RESUMO
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.
Assuntos
Padronização Corporal/genética , Proteínas Hedgehog/fisiologia , Organogênese/genética , Glândulas Paratireoides/embriologia , Timo/embriologia , Animais , Células Cultivadas , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Especificidade de Órgãos/genética , Glândulas Paratireoides/metabolismo , Transdução de Sinais/genética , Timo/metabolismoRESUMO
Medullary thymic epithelial cells (mTECs) are essential for establishing central tolerance by expressing a diverse array of self-peptides that delete autoreactive thymocytes and/or divert thymocytes into the regulatory T cell lineage. Activation of the NFκB signaling pathway in mTEC precursors is indispensable for mTEC maturation and proliferation resulting in proper medullary region formation. Here we show that the Stat3-mediated signaling pathway also plays a key role in mTEC development and homeostasis. Expression of a constitutively active Stat3 transgene targeted to the mTEC compartment increases mTEC cellularity and bypasses the requirement for signals from positively selected thymocytes to drive medullary region formation. Conversely, conditional deletion of Stat3 disrupts medullary region architecture and reduces the number of mTECs. Stat3 signaling does not affect mTEC proliferation, but rather promotes survival of immature MHCIIloCD80lo mTEC precursors. In contrast to striking alterations in the mTEC compartment, neither enforced expression nor deletion of Stat3 affects cTEC cellularity or organization. These results demonstrate that in addition to the NFkB pathway, Stat3-mediated signals play an essential role in regulating mTEC cellularity and medullary region homeostasis.
Assuntos
Diferenciação Celular/genética , Células Epiteliais/metabolismo , NF-kappa B/genética , Fator de Transcrição STAT3/biossíntese , Animais , Antígenos CD40/genética , Desenvolvimento Embrionário/genética , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento , Ativação Linfocitária , Camundongos , Fator de Transcrição STAT3/genética , Transdução de Sinais , Linfócitos T/metabolismo , Timócitos/metabolismo , Timo/crescimento & desenvolvimento , Timo/metabolismo , TransgenesRESUMO
The thymus and parathyroid glands arise from a shared endodermal primordium in the third pharyngeal pouch (3rd pp). Thymus fate is specified in the ventral 3rd pp between E9.5 and E11, whereas parathyroid fate is specified in the dorsal domain. The molecular mechanisms that specify fate and regulate thymus and parathyroid development are not fully delineated. Previous reports suggested that Tbx1 is required for thymus organogenesis because loss of Tbx1 in individuals with DiGeorge syndrome and in experimental Tbx1 deletion mutants is associated with thymus aplasia or hypoplasia. However, the thymus phenotype is likely to be secondary to defects in pharyngeal pouch formation. Furthermore, the absence of Tbx1 expression in the thymus-fated domain of the wild-type 3rd pp suggested that Tbx1 is instead a negative regulator of thymus organogenesis. To test this hypothesis, we generated a novel mouse strain in which expression of a conditional Tbx1 allele was ectopically activated in the thymus-fated domain of the 3rd pp. Ectopic Tbx1 expression severely repressed expression of Foxn1, a transcription factor that marks the thymus-fated domain and is required for differentiation and proliferation of thymic epithelial cell (TEC) progenitors. By contrast, ectopic Tbx1 did not alter the expression pattern of Gcm2, a transcription factor restricted to the parathyroid-fated domain and required for parathyroid development. Ectopic Tbx1 expression impaired TEC proliferation and arrested TEC differentiation at an early progenitor stage. The results support the hypothesis that Tbx1 negatively regulates TEC growth and differentiation, and that extinction of Tbx1 expression in 3rd pp endoderm is a prerequisite for thymus organogenesis.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Organogênese , Proteínas com Domínio T/fisiologia , Timo/embriologia , Alelos , Animais , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Endoderma , Feminino , Fatores de Transcrição Forkhead/fisiologia , Deleção de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Proteínas Nucleares/fisiologia , Células-Tronco/citologia , Proteínas com Domínio T/genética , Fatores de Tempo , Fatores de Transcrição/fisiologiaRESUMO
A critical process during thymic development of the T cell repertoire is the induction of self-tolerance. Tolerance in developing T cells is highly dependent on medullary thymic epithelial cells (mTEC), and mTEC development in turn requires signals from mature single-positive thymocytes, a bidirectional relationship termed thymus crosstalk. We show that CD28-CD80/86 and CD40-CD40L costimulatory interactions, which mediate negative selection and self-tolerance, upregulate expression of LTα, LTß, and receptor activator for NF-κB in the thymus and are necessary for medullary development. Combined absence of CD28-CD80/86 and CD40-CD40L results in profound deficiency in mTEC development comparable to that observed in the absence of single-positive thymocytes. This requirement for costimulatory signaling is maintained even in a TCR transgenic model of high-affinity TCR-ligand interactions. CD4 thymocytes maturing in the altered thymic epithelial environment of CD40/CD80/86 knockout mice are highly autoreactive in vitro and are lethal in congenic adoptive transfer in vivo, demonstrating a critical role for these costimulatory pathways in self-tolerance as well as thymic epithelial development. These findings demonstrate that cooperativity between CD28-CD80/86 and CD40-CD40L pathways is required for normal medullary epithelium and for maintenance of self-tolerance in thymocyte development.
