Thymic Epithelial Cells Research Articles

Human MHC Class II and Invariant Chain Knock-in Mice Mimic Rheumatoid Arthritis with Allele Restriction in Immune Response and Arthritis Association.

Transgenic mice expressing human major histocompatibility complex class II (MHCII) risk alleles are widely used in autoimmune disease research, but limitations arise due to non-physiologic expression. To address this, physiologically relevant mouse models are established via knock-in technology to explore the role of MHCII in diseases like rheumatoid arthritis. The gene sequences encoding the ectodomains are replaced with the human DRB1*04:01 and 04:02 alleles, DRA, and CD74 (invariant chain) in C57BL/6N mice. The collagen type II (Col2a1) gene is modified to mimic human COL2. Importantly, DRB1*04:01 knock-in mice display physiologic expression of human MHCII also on thymic epithelial cells, in contrast to DRB1*04:01 transgenic mice. Humanization of the invariant chain enhances MHCII expression on thymic epithelial cells, increases mature B cell numbers in spleen, and improves antigen presentation. To validate its functionality, the collagen-induced arthritis (CIA) model is used, where DRB1*04:01 expression led to a higher susceptibility to arthritis, as compared with mice expressing DRB1*04:02. In addition, the humanized T cell epitope on COL2 allows autoreactive T cell-mediated arthritis development. In conclusion, the humanized knock-in mouse faithfully expresses MHCII, confirming the DRB1*04:01 alleles role in rheumatoid arthritis and being also useful for studying MHCII-associated diseases.

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 Tcells. 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 Tcells. 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 Tcells and the generation of regulatory Tcells. 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.

Derivation of functional thymic epithelial organoid lines from adult murine thymus

Thymic epithelial cells (TECs) orchestrate Tcell development by imposing positive and negative selection on thymocytes. Current studies on TEC biology are hampered by the absence of long-term exvivo culture platforms, while the cells driving TEC self-renewal remain to be identified. Here, we generate long-term (>2 years) expandable 3D TEC organoids from the adult mouse thymus. For further analysis, we generated single anddouble FoxN1-P2A-Clover, Aire-P2A-tdTomato, and Cldn4-P2A-tdTomato reporter lines by CRISPR knockin. Single-cell analyses of expanding clonal organoids reveal cells with bipotent stem/progenitor phenotypes. These clonal organoids can be induced to express Foxn1 and to generate functional cortical- and Aire-expressing medullary-like TECs upon RANK ligand+ retinoic acid treatment. TEC organoids support Tcell development from immature thymocytes invitro as well as invivo upon transplantation into athymic nude mice. This organoid-based platform allows invitro study of TEC biology and offers a potential strategy for exvivo Tcell development.

Mesenchymal stem cells reverse thymus aging by reprogramming the DNA methylation of thymic epithelial cells

BackgroundA decrease in the number and activity of thymic epithelial cells (TECs) is an important factor in thymic degeneration. Mesenchymal stem cells (MSCs) treating thymic ageing is a promising strategy, but the DNA methylation modification mechanism in TECs remains unclear. MethodsAged rhesus monkeys were treated with MSCs to establish a thymic senescence model, and hematoxylin-eosin (HE) staining, immunofluorescence staining, and ELISA were performed to observe the structure and function of the thymus. TEC aging model and MSCs co-culture system were established to detect DNA methylation modification and transcriptomic changes, correlation analysis between transcription factor methylation and mRNA expression, and q-PCR, immunofluorescence staining, and Western blot were used to identified key genes. ResultsMSCs improved the structure and function of thymus in elderly macaque monkeys; reduced the expression levels of β-Gal, P16, and P21; and increased the activity of aging TECs. There were 501 genes with increased methylation in the promoter region in the treated group compared with the untreated group, among which 23 genes were involved in the negative regulation of cell growth, proliferation and apoptosis, while 591 genes had decreased methylation, among which 37 genes were associated with promoting cell growth and proliferation and inhibiting apoptosis. Furthermore, 66 genes showed a negative correlation between promoter methylation levels and gene transcription; specifically, PDE5A, DUOX2, LAMP1 and SVIL were downregulated with increased methylation, inhibiting growth and development, while POLR3G, PGF, CHTF18, KRT17, FOXJ1, NGF, DYRK3, LRP8, CDT1, PRELID1, F2R, KNTC1 and TRIM3 were upregulated with decreased methylation, promoting cell growth. ConclusionMSCs improve the structure and function of aged thymus, which involves the regulation of DNA methylation profiles and a decrease in the methylation level of the transcription factor NGF to specifically upregulate KRT17 and FOXJ1 to promote the proliferation of TECs.

