Johanna Grahammer

Molecular Markers and Targeted Therapy of Skin Rejection in Composite Tissue Allotransplantation

Skin rejection remains a major hurdle in reconstructive transplantation. We investigated molecular markers of skin rejection with particular attention to lymphocyte trafficking. Skin biopsies (n = 174) from five human hand transplant recipients were analyzed for rejection, characteristics of the infiltrate and lymphocytic adhesion markers. The cellular infiltrate predominantly comprised CD3+ T cells. CD68, Foxp3 and indoleamine 2, 3‐dioxygenase expression and the CD4/CD8 increased with severity of rejection. Lymphocyte adhesion markers were upregulated upon rejection, intercellular adhesion molecule‐1 and E‐selectin correlated best with severity of rejection. Guided by the findings, a specific E‐ and P‐selectin inhibitor was investigated for its effect on skin rejection in a rat hind limb allotransplant model. While efomycine M (weekly s.c. injection into the graft) alone had no effect, long‐term allograft survival was achieved when combined with antithymocyte globulin and tacrolimus (control group without efomycine M rejected at postoperative day [POD] 61 ± 1). Upregulation of lymphocyte trafficking markers correlates with severity of skin rejection and time after transplantation in human hand transplantation. Blocking E‐ and P‐selectin in the skin holds potential to significantly prolong limb allograft survival.

Histopathologic characterization of mild rejection (grade I) in skin biopsies of human hand allografts

Mild skin rejection is a common observation in reconstructive transplantation. To enlighten the role of this inflammatory reaction we investigated markers for cellular and antibody mediated rejection, adhesion molecules and tolerance markers. Forty‐seven skin biopsies (rejection grade I) of human hand allografts were investigated by immunohistochemistry (CD3, CD4, CD8, CD20, CD68, C4d, LFA‐1, ICAM‐1, E‐selectin, P‐selectin, VE‐cadherin, HLA‐DR, IDO, and Foxp3). Expression was read with respect to time after transplant. The infiltrate was mainly comprised of CD3+T‐lymphocytes. Among these, CD8+cells were more prominent than CD4+cells. CD20+B‐lymphocytes were sparse and CD68+macrophages were found in some, but not all samples (approximately 10% of the infiltrate). The CD4/CD8‐ratio was increased after the first year. C4d staining was mainly positive in samples at time‐points later than 1 year. Adhesion molecules LFA‐1, ICAM‐1, E‐selectin, P‐selectin, and VE‐cadherin were found upregulated, and for P‐selectin, expression increased with time after transplant. IDO expression was strongest at 3 months–1 year post‐transplant and a tendency toward more Foxp3+ cells at later time points was observed. Mild skin rejection after hand transplantation presents with a T‐cell dominated dermal cell infiltrate and upregulation of adhesion molecules. The role of C4d expression after year one remains to be elucidated.

Animal models for basic and translational research in reconstructive transplantation

Reconstructive transplantation represents a bona fide option for select patients with devastating tissue loss, which could better restore the appearance, anatomy, and function than any other conventional treatment currently available. Despite favorable outcomes, broad clinical application of reconstructive transplantation is limited by the potential side effects of chronic multidrug immunosuppression. Thus, any reconstructive measures to improve these non-life-threatening conditions must address a delicate balance of risks and benefits. Today, several exciting novel therapeutic strategies, such as the implementation of cellular therapies including bone marrow or stem cells that integrate the concepts of immune regulation with those of nerve regeneration, are on the horizon. The development of reliable and reproducible small and large animal models is essential for the study of the unique immunological and biological aspects of vascularized composite allografts and to translate such novel immunoregulatory and tolerance-inducing strategies and therapeutic concepts from the bench to bedside. This review provides an overview of the multitude of small and large animal models that have been particularly designed for basic and translational research related to reconstructive transplantation.

Mechanisms and mediators of inflammation: potential models for skin rejection and targeted therapy in vascularized composite allotransplantation.

Vascularized composite allotransplantation (VCA) is an effective treatment option for patients suffering from limb loss or severe disfigurement. However, postoperative courses of VCA recipients have been complicated by skin rejection, and long-term immunosuppression remains a necessity for allograft survival. To widen the scope of this quality-of-life improving procedure minimization of immunosuppression in order to limit risks and side effects is needed. In some aspects, the molecular mechanisms and dynamics of skin allograft rejection seem similar to inflammatory skin conditions. T cells are key players in skin rejection and are recruited to the skin via activation of adhesion molecules, cytokines, and chemokines. Blocking these molecules has not only shown success in the treatment of inflammatory dermatoses, but also prolonged graft survival in various models of solid organ transplantation. In addition to T cell recruitment, ectopic lymphoid structures within the allograft associated with chronic rejection in solid organ transplantation might contribute to the strong alloimmune response towards the skin. Selectively targeting the molecules involved offers exciting novel therapeutic options in the prevention and treatment of skin rejection after VCA.