Assuntos
Antígeno B7-1/imunologia , Antígeno B7-2/imunologia , Antígenos CD28/imunologia , Antígenos CD40/imunologia , Ligante de CD40/imunologia , Epitélio/imunologia , Tolerância a Antígenos Próprios/imunologia , Timócitos/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Células Epiteliais/imunologia , Células Matadoras Naturais/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , NF-kappa B/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia , Regulação para Cima/imunologiaRESUMO
Coactivator-associated arginine methyltransferase 1 (CARM1) is a protein arginine methyltransferase that methylates histones and transcriptional regulators. We previously reported that the absence of CARM1 partially blocks thymocyte differentiation at embryonic day 18.5 (E18.5). In this study, we find that reduced thymopoiesis in Carm1(-/-) mice is due to a defect in the fetal hematopoietic compartment rather than in the thymic stroma. To determine the cellular basis for impaired thymopoiesis, we examined the number and function of fetal liver (FL) and bone marrow cells. Despite markedly reduced cellularity of hematopoietic progenitors in E18.5 bone marrow, the number of long-term hematopoietic stem cells and downstream subsets was not reduced in Carm1(-/-) E14.5 or E18.5 FL. Nevertheless, competitive reconstitution assays revealed a deficit in the ability of Carm1(-/-) FL cells to contribute to hematopoiesis. Furthermore, impaired differentiation of Carm1(-/-) FL cells in a CARM1-sufficient host showed that CARM1 is required cell autonomously in hematopoietic cells. Coculture of Carm1(-/-) FL cells on OP9-DL1 monolayers showed that CARM1 is required for survival of hematopoietic progenitors under conditions that promote differentiation. Taken together, this report demonstrates that CARM1 is a key epigenetic regulator of hematopoiesis that affects multiple lineages at various stages of differentiation.
Assuntos
Feto/metabolismo , Hematopoese/genética , Proteína-Arginina N-Metiltransferases/genética , Timócitos/metabolismo , Animais , Medula Óssea/metabolismo , Medula Óssea/patologia , Diferenciação Celular/genética , Sobrevivência Celular/genética , Feto/embriologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Camundongos Knockout , Proteína-Arginina N-Metiltransferases/deficiência , Proteína-Arginina N-Metiltransferases/metabolismo , Receptores Notch/metabolismo , Células Estromais/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismo , Timócitos/citologia , Timo/embriologia , Timo/metabolismoRESUMO
Transposable elements (TEs) are repetitive sequences representing ~45% of the human and mouse genomes and are highly expressed by medullary thymic epithelial cells (mTECs). In this study, we investigated the role of TEs on T-cell development in the thymus. We performed multiomic analyses of TEs in human and mouse thymic cells to elucidate their role in T-cell development. We report that TE expression in the human thymus is high and shows extensive age- and cell lineage-related variations. TE expression correlates with multiple transcription factors in all cell types of the human thymus. Two cell types express particularly broad TE repertoires: mTECs and plasmacytoid dendritic cells (pDCs). In mTECs, transcriptomic data suggest that TEs interact with transcription factors essential for mTEC development and function (e.g., PAX1 and REL), and immunopeptidomic data showed that TEs generate MHC-I-associated peptides implicated in thymocyte education. Notably, AIRE, FEZF2, and CHD4 regulate small yet non-redundant sets of TEs in murine mTECs. Human thymic pDCs homogenously express large numbers of TEs that likely form dsRNA, which can activate innate immune receptors, potentially explaining why thymic pDCs constitutively secrete IFN É/ß. This study highlights the diversity of interactions between TEs and the adaptive immune system. TEs are genetic parasites, and the two thymic cell types most affected by TEs (mTEcs and pDCs) are essential to establishing central T-cell tolerance. Therefore, we propose that orchestrating TE expression in thymic cells is critical to prevent autoimmunity in vertebrates.
