Izumi Ohigashi

The Cytokine RANKL Produced by Positively Selected Thymocytes Fosters Medullary Thymic Epithelial Cells that Express Autoimmune Regulator

The thymic medulla provides a microenvironment where medullary thymic epithelial cells (mTECs) express autoimmune regulator and diverse tissue-restricted genes, contributing to launching self-tolerance. Positive selection is essential for thymic medulla formation via a previously unknown mechanism. Here we show that the cytokine RANK ligand (RANKL) was produced by positively selected thymocytes and regulated the cellularity of mTEC by interacting with RANK and osteoprotegerin. Forced expression of RANKL restored thymic medulla in mice lacking positive selection, whereas RANKL perturbation impaired medulla formation. These results indicate that RANKL produced by positively selected thymocytes is responsible for fostering thymic medulla formation, thereby establishing central tolerance.

Aire-dependent production of XCL1 mediates medullary accumulation of thymic dendritic cells and contributes to regulatory T cell development

Aire regulates medullary epithelial cell production of XCL1, a chemoattractant for XCR1-expressing thymic DCs whose presence in the medulla contributes to the generation of T reg cells.

Rank Signaling Links the Development of Invariant γδ T Cell Progenitors and Aire+ Medullary Epithelium

Summary The thymic medulla provides a specialized microenvironment for the negative selection of T cells, with the presence of autoimmune regulator (Aire)-expressing medullary thymic epithelial cells (mTECs) during the embryonic-neonatal period being both necessary and sufficient to establish long-lasting tolerance. Here we showed that emergence of the first cohorts of Aire+ mTECs at this key developmental stage, prior to αβ T cell repertoire selection, was jointly directed by Rankl+ lymphoid tissue inducer cells and invariant Vγ5+ dendritic epidermal T cell (DETC) progenitors that are the first thymocytes to express the products of gene rearrangement. In turn, generation of Aire+ mTECs then fostered Skint-1-dependent, but Aire-independent, DETC progenitor maturation and the emergence of an invariant DETC repertoire. Hence, our data attributed a functional importance to the temporal development of Vγ5+ γδ T cells during thymus medulla formation for αβ T cell tolerance induction and demonstrated a Rank-mediated reciprocal link between DETC and Aire+ mTEC maturation.

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.

Aire-expressing thymic medullary epithelial cells originate from β5t-expressing progenitor cells

The thymus provides multiple microenvironments that are essential for the development and repertoire selection of T lymphocytes. The thymic cortex induces the generation and positive selection of T lymphocytes, whereas the thymic medulla establishes self-tolerance among the positively selected T lymphocytes. Cortical thymic epithelial cells (cTECs) and medullary TECs (mTECs) constitute the major stromal cells that structurally form and functionally characterize the cortex and the medulla, respectively. cTECs and mTECs are both derived from the endodermal epithelium of the third pharyngeal pouch. However, the molecular and cellular characteristics of the progenitor cells for the distinct TEC lineages are unclear. Here we report the preparation and characterization of mice that express the recombinase Cre instead of β5t, a proteasome subunit that is abundant in cTECs and not detected in other cell types, including mTECs. By crossing β5t-Cre knock-in mice with loxP-dependent GFP reporter mice, we found that β5t-Cre–mediated recombination occurs specifically in TECs but not in any other cell types in the mouse. Surprisingly, in addition to cTECs, β5t-Cre-loxP–mediated GFP expression was detected in almost all mTECs. These results indicate that the majority of mTECs, including autoimmune regulator-expressing mTECs, are derived from β5t-expressing progenitor cells.

Lymphotoxin β Receptor Regulates the Development of CCL21-Expressing Subset of Postnatal Medullary Thymic Epithelial Cells

Medullary thymic epithelial cells (mTECs) play a pivotal role in the establishment of self-tolerance in T cells by ectopically expressing various tissue-restricted self-Ags and by chemoattracting developing thymocytes. The nuclear protein Aire expressed by mTECs contributes to the promiscuous expression of self-Ags, whereas CCR7-ligand (CCR7L) chemokines expressed by mTECs are responsible for the attraction of positively selected thymocytes. It is known that lymphotoxin signals from the positively selected thymocytes preferentially promote the expression of CCR7L rather than Aire in postnatal mTECs. However, it is unknown how lymphotoxin signals differentially regulate the expression of CCR7L and Aire in mTECs and whether CCR7L-expressing mTECs and Aire-expressing mTECs are distinct populations. In this study, we show that the majority of postnatal mTECs that express CCL21, a CCR7L chemokine, represent an mTEC subpopulation distinct from the Aire-expressing mTEC subpopulation. Interestingly, the development of CCL21-expressing mTECs, but not Aire-expressing mTECs, is impaired in mice deficient in the lymphotoxin β receptor. These results indicate that postnatal mTECs consist of heterogeneous subsets that differ in the expression of CCL21 and Aire, and that lymphotoxin β receptor regulates the development of the CCL21-expressing subset rather than the Aire-expressing subset of postnatal mTECs.