Micronodular thymoma with lymphoid stroma: As benign as it sounds.

Micronodular thymoma (MNT) is a rare subtype of thymoma (reported incidence is approximately 1-5%). We report a case received as a core biopsy from the "right lung mass" of a 31-year-old female. CT scan showed an 8.1 cm large well-defined mass lesion in the right middle lobe, causing indentation and mild compression of the right atrium. Microscopically, the biopsy showed a thymic neoplasm comprised of multiple discrete and coalescing nodules of bland epithelioid tumor cells separated by an epithelial cell-free lymphocyte-rich stroma. On immunohistochemistry, cytokeratin highlighted the thymic epithelial cells. The lymphoid cells showed a naive T-cell phenotype (TdT+ CD 3+). It is worthwhile recognizing this rare variant of thymoma as almost all the patients present with localized low stage disease with rare/no reports of recurrences or distant metastases.

Abstract 3587: Evidence that membrane progesterone receptors (mPR) play a key role in cancer metastases in most cases of metastatic cancer

Abstract An immunomodulatory protein called the progesterone induced blocking factor (PIBF) is essential for the fetal semi-allograft to escape immune surveillance. Many cancer cell lines make both mPRs for PIBF and the protein itself. The progesterone receptor (PR) antagonist mifepristone added to cancer cell line cultures down-regulated mRNA for PIBF and the PIBF protein. This IRB approved study (that required a compassionate use IND from the FDA) evaluated oral mifepristone 200mg per day (majority) or 300mg in patients with very advanced cancers with no more treatment options available. Patients would remain under the care of their primary oncologists (FDA requirement). Because the nuclear (n) PR may be protective against cancer spread, only patients with cancers negative for the nPR were treated. Patients with the following types of cancers received mifepristone: non-small cell lung cancer (NSCLC) (n=9), small cell lung cancer (SCLC) (n=2), colon (n=3), pancreatic (n=2), thymic epithelial cell (n=1), transitional cell carcinoma of the renal pelvis (n=1), malignant fibrous histiocytoma (n=1), glioblastoma multiforme stage IV (n=1), leiomyosarcoma (n=1). All 21 patients showed significant palliative benefits, especially but not limited to, marked reduction in pain and marked improvement in energy, decreased dyspnea on exertion, and return of mental clarity. For those who had repeat CT evaluation no one failed to show shrinkage and decrease in malignant lesions and 2 (leiomyosarcoma and SCLC) showed complete remission after a few months on mifepristone. Five patients (3 females, 2 males) with SCLC (1), NSCL (n=3) and fibrous osteosarcoma (n=1) survived at least 5 years with 2 dying from cancer unrelated causes (SCLC and NSCLC). Two are still alive (NSCLC with EGFR mutation and brain metastases). The male with fibrous osteosarcoma died after stopping mifepristone in favor of a new drug recommended by his oncologist. Two patients lived over 2 years. Most would have lived longer had they not stopped mifepristone. Other observations: significant palliative benefits are seen within 3 weeks of taking mifepristone before any evidence of significant tumor regression. After a period of time, some lesions (especially in the primary location) may start growing slowly, but this is not a sign of loss of drug efficacy and long good quality life is still probable if one remains on the drug. The cancer spreads rapidly if one stops the mifepristone. The usual reason for stopping the drug was intervention by the primary oncologist suggesting a new clinical trial because there has not been complete remission of the cancer or some tumor growth (usually slow) or finances. Not one patient had a side effect. Hopefully this study will generate interest in treating patients with advanced cancer with mifepristone, especially those devoid of the nPR. These data support the role of mPRs in cancer proliferation. Citation Format: Jerome H. Check, Diane Check, Brooke Neumann, Carrie Wilson. Evidence that membrane progesterone receptors (mPR) play a key role in cancer metastases in most cases of metastatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3587.