Assuntos
Proteína AIRE , Elementos de DNA Transponíveis , Camundongos , Humanos , Animais , Timo/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Timócitos/metabolismo , Células Epiteliais/metabolismo , Diferenciação Celular/genética , Camundongos Endogâmicos C57BLRESUMO
Age-related thymus involution results in decreased T-cell production, contributing to increased susceptibility to pathogens and reduced vaccine responsiveness. Elucidating mechanisms underlying thymus involution will inform strategies to restore thymopoiesis with age. The thymus is colonized by circulating bone marrow (BM)-derived thymus seeding progenitors (TSPs) that differentiate into early T-cell progenitors (ETPs). We find that ETP cellularity declines as early as 3 months (3MO) of age in mice. This initial ETP reduction could reflect changes in thymic stromal niches and/or pre-thymic progenitors. Using a multicongenic progenitor transfer approach, we demonstrate that the number of functional TSP/ETP niches does not diminish with age. Instead, the number of pre-thymic lymphoid progenitors in the BM and blood is substantially reduced by 3MO, although their intrinsic ability to seed and differentiate in the thymus is maintained. Additionally, Notch signaling in BM lymphoid progenitors and in ETPs diminishes by 3MO, suggesting reduced niche quality in the BM and thymus contribute to the early decline in ETPs. Together, these findings indicate that diminished BM lymphopoiesis and thymic stromal support contribute to an initial reduction in ETPs in young adulthood, setting the stage for progressive age-associated thymus involution.
Assuntos
Medula Óssea , Linfócitos T , Camundongos , Animais , Timo , Transdução de Sinais , Camundongos Endogâmicos C57BL , Diferenciação CelularRESUMO
Cancer-related alterations of the p53 tetramerization domain (TD) abrogate wild-type (WT) p53 function. They result in a protein that preferentially forms monomers or dimers, which are also normal p53 states under basal cellular conditions. However, their physiologic relevance is not well understood. We have established in vivo models for monomeric and dimeric p53, which model Li-Fraumeni syndrome patients with germline p53 TD alterations. p53 monomers are inactive forms of the protein. Unexpectedly, p53 dimers conferred some tumor suppression that is not mediated by canonical WT p53 activities. p53 dimers upregulate the PPAR pathway. These activities are associated with lower prevalence of thymic lymphomas and increased CD8+ T-cell differentiation. Lymphomas derived from dimeric p53 mice show cooperating alterations in the PPAR pathway, further implicating a role for these activities in tumor suppression. Our data reveal novel functions for p53 dimers and support the exploration of PPAR agonists as therapies. SIGNIFICANCE: New mouse models with TP53R342P (monomer) or TP53A347D (dimer) mutations mimic Li-Fraumeni syndrome. Although p53 monomers lack function, p53 dimers conferred noncanonical tumor-suppressive activities. We describe novel activities for p53 dimers facilitated by PPARs and propose these are "basal" p53 activities. See related commentary by Stieg et al., p. 1046. See related article by Choe et al., p. 1250. This article is highlighted in the In This Issue feature, p. 1027.
