Megadose of T cell-depleted bone marrow overcomes MHC barriers in sublethally irradiated mice.

Graft-versus-host disease (GVHD) is uniformly lethal in recipients of HLA-mismatched marrow. In patients with severe combined immunodeficiency disease, this major obstacle can be overcome by rigorous T-cell depletion before transplantation. In leukaemia patients, however, the benefit of preventing GVHD is offset by graft rejection or graft failure. Very recently, this problem was overcome by supplementing T cell-depleted bone marrow transplants with megadoses of peripheral blood stem cells collected by leukapheresis after mobilization of the donor stem cells with granulocyte colony-stimulating factor (G-CSF). In the present study, we further demonstrate in a mouse model (C57BL/6-->C3H/Hej) that escalation of bone marrow doses by four- to fivefold leads to full donor-type chimerism in sublethally irradiated (6.5 Gy) recipients. Thus, the new source of G-CSF mobilized human haematopoietic stem cells may enable extending the use of mismatched bone marrow transplants to patients with non-malignant diseases for whom supralethal conditioning is not a prerequisite.

Impulse conduction and gap junctional remodelling by endothelin-1 in cultured neonatal rat ventricular myocytes.

Endothelin-1 (ET-1) is an important contributor to ventricular hypertrophy and failure, which are associated with arrhythmogenesis and sudden death. To elucidate the mechanism(s) underlying the arrhythmogenic effects of ET-1 we tested the hypothesis that long-term (24 hrs) exposure to ET-1 impairs impulse conduction in cultures of neonatal rat ventricular myocytes (NRVM). NRVM were seeded on micro-electrode-arrays (MEAs, Multi Channel Systems, Reutlingen, Germany) and exposed to 50 nM ET-1 for 24 hrs. Hypertrophy was assessed by morphological and molecular methods. Consecutive recordings of paced activation times from the same cultures were conducted at baseline and after 3, 6 and 24 hrs, and activation maps for each time period constructed. Gap junctional Cx43 expression was assessed using Western blot and confocal microscopy of immunofluorescence staining using anti-Cx43 antibodies. ET-1 caused hypertrophy as indicated by a 70% increase in mRNA for atrial natriuretic peptide (P < 0.05), and increased cell areas (P < 0.05) compared to control. ET-1 also caused a time-dependent decrease in conduction velocity that was evident after 3 hrs of exposure to ET-1, and was augmented at 24 hrs, compared to controls (P < 0.01). ET-1 increased total Cx43 protein by approximately 40% (P < 0.05) without affecting non- phosphorylated Cx43 (NP-Cx43) protein expression. Quantitative confocal microscopy showed a approximately 30% decrease in the Cx43 immunofluorescence per field in the ET-1 group (P < 0.05) and a reduced field stain intensity (P < 0.05), compared to controls. ET-1-induced hypertrophy was accompanied by reduction in conduction velocity and gap junctional remodelling. The reduction in conduction velocity may play a role in ET-1 induced susceptibility to arrhythmogenesis.

Engraftment and development of human T and B cells in mice after bone marrow transplantation.

A model for human lymphocyte ontogeny has been developed in a normal mouse. Human bone marrow, depleted of mature T and B lymphocytes, and bone marrow from mice with severe combined immunodeficiency were transplanted into lethally irradiated BALB/c mice. Human B and T cells were first detected 2 to 4 months after transplantation and persisted for at least 6 months. Most human thymocytes (30 to 50 percent of total thymocytes) were CD3+CD4+CD8+. Human immunoglobulin was detected in some chimeras, and a human antibody response to dinitrophenol could be generated after primary and secondary immunization.

Hematopoietic stem cell transplantation for cancer therapy.