Immune tolerance and the prevention of autoimmune diseases essentially depend on thymic tissue homeostasis.

The intricate balance of immune reactions towards invading pathogens and immune tolerance towards self is pivotal in preventing autoimmune diseases, with the thymus playing a central role in establishing and maintaining this equilibrium. The induction of central immune tolerance in the thymus involves the elimination of self-reactive T cells, a mechanism essential for averting autoimmunity. Disruption of the thymic T cell selection mechanisms can lead to the development of autoimmune diseases. In the dynamic microenvironment of the thymus, T cell migration and interactions with thymic stromal cells are critical for the selection processes that ensure self-tolerance. Thymic epithelial cells are particularly significant in this context, presenting self-antigens and inducing the negative selection of autoreactive T cells. Further, the synergistic roles of thymic fibroblasts, B cells, and dendritic cells in antigen presentation, selection and the development of regulatory T cells are pivotal in maintaining immune responses tightly regulated. This review article collates these insights, offering a comprehensive examination of the multifaceted role of thymic tissue homeostasis in the establishment of immune tolerance and its implications in the prevention of autoimmune diseases. Additionally, the developmental pathways of the thymus are explored, highlighting how genetic aberrations can disrupt thymic architecture and function, leading to autoimmune conditions. The impact of infections on immune tolerance is another critical area, with pathogens potentially triggering autoimmunity by altering thymic homeostasis. Overall, this review underscores the integral role of thymic tissue homeostasis in the prevention of autoimmune diseases, discussing insights into potential therapeutic strategies and examining putative avenues for future research on developing thymic-based therapies in treating and preventing autoimmune conditions.

Mechanism study of ubiquitination in T cell development and autoimmune disease.

T cells play critical role in multiple immune processes including antigen response, tumor immunity, inflammation, self-tolerance maintenance and autoimmune diseases et. Fetal liver or bone marrow-derived thymus-seeding progenitors (TSPs) settle in thymus and undergo T cell-lineage commitment, proliferation, T cell receptor (TCR) rearrangement, and thymic selections driven by microenvironment composed of thymic epithelial cells (TEC), dendritic cells (DC), macrophage and B cells, thus generating T cells with diverse TCR repertoire immunocompetent but not self-reactive. Additionally, some self-reactive thymocytes give rise to Treg with the help of TEC and DC, serving for immune tolerance. The sequential proliferation, cell fate decision, and selection during T cell development and self-tolerance establishment are tightly regulated to ensure the proper immune response without autoimmune reaction. There are remarkable progresses in understanding of the regulatory mechanisms regarding ubiquitination in T cell development and the establishment of self-tolerance in the past few years, which holds great potential for further therapeutic interventions in immune-related diseases.

AIRE relies on Z-DNA to flag gene targets for thymic T cell tolerization.

AIRE is an unconventional transcription factor that enhances the expression of thousands of genes in medullary thymic epithelial cells and promotes clonal deletion or phenotypic diversion of self-reactive T cells1-4. The biological logic of AIRE's target specificity remains largely unclear as, in contrast to many transcription factors, it does not bind to a particular DNA sequence motif. Here we implemented two orthogonal approaches to investigate AIRE's cis-regulatory mechanisms: construction of a convolutional neural network and leveraging natural genetic variation through analysis of F1 hybrid mice5. Both approaches nominated Z-DNA and NFE2-MAF as putative positive influences on AIRE's target choices. Genome-wide mapping studies revealed that Z-DNA-forming and NFE2L2-binding motifs were positively associated with the inherent ability of a gene's promoter to generate DNA double-stranded breaks, and promoters showing strong double-stranded break generation were more likely to enter a poised state with accessible chromatin and already-assembled transcriptional machinery. Consequently, AIRE preferentially targets genes with poised promoters. We propose a model in which Z-DNA anchors the AIRE-mediated transcriptional program by enhancing double-stranded break generation and promoter poising. Beyond resolving a long-standing mechanistic conundrum, these findings suggest routes for manipulating T cell tolerance.

