Mechanisms underlying the direct programming of mouse embryonic fibroblasts to thymic epithelial cells by FOXN1.

Thymic epithelial cells (TECs) are a critical functional component of the thymus's ability to generate T cells for the adaptive immune system in vertebrates. However, no in vitro system for studying TEC function exists. Overexpressing the transcription factor FOXN1 initiates transdifferentiation of fibroblasts into TEC-like cells (iTECs) that support T cell differentiation in culture or after transplant. In this study, we characterized iTEC programming at the cellular and molecular level to determine how it proceeds and identified mechanisms that can be targeted for improving this process. These data showed that iTEC programming consisted of discrete gene expression changes that differed early and late in the process, and that iTECs upregulated markers of both cortical and medullary TEC (cTEC and mTEC) lineages. We demonstrated that promoting proliferation enhanced iTEC generation, and that Notch inhibition allowed induction of mTEC differentiation. Finally, we showed that MHCII expression was the major difference between iTECs and fetal TECs. MHCII expression was improved by co-culturing iTECs with fetal double-positive T-cells. This study supports future efforts to improve iTEC generation for both research and translational uses.

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.

Novel murine model reveals an early role for pertussis toxin in disrupting neonatal immunity to Bordetella pertussis.

The increased susceptibility of neonates to specific pathogens has previously been attributed to an underdeveloped immune system. More recent data suggest neonates have effective protection against most pathogens but are particularly susceptible to those that target immune functions specific to neonates. Bordetella pertussis (Bp), the causative agent of "whooping cough", causes more serious disease in infants attributed to its production of pertussis toxin (PTx), although the neonate-specific immune functions it targets remain unknown. Problematically, the rapid development of adult immunity in mice has confounded our ability to study interactions of the neonatal immune system and its components, such as virtual memory T cells which are prominent prior to the maturation of the thymus. Here, we examine the rapid change in susceptibility of young mice and define a period from five- to eight-days-old during which mice are much more susceptible to Bp than mice even a couple days older. These more narrowly defined "neonatal" mice display significantly increased susceptibility to wild type Bp but very rapidly and effectively respond to and control Bp lacking PTx, more rapidly even than adult mice. Thus, PTx efficiently blocks some very effective form(s) of neonatal protective immunity, potentially providing a tool to better understand the neonatal immune system. The rapid clearance of the PTx mutant correlates with the early accumulation of neutrophils and T cells and suggests a role for PTx in disrupting their accumulation. These results demonstrate a striking age-dependent response to Bp, define an early age of extreme susceptibility to Bp, and demonstrate that the neonatal response can be more efficient than the adult response in eliminating bacteria from the lungs, but these neonatal functions are substantially blocked by PTx. This refined definition of "neonatal" mice may be useful in the study of other pathogens that primarily infect neonates, and PTx may prove a particularly valuable tool for probing the poorly understood neonatal immune system.

Foxn1 overexpression promotes thymic epithelial progenitor cell proliferation and mTEC maintenance, but does not prevent thymic involution.

The transcription factor FOXN1 is essential for fetal thymic epithelial cell (TEC) differentiation and proliferation. Postnatally, Foxn1 levels vary widely between TEC subsets, from low/undetectable in putative TEC progenitors to highest in differentiated TEC subsets. Correct Foxn1 expression is required to maintain the postnatal microenvironment; premature downregulation of Foxn1 causes a rapid involution-like phenotype, and transgenic overexpression can cause thymic hyperplasia and/or delayed involution. We investigated a K5.Foxn1 transgene that drives overexpression in mouse TECs, but causes neither hyperplasia nor delay or prevention of aging-related involution. Similarly, this transgene cannot rescue thymus size in Foxn1lacZ/lacZ mice, which undergo premature involution as a result of reduced Foxn1 levels. However, TEC differentiation and cortico-medullary organization are maintained with aging in both K5.Foxn1 and Foxn1lacZ/lacZ mice. Analysis of candidate TEC markers showed co-expression of progenitor and differentiation markers as well as increased proliferation in Plet1+ TECs associated with Foxn1 expression. These results demonstrate that the functions of FOXN1 in promoting TEC proliferation and differentiation are separable and context dependent, and suggest that modulating Foxn1 levels can regulate the balance of proliferation and differentiation in TEC progenitors.

