Immunosuppressive Toxicity Research Articles

Evaluation of Porcine Versus Human Mesenchymal Stromal Cells From Three Distinct Donor Locations for Cytotherapy

Background: Mesenchymal stromal cell (MSC)-based cytotherapies fuel the hope for reduction of chronic systemic immunosuppression in allotransplantation, and our group has previously shown this capability for both swine and human cells. MSCs harvested from distinct anatomical locations may have different behavior and lead to different outcomes in both preclinical research and human trials. To provide an effective reference for cell therapy studies, we compared human and porcine MSCs from omental fat (O-ASC), subcutaneous fat (SC-ASC) and bone marrow (BM-MSC) under rapid culture expansion with endothelial growth medium (EGM).Methods: MSCs isolated from pigs and deceased human organ donors were compared for yield, viability, cell size, population doubling times (PDT), surface marker expression and differentiation potential after rapid expansion with EGM. Immunosuppressant toxicity on MSCs was investigated in vitro for four different standard immunosuppressive drugs. Immunomodulatory function was compared in mixed lymphocyte reaction assays (MLR) with/without immunosuppressive drug influence.Results: Human and porcine omental fat yielded significantly higher cell numbers than subcutaneous fat. Initial PDT was significantly shorter in ASCs than BM-MSCs and similar thereafter. Viability was reduced in BM-MSCs. Porcine MSCs were positive for CD29, CD44, CD90, while human MSCs expressed CD73, CD90 and CD105. All demonstrated confirmed adipogenic differentiation capacity. Cell sizes were comparable between groups and were slightly larger in human cells. Rapamycin revealed slight, mycophenolic acid strong and significant dose-dependent toxicity on viability/proliferation of almost all MSCs at therapeutic concentrations. No relevant toxicity was found for Tacrolimus and Cyclosporin A. Immunomodulatory function was dose-dependent and similar between groups. Immunosuppressants had no significant adverse effect on MSC immunomodulatory function.Discussion: MSCs from different harvest locations and donor species differ in terms of isolation yields, viability, PDT, and size. We did not detect relevant differences in immunomodulatory function with or without the presence of immunosuppressants. Human and pig O-ASC, SC-ASC and BM-MSC share similar immunomodulatory function in vitro and warrant confirmation in large animal studies. These findings should be considered in preclinical and clinical MSC applications.

A Novel Immunosuppressive Regiment for Cardiac Transplant Patients

Heart transplantation remains curative for end-stage heart failure. The choice between immunosuppressive regimens is guided by several principles: mitigating the risk of rejection early after transplantation, limiting drug toxicity of immunosuppressants, and minimizing over-suppression. We sought to investigate the efficacy of a novel immunosuppressive regimen targeting a lower therapeutic range (LTR) for Tacrolimus without antecedent induction therapy compared with a standard therapeutic range (STR). The protocol was implemented after 2016 and included the administration of Tacrolimus to a goal trough of 6-8 ng/mL immediately post transplant, Mycophenolate mofetil 1000-15000 mg BID, and Prednisone 1 mg/kg/day post-op divided into two doses and tapered by 5 mg Daily. A retrospective chart review was performed comparing transplant outcomes utilizing the novel LTR immunosuppression regimen compared to STR from 2008-2019. Primary outcomes included infection, bone marrow suppression and rejection within the first 3 years of transplantation. Of the 83 patients analyzed, 25 (30%) received the lower Tacrolimus dosing regimen (LDR), and 58 received the standard dosing regimen (SDR). Patients in the LDR group were younger (56.2 +/- 12.2 years vs 61.8 +/- 10.4 years) and less likely to be male (68% vs 87.9%). Rates of high risk CMV donor/recipient status was similar between the two groups. There was no statistically significant difference between the LDR group and SDR group with respect to rates of infection (44% vs 34.5%, p=0.41), bone marrow suppression (20% vs 13.8%, p=0.47), or > 2R CMR (24% vs 32.8%, p=0.42). In this single-center study we compared a conventional post-transplant immunosuppression regimen with one that utilized a lower Tacrolimus trough goal (6-8ng/mL). There was no statistical difference between the two groups in rates of rejection, bone marrow suppression, or infectious complications. We present a successful novel immunosuppression regiment that could serve to benefit patients with intolerance to higher doses of calcineurin inhibitors with no adverse outcomes. Future studies are needed to determine the impact of LTR on renal function.

B Cell Immunophenotyping and Transcriptional Profiles of Memory B Cells in Patients with Myasthenia Gravis.

