Experience with cultured thymus tissue in 105 children.

BACKGROUND: Currently, there are no approved therapies to treat congenital athymia, a condition of immune deficiency resulting in high early mortality due to infection and immune dysregulation. Multiple syndromic conditions, such as complete DiGeorge syndrome, 22q11.2 deletion syndrome, CHARGE (coloboma, heart defects, choanal atresia, growth or mental retardation, genital hypoplasia, and ear anomalies and/or deafness) syndrome, diabetic embryopathy, other genetic variants, and FOXN1 deficiency, are associated with congenital athymia. OBJECTIVE: Our aims were to study 105 patients treated with cultured thymus tissue (CTT), and in this report, to focus on the outcomes of 95 patients with treatment-naive congenital athymia. METHODS: A total of 10 prospective, single-arm open-label studies with patient enrollment from 1993 to 2020 form the basis of this data set. Patients were tested after administration of CTT for T-cell development; all adverse events and infections were recorded. RESULTS: A total of 105 patients were enrolled and received CTT (the full analysis set). Of those patients, 10 had diagnoses other than congenital athymia and/or received prior treatments. Of those 105 patients, 95 patients with treatment-naive congenital athymia were included in the efficacy analysis set (EAS). The Kaplan-Meier estimated survival rates at year 1 and year 2 after administration of CTT in the EAS were 77% (95% CI = 0.670-0.844) and 76% (95% CI = 0.657-0.834), respectively. In all, 21 patients died in the first year before developing naive T cells and 1 died in the second year after receipt of CTT; 3 subsequent deaths were not related to immunodeficiency. A few patients developed alopecia, autoimmune hepatitis, psoriasis, and psoriatic arthritis after year 1. The rates of infections, autologous graft-versus-host-disease manifestations, and autoimmune cytopenias all decreased approximately 1 year after administration of CTT. CONCLUSION: Treatment with CTT led to development of naive T cells with a 1-year survival rate of 77% and a median follow-up time of 7.6 years. Immune reconstitution sufficient to prevent infections and support survival typically develops 6 to12 months after administration of CTT.

Care of Children with DiGeorge Before and After Cultured Thymus Tissue Implantation.

BACKGROUND: Children with complete DiGeorge anomaly (cDGA) have congenital athymia plus a myriad of other challenging clinical conditions. The term cDGA encompasses children with congenital athymia secondary to 22q11.2DS, CHARGE syndrome (coloboma, heart defects, choanal atresia, growth or mental retardation, genital abnormalities, and ear abnormalities and/or deafness), and other genetic abnormalities. Some children have no known genetic defects. Since 1993, more than 100 children with congenital athymia have been treated with cultured thymus tissue implantation (CTTI). Naïve T cells develop approximately 6 to 12 months after CTTI. Most of the children had significant comorbidities such as heart disease, hypoparathyroidism, and infections requiring complex clinical care post cultured thymus tissue implantation (CTTI). OBJECTIVE: The purpose of this guidance is to assist multidisciplinary teams in caring for children with cDGA both before and after CTTI. METHODS: Thirty-one specialists, in addition to the authors, were asked to share their experience in caring for children with cDGA at Duke University Health System, before and after CTTI. These specialists included physicians, nurses, dentists, therapists, and dieticians. RESULTS: The goal of a multidisciplinary approach is to have children in the best possible condition for receiving CTTI and provide optimal care post CTTI through development of naïve T cells and beyond. The CTT (cultured thymus tissue) must be protected from high doses of steroids which can damage CTT. Organs must be protected from adverse effects of immunosuppression. CONCLUSION: Creating a multidisciplinary team and a detailed plan of care for children with cDGA is important for optimal outcomes.

Self-renewal of double-negative 3 early thymocytes enables thymus autonomy but compromises the ß-selection checkpoint.

