Thymic Stromal Cells Research Articles

The effect of thymic mesenchymal stromal cells on arginase activity and nitric oxide produced by mouse macrophages

The effect of thymic mesenchymal stromal cells on arginase activity and nitric oxide produced by mouse macrophages

Dissecting and modeling the emergent murine TEC compartment during ontogeny.

The origin of the thymic epithelium, i.e. the cortical (cTEC) and medullary (mTEC) epithelial cells, from bipotent stem cells through TEC progenitors and lineage-specific progeny still remains poorly understood. We sought to obtain an unbiased view of the incipient emergence of TEC subsets by following embryonic TEC development based on co-expression of EpCAM, CD80 and MHC class II (MHCII) on non-hematopoietic (CD45- ) thymic stromal cells in wild-type BL6 mice. Using a combination of ex vivo analysis, Re-aggregate Thymic Organ Culture (RTOC) reconstitution assays and mathematical modeling, we traced emergent lineage commitment in murine embryonic TECs. Both experimental and mathematical datasets supported the following developmental sequence: MHCII- CD80- → MHCIIlo CD80- → MHCIIhi CD80- → MHCIIhi CD80hi TECs, whereby MHCIIhi CD80- and MHCIIhi CD80hi TECs bear features of cTECs and mTECs respectively. These emergent MHCIIhi CD80- cTECs directly generate mature MHCIIhi CD80hi mTECs in vivo and in vitro, thus supporting the asynchronous model of TEC lineage commitment.

Catalase expression mediates redox regulation of autophagy and promiscuous gene expression in thymic stromal cells.

Abstract T lymphocytes develop in the thymus, where mutually inductive signaling between lymphoid progenitors and thymic stromal cells (TSCs) directs progenitors along a well-characterized differentiation program. However, the biology of stromal cells comprising the lymphopoietic thymic microenvironment remains relatively under-characterized because stromal cells are rare and difficult to isolate. Using a deconvolution technique to study gene expression essentially in situ, we previously identified a deficiency in the peroxide quenching enzyme catalase (CAT) in thymic stromal cells, and found that CAT deficiency results in high reactive oxygen species (ROS) levels in this population, eventually leading to thymic atrophy in aged mice. Here, we find that when catalase deficiency is complemented by overexpression targeted to mitochondria in transgenic mice (mCAT Tg), both ROS levels and stromal function decline in young mice relative to non-transgenic littermates. TSC transcriptome analysis reveals decreased expression of tissue-restricted antigen (TRA) and autophagy pathway genes in Cat Tg mice. Analysis of autophagy flux reporter mice ubiquitously expressing the RFP-EGFPLC3 fusion transgene also indicates diminished autophagic flux in mCAT Tg mice, particularly in TSCs. Stromal TRA expression and autophagic flux are required for self-antigen presentation, and therefore promote negative selection of potentially auto-reactive T cells. We propose that oxidative stress generated by low catalase levels in stromal cells promotes these key physiological functions in the young, steady state thymus; in contrast however, the resulting accumulated oxidative damage ultimately impairs function in the aged thymus.

Thymic Epithelial Cells.

Intrathymic T cell development is a complex process that depends upon continuous guidance from thymus stromal cell microenvironments. The thymic epithelium within the thymic stroma comprises highly specialized cells with a high degree of anatomic, phenotypic, and functional heterogeneity. These properties are collectively required to bias thymocyte development toward production of self-tolerant and functionally competent T cells. The importance of thymic epithelial cells (TECs) is evidenced by clear links between their dysfunction and multiple diseases where autoimmunity and immunodeficiency are major components. Consequently, TECs are an attractive target for cell therapies to restore effective immune system function. The pathways and molecular regulators that control TEC development are becoming clearer, as are their influences on particular stages of T cell development. Here, we review both historical and the most recent advances in our understanding of the cellular and molecular mechanisms controlling TEC development, function, dysfunction, and regeneration.

The transcriptional regulator Aire binds to and activates super-enhancers.