Myasthenia gravis (MG) is an autoimmune neuromuscular junction disorders mediated by various autoantibodies. Although most patients with MG require chronic immunosuppressive treatment to control disease activity, appropriate surveillance biomarkers that monitor disease activity or potential toxicity of immunosuppressants are yet to be developed. Herein, we investigated quantitative distribution of peripheral blood B cell subsets and transcriptional profiles of memory B cells (CD19+ CD27+) in several subgroups of MG patients classified according to the Myasthenia Gravis Foundation of America (MGFA) Clinical Classification. This study suggests potential immunologic B-cell markers that may guide treatment decision in future clinical settings.

Adipose-derived stromal cell therapy combined with a short course nonmyeloablative conditioning promotes long-term graft tolerance in vascularized composite allotransplantation.

The risks of chronic immunosuppression limit the utility of vascularized composite allotransplantation (VCA) as a reconstructive option in complex tissue defects. We evaluated a novel, clinically translatable, radiation-free conditioning protocol that combines anti-lymphocyte serum (ALS), tacrolimus, and cytotoxic T-lymphocyte-associated protein 4 immunoglobulin (CTLA4-Ig) with adipose-derived stromal cells (ASCs) to allow VCA survival without long-term systemic immunosuppression. Full-mismatched rat hind-limb-transplant recipients received tacrolimus (0.5mg/kg) for 14days and were assigned to 4 groups: controls (CTRL) received no conditioning; ASC-group received CTLA4-Ig (10mg/kg body weight i.p. postoperative day [POD] 2, 4, 7) and donor ASCs (1×106 iv, POD 2, 4, 7, 15, 28); the ASC-cyclophosphamide (CYP)-group received CTLA4-Ig, ASC plus cyclophosphamide (50mg/kg ip, POD 3); the ASC-ALS-group received CTLA4-Ig, ASCs plus ALS (500µL ip, POD 1, 5). Banff grade III or 120days were endpoints. ASCs suppressed alloresponse in vitro. Median rejection-free VCA survival was 28days in CTRL (n=7), 34 in ASC (n=6), and 27.5 in ASC-CYP (n=4). In contrast, ASC-ALS achieved significantly longer, rejection-free VCA survival in 6/7 animals (86%), with persistent mixed donor-cell chimerism, and elevated systemic and allograft skin Tregs , with no signs of acute cellular rejection. Taken together, a regimen comprised of short-course tacrolimus, repeated CTLA4-Ig and ASC administration, combined with ALS, promotes long-term VCA survival without chronic immunosuppression.

A Quick and Reliable Method to Decellularize a Gracilis Flap: A Crucial Step Toward Building a Muscle.

Tissue loss as a consequence of congenital anomalies, trauma, malignancy, or gangrene represents a major health care problem in the United States. Because younger individuals are disproportionately affected, the costs are magnified over time and the resultant individual and societal effects are tremendous. The currently available options to restore soft tissue defects are associated with donor site morbidities. Vascularized composite allotransplantation may provide form, function, and esthetics without a donor site; however, it comes with the significant risk associated with toxic immunosuppression (Biomaterials. 2015;61:246-256, Ann Plast Surg. 2015;75(1):112-116, Transplantation. 2009;88(2):203-210). Engineered tissues offer promise in finding viable alternatives to allograft and autologous tissues. In this study, we present our simple and quick method to decellularize a muscle without disrupting the vascular network integrity or the extracellular matrix. Optimizing the decellularization process is a crucial step toward creating an "off-the-shelf" flap that can be used for soft tissue reconstruction. The superficial gracilis muscle of 20 rats were harvested on their circulation and decellularized using perfusion with Krebs-Henseleit buffer and sodium dodecyl sulfate for 6 hours. These flaps were evaluated by gross morphology, histology, DNA quantification, integrity of the vascular network, scanning electron microscopy, and transmission electron microscopy. All samples were decellularized successfully as determined by DNA content and histological analysis for cellular content. The vascular network was preserved in all samples. We present a quick, simple, and affordable method to decellularize a muscle flap through the vascular network. Our proposed method is efficient and can be completed in a significantly shorter time when compared with other methods. It is also safe and does not affect integrity of tissue, and this is essential for a reliable recellularization.