Thymic epithelial cells require lipid kinase Vps34 for CD4 but not CD8 T cell selection.

The generation of a functional, self-tolerant T cell receptor (TCR) repertoire depends on interactions between developing thymocytes and antigen-presenting thymic epithelial cells (TECs). Cortical TECs (cTECs) rely on unique antigen-processing machinery to generate self-peptides specialized for T cell positive selection. In our current study, we focus on the lipid kinase Vps34, which has been implicated in autophagy and endocytic vesicle trafficking. We show that loss of Vps34 in TECs causes profound defects in the positive selection of the CD4 T cell lineage but not the CD8 T cell lineage. Utilizing TCR sequencing, we show that T cell selection in conditional mutants causes altered repertoire properties including reduced clonal sharing. cTECs from mutant mice display an increased abundance of invariant chain intermediates bound to surface MHC class II molecules, indicating altered antigen processing. Collectively, these studies identify lipid kinase Vps34 as an important contributor to the repertoire of selecting ligands processed and presented by TECs to developing CD4 T cells.

NOTCH1 signaling establishes the medullary thymic epithelial cell progenitor pool during mouse fetal development.

The cortical and medullary thymic epithelial cell (cTEC and mTEC) lineages are essential for inducing T cell lineage commitment, T cell positive selection and the establishment of self-tolerance, but the mechanisms controlling their fetal specification and differentiation are poorly understood. Here, we show that notch signaling is required to specify and expand the mTEC lineage. Notch1 is expressed by and active in TEC progenitors. Deletion of Notch1 in TECs resulted in depletion of mTEC progenitors and dramatic reductions in mTECs during fetal stages, consistent with defects in mTEC specification and progenitor expansion. Conversely, forced notch signaling in all TECs resulted in widespread expression of mTEC progenitor markers and profound defects in TEC differentiation. In addition, lineage-tracing analysis indicated that all mTECs have a history of receiving a notch signal, consistent with notch signaling occurring in mTEC progenitors. These data provide strong evidence for a requirement for notch signaling in specification of the mTEC lineage.

Embryology of the Parathyroid Glands.

The parathyroid glands are essential for regulating calcium homeostasis in the body. The genetic programs that control parathyroid fate specification, morphogenesis, differentiation, and survival are only beginning to be delineated, but are all centered around a key transcription factor, GCM2. Mutations in the Gcm2 gene as well as in several other genes involved in parathyroid organogenesis have been found to cause parathyroid disorders in humans. Therefore, understanding the normal development of the parathyroid will provide insight into the origins of parathyroid disorders.

Polycomb Repressive Complex 2 is essential for development and maintenance of a functional TEC compartment.

Thymic epithelial cells (TEC) are essential for thymocyte differentiation and repertoire selection. Despite their indispensable role in generating functional T cells, the molecular mechanisms that orchestrate TEC development from endodermal progenitors in the third pharyngeal pouch (3rd PP) are not fully understood. We recently reported that the T-box transcription factor TBX1 negatively regulates TEC development. Although initially expressed throughout the 3rd PP, Tbx1 becomes downregulated in thymus-fated progenitors and when ectopically expressed impairs TEC progenitor proliferation and differentiation. Here we show that ectopic Tbx1 expression in thymus fated endoderm increases expression of Polycomb repressive complex 2 (PRC2) target genes in TEC. PRC2 is an epigenetic modifier that represses gene expression by catalyzing trimethylation of lysine 27 on histone H3. The increased expression of PRC2 target genes suggests that ectopic Tbx1 interferes with PRC2 activity and implicates PRC2 as an important regulator of TEC development. To test this hypothesis, we used Foxn1Cre to delete Eed, a PRC2 component required for complex stability and function in thymus fated 3rd PP endoderm. Proliferation and differentiation of fetal and newborn TEC were disrupted in the conditional knockout (EedCKO) mutants leading to severely dysplastic adult thymi. Consistent with PRC2-mediated transcriptional silencing, the majority of differentially expressed genes (DEG) were upregulated in EedCKO TEC. Moreover, a high frequency of EedCKO DEG overlapped with DEG in TEC that ectopically expressed Tbx1. These findings demonstrate that PRC2 plays a critical role in TEC development and suggest that Tbx1 expression must be downregulated in thymus fated 3rd PP endoderm to ensure optimal PRC2 function.

