Biomedical subjects
T Wekerle
Publications and source records attributed to T Wekerle.
Peripheral deletion after bone marrow transplantation with costimulatory blockade has features of both activation-induced cell death and passive cell death.
Two major pathways of death of previously activated T cells have been described: activation-induced cell death can be triggered by restimulating activated T cells with high concentrations of Ag, is Fas-dependent, is not influenced by proteins of the Bcl family, and is blocked by cyclosporin A; in contrast, passive cell death is induced by the withdrawal of growth factors and activation stimuli, is Fas-independent, and is blocked by Bcl family proteins. We examined the role of these two forms of cell death in the peripheral deletion of donor-reactive host T cells after allogeneic bone marrow transplantation and costimulatory blockade with anti-CD154 plus CTLA4Ig in two murine models. The substantial decline in donor-reactive CD4 cells seen in wild-type recipients 1 wk after bone marrow transplantation with costimulatory blockade was largely inhibited in Fas-deficient recipients and in Bcl-x(L)-transgenic recipients. We observed these effects both in a model involving low-dose total body irradiation and a conventional dose of bone marrow, and in a radiation-free regimen using high-dose bone marrow transplantation. Furthermore, cyclosporin A did not completely block the deletion of donor-reactive CD4(+) T cells in recipients of bone marrow transplantation with costimulatory blockade. Thus, the deletion of donor-reactive T cells occurring early after bone marrow transplantation with costimulatory blockade has features of both activation-induced cell death and passive cell death. Furthermore, these in vivo data demonstrate for the first time the significance of in vitro results indicating that proteins of the Bcl family can prevent Fas-mediated apoptosis under certain circumstances.
Transplantation tolerance induced by mixed chimerism.
Although short- and long-term results after organ transplantation have improved considerably in recent years, morbidity and mortality rates in graft recipients remain high. The induction of lifelong donor-specific tolerance would dramatically improve outcome after organ transplantation. Although many tolerance protocols have been successful in rodent studies, most of these approaches have failed when attempted in large animals or humans. Robust tolerance, in contrast, has been demonstrated with mixed chimerism regimens not only in rodents but also in large animal models, including non-human primates. Furthermore, mixed chimerism protocols have been developed that would be feasible in cadaveric, and thus in thoracic, transplantation. The induction of mixed hematopoietic chimerism is therefore an attractive experimental approach for development of clinical tolerance protocols. One of the obstacles to widespread clinical application of this concept is the remaining toxicity of the host conditioning. Recent advances, however, have led to substantially milder protocols that could become clinically acceptable in the foreseeable future. This article provides a short overview of the basic mechanisms by which immunologic tolerance may be induced, describes the concept of mixed chimerism as a promising approach for clinical tolerance induction, and reviews recent progress in developing clinically feasible mixed chimerism protocols.
Mechanisms involved in the establishment of tolerance through costimulatory blockade and BMT: lack of requirement for CD40L-mediated signaling for tolerance or deletion of donor-reactive CD4+ cells.
We have previously shown that high levels of multiline-age mixed hematopoietic chimerism and systemic T-cell tolerance can be achieved in mice without myeloablation through the use of anti-CD40L and costimulatory blockade alone (plus CTLA4Ig) or with recipient CD8 depletion and allogeneic bone marrow transplantation. Chimeric mice permanently accept donor skin grafts (> 100 days), and rapidly reject third-party grafts. The mechanisms by which costimulatory blockade facilitates the engraftment of allogeneic hematopoietic cells have not been defined. To further understand the in vivo mechanisms by which the administration of anti-CD40L mAb facilitates the engraftment of donor bone marrow and rapidly tolerizes CD4+ T cells, we analyzed the establishment of chimerism and tolerance in CD40L -/- mice. We demonstrate here that anti-CD40L mAb treatment is required only to prevent CD40L/CD40 interactions, and that no signal to the T cell through CD40L is necessary for the induction of CD4+ tolerance. Peripheral deletion of donor-reactive CD4+ T cells occurs rapidly in CD40L -/- mice receiving bone marrow transplantation (BMT), indicating that this deletion in the presence of anti-CD40L is not due to targeting of activated CD4+ cells by the antibody. Complete CD4+ cell tolerance is observed by both skin graft acceptance and in vitro assays before deletion is complete, indicating that additional mechanisms play a role in inducing CD4+ T-cell tolerance as the result of BMT in the presence of CD40/CD40L blockade.
Mixed chimerism and transplantation tolerance.
Achieving transplantation tolerance is an important goal in the effort to reduce long-term morbidity and mortality in organ transplant recipients. Robust, lifelong, donor-specific tolerance can be reliably achieved by induction of mixed chimerism in various animal models. To date, the clinical application of these proto-cols has been impeded partly by the potential toxicity of the required host conditioning regimens and the lack of successful studies in large animals. This article reviews the progress achieved in recent years in developing considerably milder conditioning protocols in rodents, and in extending some of these models to achieve permanent mixed chimerism and tolerance in large animals. Advances in the induction of xenogeneic tolerance through mixed chimerism are also discussed.
