Thymus Transplantation Research Articles

Intra-bone Bone Marrow Transplantation in Pig-to-Nonhuman Primates for the Induction of Tolerance Across Xenogeneic Barriers.

Mixed chimerism and thymic tissue transplantation strategies have achieved xenogeneic tolerance in pig-to-mouse models, and both have been extended to pig-to-baboon models. A mixed chimerism strategy has shown promise toward inducing tolerance in allogeneic models in mice, pigs, nonhuman primates (NHP), humans, and a rat-to-mouse small animal xeno-model. However, even though α-1,3-galactosyltransferase gene knockout (GalTKO) pigs have been used as bone marrow (BM) donors, direct intravenous injection of porcine BM cells was detected for only up to 4days (peripheral macro-chimerism) in one case, and the rest lost chimerism within 2days.Recent data in allogeneic models demonstrated that direct injection of donor BM cells into recipient BM spaces (intra-bone bone marrow transplantation: IBBMTx) produces rapid reconstitution and a higher survival rate compared to i.v. injection. In order to minimize the loss of injected porcine BM peripherally before reaching the BM space, Yamada developed a xeno-specific regimen including IBBMTx coated with a collagen gel matrix in a preclinical pig-to-baboon model (Yamada IBBMTx). This strategy aims to achieve improved, persistent macro-chimerism as well as engraftment of BM across a xenogeneic barrier. The initial study published in 2015 demonstrated that this IBBMTx strategy leads to markedly prolonged peripheral macro-chimerism detectable for up to 23days. Furthermore, a more recent study using human CD47-transgenic (Tg) GalTKO pigs as xeno-donors achieved long-lasting macro-chimerism >60days with evidence of reduction of anti-pig natural antibodies (nAb). This is the longest macro-chimerism that has ever been achieved in a preclinical large animal xenotransplant model to date. In this chapter, we introduce a brief summary of our achievements in regard to successful tolerance induction by utilizing our novel strategy of IBBMTx as well as describe the step-by-step methodology of surgical and in vitro procedures that are required for this project.

Reduced positive selection of a human TCR in a swine thymus using a humanized mouse model for xenotolerance induction.

Tolerance-inducing approaches to xenotransplantation would be optimal and may be necessary for long-term survival of transplanted pig organs in human patients. The ideal approach would generate donor-specific unresponsiveness to the pig organ without suppressing the patient's normal immune function. Porcine thymus transplantation has shown efficacy in promoting xenotolerance in humanized mice and large animal models. However, murine studies demonstrate that T cells selected in a swine thymus are positively selected only by swine thymic epithelial cells, and therefore, cells expressing human HLA-restricted TCRs may not be selected efficiently in a transplanted pig thymus. This may lead to suboptimal patient immune function. To assess human thymocyte selection in a pig thymus, we used a TCR transgenic humanized mouse model to study positive selection of cells expressing the MART1 TCR, a well-characterized human HLA-A2-restricted TCR, in a grafted pig thymus. Positive selection of T cells expressing the MART1 TCR was inefficient in both a non-selecting human HLA-A2- or swine thymus compared with an HLA-A2+ thymus. Additionally, CD8 MART1 TCRbright T cells were detected in the spleens of mice transplanted with HLA-A2+ thymi but were significantly reduced in the spleens of mice transplanted with swine or HLA-A2- thymi. [Correction added on October 15, 2019, after first online publication: The missing superscript values +, -, and bright have been included in the Results section.] CONCLUSIONS: Positive selection of cells expressing a human-restricted TCR in a transplanted pig thymus is inefficient, suggesting that modifications to improve positive selection of cells expressing human-restricted TCRs in a pig thymus may be necessary to support development of a protective human T-cell pool in future patients.

