Number Of Thymic Epithelial Cells Research Articles

Recombinant FOXN1 fusion protein increases T cell generation in old mice.

T cell development in the thymus is dependent on the thymic microenvironment, in which thymic epithelial cells (TECs) are the major component. However, TECs undergo both a qualitative and quantitative loss during aging, which is believed to be the major factor responsible for age-dependent thymic atrophy. FOXN1 plays a critical role in TEC development and adult TECs maintenance. We have previously reported that intrathymic injection of a recombinant (r) protein containing murine FOXN1 and a protein transduction domain increases the number of TECs in mice, leading to enhanced thymopoiesis. However, intrathymic injection may not be an ideal choice for clinical applications. In this study, we produced a rFOXN1 fusion protein containing the N-terminal of CCR9, human FOXN1 and a protein transduction domain. When injected intravenously into 14-month-old mice, the rFOXN1 fusion protein enters the thymus and TECs, and enhances thymopoiesis, resulting in increased T cell generation in the thymus and increased number of T cells in peripheral lymphoid organ. Our results suggest that the rFOXN1 fusion protein has the potential to be used in preventing and treating T cell immunodeficiency in older adults.

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.

Administration of recombinant FOXN1 protein attenuates Alzheimer’s pathology in mice

BackgroundAlzheimer’s disease (AD) is the most common cause of dementia in older adults and characterized by progressive loss of memory and cognitive functions that are associated with amyloid-beta (Aβ) plaques and neurofibrillary tangles. Immune cells play an important role in the clearance of Aβ deposits and neurofibrillary tangles. T cells are the major component of the immune system. The thymus is the primary organ for T cell generation. T cell development in the thymus depends on thymic epithelial cells (TECs). However, TECs undergo both qualitative and quantitative loss over time. We have previously reported that a recombinant (r) protein containing FOXN1 and a protein transduction domain can increase the number of TECs and subsequently increases the number of T cells in mice. In this study we determined the ability of rFOXN1 to affect cognitive performance and AD pathology in mice. MethodsAged 3xTg-AD and APP/PS1 AD mice were injected with rFOXN1 or control protein. Cognitive performance, AD pathology, the thymic microenvironment and immune cells were then analyzed. ResultsAdministration of rFOXN1 into AD mice improves cognitive performance and reduces Aβ plaque load and phosphorylated tau in the brain. This is related to rejuvenating the aged thymic microenvironment, which results in enhanced T cell generation in the thymus, leading to increased number of T cells, especially IFNγ-producing T cells, in the spleen and the choroid plexus (CP), enhanced expression of immune cell trafficking molecules in the CP, and increased migration of monocyte-derived macrophages into the brain. Furthermore, the production of anti-Aβ antibodies in the serum and the brain, and the macrophage phagocytosis of Aβ are enhanced in rFOXN1-treated AD mice. ConclusionsOur results suggest that rFOXN1 protein has the potential to provide a novel approach to treat AD patients.

