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.

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.

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.

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.

Transdifferentiation of parathyroid cells into cervical thymi promotes atypical T-cell development.

The thoracic thymus is the primary vertebrate organ for T-cell generation. Accessory cervical thymi have also been identified in humans and mice, and shown in mice to be independent functional organs that support T-cell development. However, their origin and functional significance remain unclear. Here we show that cervical thymi in mice have following two origins: delayed differentiation of endodermal precursors and transdifferentiation of parathyroid-fated cells. Compared with thoracic thymus, parathyroid-origin cervical thymi (pCT) express low levels of the thymic epithelial cell-specific transcription factor FOXN1. Consequently, pCT form a distinct microenvironment that supports an atypical thymocyte development pathway, generating T cells with unconventional phenotypic characteristics. Our data demonstrate a transdifferentiation origin for a subset of cervical thymi, with specific functional consequences for T-cell development.

Impaired thymic selection and abnormal antigen-specific T cell responses in Foxn1(Δ/Δ) mutant mice.

BACKGROUND: Foxn1(Δ/Δ) mutant mice have a specific defect in thymic development, characterized by a block in TEC differentiation at an intermediate progenitor stage, and blocks in thymocyte development at both the DN1 and DP cell stages, resulting in the production of abnormally functioning T cells that develop from an atypical progenitor population. In the current study, we tested the effects of these defects on thymic selection. METHODOLOGY/PRINCIPAL FINDINGS: We used Foxn1(Δ/Δ); DO11 Tg and Foxn1(Δ/Δ); OT1 Tg mice as positive selection and Foxn1(Δ/Δ); MHCII I-E mice as negative selection models. We also used an in vivo system of antigen-specific reactivity to test the function of peripheral T cells. Our data show that the capacity for positive and negative selection of both CD4 and CD8 SP thymocytes was reduced in Foxn1(Δ/Δ) mutants compared to Foxn1(+/Δ) control mice. These defects were associated with reduction of both MHC Class I and Class II expression, although the resulting peripheral T cells have a broad TCR Vß repertoire. In this deficient thymic environment, immature CD4 and CD8 SP thymocytes emigrate from the thymus into the periphery. These T cells had an incompletely activated profile under stimulation of the TCR signal in vitro, and were either hypersensitive or hyporesponsive to antigen-specific stimulation in vivo. These cell-autonomous defects were compounded by the hypocellular peripheral environment caused by low thymic output. CONCLUSIONS/SIGNIFICANCE: These data show that a primary defect in the thymic microenvironment can cause both direct defects in selection which can in turn cause indirect effects on the periphery, exacerbating functional defects in T cells.

Foxn1 is required to maintain the postnatal thymic microenvironment in a dosage-sensitive manner.

The postnatal thymus is the primary source of T cells in vertebrates, and many if not all stages of thymocyte development require interactions with thymic epithelial cells (TECs). The Foxn1 gene is a key regulator of TEC differentiation, and is required for multiple aspects of fetal TEC differentiation. Foxn1 is also expressed in the postnatal thymus, but its function after birth is unknown. We generated a Foxn1 allele with normal fetal expression and thymus development, but decreased expression in the postnatal thymus. This down-regulation causes rapid thymic compartment degeneration and reduced T-cell production. TEC subsets that express higher Foxn1 levels are most sensitive to its down-regulation, in particular MHCII(hi)UEA-1(hi) medullary TECs. The requirement for Foxn1 is extremely dosage sensitive, with small changes in Foxn1 levels having large effects on thymus phenotypes. Our results provide the first evidence that Foxn1 is required to maintain the postnatal thymus. Furthermore, the similarities of this phenotype to accelerated aging-related thymic involution support the possibility that changes in Foxn1 expression in TECs during aging contribute to the mechanism of involution.

T cell development from kit-negative progenitors in the Foxn1Delta/Delta mutant thymus.

Foxn1Delta is a hypomorphic allele of the nude gene that causes arrested thymic epithelial cell differentiation and abnormal thymic architecture lacking cortical and medullary domains. T cells develop in the Foxn1Delta/Delta adult thymus to the double- and single-positive stages, but in the apparent absence of double-negative 3 (DN3) cells; however, DN3 cells are present in the fetal thymus. To investigate the origin of this seemingly contradictory phenotype, we performed an analysis of fetal and adult DN cells in these mutants. Neither adult bone marrow-derived cells nor fetal liver cells from wild-type or Rag1-/- mice were able to differentiate to the DN2 or DN3 stage in the Foxn1Delta/Delta thymus. Our data suggest that thymopoiesis in the Foxn1Delta/Delta adult thymus proceeds from CD117- atypical progenitors, while CD117+ DN1a cells are absent or blocked in their ability to differentiate to the T lineage. Wild-type cells generated by this pathway in the postnatal thymus were exported to the periphery, demonstrating that these atypical cells contributed to the peripheral T cell pool. The Foxn1Delta/Delta adult (but not fetal) thymus also preferentially supports B cell development, specifically of the B-1 type, and this phenotype correlated with reduced Notch ligand expression in the adult stroma.

Atypical memory phenotype T cells with low homeostatic potential and impaired TCR signaling and regulatory T cell function in Foxn1Delta/Delta mutant mice.

