2 infection in the allograft, technical challenges imposed by viral-mediated injury to the native lung.Here, we report the first successful lung transplantation in a patient with non-resolving COVID-19 associated acute respiratory distress syndrome in the United States.Case Report: The recipient was a 28-year old female with past medical history of neuromyelitis optica treated with mycophenolate and rituximab who developed COVID pneumonia leading acute respiratory distress syndrome.The patient was intubated for 8 days with prone prior to initiation of VV ECMO.Her ECMO course was complicated by right sided pneumothorax requiring multiple pleural tubes and the development of Serratia marcescens pneumonia with left lower lung necrosis, and a liver capsular bleed necessitating emergent exploratory laparotomy.(Figure1a, b) She received antibiotics, remdesivir, hydroxychloroquine, tocilizumab, and convalescent plasma.However there was no signs of recovery and she was listed for lung transplantation after ECMO support for 32 days .Implantation was supported with central VA ECMO, and there was severe dense vascular adhesions bilaterally with severe distortion of hilar.(Figure1c) Explanted Lungs damaged by COVID-19 were free of virus but pathology showed extensive evidence of acute interstitial inflammation with fibrosis which consistence with end-stage pulmonary fibrosis.(Figure1d, e) The patient was decannulated from VV ECMO on POD 17, and was discharged on POD 27. (Figure1f) Four months after transplantation, she is at home with oxygen saturations above 98% on room air.

Vascularized composite allograft (VCA) transplantation can restore form and function following severe craniofacial injuries, extremity amputations or massive tissue loss. The induction of transplant tolerance would eliminate the need for long-term immunosuppression, realigning the risk-benefit ratio for these life-enhancing procedures. Skin, a critical component of VCA, has consistently presented the most stringent challenge to transplant tolerance. Here, we demonstrate, in a clinically relevant miniature swine model, induction of immunologic tolerance of VCAs across MHC barriers by induction of stable hematopoietic mixed chimerism. Recipient conditioning consisted of T cell depletion with CD3-immunotoxin, and 100 cGy total body irradiation prior to hematopoietic cell transplantation (HCT) and a 45-day course of cyclosporine A. VCA transplantation was performed either simultaneously to induction of mixed chimerism or into established mixed chimeras 85-150 days later. Following withdrawal of immunosuppression both VCAs transplanted into stable chimeras (n=4), and those transplanted at the time of HCT (n=2) accepted all components, including skin, without evidence of rejection to the experimental end point 115-504 days posttransplant. These data demonstrate that tolerance across MHC mismatches can be induced in a clinically relevant VCA model, providing proof of concept for long-term immunosuppression-free survival.