Bone marrow transplantation has become well established in the treatment of malignant disorders. High-dose chemotherapy with hematopoietic stem cell support is widely used for most hematological malignancies, as well as for some solid tumors. In the light of recent developments in blood progenitor cell harvest, there have been clinical trials with autologous and allogeneic transplants. In particular, the availability of large numbers of blood stem cells, mobilized by granulocyte colony-stimulating factor and collected by leukapheresis, has made it possible to overcome histocompatibility barriers in HLA-mismatched leukemia patients. Other recent developments include new methods for blood progenitor cells mobilization and ex vivo expansion, the use of umbilical cord blood as an alternative source of stem cells, and molecular techniques that may, in the future, provide other modalities of purging tumor cells from autologous grafts.

Tolerance induction by 'megadose' transplants of CD34+ stem cells: a new option for leukemia patients without an HLA-matched donor.

Early studies in murine models and more recent clinical data in heavily pre-treated leukemia patients have shown that escalation of the dose of hematopoietic progenitor cells can overcome major genetic barriers and enable rapid and durable engraftment of haploidentical, three-locus-mismatched transplants without graft-versus-host disease. In vitro studies suggest that veto cells within the progenitor population most probably mediate this facilitating effect.

Differential binding of soybean agglutinin to human neuroblastoma cell lines: potential application to autologous bone marrow transplantation.

Normal human bone marrow cells were mixed with neuroblastoma cells from four different human cell lines, and the cell mixtures were separated by differential agglutination with soybean agglutinin (SBA). The unagglutinated cell fraction, previously shown to be highly enriched for the hematopoietic pluripotential stem cells and capable of reconstituting lethally irradiated adult patients with acute leukemia, was further fractionated by affinity chromatography on the lectin conjugated to Sepharose 6MB beads. Two independent assays, one using radiolabeling of the tumor cells and the other based on cloning of the neuroblastoma cells on agar, showed that the agglutination step alone removes 64-76% of the radiolabeled neuroblastoma cells and 85-98% of the clonogenic cells from the tumor/bone marrow cell mixture. Passage of the unagglutinated radiolabeled cells through SBA-Sepharose columns results in further purging of 28-53% of the neuroblastoma cells. Thus a combination of the two methods affords only one-log depletion for the neuroblastoma cells, compared to a three-log depletion achieved for a T-cell leukemia line CEM tested in parallel. It seems therefore that the agglutination technique, or the use of SBA-Sepharose columns, can be used only as a preliminary step for the purging of neuroblastoma cells from involved human bone marrow preparations. Staining with fluorescein isothiocyanate-conjugated SBA of nine different neuroblastoma cell lines, including the four tested in the fractionation studies, showed that more than 98% of the cells, of all the cell lines tested, specifically bind to the lectin, whereas no specific binding can be detected on the stem cell-enriched bone marrow cell fraction. However, the total number of receptors on the neuroblastoma cells is small compared to that of line CEM or normal granulocytes, which are strongly agglutinated by SBA. It seems therefore that the quantitative difference in the total number of SBA receptors is a crucial factor for purging by the agglutination technique or by affinity chromatography. Although these results show limitations to the use of both methods, this study establishes that all neuroblastoma cell lines tested express receptors for the lectin. Improved purging of neuroblastoma cells may possibly be achieved by targeting SBA-bound toxins or magnetic spheres to these receptors.

Autologous T cells control B-chronic lymphocytic leukemia tumor progression in human-->mouse radiation chimera.

