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.

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.

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.

[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.

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.

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.

Isolation of Highly Viable Thymic Epithelial Cells for Use in In Vitro and In Vivo Experiments.

Thymic epithelial cells (TECs) play multiple essential roles in T-cell development and the establishment of immune tolerance, but their isolation can be challenging, and their low viability upon isolation complicates downstream experiments. A method that allows TECs to be isolated easily and to survive afterward will be useful for elucidating key questions in TEC biology. Here, we demonstrate a simple method to isolate highly viable TECs. Primary TECs isolated using papain together with collagenase IV and DNase I survive and proliferate in vitro. Moreover, these primary TECs functionally engraft after intrathymic transplantation into recipient mice. Thus, the methods described herein will be useful for elucidating the roles of TECs and TEC subsets in T-cell development and immune tolerance.

Alcohol consumption increases susceptibility to pneumococcal pneumonia in a humanized murine HIV model mediated by intestinal dysbiosis.

Alcohol use in persons living with HIV (PLWH) worsens the severity of bacterial pneumonia. However, the exact mechanism(s) by which this occurs remain ill-defined. We hypothesized that alcohol in the setting of HIV infection decreases Streptococcus pneumoniae clearance from the lung through mechanisms mediated by the gut microbiota. Humanized BLT (bone marrow, liver, thymus) mice were infected with 1 × 104 TCID50 of HIV (BAL and JRCSF strains) via intraperitoneal (i.p.) injection. One week post-HIV infection, animals were switched to a Lieber-DeCarli 5% ethanol diet or an isocaloric control diet for 10 days. Alcohol-fed animals were also given two binges of 2 g/kg ethanol on days 5 and 10. Feces were also collected, banked, and the community structures were analyzed. Mice were then infected with 1 × 105 CFU (colony-forming units) of S. pneumoniae and were sacrificed 48 h later. HIV-infected mice had viral loads of ∼2 × 104 copies/mL of blood 1 week post-infection, and exhibited an ∼57% decrease in the number of circulating CD4+ T cells at the time of sacrifice. Fecal microbial community structure was significantly different in each of the feeding groups, as well as with HIV infection. Alcohol-fed mice had a significantly higher burden of S. pneumoniae 48 h post-infection, regardless of HIV status. In follow-up experiments, female C57BL/6 mice were treated with a cocktail of antibiotics daily for 2 weeks and recolonized by gavage with intestinal microbiota from HIV+ ethanol-fed, HIV+ pair-fed, HIV- ethanol-fed, or HIV- pair-fed mice. Recolonized mice were then infected with S. pneumoniae and were sacrificed 48 h later. The intestinal microbiota from alcohol-fed mice (regardless of HIV status) significantly impaired clearance of S. pneumoniae. Collectively, these data indicate that alcohol feeding, as well as alcohol-associated intestinal dysbiosis, compromise pulmonary host defenses against pneumococcal pneumonia. Determining whether HIV infection acts synergistically with alcohol use in impairing pulmonary host defenses will require additional study.

Immune and Genetic Features of the Chromosome 22q11.2 Deletion (DiGeorge Syndrome).

PURPOSE OF REVIEW: This review provides an update on the progress in identifying the range of immunological dysfunction seen in DiGeorge syndrome and on more recent diagnostic and treatment approaches. RECENT FINDINGS: Clinically, the associated thymic hypoplasia/aplasia is well known and can have profound effects on T cell function. Further, the humoral arm of the immune system can be affected, with hypogammaglobulinemia and poor vaccine-specific antibody response. Additionally, genetic testing utilizing chromosomal microarray demonstrates a small but significant number of 22q11 deletions that are not detectable by standard FISH testing. The recent addition of a TREC assay to newborn screening can identify a subset of infants whose severe immune defects may result from 22q11 deletion. This initial presentation now also places the immunologist in the role of "first responder" with regard to diagnosis and management of these patients. DiGeorge syndrome reflects a clinical phenotype now recognized by its underlying genetic diagnosis, chromosome 22q11.2 deletion syndrome, which is associated with multisystem involvement and variable immune defects among patients. Updated genetic and molecular techniques now allow for earlier identification of immune defects and confirmatory diagnoses, in this disorder with life-long clinical issues.

