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Yousuke Takahama

Publications and source records attributed to Yousuke Takahama.

At least 19 recordsLinked to original sources
PubMed2026

Improved protocol for isolation and characterization of human thymic epithelial cells.

Thymic epithelial cells (TECs) play a vital role in the generation of immunocompetent and immunotolerant T cells. The understanding of TEC biology in model animals such as mice has advanced thanks to the cellular and molecular analyses of purified TEC populations, including cortical TECs (cTECs) and medullary TECs (mTECs). On the other hand, studies of human TECs have fallen behind because of the lack of a well-established method for purifying TEC populations. Here, we show that using 0.5 U/mL liberase to digest thymus tissue into single cells is quicker than using collagenase D or 0.1 U/mL liberase. Thymus digestion with 0.5 U/ml liberase preserves CD205 expressed by cTECs. RNA sequencing analysis of TEC populations isolated from 0.5 U/mL liberase-digested human thymic tissue revealed the expression of molecules known as cTEC- and mTEC-specific genes in mouse thymus in each human TEC population. Our study is expected to fuel further cellular and molecular studies of human TECs, including those related to the collapse of immune tolerance in humans.

Flow cytometry↗
PubMed2007

Keratinocyte growth factor (KGF) enhances postnatal T-cell development via enhancements in proliferation and function of thymic epithelial cells.

The systemic administration of keratinocyte growth factor (KGF) enhances T-cell lymphopoiesis in normal mice and mice that received a bone marrow transplant. KGF exerts protection to thymic stromal cells from cytoablative conditioning and graft-versus-host disease-induced injury. However, little is known regarding KGF's molecular and cellular mechanisms of action on thymic stromal cells. Here, we report that KGF induces in vivo a transient expansion of both mature and immature thymic epithelial cells (TECs) and promotes the differentiation of the latter type of cells. The increased TEC numbers return within 2 weeks to normal values and the microenvironment displays a normal architectural organization. Stromal changes initiate an expansion of immature thymocytes and permit regular T-cell development at an increased rate and for an extended period of time. KGF signaling in TECs activates both the p53 and NF-kappaB pathways and results in the transcription of several target genes necessary for TEC function and T-cell development, including bone morphogenetic protein 2 (BMP2), BMP4, Wnt5b, and Wnt10b. Signaling via the canonical BMP pathway is critical for the KGF effects. Taken together, these data provide new insights into the mechanism(s) of action of exogenous KGF on TEC function and thymopoiesis.

Animals↗
PubMed2006

The lymphocyte guard-IANs: regulation of lymphocyte survival by IAN/GIMAP family proteins.

The life-or-death decision of immune cells makes an essential contribution to immune-system development and the regulation of immune responses. A new family of cell-survival regulators expressed in lymphocytes, termed immune-associated nucleotide-binding proteins (IANs) [also known as GTPase of immunity-associated proteins (GIMAPs)], has been described. The IAN/GIMAP family consists of GTP-binding proteins that share a unique primary structure and whose expression is finely regulated by T-cell receptor signals. Recent studies have shown that IAN/GIMAP family proteins crucially regulate the survival of T cells during development, selection and homeostasis, and are possibly linked to the onset of T-lymphopenia, leukemia and autoimmunity. IAN/GIMAP family proteins might also take part in mitochondrial regulation of lymphocyte apoptosis by interacting with Bcl-2 family proteins.

Animals↗
PubMed2006

Chemokines CCL19 and CCL21 promote activation-induced cell death of antigen-responding T cells.

Secondary lymphoid organs (SLOs) provide a niche for the initiation and regulation of T-cell responses, but the mechanisms have been poorly understood. We investigated the influence of chemokines CCL19 and CCL21 constitutively expressed in SLOs on activation-induced cell death (AICD) of CD4+ T cells. When paucity of lymph node T cells (plt) mutant mice lacking expression of CCL19/CCL21 were primed with OVA/CFA, both expansion of OVA-responding CD4+ T cells in the draining lymph nodes and an in vitro recall response were prolonged as compared with responses in wild-type (WT) mice. The apoptotic cell frequency among OVA-responding CD4+ T cells was similarly low in plt/plt and WT mice during the clonal expansion phase. However, during the clonal contraction phase, the frequency never increased in plt/plt mice, whereas in WT mice it continuously increased to a peak 18 days after immunization. The presence of CCL19/CCL21 during the in vitro stimulation of CD4+ T cells with anti-CD3 plus anti-CD28 significantly enhanced in vitro AICD induction of the restimulated T cells, partially through enhancing expression of Fas ligand. Our results suggest that CCL19/CCL21 produced by stromal cells and antigen-presenting cells regulate CD4+ T-cell immune responses in SLOs by promoting AICD.