B-chronic lymphocytic leukemia (B-CLL) is characterized by the clonal accumulation of CD5+ B cells. It has been suggested that CLL cells may be regulated by inhibitory and growth-promoting signals exerted by autologous T cells. We have recently described a model for human B-CLL in which peripheral blood mononuclear cells (PBMCs) are transplanted into the peritoneal cavity of lethally irradiated mice radioprotected with bone marrow from mice with severe combined immunodeficiency. In this model, adoptive transfer of low-stage PBMCs leads to marked engraftment of T cells or combined T and CLL cell engraftment, whereas infusion of high-stage PBMCs leads to dominance of CLL cells with a miniscule level of T-cell engraftment. This mutual exclusive pattern of engraftment indicated that T cells might control the expansion of tumor cells in the peritoneum of recipient BALB/c mice. In the present study, we further investigated this question and we demonstrate that in vivo T-cell depletion, using OKT3 antibody, markedly enhances the engraftment of B-CLL cells from patients with early-stage disease. In mice receiving PBMCs from 11 donors with advanced-stage disease, the results were more heterogeneous. In five patients the results were similar to those observed in early stage, whereas in two cases no CLL cell engraftment was found in the absence of T cells. The addition of purified T cells to PBMCs led to a substantial decrease of CLL engraftment in three advanced-stage cases. These results strengthen the working hypothesis that autologous T cells can actively suppress the expansion of the pathological cells in human-->mouse radiation chimera. This effect is prominent in early-stage disease, whereas in advanced stage suppressive and/or stimulatory effects may occur in different patients. The interaction of T cells with tumor cells and the potential of autologous T cell/immune-therapy in CLL can be further explored in this model.

Improved outcome with T-cell-depleted bone marrow transplantation for acute leukemia.

PURPOSE: To eliminate the risk of rejection and lower the risk of relapse after T-cell-depleted bone marrow transplants in acute leukemia patients, we enhanced pretransplant immunosuppression and myeloablation. PATIENTS AND METHODS: Antithymocyte globulin and thiotepa were added to standard total-body irradiation/cyclophosphamide conditioning. Donor bone marrows were depleted ex vivo of T lymphocytes by soybean agglutination and E-rosetting. This approach was tested in 54 consecutive patients with acute leukemia who received transplants from HLA-identical sibling donors or, in two cases, from family donors mismatched at D-DR. No posttransplant immunosuppressive treatment was given as graft-versus-host disease (GVHD) prophylaxis. RESULTS: Neither graft rejection nor GVHD occurred. Transplant-related deaths occurred in six (16.6%) of 36 patients in remission and in seven (38.8%) of 18 patients in relapse at the time of transplantation. The probability of relapse was .12 (95% confidence interval [CI], 0 to .19) for patients with acute myeloid leukemia and .28 (95% CI, .05 to .51) for patients with acute lymphoblastic leukemia who received transplants at the first or second remission. At a median follow-up of 6.9 years (minimum follow-up, 4.9 years), event-free survival for patients who received transplants while in remission was .74 (95% CI, .54 to .93) for acute myeloid leukemia patients and .59 (95% CI, .35 to .82) for acute lymphoblastic leukemia patients. All surviving patients have 100% performance status. CONCLUSION: Adding antithymocyte globulin and thiotepa to the conditioning regimen prevents rejection of extensively T-cell-depleted bone marrow. Even in the complete absence of GVHD, the leukemia relapse rate is not higher than in unmanipulated transplants.

Human/mouse radiation chimera generated from PBMC of B chronic lymphocytic leukemia patients with autoimmune hemolytic anemia produce anti-human red cell antibodies.

Previous studies performed in our laboratory have shown that B-CLL cells are involved in the production of anti-red cell auto-antibodies, providing a possible mechanism for the autoimmune hemolytic anemia occurring during the course of B-CLL. In order to confirm this hypothesis, we attempted to transfer human B-CLL with AIHA to immunodeficient mice. Peripheral blood mononuclear cells (PBMC) from 11 B-CLL patients suffering from AIHA were transplanted into the peritoneal cavity of lethally irradiated Balb/c mice reconstituted with SCID bone marrow. Chimeric mice generated from PBMC of these patients (in stage III-IV of the disease) exhibited an engraftment profile with dominance of tumor cells and minuscule levels of T cells. Eighty-five percent of the chimeric mice generated from 10 out of the 11 B-CLL patients with Coombs'-positive AIHA, produced human Ig with anti-human red cell specificity as detected by indirect anti-globulin test. In addition, anti-red cell auto-antibodies were produced in 36% of chimeric mice generated from PBMC of Coombs'-negative B-CLL. In contrast, control experiments in which splenic cells from idiopathic AIHA or PBMC from normal donors were transplanted, failed to produce anti-RBC. This in vivo model further supports the relationship between the B cell expansion and the autoimmune hemolytic process.