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.

Human immunodeficiency virus infection induces lymphoid fibrosis in the BM-liver-thymus-spleen humanized mouse model.

A major pathogenic feature associated with HIV infection is lymphoid fibrosis, which persists during antiretroviral therapy (ART). Lymphoid tissues play critical roles in the generation of antigen-specific immune response, and fibrosis disrupts the stromal network of lymphoid tissues, resulting in impaired immune cell trafficking and function, as well as immunodeficiency. Developing an animal model for investigating the impact of HIV infection-induced lymphoid tissue fibrosis on immunodeficiency and immune cell impairment is critical for therapeutics development and clinical translation. Said model will enable in vivo mechanistic studies, thus complementing the well-established surrogate model of SIV infection-induced lymphoid tissue fibrosis in macaques. We developed a potentially novel human immune system-humanized mouse model by coengrafting autologous fetal thymus, spleen, and liver organoids under the kidney capsule, along with i.v. injection of autologous fetal liver-derived hematopoietic stem cells, thus termed the BM-liver-thymus-spleen (BLTS) humanized mouse model. BLTS humanized mouse model supports development of human immune cells and human lymphoid organoids (human thymus and spleen organoids). HIV infection in BLTS humanized mice results in progressive fibrosis in human lymphoid tissues, which was associated with immunodeficiency in the lymphoid tissues, and lymphoid tissue fibrosis persists during ART, thus recapitulating clinical outcomes.

Role of Regulatory T Cells in Tumor-Bearing Mice Treated with Allo-Hematopoietic Stem Cell Transplantation Plus Thymus Transplantation.

We recently developed a new allogeneic hematopoietic stem cell transplantation method (allo-HSCT) combined with thymus transplantation (TT) from the same donor (allo-HSCT + TT). This method induces elevated T cell function with mild graft-versus-host disease (GVHD) in comparison to conventional HSCT alone and HSCT + donor lymphocyte infusion (DLI). This new method is effective against several intractable diseases, including malignant tumors, for which conventional treatments are ineffective. Regulatory T (Treg) cells play an important role in the enhanced graft-versus-tumor (GVT) effect and reduction of GVHD, thus leading to longer survival. Replacement and reduction of elevated Treg cells by donor-derived allo-Treg cells from the transplanted thymus may play one of crucial roles in the effect. This review discusses the role of Treg cells in a tumor-bearing mouse model treated with allo-HSCT + TT.

Humanized mice reveal an essential role for human hepatocytes in the development of the liver immune system.

The liver is an immunological organ with a distinct immune cell profile. Although the composition and function of liver immune cells have been widely investigated, the mechanisms regulating the development and homeostasis of the specialized immune system, especially in humans, remain largely unknown. Herein, we address this question in humanized mice (hu-mice) that were constructed by transplantation of human fetal thymus and CD34+ hematopoietic stem/progenitor cells in immunodeficient mice with or without autologous human hepatocyte engraftment. Although the levels of human immune cell reconstitution in peripheral blood and spleen were comparable between hu-mice with and without human hepatocyte engraftment, the former group showed that human immune cell reconstitution in the liver was significantly improved. Notably, human immune cells, including Kupffer cells, dendritic cells and natural killer cells, were shown to be closely colocalized with human hepatocytes in the liver. Human hepatocytes engrafted in the mouse liver were found to produce IL-3, IL-15, GM-CSF, M-CSF, MCP-1, CXCL-1 and CXCL-10, which are known to be important for immune cell development, differentiation, tissue migration and retention, and have no or poor cross-reaction between humans and mice. Furthermore, human hepatocytes were able to support human immune cell survival and expansion in an in vitro co-culture assay. This study demonstrates an essential role for hepatocytes in the development and maintenance of the liver immune cell profile. The hu-mouse model with human autologous immune cell and hepatocyte reconstitution has potential for use in studies of the pathogenesis of liver immune disorders such as hepatotropic virus infections.