T lymphocyte differentiation in the steady state is characterized by high cellular turnover whereby thymocytes do not self-renew. However, if deprived of competent progenitors, the thymus can temporarily maintain thymopoiesis autonomously. This bears a heavy cost, because prolongation of thymus autonomy causes leukemia. Here, we show that, at an early stage, thymus autonomy relies on double-negative 3 early (DN3e) thymocytes that acquire stem-cell-like properties. Following competent progenitor deprivation, DN3e thymocytes become long lived, are required for thymus autonomy, differentiate in vivo, and include DNA-label-retaining cells. At the single-cell level, the transcriptional programs of thymopoiesis in autonomy and the steady state are similar. However, a new cell population emerges in autonomy that expresses an aberrant Notch target gene signature and bypasses the ß-selection checkpoint. In summary, DN3e thymocytes have the potential to self-renew and differentiate in vivo if cell competition is impaired, but this generates atypical cells, probably the precursors of leukemia.

Defective dystrophic thymus determines degenerative changes in skeletal muscle.

In Duchenne muscular dystrophy (DMD), sarcolemma fragility and myofiber necrosis produce cellular debris that attract inflammatory cells. Macrophages and T-lymphocytes infiltrate muscles in response to damage-associated molecular pattern signalling and the release of TNF-α, TGF-ß and interleukins prevent skeletal muscle improvement from the inflammation. This immunological scenario was extended by the discovery of a specific response to muscle antigens and a role for regulatory T cells (Tregs) in muscle regeneration. Normally, autoimmunity is avoided by autoreactive T-lymphocyte deletion within thymus, while in the periphery Tregs monitor effector T-cells escaping from central regulatory control. Here, we report impairment of thymus architecture of mdx mice together with decreased expression of ghrelin, autophagy dysfunction and AIRE down-regulation. Transplantation of dystrophic thymus in recipient nude mice determine the up-regulation of inflammatory/fibrotic markers, marked metabolic breakdown that leads to muscle atrophy and loss of force. These results indicate that involution of dystrophic thymus exacerbates muscular dystrophy by altering central immune tolerance.

IFNγ and iNOS-Mediated Alterations in the Bone Marrow and Thymus and Its Impact on Mycobacterium avium-Induced Thymic Atrophy.

Disseminated infection with the high virulence strain of Mycobacterium avium 25291 leads to progressive thymic atrophy. We previously showed that M. avium-induced thymic atrophy results from increased glucocorticoid levels that synergize with nitric oxide (NO) produced by interferon gamma (IFNγ) activated macrophages. Where and how these mediators act is not understood. We hypothesized that IFNγ and NO promote thymic atrophy through their effects on bone marrow (BM) T cell precursors and T cell differentiation in the thymus. We show that M. avium infection cause a reduction in the percentage and number of common lymphoid progenitors (CLP). Additionally, BM precursors from infected mice show an overall impaired ability to reconstitute thymi of RAGKO mice, in part due to IFNγ. Thymi from infected mice present an IFNγ and NO-driven inflammation. When transplanted under the kidney capsule of uninfected mice, thymi from infected mice are unable to sustain T cell differentiation. Finally, we observed increased thymocyte death via apoptosis after infection, independent of both IFNγ and iNOS; and a decrease on active caspase-3 positive thymocytes, which is not observed in the absence of iNOS expression. Together our data suggests that M. avium-induced thymic atrophy results from a combination of defects mediated by IFNγ and NO, including alterations in the BM T cell precursors, the thymic structure and the thymocyte differentiation.

Reconstitution of a functional human thymus by postnatal stromal progenitor cells and natural whole-organ scaffolds.

The thymus is a primary lymphoid organ, essential for T cell maturation and selection. There has been long-standing interest in processes underpinning thymus generation and the potential to manipulate it clinically, because alterations of thymus development or function can result in severe immunodeficiency and autoimmunity. Here, we identify epithelial-mesenchymal hybrid cells, capable of long-term expansion in vitro, and able to reconstitute an anatomic phenocopy of the native thymus, when combined with thymic interstitial cells and a natural decellularised extracellular matrix (ECM) obtained by whole thymus perfusion. This anatomical human thymus reconstruction is functional, as judged by its capacity to support mature T cell development in vivo after transplantation into humanised immunodeficient mice. These findings establish a basis for dissecting the cellular and molecular crosstalk between stroma, ECM and thymocytes, and offer practical prospects for treating congenital and acquired immunological diseases.