Transplantation tolerance induced by "mega dose" CD34+ cell transplants.

Early studies in murine models and more recent clinical data in heavily pretreated leukemia patients have shown that escalation of hematopoietic progenitor cells can overcome major genetic barriers and enable rapid and durable engraftment of haploidentical 3-loci mismatched transplants without graft-versus-host disease. In vitro studies suggest that veto cells within the progenitors population most likely mediate this facilitating effect. Leukemia relapse is relatively low in patients with acute myeloid leukemia (AML) but is greater in adults with acute lymphoblastic leukemia (ALL). Donor NK cells most likely mediate the resistance to relapse in patients with AML who are recipients of haploidentical transplants. Immune reconstitution in adults but not in children is slow as in adult recipients of HLA matched unrelated bone marrow transplants. The "mega dose" concept was also shown recently to be useful for tolerance induction in sublethally irradiated mice, so as to effectively overcome the marked resistance presented by the large number of lymphocytes surviving the sublethal conditioning. Thus, allogeneic chimeras generated by transplantation of large doses of Sca1+Lin- cells, permanently accept allogeneic donor type skin grafts. However, the numbers required to attain this desirable goal may not be easily collected from human donors. Nonalloreactive T cells synergize with murine Sca1+Lin- cells and might, therefore, enable achievement of engraftment of haploidentical transplants in sublethally conditioned patients.

The role of donor-derived veto cells in nonmyeloablative haploidentical HSCT.

Allogeneic HSCT offers a route for achieving immune tolerance via mixed chimerism and remains the sole curative option for many nonmalignant, autoimmune and metabolic diseases. Present-day improvements of nonmyeloablative protocols are increasing the safety of HSCT, thereby widening the target population and resurrecting the interest of HSCT application as a platform for tolerance induction in organ transplantation. Using high cell doses of T-cell-depleted (TCD) grafts has been shown to circumvent graft-vs-host disease, leaving graft rejection as the main hindrance due to the robust host immunity that remains after mild conditioning. In this review we highlight the advantages of utilizing unique non-alloreactive 'veto' cells, such as anti-third party central memory CD8 T cells (Tcm), to enable induction of mixed chimerism after megadose HSCT under nonmyeloablative conditioning. Co-administration of HSCT with veto Tcm allows for induction of mixed chimerism that was successfully translated into immune tolerance, as demonstrated by engraftment of donor-type skin grafts. These veto Tcm cells have been shown to specifically eradicate anti-donor host T cells, through lymph-node sequestration and deletion, thus sparing host immunity and circumventing the need for life-long immunosuppression. Hence, data indicate that this treatment modality of combined TCD HSCT and adoptive transfer of Tcm veto cells under nonmyeloablative conditions may serve as a valuable tool for treatment of patients with a wide array of disorders such as hemoglobinopathies, autoimmune diseases and as a prelude for organ tolerance.

Haploidentical hematopoietic stem cell transplantation: state of art.

For patients with hematologic malignancies at high risk of relapse who do not have matched donors, a suitable alternative stem cell source is the HLA-haploidentical 2- or 3-loci mismatched family donor who is readily available for nearly all patients. Transplantation across the major HLA barrier is associated with strong T-cell alloreactions, which were originally manifested as a high incidence of severe GVHD and graft rejection. The present overview of the 7th symposium on haplidentical transplantation that took place at the Weizmann Institute on February 2014, shows how these obstacles to successful transplantation can now be overcome. The review also discusses the advantages and drawbacks of current options for full haplotype-mismatched transplantation and highlights innovative approaches for rebuilding immunity, reducing leukemia relapse and improving survival after transplantation. In addition, new modalities for immune tolerance induction following nonmyeloablative conditioning are discussed, showing new options for treatment of elderly patients who cannot tolerate myeloablative conditioning protocols, as well as novel strategies for immune tolerance and chimerism induction as a platform for cell therapy and organ transplantation.