MSCs-ILC2s-Tecs Axis Promotes the Recovery of Thymic Function in aGVHD

Background: Previous studies found that mesenchymal stem cells (MSCs) administrated at the acute graft versus host disease (aGVHD) phase effectively decreased the incidence and severity of chronic GVHD (cGVHD) by boosting thymic regeneration. However, the mechanism for MSCs repairing the damaged thymus caused by aGVHD is unclear. It has been reported that innate lymphoid cells (ILCs) are also important for the structural remodeling of the thymus in recipients after allogeneic bone marrow transplantation (allo-BMT). Here, we explored the mechanisms on how MSCs promote thymus recovery post allo-BMT through ILCs. Methods: We carried out studies in a aGVHD mice model of fully MHC-mismatched myeloablative bone marrow transplantation (C57BL/6 to BALB/c). MSCs or ILC2s were generated and administrated intravenously at day 7 post-BMT to the treated groups to compare therapeutic effects of GVHD with the untreated group. Clinical scores and survival were recorded once every days. Flow cytometery was employed for analyzing the number and proportion of ILCs and their subtypes (ILCs Lin-CD45+iCD3-CD127+, type 1 ILCs (ILC1s) Tbet+, type 2 ILCs (ILC2s) GATA3+, type 3 ILCs (ILC3s) RORγt+) in the thymus to assess the effect of MSCs on ILCs in the thymus at day 14 post-BMT. We analyzed the thymic T cells subsets and thymic epithelial cells (TECs) substes among these two groups to evaluate the repair effect of ILC2s for thymus. Thymic ILC2s were cultured alone or co-cultured with murine MSCs in vitro to investigate the effect and underlying mechanism of MSCs on ILC2s. Results: We found that ILC2s in the thymus are sensitive to conditioning therapy and and further impaired by aGVHD. But infusion of MSCs was effective in reducing the lethality of aGVHD and promote expansion of thymic ILC2s in aGVHD mice compared with the control group. The absolute number of ILC2s in the thymus had a significant negative correlation with GVHD scores in aGVHD mice. Then we found that ILC2s infusion significant increases in thymus size and weight, as well as the number of total thymocytes and the more organized cortical medullary structure compared to the untreated groups. The number of TECs and its proportion of thymus stroma were significantly improved, including cortical TEC and medullary TECs. As for thymocyte, ILC2s infusion significantly increased the number and proportion of CD4+CD8+T cells. In vitro study showed co-cultured ILC2s had a increased proliferation compared to ILC2s cultured alone, and anti-IL33 antibody inhibited the pro-proliferative effect of MSCs on ILC2. Then we perform in vivo validation by injecting IL33 knocked down MSCs and found that the effect of MSC in repairing the damaged thymus was reduced. And the number and proportion of thymic ILC2s were also significantly decreased in the thymus. Furthermore, treatment with rIL33 on day 7-11 also ameliorated aGVHD and promoted the proliferation of thymic ILC2s in aGVHD mice as well as MSCs infusion. Conclusion: These findings suggest that MSCs participate in thymus regeneration from aGVHD damage through ILC2s-mediated tissue recovery. The study outlined here provide a new insight into the mechanism of MSC therapy for aGVHD.

Large-Scale Isolation of Mouse Thymic Epithelial Cells.

The thymus is compartmentalized into the cortex and the medulla. Cortical and medullary thymic epithelial cells (TECs) characterize T cell-producing and T cell-selecting functions of cortical and medullary microenvironments in the thymus. Enzymatic digestion of the thymus and flow cytometric isolation of TECs and their subpopulations are useful for molecular and cellular characterization of TECs. However, the cellularity of cTECs and mTECs isolated from mouse thymus is limited. In this chapter, we describe the method for isolation of a large number of TECs using enlarged mouse thymus, which enables biochemical and proteomic analysis of TEC subpopulations.

Cytosolic Nuclear Sensor Dhx9 Controls Medullary Thymic Epithelial Cell Differentiation by p53-Mediated Pathways.

Thymic epithelial cells (TECs) critically participate in T cell maturation and selection for the establishment of immunity to foreign antigens and immune tolerance to self-antigens of T cells. It is well known that many intracellular and extracellular molecules elegantly have mastered the development of medullary TECs (mTECs) and cortical TECs (cTECs). However, the role played by NTP-dependent helicase proteins in TEC development is currently unclear. Herein, we created mice with a TEC-specific DExD/H-box helicase 9 (Dhx9) deletion (Dhx9 cKO) to study the involvement of Dhx9 in TEC differentiation and function. We found that a Dhx9 deficiency in TECs caused a significant decreased cell number of TECs, including mTECs and thymic tuft cells, accompanied by accelerated mTEC maturation but no detectable effect on cTECs. Dhx9-deleted mTECs transcriptionally expressed poor tissue-restricted antigen profiles compared with WT mTECs. Importantly, Dhx9 cKO mice displayed an impaired thymopoiesis, poor thymic T cell output, and they suffered from spontaneous autoimmune disorders. RNA-seq analysis showed that the Dhx9 deficiency caused an upregulated DNA damage response pathway and Gadd45, Cdkn1a, Cdc25, Wee1, and Myt1 expression to induce cell cycle arrest in mTECs. In contrast, the p53-dependent upregulated RANK-NF-κB pathway axis accelerated the maturation of mTECs. Our results collectively indicated that Dhx9, a cytosolic nuclear sensor recognizing viral DNA or RNA, played an important role in mTEC development and function in mice.

