Xenotransplantation of pig islets into Mexican children: were the fundamental ethical requirements to proceed with such a study really met?
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Animals↗
Biomedical subjects
Megan Sykes
Publications and source records attributed to Megan Sykes.
Vascularized thymic lobe transplantation in a pig-to-baboon model: a novel strategy for xenogeneic tolerance induction and T-cell reconstitution.
BACKGROUND: This laboratory has previously demonstrated the induction of allogeneic tolerance by vascularized thymic lobe (VTL) transplantation in miniature swine. We report here our initial attempt to induce tolerance by VTL transplantation in the clinically relevant, discordant, pig-to-baboon model of xenotransplantation. METHODS: Six baboons received xenografts of hDAF VTLs. Four of these baboons also received omental thymic tissue implants. All recipients were treated with an immunosuppressive conditioning regimen that included thymectomy, splenectomy, extracorporeal immunoadsorption of anti-alpha Gal antibodies, and T-cell depletion. Two control baboons received sham operations, of which one also received 5x10 hDAF porcine thymocytes/kg intravenously. RESULTS: Transplanted VTL grafts supported early thymopoiesis of recipient-type immature thymocytes, and facilitated engraftment of nonvascularized thymic omental implants. Recipients of the VTL grafts demonstrated donor-specific unresponsiveness in MLR assays, development of peripheral CD45RAhigh/CD4 double positive (DP) cells, and positive cytokeratin staining of thymic stroma in the grafts for 2 months following xenotransplantation. The control baboons did not show these markers of thymic reconstitution. The eventual return of Gal natural antibodies led to the destruction of graft epithelial cells and the rejection of all VTL grafts by 3 months posttransplantation. CONCLUSIONS: VTL transplantation from hDAF swine to baboons induced early thymopoiesis in the recipients and donor-specific cellular unresponsiveness in vitro. When coupled with additional strategies aimed at silencing humoral rejection, VTL transplantation may significantly prolong xenograft survival and result in long-term tolerance.
Animals↗
Maturation of human monocyte-derived dendritic cells (MoDCs) in the presence of prostaglandin E2 optimizes CD4 and CD8 T cell-mediated responses to protein antigens: role of PGE2 in chemokine and cytokine expression by MoDCs.
Prostaglandin E2 (PGE2) acts in synergy with other inflammatory stimuli such as tumor necrosis factor (TNF) to induce the maturation of migratory-type monocyte-derived dendritic cells (MoDCs). However, PGE2 has been reported to inhibit IL-12p70 production by MoDCs and to promote the generation of Th2 T cell responses. We demonstrate here that the addition of PGE2 to TNF for the maturation of MoDCs enhanced CD4 and CD8 T cell proliferative responses to neoantigen and recall antigen, and enhanced Th1-type responses. The increased stimulatory capacity of MoDCs matured with PGE2 was associated with a fully mature, migratory-type MoDC phenotype and more rapid down-regulation of the expression of inflammatory chemokines, with up-regulated expression of the constitutive chemokines TARC and MDC. In addition, although MoDCs matured with TNF and PGE2 selectively produced the inhibitory IL-12p40 subunit at steady state, they were able to produce the bioactive IL-12p70 heterodimer after stimulation with CD40 ligand and/or IFN-gamma. Despite increased IL-6 mRNA expression, MoDCs matured with PGE2 did not overcome the suppressive effects of CD4+ CD25+ T cells in allogeneic mixed lymphocyte reactions. In conclusion, MoDCs matured in the presence of PGE2 display characteristics of more efficient antigen-presenting cells that might be optimal for use in cancer vaccine-based clinical trials.
Antigen Presentation↗
Use of skin grafting to demonstrate tolerance before kidney transplantation without immunosuppression in the recipient of a previous bone marrow transplant.
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Bone Marrow Transplantation↗
Short-term immunosuppression facilitates induction of mixed chimerism and tolerance after bone marrow transplantation without cytoreductive conditioning.