Aire is a transcription factor that controls T cell tolerance by inducing the expression of a large repertoire of genes specifically in thymic stromal cells. It interacts with scores of protein partners of diverse functional classes. Here we showed that Aire and some of its partners, notably those implicated in the DNA-damage response, preferentially localized within and activated long chromatin stretches overloaded with transcriptional regulators, known as “super-enhancers”. We also identified topoisomerase 1 as a cardinal Aire partner that co-localized on super-enhancers and was required for the interaction of Aire with all of its other associates. We propose a model that entails looping of super-enhancers to efficiently deliver Aire-containing complexes to local and distal transcriptional start sites.

Thymic Stromal Lymphopoietin: To Cut a Long Story Short

Thymic stromal lymphopoietin (TSLP) was identified more than 20 years ago as a secreted factor of a mouse thymic stromal cell line; later, a human orthologue was also identified. The signaling pathway triggered by TSLP has been extensively studied, and upregulation of the cytokine itself is linked to the pathogenesis of numerous Th2-related diseases, including atopic dermatitis, asthma, allergic responses, as well as certain types of cancers. On the other hand, TSLP mediates several immune homeostatic functions in both the gut and the thymus. Thus, a paradox occurs; why is TSLP homeostatic in certain tissues and a hallmark of exacerbated Th2 responses in the aforementioned pathologies? We and others have recently shown that in humans a novel isoform exists; this is a shorter isoform of TSLP whose expression is constitutive and controlled by a separate promoter. Short TSLP isoform mediates the homeostatic functions, whereas the long isoform is expressed at low/undetectable level at steady state and upregulated during inflammation in several tissues. Here we review the most recent data concerning the differential expression of the 2 isoforms and provide a potential explanation to the paradox. TSLP is regarded as a promising target for treatment of relevant pathologies, with a number of clinical trials already underway. It is important to design new strategies aimed at leaving intact the homeostatic effects of the short isoform while targeting the inflammatory effects of the long isoform.

Age-related changes of arginase activity and nitric oxide level in phagocytes and their modulation by thymic mesenchymal stromal cells

Arginine metabolism plays an important role in the activation of phagocytes, which are responsible for initiation, regulation, and resolution of inflammation. Deterioration of phagocyte functions is thought to cause the development of inflammaging and other age-related disorders. Mesenchymal stromal cells are well known for their immunoregulatory properties and ability to modulate activation status of mononuclear phagocytes. We aimed to compare arginase activity and NO production in phagocytes from different sources in young and aged animals, and to explore the effect of syngeneic thymic mesenchymal stromal cells on these parameters in tissue-resident phagocytes. We found that arginine metabolism of tissue-resident mononuclear phagocytes from aged mice is shifted to alternative or anti-inflammatory phenotype, due to a statistically significant arginase activity increase. Syngeneic co-culture with mesenchymal stromal cells greatly stimulates arginase activity and up-regulates nitric oxide generation by tissue mononuclear phagocytes regardless of cell donor age. Such bi-directional influence may be beneficial in clinical application for simultaneous inhibition of inflammation and infectious process.

Analysis of Thymocyte Migration, Cellular Interactions, and Activation by Multiphoton Fluorescence Microscopy of Live Thymic Slices.

Thymocytes migrate through discrete compartments within the thymus, engaging in cellular interactions essential for their differentiation into functional and self-tolerant T cells. Thus, understanding the temporal and spatial behavior of thymocytes within an intact thymic microenvironment is critical for elucidating processes governing T cell development. Towards this end, we describe methods for preparing thymic explant slices, in which the migration of thymocytes through three-dimensional space can be probed using time-lapse, multiphoton fluorescence microscopy. Thymocytes, enriched for developmental subsets of interest, are labeled with cytoplasmic fluorescent dyes, and seeded onto live thymic slices that express an endogenous, stromal cell-specific fluorescent reporter. In response to chemotactic cues produced by thymic stromal cells, the labeled thymocytes migrate withinthymic microenvironments and engage in cellular interactions that recapitulate a physiological system, whichcan be readily imaged. Here we describe specimen preparation that maintains the integrity of thymic structures. We also describe imaging protocols for acquiring multiple fluorochrome channels to enable detection of thymocyte:stromal cell interactions and quantification of relative intracellular calcium levels to monitor T cell receptor activation. Parameters for quantifying motility and interaction behaviors during data analysis are also briefly described. The thymic slice is a versatile tool for probing live cell behaviors and developing novel hypotheses not readily apparent by static experimental methods.