Next generation HLA-haploidentical HSCT.

Relapse is still the major cause of failure of allogeneic stem cell transplantation in high-risk acute leukemia patients. Indeed, whoever the donor and whatever the transplantation strategy, post-transplant relapse rates are ~30%, which is hardly satisfactory. The present phase 2 study analyzed the impact of adoptive immunotherapy with naturally occurring FoxP3+ T-regulatory cells (2 × 10(6) per kg) and conventional T lymphocytes (1 × 10(6) per kg) on prevention of GvHD and leukemia relapse in 43 high-risk adults undergoing full-haplotype mismatched transplantation without any post-transplant immunosuppression. Ninety-five percent of patients achieved full-donor type engraftment. Only 6/41 patients (15%) developed ⩾ grade II acute GvHD. Specific CD4(+) and CD8(+) for opportunistic pathogens emerged significantly earlier than after standard T-cell-depleted haplo-transplantation. The probability of disease-free survival was 0.56. At a median follow-up of 46 months (range 18-65 months), only 2/41 evaluable patients have relapsed. The cumulative incidence of relapse was significantly lower than in historical controls (0.05 vs 0.21; P = 0.03). These results demonstrate that the immunosuppressive potential of Tregs can be used to suppress GvHD without loss of the benefits of GvL activity. Humanized murine models provided insights into the mechanisms underlying separation of GvL from GvHD.

The Trimera mouse: generating human monoclonal antibodies and an animal model for human diseases.

Monoclonal antibodies of human origin may have great therapeutic value in the treatment of cancer, autoimmune disorders and viral or bacterial infections. Several methods for generating human monoclonal antibodies exist; some are based on the transplantation of a functioning human immune system into severe combined immunodeficient (scid) mice or into Trimera mice, which are mice that have been lethally irradiated and radioprotected by transplantation of bone-marrow cells from scid mice. Trimera mice could be also used to develop animal models for human diseases by transplanting infected human tissue fragments and for creating models for cell therapy.

Haploidentical stem cell transplantation in leukemia.

In acute leukemia patients, infusing a megadose of extensively T-cell-depleted hematopoietic stem cells after an immuno-myeloablative conditioning regimen ensures sustained engraftment of full-haplotype mismatched transplants without graft-vs-host disease. Besides the conditioning regimen and the megadose of stem cells donor natural killer cell alloreactivity also plays a role in facilitating engraftment and in preventing relapse. Since our first successful pilot study, our efforts have concentrated on developing new conditioning regimens, optimizing the graft processing and improving the post-transplant immunological recovery. The results we have so far achieved in 112 very high-risk acute leukemia patients show that haploidentical transplantation is now a clinical reality. Because virtually all patients in need of a hematopoietic stem cell transplant have a full-haplotype mismatched donor, who is immediately available, a T-cell depleted mismatched transplant should be offered, not as a last resort, but as a viable option to high risk acute leukemia patients who do not have, or cannot find, a matched donor.

Induction of prolonged tolerance to third-party skin grafts following fully allogeneic bone marrow transplantation in mice.

