Anette Wu

Tolerance to musculoskeletal allografts with transient lymphocyte chimerism in miniature swine

Background. Although transplantation of musculoskeletal allografts in humans is technically feasible, the adverse effects of long-term immunosuppression subject the patient to high risks for correcting a non-life-threatening condition. Achieving immunologic tolerance to musculoskeletal allografts, without the need for chronic immunosuppression, could expand the clinical application of limb tissue allografting. Tolerance to musculoskeletal allografts has been accomplished previously in miniature swine in our laboratory. Although stable, mixed chimerism has been suggested as the mechanism underlying long-term tolerance in a rat limb model, the mechanism of this tolerance induction has not been established. This report explores the possible relationship between hematopoietic chimerism and tolerance to musculoskeletal allografts in swine. Methods. Twelve miniature swine underwent vascularized musculoskeletal allograft transplantation from histocompatibility complex (MHC) matched, minor antigen-mismatched donors. Eight animals received a 12-day course of cyclosporine, one of which was excluded due to subtherapeutic levels. Four recipients were not immunosuppressed. Serial biopsies to assess graft viability and flow cytometry to assess chimerism were performed. Donor and third-party skin grafts were placed on recipients with surviving allografts greater than 100 days to validate tolerance. Results. Both groups developed early peripheral chimerism, but this chimerism became undetectable by postoperative day 19 in the cyclosporine group and by day 13 in the control group. Animals receiving cyclosporine developed permanent tolerance to their allografts, whereas those not receiving cyclosporine rejected their allografts in 6–9 weeks. Animals demonstrating tolerance to their bone allografts also demonstrated prolonged donor skin graft survival. Conclusions. Induction of tolerance to musculoskeletal allografts can be achieved in the MHC matched swine. Although hematopoietic chimerism is present in the immediate postoperative period, persistent, long-term chimerism does not seem to be necessary for maintenance of such tolerance.

Xenogeneic thymus transplantation in a pig-to-baboon model.

Background. We have tested whether fetal porcine thymic tissue transplantation can lead to tolerance across a discordant (pig-to-baboon) xenogeneic barrier. Methods. Six baboons underwent a conditioning regimen with thymectomy, splenectomy, and anti-monkey CD3 antibody conjugated to a diphtheria toxin binding site mutant (FN18-CRM9). Porcine fetal or neonatal thymic tissue was transplanted into three baboons. Three control baboons received either no transplanted pig tissue (n=1) or adult pig lymph node (n=2). Cellular responses and skin xenografts were used to test for tolerance. Results. Experimental baboons: After T-cell depletion and thymic transplantation, recovery of thymus-dependent naïve-type CD4 cells (CD4/CD45RAhigh) and in vitro xenogeneic hyporesponsiveness were observed. No sensitization of alpha-galactosyl antibody responses was observed. The thymic grafts survived up to 48 days. Porcine skin xenografts were performed in two of these animals with survival of 22 and 24 days. Control baboons: Only two of these animals were completely T-cell depleted, and both failed to recover thymus-dependent T cells (CD4/CD45RAhigh). In one animal, general in vitro hyporesponsiveness was observed, with subsequent death from infection. The second animal demonstrated delayed recovery of T cells and prolonged general hyporesponsiveness in vitro. Neither animal demonstrated prolongation of porcine skin grafts compared with allografts (both rejected by day 13). Conclusions. Porcine thymic tissue is able to induce xenogeneic hyporesponsiveness. More efficient thymic engraftment may allow this approach to induce xenograft tolerance.

Effect of ex vivo Expanded Recipient Regulatory T Cells on Hematopoietic Chimerism and Kidney Allograft Tolerance Across MHC Barriers in Cynomolgus Macaques