A Key Role for Inhibins in Dendritic Cell Maturation and Function.

Inhibins are members of the TGFβ superfamily, which regulate many cellular processes including differentiation, proliferation, survival and apoptosis. Although initially described as hormones regulating the hypothalamus-pituitary-gonadal axis, based on their ability to antagonize Activins, our group has recently reported that they play a role in thymocyte differentiation and survival, as well as in thymic stromal cell maturation and nTreg generation. Here, we used Inhibin knock out mice (Inhα-/-) to investigate the role of Inhibins in peripheral dendritic cell maturation and function. We first demonstrated that LPS treated Inhα+/+ bone marrow derived dendritic cells (BMDC) were capable to produce significant levels of Inhibin A. Interestingly, Inhα-/- BMDC showed reduced MHCII and CD86 upregulation and increased PD-L1 expression in response to LPS compared to Inhα+/+, which correlated with reduced ability to induce proliferation of allogeneic T cells. The “semi-mature” phenotype displayed by Inhα-/- mBMDC correlated with increased levels of IL-10 and slightly decreased IL-6 production after LPS stimulation. In addition, Inhα-/- mBMDC showed impaired migration towards CCL19 and CCL21, assessed by in vitro chemotaxis and in vivo competitive homing experiments, despite their normal CCR7 expression. Furthermore, in vivo LPS-induced DC maturation was also diminished in Inhα-/- mice, specially within the LC (CD207+ CD11b+ CD103-) subpopulation. Finally, analysis of delayed type hypersensitivity responses in Inhα-/- mice, showed reduced ear swelling as a result of reduced cellular infiltration in the skin, correlating with impaired homing of CD207+ DCs to the draining lymph nodes. In summary, our data demonstrate for the first time that Inhibins play a key role in peripheral DC maturation and function, regulating the balance between immunity and tolerance.

Alloreactive Natural Killer Cells Initiate a Unique Cellular and Molecular Pathway That Greatly Accelerates Immune Reconstitution after Allogeneic Bone Marrow Transplantation