Replacing defective thymus function.

PURPOSE OF REVIEW: Transplantation of cultured postnatal allogeneic thymus has been successful for treating athymia, mostly associated with complete DiGeorge syndrome, for more than 20 years. Advances in molecular genetics provide opportunities for widening the range of athymic conditions that can be treated while advances in cell culture and organ/tissue regeneration may offer the prospect of alternative preparations of thymic tissue. There are potential broader applications of this treatment outside congenital athymia. RECENT FINDINGS: At the same time as further characterization of the cultured thymus product in terms of thymic epithelial cells and lymphoid composition, preclinical studies have looked at de-novo generation of thymic epithelial cells from stem cells and explored scaffolds for delivering these as three-dimensional structures. In the era of newborn screening for T-cell lymphopaenia, a broadening range of defects leading to athymia is being recognized and new assays should allow differentiation of these from haematopoietic cell defects, pending their genetic/molecular characterization. Evidence suggests that the tolerogenic effect of transplanted thymus could be exploited to improve outcomes after solid organ transplantation. SUMMARY: Thymus transplantation, the accepted standard treatment for complete DiGeorge syndrome is also appropriate for other genetic defects leading to athymia. Improved strategies for generating thymus may lead to better outcomes and broader application of this treatment.

Cell Competition, the Kinetics of Thymopoiesis, and Thymus Cellularity Are Regulated by Double-Negative 2 to 3 Early Thymocytes.

Cell competition in the thymus is a homeostatic process that drives turnover. If the process is impaired, thymopoiesis can be autonomously maintained for several weeks, but this causes leukemia. We aimed to understand the effect of cell competition on thymopoiesis, identify the cells involved, and determine how the process is regulated. Using thymus transplantation experiments, we found that cell competition occurs within the double-negative 2 (DN2) and 3 early (DN3e) thymocytes and inhibits thymus autonomy. Furthermore, the expansion of DN2b is regulated by a negative feedback loop that is imposed by double-positive thymocytes and determines the kinetics of thymopoiesis. This feedback loop affects the cell cycle duration of DN2b, in a response controlled by interleukin 7 availability. Altogether, we show that thymocytes do not merely follow a pre-determined path if provided with the correct signals. Instead, thymopoiesis dynamically integrates cell-autonomous and non-cell-autonomous aspects that fine-tune normal thymus function.

Cultured thymus tissue implantation promotes donor-specific tolerance to allogeneic heart transplants.

Eighty-six infants born without a thymus have been treated with allogeneic cultured thymus tissue implantation (CTTI). These infants, who lack T cells and are profoundly immunodeficient at birth, after CTTI from an unmatched donor develop T cells similar to those of recipient that are tolerant to both their own major histocompatibility antigens and those of the donor. We tested use of CTTI with the goal of inducing tolerance to unmatched heart transplants in immunocompetent rats. We thymectomized and T cell-depleted Lewis rats. The rats were then given cultured thymus tissue from F1 (Lewis × Dark Agouti ) under the kidney capsule and vascularized Dark Agouti (DA) heart transplants in the abdomen. Cyclosporine was administered for 4 months. The control group did not receive CTTI. Recipients with CTTI showed repopulation of naive and recent thymic emigrant CD4 T cells; controls had none. Recipients of CTTI did not reject DA cardiac allografts. Control animals did not reject DA grafts, due to lack of functional T cells. To confirm donor-specific unresponsiveness, MHC-mismatched Brown Norway (BN) hearts were transplanted 6 months after the initial DA heart transplant. LW rats with LWxDA CTTI rejected the third-party BN hearts (mean survival time 10 days); controls did not. CTTI recipients produced antibody against third-party BN donor but not against the DA thymus donor, demonstrating humoral donor-specific tolerance. Taken together, F1(LWxDA) CTTI given to Lewis rats resulted in specific tolerance to the allogeneic DA MHC expressed in the donor thymus, with resulting long-term survival of DA heart transplants after withdrawal of all immunosuppression.