The critical role of mouse CD4+ cells in the rejection of highly disparate xenogeneic pig thymus grafts.
Long-term survival of fetal pig thymus (FP THY) grafts and efficient repopulation of mouse CD4+ T cells is achieved in thymectomized (ATX) B6 mice that receive T and NK cell depletion by injection of a cocktail of mAbs (GK1.5, 2.43, 30-H12, and PK136) and fetal pig thymus/liver (FP THY/LIV) grafts. The requirement for each mAb in this conditioning regimen in order to avoid the rejection of FP THY grafts has not yet been defined. In our present studies, CD4 cell-depleted ATX B6 mice and euthymic MHC class II-deficient (IIKO) mice were employed to investigate the role of mouse CD4+ cells in the rejection of FP THY grafts in vivo. After grafting FP THY/LIV to CD4+ cell-depleted ATX B6 mice, efficient repopulation of mouse CD4+ T cells was observed in the periphery. However, only two of four mice had remaining FP THY grafts by 17 weeks post-implantation, and these were of poor quality, whereas four of four T and NK cell-depleted ATX B6 mice had well-developed FP THY grafts. Furthermore, three of four FP THY/LIV-grafted, CD4+ cell-depleted ATX B6 mice rejected donor MHC-matched pig skin grafts. In contrast, three of three FP THY/LIV grafted, T and NK cell-depleted, ATX B6 mice accepted donor MHC-matched pig skin grafts, suggesting that optimal tolerance to xenogeneic pig antigens was not achieved in mice conditioned only with anti-CD4 mAb. ATX B6 mice treated with only anti-CD8 mAb rejected FP THY completely by 6 weeks post-grafting, a time when CD4+ cell-depleted ATX B6 mice had well-vascularized FP THY grafts. In addition, when euthymic IIKO mice were pre-treated with the standard conditioning regimen that includes four different mAbs, FP THY grafts survived and supported the repopulation of mouse CD4+ T cells in the periphery, while high levels of mouse CD8+ T cells developed in host thymi. These studies suggest that mouse CD4+ T cells play a critical role in the acute rejection of xenogeneic FP THY grafts. Without help from CD4+ cells, mouse CD8+ cells, NK, NK/T, and TCR(gamma/delta)+ T cells do not mediate acute rejection of FP THY grafts. Furthermore, our results suggest that other cell subsets besides CD4+ T cells play a role in the delayed rejection of highly disparate xenogeneic FP THY grafts.
Allogeneic bone marrow transplantation with co-stimulatory blockade induces macrochimerism and tolerance without cytoreductive host treatment.
Allogeneic bone marrow transplantation (in immunocompetent adults) has always required cytoreductive treatment of recipients with irradiation or cytotoxic drugs to achieve lasting engraftment at levels detectable by non-PCR-based techniques ('macrochimerism' or 'mixed chimerism'). Only syngeneic marrow engraftment at such levels has been achieved in unconditioned hosts. This requirement for potentially toxic myelosuppressive host pre-conditioning has precluded the clinical use of allogeneic bone marrow transplantation for many indications other than malignancies, including tolerance induction. We demonstrate here that treatment of naive mice with a high dose of fully major histocompatibility complex-mismatched allogeneic bone marrow, followed by one injection each of monoclonal antibody against CD154 and cytotoxic T-lymphocyte antigen 4 immunoglobulin, resulted in multi-lineage hematopoietic macrochimerism (of about 15%) that persisted for up to 34 weeks. Long-term chimeras developed donor-specific tolerance (donor skin graft survival of more than 145 days) and demonstrated ongoing intrathymic deletion of donor-reactive T cells. A protocol of high-dose bone marrow transplantation and co-stimulatory blockade can thus achieve allogeneic bone marrow engraftment without cytoreduction or T-cell depletion of the host, and eliminates a principal barrier to the more widespread use of allogeneic bone marrow transplantation. Although efforts have been made to minimize host pre-treatment for allogeneic bone marrow transplantation for tolerance induction, so far none have succeeded in eliminating pre-treatment completely. Our demonstration that this can be achieved provides the rationale for a safe approach for inducing robust transplantation tolerance in large animals and humans.
Anti-CD154 or CTLA4Ig obviates the need for thymic irradiation in a non-myeloablative conditioning regimen for the induction of mixed hematopoietic chimerism and tolerance.