One outstanding issue in allogeneic hematopoietic transplantation is impaired immune reconstitution. As the primary site of T cell development, the thymus plays a key role in the generation of a strong yet self-tolerant adaptive immune response, essential in the face of the potential threat from pathogens or neoplasia. Allogeneic hematopoietic transplantation may acutely damage the thymus through the chemo or radiotherapy, antibody therapy of the conditioning regime, infections acquired by the immunosuppressed patient, and thymic graft versus host disease. To date, attempts to improve thymic reconstitution have been disappointing. Pre-clinical experiments and pilot clinical trials tried to assess the role of a variety of therapeutic approaches, such as transfer of lymphoid progenitor cells, thymic grafts, or enhancement of thymopoiesis by administration of hormonal or cytokine/growth factor-based therapies, such as sex-steroid blockade, and IL-7, IL-22, KGF, or Flt-3 ligand administration (reviewed in Chaudhry et al., Immunol Rev. 2016). In mouse MHC mismatched transplantation models (F1 H-2d/b→parent H-2b), we previously found that infusion of donor versus recipient alloreactive NK cells eradicated recipient-type lympho-hematopoietic lineage cells, thereby enhancing engraftment, protecting from GvHD and eradicating leukemia (Ruggeri et al., Science 2002). Here, in the same models we show that infusion of alloreactive NK cells greatly accelerates the post-hematopoietic transplant recovery of donor-type immune cells, i.e., dendritic cells (DCs) (p<0.001), B lineage cells (p<0.001) and thymocytes (p<0.001) and maturation to B (p<0.001) and T cells (p<0.001). By the use of recipient chimeric mice displaying different tissue (i.e., hematopoietic vs non-hematopoietic) susceptibility to donor alloreactive NK cell killing, we show that a specific interaction between donor alloreactive NK cells and recipient DCs is responsible for the accelerated immune rebuilding. We find that donor-versus-recipient alloreactive NK cells trigger recipient DCs to synthesize a protein factor in a DNA translation-depended fashion (i.e., blocking DNA transcription in DCs abrogated the DC ability to produce the factor), and release it. Infusion of NK/DC co-culture supernatants containing this factor induced bone marrow and thymic stromal cells to produce IL-7 (p<0.001) and c-Kit ligand (p<0.001) and, thereby, the extraordinarily accelerated maturation of donor DCs, B- and T-cell precursors. Interestingly, in vitro experiments with human thymic stromal cells that support human thymocyte proliferation and differentiation demonstrated the exact same mechanisms. Supernatants from human alloreactive NK cell clones and human (HLA-class I KIR ligand mismatched) allogeneic DCs induced IL-7 production by human thymic stromal cells which in turn supported accelerated proliferation and maturation of human thymocytes (p<0.001). The murine and the human "immune rebuilding" factors displayed biochemical similarities as they both are highly hydrophobic 12KDa molecular weight proteins. Mass spectrometry analysis by stable isotope labeling with amino acids in cell culture (SILAC) identified Beta-2 Microglobulin (B2M) as the newly synthesized protein sharing the above biochemical features and present both in murine and human samples. B2M-KO mice used as recipients of MHC mismatched bone marrow transplant and given donor versus recipient alloreactive NK cells were unable to undergo accelerated immune rebuilding. However, their defect was repaired and accelerated rebuilding of donor-type DCs (p<0.001), B lineage cells (p<0.001) and thymocytes (p<0.001) was restored by the administration of culture supernatants obtained from alloreactive NK cells and wild-type (non-KO) MHC mismatched DCs. Finally, RNA interference experiments that silenced the B2M gene in human DCs resulted in loss of biological activity of supernatants obtained from alloreactive NK cells and B2M-silenced DCs. B2M plays a key role in the immune system as it is known to be part of MHC class I molecules. However, its role in signaling for immune precursor cell development has never been recognized. Here we report the discovery of a novel cellular and molecular pathway initiated by alloreactive NK cells and mediated by B2M that leads to greatly accelerated rebuilding of B and T cells after hematopoietic transplantation. DisclosuresNo relevant conflicts of interest to declare.

Potentiation of directed osteogenic differentiation of thymic multipotent stromal cells by prior co-cultivation with thymocytes

It is known that multipotent stromal cells (MSCs) and thymocytes possess membrane affinity and interaction in the thymic niches that is essentially important for thymocytes differentiation. However there are no data about possible influence of intercellular contacts in the reverse direction: from the thymocytes to the MSCs.Materials and methods. The MSCs were obtained from the thymuses of С57ВL mice, using the explants technique, and cultivated under standard conditions during 8-12 passages. Thymocytes or bone marrow cells (106) were added to 4×104 MSCs for 24 hours. Thereafter they were eliminated and standard culture medium was changed by osteogenic or adipogenic differentiation medium and cultured during 10 days. After fixation the cells were stained by 1 % alizarin red S solution or 0.2 % solution of oil red О respectively. After extraction of the stains with 10 % acetic acid or isopropyl alcohol the optic density of extracts at 520 nm was measured.Results. We found that thymic multipotent stromal cells of the C57BL mice were effectively differentiated in vitro into the osteogenic and adipogenic lineages in the appropriate differentiation media that was evidenced by alizarin red and oil red staining of cell cultures. According to the results of measurement of optic density of the dye extracts, it was found that effectiveness of thymic MSCs differentiation into the osteogenic lineage after prior short-term co-cultivation with the thymocytes is increased.Conclusions. The contact of thymic stromal cells with thymocytes but not with bone marrow cells in the previous 24 hours potentiates the osteogenic differentiation and has no effect on the adipogenic cells maturation.