Abstract Introduction Reduced and full intensity conditioning (RIC, FIC) regimens employing intravenous (i.v.) busulfan plus fludarabine (Bu-Flu) have resulted in improved treatment related mortality and comparable overall survival in patients undergoing allogeneic HCT for AML/MDS who are not candidates for more intense regimens. However, relapse after HCT remains a leading cause of treatment failure after such conditioning regimens. Methods In Order to assess relapse following allogeneic HCT for AML/MDS, a retrospective analysis was performed to evaluate the outcomes of 55 consecutive patients with AML/MDS (49/6) who received i.v. Bu-Flu based conditioning. Blood samples were collected post HCT in a subset of those patients (30 patients). Serum values of 42 biological markers were measured at day 30 post HCT (2/30 patients were day 60 samples) using multiplex Luminex assay. Patients characteristic are shown in Table-1. Patients received single daily dose of iv Bu 3.2 mg/kg for 2 days (RIC, Bu2-Flu) or 4 days (FIC, Bu4-Flu) based on age, older or younger than 65 respectively. Fludarabine was given as a single daily dose of 40 mg/Kg for 4 days. Graft versus host disease prophylaxis was Tacrolimus/Methotrexate in FIC recipients and Tacrolimus/Mycophenolate in RIC recipients. Low dose thymoglobulin of 4.5 mg/kg was used in unrelated donor HCT recipients. Results With a median follow up of 18 month, the overall survival (OS) at 1 & 2 years was 73 ± 6% and 67 ± 7%, respectively, (Fig 1). Similarly, disease free survival at 1 and 2 years was 64 ± 7%. As expected, there was low cumulative incidence of treatment related mortality of 8 ± 3% at 1 and 2 years while the cumulative incidence of relapse was 28.0 ± 3% and 31± 2% at 1 and 2 years respectively, (Fig 2). Cumulative incidence of grade II-IV acute GVHD was 54% with grade III-IV of 25% at day 100. Cumulative incidence of chronic GVHD was 49, 54% at 1 and 2 years respectively. In a subset of patients where chemokine analysis was performed (30 patients), only MCP-1 levels at day 30 post HCT were predictive of relapse out of the 42 biological markers tested. The 7 out of 30 patients who relapsed in this subset (23%) had higher mean level of MCP-1 at day 30 of 537, SD ±213 versus 324, SD ± 160, P=0.007, (Fig 3). MCP-1 was predictive of leukemic relapse 82 days in advance on average prior to overt hematological relapse. Full chimerism (>95%) was detected at Day 30 in 5/7 patients who relapsed in the biological marker group. Conclusion Bu-Flu based conditioning regimens result in improved OS in patients with AML/MDS but do not impact relapse rate after allogeneic HCT. Serum MCP-1 levels in the early post-transplant period were predictive of relapse in subset of patients where post HCT biomarkers were available. Future larger studies may find potential role of MCP-1 in predicting relapse in patients at risk after HCT with Bu-Flu for AML/MDS. Disclosures: No relevant conflicts of interest to declare.

AMERICAN SOCIETY OF PLASTIC SURGEONS PLASTIC & RECONSTRUCTIVE SURGERY PRS GLOBAL OPEN ASPS EDUCATION NETWORK AMERICAN SOCIETY OF PLASTIC SURGEONS PLASTIC & RECONSTRUCTIVE SURGERY PRS GLOBAL OPEN ASPS EDUCATION NETWORK

Introduction: Loss of chimerism is an undesirable outcome of allogeneic hematopoietic cell transplantation (HCT) following reduced intensity conditioning (RIC). Understanding the nature of cellular and humoral immune responses to HCT following graft loss could lead to improved retransplantation strategies. We investigated the immunological responses following graft loss in miniature swine recipients of haploidentical HCT. Methods: Recipients underwent RIC consisting of 100cGy total body irradiation, partial T cell depletion using porcine CD3 immunotoxin, and a 45 day course of cyclosporin A. Proliferative responses to donor cells were assessed by mixed lymphocyte reactivity (MLR) and phenotyped using carboxyfluorescein succinimidyl ester (CFSE) and flow cytometry. Cell mediated cytotoxic responses and antibody mediated cellular cytotoxicity responses to donor cells were also assessed. Results: During the period of transient chimerism, no donor specific cellular or antibody cytotoxic responses could be detected. Following loss of peripheral blood chimerism, anti-donor cellular responses returned without evidence of sensitization and without detectable anti-donor antibody. Re-exposure to donor hematopoietic cells following graft loss, however, induced a sensitized anti-donor cellular proliferative response. No anti-donor antibodies could be detected despite this sensitization to donor cells. Attempts to intentionally immunize these animals to induce anti-donor allo-antibodies by donor skin grafting or injecting donor peripheral blood mononuclear cells subcutaneously were unsuccessful despite normal induced antibody responses to KLH immunization. In contrast, unconditioned animals exposed repeatedly to similar doses of haploidentical donor cells develop strong anti-donor antibody responses. Conclusions: The studies presented could have important implications for design of treatment strategies to overcome HVG responses and improve the outcome of re-transplantation following graft loss.