BackgroundInfusion of recipient regulatory T (Treg) cells promotes durable mixed hematopoietic chimerism and allograft tolerance in mice receiving allogeneic bone marrow transplant (BMT) with minimal conditioning. We applied this strategy in a Cynomolgus macaque model. MethodsCD4+ CD25high Treg cells that were polyclonally expanded in culture were highly suppressive in vitro and maintained high expression of FoxP3. Eight monkeys underwent nonmyeloablative conditioning and major histocompatibility complex mismatched BMT with or without Treg cell infusion. Renal transplantation (from the same BMT donor) was performed 4 months post-BMT without immunosuppression to assess for robust donor-specific tolerance. ResultsTransient mixed chimerism, without significant T cell chimerism, was achieved in the animals that received BMT without Treg cells (N = 3). In contrast, 2 of 5 recipients of Treg cell+ BMT that were evaluable displayed chimerism in all lineages, including T cells, for up to 335 days post-BMT. Importantly, in the animal that survived long-term, greater than 90% of donor T cells were CD45RA+ CD31+, suggesting they were new thymic emigrants. In this animal, the delayed (to 4 months) donor kidney graft was accepted more than 294 days without immunosuppression, whereas non–Treg cell BMT recipients rejected delayed donor kidneys within 3 to 4 weeks. Early CMV reactivation and treatment was associated with early failure of chimerism, regardless of Treg cell administration. ConclusionsOur studies provide proof-of-principle that, in the absence of early CMV reactivation (and BM-toxic antiviral therapy), cotransplantation of host Treg cell can promote prolonged and high levels of multilineage allogeneic chimerism and robust tolerance to the donor.

Stable mixed hematopoietic chimerism permits tolerance of vascularized composite allografts across a full major histocompatibility mismatch in swine

This study tested the hypothesis that vascularized composite allografts (VCA) could be accepted in a robust model of hematopoietic chimerism by injecting allogeneic bone marrow cells (BMC) into swine fetuses. Outbred Yorkshire sows and boars were screened to ensure the absence of the major histocompatibility (MHC) allele SLAcc of inbred MGH miniature swine and then mated. Bone marrow harvested from an SLAcc swine donor was T‐cell depleted and injected intravenously into the fetuses between days 50–55 of gestation. After birth, the piglets were studied with flow cytometry to detect donor cells and mixed lymphocyte reactions (MLR) and cell‐mediated lympholysis (CML) assays to assess their response to donor. Donor‐matched VCAs from SLAcc donors were performed on four chimeric and two nonchimeric swine. The results showed donor cell engraftment and multilineage macrochimerism after the in utero transplantation of adult BMC, and chimeric animals were unresponsive to donor antigens in vitro. Both control VCAs were rejected by 21 days and were alloreactive. Chimeric animals accepted the VCAs and never developed antidonor antibodies or alloreactivity to donor. These results confirm that the intravascular, in utero transplantation of adult BMC leads to donor cell chimerism and donor‐specific tolerance of VCAs across a full MHC barrier in this animal model.

Xenogeneic thymic replacement to achieve immune restoration in HIV infection

Abstract The thymus is a target of human immunodeficiency virus (HIV)-1 infection in humans, and destruction of thymic progenitors and of the thymic microenvironment by HIV may preclude the generation of functional naive T cells to repopulate the peripheral CD4 T cell pool in HIV-infected people. Xenogeneic thymic grafting might provide a useful strategy for overcoming this obstacle, as xenogeneic thymi might resist HIV infection, and xenogeneic donors could provide essentially unlimited amounts of thymic tissue. We have recently demonstrated that a highly disparate (porcine) xenogeneic thymus graft can be used to replace the host thymus to reconstitute CD4 + T cells in thymectomized, T cell-depleted mice, and that these CD4 + T cells that repopulate the periphery after developing in porcine thymic xenografts are fully functional, confer resistance to opportunistic infections, and show specific tolerance to the porcine donor and the recipient. Studies in immunodeficient mice show that porcine thymic tissue is likewise capable of generating mature, functional human T cells from hematopoietic progenitors, and that these human cells are specifically tolerant of the porcine donor. Preliminary studies suggest that porcine thymic tissue may confer resistance to the destructive effects of HIV-1 infection on human thymopoiesis. Attempts to apply this approach in nonhuman primates, including simian immunodeficiency virus (SIV)-infected rhesus monkeys, have demonstrated the importance of overcoming the powerful recipient anti-pig T cell response in order to avoid rejection of porcine thymic grafts. However, preliminary results provide encouragement that a tolerant primate T cell repertoire could be generated in a porcine thymic xenograft if this initial immune response were fully prevented.

Regulatory cells in the maintenance phase of tolerance in miniature swine: coculture cell mediated lympholysis assay as approach for a tolerance assay

that CM/IFA-injected animals grafted with MHC class II KO hearts experienced significant prolongation of heart survival as compared to untreated mice (11 1 d vs 24 2 d). We conclude that modulation of CM response can on its own achieve graft prolongation when CD4 alloresponse is oligoclonal, i.e. mediated via indirect allorecognition. Our findings have important implications for the design of future therapies in heart transplantation.