Understanding the Thymic Microenvironment: the Cellular and Molecular Basis of T Cell Development

Objectives: The thymus is a primary lymphoid organ that provides specialized microenvironment for T cell development. A variety of thymic stromal cells form the thymic tissue architecture and critically regulate the development and repertoire selection of T cells. Methods: We reviewed historical and recent studies on thymic stromal cells, especially focusing on the well-characterized functions of thymic epithelial cells (TECs) and the significance of as yet less characterized non-TEC thymic stromal cells and hematopoietic antigen-presenting cells in the regulation of T cell development. Results: Cortical TECs (cTECs) induce positive selection of diverse and functional T cells, while medullary TECs (mTECs) establish T cell tolerance via the negative selection of auto-reactive T cells and their conversion into regulatory T cells. These modes of T cell tolerance induction are also mediated by hematopoietic antigen-presenting cells such as dendritic cells and thymic B cells. Thymic mesenchymal cells, a prominent component of non-TEC thymic stromal cells, support the development and maintenance of TECs and thereby T cell production. Conclusion: Understanding the cellular and molecular basis for thymic stromal subsets will provide invaluable information toward in vivo reconstitution of the thymic microenvironment for future therapeutic applications.

Thymocytes in Lyve1-CRE/S1pr1f/f Mice Accumulate in the Thymus due to Cell-Intrinsic Loss of Sphingosine-1-Phosphate Receptor Expression.

T cell emigration from the thymus is essential for immunological homeostasis. While stromal cell-produced sphingosine-1-phosphate (S1P) has been shown to promote thymocyte egress via the S1P receptor, S1PR1, the significance of S1P/S1PR1 signaling in the thymic stromal cells that surround T cells remains unclear. To address this issue, we developed conditional knockout mice (Lyve1-CRE/S1pr1f/f mice) in which S1pr1 was selectively targeted in cells expressing the lymphatic endothelial cell marker, Lyve1. In these mice, T cells were significantly reduced in secondary lymphoid tissues, and CD62L+ mature CD4 and CD8 single-positive (SP) T cells accumulated in the medulla failed to undergo thymus egress. Using a Lyve1 reporter strain in which Lyve1 lineage cells expressed tdTomato fluorescent protein, we unexpectedly found that a considerable proportion of the thymocytes were fluorescently labeled, indicating that they belonged to the Lyve1 lineage. The CD4 and CD8 SP thymocytes in Lyve1-CRE/S1pr1f/f mice exhibited an egress-competent phenotype (HSAlow, CD62Lhigh, and Qa-2high), but were CD69high and lacked S1PR1 expression. In addition, CD4 SP thymocytes from these mice were unable to migrate to the periphery after their intrathymic injection into wild-type (WT) mice. In contrast, WT T cells could migrate to the periphery in both WT and Lyve1-CRE/S1pr1f/f thymuses. These results demonstrated that thymocyte egress is mediated by T cell-expressed, but not stromal cell-expressed, S1PR1 and caution against using the Lyve1-CRE system for selectively gene deletion in lymphatic endothelial cells.

Lymphotoxin β Receptor Controls T Cell Progenitor Entry to the Thymus.