We have previously reported that transplantation (Tx) of prevascularized donor islets as composite Islet-Kidneys (IK) reversed diabetic hyperglycemia in miniature swine, while similar numbers of free islets did not. In order to enhance the IK strategy's clinical applicability, we attempted to induce tolerance of both islets and kidneys in non-human primates (NHP). Our tolerance strategy included hematopoietic cell transplantation (HCTx), 100cGy-Total Body Irradiation, T-cell depletion and 45 days of CyA (ITC). Methods: IKs were prepared by isolating islets from 70% partial pancreatectomies and injecting them beneath the autologous renal capsule of five rhesus monkey donors (Bwt 4.2-6kg) 3 weeks prior to HCTx. Haploidentical HCTx was performed following GCSF mobilization and leukapheresis of the donors and ITC conditioning of the recipients. Four IKs were transplanted to HCT recipients (Animal 1, 2, 3 and 4) and one was used for histology. Animal 1 received an allogeneic kidney from the same IK donor with an ITC +HCTx regimen 211 days prior to IK Tx. No immunosuppression was given at the time of IK Tx. Animals 2, 3 and 4 received IKs 3 weeks after HCTx with an ITC regimen. IDDM was induced by STZ at 80mg/kg intravenously before IK-Tx. Results: IK preparation was successful in all monkeys (24000˜45000IE). Greater than 20U/day of insulin was required to maintain BS < 200mg/dl following STZ induced IDDM. Animal 1 accepted the donor kidney with ITC+HCT regimen without rejection. Following IK-Tx without further immunosuppression, Animal 1 accepted an IK from the same donor 209 days following HCT and maintained IK >248 days. Only 1-3U of insulin every other day was required to maintain BS < 180mg. Animals 2 and 4 both received IKs with an HCT+ITC regimen. Both animals required 4-5U/day of insulin during a CyA period, but insulin requirement markedly decreased 1 to 3U daily. Animal 2 was sacrificed at POD348, and Animal 4 is currently on POD159 without any immunosuppression and requires only 1-2U of insulin per day. Animal 3 unfortunately died on POD68, but did not require any insulin treatment. Conclusions: We successfully induced tolerance of allogeneic islets as a part of lifesupporting composite IK grafts with HCTx. These results demonstrate the feasibility of composite IK-Tx with a tolerance regimen in NHP, with possible clinical applicability for the cure of diabetic nephropathy.

AMERICAN SOCIETY OF PLASTIC SURGEONS PLASTIC & RECONSTRUCTIVE SURGERY PRS GLOBAL OPEN ASPS EDUCATION NETWORK AMERICAN SOCIETY OF PLASTIC SURGEONS PLASTIC & RECONSTRUCTIVE SURGERY PRS GLOBAL OPEN ASPS EDUCATION NETWORK

Toxicities and complications associated with hematopoietic cell transplantation (HCT) currently limit this potentially curative therapy for malignant and non-malignant blood disorders in the clinic. Similar to outcomes in patients, miniature swine develop severe GVHD, infectious complications or hematopioetic failure following standard clinical myeloablative conditioning and HCT. Miniature swine recipients pre-treated with a low-dose (100cGy) of total body irradiation (TBI), partial T-cell depletion using a CD3-immunotoxin prior to HCT and a 45 day course of Cyclosporine A (referred to as ITC non myeloablative conditioning) following high-dose haploidentical-HCT (5-15X109 PBMCs/kg) exhibit stable engraftment and multilineage chimerism. We have observed a wide variability in engraftment outcomes depending on the occurrence of adverse reactions (fever, loss of appetite, bloody diarrhea, shedding of the GI lining and development of disseminated intravascular coagulopathy [DIC]) in the donor ("DIC Donor") following cytokine mobilization at the time of HC donation. ITC recipients (n = 12) that received haploidentical HCT from donors that remained healthy ("Healthy Donors") during IL-3 and stem cell factor (SCF) mobilization, engrafted with multilineage chimerism, did not develop GVHD and did not require any blood product support. In contrast, ITC recipients (n = 11) that received a stem cell product from a DIC Donor mobilized with IL3 and SCF had adverse outcomes. All recipients (11/11) lost peripheral blood myeloid chimerism (i.e. lack of donor stem cell engraftment). Also, all animals that received an HCT graft from DIC Donors developed moderate-severe thrombocytopenia (< 100,000 PLT/uL) and 73% (8/11) developed severe and life threatening thrombocytopenia (under/equal 20,000 PLT/uL) which required blood product support. One animal developed grade IV acute GVHD on day 30 post HCT. In contrast only 2/12 (16%) of animals receiving a transplant from Healthy Donors had thrombocytopenia with levels falling under 100K, none required blood product support and none developed GVHD. These data suggest that engraftment outcomes and post-transplant complications are greatly influenced by the health status of the donor prior to collection of cells by leukapheresis. We are currently investigating the causes of possible factors responsible for the poor engraftment outcomes.