Indispensable epigenetic control of thymic epithelial cell development and function by polycomb repressive complex 2

Thymic T cell development and T cell receptor repertoire selection are dependent on essential molecular cues provided by thymic epithelial cells (TEC). TEC development and function are regulated by their epigenetic landscape, in which the repressive H3K27me3 epigenetic marks are catalyzed by polycomb repressive complex 2 (PRC2). Here we show that a TEC-targeted deficiency of PRC2 function results in a hypoplastic thymus with reduced ability to express antigens and select a normal repertoire of T cells. The absence of PRC2 activity reveals a transcriptomically distinct medullary TEC lineage that incompletely off-sets the shortage of canonically-derived medullary TEC whereas cortical TEC numbers remain unchanged. This alternative TEC development is associated with the generation of reduced TCR diversity. Hence, normal PRC2 activity and placement of H3K27me3 marks are required for TEC lineage differentiation and function and, in their absence, the thymus is unable to compensate for the loss of a normal TEC scaffold.

Intrathymic Selection and Defects in the Thymic Epithelial Cell Development.

Intimate interactions between thymic epithelial cells (TECs) and thymocytes (T) have been repeatedly reported as essential for performing intrathymic T-cell education. Nevertheless, it has been described that animals exhibiting defects in these interactions were capable of a proper positive and negative T-cell selection. In the current review, we first examined distinct types of TECs and their possible role in the immune surveillance. However, EphB-deficient thymi that exhibit profound thymic epithelial (TE) alterations do not exhibit important immunological defects. Eph and their ligands, the ephrins, are implicated in cell attachment/detachment and govern, therefore, TEC–T interactions. On this basis, we hypothesized that a few normal TE areas could be enough for a proper phenotypical and functional maturation of T lymphocytes. Then, we evaluated in vivo how many TECs would be necessary for supporting a normal T-cell differentiation, concluding that a significantly low number of TEC are still capable of supporting normal T lymphocyte maturation, whereas with fewer numbers, T-cell maturation is not possible.

Mesenchymal Stem Cells Improve the Structure and Function of the Graft-Versus-Host Disease Receptor Thymus: CCR9 Plays an Important Role in Its Homing Thymus