Animals↗
PubMed2006

Coordination between CCR7- and CCR9-mediated chemokine signals in prevascular fetal thymus colonization.

Thymus seeding by T-lymphoid progenitor cells is a prerequisite for T-cell development. However, molecules guiding thymus colonization and their roles before and after thymus vascularization are unclear. Here we show that mice doubly deficient for chemokine receptors CCR7 and CCR9 were defective specifically in fetal thymus colonization before, but not after, thymus vascularization. The defective prevascular fetal thymus colonization was followed by selective loss of the first wave of T-cell development generating epidermal Vgamma3(+) gammadelta T cells. Unexpectedly, CCL21, a CCR7 ligand, was expressed not by Foxn1-dependent thymic primordium but by Gcm2-dependent parathyroid primordium, whereas CCL25, a CCR9 ligand, was predominantly expressed by Foxn1-dependent thymic primordium, revealing the role of the adjacent parathyroid in guiding fetal thymus colonization. These results indicate coordination between Gcm2-dependent parathyroid and Foxn1-dependent thymic primordia in establishing CCL21/CCR7- and CCL25/CCR9-mediated chemokine guidance essential for prevascular fetal thymus colonization.

Animals↗
PubMed2006

IAN family critically regulates survival and development of T lymphocytes.

The IAN (immune-associated nucleotide-binding protein) family is a family of functionally uncharacterized GTP-binding proteins expressed in vertebrate immune cells and in plant cells during antibacterial responses. Here we show that all eight IAN family genes encoded in a single cluster of mouse genome are predominantly expressed in lymphocytes, and that the expression of IAN1, IAN4, and IAN5 is significantly elevated upon thymic selection of T lymphocytes. Gain-of-function experiments show that the premature overexpression of IAN1 kills immature thymocytes, whereas short hairpin RNA-mediated loss-of-function studies show that IAN4 supports positive selection. The knockdown of IAN5 perturbs the optimal generation of CD4/CD8 double-positive thymocytes and reduces the survival of mature T lymphocytes. We also show evidence suggesting that IAN4 and IAN5 are associated with anti-apoptotic proteins Bcl-2 and Bcl-xL, whereas IAN1 is associated with pro-apoptotic Bax. Thus, the IAN family is a novel family of T cell-receptor-responsive proteins that critically regulate thymic development and survival of T lymphocytes and that potentially exert regulatory functions through the association with Bcl-2 family proteins.

Animals↗
PubMed2006

CCR7-dependent cortex-to-medulla migration of positively selected thymocytes is essential for establishing central tolerance.

Immature CD4+CD8+ thymocytes, which are generated in the thymic cortex, are induced upon positive selection to differentiate into mature T lymphocytes and relocate to the thymic medulla. It was recently shown that a chemokine signal via CCR7 is essential for the cortex-to-medulla migration of positively selected thymocytes in the thymus. However, the role of the cortex-to-medulla migration in T cell development and selection has remained unclear. The present study shows that the developmental kinetics and the thymic export of mature thymocytes were undisturbed in adult mice lacking CCR7 or its ligands (CCR7L). The inhibition of sphingosine-1-phosphate-mediated lymphocyte egress from the thymus led to the accumulation of mature thymocytes in the cortex of CCR7- or CCR7L-deficient mice, unlike the accumulation in the medulla of normal mice, thereby suggesting that mature thymocytes may be exported directly from the cortex in the absence of CCR7 signals. However, the thymocytes that were generated in the absence of CCR7 or CCR7L were potent in causing autoimmune dacryoadenitis and sialadenitis in mice and were thus incapable of establishing central tolerance to organ-specific antigens. These results indicate that CCR7-mediated cortex-to-medulla migration of thymocytes is essential for establishing central tolerance rather than for supporting the maturation or export of thymocytes.

Animals↗
PubMed2006

Journey through the thymus: stromal guides for T-cell development and selection.

Lympho-stromal interactions in multiple microenvironments within the thymus have a crucial role in the regulation of T-cell development and selection. Recent studies have implicated that chemokines that are produced by thymic stromal cells have a pivotal role in positioning developing T cells within the thymus. In this Review, I discuss the importance of stroma-derived chemokines in guiding the traffic of developing thymocytes, with an emphasis on the processes of cortex-to-medulla migration and T-cell-repertoire selection, including central tolerance.

Animals↗
PubMed2006

Cellular and molecular events during early thymus development.

