Generating an organ-deficient animal model using a multi-targeted CRISPR-Cas9 system.

Gene-knockout animal models with organ-deficient phenotypes used for blastocyst complementation are generally not viable. Animals need to be maintained as heterozygous mutants, and homozygous mutant embryos yield only one-fourth of all embryos. In this study, we generated organ-deficient embryos using the CRISPR-Cas9-sgRNAms system that induces cell death with a single-guide RNA (sgRNAms) targeting multiple sites in the genome. The Cas9-sgRNAms system interrupted cell proliferation and induced cell ablation in vitro. The mouse model had Cas9 driven by the Foxn1 promoter with a ubiquitous expression cassette of sgRNAms at the Rosa26 locus (Foxn1Cas9; Rosa26_ms). It showed an athymic phenotype similar to that of nude mice but was not hairless. Eventually, a rat cell-derived thymus in an interspecies chimera was generated by blastocyst complementation of Foxn1Cas9; Rosa26_ms mouse embryos with rat embryonic stem cells. Theoretically, a half of the total embryos has the Cas9-sgRNAms system because Rosa26_ms could be maintained as homozygous.

Dynamics of somatostatin 4 receptor expression during chronic-stress loading and its potential as a chronic-stress marker.

Chronic stress has been implicated in mental illnesses and depressive behaviors. Somatostatin 4 receptor (SSTR4) has been shown to mediate anxiolytic and depression-like effects. Here, we aimed to explore the potential of SSTR4 as a diagnostic marker for chronic stress in mice. The mice were divided into single stress, chronic restraint stress, and control groups, and Sstr4 mRNA expression in the pituitary, lungs, and thymus, its protein expression in the thymus, were analyzed. Compared to controls, Sstr4 mRNA expression decreased significantly in the pituitary gland of the chronic and single-stress groups (P = 0.0181 and 0.0022, respectively) and lungs of the single-stress group (P = 0.0124), whereas it significantly increased in the thymus of the chronic-stress group (P = 0.0313). Thymic SSTR4 expression did not decrease significantly in stress groups compared to that in the control group (P = 0.0963). These results suggest that SSTR4 expression fluctuates in response to stress. Furthermore, Sstr4 mRNA expression dynamics in each organ differed based on single or chronic restraint stress-loading periods. In conclusion, this study suggests that investigating SSTR4 expression in each organ could allow for its use as a stress marker to estimate the stress-loading period and aid in diagnosing chronic stress.

Aire mediates tolerance to insulin through thymic trimming of high-affinity T cell clones.

Insulin is a central autoantigen in the pathogenesis of T1D, and thymic epithelial cell expression of insulin under the control of the Autoimmune Regulator (Aire) is thought to be a key component of maintaining tolerance to insulin. In spite of this general working model, direct detection of this thymic selection on insulin-specific T cells has been somewhat elusive. Here, we used a combination of highly sensitive T cell receptor transgenic models for detecting thymic selection and sorting and sequencing of Insulin-specific CD4+ T cells from Aire-deficient mice as a strategy to further define their selection. This analysis revealed a number of unique t cell receptor (TCR) clones in Aire-deficient hosts with high affinity for insulin/major histocompatibility complex (MHC) ligands. We then modeled the thymic selection of one of these clones in Aire-deficient versus wild-type hosts and found that this model clone could escape thymic negative selection in the absence of thymic Aire. Together, these results suggest that thymic expression of insulin plays a key role in trimming and removing high-affinity insulin-specific T cells from the repertoire to help promote tolerance.

Foxo3 regulates cortical and medullary thymic epithelial cell homeostasis with implications in T cell development.