Histomorphometric evaluation of ischemia-reperfusion injury and the effect of preservation solutions histidine-tryptophan-ketoglutarate and University of Wisconsin in limb transplantation.

Background The effect of cold ischemia (CI) in vascularized composite allotransplantation is unknown. We herein assess tissue-specific damage, acceptable CI time, and the effect of preservation solutions in a syngenic rat hindlimb transplant model. Methods Lewis rat limbs were flushed and stored for 2, 10, or 30 hr CI in saline, histidine-tryptophan-ketoglutarate or University of Wisconsin preservation solution before transplantation. Morphologic alterations, inflammation, and damage of the individual tissues were analyzed on day 10 using histomorphology, confocal, light, and transmission-electron microscopy. Results Two-hour CI led to mild inflammation of tissues on day 10, whereas 10-hr and 30-hr CI resulted in massive inflammation and tissue damage. Although muscle was mainly affected after prolonged CI (≥10 hr), nerve was affected in all CI groups. A perineural cell infiltrate, hypercellular appearance, pronounced vacuolization, and mucoid degeneration, appearing as Wallerian degeneration, were observed. Staining with propidium iodide and Syto 16 revealed a decrease in viable muscle cell nuclei in the anterior tibial muscle on day 10 in all groups, which was most pronounced in 10-hr and 30-hr CI animals. Transmission-electron microscopy indicated that a large number of mitochondria were degenerated in the 10-hr and 30-hr CI groups. Histidine-tryptophan-ketoglutarate preservation solution slightly decreased inflammation and tissue damage compared to University of Wisconsin-treated and saline-treated animals, especially in skin and muscle when CI times did not exceed 10 hr. Conclusion Severe inflammation and tissue damage are observed after prolonged CI in muscle and nerve. Ischemia times in vascularized composite allotransplantation should be kept as short as possible and certainly below 10 hr.

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.

Benefits and Limitations of Belatacept in 4 Hand Transplanted Patients

Belatacept (cytotoxic T‐lymphocyte–associated protein 4 Ig) is an emerging treatment in kidney transplantation. Lack of nephrotoxicity and possibly an inhibitory effect on the development of donor‐specific antibodies (DSAs) make it an interesting agent in hand transplantation. To reduce calcineurin inhibitor immunosuppression and preserve kidney function, we have added belatacept to the therapeutic regimen of 4 hand‐transplanted patients at month 4 and at 6, 9, and 13 years after hand–forearm transplantation. Patients received 5 mg/kg belatacept every 2 weeks, and the dosing interval was extended to 4 weeks after 5 applications. Belatacept was initially well tolerated in all cases. Two patients were weaned to a low‐dose tacrolimus monotherapy together with monthly belatacept applications. One patient is taking belatacept with lowered tacrolimus and sirolimus trough levels. A fourth patient had significant levels of DSAs at time of conversion and progressed to a severe necrotizing rejection early despite an unaltered baseline immunosuppression. Finger skin necrosis and histologic signs of severe chronic allograft vasculopathy eventually led to amputation of the graft. Implementation of belatacept can be beneficial in hand transplantation. However, our findings indicated both potential and caution and reflection of the immunologic state at the time of conversion.

Targeting the Kv1.3 potassium channel for immunosuppression in vascularized composite allotransplantation – a pilot study

Kv1.3‐channels are critically involved in activation and function of effector memory T cells. Blocking Kv1.3‐channels was investigated for its effect on skin rejection in a rat limb‐transplantation‐model. Animals received the Kv1.3‐blocker correolide C systemically or locally as intra‐graft‐treatment in combination with tacrolimus. Systemic (intraperitoneal) administration of correolide C resulted in slight, but significant prolongation of allograft survival compared with untreated and placebo treated controls. In 4/6 correolide C treated animals, histology showed an intact epidermis and a mild infiltrate by day 10. High correolide C plasma trough levels correlated with prolonged allograft survival. A decrease in CD4+ and CD8+ effector memory T cells was observed in allograft skin, peripheral blood and the spleen on day 5. When applied subcutaneously in combination with systemic tacrolimus (30 days+/−anti‐lymphocyte serum) detectable, but insignificant prolongation of graft survival was achieved. 2/5 animals showed an intact epidermis and a mild infiltrate until day 45. Tapering systemic tacrolimus and weaning on day 50 resulted in rejection by day 55, regardless of local correolide C treatment. Subcutaneous injection did not lead to systemic plasma levels. The Kv1.3‐channel is a potential drug target worth exploring in more detail for immunosuppression in vascularized composite allotransplantation.

