Cutaneous leukocyte lineages in tolerant large animal and immunosuppressed clinical vascularized composite allograft recipients.

Vascularized composite allografts (VCAs) can restore fully functional anatomic units in patients with limb amputations or severe facial tissue loss. However, acute rejection of the skin is frequently observed and underscores the importance of developing tolerance induction protocols. In this study, we have characterized the skin immune system in VCAs.

Topical Delivery of Immunosuppression to Prolong Xenogeneic and Allogeneic Split-Thickness Skin Graft Survival.

Cadaveric skin allograft is the current standard of treatment for temporary coverage of large burn wounds. Porcine xenografts are viable alternatives but undergo α-1,3-galactose (Gal)-mediated hyperacute rejection and are lost by post-operative day (POD) 3 because of naturally occurring antibodies to Gal in primate recipients. Using baboons, we previously demonstrated that xenografts from GalT-KO swine (lacking Gal) provided wound coverage comparable with allografts with systemic immunosuppression.

Penis Transplantation: First US Experience.

Objective: We describe the first successful penis transplant in the United States in a patient with a history of subtotal penectomy for penile cancer.

Background: Penis transplantation represents a new paradigm in restoring anatomic appearance, urine conduit, and sexual function after genitourinary tissue loss. To date, only 2 penis transplants have been performed worldwide.

Genetically modified porcine split-thickness skin grafts as an alternative to allograft for provision of temporary wound coverage: preliminary characterization.

Temporary coverage of severely burned patients with cadaver allograft skin represents an important component of burn care, but is limited by availability and cost. Porcine skin shares many physical properties with human skin, but is susceptible to hyperacute rejection due to preformed antibodies to α-1,3-galactose (Gal), a carbohydrate on all porcine cells. Our preliminary studies have suggested that skin grafts from α-1,3-galactosyltransferase knock out (GalT-KO) miniature swine might provide temporary wound coverage comparable to allografts, since GalT-KO swine lack this carbohydrate.

The unique immunobiology of the skin: implications for tolerance of vascularized composite allografts.

Purpose Of Review: Vascularized composite allograft (VCA) transplantation restores function and form following major soft tissue and musculoskeletal injury. Lifelong immunosuppression is necessary for graft function and survival but acute skin-targeted rejection episodes remain common. We review recent advances in skin immunobiology, emphasizing findings in clinical and experimental VCAs.

Lack of cross-sensitization between α-1,3-galactosyltransferase knockout porcine and allogeneic skin grafts permits serial grafting.

Background: The current standard of care for burns requiring operative treatment consists of early burn excision and autologous split-thickness skin grafting. However, in large burns, sufficient donor sites may not be available to achieve total coverage, necessitating temporary coverage with allogeneic human cadaver skin grafts or synthetic skin substitutes. A previous study from this laboratory demonstrated that skin grafts from alpha-1,3 galactosyltransferase knockout (GalT-KO) miniature swine enjoyed survival comparable to that of allogeneic skin grafts in baboons.

Vascularized composite allotransplantation: towards tolerance and the importance of skin-specific immunobiology.

Purpose Of Review: Vascularized composite allotransplantation (VCA) is increasingly utilized in the restoration of complex injuries and tissue loss. Acute skin-targeted rejection episodes are common and concerns remain regarding the risks of conventional immunosuppression. We review current immunosuppressive regimens for VCA, progress with immunomodulatory and tolerance protocols, and highlight recent advances in cutaneous immunobiology which will have significant implications for future development in the field.

Tolerance induction strategies in vascularized composite allotransplantation: mixed chimerism and novel developments.

Since the start of the clinical vascularized composite allotransplantation (VCA) era over a decade ago this field has witnessed significant developments in both basic and translational research. Transplant tolerance, defined as rejection-free acceptance of transplanted organs or tissues without long-term immunosuppression, holds the potential to revolutionize the field of VCA by removing the need for life-long immunosuppression. While tolerance of organ and vascularized composite transplants may be induced in small animal models by a variety of protocols, only mixed-chimerism-based protocols have successfully bridged the gap to preclinical study and to clinical trial in solid organ transplantation to date.

B- and T-lymphocyte attenuator targeting protects against the acute phase of graft versus host reaction by inhibiting donor anti-host cytotoxicity.

Background: B- and T-lymphocyte attenuator (BTLA) functions as a coinhibitory/costimulatory molecule that belongs to the immunoglobulin superfamily and exhibits a pattern of expression restricted to the hematopoietic compartment. Engagement of BTLA by its ligand, herpes virus entry mediator (HVEM), delivers negative signals to T cells, whereas engagement of HVEM receptor on T cells by surface BTLA expressed on other immune cells costimulates T activation. Previous work has reported that parental donor BTLA knock-out or HVEM knock-out T cells adoptively transferred into nonirradiated F1 recipient mice survived poorly, and the rejection of host hematopoietic cells was attenuated compared with F1 recipients receiving wild-type T cells.

Hematopoietic stem cell transplantation for the treatment of autoimmunity in type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disease that leads to the destruction of the insulin-producing pancreatic b cells. While there is no current cure, recent work in the field of allogeneic hematopoietic stem cell transplantation (HSCT) and the induction of mixed chimerism, a state in which multilineage hematopoietic populations of both recipient and donor co-exist, has demonstrated that it is possible to provide protection from disease onset, as well as reverse the autoimmune state in spontaneously diabetic mice. Furthermore, the establishment of mixed chimerism induces donor-specific tolerance, providing the potential to normalize glucose regulation via pancreatic islet transplantation without the requirement of life-long immunosuppression.

A CD8 T cell-intrinsic role for the calcineurin-NFAT pathway for tolerance induction in vivo.