Within the thymus, thymic epithelial cells (TECs) create dedicated microenvironments for T cell development and selection. Considering that TECs are sensitive to distinct pathophysiological conditions, uncovering the molecular elements that coordinate their thymopoietic role has important fundamental and clinical implications. Particularly, medullary thymic epithelial cells (mTECs) play a crucial role in central tolerance. Our previous studies, along with others, suggest that mTECs depend on molecular factors linked to genome-protecting pathways, but the precise mechanisms underlying their function remain unknown. These observations led us to examine the role of Foxo3, as it is expressed in TECs and involved in DNA damage response. Our findings show that mice with TEC-specific deletion of Foxo3 (Foxo3cKO) displayed a disrupted mTEC compartment, with a more profound impact on the numbers of CCL21+ and thymic tuft mTEClo subsets. At the molecular level, Foxo3 controls distinct functional modules in the transcriptome of cTECs and mTECs under normal conditions, which includes the regulation of ribosomal biogenesis and DNA damage response, respectively. These changes in the TEC compartment resulted in a reduced total thymocyte cellularity and specific changes in regulatory T cell and iNKT cell development in the Foxo3cKO thymus. Lastly, the thymic defects observed in adulthood correlated with mild signs of altered peripheral immunotolerance in aged Foxo3cKO mice. Moreover, the deficiency in Foxo3 moderately aggravated the autoimmune predisposition observed in Aire-deficient mice. Our findings highlight the importance of Foxo3 in preserving the homeostasis of TECs and in supporting their role in T cell development and tolerance.

Functionally diverse thymic medullary epithelial cells interplay to direct central tolerance.

Medullary thymic epithelial cells (mTECs) are essential for the establishment of self-tolerance in T cells. Promiscuous gene expression by a subpopulation of mTECs regulated by the nuclear protein Aire contributes to the display of self-genomic products to newly generated T cells. Recent reports have highlighted additional self-antigen-displaying mTEC subpopulations, namely Fezf2-expressing mTECs and a mosaic of self-mimetic mTECs including thymic tuft cells. In addition, a functionally different subset of mTECs produces chemokine CCL21, which attracts developing thymocytes to the medullary region. Here, we report that CCL21+ mTECs and Aire+ mTECs non-redundantly cooperate to direct self-tolerance to prevent autoimmune pathology by optimizing the deletion of self-reactive T cells and the generation of regulatory T cells. We also detect cooperation for self-tolerance between Aire and Fezf2, the latter of which unexpectedly regulates thymic tuft cells. Our results indicate an indispensable interplay among functionally diverse mTECs for the establishment of central self-tolerance.

Whole-transcriptome profiling reveals potential biomarkers for the reversal of thymic epithelial cell senescence by umbilical cord mesenchymal stem cells.

BACKGROUND: Reduced numbers and dysfunction of thymic epithelial cells (TECs) are important factors of thymic degeneration. Previous studies have found that umbilical cord mesenchymal stem cells (UCMSCs) reverse the structure and function of the senescent thymus in vivo. However, the transcriptomic regulation mechanism is unclear. METHODS: TECs were cultured with H2O2 for 72 hours to induce senescence. UCMSCs were cocultured with senescent TECs for 48 hours to detect SA-ß-gal, P16 and Ki67. The cocultured TECs were collected for lncRNA, mRNA and miRNA sequencing to establish a competitive endogenous regulatory network (ceRNA). And RT-qPCR, immunofluorescence staining, and western blot were used to identified key genes. RESULTS: Our results showed that H2O2 induced TEC aging and that UCMSCs reversed these changes. Compared with those in aged TECs, 2260 DE mRNAs, 1033 DE lncRNAs and 67 DE miRNAs were differentially expressed, and these changes were reversed by coculturing the cells with UCMSCs. Differential mRNA enrichment analysis of ceRNA regulation revealed that the PI3K-AKT pathway was a significant signaling pathway. UCMSC coculture upregulated VEGFA, which is the upstream factor of the PI3K-AKT signaling pathway, and the expression of the key proteins PI3K and AKT. Thus, the expression of the cell cycle suppressor P27, which is downstream of the PI3K-AKT signaling pathway, was downregulated, while the expression of the cell cycle regulators CDK2 and CCNE was upregulated. CONCLUSION: UCMSC coculture upregulated the expression of VEGFA, activated the PI3K-AKT signaling pathway, increased the expression of CDK2 and CCNE, decreased the expression of P27, and promoted the proliferation of TECs.

CARD11 regulates the thymic Treg development in an NF-κB-independent manner.