Serial progression of cortical and medullary thymic epithelial microenvironments.

Thymic epithelial cells (TECs) provide key instructive signals for T‐cell differentiation. Thymic cortical (cTECs) and medullary (mTECs) epithelial cells constitute two functionally distinct microenvironments for T‐cell development, which derive from a common bipotent TEC progenitor. While seminal studies have partially elucidated events downstream of bipotent TECs in relation to the emergence of mTECs and their progenitors, the control and timing of the emergence of the cTEC lineage, particularly in relation to that of mTEC progenitors, has remained elusive. In this review, we describe distinct models that explain cTEC/mTEC lineage divergence from common bipotent progenitors. In particular, we summarize recent studies in mice providing evidence that mTECs, including the auto‐immune regulator+ subset, derive from progenitors initially endowed with phenotypic properties typically associated with the cTEC lineage. These observations support a novel “serial progression” model of TEC development, in which progenitors serially acquire cTEC lineage markers, prior to their commitment to the mTEC differentiation pathway. Gaining a better understanding of the phenotypic properties of early stages in TEC progenitor development should help in determining the mechanisms regulating cTEC/mTEC lineage development, and in strategies aimed at thymus reconstitution involving TEC therapy.

Cytokine crosstalk for thymic medulla formation

The medullary microenvironment of the thymus plays a crucial role in the establishment of self-tolerance through the deletion of self-reactive thymocytes and the generation of regulatory T cells. Crosstalk or bidirectional signal exchanges between developing thymocytes and medullary thymic epithelial cells (mTECs) contribute to the formation of the thymic medulla. Recent studies have identified the molecules that mediate thymic crosstalk. Tumor necrosis factor superfamily cytokines, including RANKL, CD40L, and lymphotoxin, produced by positively selected thymocytes and lymphoid tissue inducer cells promote the proliferation and differentiation of mTECs. In return, CCR7 ligand chemokines produced by mTECs facilitate the migration of positively selected thymocytes to the medulla. The cytokine crosstalk between developing thymocytes and mTECs nurtures the formation of the thymic medulla and thereby regulates the establishment of self-tolerance.

UNDIMINISHED REGULATORY T CELLS IN THE THYMUS OF PATIENTS WITH MYASTHENIA GRAVIS

Objective: The thymus has been implicated as a possible site of origin that triggers autoimmunity in myasthenia gravis (MG). Although several groups have suggested that the decrease in the number of regulatory T (Treg) cells contributes to the onset of MG, the exact role of Treg cells in MG remains unclear. To address this point, we examined the number and distribution of Treg cells in a large number of patients with MG. Methods: Immunohistofluorescence analysis of Foxp3 along with CD4 and CD8 was performed in thymic sections of MG (+) (n = 24) and MG (−) patients (n = 27). Circulating CD4+CD25+ cells in the peripheral blood of patients with MG (n = 15) and age-matched healthy subjects (n = 15) were also analyzed. Results: Foxp3+CD4+CD8− cells were predominantly found in the thymic medulla and their number declined with age. There was no significant difference in the number or the distribution of Foxp3+CD4+CD8− cells in the thymus between MG (+) and MG (−) patients. The number of circulating CD4+CD25+ cells in the peripheral blood of patients with MG was not significantly altered compared to that in healthy subjects. Conclusion: The cellularity of Treg cells in the thymus and circulation is not diminished in patients with myasthenia gravis.

Generation of diversity in thymic epithelial cells

In the thymus, diverse populations of thymic epithelial cells (TECs), including cortical and medullary TECs and their subpopulations, have distinct roles in coordinating the development and repertoire selection of functionally competent and self-tolerant T cells. Here, we review the expanding diversity in TEC subpopulations in relation to their functions in T cell development and selection as well as their origins and development.