Oral manifestations of allograft recipients immediately before and after kidney transplantation

Objective: To identify the oral lesions of individuals with kidney disease immediately before and shortly after kidney transplantation, taking into account the immunosuppressive regimen, antiviral prophylaxis and type of transplantation.Methods: A prospective observational cohort study was carried from January 2017 to January 2018. Eighty individuals aged 18 years or older who were admitted for kidney transplantation were eligible to participate. Clinical data regarding medical history, immunosuppressive therapy, antiviral prophylaxis, laboratorial data and oral examination were performed by the same trained researcher, in three different moments: 24 hours before transplantation (1st time point), 15–20 days (2nd time point) and 45–60 days (3rd time point) after transplantation.Results: In the first, second and third time points, it was found that 3.7% (3/80), 23.7% (18/76) and 25.7% (19/74) of the participants showed oral soft tissue lesions. Ulcers and candidiasis were the most frequent oral lesions, and they were associated with the use of everolimus (p = .005) and azathioprine (p = .034), respectively. Less patients reported xerostomia after transplantation than before (p < .001).Conclusions: Oral lesions are common in the short term after renal transplantation and are particularly related to both toxicities of immunosuppressive drugs and immunosuppression.

Induced pluripotent stem cell-derived mesenchymal stem cells prolong hind limb survival in a rat vascularized composite allotransplantation model.

The reduction of systemic immunosuppressive agents is essential for the expansion of vascularized composite allotransplantation (VCA) in a clinical setting. The purpose of this study is to compare human-induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs) with four other types of mesenchymal stem cells (human bone marrow-derived MSCs [BMMSCs], human adipose-derived MSCs [ADMSCs], rat BMMSCs, and rat ADMSCs) in vitro, and to investigate the in vivo immunomodulatory effect of iMSCs in a rat VCA model. One Brown Norway (BN) rat, 2 Lewis (LEW) rats, and 1 Wistar rat were used in the mixed lymphocyte reaction (MLR), and 9 BN rats and 3 LEW rats (for donors), and 24 LEW rats (for recipients) were used in the VCA model. The abovementioned five types of MSCs were imaged to examine their morphology and were also tested for suppressor function using a MLR. The 24 recipient LEW rats were divided randomly into four groups, and subjected to orthotopic hind limb transplantation. The three control groups were the Iso group, in which transplantation was performed on from three to six LEW rats without immunosuppressive treatment (n = 6); the FK group, in which transplantation was performed from BN rats to LEW rats and recipient rats were treated with tacrolimus alone (FK 506, 0.2 mg/kg, days 0-6 postoperatively, intraperitoneally) (n = 6); and the UT group, in which transplantation was performed from BN rats to LEW rats without any immunosuppressive treatment (n = 6). The experimental group was the iMSC group, in which transplantation was performed from BN rats to LEW rats and recipient rats were treated with tacrolimus (FK 506, 0.2 mg/kg, days 0-6 postoperatively, intraperitoneally) and injected with iMSCs (2 × 106 cells, day 7, intravenously) (n = 6). Hind limb survival was assessed by daily inspection of gross appearance until 50 days postoperatively. Histology of the skin and muscle biopsy were investigated on day 14 postoperatively. A time series of the plasma cytokine level (before transplantation, and at 10, 14, and 17 days after transplantation) was also analyzed. The size of adherent and trypsinized iMSCs was 67.5 ± 8.7 and 9.5 ± 1.1 μm, respectively, which was the smallest among the five types of MSCs (p < .01). The absorbance in MLR was significantly smaller with rat ADMSCs (p = .0001), human iMSCs (p = .0006), rat BMMSCs (p = .0014), human ADMSCs (p = .0039), and human BMMSCs (p = .1191) compared to without MSCs. In vivo, iMSC treatment prolonged hind limb survival up to 12.7 days in macroscopic appearance, which is significantly longer than that of the FK group (p < .01). Histology of the skin and muscle biopsy revealed that mononuclear cell infiltration was significantly reduced by iMSC injection (p < .01). iMSC treatment also affected proinflammatory cytokines (interferon-gamma (IFNγ) and tumor necrosis factor α (TNFα)) and the anti-inflammatory cytokine (interleukin-10 (IL-10)) of the recipient plasma. The IFNγ levels at Δ14 and the TNFα levels at Δ14 and Δ17 of the iMSC group were significantly lower than those of the FK group (p = .0226, .0004, and .004, respectively). The IL-10 levels at Δ10 and Δ14 of the iMSC group were significantly higher than those of the FK group (p = .0013 and .0374, respectively). iMSCs induce T cell hyporesponsiveness to prolong hind limb survival in a rat VCA model. This immunomodulatory property against acute rejection could provide one of the promising strategies capable of enabling the toxicities of immunosuppressants to be avoided in clinical settings.