BACKGROUND: Thymic irradiation (TI) or repeated administration of T cell-depleting monoclonal antibodies (TCD mAbs) is required in a previously described non-myeloablative regimen allowing allogeneic marrow engraftment with stable mixed chimerism and tolerance. As both treatments might be associated with toxicity in the clinical setting, we evaluated whether T-cell costimulatory blockade could be used to replace them. METHODS: C57BL/6 mice received depleting anti-CD4 and anti-CD8 mAbs on day -5, 3 Gy whole body irradiation (day 0), and 15x10(6) fully MHC-mismatched, B10.A bone marrow cells. In addition, hosts were injected with an anti-CD154 mAb (day 0) and/or CTLA4Ig (day +2). Chimerism in peripheral blood was followed by flow cytometric (FACS) analysis, and tolerance was assessed by skin grafting, and also by mixed lymphocyte reaction (MLR) and cell-mediated lympholysis (CML) assays. The frequency of certain Vbeta families was determined by FACS to assess deletion of donor-reactive T cells. RESULTS: Chimerism was transient and tolerance was not present in animals receiving TCD mAbs on day -5 without costimulatory blockade. The addition of anti-CD154 and CTLA4Ig, alone or in combination, reliably permitted induction of high levels of stable (>6 months) multi-lineage chimerism, with specific tolerance to skin grafts and donor antigens by MLR and CML assays. Long-term chimeras showed deletion of donor-reactive CD4+ peripheral blood lymphocytes, splenocytes, and mature thymocytes. Administration of TCD mAbs only 1 day before bone marrow transplantation plus anti-CD154 also allowed induction of permanent chimerism and tolerance. CONCLUSIONS: One injection of anti-CD154 or CTLA4Ig overcomes the need for TI or prolonged host TCD in a preclinical model for the induction of mixed chimerism and deletional tolerance and thus further decreases the toxicity of this protocol. Achievement of tolerance with conditioning given over 24 hr suggests applicability to cadaveric organ transplantation.
Mixed chimerism as an approach for the induction of transplantation tolerance.
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Stable prodrugs of n-butyric acid: suppression of T cell alloresponses in vitro and prolongation of heart allograft survival in a fully allogeneic rat strain combination.
n-Butyric acid has previously been shown in vitro to suppress T cell alloresponses and beyond that to induce a state of alloantigen-specific hyporesponsiveness suggesting a potential relevance for suppressing alloresponses also in vivo. The clinical use of butyrate salt derivatives, however, is limited by an extremely short half-life due to rapid metabolism. This prompted us to investigate the effect of butyric acid derivatives with prolonged residence time in vivo on T cell alloresponses in vitro and further to explore the immunosuppressive capacity of esterified n-butyric acid in vivo. First, the effect of three butyric acid esters, i.e. glucose pentabutyrate, diacetone glucose butyrate and tributyrin on T cell proliferation in a human mixed lymphocyte culture (MLC) was evaluated. All three derivatives were found to inhibit T cell alloresponses in a concentration-dependent manner. Based on the ED50 values, glucose pentabutyrate was found to be most effective in inhibiting T cell alloreactivity in vitro (11 microM), followed by diacetone glucose butyrate (122 microM), tributyrin (146 microM) and sodium butyrate (539 microM). Because of its favourable in vitro properties, glucose pentabutyrate was chosen for in vivo experiments. To test the effect of this compound on allograft survival in vivo, in the second part of this study, heterotopic heart transplants were performed in a high responder fully allogeneic rat strain combination (Brown Norway to Lewis strain rats). We found that intraperitoneal (i.p.) injection of glucose pentabutyrate at 500 mg/kg/day (day 0 and daily up to 12 days posttransplant) induced a significant prolongation of allograft survival as compared to animals treated with vehicle (glycerol formal, i.p.) alone (14.1+/-6.3 versus 9.6+/-3.2 days, p = 0.036), whereby at lower dosage (100 mg/kg/day) no such effect was observed (10.2+/-2.1 days, p = 0.21). Our findings suggest that stable prodrugs of n-butyric acid might have potential clinical relevance for inhibiting alloresponses in vivo.
Extrathymic T cell deletion and allogeneic stem cell engraftment induced with costimulatory blockade is followed by central T cell tolerance.
A reliable, nontoxic method of inducing transplantation tolerance is needed to overcome the problems of chronic organ graft rejection and immunosuppression-related toxicity. Treatment of mice with single injections of an anti-CD40 ligand antibody and CTLA4Ig, a low dose (3 Gy) of whole body irradiation, plus fully major histocompatibility complex-mismatched allogeneic bone marrow transplantation (BMT) reliably induced high levels (>40%) of stable (>8 mo) multilineage donor hematopoiesis. Chimeric mice permanently accepted donor skin grafts (>100 d), and rapidly rejected third party grafts. Progressive deletion of donor-reactive host T cells occurred among peripheral CD4(+) lymphocytes, beginning as early as 1 wk after bone marrow transplantation. Early deletion of peripheral donor-reactive host CD4 cells also occurred in thymectomized, similarly treated marrow recipients, demonstrating a role for peripheral clonal deletion of donor-reactive T cells after allogeneic BMT in the presence of costimulatory blockade. Central intrathymic deletion of newly developing T cells ensued after donor stem cell engraftment had occurred. Thus, we have shown that high levels of chimerism and systemic T cell tolerance can be reliably achieved without myeloablation or T cell depletion of the host. Chronic immunosuppression and rejection are avoided with this powerful, nontoxic approach to inducing tolerance.