The recruitment of lymphoid progenitors to the thymus is essential to sustain T cell production throughout life. Importantly, it also limits T lineage regeneration following bone marrow transplantation, and so contributes to the secondary immunodeficiency that is caused by delayed immune reconstitution. Despite this significance, the mechanisms that control thymus colonization are poorly understood. In this study, we show that in both the steady-state and after bone marrow transplant, lymphotoxin β receptor (LTβR) controls entry of T cell progenitors to the thymus. We show that this requirement maps to thymic stroma, further underlining the key importance of this TNFR superfamily member in regulation of thymic microenvironments. Importantly, analysis of the requirement for LTβR in relationship to known regulators of thymus seeding suggests that it acts independently of its regulation of thymus-homing chemokines. Rather, we show that LTβR differentially regulates intrathymic expression of adhesion molecules known to play a role in T cell progenitor entry to the thymus. Finally, Ab-mediated in vivo LTβR stimulation following bone marrow transplant enhances initial thymus recovery and boosts donor-derived T cell numbers, which correlates with increased adhesion molecule expression by thymic stroma. Collectively, we reveal a novel link between LTβR and thymic stromal cells in thymus colonization, and highlight its potential as an immunotherapeutic target to boost T cell reconstitution after transplantation.

The role of thymic stromal lymphopoietin on mast cell-mediated allergic inflammatory reactions

Thymic stromal lymphopoietin (TSLP) is a novel interleukin (IL)-7-like cytokine and was originally discovered in the supernatant of a murine thymic stromal cell line. TSLP signal initiates via complex of the TSLP receptor and the IL-7 receptor α chain. TSLP expression is closely connected with many diseases such as atopic dermatitis, allergic rhinitis, asthma, inflammatory arthritis, eosinophilic esophagitis, rheumatoid arthritis, inflammatory bowel diseases, and cancer. In this review, I discussed biological roles of TSLP on mast cell-mediated allergic responses. In addition, this review summarizes the effective drugs in allergic-inflammatory reactions induced by TSLP on mast cells.

FOXN1 in thymus organogenesis and development.

Development of the primary T‐cell repertoire takes place in the thymus. The linked processes of T‐cell differentiation and T‐cell repertoire selection each depend on interactions between thymocytes and thymic stromal cells; in particular, with the epithelial cells of the cortical and medullary thymic compartments (cortical and medullary thymic epithelial cells; cTECs and mTECs, respectively). The importance of the thymic epithelial cell lineage in these processes was revealed in part through analysis of nude (nu/nu) mice, which are congenitally hairless and athymic. The nude phenotype results from null mutation of the forkhead transcription factor FOXN1, which has emerged as a pivotal regulator both of thymus development and homeostasis. FOXN1 has been shown to play critical roles in thymus development, function, maintenance, and even regeneration, which positions it as a master regulator of thymic epithelial cell (TEC) differentiation. In this review, we discuss current understanding of the regulation and functions of FOXN1 throughout thymus ontogeny, from the earliest stages of organogenesis through homeostasis to age‐related involution, contextualising its significance through reference to other members of the wider Forkhead family.

Induction of epithelial to mesenchymal transition (EMT) and inhibition on adipogenesis: Two different sides of the same coin? Feasible roles and mechanisms of transforming growth factor β1 (TGF-β1) in age-related thymic involution.

Age-related thymic involution is characterized by a loss of thymic epithelial cells (TECs) and a concomitant increase in adipocytes, but the mechanisms involved in thymic adipogenesis are still not clear. Transforming growth factor β1 (TGF-β1) is a pleiotropic cytokine that has been reported to be up-regulated with age in thymic stromal cells in both human and mouse. However, the exact role of TGF-β1 in age-related thymic involution remains to be further elucidated. On the basis of previous findings, we propose a novel hypothesis that TGF-β1 functions a dual role in age-related thymic involution. On one hand, up-regulation of TGF-β1 promotes epithelial to mesenchymal transition (EMT) process in TECs via activating forkhead box protein C2 (FoxC2). On the other hand, TGF-β1 inhibits the transdifferentiation of EMT-derived mesenchymal cells to adipocytes in the thymus. If confirmed, our hypothesis will not only provide further evidence supporting that the transdifferentiation of TECs into pre-adipocytes represents a source of thymic adiposity during age-related thymic involution, but also uncover a unique role of TGF-β1 in the transdifferentiation of TECs into pre-adipocytes. Collectively, the inhibition of TGF-β1 may serve as a strategy to hinder age-related thymic involution or even to restore thymic function in the elderly.