P3.55: Allointestine colon conduit urinary diversion in combined intestine and kidney transplant: A case report

Introduction: In intestinal transplant candidates, the incidence of renal failure is high and is associated with increased mortality. Additionally previous surgeries and loss of abdominal domain may make standard kidney transplant with ureterocystostomy impossible. . Methods: We present an extreme case of combined small intestine, colon, and kidney transplant in a patient with short gut syndrome due to radiation enteritis, severe loss of abdominal domain, failed previous kidney transplant, and absence of native urinary system. Results: Our patient is a forty years old female with a past history of bladder Rabdomyosarcoma as an infant, who underwent bladder resection and post resection radiation. She developed radiation enteritis, renal failure, and eventually received a living related kidney transplant. Her renal allograft subsequently failed due to multiple urinary obstructions despite multiple revision surgeries, as well as chronic immunosuppression toxicity. As a result, she had short gut syndrome and renal failure with no feasible route of urinary drainage. In addition, she had severe loss of abdominal domain due to abnormal abdominal development from the radiation she received as an infant. She had multiple complications of her short gut syndrome and she underwent a simultaneous small intestine, colon, and kidney transplant. Donor was an eight years old female (132 cm, 23.4 kg), who became brain dead after severe exacerbation of asthma attack. Since there were no viable routes for urinary drainage, as there were no viable native intestines, and native and previous transplanted kidney ureter had been obstructed, a 10 cm allograft colon segment was used as a urinary conduit. The patient also underwent a complex abdominal closure with pedicled left anterior lateral thigh perforator flap by the plastic surgery team. One hundred days post transplant the patient is parenteral nutritional independent with normal renal function and her transplant colon conduit is healthy and functional. Conclusion: In extreme cases of kidney transplant in intestinal transplant recipient patients with no feasible urinary drainage path, transplant intestine can successfully be used as a conduit. In review of literature, and to the best of our knowledge, this is the first description of utilization of allograft intestine as a urinary conduit.

Management of Diabetes Mellitus in Normal Renal Function, Renal Dysfunction and Renal Transplant Recipients, Focusing on Glucagon-Like Peptide-1 Agonist: A Review Based upon Current Evidence

Diabetes Mellitus (DM) is a leading cause of both Cardiovascular Disease (CVD) and End-stage Renal Disease (ESRD). After 2008, there has been much evidence presented, and recently the guidelines for sugar control have changed to focus on being more disease orientated. GLP-1 Receptor Agonists (GLP-1R) and sodium glucose cotransporter-2 inhibitors are suggested as the first line towards fighting all DM, CVD and ESRD. However, the benefits of GLP-1R in organ transplantation recipients remain very limited. No clinical trials have been designed for this particular population. GLP-1R, a gastrointestinal hormone of the incretin family, possesses antidiabetic, antihypertensive, anti-inflammatory, anti-apoptotic and immunomodulatory actions. There are few drug–drug interactions, with delayed gastric emptying being the major concern. The trough level of tacrolimus may not be significant but should still be closely monitored. There are some reasons which support GLP-1R in recipients seeking glycemic control. Post-transplant DM is due to an impaired β-cell function and glucose-induced glucagon suppression during hyperglycemia, which can be reversed by GLP-1R. GLP-1R infusion tends to relieve immunosuppressant related toxicity. Until now, in some cases, glycemic control and body weight reduction can be anticipated with GLP-1R. Additional renal benefits have also been reported. Side effects of hypoglycemia and gastrointestinal discomfort were rarely reported. In conclusion, GLP-1R could be implemented for recipients while closely monitoring their tacrolimus levels and any potential side effects. Any added benefits, in addition to sugar level control, still require more well-designed studies to prove their existence.

260-OR: Encapsulation of Islets with Tannic Acid Enhances Islet Function

Islet transplantation can restore glucose homeostasis in patients with type 1 diabetes (T1D), however, limited islet availability, toxicity of immunosuppressants, and poor graft survival impede clinical application. We hypothesize that islet encapsulation with coatings consisting of tannic acid (TA), an immunomodulatory antioxidant, and poly(N-vinylpyrrolidone) (PVPON), will provide immunoprotection and maintain β-cell function after transplantation. (PVPON/TA)-encapsulated syngeneic islets (n=13) significantly delayed graft failure without immunosuppression after transplantation under the kidney capsule into diabetic non-obese diabetic (NOD) mice by more than 30 days compared to non-encapsulated grafts (n= 13; p=0.0021). Histology of islet engrafted kidneys demonstrated increased insulin, glucagon, and somatostatin positivity within encapsulated grafts compared to non-encapsulated grafts and was corroborated with enhanced glucose responsiveness following an intraperitoneal glucose tolerance test (n=9, p=0.0032). To define the immunoprotective properties of (PVPON/TA) coatings, flow cytometric analysis of islet grafts at day 7 post-transplantation displayed fewer MHC-II expressing cells compared to non-encapsulated controls. Gene expression of islet grafts at 5 days post-transplant displayed a significant increase in Ins2 (p&amp;lt;0.05), Arg1 (p&amp;lt;0.0001), and a decrease in Ccl5 (p=0.0006), Cxcl10 (p=0.043), and Grzmb (p=0.032) mRNA accumulation in encapsulated islet grafts in contrast to controls, suggesting that (PVPON/TA)-encapsulation reduces proinflammatory immune responses and elicits a shift toward an anti-inflammatory phenotype. To address limited islet availability, encapsulation of neonatal porcine islets restored euglycemia in diabetic NOD.scidmice for more than 200 days after xenotransplantation. Our results support the hypothesis that (PVPON/TA) coatings are immunomodulatory and may prolong islet graft function following transplantation. Disclosure J. Barra: None. V. Kozlovskaya: None. E. Kharlampieva: None. G. Korbutt: None. H.M. Tse: None. Funding American Diabetes Association (7-12-CD-11 to H.M.T.); JDRF (1-SRA-2015-42-A-N)

