Madeline Fryer

Exploring cell-based tolerance strategies for hand and face transplantation

Broader clinical application of reconstructive hand and face transplantation is hindered by the need for lifelong immunosuppression for allograft maintenance. In this review, we summarize various cell-based approaches to tolerance induction currently under investigation in both clinical and pre-clinical models to alleviate the need for chronic immunosuppression. These include strategies to induce mixed hematopoietic chimerism, therapy with T and B regulatory cells, regulatory macrophages, tolerogenic dendritic cells, and mesenchymal stem cells. The vascularized, intragraft bone components inherent to reconstructive transplants serve as a continuous source of donor-derived hematopoietic cells, and make hand and face transplants uniquely well suited for cell-based approaches to tolerance that may ultimately tilt the risk–benefit balance for these life-changing, but not life-saving, procedures.

Murine Full-thickness Skin Transplantation

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited application to humans, skin transplantation in mice has been widely employed for transplantation research. The procedure is easy to learn and perform, and it does not require delicate microsurgical techniques nor extensive training. Moreover, graft rejection in this model occurs in a very reproducible immunological reaction and is easily monitored by direct inspection and palpation. In addition, secondary skin transplantation with donor-matched or third-party skin grafts can be performed on more complex transplant models as an alternative and uncomplicated method to assess donor-specific tolerance. The complications are low and are in general limited to anesthesia overdose or respiratory distress after the procedure. Graft failure, on the other hand, occurs commonly as a result of poor preparation of the graft, incorrect positioning in the graft bed, or inappropriate placement of the bandage. In this article, we present a protocol for full-thickness skin transplantation in mice and describe the important steps necessary for a successful procedure.

Ex vivo Expanded Regulatory T cells Combined with Short-term Costimulation Blockade Prevent Rejection of Vascularized Composite Allografts

Background Reconstructive transplantation represents a valid therapeutic option after devastating tissue loss. Routine clinical application, however, is hampered by the toxicity of long-term maintenance immunosuppression. The current study investigated a novel approach using ex vivo expanded regulatory T cells combined with a short-term immunomodulatory strategy in a murine hind limb transplantation model. Methods Fully MHC-mismatched orthotopic hind limb transplants were performed from Balb/C to C57BL/6 mice. Recipients in the experimental groups received a combination regimen consisting of 0.5mg CTLA4 Ig on day 0, 2, 4 and 6 post-transplant, 20mg/kg anti-Thy 1.2 mAb on POD-1, and 1mg/kg Rapamycin (POD 0-9), and in one group, 1 wk expanded CD4+CD25+ Treg cells. Allograft survival was monitored and flow cytometric analysis was performed to evaluate mixed chimerism and clonal deletion of alloreactive T cells. Treg activity was assessed in vitro using suppression assays in order to support and supplement in vivo data. Results Combination of T cell depletion and CTLA4-Ig plus short-course of Rapamycin increased VCA survival significantly while untreated controls rejected allografts (MST 105 days; Untreated, MST 9 days; CTLA4 Ig only, MST 17 days, Rapamycin, MST 20 days; T cell depletion, 20 days; p<0.01[GB1]). [GB2] Mixed chimerism was detected in recipients receiving this combined treatment protocol with 5.013 ± 1.23 % of CD11b+ cells being donor-derived on POD 55. V&bgr; – TCR staining profiles in recipients after full treatment showed 1.570 ± 0.3700 % of &ngr;&bgr;5+CD4+ T cells, while naïve C57BL/6 express 3.567 ± 0.3690 % of &ngr;&bgr;5+CD4+ T cells, suggesting the actuation of central deletion of developing donor-reactive T cells. In order to further prolong allograft survival, one week expanded Tregs were then included in the combination therapy. The suppressive activity of the CD4+CD25+ Tregs was confirmed with in vitro suppression assays. The addition of ex vivo expanded regulatory T cells further increased VCA survival to >200 days and induced long-term stable mixed chimerism[GB3] with 16.7±1.5 % of CD11b cells being donor-derived on POD 55 after administration of expanded Treg cells. Conclusion The combination of T cell depletion, costimulation blockade, and a short-course of Rapamycin prevents VCA rejection and significantly prolongs graft survival without the need for myeloablative conditioning or maintenance therapy. Moreover, regulatory T cells added in the early post transplant period further optimize immune regulation by inducing sustained mixed chimerism.