Fully allogeneic C57BL/6-->BALB/c chimeras were grafted at different intervals after bone marrow transplantation with C3H/HeJ skin grafts. We found that the donor-type and host-type skin grafts were always permanently accepted by the allogeneic chimeras, whereas the acceptance or rejection of third-party grafts was dependent on the timing of skin grafting: if grafted later then 3.5 weeks after BMT, they were rejected; if grafted earlier they were accepted for prolonged periods (over 100 days). We further show that 77% of the C57BL/6-->BALB/c chimeras that had been grafted with C3H/HeJ skin within the first 2 weeks post-BMT were still holding the graft 15 weeks after transplantation, as opposed to 11% of the chimeras that had been grafted 8.5 weeks post-BMT. Syngeneic BALB/c-->BALB/c chimeras, in contrast to allogeneic chimeras, promptly rejected the third-party skin grafts even when these grafts were placed within the first 3 weeks after BMT. Thymocytes and splenocytes from allogeneic chimeras that accepted the third-party skin grafts were able to mount relatively strong mixed lymphocytes reactions against the third-party antigens. Grafting of secondary skin grafts from the third-party donor on chimeras that had already accepted such skin grafts led to a prompt rejection of both third-party grafts but not of donor-type skin grafts, suggesting that the immune capacity to reject third-party skin grafts has already been attained in such chimeras. It is therefore suggested that the prolonged tolerance to third-party skin grafts may be associated with the origin of the bone marrow--derived cells in the skin graft.

Donor-type chimerism determination by competitive polymerase chain reaction (PCR) in a primate model for bone marrow transplantation.

BACKGROUND: Evaluation of the outcome of successful bone marrow transplantation (BMT) and in-depth studies of transplantation biology rely increasingly on accurate detection of donor origin cells in the transplanted recipients. This study describes a quantitative competitive polymerase chain reaction (PCR) assay for accurate evaluation of chimerism after allogeneic BMT in a cynomologous primate model, based on detection of monkey Y-specific DNA. METHODS: A competitor standard was generated via PCR using a mutagenic primer that makes the competitor DNA 22 bp less than the wild type monkey Y-specific DNA. The mutated form can still be amplified by the primer pair for the detection of monkey Y-specific DNA. A fixed amount of sample subjected to chimerism detection was co-amplified with a range of competitor DNA using a touch down program and hot start PCR technique. The PCR products were analyzed by computing densitometry. The ratio of competitor/target (Y-specific) DNA for each sample pair was calculated. RESULTS: Using DNAs prepared from an artificial mixture of male and female cells, a set of standard curves has been obtained and the sensitivity of the established quantitative PCR was found to be 25 pg of male DNA, which corresponds approximately to 0.005 fg competitor DNA. A DNA sample taken from a female monkey, transplanted with purified CD34+ stem cells from a male monkey donor 26 days after BMT, was subjected to the competitive PCR with 10% male DNA as a control; the level of male DNA in this sample was calculated to be around 50%. CONCLUSIONS: This quantitative PCR assay offers both a high degree of specificity as well as a very accurate and sensitive evaluation of chimerism in a sex-mismatched monkey BMT model.

Engraftment of human kidney tissue in rat radiation chimera: I. A new model of human kidney allograft rejection.

BACKGROUND: We have recently shown that lethally irradiated normal strains of mice and rats, reconstituted with bone marrow from severe combined immune deficiency (SCID) mice, can be engrafted with human peripheral blood mononuclear cells (PBMC). METHODS: The feasibility of transplanting human renal tissue under the kidney capsule of the SCID/Lewis and SCID/nude radiation chimera and the effects of intraperitoneal infusion of allogeneic human PBMC on the human renal implants were investigated by histology, electron microscopy, immunohistochemistry, and fluorescence-activated cell sorter analysis. RESULTS: Sequential evaluation of the human renal implants from 10 days to 2 months after transplantation showed that human parenchymal elements survive in the implants up to 2 months after transplantation. The overall architecture of the transplanted kidney tissue and the normal structure of individual cells in the glomeruli and tubuli were preserved. Infusion of allogeneic human PBMC after kidney implantation resulted in patchy cellular infiltrates, composed mainly of activated human T cells, and led to prompt rejection of the human renal tissue, whereas no signs of inflammation were observed in human renal implants of chimeric rats that did not receive human PBMC. Treatment with OKT3 antibody, anti-human CD25 antibody, or CTLA4Ig fusion protein in vivo ameliorated the rejection process. CONCLUSIONS: Human adult kidney fragments transplanted into SCID-like rats transiently retain competent parenchymal structures. When these grafts are combined with allogeneic human PBMC, acute cellular rejection develops. We suggest that this chimeric model might be useful for the investigation of the effects of experimental manipulation on the kinetics of the inflammatory response during human renal allograft rejection.