Induction and Scoring of Graft-Versus-Host Disease in a Xenogeneic Murine Model and Quantification of Human T Cells in Mouse Tissues using Digital PCR.

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological deficiencies and malignancies. Acute GVHD occurs when donor T cells recognize host tissues as a foreign antigen and mount an immune response to the host. Current treatments involve toxic immunosuppressive drugs that render patients susceptible to infection and recurrence. Thus, there is ongoing research to provide an acute GVHD therapy that can effectively target donor T cells and reduce side effects. Much of this pre-clinical work uses the xenogenic GVHD (xenoGVHD) murine model that allows for testing of immunosuppressive therapies on human cells rather than murine cells in an in vivo system. This protocol outlines how to induce xenoGVHD and how to blind and standardize clinical scoring to ensure consistent results. Additionally, this protocol describes how to use digital PCR to detect human T cells in mouse tissues, which can subsequently be used to quantify efficacy of tested therapies. The xenoGVHD model not only provides a model to test GVHD therapies but any therapy that can suppress human T cells, which could then be applied to many inflammatory diseases.

Therapeutic management with biological anti-TNF-α agent in severe psoriasis associated with chronic hepatitis B: A case report.

Systemic therapy in patients with concurrent psoriasis and chronic hepatitis B is a challenging task for both dermatologists and gastroenterologists since there is a high risk for hepatitis B virus (HBV) reactivation and hepatic toxicity under biological therapy. The therapeutic management of a patient with psoriasis and infection with the HBV is a challenge as the classical systemic treatment (methotrexate, acitretin, cyclosporine) shows a high risk of immunosuppression and/or hepatic toxicity and the biological therapy is endangered by the possibility of HBV reactivation. We present the case of a patient with moderate-severe psoriasis and chronic hepatitis B for whom we assessed the risk-benefit relation and considered useful to initiate the anti-TNF therapy concomitantly with the antiviral therapy with entecavir. The therapeutic algorithm included initiation of anti-TNF therapy with etanercept 2×50 mg/week combined with entecavir, an antiviral treatment administered continuously since the diagnosis of the HBV hepatitis, with hepatic function and viral load monitoring. After 3 months of therapy with etanercept the patient was given a dose of etanercept of 50 mg/week combined with entecavir 0.5 mg/day which he continued until week 36 when psoriatic lesions had cleared (PASI=0.6; DLQI=0). No adverse effects were registered and there was no evidence of HBV viral replication or changes in viral markers. We wish to emphasize that the use of etanercept in a patient with psoriasis and hepatitis B is a successful therapeutic alternative which may be safely used concomitantly with entecavir, with regular monitoring of viral load and hepatic function tests.

Characterizing the pharmacokinetics and pharmacodynamics of immunosuppressant medicines and patient outcomes in elderly renal transplant patients.