Previous studies have indicated that blockade of signaling through the T-cell receptor (TCR)/calcineurin/nuclear factor of activated T cells (NFAT) pathway impairs transplantation tolerance induced with anti-CD154 antibody. By using an allogeneic bone marrow transplantation model, we examined the role of the TCR/calcineurin/NFAT pathway for tolerance induction with anti-CD154. Calcineurin blockade by cyclosporine A led to a failure of CD8 but not CD4 tolerance, and experiments in NFAT1(-/-) mice replicated this effect.

CTLA-4 on alloreactive CD4 T cells interacts with recipient CD80/86 to promote tolerance.

Although the inhibitory receptor CTLA-4 (CD152) has been implicated in peripheral CD4 T-cell tolerance, its mechanism of action remains poorly defined. We analyzed mechanisms of CD4 cell tolerance in a model of tolerance induction involving establishment of mixed hematopoietic chimerism in recipients of fully MHC-mismatched allogeneic bone marrow cells with anti-CD154 mAb. Animals lacking CD80 and CD86 failed to achieve chimerism.

Rapid deletional peripheral CD8 T cell tolerance induced by allogeneic bone marrow: role of donor class II MHC and B cells.

Mixed chimerism and donor-specific tolerance are achieved in mice receiving 3 Gy of total body irradiation and anti-CD154 mAb followed by allogeneic bone marrow (BM) transplantation. In this model, recipient CD4 cells are critically important for CD8 tolerance. To evaluate the role of CD4 cells recognizing donor MHC class II directly, we used class II-deficient donor marrow and were not able to achieve chimerism unless recipient CD8 cells were depleted, indicating that directly alloreactive CD4 cells were necessary for CD8 tolerance.

Central tolerance to myogenic cell transplants does not include muscle neoantigens.

Background: Duchenne muscular dystrophy is a fatal genetic disease caused by lack of dystrophin. Myogenic cell transplantation (MT), a potential therapy for Duchenne muscular dystrophy, can restore dystrophin expression in muscles. Because allogeneic MT is highly resistant to peripheral tolerance, we proposed to induce central tolerance.

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.

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.

Tolerance in mixed chimerism - a role for regulatory cells?

The establishment of mixed hematopoietic chimerism induces life-long donor-specific organ graft tolerance while obviating the need for chronic immunosuppression. Recent advances have dramatically reduced the conditioning toxicity required to achieve mixed chimerism. We argue that the achievement of high levels of donor chimerism ensures life-long deletion of donor-reactive T cells, precluding and obviating the need for regulatory mechanisms in the maintenance of tolerance.

Mechanisms of early peripheral CD4 T-cell tolerance induction by anti-CD154 monoclonal antibody and allogeneic bone marrow transplantation: evidence for anergy and deletion but not regulatory cells.

Anti-CD154 (CD40L) monoclonal antibody (mAb) plus bone marrow transplantation (BMT) in mice receiving CD8 cell-depleting mAb leads to long-term mixed hematopoietic chimerism and systemic donor-specific tolerance through peripheral and central deletional mechanisms. However, CD4(+) T-cell tolerance is demonstrable in vitro and in vivo rapidly following BMT, before deletion of donor-reactive CD4 cells is complete, suggesting the involvement of other mechanisms. We examined these mechanisms in more detail.

Earlier low-dose TBI or DST overcomes CD8+ T-cell-mediated alloresistance to allogeneic marrow in recipients of anti-CD40L.

Treatment with a single injection of anti-CD40L (CD154) monoclonal antibody (mAb) and fully mismatched allogeneic bone marrow transplant (BMT) allows rapid tolerization of CD4+ T cells to the donor. The addition of in vivo CD8 T-cell depletion leads to permanent mixed hematopoietic chimerism and tolerance. We now describe two approaches that obviate the requirement for CD8 T-cell depletion by rapidly tolerizing recipient CD8 T cells in addition to CD4 cells.

Lack of role for CsA-sensitive or Fas pathways in the tolerization of CD4 T cells via BMT and anti-CD40L.

Anti-CD40L mAb plus bone marrow transplantation (BMT) and recipient CD8 T-cell depletion permits long-term mixed hematopoietic chimerism and systemic donor-specific tolerance to be achieved across full MHC barriers. Initial tolerance is characterized by peripheral deletion of donor-reactive CD4 cells. In regimens using costimulatory blockade without BMT to achieve allograft survival, cyclosporine inhibited graft survival, suggesting that the combination may not be clinically applicable.

The influence of immunosuppressive drugs on tolerance induction through bone marrow transplantation with costimulation blockade.

We recently developed a murine protocol for the induction of allogeneic mixed chimerism and tolerance employing nonmyeloablative total body irradiation (TBI), standard-dose bone marrow transplantation (BMT), and costimulation blockade (cobl) with an anti-CD154 monoclonal antibody (mAb) plus CTLA4Ig. We now evaluated whether a short course (1 month) of immunosuppressive drugs, which would be ethically required in the clinical setting of organ transplantation to prevent graft loss in case tolerance is not achieved, interferes with tolerance induced with this regimen. Our results show that calcineurin inhibitors (cyclosporin A [CyA] or tacrolimus [FK]) inhibit development of long-term chimerism and abrogate tolerance induction in this model.

Mechanisms of transplant tolerance induction using costimulatory blockade.

The potential use of costimulation-blocking reagents to induce transplantation tolerance has recently created considerable excitement. Recent evidence has begun to delineate the mechanisms by which these powerful effects occur. It has become increasingly clear, firstly, that T cell costimulation is mediated by a delicate network of signaling pathways and, secondly, that interference with these systems can lead to numerous different tolerance mechanisms, including immune regulation, anergy and deletion.