Introduction: CARD11 is a lymphoid lineage-specific scaffold protein regulating the NF-κB activation downstream of the antigen receptor signal pathway. Defective CARD11 function results in abnormal development and differentiation of lymphocytes, especially thymic regulatory T cells (Treg). Method: In this study, we used patients' samples together with transgenic mouse models carrying pathogenic CARD11 mutations from patients to explore their effects on Treg development. Immunoblotting and a GFP receptor assay were used to evaluate the activation effect of CARD11 mutants on NF-κB signaling. Then the suppressive function of Tregs carrying distinct CARD11 mutations was measured by in vitro suppression assay. Finally, we applied the retroviral transduced bone marrow chimeras to rescue the Treg development in an NF-κB independent manner. Results and discuss: We found CARD11 mutations causing hyper-activated NF-κB signals also gave rise to compromised Treg development in the thymus, similar to the phenotype in Card11 deficient mice. This observation challenges the previous view that CARD11 regulates Treg lineage dependent on the NF-kB activation. Mechanistic investigations reveal that the noncanonical function CARD11, which negatively regulates the AKT/ FOXO1 signal pathway, is responsible for regulating Treg generation. Moreover, primary immunodeficiency patients carrying CARD11 mutation, which autonomously activates NF-κB, also represented the reduced Treg population in their peripheral blood. Our results propose a new regulatory function of CARD11 and illuminate an NF-κB independent pathway for thymic Treg lineage commitment.

Deciphering the developmental trajectory of tissue-resident Foxp3+ regulatory T cells.

Foxp3+ TREG cells have been at the focus of intense investigation for their recognized roles in preventing autoimmunity, facilitating tissue recuperation following injury, and orchestrating a tolerance to innocuous non-self-antigens. To perform these critical tasks, TREG cells undergo deep epigenetic, transcriptional, and post-transcriptional changes that allow them to adapt to conditions found in tissues both at steady-state and during inflammation. The path leading TREG cells to express these tissue-specialized phenotypes begins during thymic development, and is further driven by epigenetic and transcriptional modifications following TCR engagement and polarizing signals in the periphery. However, this process is highly regulated and requires TREG cells to adopt strategies to avoid losing their regulatory program altogether. Here, we review the origins of tissue-resident TREG cells, from their thymic and peripheral development to the transcriptional regulators involved in their tissue residency program. In addition, we discuss the distinct signalling pathways that engage the inflammatory adaptation of tissue-resident TREG cells, and how they relate to their ability to recognize tissue and pathogen-derived danger signals.

Transposable elements regulate thymus development and function.

Transposable elements (TEs) are repetitive sequences representing ~45% of the human and mouse genomes and are highly expressed by medullary thymic epithelial cells (mTECs). In this study, we investigated the role of TEs on T-cell development in the thymus. We performed multiomic analyses of TEs in human and mouse thymic cells to elucidate their role in T-cell development. We report that TE expression in the human thymus is high and shows extensive age- and cell lineage-related variations. TE expression correlates with multiple transcription factors in all cell types of the human thymus. Two cell types express particularly broad TE repertoires: mTECs and plasmacytoid dendritic cells (pDCs). In mTECs, transcriptomic data suggest that TEs interact with transcription factors essential for mTEC development and function (e.g., PAX1 and REL), and immunopeptidomic data showed that TEs generate MHC-I-associated peptides implicated in thymocyte education. Notably, AIRE, FEZF2, and CHD4 regulate small yet non-redundant sets of TEs in murine mTECs. Human thymic pDCs homogenously express large numbers of TEs that likely form dsRNA, which can activate innate immune receptors, potentially explaining why thymic pDCs constitutively secrete IFN ɑ/ß. This study highlights the diversity of interactions between TEs and the adaptive immune system. TEs are genetic parasites, and the two thymic cell types most affected by TEs (mTEcs and pDCs) are essential to establishing central T-cell tolerance. Therefore, we propose that orchestrating TE expression in thymic cells is critical to prevent autoimmunity in vertebrates.

The role of quiescent thymic progenitors in TAL/LMO2-induced T-ALL chemotolerance.