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.

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.

Characterizing the Thymic Epithelial Cell Network Structure by Fluorescence Whole Slide Scanning and 3D SEM Array Tomography

Characterizing the Thymic Epithelial Cell Network Structure by Fluorescence Whole Slide Scanning and 3D SEM Array Tomography

B cells orchestrate tolerance to the neuromyelitis optica autoantigen AQP4

Neuromyelitis optica is a paradigmatic autoimmune disease of the central nervous system, in which the water-channel protein AQP4 is the target antigen1. The immunopathology in neuromyelitis optica is largely driven by autoantibodies to AQP42. However, the T cell response that is required for the generation of these anti-AQP4 antibodies is not well understood. Here we show that B cells endogenously express AQP4 in response to activation with anti-CD40 and IL-21 and are able to present their endogenous AQP4 to T cells with an AQP4-specific T cell receptor (TCR). A population of thymic B cells emulates a CD40-stimulated B cell transcriptome, including AQP4 (in mice and humans), and efficiently purges the thymic TCR repertoire of AQP4-reactive clones. Genetic ablation of Aqp4 in B cells rescues AQP4-specific TCRs despite sufficient expression of AQP4 in medullary thymic epithelial cells, and B-cell-conditional AQP4-deficient mice are fully competent to raise AQP4-specific antibodies in productive germinal-centre responses. Thus, the negative selection of AQP4-specific thymocytes is dependent on the expression and presentation of AQP4 by thymic B cells. As AQP4 is expressed in B cells in a CD40-dependent (but not AIRE-dependent) manner, we propose that thymic B cells might tolerize against a group of germinal-centre-associated antigens, including disease-relevant autoantigens such as AQP4.

Somatic variant profiling of a thymoma in Good syndrome

Good syndrome (GS) is a combined immunodeficiency that is associated with thymomas. The cause of the reduction in B-cells in patients with GS may be multifactorial and may include dysregulated T-cell responses. It has been proposed that tumorigenesis in a normal thymus alters thymic epithelial cell function, which leads to attenuated elimination of T-cells autoreactive to B-cells. Although the comprehensive genetic analysis of thymoma has been performed and reported in many articles, the comprehensive genetic analysis specified for GS-related thymoma has not been reported. Herein, we report comprehensive genetic analysis of a thymoma taken from a patient with GS. Oncogenesis-associated genes that may contribute to thymoma development were detected. Additionally, alteration of VCAM1, which is required in the interaction between T-cells and thymic epithelial cells, was observed. Aberrantly expressed VCAM1 in thymic epithelial cells may decrease the efficacy of negative of selection autoreactive T-cells and contribute to autoimmunity to B-cells.

Ehf and Fezf2 regulate late medullary thymic epithelial cell and thymic tuft cell development.

Thymic epithelial cells are indispensable for T cell maturation and selection and the induction of central immune tolerance. The self-peptide repertoire expressed by medullary thymic epithelial cells is in part regulated by the transcriptional regulator Aire (Autoimmune regulator) and the transcription factor Fezf2. Due to the high complexity of mTEC maturation stages (i.e., post-Aire, Krt10+ mTECs, and Dclk1+ Tuft mTECs) and the heterogeneity in their gene expression profiles (i.e., mosaic expression patterns), it has been challenging to identify the additional factors complementing the transcriptional regulation. We aimed to identify the transcriptional regulators involved in the regulation of mTEC development and self-peptide expression in an unbiased and genome-wide manner. We used ATAC footprinting analysis as an indirect approach to identify transcription factors involved in the gene expression regulation in mTECs, which we validated by ChIP sequencing. This study identifies Fezf2 as a regulator of the recently described thymic Tuft cells (i.e., Tuft mTECs). Furthermore, we identify that transcriptional regulators of the ELF, ESE, ERF, and PEA3 subfamily of the ETS transcription factor family and members of the Krüppel-like family of transcription factors play a role in the transcriptional regulation of genes involved in late mTEC development and promiscuous gene expression.