Background: Insufficient thymic function of allogeneic hematopoietic stem cell transplantation (allo-HSCT) receptors results in continuous production of alloreactive T cells, which leads to the development of graft-versus-host disease (GVHD), especially chronic GVHD (cGVHD). We have previously found that patients with acute GVHD (aGVHD) treated with mesenchymal stem cells (MSCs) have increased thymic output and decreased incidence of cGVHD, thus hypothesized that MSCs may reduce the incidence of cGVHD by remodeling the thymus. Chemokine receptor 9 (CCR9), the receptor that specifically guides migration of T-lineage precursors into thymus, is also expressed on MSCs, and thus may be a key factor mediating MSCs homing to the thymus. This in turn allows MSCs to reduce GVHD by repairing thymus tissue structure and saving thymus function. Methods: We carried out studies in a murine GVHD model of fully MHC-mismatched myeloablative bone marrow transplantation (C57BL/6 to BALB/c), a model that can observe the prolongation of aGVHD to cGVHD. We randomly divided GVHD mice into four groups, including three MSCs treated groups and one untreated group. CCR9 over-expressed (MSC/CCR9+), knocked-down (MSC/CCR9-) and empty-load MSCs (MSC/Control) were generated and administrated intravenously at dose of 5 × 105 cells/infusion at 7th and 21th day post HCT to the treated groups respectively to compare their thymic homing ability, and therapeutic effects of GVHD with the untreated group. Clinical scores were recorded once every five days to evaluate GVHD symptoms. Mice of MSCs treated groups and the untreated group were sacrificed at 30d, 45d and 60d after HCT. Thymuses of each group were collected and assessed for size and weight before being manufactured into frozen sections or thymic single-cell suspension. We then analyzed the number and distribution of MSCs in the thymus of the treated groups to assess the role of CCR9 in thymic homing, and analyzed the expression of thymic T cells subsets (CD4+CD8-, CD4-CD8+, CD4+CD8+ T, CD4+CD25+Foxp3+Tregs), thymic epithelial cells (TECs) substes (CD45-CD326+Ly51+ cortical TECs and CD45-CD326+UEA-1+ medullary TECs) and the level of T cell receptor rearrangement excision circles (TRECs) in thymus among the four groups to evaluate the repair effect of MSCs for thymus. Radiation-pretreated murine TECs were cultured alone or co-cultured with murine MSCs in vitro to assess the effect of MSCs on damaged TECs. Results: The infusion of MSC/CCR9+ potently alleviated the clinical signs of GVHD and prolonged the survival of GVHD mice (P<0.05 versus MSC/CCR9- and untreated group). Significant increases in thymus size and weight were observed in the MSC/CCR9+ group, as well as the number of total thymocytes and the more organized cortical medullary structure compared to the other groups. MSCs enter the thymus from the microvascular region at the cortex-medium junction. MSC/CCR9+ were found to appear in the cortex-medium junction of thymus in a greater amount 24 hours after the first infusion, then distribute throughout the whole thymus and relocate in proximity with TECs 48 hours thereafter. MSC/Control could be observed in the cortical and cortex-medium junction, whereas MSC/CCR9- was observed only in the cortex-medium junction with a small amount of distribution. Immunofluorescence of thymus frozen sections showed that, compared with other groups, TECs had decreased apoptosis and significantly increased proliferation and maturation levels in MSC/CCR9+ group, indicating MSCs potently repaired injured TECs and promoted their proliferation and maturation. The number of TECs and its proportion of thymus stroma were significantly improved, including cortical TEC and medullary TECs. As for thymocyte, MSC/CCR9+ infusion significantly increased the number and proportion of CD4+CD8+T cells and Tregs, which were reported deficiency in GVHD thymus. Furthermore, MSC/CCR9+ administration resulted in a remarkable increase in the levels of TRECs in the thymocyte at 45d and 60d after HCT (P<0.05 versus MSC/CCR9- and untreated group). In vitro study showed co-cultured TECs had a decreased apoptosis and increased proliferation compared to TECs cultured alone. Conclusion: This study demonstrates that CCR9 plays an important role in guiding migration of MSCs to thymus and thus highly intensify their issue repair and immunomodulatory effect to rescue thymus function in GVHD model. Disclosures No relevant conflicts of interest to declare.

IL-22 Accelerates Thymus Regeneration via Stat3/Mcl-1 and Decreases Chronic Graft-versus-Host Disease in Mice after Allotransplants.

High-dose chemotherapy and/or radiation given before an allogeneic hematopoietic cell transplantation severely damage thymic epithelial cells (TECs), resulting in poor post-transplant immune recovery. IL-22 mediates recovery of TECs via a proregenerative effect, but the precise mechanism by which this occurs is unknown. In this study, we found IL-22 improved thymus recovery after damage from irradiation in association with increased number of TECs. This effect was blocked by ruxolitinib, a JAK1/JAK2 inhibitor. IL-22 increased the number of TECs via a Stat3-dependent signaling in the mTEC1 murine thymic epithelial cell line. This, in turn, upregulated transcription of myeloid cell leukemia sequence 1 (Mcl1), resulting in increased number of TECs. Similar effects were seen in irradiated mice given IL-22. Defects in IL-22 resulted in delayed thymus recovery in irradiated mice and had an impact on levels of thymus function-related genes such as Foxn1, Aire, and Kgf. In mice, post-transplant use of IL-22 improved repair of TECs, increased the numbers of thymus T cells, increased the intrathymic levels of Aire, and increased the proportion of natural regulatory T cells, resulting in decreased severity of chronic graft-versus-host disease (GVHD). Our data highlight the critical role of the IL-22/Stat3/Mcl-1 pathway in the regeneration of TECs after damage from irradiation in mice and highlight circumstances where normalizing thymus T cell function with IL-22 decreases GVHD after allotransplants.