Leto Barone, AA MD; Torabi, R; Randolph, MA; Duran-Struuck, R; Hawley, RJ; Horner, BM; Matar, A; Crepeau, R; Albritton, A; Mallard, C; Tang, Y; Tena, A; Scalea, JR; Kurtz, JM; Huang, CA; Sachs, DH; Cetrulo, CL Author Information

Leto Barone, A.A.; Torabi, R.; Randolph, M.A.; Leonard, D.A.; Mallard, C.; Albritton, A.; Duran-Struuck, R.; Matar, A.; Crepeau, R.; Tang, Y.; Scalea, J.R.; Wang, Z.; Tena, A.; Hawley, R.J.; Kurtz, J.M.; Huang, C.A.; Sachs, D.H.; Cetrulo, C.L. Jr. Author Information

Pathiraja, V. A.1; Lutton, B.2; Wikiel, K.2; Duran-Struuck, R.2; Matar, A.2; Crepeau, R.2; Wamala, I.2; Teague, A.2; Wu, A.2; Huang, C. A.2; Sachs, D. H.2 Author Information

Haploidentical HCT (haplo-HCT) is a potent and potentially curative therapy for a number of lymphohematopoietic neoplasms. Haplo-HCT has been limited by toxic conditioning regimens and the development of lethal graft-versus-host disease (GVHD). Similar to what has been observed in the clinic, myeloablative HCT outcomes in the pig have a high incidence of GVHD. Less toxic, non-myeloablative haplo-HCTs developed in our laboratory resulted in a greatly reduced incidence and intensity of GvHD. However, post-transplantation lymphoproliferative disease (PTLD) was often observed following these novel reduced intensity conditioning protocols. Haplo-HCT conditioning with 700-1000 cGy thymic irradiation (TI), CD3 immunotoxin and 30-60 days of cyclosporine had a 6% incidence of GVHD (2 of 32) but was complicated by a 33% incidence of PTLD (11 of 32). Of these 11 pigs, 2 resolved their PTLD after cyclosporine was discontinued but then developed GVHD. When thymic irradiation was eliminated from the protocol, no PTLD was observed and GVHD was only observed in 2 of 24 (8.3%) transplanted pigs. Unfortunately this very mild preparatory regimen without TI failed to consistently achieve successful long term engraftment. In an attempt to decrease the incidence of PTLD, avoid GVHD and increase haplo-HCT engraftment outcomes, 100 cGy of total body irradiation (TBI) was introduced to the protocol. Of 46 haplo-HCT recipients, only 4 (<10%) developed PTLD and 2/46 animals developed GVHD, one of which recovered spontaneously. We also attempted to identify serum markers for diagnosing PTLD in our pigs. As it is observed in humans, we identified lactate dehydrogenase (LDH), to be elevated in pigs prior to onset of PTLD. In support of these findings, three porcine PTLD tumor cell lines harvested from animals and passaged in vitro demonstrate higher LDH levels compared to naïve PBMCs. We conclude that: (1) our current haplo-HCT protocol utilizing 100 cGy TBI, porcine CD3 immunotoxin and 45 days of cyclosporine is relatively safe and significantly reduces the incidence of PTLD; (2) stable engraftment and multilineage chimerism can be achieved with minimal GVHD; and (3) LDH is a clinically relevant serum marker for the diagnosis of PTLD in the pig. These findings reinforce the pig model as a valuable tool for HCT translational studies.