Preliminary study on tolerance induction by combined of rat thymus and metanephros anlages transplantation preconditioned with cyclophosphamide protocol

Objective To investigate whether allo-or xenotransplantation tolerance can be induced by transplanted thymus combined with metanephros primordia, from rats to cyclophosphamide (CP) pretreated recipients. Methods Two pairs of thymus and metanephros primordia were extracted from the Lewis rats on embryonic day16 (E16) and implanted into experiment (Ⅰ) group or control (Ⅱ, Ⅲ, Ⅳ) group renal capsules and omenta of thymectomized recipients, which had been pretreated with CP (80 mg/kg in BN rats and 200 mg/kg in C57BL/6 mice, ip, q6d×4). Post-transplantation, subcutaneous injection (sc, qd×12 d) of cyclosporine [8 mg/(kg·d) in BN rat and 12 mg/(kg·d) in C57BL/6 mouse] were simultaneously administrated till the 13th day. Histopathological examination of thymus and metanephros grafts and functional of developed metanephroi were performed post-transplantation. Rat seruminterleukin (IL)-2 and interferon-γ (IFN-γ) levels, one-way mixed lymphocyte reaction (MLR) and skin transplantation tests were measured. Results On the 14th day post-xenotransplantation, the thymus and metanephros primordia of Lewis rats were not found in the C57BL/6 mouse. On the 21st day after allotransplantation, the thymus and metanephros of some experimental (Ⅰ) group developed well, displaying partial urinary function. In experimental (Ⅰ) group, Banff rejection score (χ2=6.316, P 0.05) and SI had no significant difference (F=0.920, P>0.05). 5-7 weeks post-transplantation, acute rejection of metanephros and thymus deteriorated and connective tissue scars gradually formed. Conclusion Using the CP pretreatment protocol, E16 Lewis thymus and metanephros anlagens xenografted into C57BL/6 mice failed to grow and function, whereas fail to induce allogeneic immune tolerance but can grow and develop in the peritoneal cavity of BN rat, produce donor-specific immune hyporesponsiveness. Key words: Embryo; Thymus Primordia; Metanephros primordia; Transplantation; Tolerance

Renal Subcapsular Transplantation of 2'-Deoxyguanosine-Treated Murine Embryonic Thymus in Nude Mice.

The thymus is an important central immune organ, which plays an essential role in the development and differentiation of T cells. Thymus transplantation is an important method for investigating thymic epithelial cell function and T cells maturation in vivo. Here we will describe the experimental methods used within our laboratory to transplant 2'-deoxyguanosine (to deplete donor's lymphocytes) treated embryonic thymus into the renal capsule of an athymic nude mouse. This method is both simple and efficient and does not require special skills or devices. The results obtained via this simple method showed that transplanted thymus can effectively support the recipient's T cells production. Additionally, several key points with regards to the protocol will be further elucidated.

De novo generation of a functional human thymus from induced pluripotent stem cells

A proper functional thymus is required for generation of T cell–mediated immunity. This is dramatically illustrated by patients lacking a thymus, such as children with complete DiGeorge syndrome, which is fatal if left untreated.1-3 Therapeutic options for such patients are limited and confined to transplantation of small fragments from allogeneic neonatal thymi.

Gene Modification and Three-Dimensional Scaffolds as Novel Tools to Allow the Use of Postnatal Thymic Epithelial Cells for Thymus Regeneration Approaches.

Defective functionality of thymic epithelial cells (TECs), due to genetic mutations or injuring causes, results in altered T‐cell development, leading to immunodeficiency or autoimmunity. These defects cannot be corrected by hematopoietic stem cell transplantation (HSCT), and thymus transplantation has not yet been demonstrated to be fully curative. Here, we provide proof of principle of a novel approach toward thymic regeneration, involving the generation of thymic organoids obtained by seeding gene‐modified postnatal murine TECs into three‐dimensional (3D) collagen type I scaffolds mimicking the thymic ultrastructure. To this end, freshly isolated TECs were transduced with a lentiviral vector system, allowing for doxycycline‐induced Oct4 expression. Transient Oct4 expression promoted TECs expansion without drastically changing the cell lineage identity of adult TECs, which retain the expression of important molecules for thymus functionality such as Foxn1, Dll4, Dll1, and AIRE. Oct4‐expressing TECs (iOCT4 TEC) were able to grow into 3D collagen type I scaffolds both in vitro and in vivo, demonstrating that the collagen structure reproduced a 3D environment similar to the thymic extracellular matrix, perfectly recognized by TECs. In vivo results showed that thymic organoids transplanted subcutaneously in athymic nude mice were vascularized but failed to support thymopoiesis because of their limited in vivo persistence. These findings provide evidence that gene modification, in combination with the usage of 3D biomimetic scaffolds, may represent a novel approach allowing the use of postnatal TECs for thymic regeneration. stem cells translational medicine 2019;8:1107–1122

Long-term survival and differentiation of human thymocytes in human thymus-grafted immunodeficient mice.