BACKGROUND: Induction of mixed chimerism and tolerance usually requires cytoreduction or transplantation of high numbers of bone marrow cells (BMC). However, such protocols have only a suboptimal success rate and, more importantly, equivalent numbers of BMC cannot be routinely obtained in the clinical setting. The authors therefore evaluated whether a short-course of immunosuppression (IS) given in addition to co-stimulation blockade would facilitate chimerism induction and allow reduction of the minimally required number of BMC without cytoreduction. METHODS: B6 mice received 200, 100, or 50 x 10 unseparated BMC from Balb/c donors plus an anti-CD40L monoclonal antibody (mAb) and CTLA4Ig (without irradiation or cytotoxic drugs). Some groups were treated additionally with IS (rapamycin, methylprednisolone, and mycophenolate mofetil for 4 weeks after bone marrow transplantation), donor-specific transfusion (DST), or anti-OX40L mAb, as indicated. RESULTS: IS led to long-term multilineage chimerism in 9 of 10 mice receiving 200 x 10 BMC (without IS, 1 of 4; P<0.05), in all mice (n=10) receiving 100 x 10 (without IS, 6 of 9; P<0.05), and notably in 9 of 10 mice treated with 50 x 10 BMC (without IS, 4 of 10; P<0.05). With transient IS, donor skin grafts were accepted longer than 170 days in 9 of 10 mice receiving 200 x 10 (without IS, 0 of 5 mice; P<0.05), all mice receiving 100 x 10 (without IS, 6 of 9; P<0.05), and 6 of 11 mice receiving 50 x 10 BMC (without IS, 4 of 10). The use of DST or anti-OX40L mAb had no beneficial effect. CONCLUSIONS: Transient IS significantly improves rates of chimerism and donor skin graft survival, and allows lasting mixed chimerism after transplantation of only 50 x 10 BMC. Thus, IS might help in the further development of noncytoreductive chimerism protocols.
Abatacept↗
Mechanisms of the antitumor responses and host-versus-graft reactions induced by recipient leukocyte infusions in mixed chimeras prepared with nonmyeloablative conditioning: a critical role for recipient CD4+ T cells and recipient leukocyte infusion-derived IFN-gamma-producing CD8+ T cells.
Surprisingly, antitumor responses can occur in patients who reject donor grafts following nonmyeloablative hemopoietic cell transplantation. In murine mixed chimeras prepared with nonmyeloablative conditioning, we previously showed that recipient leukocyte infusions (RLI) induced loss of donor chimerism, IFN-gamma production, and antitumor responses against host-type tumors. However, the mechanisms behind this phenomenon remain to be determined. We now demonstrate that the effects of RLI are mediated by distinct and complex mechanisms. Donor marrow rejection is induced by RLI-derived alloactivated T cells, which activate non-RLI-derived, recipient IFN-gamma-producing cells. RLI-derived CD8 T cells induce the production of IFN-gamma by both RLI and non-RLI-derived recipient cells. The antitumor responses of RLI involve mainly RLI-derived IFN-gamma-producing CD8 T cells and recipient-derived CD4 T cells and do not involve donor T cells. The pathways of donor marrow and tumor rejection lead to the development of tumor-specific cell-mediated cytotoxic responses that are not due to bystander killing by alloreactive T cells.
Animals↗
Roles of deletion and regulation in creating mixed chimerism and allograft tolerance using a nonlymphoablative irradiation-free protocol.
The induction of mixed chimerism (MC) is a powerful and effective means to achieve transplantation tolerance in rodent models. Host conditioning with irradiation or cytotoxic drugs has been used in many protocols for chimeric induction across allogeneic barriers. The deletion of alloreactive T cell clones has been described as the main mechanism responsible for the induction of a stable MC. In this study, we demonstrate that a stable MC and skin allograft tolerance can be established across MHC barriers by a noncytotoxic, irradiation-free approach using costimulation blockade plus rapamycin treatment. By using an adoptive transfer model of skin allograft and using specific Vbeta TCR probes, we demonstrated that deletion of donor-reactive cytopathic T cell clones is indeed profound in tolerant hosts. Nonetheless, the challenge of tolerant mixed chimeras with 5 million mononuclear leukocytes (MNL) from naive syngeneic mice was neither able to abolish the stable MC nor to trigger skin allograft rejection, a hallmark of peripheral, not central tolerance. Furthermore, in an adoptive transfer model, MNLs harvested from tolerant hosts significantly inhibited the capacity of naive MNLs to reject same donor, but not third-party, skin allografts. Moreover, when we transplanted skin allografts from stable tolerant chimeras onto syngeneic immune-incompetent mice, graft-infiltrating T cells migrated from the graft site, expanded in the new host, and protected allografts from acute rejection by naive syngeneic MNLs. In this model, both deletional and immunoregulatory mechanisms are active during the induction and/or maintenance of allograft tolerance through creation of MC using a potentially clinically applicable regimen.