Predicted miRNA-mRNA-mediated posttranscriptional control associated with differences in cervical and thoracic thymus function.

A secondary cervical thymus (CT) is present in the neck region in about 50% of human and mice. CT in mice is an independent and functional organ, which can be colonized by T lymphocyte progenitors and generate thymocytes that are selected by the T cell receptor repertoire following the positive and negative selection. However, CT and the main thoracic thymus (TT) have been shown in mice to have significant functional differences. In this study, we use transcriptional profiling to compare mRNA or miRNAs expression patterns in murine CT and TT. We used these data to perform functional enrichment of the expression signatures and reconstruction of posttranscriptional miRNA-mRNA interaction networks. For this purpose, we compared the transcriptome profiling of paired RNA samples of whole CTs, TTs and parathyroid gland (PT), which was used as an external group, from Foxn1-GFP;Pth-Cre;R26dTomato transgenic mice that differentially label CT and TT. As expected, CT and TT featured comprehensive transcriptome similarity and this suggests that these organs are subjected to correlated transcriptional control. Nevertheless, significant differences were also observed between TT and CT, characterized by 107 differentially expressed (DE) mRNAs, and in 13 DE miRNAs, that in turn established interactions. These results suggest that functional similarity between TT and CT is reflected in their transcriptional activity and that CT functional uniqueness might be under posttranscriptional control.

Thymic epithelial cell-derived signals control B progenitor formation and proliferation in the thymus by regulating Let-7 and Arid3a.

The postnatal thymus is an efficient microenvironment for T cell specification and differentiation. B cells are also present in the thymus and have been recently shown to impact T cell selection, however, the mechanisms controlling B cell development in the thymus are largely unknown. In Foxn1lacZ mutant mice, down-regulation of Foxn1 expression in thymic epithelial cells beginning 1 week after birth caused a dramatic reduction of T progenitors and an increase of B cell progenitors. This time point is coincident with the switch from fetal to adult-type hematopoietic stem cells (HSCs), which is regulated by the Lin28-Let7 system. We hypothesize that the thymic environment might regulate this process to suppress fetal-type B cell development in the thymus. In this study we show that in the Foxn1lacZ thymus, although the down-regulation of Lin28 in thymocytes was normal, up-regulation of Let-7 was impaired. The failure to up-regulate Let-7 caused a transient increase of Arid3a in B precursors, which is known to promote fetal-type B cell fate. Over-expression of Lin28a in HSCs also reduced Let-7 and promoted Arid3a expression in BM and thymic B progenitors, increasing B cell production in the thymus. The level of Let-7 in thymic B progenitors was up regulated by in vitro co-culture with IL15, Vitamin-D3, and retinoic acid, thus down-regulating Arid3a to promote B cell differentiation. All of these signals were produced in thymic epithelial cells (TECs) related to Let-7 expression in thymic B progenitors, and down-regulated in Foxn1lacZ mutants. Our data show that signals provided by TEC control thymic B cell development by up-regulating Let-7, suppressing Arid3a expression in intrathymic progenitor B cells to limit their proliferation during the neonatal to adult transition.

Thymic B cell development is controlled by the B potential of progenitors via both hematopoietic-intrinsic and thymic microenvironment-intrinsic regulatory mechanisms.