Split tolerance in a murine model of heterotopic En bloc chest wall transplantation

Background: Congenital and acquired chest wall deformities represent a significant challenge to functional reconstruction and may impact feasibility of heart transplantation for patients with end-stage organ failure. In the recent past, the concept of replacing like-with-like tissue by using vascularized composite allografts (VCA) has been enthusiastically employed for reconstruction of complex tissue defects. Methods: In this study, we introduce a novel murine model for en bloc chest wall, heart, and thymus transplantation and thereby the use of complex tissue allografts for reconstruction of both chest wall defects and also end-stage organ failure. Additionally, this model allows us to study the features of combined vascularized bone marrow (VBM), thymus, and heart transplantation on allograft survival and function. Heterotopic chest wall, thymus, and heart transplants were performed in untreated syngeneic and allogeneic combinations and in allogeneic combinations treated with costimulation blockade (CTLA4-Ig and MR-1). Results: Indefinite (ie, 150 d, N = 3) graft survival was observed in syngeneic controls. In untreated recipients of allogeneic grafts, the skin component was rejected after 10 (±1) days, whereas rejection of the heart occurred after 13 (± 1) days (N = 3). Costimulation blockade treatment prolonged survival of the heart and chest wall component (130 d, N = 3) as well as the VBM niche as evidenced by donor-specific chimerism (average: 2.35 ± 1.44%), whereas interestingly, the skin component was rejected after 13 (±1) days. Conclusion: Thus, this novel microsurgical model of VCA combined with solid organ transplantation is technically feasible and results in split tolerance when treated with costimulatory blockade.

2548: Tolerance induction to vascularized composite allografts by costimulation blockade

2548: Tolerance induction to vascularized composite allografts by costimulation blockade Byoung Chol Oh, DVM, PhD, Georg Furtmuller, MD, Madeline Fryer, BS, Johanna Grahammer, Stefan Schneeberger, Damon Cooney, MD, PhD, W. P. Andrew Lee, MD, Giorgio Raimondi, PhD, and Gerald Brandacher, MD Johns Hopkins University School of Medicine, Vascularized Composite Allotransplantation (VCA) Laboratory, Baltimore, MD, USA; Dept. of Visceral, Transplant and Thoracic Surgery, Innsbruck Medical University, Innsbruck, Austria Background Vascularized composite allotransplantation (VCA) has become a valid therapeutic option after devastating tissue loss While costimulation blockade (CoB) has shown considerable efficacy in preventing rejection, its efficacy in VCA remains poorly explored Here we investigated the immunoregulatory potential of CoB in a novel murine model of hind limb transplantation. Methods Fully MHC-mismatched allogeneic, orthotopic hind limb transplants were performed from Balb/c to C57BL/6 mice Recipient animals received combinations of total body irradiation (TBI), CTLA4-Ig, and anti-CD154 mAb (CoB) Mixed chimerism, clonal deletion of alloreactive T cells, and cytokine production were assessed by flow cytometry. Results CoB treated recipients showed increased survival compared to untreated and CTLA4-Ig only groups (Mean survival time [MST] 82 days) Adding non-myeloablative TBI to CoB enabled indefinite graft survival (MST,>210 days) Mixed chimerism induced by donorderived bone marrow (BM) was detected in the CoB treated group, and was even higher in TBICCoB treated recipients Decreased v̂I211C and v̂I25CCD4C T cells were detected in both groups treated with either CoB or TBICCoB, suggesting central thymic deletion of donor reactive T cells Donor specific tolerance was confirmed in long-term survivors (TBICCoB group) by acceptance of donor matched secondary skin grafts and rejection of third party ones In long term survivors treated with TBICCoB, decreased T cell responsiveness and increased graft-infiltrating regulatory T cells were detected on POD50. Conclusion Our results show that CoB enables the tolerogenicity of BM components carried by VCA, but requires TBI to establish durable donor-specific tolerance. CONTACT Byoung Chol Oh, DVM, PhD boh3@jhmi.edu

2546: Non-cytoreductive immunosuppression prevents rejection of vascularized composite allografts

