SOCS3 Expression by Thymic Stromal Cells Is Required for Normal T Cell Development

The suppressor of cytokine signaling 3 (SOCS3) is a major regulator of immune responses and inflammation as it negatively regulates cytokine signaling. Here, the role of SOCS3 in thymic T cell formation was studied in Socs3fl/flActin-creER mice (Δsocs3) with a tamoxifen inducible and ubiquitous Socs3 deficiency. Δsocs3 thymi showed a 90% loss of cellularity and altered cortico-medullary organization. Thymocyte differentiation and proliferation was impaired at the early double negative (CD4-CD8-) cell stage and apoptosis was increased during the double positive (CD4+CD8+) cell stage, resulting in the reduction of recent thymic emigrants in peripheral organs. Using bone marrow chimeras, transplanting thymic organoids and using mice deficient of SOCS3 in thymocytes we found that expression in thymic stromal cells rather than in thymocytes was critical for T cell development. We found that SOCS3 in thymic epithelial cells (TECs) binds to the E3 ubiquitin ligase TRIM 21 and that Trim21−/− mice showed increased thymic cellularity. Δsocs3 TECs showed alterations in the expression of genes involved in positive and negative selection and lympho-stromal interactions. SOCS3-dependent signal inhibition of the common gp130 subunit of the IL-6 receptor family was redundant for T cell formation. Together, SOCS3 expression in thymic stroma cells is critical for T cell development and for maintenance of thymus architecture.

Metabolic Regulation of Thymic Epithelial Cell Function

The thymus is the primary site of T lymphocyte development, where mutually inductive signaling between lymphoid progenitors and thymic stromal cells directs the progenitors along a well-characterized program of differentiation. Although thymic stromal cells, including thymic epithelial cells (TECs) are critical for the development of T cell-mediated immunity, many aspects of their basic biology have been difficult to resolve because they represent a small fraction of thymus cellularity, and because their isolation requires enzymatic digestion that induces broad physiological changes. These obstacles are especially relevant to the study of metabolic regulation of cell function, since isolation procedures necessarily disrupt metabolic homeostasis. In contrast to the well-characterized relationships between metabolism and intracellular signaling in T cell function during an immune response, metabolic regulation of thymic stromal cell function represents an emerging area of study. Here, we review recent advances in three distinct, but interconnected areas: regulation of mTOR signaling, reactive oxygen species (ROS), and autophagy, with respect to their roles in the establishment and maintenance of the thymic stromal microenvironment.

Inborn errors of thymic stromal cell development and function

As the primary site for T cell development, the thymus is responsible for the production and selection of a functional, yet self-tolerant T cell repertoire. This critically depends on thymic stromal cells, derived from the pharyngeal apparatus during embryogenesis. Thymic epithelial cells, mesenchymal and vascular elements together form the unique and highly specialised microenvironment required to support all aspects of thymopoiesis and T cell central tolerance induction. Although rare, inborn errors of thymic stromal cells constitute a clinically important group of conditions because their immunological consequences, which include autoimmune disease and T cell immunodeficiency, can be life-threatening if unrecognised and untreated. In this review, we describe the molecular and environmental aetiologies of the thymic stromal cell defects known to cause disease in humans, placing particular emphasis on those with a propensity to cause thymic hypoplasia or aplasia and consequently severe congenital immunodeficiency. We discuss the principles underpinning their diagnosis and management, including the use of novel tools to aid in their identification and strategies for curative treatment, principally transplantation of allogeneic thymus tissue.

Exosomes derived from statin-modified bone marrow dendritic cells increase thymus-derived natural regulatory T cells in experimental autoimmune myasthenia gravis

Background The thymus plays an essential role in the pathogenesis of myasthenia gravis (MG). In patients with MG, natural regulatory T cells (nTreg), a subpopulation of T cells that maintain tolerance to self-antigens, are severely impaired in the thymuses. In our previous study, upregulated nTreg cells were observed in the thymuses of rats in experimental autoimmune myasthenia gravis after treatment with exosomes derived from statin-modified dendritic cells (statin-Dex). Methods We evaluated the effects of exosomes on surface co-stimulation markers and Aire expression of different kinds of thymic stromal cells, including cTEC, mTEC, and tDCs, in EAMG rats. The isolated exosomes were examined by western blot and DLS. Immunofluorescence was used to track the exosomes in the thymus. Flow cytometry and western blot were used to analyze the expression of co-stimulatory molecules and Aire in vivo and in vitro. Results We confirmed the effects of statin-Dex in inducing Foxp3+ nTreg cells and found that both statin-Dex and DMSO-Dex could upregulate CD40 but only statin-Dex increased Aire expression in thymic stromal cells in vivo. Furthermore, we found that the role of statin-Dex and DMSO-Dex in the induction of Foxp3+ nTreg cells was dependent on epithelial cells in vitro. Conclusions We demonstrated that statin-Dex increased expression of Aire in the thymus, which may further promote the Foxp3 expression in the thymus. These findings may provide a new strategy for the treatment of myasthenia gravis.

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. Disclosures No relevant conflicts of interest to declare.