Co-transplantation of Vascularized Thymic Graft with Kidney in Pig-to-Nonhuman Primates for the Induction of Tolerance Across Xenogeneic Barriers.

Using advanced gene editing technologies, xenotransplantation from multi-transgenic alpha-1,3-galactosyltransferase knockout pigs has demonstrated marked prolongation of renal xenograft survival, ranging from days to greater than several months for life-supporting kidneys and >2 years in a heterotopic non-life-supporting cardiac xenograft model. However, continuous administration of multiple immunosuppressive drugs continues to be required, and attempts to taper immunosuppression have been unsuccessful. These data are consistent with previous reports indicating that the human-anti-porcine T cell response is similar or stronger than that across allogeneic barriers. Due to the strength of both the innate and adaptive immune responses in xenotransplantation, the level of continuous immunosuppression needed to control these responses and prolong xenograft survival has been associated with prohibitive morbidity and mortality. These facts provide compelling rationale to pursue a clinically applicable strategy for the induction of tolerance.Mixed chimerism and thymic tissue transplantation have both achieved xenogeneic tolerance in pig-to-mouse models, and both have recently been extended to pig-to-baboon models. Although these strategies are promising in small animal models, neither direct intravenous injection of porcine bone marrow cells nor direct fetal thymic tissue transplantation into recipients was able to achieve >2 days chimerism following BM Tx or the engraftment of thymic tissues across xenogeneic barriers in pig-to-nonhuman primate models. Several innovative procedures have been largely developed by Kazuhiko Yamada to overcome these failures. These include vascularized thymic transplantation, combined with either thymokidney (TK) or vascularized thymic lobe (VTL) transplantation. Utilizing the strategy of transplanting vascularized thymic grafts with kidney from the same GalT-KO donor without further gene modification, we have achieved longer than 6 months survival of life-supporting kidneys in a baboon. Notably, the recipient became donor specific unresponsive and developed new thymic emigrants. In this chapter, we introduce a brief summary of our achievements to date toward the successful induction of tolerance by utilizing our novel strategy of vascularized thymic transplantation (including thymokidney transplantation), as well as describe the step-by-step methodology of surgical and in vitro procedures which are required for this experiment.

Rapid thymectomy of NSG mice to analyze the role of native and grafted thymi in humanized mice.

We developed a rapid method to remove the native mouse thymus from NSG mice, which allowed us to compare the behavior of human immune cells in the presence or absence of human T cells in human immune system mice. Removing the native mouse thymus is critical for studies of human thymopiesis in grafted thymic tissue in humanized mice.

The Tumor-Immune Response Is Not Compromised by Mesenchymal Stromal Cells in Humanized Mice.

Therapeutic uses of mesenchymal stromal cells (MSCs) have emerged over the past decade. Yet, their effect on tumor growth remains highly debated, particularly in an immune competent environment. In this study, we wanted to investigate the impact of human umbilical cord-derived MSCs (hUC-MSCs) on tumor growth in humanized mice generated by the human adoptive transfer of PBMCs or the cotransplantation of hematopoietic stem cells and human thymic tissue (human BLT [Hu-BLT]). Our results showed that the growth and immune rejection of engineered human fibroblastic tumors was not altered by the injection of hUC-MSCs in immune-deficient or humanized mice, respectively. This was observed whether tumor cells were injected s.c. or i.v. and independently of the injection route of the hUC-MSCs. Moreover, only in Hu-BLT mice did hUC-MSCs have some effects on the tumor-immune infiltrate, yet without altering tumor growth. These results demonstrate that hUC-MSCs do not promote fibroblastic tumor growth and neither do they prevent tumor infiltration and rejection by immune cells in humanized mice.