The thymic stromal compartment consists of several cell types that collectively enable the attraction, survival, expansion, migration, and differentiation of T-cell precursors. The thymic epithelial cells constitute the most abundant cell type of the thymic microenvironment and can be differentiated into morphologically, phenotypically, and functionally separate subpopulations of the postnatal thymus. All thymic epithelial cells are derived from the endodermal lining of the third pharyngeal pouch. Very soon after the formation of a thymus primordium and prior to its vascularization, thymic epithelial cells orchestrate the first steps of intrathymic T-cell development, including the attraction of lymphoid precursor cells to the thymic microenvironment. The correct segmentation of pharyngeal epithelial cells and their subsequent crosstalk with cells in the pharyngeal arches are critical prerequisites for the formation of a thymus anlage. Mutations in several transcription factors and their target genes have been informative to detail some of the complex mechanisms that control the development of the thymus anlage. This review highlights recent findings related to the genetic control of early thymus organogenesis and provides insight into the molecular basis by which lymphocyte precursors are attracted to the thymus.

Animals↗
PubMed2006

[Central tolerance and autoimmune diseases].

Central tolerance is established by the repertoire selection of immature T lymphocytes in the thymus, avoiding autoimmune responses to self-antigens. Differential ligand-TCR interactions that result in positive and negative selection initiate differential intracellular signals that, in turn, lead to the survival-or-death decision of immature thymocytes. TCR signal dysregulation due to the mutation of ZAP-70 or defective apoptosis of autoreactive thymocytes due to the deficiency of pro-apoptotic protein Bim impair tolerance and cause autoimmunity. Thymic repertoire selection also induces the development of CD25(+)CD4(+) regulatory T cells, which play important roles for maintaining peripheral tolerance. Furthermore, the establishment of central tolerance requires the development of thymic medulla that is mediated by the activation of NF-kappaB signaling pathway, promiscuous expression of tissue-specific self-antigens by medullary epithelial cells that is regulated by AIRE, and cortex-to-medulla migration of developing thymocytes that is regulated by CCR7-mediated chemokine signals.

Animals↗
PubMed2005

Development of autoimmunity against transcriptionally unrepressed target antigen in the thymus of Aire-deficient mice.

Autoimmune regulator (AIRE) gene mutation is responsible for the development of organ-specific autoimmune disease with monogenic autosomal recessive inheritance. Although Aire has been considered to regulate the elimination of autoreactive T cells through transcriptional control of tissue-specific Ags in thymic epithelial cells, other mechanisms of AIRE-dependent tolerance remain to be investigated. We have established Aire-deficient mice and examined the mechanisms underlying the breakdown of self-tolerance. The production and/or function of immunoregulatory T cells were retained in the Aire-deficient mice. The mice developed Sjogren's syndrome-like pathologic changes in the exocrine organs, and this was associated with autoimmunity against a ubiquitous protein, alpha-fodrin. Remarkably, transcriptional expression of alpha-fodrin was retained in the Aire-deficient thymus. These results suggest that Aire regulates the survival of autoreactive T cells beyond transcriptional control of self-protein expression in the thymus, at least against this ubiquitous protein. Rather, Aire may regulate the processing and/or presentation of self-proteins so that the maturing T cells can recognize the self-Ags in a form capable of efficiently triggering autoreactive T cells. With the use of inbred Aire-deficient mouse strains, we also demonstrate the presence of some additional factor(s) that determine the target-organ specificity of the autoimmune disease caused by Aire deficiency.

Animals↗
PubMed2005

Controlling the thymic microenvironment.

T-cell development in the thymus is a stepwise process, mediated by a variety of stromal cells in different regions of the organ. Although the cellular composition of the thymic microenvironment has been known for over a decade, the molecular cues that govern its formation are only beginning to be understood. Stromal-derived chemokines attract T-cell precursors to the thymus and direct maturing thymocytes to appropriate niches for their further development. Reciprocal signals from developing T cells provide crosstalk that is essential for establishment and maintenance of the thymic microenvironment. Elucidation of the molecular players involved and their context within the organ is the challenge for the field today. This knowledge could then be translated to clinical restoration of thymic function and T-cell reconstitution.

Animals↗
PubMed2005

Development of T-lymphocytes in mouse fetal thymus organ culture.

Fetal thymus organ culture (FTOC) is a unique and powerful culture system that allows intrathymic T-lymphocyte development in vitro. T-cell development in FTOC well represents fetal thymocyte development in vivo. Here, we describe the basic method for FTOC as well as several related techniques, including the reconstitution of thymus lobes with T-lymphoid progenitor cells, high-oxygen submersion culture, time-lapse visualization of thymic emigration, reaggregation culture, and retrovirus-mediated gene transfer to developing thymocytes in FTOC.

Animals↗
PubMed2004

The role of CCL21 in recruitment of T-precursor cells to fetal thymi.