Relapse in T-cell acute lymphoblastic leukemia (T-ALL) may signify the persistence of leukemia-initiating cells (L-ICs). Ectopic TAL1/LMO expression defines the largest subset of T-ALL, but its role in leukemic transformation and its impact on relapse-driving L-ICs remain poorly understood. In TAL1/LMO mouse models, double negative-3 (DN3; CD4-CD8-CD25+CD44-) thymic progenitors harbored L-ICs. However, only a subset of DN3 leukemic cells exhibited L-IC activity, and studies linking L-ICs and chemotolerance are needed. To investigate L-IC heterogeneity, we used mouse models and applied single-cell RNA-sequencing and nucleosome labeling techniques in vivo. We identified a DN3 subpopulation with a cell cycle-restricted profile and heightened TAL1/LMO2 activity, that expressed genes associated with stemness and quiescence. This dormant DN3 subset progressively expanded throughout leukemogenesis, displaying intrinsic chemotolerance and enrichment in genes linked to minimal residual disease. Examination of TAL/LMO patient samples revealed a similar pattern in CD7+CD1a- thymic progenitors, previously recognized for their L-IC activity, demonstrating cell cycle restriction and chemotolerance. Our findings substantiate the emergence of dormant, chemotolerant L-ICs during leukemogenesis, and demonstrate that Tal1 and Lmo2 cooperate to promote DN3 quiescence during the transformation process. This study provides a deeper understanding of TAL1/LMO-induced T-ALL and its clinical implications in therapy failure.

Kidins220 and Aiolos promote thymic iNKT cell development by reducing TCR signals.

Development of T cells is controlled by the signal strength of the TCR. The scaffold protein kinase D-interacting substrate of 220 kilodalton (Kidins220) binds to the TCR; however, its role in T cell development was unknown. Here, we show that T cell-specific Kidins220 knockout (T-KO) mice have strongly reduced invariant natural killer T (iNKT) cell numbers and modest decreases in conventional T cells. Enhanced apoptosis due to increased TCR signaling in T-KO iNKT thymocytes of developmental stages 2 and 3 shows that Kidins220 down-regulates TCR signaling at these stages. scRNA-seq indicated that the transcription factor Aiolos is down-regulated in Kidins220-deficient iNKT cells. Analysis of an Aiolos KO demonstrated that Aiolos is a downstream effector of Kidins220 during iNKT cell development. In the periphery, T-KO iNKT cells show reduced TCR signaling upon stimulation with α-galactosylceramide, suggesting that Kidins220 promotes TCR signaling in peripheral iNKT cells. Thus, Kidins220 reduces or promotes signaling dependent on the iNKT cell developmental stage.

Developmental conversion of thymocyte-attracting cells into self-antigen-displaying cells in embryonic thymus medulla epithelium.

Thymus medulla epithelium establishes immune self-tolerance and comprises diverse cellular subsets. Functionally relevant medullary thymic epithelial cells (mTECs) include a self-antigen-displaying subset that exhibits genome-wide promiscuous gene expression promoted by the nuclear protein Aire and that resembles a mosaic of extrathymic cells including mucosal tuft cells. An additional mTEC subset produces the chemokine CCL21, thereby attracting positively selected thymocytes from the cortex to the medulla. Both self-antigen-displaying and thymocyte-attracting mTEC subsets are essential for self-tolerance. Here, we identify a developmental pathway by which mTECs gain their diversity in functionally distinct subsets. We show that CCL21-expressing mTECs arise early during thymus ontogeny in mice. Fate-mapping analysis reveals that self-antigen-displaying mTECs, including Aire-expressing mTECs and thymic tuft cells, are derived from CCL21-expressing cells. The differentiation capability of CCL21-expressing embryonic mTECs is verified in reaggregate thymus experiments. These results indicate that CCL21-expressing embryonic mTECs carry a developmental potential to give rise to self-antigen-displaying mTECs, revealing that the sequential conversion of thymocyte-attracting subset into self-antigen-displaying subset serves to assemble functional diversity in the thymus medulla epithelium.

The thymocyte-specific RNA-binding protein Arpp21 provides TCR repertoire diversity by binding to the 3'-UTR and promoting Rag1 mRNA expression.