Aim: Thymus transplants have produced encouraging clinical outcomes in achieving thymopoiesis and T-cell development. This study was aimed to investigate whether human thymus contains self-renewing lymphoid progenitors capable of maintaining long-term T-cell development. Materials & methods: Immunodeficient mice were transplanted with human thymic tissue along with autologous GFP-expressing or allogeneic CD34+ cells and followed for human thymopoiesis and T-cell development from the thymic progenitors versus CD34+ cells, which can be distinguished by GFP or HLA expression. Results: In both models, long-term thymopoiesis and T-cell development from the thymic grafts were detected. In these mice, human thymic progenitor-derived T cells including CD45RA+CD31+CD4+ new thymic emigrants were persistently present in the periphery throughout the observation period (32weeks). Conclusion: The results indicate that human thymus contains long-lived lymphoid progenitors that can maintain durable thymopoiesis and T-cell development.

Graft plasmacytoid dendritic cells improve thymic function in allogeneic hematopoietic cell transplantation

Abstract Allogeneic hematopoietic stem cell transplantation(allo-HSCT) is a well established cure for a variety of hematologic diseases. However, graft-versus-host disease(GVHD) remain the primary obstacles to successful transplant. GVHD results from donor T-cell recognition of a genetically disparate recipient. Thymus has been recognized as a recipient tissue that can be severely damaged by GVHD. Plasmacytoid dendritic cells(pDCs) is a distinct subset of DC, which have both immunogenic and tolerogenic effect in allo-HSCT, licensed by the microenvironment to respond to the milieu in which they migrate. Impaired thymic DC has been the major causation of dysfunctional thymic negative selection, thus generating allo-reactive T cells and GVHD. To further demonstrate pDCs from the graft are migrating to thymus to protecting host from GVHD. We transplanted B10.BR mouse with pDCs from MHC mismatched GFP-labeled C57/B6 mouse, along with hematopoietic stem cells and T-cells from normal C57/B6 mouse. The results from the confocal imaging suggest that pDCs are migrating to thymus 14 days after allo-HSCT. CCR9 expression is important for pDCs migrating to thymus. We transplated mice with either wild type pDCs or CCR9 knock out pDCs. Survival rate is significantly lower in group transplanted with CCR9-knockout pDCs suggesting thymus is a critical place for pDCs to induce tolerogenic response. We also tranplanted wild tipe pDCs with thymectomized host and normal host. Thymectomy group have better survival rate compared to non-thymectomized group, further confirming the association between pDCs and thymus. Improved thymic function post transplant with pDCs from the graft may have a potential to decrease GVHD.

T Cell Acute Lymphoblastic Leukemia as a Consequence of Thymus Autonomy.

Thymus autonomy is the capacity of the thymus to maintain T lymphocyte development and export independently of bone marrow contribution. Prolonging thymus autonomy was shown to be permissive to the development of T cell acute lymphoblastic leukemia (T-ALL), similar to the human disease. In this study, performing thymus transplantation experiments in mice, we report that thymus autonomy can occur in several experimental conditions, and all are permissive to T-ALL. We show that wild type thymi maintain their function of T lymphocyte production upon transplantation into recipients with several genotypes (and corresponding phenotypic differences), i.e., Rag2 - / - γc - / -, γc - / -, Rag2 - / - IL-7rα - / -, and IL-7rα - / - We found that the cellularity of the thymus grafts is influenced exclusively by the genotype of the host, i.e., IL-7rα-/- versus γc -/- Nonetheless, the difference in cellularity detected in thymus autonomy bore no impact on onset, incidence, immunophenotype, or pathologic condition of T-ALL. In all tested conditions, T-ALL reached an incidence of 80%, demonstrating that thymus autonomy bears a high risk of leukemia. We also analyzed the microbiota composition of the recipients and their genetic background, but none of the differences found influenced the development of T-ALL. Taken together, our data support that IL-7 drives cellular turnover non-cell autonomously, which is required for prevention of T-ALL. We found no influence for T-ALL in the specific combination of the genotypic mutations tested (including the developmental block caused by Rag deficiency), in microbiota composition, or minor differences in the genetic background of the strains.