Assuntos
Síndrome de Li-Fraumeni , Animais , Camundongos , Síndrome de Li-Fraumeni/genética , Síndrome de Li-Fraumeni/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ativação Transcricional , Receptores Ativados por Proliferador de Peroxissomo/genética , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Morte CelularRESUMO
One of the earliest hallmarks of immune aging is thymus involution, which not only reduces the number of newly generated and exported T cells, but also alters the composition and organization of the thymus microenvironment. Thymic T-cell export continues into adulthood, yet the impact of thymus involution on the quality of newly generated T-cell clones is not well established. Notably, the number and proportion of medullary thymic epithelial cells (mTECs) and expression of tissue-restricted antigens (TRAs) decline with age, suggesting the involuting thymus may not promote efficient central tolerance. Here, we demonstrate that the middle-aged thymic environment does not support rapid motility of medullary thymocytes, potentially diminishing their ability to scan antigen presenting cells (APCs) that display the diverse self-antigens that induce central tolerance. Consistent with this possibility, thymic slice assays reveal that the middle-aged thymic environment does not support efficient negative selection or regulatory T-cell (Treg) induction of thymocytes responsive to either TRAs or ubiquitous self-antigens. This decline in central tolerance is not universal, but instead impacts lower-avidity self-antigens that are either less abundant or bind to TCRs with moderate affinities. Additionally, the decline in thymic tolerance by middle age is accompanied by both a reduction in mTECs and hematopoietic APC subsets that cooperate to drive central tolerance. Thus, age-associated changes in the thymic environment result in impaired central tolerance against moderate-avidity self-antigens, potentially resulting in export of increasingly autoreactive naive T cells, with a deficit of Treg counterparts by middle age.
Assuntos
Células Apresentadoras de Antígenos , Tolerância Central , Células Apresentadoras de Antígenos/metabolismo , Autoantígenos/metabolismo , Células Epiteliais/metabolismo , Linfócitos T Reguladores , Timócitos , TimoRESUMO
Thymic epithelial cells (TECs) and hematopoietic antigen presenting cells (HAPCs) in the thymus microenvironment provide essential signals to self-reactive thymocytes that induce either negative selection or generation of regulatory T cells (Treg), both of which are required to establish and maintain central tolerance throughout life. HAPCs and TECs are comprised of multiple subsets that play distinct and overlapping roles in central tolerance. Changes that occur in the composition and function of TEC and HAPC subsets across the lifespan have potential consequences for central tolerance. In keeping with this possibility, there are age-associated changes in the cellular composition and function of T cells and Treg. This review summarizes changes in T cell and Treg function during the perinatal to adult transition and in the course of normal aging, and relates these changes to age-associated alterations in thymic HAPC and TEC subsets.
Assuntos
Envelhecimento/imunologia , Tolerância Central , Timo/imunologia , Fatores Etários , Células Apresentadoras de Antígenos/imunologia , Células Epiteliais/imunologia , Humanos , Linfócitos T Reguladores/imunologiaRESUMO
Embryos that are homozygous for Splotch, a null allele of Pax3, have a severe neural crest cell (NCC) deficiency that generates a complex phenotype including spina bifida, exencephaly and cardiac outflow tract abnormalities. Contrary to the widely held perception that thymus aplasia or hypoplasia is a characteristic feature of Pax3(Sp/Sp) embryos, we find that thymic rudiments are larger and parathyroid rudiments are smaller in E11.5-12.5 Pax3(Sp/Sp) compared to Pax3(+/+) embryos. The thymus originates from bilateral third pharyngeal pouch primordia containing endodermal progenitors of both thymus and parathyroid glands. Analyses of Foxn1 and Gcm2 expression revealed a dorsal shift in the border between parathyroid- and thymus-fated domains at E11.5, with no change in the overall cellularity or volume of each shared primordium. The border shift increases the allocation of third pouch progenitors to the thymus domain and correspondingly decreases allocation to the parathyroid domain. Initial patterning in the E10.5 pouch was normal suggesting that the observed change in the location of the organ domain interface arises during border refinement between E10.5 and E11.5. Given the well-characterized NCC defects in Splotch mutants, these findings implicate NCCs in regulating patterning of third pouch endoderm into thymus- versus parathyroid-specified domains, and suggest that organ size is determined in part by the number of progenitor cells specified to a given fate.