Fundamental parameters of the developing thymic epithelium in the mouse

The numbers of thymic epithelial cells (TECs) and thymocytes steadily increase during embryogenesis. To examine this dynamic, we generated several TEC-specific transgenic mouse lines, which express fluorescent proteins in the nucleus, the cytosol and in the membranes under the control of the Foxn1 promoter. These tools enabled us to determine TEC numbers in tissue sections by confocal fluorescent microscopy, and in the intact organ by light-sheet microscopy. Compared to histological procedures, flow cytometric analysis of thymic cellularity is shown to underestimate the numbers of TECs by one order of magnitude; using enzymatic digestion of thymic tissue, the loss of cortical TECs (cTECs) is several fold greater than that of medullary TECs (mTECs), although different cTEC subsets appear to be still present in the final preparation. Novel reporter lines driven by Psmb11 and Prss16 promoters revealed the trajectory of differentiation of cTEC-like cells, and, owing to the additional facility of conditional cell ablation, allowed us to follow the recovery of such cells after their depletion during embryogenesis. Multiparametric histological analyses indicate that the new transgenic reporter lines not only reveal the unique morphologies of different TEC subsets, but are also conducive to the analysis of the complex cellular interactions in the thymus.

Retinoic Acid Signaling in Thymic Epithelial Cells Regulates Thymopoiesis.

Despite the essential role of thymic epithelial cells (TEC) in T cell development, the signals regulating TEC differentiation and homeostasis remain incompletely understood. In this study, we show a key in vivo role for the vitamin A metabolite, retinoic acid (RA), in TEC homeostasis. In the absence of RA signaling in TEC, cortical TEC (cTEC) and CD80loMHC class IIlo medullary TEC displayed subset-specific alterations in gene expression, which in cTEC included genes involved in epithelial proliferation, development, and differentiation. Mice whose TEC were unable to respond to RA showed increased cTEC proliferation, an accumulation of stem cell Ag-1hi cTEC, and, in early life, a decrease in medullary TEC numbers. These alterations resulted in reduced thymic cellularity in early life, a reduction in CD4 single-positive and CD8 single-positive numbers in both young and adult mice, and enhanced peripheral CD8+ T cell survival upon TCR stimulation. Collectively, our results identify RA as a regulator of TEC homeostasis that is essential for TEC function and normal thymopoiesis.

A critical epithelial survival axis regulated by MCL-1 maintains thymic function

Abstract T cell differentiation is governed by interactions with thymic epithelial cells (TECs) and defects in this process undermine immune function. Age-related thymic involution is recognised as a potentially fatal brake on immune recovery following haematopoietic stem/progenitor cell transplantation and cancer treatments. To uncover new strategies to restore thymic function, we determined the molecular mechanisms that control life and death decisions in TEC. We recently published that while TEC-specific deletion of pro-survival proteins BCL-2 and BCL-XL did not cause gross abnormalities, MCL-1 deficiency impacted on TEC as early as E15.5, resulting in early thymic atrophy, T cell lymphopenia, with near complete loss of thymic tissue by 2 months of age. MCL-1 was continually required for the survival of medullary TEC, including autoimmune regulator (AIRE) expressing TECs and the maintenance of overall thymic architecture. A screen of TEC trophic factors in organ cultures showed that epidermal growth factor upregulated MCL-1 via the MAPK/ERK kinase activity, providing a molecular mechanism for the support of TEC survival. However, these findings do not imply that the intrinsic pathway of apoptosis necessarily controls TEC death during homeostasis. Therefore, we removed essential effectors of this pathway, BAX and BAK, specifically in TECs and found that there was a specific increase in a medullary TEC subset leading to an overall increase in TEC numbers both in young and old mice. This signalling axis governing TEC survival, death and thymic function represent a compelling strategy for protecting the thymus during blood cancer treatment to prevent immunodeficiency, without counterproductive survival of haematopoietic lineages.