Newborn Screening for Severe Combined Immunodeficiency and T-cell Lymphopenia in California, 2010-2017.

Newborn screening for severe combined immunodeficiency (SCID) was instituted in California in 2010. In the ensuing 6.5 years, 3 252 156 infants in the state had DNA from dried blood spots assayed for T-cell receptor excision circles (TRECs). Abnormal TREC results were followed-up with liquid blood testing for T-cell abnormalities. We report the performance of the SCID screening program and the outcomes of infants who were identified. Data that were reviewed and analyzed included demographics, nursery summaries, TREC and lymphocyte flow-cytometry values, and available follow-up, including clinical and genetic diagnoses, treatments, and outcomes. Infants with clinically significant T-cell lymphopenia (TCL) were successfully identified at a rate of 1 in 15 300 births. Of these, 50 cases of SCID, or 1 in 65 000 births (95% confidence interval 1 in 51 000-1 in 90 000) were found. Prompt treatment led to 94% survival. Infants with non-SCID TCL were also identified, diagnosed and managed, including 4 with complete DiGeorge syndrome who received thymus transplants. Although no cases of typical SCID are known to have been missed, 2 infants with delayed-onset leaky SCID had normal neonatal TREC screens but came to clinical attention at 7 and 23 months of age. Population-based TREC testing, although unable to detect immune defects in which T cells are present at birth, is effective for identifying SCID and clinically important TCL with high sensitivity and specificity. The experience in California supports the rapid, widespread adoption of SCID newborn screening.

Effects of Birth Season and Thymus Transplantation on Experimental Animal Longevity

It is demonstrated in this study that a decrease in the rate of irreversible age-related thymus atrophy affects animal longevity. It was observed that the life span of rats may be increased by thymus transplantation from young animals into the immune privileged regions of ageing rats. It has been shown that the season of birth has effects on animal longevity, namely, rats born in the spring–summer period live shorter lives than those born in autumn or winter.

Clinical and immunophenotypic characteristics of patients with chromosome 22q11.2 deletion syndrome: a single institution's experience.

Aim:The aim of this study was to identify the clinical and immunologic features of patients with 22q11.2 deletion syndrome who were followed up in our clinic. Thus, it is aimed to identify the syndrome early, choose the right treatment options according to humoral and cellular immunologic analysis, and enlighten how to follow up these kinds of patients with immunodeficiencies.Material and Methods:We retrospectively collected data by reviewing the files of 11 patients with 22q11.2 deletion syndrome who were followed up in our clinic between January 2003 and January 2015. The diagnoses were based on the patients’ clinical, genetic, and immunologic features. Demographic features, family history, initial symptoms on admission, physical findings, and results of immunologic studies of the patients. Age of diagnosis, treatment options, and clinical follow-up were evaluated.Results:The patients’ diagnosis age ranged from 1-11 months and the most common symptoms of admission were cardiac murmur and atypical facial appearance, which were detected during a routine physical examination. All patients had cardiac anomalies, and four patients had a history of cardiovascular surgery. Eight patients (72.7%) had a history of severe infection; recurrent lower respiratory tract infections were reported in six patients (54.5%), pulmonary tuberculosis in one patient (9.1%), and moniliasis resistant to treatment was detected in one patient. None of the patients required intravenous immunoglobulin replacement therapy, and antibiotic prophylaxis was administered to two patients with lymphopenia.Conclusion:22q11.2 deletion syndrome is a multi-systemic disorder that should be evaluated by a multidisciplinary team. It should be kept in mind for patients with neonatal hypocalcemic tetany or recurrent infections or atypical facial appearance with cardiac anomalies. Early diagnosis should lead to immunologic analysis and enable the choice of treatment. Preventive measures against infection is recommended for the patients with incomplete immunodeficiency, and thymus transplantation is recommended for patients with complete immunodeficiency.