During embryonic development, T-lymphoid precursor cells colonize the thymus. Chemoattraction by the fetal thymus is thought to mediate T-precursor cell colonization. However, the molecules that attract T-precursor cells to the thymus remain unclear. By devising time-lapse visualization in culture, the present results show that alymphoid fetal thymus lobes attract T-precursor cells from fetal liver or fetal blood. CD4(-)CD8(-)CD25(-)CD44+ fetal thymocytes retained the activity to specifically re-enter the thymus. The attraction was predominantly due to I-A-expressing thymic epithelial cells and was mediated by pertussis toxin-sensitive G-protein signals. Among the chemokines produced by the fetal thymus, CCL21, CCL25, and CXCL12 could attract CD4(-)CD8(-)CD25(-)CD44+ fetal thymocytes. However, fetal thymus colonization was markedly diminished by neutralizing antibodies specific for CCL21 and CCL25, but not affected by anti-CXCL12 antibody. Fetal thymus colonization was partially defective in CCL21-deficient plt/plt mice and was further diminished by anti-CCL25 antibody. These results indicate that CCL21 is involved in the recruitment of T-cell precursors to the fetal thymus and suggest that the combination of CCL21 and CCL25 plays a major role in fetal thymus colonization.

Animals↗
PubMed2004

Increased frequency of aberrant V(D)J recombination products in core RAG-expressing mice.

RAG1 and RAG2 play a central role in V(D)J recombination, a process for antigen receptor gene assembly. The truncated 'core' regions of RAGs are sufficient to catalyze the recombination reaction, although with lower joining efficiency than full-length proteins. To investigate the role of the non-core regions of RAGs in the end-joining phase of antigen receptor rearrangement, we analyzed recombination products isolated from core RAG1 and core RAG2 knock-in mice. Here, we report that the truncation of RAGs increases the frequency of aberrant recombination in vivo. Signal joints (SJs) associated with V-to-D recombination of core RAG1 knock-in mice were normal, whereas those of core RAG2 knock-in mice were highly imprecise, containing large deletions and additions, and in some cases coding sequences. In contrast, we found an elevated level of imprecise D-to-J associated SJs for both core RAG1- and RAG2-expressing mice. Likewise, sequences of coding joints (CJs) were also affected by the expression of core RAGs. Finally, sequences found at the junctions of rearranged T-cell receptor loci were highly influenced by differences in rearranging recombination signal sequence pairs. We provide the first evidence that the non-core regions of RAGs have critical functions in the proper assembly and resolution of recombination intermediates in endogenous antigen receptor loci.

Animals↗
PubMed2004

CCR7 signals are essential for cortex-medulla migration of developing thymocytes.

Upon TCR-mediated positive selection, developing thymocytes relocate within the thymus from the cortex to the medulla for further differentiation and selection. However, it is unknown how this cortex-medulla migration of thymocytes is controlled and how it controls T cell development. Here we show that in mice deficient for CCR7 or its ligands mature single-positive thymocytes are arrested in the cortex and do not accumulate in the medulla. These mutant mice are defective in forming the medullary region of the thymus. Thymic export of T cells in these mice is compromised during the neonatal period but not in adulthood. Thymocytes in these mice show no defects in maturation, survival, and negative selection to ubiquitous antigens. TCR engagement of immature cortical thymocytes elevates the cell surface expression of CCR7. These results indicate that CCR7 signals are essential for the migration of positively selected thymocytes from the cortex to the medulla. CCR7-dependent cortex-medulla migration of thymocytes plays a crucial role in medulla formation and neonatal T cell export but is not essential for maturation, survival, negative selection, and adult export of thymocytes.

Animals↗
PubMed2004

NF-kappa B-inducing kinase establishes self-tolerance in a thymic stroma-dependent manner.

Physical contact between thymocytes and the thymic stroma is essential for T cell maturation and shapes the T cell repertoire in the periphery. Stromal elements that control these processes still remain elusive. We used a mouse strain with mutant NF-kappaB-inducing kinase (NIK) to examine the mechanisms underlying the breakdown of self-tolerance. This NIK-mutant strain manifests autoimmunity and disorganized thymic structure with abnormal expression of Rel proteins in the stroma. Production of immunoregulatory T cells that control autoreactive T cells was impaired in NIK-mutant mice. The autoimmune disease seen in NIK-mutant mice was reproduced in athymic nude mice by grafting embryonic thymus from NIK-mutant mice, and this was rescued by supply of exogenous immunoregulatory T cells. Impaired production of immunoregulatory T cells by thymic stroma without normal NIK was associated with altered expression of peripheral tissue-restricted Ags, suggesting an essential role of NIK in the thymic microenvironment in the establishment of central tolerance.

Animals↗