[Contribution of animal models to HIV research]. / Apport des modèles animaux dans la recherche sur le VIH.

Even today, despite triple therapy, the epidemic of the human immunodeficiency virus (HIV) is a major public health problem. In this perspective, continuous research is essential for the development of curative and vaccinal approaches. Animal models contribute to the implementation of new therapeutic and preventive strategies. We present here the characteristics of major animal models of HIV, which are non-human primates (SIV or SHIV-infected macaques and natural hosts of SIV), as well as different humanized mouse models and their advances. We will also list how they have already allowed, and still allow today, to broaden our knowledge on the physiopathology of HIV infection, tissue distribution of the virus, viral reservoirs, immunological responses against the virus in the very early infection stages and at the tissue level, but also in the development of vaccine candidates (RhCMV, broad-spectrum antibodies, etc…) and clinical trials for a cure. The advantages and limitations of the different animal models will be described. While continuing research on alternative methods, refinement or reduction of the animal model, a good knowledge of the specificities of each animal model allows an adequate use in relation to the scientific questions addressed.

Therapeutic targeting of Notch signaling and immune checkpoint blockade in a spontaneous, genetically heterogeneous mouse model of T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic cancer derived from the malignant transformation of T-cell progenitors. Outcomes remain poor for T-ALL patients who have either primary resistance to standard-of-care chemotherapy or disease relapse. Notably, there are currently no targeted therapies available in T-ALL. This lack of next-generation therapies highlights the need for relevant preclinical disease modeling to identify and validate new targets and treatment approaches. Here, we adapted a spontaneously arising, genetically heterogeneous, thymic transplantation-based murine model of T-ALL, recapitulating key histopathological and genetic features of the human disease, to the preclinical testing of targeted and immune-directed therapies. Genetic engineering of the murine Notch1 locus aligned the spectrum of Notch1 mutations in the mouse model to that of human T-ALL and confirmed aberrant, recombination-activating gene (RAG)-mediated 5' Notch1 recombination events as the preferred pathway in murine T-ALL development. Testing of Notch1-targeting therapeutic antibodies demonstrated T-ALL sensitivity to different classes of Notch1 blockers based on Notch1 mutational status. In contrast, genetic ablation of Notch3 did not impact T-ALL development. The T-ALL model was further applied to the testing of immunotherapeutic agents in fully immunocompetent, syngeneic mice. In line with recent clinical experience in T-cell malignancies, programmed cell death 1 (PD-1) blockade alone lacked anti-tumor activity against murine T-ALL tumors. Overall, the unique features of the spontaneous T-ALL model coupled with genetic manipulations and the application to therapeutic testing in immunocompetent backgrounds will be of great utility for the preclinical evaluation of novel therapies against T-ALL.

B Cell-Mediated Antigen Presentation through MHC Class II Is Dispensable for Atherosclerosis Progression.

Depletion of B cells attenuates plaque development and modulates T cell responses in mouse models of atherosclerosis, suggesting that Ag presentation by B cells may promote disease progression. Thus, we set out to determine the role of B cell-mediated MHC class II (MHC II) Ag presentation during atherosclerotic plaque development. We developed murine conditional MHC II deletion and expression systems under control of the B cell-restricted CD19 promoter in an experimental model of atherosclerosis. Mice lacking MHC II expression only on B cells exhibited systemic shifts in germinal center and marginal zone B cell populations, leading to a reduced Ab response compared with littermate control animals. However, all populations were present and normal cholesterol uptake was detected in the plasma following high-fat diet treatment. In a second model, in which conditional expression of MHC II is limited only to B cells, showed similar overall cellularity characteristics compared with mice with complete MHC II deficiency. High-fat diet feeding showed no major changes in atherosclerotic plaque size or plaque cellular content in either conditional deletion or conditional expression approaches, compared with control animals. By testing the necessity and sufficiency of MHC II on B cells in the progression of atherosclerosis, we determine that MHC II on B cells does not directly regulate lesion development in murine models.