Foxn1Delta/Delta mutants have a block in thymic epithelial cell differentiation at an intermediate progenitor stage, resulting in reduced thymocyte cellularity and blocks at the double-negative and double-positive stages. Whereas naive single-positive thymocytes were reduced >500-fold in the adult Foxn1Delta/Delta thymus, peripheral T cell numbers were reduced only 10-fold. The current data shows that Foxn1Delta/Delta peripheral T cells had increased expression of activation markers and the ability to produce IL-2 and IFN-gamma. These cells acquired this profile immediately after leaving the thymus as early as the newborn stage and maintained high steady-state proliferation in vivo but decreased proliferation in response to TCR stimulation in vitro. Single-positive thymocytes and naive T cells also had constitutively low alphabetaTCR and IL7R expression. These cells also displayed reduced ability to undergo homeostatic proliferation and increased rates of apoptosis. Although the frequency of Foxp3+CD4+CD25+ T cells was normal in Foxn1Delta/Delta mutant mice, these cells failed to have suppressor function, resulting in reduced regulatory T cell activity. Recent data from our laboratory suggest that T cells in the Foxn1Delta/Delta thymus develop from atypical progenitor cells via a noncanonical pathway. Our results suggest that the phenotype of peripheral T cells in Foxn1Delta/Delta mutant mice is the result of atypical progenitor cells developing in an abnormal thymic microenvironment with a deficient TCR and IL7 signaling system.

Specific expression of lacZ and cre recombinase in fetal thymic epithelial cells by multiplex gene targeting at the Foxn1 locus.

BACKGROUND: Thymic epithelial cells (TECs) promote thymocyte maturation and are required for the early stages of thymocyte development and for positive selection. However, investigation of the mechanisms by which TECs perform these functions has been inhibited by the lack of genetic tools. Since the Foxn1 gene is expressed in all presumptive TECs from the early stages of thymus organogenesis and broadly in the adult thymus, it is an ideal locus for driving gene expression in differentiating and mature TECs. RESULTS: We generated two knock-in alleles of Foxn1 by inserting IRES-Cre or IRES-lacZ cassettes into the 3' UTR of the Foxn1 locus. We simultaneously electroporated the two targeting vectors to generate the two independent alleles in the same experiment, demonstrating the feasibility of multiplex gene targeting at this locus. Our analysis shows that the knockin alleles drive expression of Cre or lacZ in all TECs in the fetal thymus. Furthermore, the knockin alleles express Cre or lacZ in a Foxn1-like pattern without disrupting Foxn1 function as determined by phenotype analysis of Foxn1 knockin/Foxn1 null compound heterozygotes. CONCLUSION: These data show that multiplex gene targeting into the 3' UTR of the Foxn1 locus is an efficient method to express any gene of interest in TECs from the earliest stage of thymus organogenesis. The resulting alleles will make possible new molecular and genetic studies of TEC differentiation and function. We also discuss evidence indicating that gene targeting into the 3' UTR is a technique that may be broadly applicable for the generation of genetically neutral driver strains.

Peripheral CD4 T cells rapidly accumulate at the host: parasite interface during an inflammatory Th2 memory response.

Memory peripheral Th2 immune responses to infectious pathogens are not well studied due to the lack of suitable models and the difficulty of assessing Th2 cytokine expression at sites of inflammation. We have examined the localized immune response to a nematode parasite that encysts in the small intestine. An unexpected architecture was observed on day 4 of the memory response, with granulocytes and macrophages infiltrating the cyst and CD4(+), TCR-alphabeta(+) T cells surrounding the cyst. Laser capture microdissection analysis showed a pronounced CD4-dependent Th2 cytokine pattern at the cyst region only during the memory response, demonstrating that the Th2 memory response is readily distinguished from the primary response by the rapid accumulation of Th2 effector cells at the host:parasite interface.

The role of OX40 ligand interactions in the development of the Th2 response to the gastrointestinal nematode parasite Heligmosomoides polygyrus.

In these studies, we examined the effects of OX40 ligand (OX40L) deficiency on the development of Th2 cells during the Th2 immune response to the intestinal nematode parasite Heligmosomoides polygyrus. Elevations in IL-4 production and total and Ag-specific serum IgE levels were partially inhibited during both the primary and memory immune responses to H. polygyrus in OX40L(-/-) mice. The host-protective memory response was compromised in OX40L(-/-) mice, as decreased worm expulsion and increased egg production were observed compared with H. polygyrus-inoculated OX40L(+/+) mice. To further examine the nature of the IL-4 defect during priming, adoptively transferred DO11.10 T cells were analyzed in the context of the H. polygyrus response. Although Ag-specific T cell IL-4 production was reduced in the OX40L(-/-) mice following immunization with OVA peptide plus H. polygyrus, Ag-specific T cell expansion, cell cycle progression, CXCR5 expression, and migration were comparable between OX40L(+/+) and OX40L(-/-) mice inoculated with OVA and H. polygyrus. These studies suggest an important role for OX40/OX40L interactions in specifically promoting IL-4 production, as well as associated IgE elevations, in Th2 responses to H. polygyrus. However, OX40L interactions were not required for serum IgG1 elevations, increases in germinal center formation, and Ag-specific Th2 cell expansion and migration to the B cell zone.