BACKGROUND: Hematopoietic stem cells (HSCs) derived from birth through adult possess differing differentiation potential for T or B cell fate in the thymus; neonatal bone marrow (BM) cells also have a higher potential for B cell production in BM compared to adult HSCs. We hypothesized that this hematopoietic-intrinsic B potential might also regulate B cell development in the thymus during ontogeny. METHODS: Foxn1lacZ mutant mice are a model in which down regulation of a thymic epithelial cell (TEC) specific transcription factor beginning one week postnatal causes a dramatic reduction of thymocytes production. In this study, we found that while T cells were decreased, the frequency of thymic B cells was greatly increased in these mutants in the perinatal period. We used this model to characterize the mechanisms in the thymus controlling B cell development. RESULTS: Foxn1lacZ mutants, T cell committed intrathymic progenitors (DN1a,b) were progressively reduced beginning one week after birth, while thymic B cells peaked at 3-4 weeks with pre-B-II progenitor phenotype, and originated in the thymus. Heterochronic chimeras showed that the capacity for thymic B cell production was due to a combination of higher B potential of neonatal HSCs, combined with a thymic microenvironment deficiency including reduction of DL4 and increase of IL-7 that promoted B cell fate. CONCLUSION: Our findings indicate that the capacity and time course for thymic B-cell production are primarily controlled by the hematopoietic-intrinsic potential for B cells themselves during ontogeny, but that signals from TECs microenvironment also influence the frequency and differentiation potential of B cell development in the thymus.

Production of BMP4 by endothelial cells is crucial for endogenous thymic regeneration.

The thymus is not only extremely sensitive to damage but also has a remarkable ability to repair itself. However, the mechanisms underlying this endogenous regeneration remain poorly understood, and this capacity diminishes considerably with age. We show that thymic endothelial cells (ECs) comprise a critical pathway of regeneration via their production of bone morphogenetic protein 4 (BMP4) ECs increased their production of BMP4 after thymic damage, and abrogating BMP4 signaling or production by either pharmacologic or genetic inhibition impaired thymic repair. EC-derived BMP4 acted on thymic epithelial cells (TECs) to increase their expression of Foxn1, a key transcription factor involved in TEC development, maintenance, and regeneration, and its downstream targets such as Dll4, a key mediator of thymocyte development and regeneration. These studies demonstrate the importance of the BMP4 pathway in endogenous tissue regeneration and offer a potential clinical approach to enhance T cell immunity.

Sublethal Total Body Irradiation Causes Long-Term Deficits in Thymus Function by Reducing Lymphoid Progenitors.

Total body irradiation (TBI) damages hematopoietic cells in the bone marrow and thymus; however, the long-term effects of irradiation with aging remain unclear. In this study, we found that the impact of radiation on thymopoiesis in mice varied by sex and dose but, overall, thymopoiesis remained suppressed for ≥12 mo after a single exposure. Male and female mice showed a long-term dose-dependent reduction in thymic cKit+ lymphoid progenitors that was maintained throughout life. Damage to hematopoietic stem cells (HSCs) in the bone marrow was dose dependent, with as little as 0.5 Gy causing a significant long-term reduction. In addition, the potential for T lineage commitment was radiation sensitive with aging. Overall, the impact of irradiation on the hematopoietic lineage was more severe in females. In contrast, the rate of decline in thymic epithelial cell numbers with age was radiation-sensitive only in males, and other characteristics including Ccl25 transcription were unaffected. Taken together, these data suggest that long-term suppression of thymopoiesis after sublethal irradiation was primarily due to fewer progenitors in the BM combined with reduced potential for T lineage commitment. A single irradiation dose also caused synchronization of thymopoiesis, with a periodic thymocyte differentiation profile persisting for at least 12 mo postirradiation. This study suggests that the number and capability of HSCs for T cell production can be dramatically and permanently damaged after a single relatively low TBI dose, accelerating aging-associated thymic involution. Our findings may impact evaluation and therapeutic intervention of human TBI events.