Adoptive Transfer↗
Treatment of severe autoimmune disease by stem-cell transplantation.
Transplantation of haematopoietic stem cells--cells capable of self renewing and reconstituting all types of blood cell--can treat numerous lethal diseases, including leukaemias and lymphomas. It may now be applicable for the treatment of severe autoimmune diseases, such as therapy-resistant rheumatoid arthritis and multiple sclerosis. Studies in animal models show that the transfer of haematopoietic stem cells can reverse autoimmunity, and several mechanistic pathways may explain this phenomenon. The outcome of ongoing clinical trials, as well as of studies in patients and animal models, will help to determine the role that stem-cell transplantation can play in the treatment of autoimmune diseases.
Animals↗
Commentary: World Health Assembly resolution 57.18 on xenotransplantation.
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Animals↗
Mixed hematopoietic chimerism for the simultaneous induction of T and B cell tolerance.
Nonmyeloablative induction of mixed hematopoietic chimerism provides a strategy for inducing T cell tolerance across allogeneic and xenogeneic barriers. We have utilized alpha1-3Gal transferase (GalT) knockout mice, which, like humans, produce anti-Gal natural antibodies, to investigate the ability of mixed chimerism to tolerize B cells producing antibodies of this important specificity, which limits xenotransplantation by causing hyperacute and delayed xenograft rejection. Mixed allogeneic or xenogeneic chimerism indeed tolerizes both preexisting anti-Gal-producing B cells and those developing de novo after establishment of mixed chimerism, even in presensitized mice. We present evidence that different mechanisms are involved in the tolerization of the preexisting and newly-developing antibody-secreting cells.
Animals↗
Early regulation of CD8 T cell alloreactivity by CD4+CD25- T cells in recipients of anti-CD154 antibody and allogeneic BMT is followed by rapid peripheral deletion of donor-reactive CD8+ T cells, precluding a role for sustained regulation.
While acquisition of regulatory function by CD4+CD25- T cells has been reported following antigenic stimulation, "naturally occurring" regulatory CD4+ T cells (Treg) are believed to express CD25. We examined the mechanisms involved in peripheral CD8 T cell tolerance by induction of mixed chimerism using non-myeloablative conditioning with low-dose (3 Gy) total body irradiation and anti-CD154 antibody. Recipient CD4+ T cells were initially required for the induction of CD8 cell tolerance, but were not needed beyond 2 weeks. Depletion of CD25+ Treg prior to bone marrow transplantation and blockade of IL-2 with neutralizing antibody did not impede tolerance induction. Tolerance was dependent on CTLA4, but not on IFN-gamma. In C57BL/6 mice containing a fraction of 2C TCR transgenic CD8+ T cells, which recognize the MHC class I alloantigen Ld, induction of chimerism with L(d+), but not Ld-, bone marrow cells led to deletion of peripheral 2C+ CD8+ cells within 1 week in peripheral blood and spleen. Complete deletion required the presence of recipient CD4+ T cells. Thus, a novel, rapid form of regulation by CD4+CD25- T cells permits initial CD8 T cell tolerance in this model. Rapid peripheral deletion of donor-specific CD8 T cells precludes an ongoing requirement for CD4 T cell-mediated regulation.
Animals↗
Role of VLA-4 and VLA-5 in ex vivo maintenance of human and pig hematopoiesis in human stroma-supported long-term cultures.