Cellularity of Thymic Epithelial Cells in the Postnatal Mouse.

The molecular and cellular biology of thymic epithelial cells (TECs) often relies on the analysis of TECs isolated in enzymatically digested single-cell suspensions derived from mouse thymus. Many independent studies have reported that the estimated cellularity of total TECs isolated from one adult mouse is on the order of up to 105 However, these numbers appear extremely small given that the cellularity of total thymocytes exceeds 108 and that TECs play multiple roles in thymocyte development and repertoire formation. In the present study, we aimed to measure the numbers of β5t-expressing cortical TECs and Aire-expressing medullary TECs in postnatal mouse thymus in situ without enzymatic digestion. The numbers of these TECs were manually counted in individual thymic sections and were three-dimensionally summed throughout the entire thymic lobes. The results show that the cellularity of total TECs in one 5-wk-old female mouse exceeds 106, containing ∼9 × 105 β5t+ cortical TECs and ∼1.1 × 106 Aire+ medullary TECs. These results suggest that the use of conventional enzymatic digestion methods for the isolation of TECs may have resulted in the underestimation of the cellularity, and possibly the biology, of TECs.

Targeted deletion of c-Met in thymic epithelial cells leads to an autoimmune phenotype.

Hepatocyte growth factor (HGF) and its receptor c-Met signaling have been implicated in regulating various types of cells including epithelial cells. We have previously reported that c-Met is expressed by thymic epithelial cells (TECs), and that in vivo administration of hybrid cytokines containing IL-7 and the beta- or alpha-chain of HGF significantly increase the number of TECs. In order to study the role of c-Met signaling in TECs, we generated conditional knockout (cKO) mice in which c-Met was specifically deleted in TECs using a Foxn1-Cre transgene. We show here that c-Met deficiency in TECs results in age-progressive reduction in TEC number and reduced number of regulatory T cells. Consequently, c-Met TEC cKO mice displayed an autoimmune phenotype. Thus, c-Met signaling in TECs is important for the maintenance of TECs and immune self-tolerance.

Genetic and non-genetic determinants of thymic epithelial cell number and function

The thymus is the site of T cell development in vertebrates. In general, the output of T cells is determined by the number of thymic epithelial cells (TECs) and their relative thymopoietic activity. Here, we show that the thymopoietic activity of TECs differs dramatically between individual mouse strains. Moreover, in males of some strains, TECs perform better on a per cell basis than their counterparts in females; in other strains, this situation is reversed. Genetic crosses indicate that TEC numbers and thymopoietic capacity are independently controlled. Long-term analysis of functional parameters of TECs after castration provides evidence that the number of Foxn1-expressing TECs directly correlates with thymopoietic activity. Our study highlights potential complications that can arise when comparing parameters of TEC biology across different genetic backgrounds; these could affect the interpretation of the outcomes of interventions aimed at modulating thymic activity in genetically diverse populations, such as humans.

MTORC2 in Thymic Epithelial Cells Controls Thymopoiesis and T Cell Development.

Thymic epithelial cells (TECs) play important roles in T cell generation. Mechanisms that control TEC development and function are still not well defined. The mammalian or mechanistic target of rapamycin complex (mTORC)2 signals to regulate cell survival, nutrient uptake, and metabolism. We report in the present study that mice with TEC-specific ablation of Rictor, a critical and unique adaptor molecule in mTORC2, display thymic atrophy, which accompanies decreased TEC numbers in the medulla. Moreover, generation of multiple T cell lineages, including conventional TCRαβ T cells, regulatory T cells, invariant NKT cells, and TCRγδ T cells, was reduced in TEC-specific Rictor-deficient mice. Our data demonstrate that mTORC2 in TECs is important for normal thymopoiesis and efficient T cell generation.

FOXN1 recombinant protein enhances T-cell regeneration after hematopoietic stem cell transplantation in mice.