This review examines what is currently known about the pharmacokinetics and pharmacodynamics of commonly prescribed immunosuppressant medicines, tacrolimus, cyclosporine, mycophenolate and prednisolone, in elderly renal transplant recipients, and reported patient outcomes in this cohort. Renal transplantation is increasing rapidly in the elderly, however, currently, long-term patient outcomes are relatively poor compared to younger adults. Some studies have suggested that elderly recipients may have higher dose-adjusted exposure and/or lower clearance of the calcineurin inhibitors tacrolimus and cyclosporine; with one study reporting up to 50% reduction in tacrolimus exposure in the elderly. Elderly transplant recipients do not appear to have higher dosage-adjusted exposure to mycophenolic acid (MPA). The effects of ageing on the pharmacokinetics of prednisolone are unknown. Only one study has examined how aging effects drug target enzymes, reporting no difference in baseline inosine 5'-monophosphate dehydrogenase (IMPDH) activity and MPA-induced IMPDH activity in elderly compared to younger adult renal transplant recipients. In elderly transplant recipients, immunosenescence likely lowers the risk of acute rejection, but increases the risk of drug-related adverse effects. Currently, the three main causes of death in elderly renal transplant recipients are cardiovascular disease, infection and malignancy. One study has showed that renal transplant recipients aged over 65 years are seven times more likely to die with a functioning graft compared with young adults (aged 18-29 years). This suggests that an optimal balance between immunosuppressant medicine efficacy and toxicity is not achieved in elderly recipients, and further studies are needed to foster long-term graft and patient survival. Lower maintenance immunosuppressant targets in elderly recipients may decrease patient susceptibility to drug side effects, however, further studies are required and appropriate targets need to be established.

Abstract 3: Ex Vivo Expanded Regulatory T Cells Combined With Short-term Costimulation Blockade Prevent Rejection Of Vascularized Composite Allografts

PURPOSE: 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 investigates 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 their 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). 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. Vβ - TCR staining profiles in recipients after full treatment showed 1.570 ± 0.3700 % of νβ5+CD4+ T cells, while naïve C57BL/6 express 3.567 ± 0.3690 % of νβ5+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 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.

Engineering advanced cancer therapies with synthetic biology.

Engineered immune-cell-based cancer therapies have demonstrated robust efficacy in B cell malignancies, but challenges such as the lack of ideal targetable tumour antigens, tumour-mediated immunosuppression and severe toxicity still hinder their therapeutic efficacy and broad applicability. Synthetic biology can be used to overcome these challenges and create more robust, effective adaptive therapies that enable the specific targeting of cancer cells while sparing healthy cells. In this Progress article, we review recently developed gene circuit therapies for cancer using immune cells, nucleic acids and bacteria as chassis. We conclude by discussing outstanding challenges and future directions for realizing these gene circuit therapies in the clinic.

Fetal Toxicity of Immunosuppressive Drugs in Pregnancy.

Women affected by autoimmune diseases, organ transplantation, or neoplasia need to continue immunosuppressive treatment during pregnancy. In this setting, not only a careful planning of pregnancy, but also the choice of drugs is critical to preventing maternal complications and minimizing the fetal risks. Some immunosuppressive drugs are teratogenic and should be replaced even before the pregnancy, while other drugs need to be managed with caution to prevent fetal risks, including miscarriage, intrauterine growth restriction, prematurity, and low birth weight. In particular, the increasing use of biologic agents raises the question of their compatibility with reproduction. In this review we present data on the indication and safety in pregnancy of the most frequently used immunosuppressive drugs.

A Phase II Study of Decitabine Followed By Allogeneic Hematopoietic Stem Cell Transplantation in High-Risk Patients with Myeloid Malignancies