Lung transplantation for primary pulmonary hypertension and giant pulmonary artery aneurysm.
We report the case of an 18-year-old patient with a giant pulmonary artery aneurysm and primary pulmonary hypertension who was successfully treated with bilateral lung transplantation and complete reconstruction of the pulmonary artery.
Mixed chimerism for the induction of tolerance: potential applicability in clinical composite tissue grafting.
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Hematopoietic chimerism and tolerance of T cells, B cells, and NK cells.
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Intrahepatic splenic tissue in a patient with recurrent idiopathic thrombocytopenic purpura.
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Separate regulation of peripheral hematopoietic and thymic engraftment.
Long-term multilineage chimerism, indicating pluripotent hematopoietic stem cell engraftment, was achieved in an Ly5-congenic strain combination without irradiation or other host conditioning when a large number of donor marrow cells (1.4-2x10(8)) was administered. However, the initial (2-4 weeks posttransplantation) percentages of T and B lymphocytes of donor origin were markedly lower than those of myeloid lineages. Steady-state levels of donor and host repopulation of all lineages were reached by 7 to 15 weeks posttransplantation and remained relatively constant for at least 41 weeks. B cell chimerism was similar to that seen in myeloid lineages at steady state. In contrast, long-term donor representation in the T cell lineage was much lower than in the B cell or myeloid lineages. Host treatment with depleting anti-T cell monoclonal antibodies increased the donor contribution to early T cell repopulation, but long-term T cell chimerism was still significantly lower in all lymphohematopoietic tissues, including the thymus, than B cell or myeloid cell chimerism. Pretreatment of hosts with 3.5 Gy of local irradiation to the thymic region further increased the donor contribution to initial T cell repopulation, which equaled that of other lineages at 4 to 7 weeks. However, donor representation in the T cell lineage declined by the time steady-state chimerism was attained and was lower than donor representation in the B cell or myeloid lineages. A higher dose of thymic irradiation (7 Gy) led to a reduction in this discrepancy, so that levels of donor thymopoiesis and hematopoiesis in other lineages were similar by 23 to 27 weeks posttransplantation. The differential contribution of adult donor marrow to long-term, steady-state thymopoiesis vs. hematopoiesis in other lineages under certain conditions in this competitive repopulation assay suggests that functionally distinguishable progenitors are responsible for these activities.
Downsizing of the donor lung: peripheral segmental resections and lobar transplantation.
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Incidence and outcome of major non-pulmonary surgical procedures in lung transplant recipients.
OBJECTIVE: Pulmonary transplant recipients are at high risk from various conditions requiring surgical intervention. As little is known about their exact incidence and course, we examined such procedures in detail. METHODS AND PATIENTS: We have retrospectively analyzed major nonpulmonary surgical procedures performed in 124 consecutive patients who received an isolated lung transplant at the University of Vienna between 1989 and December 1995. Twenty-two patients underwent a total of 28 major interventions (22/124 = 17.7%), resulting in an incidence of one procedure every 5.8 patient years of follow-up. The mean interval between transplantation and intervention was 17.9 months (range 3 days to 62 months) with six interventions being carried out during the first month after transplantation. Fourteen emergency operations were performed, the remaining 14 procedures were carried out electively. Overall, 15 abdominal procedures, four thoracic, four orthopedic, two gynecological, one neurosurgical, one urological and one plastic surgery were performed. RESULTS: There was no intraoperative death. Perioperatively, five surgery related deaths were observed (5/28, related mortality 17.9%) with multiple organ failure as the cause of death in all cases. All of these deaths followed emergency operations (5/14 = 35.7%) and all were observed in patients with septic abdominal complications. In contrast, even very extensive procedures were performed electively without related mortality (0/14, P = 0.02). During the first month after transplantation, major surgery was associated with a 50% (3/6) mortality, for late interventions mortality was 9.1% (2/22; P = 0.047). CONCLUSIONS: Pulmonary transplant recipients showed a high incidence of conditions requiring surgical intervention. As expected, septic complications, especially during the immediate post transplant period, carried a very poor prognosis. However, it was reassuring to observe that even extensive surgical procedures could be performed safely without associated mortality in the elective setting.