Engraftment of human kidney tissue in rat radiation chimera: II. Human fetal kidneys display reduced immunogenicity to adoptively transferred human peripheral blood mononuclear cells and exhibit rapid growth and development.

BACKGROUND: Transplantation of human kidney tissue under the kidney capsule of immunodeficient animals (severe combined immunodeficiency [SCID]/Lewis and SCID/nude chimeric rats), and the subsequent intraperitoneal infusion of allogeneic human peripheral blood mononuclear cells (PBMC), results in a rapid and consistent human renal allograft rejection. We investigated the consequences of grafting human fetal kidney fragments instead of the adult tissue. METHODS: The development of human fetal kidney tissue and its interaction with allogeneic human PBMC in chimeric rats were analyzed by histology, immunohistochemistry, and in situ hybridization. RESULTS: We report successful establishment of human fetal kidney to SCID/Lewis and SCID/nude chimeric rats. The intrarenal human fetal renal implants displayed rapid growth and maintained numerous developing glomeruli and tubular structures up to 4 months after transplantation. In contrast to the adult human kidney, infusion of allogeneic human PBMC resulted in either minimal human T-cell infiltration or abundant nonrejecting T-cell infiltrates, characterized by a reduced number of T cells of the CD45RO+ or HLA-DR+ subsets, both leading to less tissue destruction as well as to continued growth of the human fetal renal tissue. This observation was found to be related to the reduced protein expression of tissue HLA class I and II, intercellular adhesion molecule 1, and vascular adhesion molecule 1 in the fetal grafts compared with the adult grafts. Lack of tissue expression of Fas ligand in the fetal grafts suggests that the latter does not contribute to the delayed rejection of human fetal kidneys. CONCLUSIONS: Our model should be useful for the study of human fetal renal development and the human alloresponse against fetal tissue.

In vivo modulation of the allogeneic immune response by human fetal kidneys: the role of cytokines, chemokines, and cytolytic effector molecules.

BACKGROUND: We have recently demonstrated that human fetal renal tissue, implanted under the kidney capsule of severe immunodeficient rats, escapes early destruction by intraperitoneal infusion of allogeneic human peripheral blood mononuclear cells, compared with the rapid rejection of implants of human adult kidney tissue. Variable amounts of human mononuclear infiltrates were seen in the transplanted fetal kidney, however, prolonged survival of the fetal tissue (maintenance of graft architecture and significant growth) was independent of the cellular infiltrate. METHODS: We have used this experimental model to sequentially analyze transcript levels of interferon-gamma and interleukin (IL)-2 (T helper 1 cytokines), IL-4 and IL-10 (T helper 2 cytokines), RANTES, MIP1beta (beta chemokines) and their receptor CCR5, and Fas ligand (cytolytic effector molecule). Analysis was performed by reverse transcriptase-polymerase chain reaction, in both fetal and adult kidney grafts, after infusion of allogeneic human peripheral blood mononuclear cells. RESULTS: Transcript levels of interferon-gamma and IL-2 in the fetal grafts were markedly reduced throughout follow-up, compared with those observed in the adult implants. Peak levels of these cytokines appeared late in the rejection process. Concomitant with these findings, IL-4 mRNA was up-regulated during the early phase, whereas IL-10 mRNA persisted throughout the rejection process, indicating that a T helper 2 bias occurred in the fetal grafts. In addition, RANTES (after an early peak), MIP1beta, CCR5, and Fas ligand mRNA levels were suppressed in the fetal grafts compared with those in the adult grafts. CONCLUSIONS: These findings indicate that the immune response of kidney rejection is dependent on whether the target organ is of fetal or adult origin, and suggest that an allogeneic immune system mounts a T helper 2-biased response when the target organ is of fetal origin.