Xenotransplantation tolerance: applications for recent advances in modified swine.

The aim of this study was to review the recent progress in xenotransplantation achieved through genetic engineering and discuss the potential of tolerance induction to overcome remaining barriers to extended xenograft survival. The success of life-saving allotransplantation has created a demand for organ transplantation that cannot be met by the supply of human organs. Xenotransplantation is one possible solution that would allow for a nearly unlimited supply of organs. Recent genetic engineering of swine has decreased the reactivity of preformed antibodies to some, but not all, potential human recipients. Experiments using genetically modified swine organs have now resulted in survival of life-supporting kidneys for over a year. However, the grafts show evidence of antibody-mediated rejection on histology, suggesting additional measures will be required for further extension of graft survival. Tolerance induction through mixed chimerism or thymic transplantation across xenogeneic barriers would be well suited for patients with a positive crossmatch to genetically modified swine or relatively negative crossmatches to genetically modified swine, respectively. This review highlights the current understanding of the immunologic processes in xenotransplantation and describes the development and application of strategies designed to overcome them from the genetic modification of the source animal to the induction of tolerance to xenografts.

Thymic Epithelial Cell Support of Thymopoiesis Does Not Require Klotho.

Age-related thymic involution is characterized by a decrease in thymic epithelial cell (TEC) number and function parallel to a disruption in their spatial organization, resulting in defective thymocyte development and proliferation as well as peripheral T cell dysfunction. Deficiency of Klotho, an antiaging gene and modifier of fibroblast growth factor signaling, causes premature aging. To investigate the role of Klotho in accelerated age-dependent thymic involution, we conducted a comprehensive analysis of thymopoiesis and peripheral T cell homeostasis using Klotho-deficient (Kl/Kl) mice. At 8 wk of age, Kl/Kl mice displayed a severe reduction in the number of thymocytes (10-100-fold reduction), especially CD4 and CD8 double-positive cells, and a reduction of both cortical and medullary TECs. To address a cell-autonomous role for Klotho in TEC biology, we implanted neonatal thymi from Klotho-deficient and -sufficient mice into athymic hosts. Kl/Kl thymus grafts supported thymopoiesis equivalently to Klotho-sufficient thymus transplants, indicating that Klotho is not intrinsically essential for TEC support of thymopoiesis. Moreover, lethally irradiated hosts given Kl/Kl or wild-type bone marrow had normal thymocyte development and comparably reconstituted T cells, indicating that Klotho is not inherently essential for peripheral T cell reconstitution. Because Kl/Kl mice have higher levels of serum phosphorus, calcium, and vitamin D, we evaluated thymus function in Kl/Kl mice fed with a vitamin D-deprived diet. We observed that a vitamin D-deprived diet abrogated thymic involution and T cell lymphopenia in 8-wk-old Kl/Kl mice. Taken together, our data suggest that Klotho deficiency causes thymic involution via systemic effects that include high active vitamin D levels.

Engineering Regenerative Thymic Tissues to Restore Long-Term T Cell Lymphopoiesis