Subcutaneous administration of a neutralizing IL-1β antibody prolongs limb allograft survival

Cytokine‐expression profiles revealed IL‐1ß highly upregulated in rejecting skin of limb allografts. We investigate the effect of intragraft treatment with a neutralizing IL‐1β antibody in limb transplantation. Following allogenic hind‐limb transplantation, Lewis rats were either left untreated or treated with anti‐lymphocyte serum + tacrolimus (baseline) ; baseline immunosuppression + anti‐IL‐1β (1 mg/kg once/week, 6‐8 subcutaneous injections) into the transplanted or contralateral limb. Endpoint was rejection grade III or day 100. Graft rejection was assessed by histology, immunohistochemistry, flow cytometry phenotyping of immune cells, and monitoring cytokine expression. Anti‐IL‐1β injections into the allograft or contralateral limb resulted in a significant delay of rejection onset (controls: 58.60 ± 0.60; group 3: 75.80 ± 10.87, P = .044; group 4: 73.00 ± 6.49, P = .008) and prolongation of graft survival (controls: 64.60 ± 0.87; group 3: 86.60 ± 5.33, P = .002; group 4: 93.20 ± 3.82, P = .002), compared to controls. Although the phenotype of the graft infiltrating immune cells did not differ between groups, significantly decreased skin protein levels of IL‐1β, IL‐4, IL‐13, IP‐10, MCP‐1, and MCP‐3 in long‐term‐survivors indicate an overall decrease of chemoattraction and infiltration of immune cells as the immunosuppressive mechanism of anti‐IL‐1β. Inhibition of IL‐1β with short‐term systemic immunosuppression prolongs limb allograft survival and represents a promising target for immunosuppression in extremity transplantation.

2555: Belatacept in hand transplantation - Effectiveness and pitfalls

2555: Belatacept in hand transplantation Effectiveness and pitfalls Johanna Grahammer, MD, Bettina Zelger, MD, Bernhard Zelger, MD, Annelies Muehlbacher, MD, Dietmar Oefner, MD, Stefan Schneeberger, MD, and Annemarie Weissenbacher, MD Dept. of Visceral Transplant and Thoracic Surgery, Innsbruck Medical University, Innsbruck, Austria; Oxford Transplant Center, Oxford, UK Study purpose Belatacept (CTLA4Ig) is an emerging treatment in solid organ transplantation Effects on the development of donor specific antibodies (DSA) as well as its clinical safety in challenging immunological settings have yet to be explored. Methods Three hand transplanted patients have been converted to a Belatacept-based immunosuppressive regimen at 4 months, 6 y and 9 y after unilateral or bilateral hand and forearm transplantation Patients have received 5 mg/kg Belatacept every 2 weeks, the dosing interval was then extended to 4 weeks after 5 applications All 3 patients were kept on their baseline immunosuppressive medication, consisting of a CNI (Patients A, B, C) or mTOR inhibitor (Patients A and B) plus steroids (Patients A and B) and CellCept (Patient B). Results No adverse effects of Belatacept have been noted so far Patient C, who received Belatacept 4 months after transplantation, can successfully be kept on Tacrolimus monotherapy with a low trough level of »4–5 ng/ml This patient has never developed donorspecific antibodies, and displays normal histologic findings Patient A, who had previously developed DSA but was in a stable immunological state at the time of conversion, is now successfully tapered from baseline immunosuppression without evidence of rejection Patient B, who had DSA at the time of conversion, showed an increase of DSA and worsening graft appearance despite stable levels of his baseline immunosuppression, and needed escalation of his immunosuppression. Discussion The addition of Belatacept to an immunosuppressive regimen can be beneficial in hand transplantation However, our patients showed variable results depending on the immunological state at the time of conversion The application of Belatacept as a “œrescue” medication has to be discussed critically. CONTACT Johanna Grahammer, MD johanna.grahammer@tirol-kliniken.at