The regulation of thymocyte development by RNA-binding proteins (RBPs) is largely unexplored. We identify 642 RBPs in the thymus and focus on Arpp21, which shows selective and dynamic expression in early thymocytes. Arpp21 is downregulated in response to T cell receptor (TCR) and Ca2+ signals. Downregulation requires Stim1/Stim2 and CaMK4 expression and involves Arpp21 protein phosphorylation, polyubiquitination and proteasomal degradation. Arpp21 directly binds RNA through its R3H domain, with a preference for uridine-rich motifs, promoting the expression of target mRNAs. Analysis of the Arpp21-bound transcriptome reveals strong interactions with the Rag1 3'-UTR. Arpp21-deficient thymocytes show reduced Rag1 expression, delayed TCR rearrangement and a less diverse TCR repertoire. This phenotype is recapitulated in Rag1 3'-UTR mutant mice harboring a deletion of the Arpp21 response region. These findings show how thymocyte-specific Arpp21 promotes Rag1 expression to enable TCR repertoire diversity until signals from the TCR terminate Arpp21 and Rag1 activities.

Transcriptional profile of human thymus reveals IGFBP5 is correlated with age-related thymic involution.

Thymus is the main immune organ which is responsible for the production of self-tolerant and functional T cells, but it shrinks rapidly with age after birth. Although studies have researched thymus development and involution in mouse, the critical regulators that arise with age in human thymus remain unclear. We collected public human single-cell transcriptomic sequencing (scRNA-seq) datasets containing 350,678 cells from 36 samples, integrated them as a cell atlas of human thymus. Clinical samples were collected and experiments were performed for validation. We found early thymocyte-specific signaling and regulons which played roles in thymocyte migration, proliferation, apoptosis and differentiation. Nevertheless, signaling patterns including number, strength and path completely changed during aging, Transcription factors (FOXC1, MXI1, KLF9, NFIL3) and their target gene, IGFBP5, were resolved and up-regulated in aging thymus and involved in promoting epithelial-mesenchymal transition (EMT), responding to steroid and adipogenesis process of thymic epithelial cell (TECs). Furthermore, we validated that IGFBP5 protein increased at TECs and Hassall's corpuscle in both human and mouse aging thymus and knockdown of IGFBP5 significantly increased the expression of proliferation-related genes in thymocytes. Collectively, we systematically explored cell-cell communications and regulons of early thymocytes as well as age-related differences in human thymus by using both bioinformatic and experimental verification, indicating IGFBP5 as a functional marker of thymic involution and providing new insights into the mechanisms of thymus involution.

Thymic-Epithelial-Cell-Dependent Microenvironment Influences Proliferation and Apoptosis of Leukemic Cells.

T-cell acute lymphoblastic leukemia (T-ALL) is a hematological cancer characterized by the infiltration of immature T-cells in the bone marrow. Aberrant NOTCH signaling in T-ALL is mainly triggered by activating mutations of NOTCH1 and overexpression of NOTCH3, and rarely is it linked to NOTCH3-activating mutations. Besides the known critical role of NOTCH, the nature of intrathymic microenvironment-dependent mechanisms able to render immature thymocytes, presumably pre-leukemic cells, capable of escaping thymus retention and infiltrating the bone marrow is still unclear. An important challenge is understanding how leukemic cells shape their tumor microenvironment to increase their ability to infiltrate and survive within. Our previous data indicated that hyperactive NOTCH3 affects the CXCL12/CXCR4 system and may interfere with T-cell/stroma interactions within the thymus. This study aims to identify the biological effects of the reciprocal interactions between human leukemic cell lines and thymic epithelial cell (TEC)-derived soluble factors in modulating NOTCH signaling and survival programs of T-ALL cells and TECs. The overarching hypothesis is that this crosstalk can influence the progressive stages of T-cell development driving T-cell leukemia. Thus, we investigated the effect of extracellular space conditioned by T-ALL cell lines (Jurkat, TALL1, and Loucy) and TECs and studied their reciprocal regulation of cell cycle and survival. In support, we also detected metabolic changes as potential drivers of leukemic cell survival. Our studies could shed light on T-cell/stroma crosstalk to human leukemic cells and propose our culture system to test pharmacological treatment for T-ALL.

Loss of thymocyte competition underlies the tumor suppressive functions of the E2a transcription factor in T-ALL.