The thymus is a primary lymphoid organ that plays a critical role in the development of adaptive T cell immunity and central tolerance. Bone marrow-derived lymphoid progenitor cells migrate into the thymus and interact with thymic epithelial cells (TECs) through sequential positive and negative selection to mature. Thymus-educated mature T cells express a diverse, MHC-restricted and self-tolerant T cell receptor (TCR) repertoire that protects against infection and prevents autoimmunity. Patients born with congenital thymic aplasia, due to 22q11 Deletion Syndrome, or mutations in TBX1, FOXN1 or CHD7, present with complete absence of T cells and a severe combined immunodeficiency (SCID)-like phenotype. Bone marrow transplantation does not cure the thymic defect in these patients and severe infections occur within the first year of life if left untreated. Allogenic thymus transplantation has provided proof of principle that HLA-unmatched pediatric donor thymic tissues can lead to successful immune reconstitution with the emergence of a diverse TCR V-beta repertoire. However, post-transplant organ-specific autoimmunity remains a major concern. Currently allogeneic thymus transplantation is no longer available in the US leaving a deadly therapeutic void for patients born without thymus. Patient-specific or histocompatible thymic tissues derived from pluripotent stem cells could address the critically unmet need, and also a broader range of clinical applications including immune reconstitution post hematopoietic stem cell transplantation (HSCT) and tolerance induction for solid donor organs.The thymus contains two major non-hematological components: the thymic stromal cells and the extracellular matrix (ECM). The thymic stromal layer is composed of thymic epithelial cells and mesenchymal cells. The thymic ECM forms a three-dimensional (3D) network to provide physical support and nutrition to thymic stromal cells.Methods: To address the need for histocompatible regenerative thymic tissues, we aim to differentiate fully functional thymic epithelial progenitor cells (TEPCs) from human pluripotent stem cells (hPSCs) and further generate 3D transplantable organoids using engineered matrix proteins that mimic the native thymic microenvironment.Results: We have developed a novel platform to generate hPSC-derived TEPCs by dissecting the key signaling pathways that govern human thymic ontogeny. These hPSC-derived TEPCs express the defining markers of TEPC-fate, such as FoxN1, Cytokeratin 8, Cytokeratin 5, Delta-like Canonical Notch Ligand 4 (DLL4) and MHC class II. Previous studies have shown FoxN1 to be the master regulator controlling thymic development, however, little is known about its regulatory network. Elucidating and validating the factors that initiate and maintain FoxN1 expression is the key to successfully engineer sustainable thymic tissues. We have identified a combination of morphogens that can maintain the expression of FoxN1, DLL4 and AIRE of primary TECs in culture. To gain insight into the composition of primary thymic ECM proteins and adapt their characteristics beyond the features of commercially available 3D hydrogels, we analyzed a series of human fetal thymic tissues using whole transcriptome analysis. Our current work focuses on adapting our 2D culture protocol to sustain hPSC-TEPCs in 3D matrix-based organoids. Ongoing studies test the capacity of hPSC-TECPs to promote T cell maturation and the development of a diverse TCR repertoire in an athymic xenograft mouse model (NSG-FoxN1null).Conclusions: hPSC can be differentiated in vitro into TEPC-fate and developed into thymic organoids using custom-designed protein matrices. Studies to test sustainability and functionality of the engineered thymic organoids in vivo are currently under way. DisclosuresNo relevant conflicts of interest to declare.

DIAGNOSIS AND MANAGEMENT OF AN INFANT WITH ATYPICAL COMPLETE DIGEORGE IMMUNOLOGIC PHENOTYPE

Introduction The diagnosis of complete DiGeorge syndrome is seen ≤1.5% of patients with 22q11.2 syndrome. We present a rare case of an infant with CHARGE syndrome with atypical complete DiGeorge immunologic phenotype. Case Description A 3-day old Caucasian male was referred for absent TRECs for SCID. He had known arch hypoplasia prior to delivery. Following delivery, a tracheoesophageal fistula was identified. Additional congenital anomalies included large patent ductus arteriosus, bilateral iris colobomas, dysplastic ears, microphallus, cryptorchidism, and bilateral hydronephrosis. His initial lymphocyte enumeration demonstrated absent T-cells, normal number of B-cells and NK-cells. Lymphocyte proliferation to mitogens was absent. Targeted sequencing demonstrated a new variant in CHD7c.2698-11A>G predicted to affect splicing. SCID genetic panel and chromosomal microarray were normal. Based on workups and congenital anomalies, he was diagnosed with CHARGE syndrome with a complete DiGeorge phenotype. At 5 months of age, he developed Omenn syndrome-like manifestations with diffuse non-blanching erythematous macular rash over his body. Serial lymphocyte subsets demonstrated the development of T-cells expressing HLA-DR indicating the emergence of activated T-cells (CD3 269/cumm; CD3+HLA-DR 258/cumm, 96% of total T-cells). Spectratyping confirmed the presence of oligoclonal-T-cells. Therefore, immunosuppression with cyclosporin and prednisolone was given for treatment of self-reactive oligoclonal-T-cells with resolution of rashes. He is currently awaiting thymus transplantation, and receiving IVIG, and antimicrobial prophylaxis. Discussion Although not yet approved by FDA, thymus transplantation is the preferred treatment for complete DiGeorge syndrome. Additionally, close monitoring of activated self-reactive oligoclonal-T-cells symptoms with proper immunosuppressive treatment is crucial while patient is waiting for thymus transplantation.