Assuntos
Fatores de Transcrição Box Pareados/genética , Glândulas Paratireoides/embriologia , Timo/embriologia , Animais , Padronização Corporal , Embrião de Mamíferos , Desenvolvimento Embrionário , Indução Embrionária , Fatores de Transcrição Forkhead/genética , Camundongos , Camundongos Mutantes , Proteínas Nucleares/genética , Fator de Transcrição PAX3 , Glândulas Paratireoides/citologia , Timo/citologia , Fatores de Transcrição/genéticaRESUMO
IL-7 is a cytokine that is required for T-cell development and homeostasis as well as for lymph node organogenesis. Despite the importance of IL-7 in the immune system and its potential therapeutic relevance, questions remain regarding the sites of IL-7 synthesis, specific cell types involved and molecular mechanisms regulating IL-7 expression. To address these issues, we generated two bacterial artificial chromosome (BAC) transgenic mouse lines in which IL-7 regulatory elements drive expression of either Cre recombinase or a human CD25 (hCD25) cell surface reporter molecule. Expression of the IL-7.hCD25 BAC transgene, detected by reactivity with anti-hCD25 antibody, mimicked endogenous IL-7 expression. Fetal and adult tissues from crosses between IL-7.Cre transgenic mice and Rosa26R or R26-EYFP reporters demonstrated X-gal or YFP staining in tissues known to express endogenous IL-7 at some stage during development. These transgenic lines provide novel genetic tools to identify IL-7 producing cells in various tissues and to manipulate gene expression selectively in IL-7 expressing cells.
Assuntos
Integrases/metabolismo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Interleucina-7/metabolismo , Animais , Animais Recém-Nascidos , Cromossomos Artificiais Bacterianos/genética , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Imuno-Histoquímica , Integrases/genética , Subunidade alfa de Receptor de Interleucina-2/genética , Interleucina-7/genética , Masculino , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Timo/citologia , Timo/embriologia , Timo/metabolismoRESUMO
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.
Assuntos
Células Epiteliais/citologia , Complexo Repressor Polycomb 2/metabolismo , Timo/citologia , Animais , Diferenciação Celular , Linhagem da Célula , Deleção de Genes , Regulação da Expressão Gênica , Histonas/metabolismo , Metilação , Camundongos , Complexo Repressor Polycomb 2/deficiência , Complexo Repressor Polycomb 2/genética , Proteínas com Domínio T/genéticaRESUMO
Humans with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), a T cell-driven autoimmune disease caused by impaired central tolerance, are susceptible to chronic fungal infection and esophageal squamous cell carcinoma (ESCC). However, the relationship between autoreactive T cells and chronic fungal infection in ESCC development remains unclear. We find that kinase-dead Ikkα knockin mice develop APECED-like phenotypes, including impaired central tolerance, autoreactive T cells, chronic fungal infection, and ESCCs expressing specific human ESCC markers. Using this model, we investigated the link between ESCC and fungal infection. Autoreactive CD4 T cells permit fungal infection and incite tissue injury and inflammation. Antifungal treatment or autoreactive CD4 T cell depletion rescues, whereas oral fungal administration promotes, ESCC development. Inhibition of inflammation or epidermal growth factor receptor (EGFR) activity decreases fungal burden. Fungal infection is highly associated with ESCCs in non-autoimmune human patients. Therefore, autoreactive T cells and chronic fungal infection, fostered by inflammation and epithelial injury, promote ESCC development.
Assuntos
Carcinoma de Células Escamosas/etiologia , Carcinoma de Células Escamosas/patologia , Neoplasias Esofágicas/etiologia , Neoplasias Esofágicas/patologia , Poliendocrinopatias Autoimunes/complicações , Animais , Autoimunidade , Linfócitos T CD4-Positivos/imunologia , Candidíase/complicações , Carcinogênese , Modelos Animais de Doenças , Receptores ErbB/metabolismo , CamundongosRESUMO
Immunofluorescence analysis of thymic tissue sections is an indispensable technique for visualizing spatial relationships among thymocyte and stromal cell subsets. The thymus is organized into distinct microenvironmental zones in which particular thymic epithelial cell (TEC) subsets support specific stages of thymocyte maturation. Conversely, thymocytes and lymphoid tissue inducer cells support functional maturation of TECs. The composition and organization of TECs change during ontogeny to generate a maximally functional organ in the young adult. Deterioration of thymic architecture and stromal organization occurs with age as the thymus undergoes involution. Such changes can be monitored by immunofluorescent staining of thymic sections obtained at different ages throughout the life-span. Here we describe methods to generate frozen or paraffin-embedded thymic tissue sections for multicolor immunofluorescence staining using antibodies to surface and/or cytoplasmic antigens.