OBJECTIVE: The advantage of recipient hematopoiesis over that of xenogeneic donors poses a fundamental obstacle to the induction of xenograft tolerance through mixed hematopoietic chimerism. Here we explore the role of beta1 integrins in maintenance of human vs porcine hematopoiesis within a human hematopoietic environment. METHODS: Porcine and human c-kit+ bone marrow cells were purified and cultured on human bone marrow stroma for 6 weeks. The role of VLA-4 and VLA-5 in the maintenance of porcine vs human hematopoiesis in this human stroma-supported long-term bone marrow culture (LTBMC) system was evaluated by using blocking mAbs that bind to both species. RESULTS: Blocking VLA-4 with HP2/1 inhibited both human and porcine hematopoiesis, whereas anti-VLA-5 (SAM-1) suppressed the function of human, but not porcine, hematopoietic cells. In mixed LTBMC of porcine and human cells on a human stroma, porcine hematopoietic cells were at a competitive disadvantage, as seen by a rapid decline in cellularity, including clonogenic progenitors. This disadvantage was substantially overcome by the addition of SAM-1. Furthermore, human, but not porcine, cell adhesion to human fibronectin was inhibited by arginine-glycine-aspartic acid (RGD) peptides. CONCLUSION: Taken together, these results indicate that VLA-4 plays critical role for porcine hematopoiesis in a human hematopoietic environment, and raise the possibility that porcine VLA-5 might be unable to bind the respective human ligand and/or to initiate adequate post-ligand-binding signaling. Thus, VLA-5 may provide a potential target for developing approaches to improve porcine hematopoiesis in human recipients.
Animals↗
T-cell P/E-selectin ligand alpha(1,3)fucosylation is not required for graft-vs-host disease induction.
OBJECTIVE: Recognition of E- and P-selectins on vascular endothelium by their leukocyte glycoprotein counterreceptor P-selectin glycoprotein ligand-1 (PSGL-1) initiates and sustains leukocyte rolling, culminating in extravasation of lymphocytes from blood into organs. PSGL-1 is rendered functional by terminal glycosylation steps, which occur mainly in activated Th1 but not Th2 cells. alpha(1,3)Fucosyltransferases IV and VII control this glycosylation pathway. Mice lacking these transferases (Fuc-TIV(-/-)/Fuc-TVII(-/-)) lack functional E- and P-selectin ligands. We hypothesized that Fuc-TIV(-/-)/Fuc-TVII(-/-) donor T cells might have reduced capacity to roll on vessels of inflamed target tissues and mediate graft-vs-host disease (GVHD). MATERIALS AND METHODS: We compared the ability of Fuc-TIV(-/-)/Fuc-TVII(-/-) and wild-type (WT) C57BL/6 (B6) spleen cells (SPCs) to produce GVHD in lethally irradiated major histocompatibility complex (MHC) haplotype-mismatched B6D2F1 recipients. Clinical GVHD, GVHD pathology in target organs, memory phenotype conversion, proliferation of donor T cells, and tissue and serum cytokine expression were examined. RESULTS: Surprisingly, clinical GVHD was not reduced in lethally irradiated mice receiving full haplotype MHC mismatched or matched Fuc-TIV(-/-)/Fuc-TVII(-/-) SPCs compared to those receiving WT SPCs. GVHD pathology in target organs, memory phenotype conversion, and proliferation of donor T cells were similar in both groups. However, reduced interferon-gamma was detected in liver and lung, and serum levels of tumor necrosis factor-alpha were higher in mice receiving Fuc-TIV(-/-)/Fuc-TVII(-/-) SPCs compared with WT SPCs. CONCLUSIONS: These results suggest that donor T cells, including Th1, are capable of trafficking to GVHD target tissues independently of P- and E- selectin ligand in conditioned hosts.
Animals↗
Host thymectomy and cyclosporine lead to unstable skin graft tolerance after class I mismatched allogeneic neonatal thymic transplantation in mice.