A 10 year experience of the management of severe hypocalcaemia associated with thymus transplantation in a United Kingdom tertiary centre

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

The Case for Cardiac Xenotransplantation in Neonates: Is Now the Time to Reconsider Xenotransplantation for Hypoplastic Left Heart Syndrome?

Neonatal cardiac transplantation for hypoplastic left heart syndrome (HLHS) is associated with excellent long-term survival compared to older recipients. However, heart transplantation for neonates is greatly limited by the critical shortage of donor hearts, and by the associated mortality of the long pre-transplant waiting period. This led to the development of staged surgical palliation as the first-line surgical therapy for HLHS. Recent advances in genetic engineering and xenotransplantation have provided the potential to replicate the excellent results of neonatal cardiac allotransplantation while eliminating wait-list-associated mortality through genetically modified pig-to-human neonatal cardiac xenotransplantation. The elimination of the major pig antigens in addition to the immature B-cell response in neonates allows for the potential to induce B-cell tolerance. Additionally, the relatively mature neonatal T-cell response could be reduced by thymectomy at the time of operation combined with donor-specific pig thymus transplantation to "reprogram" the host's T-cells to recognize the xenograft as host tissue. In light of the recent significantly increased graft survival of genetically-engineered pig-to-baboon cardiac xenotransplantation, we propose that now is the time to consider devoting research to advance the potential clinical application of cardiac xenotransplantation as a treatment option for patients with HLHS. Employing cardiac xenotransplantation could revolutionize therapy for complex congenital heart defects and open a new chapter in the field of pediatric cardiac transplantation.

Thymus autonomy as a prelude to leukemia.

Cell competition in the thymus promotes turnover and functions as a tumor suppressor by inhibiting leukemia. Using thymus transplantation experiments, we have shown that the presence of T lymphocyte precursors, recently seeding the thymus, promotes the clearance of precursors with a longer time of thymus residency. If cell competition is impaired and no cells seed the thymus, the organ is capable of sustaining T lymphocyte production, a state termed thymus autonomy. However, we observed consistently that prolonged autonomy is permissive to the emergence of T cell acute lymphoblastic leukemia (T-ALL). This resembled the onset of T-ALL in patients treated by gene therapy for X-linked severe combined immunodeficiency (SCID-X1). Following treatment, thymus activity was established, with T lymphocyte production, although no bone marrow contribution was detected. However, some patients developed T-ALL. The favored explanation for malignant transformation was considered to be genotoxicity due to integration of the retroviral vector next to oncogenes, thereby activating them ectopically. Although plausible, we consider an alternative, mutually nonexclusive explanation: that any condition enabling prolonged thymus autonomy will promote leukemogenesis. In support of this view, two independent studies have recently shown that the efficacy of reconstitution of the bone marrow in the context of SCID-X1 dramatically influences the outcome of treatment, and that lymphoid malignancies emerge following transplantation of a small number of healthy progenitors. Here, we discuss the most recent data in light of our own studies in thymopoiesis and the conditions that trigger malignant transformation of thymocytes in various experimental and clinical settings.

Transplant Tolerance: Current Insights and Strategies for Long-Term Survival of Xenografts.

Xenotransplantation is an attractive solution to the problem of allograft shortage. However, transplants across discordant species barriers are subject to vigorous immunologic and pathobiologic hurdles, some of which might be overcome with the induction of immunologic tolerance. Several strategies have been designed to induce tolerance to a xenograft at both the central (including induction of mixed chimerism and thymic transplantation) and peripheral (including adoptive transfer of regulatory cells and blocking T cell costimulation) levels. Currently, xenograft tolerance has been well-established in rodent models, but these protocols have not yet achieved similar success in nonhuman primates. This review will discuss the major barriers that impede the establishment of immunological tolerance across xenogeneic barriers and the potential solution to these challenges, and provide a perspective on the future of the development of novel tolerance-inducing strategies.