Assuntos
Imunofluorescência , Células Estromais/metabolismo , Subpopulações de Linfócitos T/metabolismo , Timócitos/metabolismo , Timo/citologia , Animais , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Imuno-Histoquímica , Camundongos , Células Estromais/citologia , Subpopulações de Linfócitos T/citologia , Timócitos/citologiaRESUMO
The mouse Polycomb-group gene, embryonic ectoderm development (eed), appears to regulate cellular growth and differentiation in a developmental and tissue specific manner. During embryogenesis, eed regulates axial patterning, whereas in the adult eed represses proliferation of myeloid and B cell precursors. The present report demonstrates two novel functional activities of eed: alteration of thymocyte maturation and suppression of thymic lymphoma development. Mice that inherit the viable hypomorphic 17Rn5(1989SB) eed allele sustain a partial developmental block at or before the CD4(-)CD8(-)CD44(-)CD25(+) stage of thymocyte differentiation. Furthermore, mice that are homozygous or heterozygous for the hypomorphic eed allele have an increased incidence and decreased latency of N-methyl-N-nitrosourea-induced thymic lymphoma compared to wild-type littermates. These findings support the notion that Polycomb-group genes exert pleiotropic effects dictated by developmental stage and cellular context.
Assuntos
Carcinógenos/toxicidade , Linfoma de Células T/genética , Proteínas Repressoras/metabolismo , Linfócitos T/citologia , Animais , Diferenciação Celular/genética , Linfoma de Células T/induzido quimicamente , Linfoma de Células T/prevenção & controle , Metilnitrosoureia/toxicidade , Camundongos , Camundongos Mutantes , Mutagênese , Complexo Repressor Polycomb 2 , Proteínas Repressoras/genéticaRESUMO
The thymus is composed of multiple stromal elements comprising specialized stromal microenvironments responsible for the development of self-tolerant and self-restricted T cells. Here, we investigated the ontogeny and maturation of the thymic vasculature. We show that endothelial cells initially enter the thymus at E13.5, with PDGFR-ß(+) mesenchymal cells following at E14.5. Using an allelic series of the thymic epithelial cell (TEC) specific transcription factor Foxn1, we showed that these events are delayed by 1-2 days in Foxn1 (Δ/Δ) mice, and this phenotype was exacerbated with reduced Foxn1 dosage. At subsequent stages there were fewer capillaries, leaky blood vessels, disrupted endothelium - perivascular cell interactions, endothelial cell vacuolization, and an overall failure of vascular organization. The expression of both VEGF-A and PDGF-B, which are both primarily expressed in vasculature-associated mesenchyme or endothelium in the thymus, were reduced at E13.5 and E15.5 in Foxn1 (Δ/Δ) mice compared with controls. These data suggest that Foxn1 is required in TECs both to recruit endothelial cells and for endothelial cells to communicate with thymic mesenchyme, and for the differentiation of vascular-associated mesenchymal cells. These data show that Foxn1 function in TECs is required for normal thymus size and to generate the cellular and molecular environment needed for normal thymic vascularization. These data further demonstrate a novel TEC-mesenchyme-endothelial interaction required for proper fetal thymus organogenesis.
Assuntos
Vasos Sanguíneos/citologia , Comunicação Celular , Células Epiteliais/citologia , Timo/irrigação sanguínea , Timo/citologia , Alelos , Animais , Vasos Sanguíneos/fisiologia , Movimento Celular , Colágeno Tipo IV/metabolismo , Endotélio/irrigação sanguínea , Endotélio/citologia , Endotélio/embriologia , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Mesoderma/citologia , Camundongos , Neovascularização Fisiológica , Crista Neural/citologia , Fluxo Sanguíneo Regional , Células Estromais/citologia , Timo/embriologiaRESUMO
Thymic involution during aging is a major cause of decreased production of T cells and reduced immunity. Here we show that inactivation of Rb family genes in young mice prevents thymic involution and results in an enlarged thymus competent for increased production of naive T cells. This phenotype originates from the expansion of functional thymic epithelial cells (TECs). In RB family mutant TECs, increased activity of E2F transcription factors drives increased expression of Foxn1, a central regulator of the thymic epithelium. Increased Foxn1 expression is required for the thymic expansion observed in Rb family mutant mice. Thus, the RB family promotes thymic involution and controls T cell production via a bone marrow-independent mechanism, identifying a novel pathway to target to increase thymic function in patients.