BACKGROUND: Our laboratory has demonstrated that xenogeneic porcine thymus tissue grafted in thymectomized (ATX) and T cell-depleted mice induces donor-specific tolerance. Recipient thymectomy is essential for the success of tolerance induction. In contrast, studies in pigs grafted with non-vascularized allogeneic class I mismatched thymus tissue under the cover of CyA have shown that removal of host thymus is detrimental to thymic graft survival. To determine the requirements for nonvascularized allogeneic class I-mismatched thymic engraftment in mice, we performed thymic allotransplantation under the cover of CyA. MATERIALS AND METHODS: Euthymic and ATX B10.MBR mice received class I mismatched B10.AKM neonatal mouse thymus (NMTHY) tissue under the kidney capsule with or without a short course of CyA. The grafts were allowed to engraft for two and a half months before exploratory laparotomy was performed to evaluate them. Three months after the thymic transplant, mice were challenged with donor-specific skin grafts to assess tolerance. One month after donor-specific skin grafting, they received third party B10.BR skin grafts. Cellular anti-donor immune responses were studied at the time of euthanasia. RESULTS: CyA-treated ATX and euthymic control mice showed good engraftment of the allogeneic thymic tissue at the time of exploratory laparotomy, whereas non-CyA-treated ATX and euthymic controls had rejected the grafts. The CyA-treated ATX B10.MBR mice accepted donor-specific skin grafts, but rejected them following a challenge with third party B10.BR skin grafts. Untreated ATX and euthymic mice and 6 of 7 CyA-treated euthymic mice rejected donor skin within 15 days. Mixed lymphocyte reactions did not show an increased anti-donor response, but CML clearly showed sensitization and increased killing activity against donor-type targets in these mice. CONCLUSION: Allogeneic thymic transplantation across a class I MHC barrier under the cover of CyA induces a metastable state of tolerance in mice. To achieve this state, ATX of the recipient is required.
Animals↗
Protecting the host naturally.
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Animals↗
Organ transplantation--how much of the promise has been realized?
Since the introduction of organ transplantation into medical practice, progress and optimism have been abundant. Improvements in immunosuppressive drugs and ancillary care have led to outstanding short-term (1--3-year) patient and graft survival rates. This success is mitigated by several problems, including poor long-term (>5-year) graft survival rates, the need for continual immunosuppressive medication and the discrepancy between the demand for organs and the supply. Developing methods to induce transplant tolerance, as a means to improve graft outcomes and eliminate the requirement for immunosuppression, and expanding the pool of organs for transplantation are the major challenges of the field.
Animals↗
Anti-tumour response despite loss of donor chimaerism in patients treated with non-myeloablative conditioning and allogeneic stem cell transplantation.
Based on a murine model, we conducted a series of trials of m-myeloablative human leucocyte antigen (HLA)-matched or mismatched related donor stem cell transplantation (SCT) with the intention of inducing mixed chimaerism (MC), then administering prophylactic donor lymphocyte infusions (DLIs), for the treatment of advanced haematologic malignancies. Preparative therapy consisted of cyclophosphamide, equine anti-thymocyte globulin (ATG) or MEDI-507 (an anti-CD2 monoclonal antibody) for in-vivo T-cell depletion, thymic irradiation on day -1 and cyclosporine alone for graft-versus-host disease (GVHD) prophylaxis. DLIs were given as early as 5 weeks post-SCT in patients with MC without evidence of GVHD. Twenty-two patients ultimately lost their graft (<1% donor cells) that could no be rescued by DLIs. Nine of 22 (41%) patients who lost donor chimaerism achieved an objective response, including three patients who showed evidence of disease regression following DLI, despite continued absence of macrochimaerism. Six patients were alive at 2.5-5.5 years following SCT, including four in continuous complete remission. In summary, it is possible to achieve sustained remission in patients with chemorefractory malignancies following non-myeloablative allogeneic SCT, even in the absence of sustained donor macrochimaerism; DLI may contribute to an ongoing anti-tumour effect in these patients. Immunological mechanisms that correlated with rejection of the graft may have a role in anti-tumour responses via a cell or cytokine-mediated pathway.
Adult↗
Induction of mixed vs full chimerism to potentiate GVL effects after bone-marrow transplantation.
Graft-vs-host (GVH) alloresponses mediated by delayed donor lymphocyte infusions (DLI) can occur in the absence of GVHD (graft-vs-host disease). These GVH responses are confined to the lymphohematopoietic system and mediate graft-vs-leukemia (GVL) reactions without causing GVHD. Although interaction of donor T-cells with host-derived antigen-presenting cells (APC) is central to the development of GVHD, we were able to show that this T-cell/APC interaction is also critical for the induction of GVL, which can occur in the absence of GVHD if donor T-cell administration is delayed to a time point after bone-marrow transplant (BMT) when conditioning-induced inflammation has subsided. Induction of mixed hematopoietic chimerism using the method described below allows analysis of these interactions between host APC and donor T-cells.
Animals↗