Increased antioxidant activity mitigates age-associated dysfunction of thymic stromal cells in aged mice.

Abstract Age-associated thymic atrophy results in diminished production of new T lymphocytes and a concomitant decrease in responsiveness to new pathogens and vaccines. In addition to loss of thymic size with age, we have shown that critical stromal functions, including tissue-restricted antigen (TRA) expression, are diminished with age. We previously identified deficiency of the hydrogen peroxide quenching enzyme catalase (CAT) in thymic stromal cells as a key cause of thymic atrophy during aging, and established that thymic atrophy can be mitigated by genetic or dietary complementation of catalase antioxidant activity. Here, we find that in addition to maintaining thymic mass with age, long-term increases in catalase activity may mitigate age-associated loss of stromal function. Our preliminary studies indicate that life-long overexpression of catalase in mitochondria of transgenic mice (mCAT Tg) results in increased TRA expression, mitigated acquisition of an aged global gene expression signature, and increased maintenance of cortico-medullary organization in aged mCAT Tg mice relative to non-transgenic littermates.

Catalase deficiency in thymic stromal cells promotes key stromal functions in the young, steady state mouse thymus

Abstract T lymphocytes develop in the thymus, where mutually inductive signaling between lymphoid progenitors and thymic stromal cells directs the progenitors along a well-characterized program of differentiation. However, the biology of the stromal cells comprising the lymphopoietic thymic microenvironment remains relatively under-characterized because stromal cells are rare and difficult to isolate. Using a deconvolution technique to study gene expression essentially in situ, we previously identified a deficiency in the peroxide quenching enzyme catalase (CAT) in thymic stromal cells, and found that CAT deficiency results in high reactive oxygen (ROS) levels in this population, eventually leading to thymic atrophy. Here, we address the possibility that high ROS levels serve physiological functions in stromal cells in the young, steady state thymus. Our preliminary data indicate that when catalase deficiency is complemented by overexpression targeted to mitochondria in transgenic mice (mCAT Tg), both ROS levels and stromal function are diminished in young mice relative to non-transgenic littermates. We find evidence of diminished tissue-restricted antigen (TRA) expression and autophagy in mCAT Tg mice, both of which are critical for establishing a self-tolerant T cell repertoire. We propose that a high ROS environment in stromal cells promotes promiscuous gene expression and autophagy critical for stromal induction of T cell tolerance in the young thymus, while accumulated oxidative damage ultimately undermines healthy thymus function.

Generation of functional mini thymus-like units with self-assembling EAK16-II/EAKIIH6 hydrogel

Abstract A functional thymus is crucial to produce mature, self-tolerant T cells. Crosstalk between the residing thymic stromal cells, especially the predominant population of thymic epithelial cells (TECs), and the developing thymocytes is essential for thymopoiesis. The survival and proliferation of TECs depend on a unique 3 dimensional (3-D) configuration of the thymus microenvironment. Disorganization of TECs results in a loss of gene expressions essential for TEC growth and survival, as well as inability to produce functional T cells. Recapitulating the property of thymus has been proven to be challenging. In our study, we have incorporated EAK16-II, a low molecular weight peptide that self-assembles into beta-sheet fibrils, and its histidinylated analogue, to induce 3-D aggregation of TECs. When the adaptor complex comprised of anti-EpCAM IgG, anti-His and recombinant protein A/G molecules were mixed with TECs in the hydrogel, TECs were captured into small clusters. TECs cultured in this condition maintained their molecular properties and were viable up to 2 weeks. Furthermore, when the TEC clusters in the hydrogel were transplanted into athymic nude mice, T cell development was observed, and these newly generated T cells proliferated upon stimulation with allogenic cells. These results demonstrate that self-assembling EAK16-II/EAKIIH6 system with addition of tri-component adaptor complex may be a useful tool to organize TECs in a 3-D-like structure to support T cell development, suggesting a possibility to generate injectable mini thymus-like units to restore adaptive immune system.