Assuntos
Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Inativação Gênica , Genes do Retinoblastoma , Linfócitos T/fisiologia , Timo/fisiologia , Animais , Fatores de Transcrição E2F/genética , Fatores de Transcrição E2F/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Epitélio/metabolismo , Epitélio/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Linfócitos T/metabolismo , Timo/metabolismoRESUMO
Medullary thymic epithelial cells (mTECs) play an essential role in establishing central tolerance due to their unique capacity to present a diverse array of tissue restricted Ags that induce clonal deletion of self-reactive thymocytes. One mTEC subset expresses keratin 5 (K5) and K14, but fails to bind Ulex europaeus agglutinin-1 (UEA-1) lectin. A distinct mTEC subset binds UEA-1 and expresses K8, but not K5 or K14. Development of both mTEC subsets requires activation of the noncanonical NF-kappaB pathway. In this study, we show that mTEC development is severely impaired and autoimmune manifestations occur in mice that are deficient in IkappaB kinase (IKK)alpha, a required intermediate in the noncanonical NF-kappaB signaling pathway. Introduction of an IKKalpha transgene driven by a K5 promoter restores the K5(+)K14(+) mTEC subset in IKKalpha(-/-) mice. Unexpectedly, the K5-IKKalpha transgene also rescues the UEA-1 binding mTEC subset even though K5 expression is not detectable in these cells. In addition, expression of the K5-IKKalpha transgene ameliorates autoimmune symptoms in IKKalpha(-/-) mice. These data suggest that 1) medulla formation and central tolerance depend on activating the alternative NF-kappaB signaling pathway selectively in K5-expressing mTECs and 2) the K5-expressing subset either contains immediate precursors of UEA-1 binding cells or indirectly induces their development.
Assuntos
Células Epiteliais/metabolismo , Quinase I-kappa B/metabolismo , Tolerância Imunológica/imunologia , Queratina-5/metabolismo , Timo/imunologia , Timo/metabolismo , Transgenes/genética , Animais , Diferenciação Celular , Células Dendríticas/metabolismo , Regulação da Expressão Gênica , Quinase I-kappa B/deficiência , Quinase I-kappa B/genética , Queratina-5/genética , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas/genética , Timo/citologia , Timo/embriologiaRESUMO
In the past 20 yr, several inbred strains have been derived from SENCAR outbred mice. These strains display different susceptibility to the induction of papillomas and progression to squamous cell carcinomas (SCC) in the skin after chemical carcinogenesis. In the present study, we showed that one of these strains SENCARB/Pt was highly susceptible to the development of N-methyl-N-nitrosourea (MNU)- and 7,12-dimethylbenz[a]anthracene (DMBA)-induced lymphomas. In contrast, the SSIN/Sprd inbred strain is completely resistant to T-cell lymphomagenesis by both carcinogens. Within 175 d after a single injection of 75 mg/kilogram body weight (kbw) of MNU, SENCARB/Pt mice exhibited a 91.6% incidence of lymphoma. In addition, during an independent tumorigenesis study with repeated doses of intragastric DMBA, SENCARB/Pt mice showed an incidence of 75% lymphoma development 300 d after the last treatment. Histopathological and flow cytometric parameters indicated that the lymphomas were of the T-cell lineage. In order to study the genetics of MNU-induced tumorigenesis, we generated F1 hybrid mice between SSIN/Sprd and SENCARB/Pt mice. Tumor incidence in MNU-injected F1 mice suggested that the high tumor incidence is a dominant trait. Loss of heterozygosity (LOH) analysis in these tumor samples revealed allelic imbalances on chromosomes 15 and 19. Given that these inbred strains are closely related, it is likely that a relatively small number of loci are responsible for the observed differences in susceptibility. Therefore, these SENCAR inbred strains constitute important new tools to study the genetic basis of resistance and susceptibility to chemically induced thymic lymphoma formation.