MiCASA is a new method for quantifying cellular organization.

While many tools exist for identifying and quantifying individual cell types, few methods are available to assess the relationships between cell types in organs and tissues and how these relationships change during aging or disease states. We present a quantitative method for evaluating cellular organization, using the mouse thymus as a test organ. The thymus is the primary lymphoid organ responsible for generating T cells in vertebrates, and its proper structure and organization is essential for optimal function. Our method, Multitaper Circularly Averaged Spectral Analysis (MiCASA), identifies differences in the tissue-level organization with high sensitivity, including defining a novel type of phenotype by measuring variability as a specific parameter. MiCASA provides a novel and easily implemented quantitative tool for assessing cellular organization.

Late Effects of Exposure to Ionizing Radiation and Age on Human Thymus Morphology and Function.

The thymus is essential for proper development and maintenance of a T-cell repertoire that can respond to newly encountered antigens, but its function can be adversely affected by internal factors such as pregnancy and normal aging or by external stimuli such as stress, infection, chemotherapy and ionizing radiation. We have utilized a unique archive of thymus tissues, obtained from 165 individuals, exposed to the 1945 atomic bomb blast in Hiroshima, to study the long-term effects of receiving up to ∼3 Gy dose of ionizing radiation on human thymus function. A detailed morphometric analysis of thymus activity and architecture in these subjects at the time of their natural deaths was performed using bright-field immunohistochemistry and dual-color immunofluorescence and compared to a separate cohort of nonexposed control subjects. After adjusting for age-related effects, increased hallmarks of thymic involution were observed histologically in individuals exposed to either low (5-200 mGy) or moderate-to-high (>200 mGy) doses of ionizing radiation compared to unirradiated individuals (<5 mGy). Sex-related differences were seen when the analysis was restricted to individuals under 60 years of attained age at sample collection, but were not observed when comparing across the entire age range. This indicates that while females undergo slower involution than males, they ultimately attain similar phenotypes. These findings suggest that even low-dose-radiation exposure can accelerate thymic aging, with decreased thymopoiesis relative to nonexposed controls evident years after exposure. These data were used to develop a model that can predict thymic function during normal aging or in individuals therapeutically or accidentally exposed to radiation.

Tissue-specific roles for sonic hedgehog signaling in establishing thymus and parathyroid organ fate.

The thymus and parathyroids develop from third pharyngeal pouch (3rd pp) endoderm. Our previous studies show that Shh null mice have smaller, aparathyroid primordia in which thymus fate specification extends into the pharynx. SHH signaling is active in both dorsal pouch endoderm and neighboring neural crest (NC) mesenchyme. It is unclear which target tissue of SHH signaling is required for the patterning defects in Shh mutants. Here, we used a genetic approach to ectopically activate or delete the SHH signal transducer Smo in either pp endoderm or NC mesenchyme. Although no manipulation recapitulated the Shh null phenotype, manipulation of SHH signaling in either the endoderm or NC mesenchyme had direct and indirect effects on both cell types during fate specification and organogenesis. SHH pathway activation throughout pouch endoderm activated ectopic Tbx1 expression and partially suppressed the thymus-specific transcription factor Foxn1, identifying Tbx1 as a key target of SHH signaling in the 3rd pp. However, ectopic SHH signaling was insufficient to expand the GCM2-positive parathyroid domain, indicating that multiple inputs, some of which might be independent of SHH signaling, are required for parathyroid fate specification. These data support a model in which SHH signaling plays both positive and negative roles in patterning and organogenesis of the thymus and parathyroids.

Temporal and spatial requirements for Hoxa3 in mouse embryonic development.