A prolonged period of T-cell recovery is the major challenge in hematopoietic stem cell transplantation (HSCT). Thymic epithelial cells (TECs) are the major component of the thymic microenvironment for T-cell generation. However, TECs undergo degeneration over time. FOXN1 plays a critical role in TEC development and is required to maintain adult TECs for thymopoiesis. To investigate the potential application of FOXN1, we have cloned and expressed recombinant FOXN1 protein (rFOXN1) that was fused with cell-penetrating peptides. We show here that the rFOXN1 protein can translocate from the cell surface into the cytoplasm and nucleus. Administration of rFOXN1 into both congenic and allogeneic HSCT recipient mice increased the number of TECs, resulting in enhanced thymopoiesis that led to an increased number of functional Tcells in the periphery. The increased number of TECs is due to the enhanced survival and proliferation of TECs. Our results suggest that rFOXN1 has the potential to be used in enhancing T-cell regeneration in patients following HSCT.

Doxycycline Protects Thymic Epithelial Cells from Mitomycin C-Mediated Apoptosis In Vitro via Trx2-NF-κB-Bcl-2/Bax Axis

Background/Aims: Age-associated and stress-induced involution of the thymus is accompanied by reduced numbers of thymic epithelial cells (TECs) and severe reduction in peripheral T cell repertoire specificities. These events seriously affect immune function, but the mechanisms involved are unclear. Our preliminary findings showed that doxycycline (Dox) could drive the proliferation of a TEC line (MTEC1 cells) partially via the MAPK signaling pathway. Dox can also up-regulate IL-6 and GM-CSF expression via the NF-κB and MAPK/ERK pathways. Herein, we investigate the effects and mechanisms used by Dox that protect against mitomycin C (MMC)-induced MTEC1 cell apoptosis. Methods: MTEC1 cells were treated with Dox, MMC, and Dox plus MMC for different amounts of time. The expression of Trx2, NF-κB, Bcl-2, and Bax proteins were then detected by western blotting. Results: Our findings show that Dox protects MTEC1 cells from MMC-induced apoptosis. Dox up-regulated the expression of Trx2 and promoted NF-κB phosphorylation. Meanwhile, Dox also increased the expression of Bcl-2, partially reduced the expression of Bax, and normalized the ratio of Bcl-2 to Bax. Conclusion: Dox exerts an anti-apoptosis function via the NF-κB-Bcl-2/Bax and Trx2-ASK1/JNK pathways in vitro. Therefore, Dox may represent a drug that could be used to attenuate thymic senescence, rescue thymic function, and promote T cell reconstitution.

Regenerative capacity of adult cortical thymic epithelial cells

Involution of the thymus is accompanied by a decline in the number of thymic epithelial cells (TECs) and a severely restricted peripheral repertoire of T-cell specificities. TECs are essential for T-cell differentiation; they originate from a bipotent progenitor that gives rise to cells of cortical (cTEC) and medullary (mTEC) phenotypes, via compartment-specific progenitors. Upon acute selective near-total ablation during embryogenesis, regeneration of TECs fails, suggesting that losses from the pool of TEC progenitors are not compensated. However, it is unclear whether this is also true for the compartment-specific progenitors. The decline of cTECs is a prominent feature of thymic involution. Because cTECs support early stages of T-cell development and hence determine the overall lymphopoietic capacity of the thymus, it is possible that the lack of sustained regenerative capacity of cTEC progenitor cells underlies the process of thymic involution. Here, we examine this hypothesis by cell-type-specific conditional ablation of cTECs. Expression of the human diphtheria toxin receptor (hDTR) gene under the regulatory influence of the chemokine receptor Ccx-ckr1 gene renders cTECs sensitive to the cytotoxic effects of diphtheria toxin (DT). As expected, DT treatment of preadolescent and adult mice led to a dramatic loss of cTECs, accompanied by a rapid demise of immature thymocytes. Unexpectedly, however, the cTEC compartment regenerated after cessation of treatment, accompanied by the restoration of T-cell development. These findings provide the basis for the development of targeted interventions unlocking the latent regenerative potential of cTECs to counter thymic involution.