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 (32 weeks). Conclusion: The results indicate that human thymus contains long-lived lymphoid progenitors that can maintain durable thymopoiesis and T-cell development.

Restoring thymic function: Then and now.

Thymic vulnerability, a leading cause of defective immunity, was discovered decades ago. To date, several strategies have been investigated to unveil any immunorestorative capacities they might confer. Studies exploiting castration, transplantation, adoptive cell therapies, hormones/growth factors, and cytokines have demonstrated enhanced in vitro and in vivo thymopoiesis, albeit with clinical restrictions. In this review, we will dissect the thymus on a physiological and pathological level and discuss the pros and cons of several strategies esteemed thymotrophic from a pre-clinical perspective. Finally, we will shed light on interleukin (IL)-21, a pharmacologically-promising cytokine with a significant thymotrophic nature, and elaborate on its potential clinical efficacy and safety in immune-deficient subjects.

Evaluation of a TGN1412 analogue using in vitro assays and two immune humanized mouse models.

Cytokine release syndrome (CRS) is a serious and potentially life-threatening complication typically associated with biological drug products. Pre-clinical testing in vitro and in vivo studies using non-human primates had failed to reliably predict CRS. To determine if bone marrow-thymus-liver (BLT) humanized mice with a fully engrafted human immune system or a CD34-humanized mouse model could predict CRS, we tested an anti-CD28 monoclonal antibody (mAb) similar to TGN1412. This TGN1412 analogue (TGN1412A) was initially tested in vitro and found to produce significant dose-dependent increases in cytokine production. For in vivo studies, adalimumab, an anti-tumor necrosis factor-alpha antibody known not to cause CRS, served as a negative control. We evaluated immune cell activation and cytokine expression in three independent experiments. In BLT humanized mice, significant increases in levels of human cytokines were identified in animals treated with anti-CD28 mAb. As expected, CD28+ cell detection was strongly reduced in the anti-CD28 treated group. Increased T cell activation was also observed. The control group did not show reductions in CD28+ T-cells and did not experience increased cytokine levels. Responses by CD34-humanized mice showed no significant differences between adalimumab and anti-CD28 treatment at doses used to test BLT-humanized mice. These results suggest that the TGN1412A produces similar results in vitro to the original TGN1412 monoclonal antibody. The BLT immune humanized mice but not the CD34 humanized mice produce both robust and specific cytokine responses and may represent a pre-clinical model to identify CRS.

BLT-Immune Humanized Mice as a Model for Nivolumab-Induced Immune-Mediated Adverse Events: Comparison of the NOG and NOG-EXL Strains.

Checkpoint inhibitors represent a new class of therapeutics in the treatment of cancer that has demonstrated remarkable clinical effectiveness. However, some patients have experienced serious immune-mediated adverse effects including pneumonitis, hepatitis, colitis, nephritis, dermatitis, encephalitis, and adrenal or pituitary insufficiency. These adverse events were not predicted by nonclinical studies. To determine if bone marrow-liver-thymus (BLT) immune humanized mice could demonstrate these adverse effects, we studied the effect of nivolumab on 2 strains of BLT-humanized mice, NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac (NOG) and NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(SV40/HTLV-IL3, CSF2)10-7Jic/JicTac (NOG-EXL). Mice were treated with 2.5, 5.0, or 10.0 mg/kg nivolumab or saline twice weekly for 28 days. BLT-NOG mice had significantly reduced survival compared with BLT-NOG-EXL mice. In spite of the difference in survival, both BLT-humanized strains showed adverse reactions similar to those reported in humans, including pneumonitis and hepatitis, with nephritis, dermatitis and adrenalitis also noted in some individuals. Additional histopathologic findings included pancreatic atrophy, myositis, and osteomyelitis in some animals. T-cell activation increased with concomitant loss of PD-1 detection. These findings show that BLT immune humanized mice can demonstrate immune-mediated adverse effects of antiPD1 therapy, and may represent a model that can be used to better understand toxicity of this class of drugs.