Introduction:Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative therapy for myelodysplastic syndrome (MDS) and high-risk acute myeloid leukemia (AML). Patients with high-risk disease have a markedly increased risk of relapse and death following transplant (Armand et al, Blood, 2014). Those who remain disease-free are at risk of severe morbidity from graft-versus- host-disease (GvHD). These issues highlight the importance of improved allo-HSCT platforms designed to reduce relapse rate without increasing risk of GvHD. Decitabine has minimal non-hematologic toxicity and proven efficacy in myeloid diseases (Blum et al, PNAS, 2010). Use of post-transplant cyclophosphamide has demonstrated improved rates of GvHD following allo-HSCT using haplo-identical donors (Bashey et al, JCO, 2013). No studies have reported on outcomes in patients undergoing decitabine immediately prior to transplantation followed by post-transplant cyclophosphamide (PTCy). We hypothesized that the combination of decitabine induction prior to transplant and PTCy would be safe and result in improved disease control with low rates of GVHD, translating into improved survival in a high-risk transplant cohort.MethodsIn this single-arm, single institution trial, eligible patients received 10 days of IV decitabine at 20mg/m2 no sooner than 24 days and no later than 17 days prior to conditioning. Myeloablative conditioning included fludarabine (50mg/m2 day-5-2), busulfan (IV 3.2mg/kg/day -5-2), and 4 Gy total body irradiation on day -1. Patients above age 65 received a 25% busulfan dose reduction. Patients received a fully or partially matched related bone marrow graft on day 0. GvHD prophylaxis included 50mg/kg of IV cyclophosphamide on day +3-4. Patients with fully matched donors received only PTCy while those with partially matched donors also received mycophenolate mofetil through day +35 and tacrolimus through day +180.ResultsWe enrolled 20 patients, fifteen patients with AML and 5 with MDS. The cohort had a median age of 64 (29-73) and was predominantly male (14/20, 70%). Eight (40%) patients scored as high risk by the HSCT comorbidity index. Eighteen patients (90%) had a high or very high-risk score by the refined disease risk index.All patients received decitabine and 18/20 (90%) underwent transplantation; 2 patients did not receive a transplant due to infectious complications. The majority of patients received a haplo-identical graft (13/18, 72%), and the remaining 5 received a matched related graft.Outcomes are reported in table 2 and figure 1. There were no engraftment failures. Five patients, 3 MDS and 2 AML, are long-term survivors with median follow-up over 3 years. One patient developed donor derived MDS and required a second transplant. Most transplanted patients (13/18, 72%) survived to day 100 with a median post-transplant survival of 138 days. There were 15 deaths on study with the majority due to underlying disease. Six patients (6/20, 30%) died of infectious complications or did not receive a transplant due to infection. Incidence of grade 3-4 acute GvHD was low among those surviving at least 40 days from transplant (3/17, 17%). There were also low rates of chronic GvHD among the 12 patients alive without ongoing GvHD at day 100 (2/12, 17%).ConclusionsDecitabine induction followed by myeloablative conditioning in this high-risk population resulted in a high treatment related mortality of 40%. Still, outcomes fell into an expected range for high-risk myeloid disease in an elderly and comorbid population. Based on expected outcomes for high-risk patients from the literature (Armand et al, Blood, 2014), decitabine did not markedly improve overall survival outcomes, recognizing that no direct comparisons are available in our limited study population. Decitabine may increase the risk of peri-transplant infections by contributing to a cumulative immunologic insult combined with disease-related immunosuppression and transplant-related toxicity, highlighting the importance of strict vigilance for infections within this setting. Diligent monitoring may improve infectious outcomes as shown in the second half of the cohort; only two out of the latter 10 patients on protocol died of treatment related complications. There were no cases of engraftment failure. Rates of acute and chronic GvHD using a PTCy platform were low and support other studies reporting this benefit. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Posterior Reversible Encephalopathy Syndrome in Pediatric Oncology and Post Bone Marrow Transplant: Single Center Experience and Systematic Review

BackgroundPosterior reversible encephalopathy syndrome (PRES) is a clinical radiologic disease described initially over two decades ago. This usually reversible entity is becoming more recognized and reported due to the increased index of suspicion and subsequent early brain imaging. It is described in pediatric oncology and post bone marrow transplant (BMT) patients mainly as case reports and case series. The aim of this single institution retrospective study is to describe a cohort of pediatric patients with oncologic diseases and post BMT and incorporate them in a systematic review of the literature to evaluate for differences in risk factors for developing PRES in patients post BMT for oncologic diseases compared to those transplanted for a benign disorder.MethodsWe retrospectively analyzed 11 patients admitted to a single center in Saudi Arabia and conducted a systematic review of previously published case reports and case series from January 1996 to December 2016.ResultsA total of 61 case reports and series were evaluated, 42 papers met the inclusion criteria in addition to our 11 patients for a total of 124 patients of which 27 were post BMT. The post BMT patients were divided by the indication of BMT into malignant and benign groups (14 (53.8%) and 12 (46.2%), respectively). One patient had missing data on the indication of BMT and was excluded. PRES was more common in males 69 / 120 (57.5%) in general, however, more common in females 17 / 27 post BMT (63%, p 0.015). The median age of presentation was 9.5 years (range 1.4 to 16 years) and PRES occurred on a median day +42 post BMT (range +2 to +144 days), and seemed to occur earlier in patients with benign diseases compared to malignant (median day +24 and day +45 respectively, p 0.104).We studied immunosuppression, hypertension history, and acute elevation of blood pressure (BP) as risk factors for developing PRES. There were 25 patients on immunosuppression; 20 (80%) were on cyclosporine (CsA) and 5 (20%) on tacrolimus. There were only 10 patients post BMT with reported presence or absence of toxicity of immunosuppression, 8 in the benign group, of which 3 (37.5%) patients had toxic levels of CsA, and 2 in the malignant group both with reported elevated CsA levels while other reviews showed PRES occurred independent of CsA level. Four patients were resumed on the same immunosuppression after the resolution of PRES, 2 developed recurrences and both were from the malignant group. The presence of history of hypertension as a risk factor was more common in the post BMT group compared to the non BMT group (9 / 13 (69.2%), 14 of 62 (22.6%) respectively, p 0.001), particularly in patients of the malignant group compared to the benign, but not statistically significant (5 of 6 (83.3%), 4 of 7 (57.1%) respectively, p 0.308). Acute hypertension was noted in all the patients with malignant disease who reported the BP, however, was only in 9 / 12 (75%) patients with benign disease. We were not able to evaluate hypomagnesemia as a risk factor due to the deficiency in reporting electrolytes in the case series, possibly due to the diverse specialties describing the disease.MRI was the diagnostic modality of choice. In the post BMT group, 18 / 26 patients (69.2%) had involvement of areas other than the parietal and occipital lobe on imaging, most commonly the temporal lobe. All the patients resolved from the acute episode of PRES. MRI was repeated in 10 patients at a median of 4 weeks (range 0.4 to 44 weeks), 5 patients had complete resolution of the MRI findings and 5 showed improvement in findings, with no differences in the two groups.Recurrence of PRES occurred in 4 / 26 patients (15.4%). There were no differences in the recurrence in relation to the underlying cause of BMT, clinical presentation, or imaging. There were 9 deaths (34.6%), none were due to PRES nor related to recurrence.ConclusionOur review showed the risk of PRES post BMT increased in females, the presence of history of hypertension, and immunosuppressive therapy, particularly CsA, which is similar to the literature. The time of presentation of PRES and the weight of each risk factor appears to be different in relation to the cause of BMT, however, the study is limited by the small number and reporting bias. The risk factor differences may be better elicited in a large cohort study. DisclosuresNo relevant conflicts of interest to declare.