2546: Non-cytoreductive immunosuppression prevents rejection of vascularized composite allografts Byoung Chol Oh, DVM, PhD, Georg Furtmuller, MD, Marcos Iglesias, PhD, Madeline Fryer, BS, Giorgio Raimondi, PhD, Damon Cooney, MD, PhD, W. P. Andrew Lee, MD, and Gerald Brandacher, MD Johns Hopkins University School of Medicine, Vascularized Composite Allotransplantation (VCA) Laboratory, Baltimore, MD, USA Background Reconstructive transplantation represents a valid therapeutic option after devastating tissue loss such as an extremity or face Immunosuppression-free donorspecific immunological tolerance has been successfully achieved in the setting of solid organ transplantation through mixed chimerism Routine clinical application of this approach, however, is hampered by the toxicity of the cytoreductive recipient conditioning The current study investigated a novel non-cytoreductive immunosuppressive strategy in a murine model of hind limb transplantation. Methods Fully MHC-mismatched allogeneic, orthotopic hind limb transplants were performed from Balb/C to C57BL/6 mice Recipient animals in the experimental groups received no treatment (Group 1); 025 mg CTLA4 Ig on postoperative days (POD) 0, 2, 4, and 6 (Group 2); 20 mg/kg anti-T cells (anti-Thy 12 mAb) on POD-1 plus CTLA4-Ig and 1 mg/kg Rapamycin (POD0-9) (Group 3) Flow cytometric analysis was performed to evaluate mixed chimerism and clonal deletion of alloreactive T cells. Results The CTLA4 Ig treated group showed increased graft survival compared to non-treated controls (mean survival time [MST] 17 d and 8 d, respectively, p < 001) Interestingly, combination of T cell depletion and CTLA4 Ig plus short-course of Rapamycin increased VCA survival significantly (MST 105 days; p < 001) Donor derived mixed chimerism was detected in recipients receiving the combined treatment protocol with 50 § 12 % of donor derived CD11bC cells on POD 55 VÎ – TCR staining profiles in recipients after combined treatment showed 16 § 04 % of Î=2Î25CCD4C T cells, while nae Ave C57BL/6 express 36 § 04 % of Î=2Î 25CCD4C T cells, suggesting central deletion of donor-reactive T cells Also, we found infiltrating Foxp3C regulatory T cells expressing donor derived marker (H-2K) at POD60 on combined treatment representing donor derived T reg cells were infiltrated. Conclusion This study shows that combination of T cell depletion and costimulation blockade and short-course of Rapamycin prevents VCA rejection and significantly prolongs graft survival Also, these data show that a clinically acceptable non-cytoreductive strategy could be applied in VCA to avoid long-term maintenance immunosuppression. CONTACT Gerald Brandacher, MD gbranda2@jhmi.edu

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression must be pursued. Employing vascularized bone marrow transplantation and co-transplantation of the thymus have shown promise in this regard in various animal models. Vascularized bone marrow transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and the incorporation of thymus tissue with vascularized bone marrow transplantation has shown to increase T-cell chimerism ultimately playing a supportive role in the induction of immune regulation. The combination of solid organ and vascularized composite allotransplantation can uniquely combine these strategies in the form of a novel transplant model. Murine models serve as an excellent paradigm to explore the mechanisms of acute and chronic rejection, chimerism, and tolerance induction, thus providing the foundation to propagate superior allograft survival strategies for larger animal models and future clinical application. Herein, we developed a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique. The experience in syngeneic and allogeneic transplant settings is described for future broader immunological investigations via an instructional manuscript and video supplement.

Abstract 18: Post Transplantation Cyclophosphamide Promotes Robust Immune Tolerance after Reconstructive Transplantation

22 hypothesize that IUSCT of gene modified hASCs will result in sustained FVIII transgene expression. The success of the first human IUSCT has been shown in a severe combined immunodeficiency patient that utilized paternal bone marrow (BM) derived stem cells. However, BM stem cell isolation is invasive and has a low quantitative yield. Adipose derived stem cell harvesting is minimally invasive and has a high stem cell count per gram. Here we report the safety and survivability of IUSCT of hASCs into a C57BL/6 murine model.

Inhibition of JAK/STAT Signaling Synergizes with CTLA4-Ig to Promote Transplant Survival

Murine T cell activation was assessed through a CFSE proliferation assay. Activation in an inflamed environment was simulated through addition of supernatant (MATSup) from maturing dendritic cells. Jak inhibition was exerted with the inhibitor Tofacitinib (Tofa). Differential expression of DC maturation markers in response to LPS C/¡ Tofa was analyzed by flow cytometry. In vivo synergism between Tofa and CTLA4-Ig was tested in B6 to BALB/c heart and skin graft models.

Costimulatory Blockade in Combination with Donor Bone Marrow Infusion Induces Immune Tolerance Across A Full MHC Barrier In A Translational Large Animal Vascularized Composite Allotransplantation Model

Fully MHCand gender mismatched MGH miniature swine underwent heterotopic hind-limb transplantation. Recipient animals received a short course of tacrolimus monotherapy, C/¡ donor BM infusion (60x10cells/kg), and CTLA4Ig (abatacept). Short course tacrolimus only and untreated animals served as controls. Chimerism was assessed by SRY-gene PCR analysis. Alloreactivity against donor antigens was assessed in vitro using CFSE-based Mixed Lymphocyte Reaction (CFSE-MLR) assays. Robust immune tolerance in vivo was assessed by secondary skin grafting. Tolerance maintenance upon allograft removal was tested in vitro by CFSE-MLR. Donor-specific antibodies (DSA) production was assessed using flow cytometry. Results