Hoxa3(null) mice have severe defects in the development of pharyngeal organs including athymia, aparathyroidism, thyroid hypoplasia, and ultimobranchial body persistence, in addition to defects of the throat cartilages and cranial nerves. Some of the structures altered in the Hoxa3(null) mutant embryos are anterior to the described Hoxa3 gene expression boundary: the thyroid, soft palate, and lesser hyoid horn. All of these structures develop over time and through the interactions of multiple cell types. To investigate the specific cellular targets for HOXA3 function in these structures across developmental time, we performed a comprehensive analysis of the temporal and tissue-specific requirements for Hoxa3, including a lineage analysis using Hoxa3(Cre). The combination of these approaches showed that HOXA3 functions in both a cell autonomous and non-cell autonomous manner during development of the 3rd and 4th arch derivatives, and functions in a neural crest cell (NCC)-specific, non-cell autonomous manner for structures that were Hoxa3-negative by lineage tracing. Our data indicate that HOXA3 is required for tissue organization and organ differentiation in endodermal cells (in the tracheal epithelium, thymus, and parathyroid), and contributes to organ migration and morphogenesis in NCCs. These data provide a detailed picture of where and when HOXA3 acts to promote the development of the diverse structures that are altered in the Hoxa3(null) mutant. Data presented here, combined with our previous studies, indicate that the regionally restricted defects in Hoxa3 mutants do not reflect a role in positional identity (establishment of cell or tissue fate), but instead indicate a wider variety of functions including controlling distinct genetic programs for differentiation and morphogenesis in different cell types during development.

An interlaboratory comparison of dosimetry for a multi-institutional radiobiological research project: Observations, problems, solutions and lessons learned.

PURPOSE: An interlaboratory comparison of radiation dosimetry was conducted to determine the accuracy of doses being used experimentally for animal exposures within a large multi-institutional research project. The background and approach to this effort are described and discussed in terms of basic findings, problems and solutions. METHODS: Dosimetry tests were carried out utilizing optically stimulated luminescence (OSL) dosimeters embedded midline into mouse carcasses and thermal luminescence dosimeters (TLD) embedded midline into acrylic phantoms. RESULTS: The effort demonstrated that the majority (4/7) of the laboratories was able to deliver sufficiently accurate exposures having maximum dosing errors of ≤5%. Comparable rates of 'dosimetric compliance' were noted between OSL- and TLD-based tests. Data analysis showed a highly linear relationship between 'measured' and 'target' doses, with errors falling largely between 0 and 20%. Outliers were most notable for OSL-based tests, while multiple tests by 'non-compliant' laboratories using orthovoltage X-rays contributed heavily to the wide variation in dosing errors. CONCLUSIONS: For the dosimetrically non-compliant laboratories, the relatively high rates of dosing errors were problematic, potentially compromising the quality of ongoing radiobiological research. This dosimetry effort proved to be instructive in establishing rigorous reviews of basic dosimetry protocols ensuring that dosing errors were minimized.

Optimization of Single- and Dual-Color Immunofluorescence Protocols for Formalin-Fixed, Paraffin-Embedded Archival Tissues.

Performance of immunofluorescence staining on archival formalin-fixed paraffin-embedded human tissues is generally not considered to be feasible, primarily due to problems with tissue quality and autofluorescence. We report the development and application of procedures that allowed for the study of a unique archive of thymus tissues derived from autopsies of individuals exposed to atomic bomb radiation in Hiroshima, Japan in 1945. Multiple independent treatments were used to minimize autofluorescence and maximize fluorescent antibody signals. Treatments with NH3/EtOH and Sudan Black B were particularly useful in decreasing autofluorescent moieties present in the tissue. Deconvolution microscopy was used to further enhance the signal-to-noise ratios. Together, these techniques provide high-quality single- and dual-color fluorescent images with low background and high contrast from paraffin blocks of thymus tissue that were prepared up to 60 years ago. The resulting high-quality images allow the application of a variety of image analyses to thymus tissues that previously were not accessible. Whereas the procedures presented remain to be tested for other tissue types and archival conditions, the approach described may facilitate greater utilization of older paraffin block archives for modern immunofluorescence studies.