Study for Comparison of CD40-mAb and CD40L-Ab Costimulation Blockade after Life-Supporting Orthotopic Cardiac Xenotransplantation of Multi-Transgenic Pig Hearts into Baboons with a Worldwide First Successful Long-Term Survival

Objectives Life-supporting orthotopic cardiac xenotransplantations (OHXTx) in a pig-to-baboon model with GalT-KO/hCD46/hTM transgenic donor pigs were performed with a compatible, non-toxic immunosuppression (IS) based on CD40mAb co-stimulation blockade in group G1 and in a group G2 with a new pasylated, non-thrombogenic CD40L-Ab. Chemical procedure of PASylation prolongs plasma half-life by 170-fold to several days. Primary aims were to realize a constant preclinical long-term survival in a life-supporting cardiac xenotransplantation model and to prevent perioperative cardiac xenograft dysfunction (PCXD) as a kind of cardiac low output. Methods OHXTx according to the technique of Lower and Shumway were performed in 11 baboons with transgenic pig hearts. The new IS consisted of a recombinant mouse-rhesus chimeric CD40 (clone 2C10R4) antibody in G1 (n=7) or a PASylated Fab-CD40L antibody (XL-protein/Wacker-Chemie) in G2 (n=4) combined with ATG, rituximab, MMF (no tacrolimus or cyclosporine!) and steroids. Results Survival in G1 with CD40mAb were 3, 1, 30, 1, 1** and 27 day(s) with 2 cases of PCXD. Using CD40L-Ab in G2 PCXD was observed in 1 case and the recipients survived 1, 18 and 40 days. Ischemic time of the hearts ranged from 112-128 min. Primary cause of death mostly were renal and hepatic failure, one died of a neurological deficit**, another after hematothorax, but no hyperacute or delayed xenograft rejection occurred. After several weeks a pig donor organ (over)growth was found. All long-term surviving baboons were in good general conditions until to the last days. Mean survival in G1 (CD40Ab) was 10.5+/-4.90 days and in G2 (Fab-CD40L) 19.7+/-7.84 days and without PCXD and brain damage** 20+/-6.55 days in G1 and 29+/-7.78 days in G2. In a preliminary modified subgroup (unpublished data und matter of a patent) one baboon with CD40L-Ab survived 50 days and another baboon (B67 with CD40Ab) reached and was successfully terminated at the endpoint of study on day 90 with no signs of rejection of the xenograft. Conclusion Co-stimulation blockade with CD40L antibody tended to prolong survival time after orthotopic cardiac xenotransplantation, but with so far incomplete groups statistically not significant. In total survival in this important life-supporting orthotopic model was significantly prolonged with a less toxic IS and better quality of life. This is an important milestone and progress on the way to a long-term survival of 2 -3 months, which is necessary for first clinical cardiac xenotransplantations. This aim was now first time worldwide reached with our last 90-days surviving baboon B67 in the last subgroup and must be replicated another 5 times before starting a clinical study.

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. Vβ – TCR staining profiles in recipients after full treatment showed 1.570 ± 0.3700 % of νβ5+CD4+ T cells, while naïve C57BL/6 express 3.567 ± 0.3690 % of νβ5+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.