Mitochondrial protein C15ORF48 is a stress-independent inducer of autophagy that regulates oxidative stress and autoimmunity

Autophagy is primarily activated by cellular stress, such as starvation or mitochondrial damage. However, stress-independent autophagy is activated by unclear mechanisms in several cell types, such as thymic epithelial cells (TECs). Here we report that the mitochondrial protein, C15ORF48, is a critical inducer of stress-independent autophagy. Mechanistically, C15ORF48 reduces the mitochondrial membrane potential and lowers intracellular ATP levels, thereby activating AMP-activated protein kinase and its downstream Unc-51-like kinase 1. Interestingly, C15ORF48-dependent induction of autophagy upregulates intracellular glutathione levels, promoting cell survival by reducing oxidative stress. Mice deficient in C15orf48 show a reduction in stress-independent autophagy in TECs, but not in typical starvation-induced autophagy in skeletal muscles. Moreover, C15orf48–/– mice develop autoimmunity, which is consistent with the fact that the stress-independent autophagy in TECs is crucial for the thymic self-tolerance. These results suggest that C15ORF48 induces stress-independent autophagy, thereby regulating oxidative stress and self-tolerance.

Immune Modulation with RANKL Blockade through Denosumab Treatment in Patients with Cancer.

Denosumab is a fully human mAb that binds receptor activator of NFκB ligand (RANKL). It is routinely administered to patients with cancer to reduce the incidence of new bone metastasis. RANK-RANKL interactions regulate bone turnover by controlling osteoclast recruitment, development, and activity. However, these interactions also can regulate immune cells including dendritic cells and medullary thymic epithelial cells. Inhibition of the latter results in reduced thymic negative selection of T cells and could enhance the generation of tumor-specific T cells. We examined whether administering denosumab could modify modulate circulating immune cells in patients with cancer. Blood was collected from 23 patients with prostate cancer and 3 patients with renal cell carcinoma, all of whom had advanced disease and were receiving denosumab, prior to and during denosumab treatment. Using high-dimensional mass cytometry, we found that denosumab treatment by itself induced modest effects on circulating immune cell frequency and activation. We also found minimal changes in the circulating T-cell repertoire and the frequency of new thymic emigrants with denosumab treatment. However, when we stratified patients by whether they were receiving chemotherapy and/or steroids, patients receiving these concomitant treatments showed significantly greater immune modulation, including an increase in the frequency of natural killer cells early and classical monocytes later. We also saw broad induction of CTLA-4 and TIM3 expression in circulating lymphocytes and some monocyte populations. These findings suggest that denosumab treatment by itself has modest immunomodulatory effects, but when combined with conventional cancer treatments, can lead to the induction of immunologic checkpoints. See related Spotlight by Nasrollahi and Davar, p. 383.

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.

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.

Thymosin β4 Regulates the Differentiation of Thymocytes by Controlling the Cytoskeletal Rearrangement and Mitochondrial Transfer of Thymus Epithelial Cells.

The thymus is one of the most crucial immunological organs, undergoing visible age-related shrinkage. Thymic epithelial cells (TECs) play a vital role in maintaining the normal function of the thymus, and their degeneration is the primary cause of age-induced thymic devolution. Thymosin β4 (Tβ4) serves as a significant important G-actin sequestering peptide. The objective of this study was to explore whether Tβ4 influences thymocyte differentiation by regulating the cytoskeletal rearrangement and mitochondrial transfer of TECs. A combination of H&E staining, immunofluorescence, transmission electron microscopy, RT-qPCR, flow cytometry, cytoskeletal immunolabeling, and mitochondrial immunolabeling were employed to observe the effects of Tβ4 on TECs' skeleton rearrangement, mitochondrial transfer, and thymocyte differentiation. The study revealed that the Tβ4 primarily regulates the formation of microfilaments and the mitochondrial transfer of TECs, along with the formation and maturation of double-negative cells (CD4-CD8-) and CD4 single-positive cells (CD3+TCRβ+CD4+CD8-) thymocytes. This study suggests that Tβ4 plays a crucial role in thymocyte differentiation by influencing the cytoskeletal rearrangement and mitochondrial transfer of TECs. These effects may be associated with Tβ4's impact on the aggregation of F-actin. This finding opens up new avenues for research in the field of immune aging.