T lymphocyte acute lymphoblastic leukemia (T-ALL) is frequently associated with increased expression of the E protein transcription factor inhibitors TAL1 and LYL1. In mouse models, ectopic expression of TAL1 or LYL1 in T cell progenitors, or inactivation of E2A, is sufficient to predispose mice to develop T-ALL. How E2A suppresses thymocyte transformation is currently unknown. Here, we show that early deletion of E2a, prior to the DN3 stage, was required for robust leukemogenesis and was associated with alterations in thymus cellularity, T cell differentiation, and gene expression in immature CD4+CD8+ thymocytes. Introduction of wild-type thymocytes into mice with early deletion of E2a prevented leukemogenesis, or delayed disease onset, and impacted the expression of multiple genes associated with transformation and genome instability. Our data indicate that E2A suppresses leukemogenesis by promoting T cell development and enforcing inter-thymocyte competition, a mechanism that is emerging as a safeguard against thymocyte transformation. These studies have implications for understanding how multiple essential regulators of T cell development suppress T-ALL and support the hypothesis that thymocyte competition suppresses leukemogenesis.

Assembling the thymus medulla: Development and function of epithelial cell heterogeneity.

The thymus is a unique primary lymphoid organ that supports the production of self-tolerant T-cells essential for adaptive immunity. Intrathymic microenvironments are microanatomically compartmentalised, forming defined cortical, and medullary regions each differentially supporting critical aspects of thymus-dependent T-cell maturation. Importantly, the specific functional properties of thymic cortical and medullary compartments are defined by highly specialised thymic epithelial cells (TEC). For example, in the medulla heterogenous medullary TEC (mTEC) contribute to the enforcement of central tolerance by supporting deletion of autoreactive T-cell clones, thereby counterbalancing the potential for random T-cell receptor generation to contribute to autoimmune disease. Recent advances have further shed light on the pathways and mechanisms that control heterogeneous mTEC development and how differential mTEC functionality contributes to control self-tolerant T-cell development. Here we discuss recent findings in relation to mTEC development and highlight examples of how mTEC diversity contribute to thymus medulla function.

Expression of the transcription factor Klf6 by thymic epithelial cells is required for thymus development.

Thymic epithelial cells (TEC) control T cell development and play essential roles in establishing self-tolerance. By using Foxn1-Cre-driven ablation of Klf6 gene in TEC, we identified Klf6 as a critical factor in TEC development. Klf6 deficiency resulted in a hypoplastic thymus-evident from fetal stages into adulthood-in which a dramatic increase in the frequency of apoptotic TEC was observed. Among cortical TEC (cTEC), a previously unreported cTEC population expressing the transcription factor Sox10 was relatively expanded. Within medullary TEC (mTEC), mTEC I and Tuft-like mTEC IV were disproportionately decreased. Klf6 deficiency altered chromatin accessibility and affected TEC chromatin configuration. Consistent with these defects, naïve conventional T cells and invariant natural killer T cells were reduced in the spleen. Late stages of T cell receptor-dependent selection of thymocytes were affected, and mice exhibited autoimmunity. Thus, Klf6 has a prosurvival role and affects the development of specific TEC subsets contributing to thymic function.

Lin28 regulates thymic growth and involution and correlates with MHCII expression in thymic epithelial cells.

Thymic epithelial cells (TECs) are essential for T cell development in the thymus, yet the mechanisms governing their differentiation are not well understood. Lin28, known for its roles in embryonic development, stem cell pluripotency, and regulating cell proliferation and differentiation, is expressed in endodermal epithelial cells during embryogenesis and persists in adult epithelia, implying postnatal functions. However, the detailed expression and function of Lin28 in TECs remain unknown. In this study, we examined the expression patterns of Lin28 and its target Let-7g in fetal and postnatal TECs and discovered opposing expression patterns during postnatal thymic growth, which correlated with FOXN1 and MHCII expression. Specifically, Lin28b showed high expression in MHCIIhi TECs, whereas Let-7g was expressed in MHCIIlo TECs. Deletion of Lin28a and Lin28b specifically in TECs resulted in reduced MHCII expression and overall TEC numbers. Conversely, overexpression of Lin28a increased total TEC and thymocyte numbers by promoting the proliferation of MHCIIlo TECs. Additionally, our data strongly suggest that Lin28 and Let-7g expression is reliant on FOXN1 to some extent. These findings suggest a critical role for Lin28 in regulating the development and differentiation of TECs by modulating MHCII expression and TEC proliferation throughout thymic ontogeny and involution. Our study provides insights into the mechanisms underlying TEC differentiation and highlights the significance of Lin28 in orchestrating these processes.