic recombinase activating gene-deficient (R-) mice restores normal blood glucose levels within 5 weeks. Adoptive transfer of in vitro-stimulated human peripheral blood mononuclear cells into R- mice before islet cell transplantation leads to acute cellular rejection of porcine ICCs. The first human cells observed to infiltrate rejecting grafts are CD4+ T-cells. Although CD8+ T-cells are observed within the grafts at later time points, CD4+ T-cells predominate until the graft is destroyed. Adoptive transfer of purified human CD4+ T-cells before ICC transplantation is sufficient to cause acute cellular rejection. These data demonstrate that human CD4+ T-cells play a critical role in porcine ICC xenograft rejection.

A new murine model of islet xenograft rejection: graft destruction is dependent on a major histocompatibility-specific interaction between T-cells and macrophages.

A new murine model of porcine islet-like cell cluster (ICC) xenograft rejection, avoiding interference of unspecific inflammation, was introduced and used to investigate rejection mechanisms. Athymic (nu/nu) mice were transplanted with syngeneic, allogeneic, or xenogeneic islets under the kidney capsule. After the original transplantation, immune cells in porcine ICC xenografts undergoing rejection in native immunocompetent mice were transferred to the peritoneal cavity of the athymic mice. At defined time points after transfer, the primary grafts were evaluated by immunohistochemistry and real-time quantitative RT-PCR to estimate cytokine and chemokine mRNA expression. Transfer of immunocompetent cells enabled athymic (nu/nu) mice to reject a previously tolerated ICC xenograft only when donor and recipient were matched for major histocompatibility complex (MHC). In contrast, allogeneic and syngeneic islets were not rejected. The ICC xenograft rejection was mediated by transferred T-cells. The main effector cells, macrophages, were shown to be part of a specific immune response. By day 4 after transplantation, there was an upreglation of both Th1- and Th2-associated cytokine transcripts. The transferred T-cells were xenospecific and required MHC compatibility to induce rejection. Interaction between the TCR of transferred T-cells and MHC on host endothelial cells and/or macrophages seems necessary for inducing ICC xenograft rejection.

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.

Conditioning of neonatal pigs using low-dose chemotherapy and murine fetal tissue before murine hybridoma transplantation.

Immunosuppression, myeloablation, and the use of immunologically immature tissue can overcome major histocompatibility complex barriers by inducing tolerance. With the goal of inducing tolerance to BALB/c-derived murine hybridoma cells producing the 4C6 monoclonal antibody (mAb), we transplanted BALB/c fetal tissue into neonatal pigs during a regimen of low-dose conditioning with busulfan and cyclophosphamide. After the tolerance induction phase, animals received intraperitoneal injections of 4C6 mAb hybridoma cells. Evidence of persistence of injected cells over time was sought by molecular analysis of peripheral blood for the presence of mouse genomic sequences and circulating 4C6 mAb. Persistence of donor polymerase chain reaction signal during the entire duration of the study, detectable mAb titer for 6 weeks, and a twofold increase of mAb concentration after a boost hybridoma infusion was observed in one animal receiving six consecutive administrations of the conditioning regimen. Our model has the distinctive advantage of allowing functional monitoring of engrafted cells for studying tolerance induction strategies. In addition, this model could be the basis for approaches aimed at producing mAbs in tolerized large animals.

The efficacy of CD40 ligand blockade in discordant pig-to-rat islet xenotransplantation is correlated with an immunosuppressive effect of immunoglobulin.

BACKGROUND: The authors' aim was to evaluate the efficacy of immunosuppression with monoclonal anti-CD40 ligand antibodies (aCD40L) or nonspecific polyclonal intravenous immunoglobulin (IVIG) in the pig-to-rat islet xenotransplantation model. METHODS: Fetal porcine islet-like cell clusters were transplanted under the kidney capsule of nondiabetic rats. All antibodies were administered alone or in combination with cyclosporine A (CsA). In addition, some animals were administered antibodies plus tacrolimus (TAC) or sirolimus (SIR). Twelve days after transplantation, islet xenograft survival and rejection were evaluated using immunohistochemistry. RESULTS: aCD40L plus CsA had a pronounced inhibitory effect on islet xenograft rejection for up to 12 days after transplantation. Unexpectedly, treatment with a monoclonal control antibody (anti-keyhole limpet hemocyanin [aKLH]) plus CsA had a similar inhibitory effect. Furthermore, a similar inhibition of islet xenograft rejection was observed also in animals administered IVIG plus CsA. Monotherapy with aCD40L, aKLH, IVIG, or CsA had no effect on the rejection process. Also, when aCD40L or aKLH was administered together with TAC, islet xenograft rejection was inhibited. There was no marked difference compared with rats treated with aCD40L or aKLH and CsA. Immunosuppression with aCD40L or aKLH in combination with SIR also inhibited pig-to-rat islet xenograft rejection, but the protective effect was not as pronounced. CONCLUSIONS: Immunosuppression with high doses of antibodies, monoclonal or polyclonal, in combination with CsA or TAC inhibits pig-to-rat islet xenograft rejection. No specific effect of co-stimulatory blockade with aCD40L could be observed. Instead, the results indicate a nonspecific immunosuppressive effect of high doses of antibodies in this model.

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.

Fetal islet xenotransplantation in rodents and primates.

Beta cell replacement in IDDM by transplantation of either isolated adult islets of Langerhans or of proliferating immature islet tissue from fetal pancreas are potential ways of curing this disease. Because of the dearth of human cadaver donors adult allogeneic islets are scarce and in most Western societies availability of human fetal tissue of suitable maturity is also uncommon. The use of xenogeneic islets from domestic species already widely used for human consumption, e.g. pigs, could overcome this scarcity but xenogeneic tissues are faced with major problems of graft rejection. Hyperacute rejection (HAR) is the main cause of destruction of immediately vascularised xenografts and is caused by the interaction of natural cross-reactive antibodies with donor endothelial cells. Neovascularized islet grafts do not have donor EC as the target for HAR and are not subjected to this problem but are still acutely rejected. The mechanism of this destruction is still poorly understood but is clearly T cell dependent. However, current immunosuppression that is usually adequate for control of allograft rejection generally does not prevent xenograft rejection. A better understanding of the ways in which xenoantigens are recognised and of the nature of the immune response they initiate is fundamental to the development of appropriate strategies for the safe and effective control of xenograft rejection. The studies summarized herein describe the response of mice and primates to a challenge with fetal pig pancreas grafts. The rejection response that develops is different from that seen against a challenge with fetal allogeneic islets. Although the xenograft response is highly T cell dependent the actual effectors of graft damage appear to be different from those that provoke allograft destruction and include macrophages and granulocytes, particularly eosinophils, and possibly non-classical T cells.

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.