Yusuke Shimo

The Tumor Necrosis Factor Family Receptors RANK and CD40 Cooperatively Establish the Thymic Medullary Microenvironment and Self-Tolerance

Medullary thymic epithelial cells (mTECs) establish T cell self-tolerance through the expression of autoimmune regulator (Aire) and peripheral tissue-specific self-antigens. However, signals underlying mTEC development remain largely unclear. Here, we demonstrate crucial regulation of mTEC development by receptor activator of NF-kappaB (RANK) and CD40 signals. Whereas only RANK signaling was essential for mTEC development during embryogenesis, in postnatal mice, cooperation between CD40 and RANK signals was required for mTEC development to successfully establish the medullary microenvironment. Ligation of RANK or CD40 on fetal thymic stroma in vitro induced mTEC development in a tumor necrosis factor-associated factor 6 (TRAF6)-, NF-kappaB inducing kinase (NIK)-, and IkappaB kinase beta (IKKbeta)-dependent manner. These results show that developmental-stage-dependent cooperation between RANK and CD40 promotes mTEC development, thereby establishing self-tolerance.

Lymphotoxin Signal Promotes Thymic Organogenesis by Eliciting RANK Expression in the Embryonic Thymic Stroma

It has recently become clear that signals mediated by members of the TNFR superfamily, including lymphotoxin-β receptor (LTβR), receptor activator for NF-κB (RANK), and CD40, play essential roles in organizing the integrity of medullary thymic epithelial cells (mTECs) required for the establishment of self-tolerance. However, details of the mechanism responsible for the unique and cooperative action of individual and multiple TNFR superfamily members during mTEC differentiation still remain enigmatic. In this study, we show that the LTβR signal upregulates expression of RANK in the thymic stroma, thereby promoting accessibility to the RANK ligand necessary for mTEC differentiation. Cooperation between the LTβR and RANK signals for optimal mTEC differentiation was underscored by the exaggerated defect of thymic organogenesis observed in mice doubly deficient for these signals. In contrast, we observed little cooperation between the LTβR and CD40 signals. Thus, the LTβR signal exhibits a novel and unique function in promoting RANK activity for mTEC organization, indicating a link between thymic organogenesis mediated by multiple cytokine signals and the control of autoimmunity.

Limitation of immune tolerance–inducing thymic epithelial cell development by Spi-B–mediated negative feedback regulation

Akiyama et al. show that transcription factor Spi-B is up-regulated by RANKL to trigger mTEC differentiation. Osteoprotegerin is also induced by this signaling pathway and acts as a negative feedback loop to attenuate mTEC development and thymic T reg cells.

TRAF6 directs commitment to regulatory T cells in thymocytes

Regulatory T cells (Tregs), a subset of CD4+ helper T cells, are crucial for immunological self‐tolerance. Defect in development or function of Tregs results in autoimmune disease in human and mice. Whereas it is known that Tregs mainly develop in the thymus, the molecular mechanism underlying development of Treg is not fully understood. TRAF6‐deficient mice showed a severe defect in the Treg development in thymus. In vitro fetal thymic organ culture experiments indicated that the defect is ascribed to the absence of TRAF6 in thymic cells. Moreover, mixed fetal liver transfer experiments revealed that the development of Foxp3+ cells differentiated from Traf6–/– hematopoietic cells was specifically impaired in the thymus, indicating cell‐intrinsic requirement for TRAF6 in the Treg development. On the other hand, TRAF6 is not required for the development of conventional CD4+ T cell. In addition, TGFβ‐dependent induction of Foxp3 in CD4+ T cells in vitro was not impaired by the absence of TRAF6. Overall, our data indicate that TRAF6 plays an essential role on the commitment of immature thymocytes to thymic Tregs in cell‐intrinsic fashion.

Developmental Stage-Dependent Collaboration between the TNF Receptor-Associated Factor 6 and Lymphotoxin Pathways for B Cell Follicle Organization in Secondary Lymphoid Organs

Signal transduction pathways regulating NF-κB activation essential for microenvironment formation in secondary lymphoid organs remain to be determined. We investigated the effect of a deficiency of TNFR-associated factor 6 (TRAF6), which activates the classical NF-κB pathway, in splenic microenvironment formation. Two-week-old TRAF6-deficient mice showed severe defects in B cell follicle and marginal zone formation, similar to mutant mice defective in lymphotoxin (Lt) β receptor (LtβR) signal induction of nonclassical NF-κB activation. However, analysis revealed a TRAF6 role in architecture formation distinct from its role in the early neonatal Lt signaling pathway. LtβR signal was essential for primary B cell cluster formation with initial differentiation of follicular dendritic cells (FDCs) in neonatal mice. In contrast, TRAF6 was dispensable for progression to this stage but was required for converting B cell clusters to B cell follicles and maintaining FDCs through to later stages. Fetal liver transfer experiments suggested that TRAF6 in radiation-resistant cells is responsible for follicle formation. Despite FDC-specific surface marker expression, FDCs in neonatal TRAF6-deficient mice had lost the capability to express CXCL13. These data suggest that developmentally regulated activation of TRAF6 in FDCs is required for inducing CXCL13 expression to maintain B cell follicles.

TRAF6 negatively regulates the Jak1-Erk pathway in interleukin-2 signaling.

Tumor necrosis factor receptor–associated factor 6 (TRAF6) plays a critical role in establishing both innate and acquired immune responses by mediating signals from the TNF superfamily, the TLR/IL‐1R family, and the T‐cell receptor. Here, we report a previously unidentified function of TRAF6 in IL‐2 signaling. CD3/CD28 stimulation‐induced proliferation and Il2 mRNA expression in Traf6−/−CD4+ T cells were dramatically enhanced. This enhancement is likely due to hyperactive IL‐2 signaling, in which activation of the Jak1‐Erk pathway was enhanced and the subsequent Fos gene expression was up‐regulated. To elucidate the molecular mechanisms of the enhanced activation of Jak1, IL‐2 signaling was reconstituted in mouse embryonic fibroblast (MEF) cells to investigate the interaction between TRAF6 and the TRAF6‐binding site that overlaps with the Jak1‐binding site present in the IL‐2R β‐chain. The Jak1‐Erk pathway was activated upon IL‐2 stimulation in Traf6−/−MEF cells, while a β‐chain mutation that inactivates TRAF6 binding but retains Jak1 binding abrogated the TRAF6‐dependent reduction in IL‐2 signaling. These results indicate that the binding of TRAF6 to the TRAF6‐binding site of the β‐chain negatively regulates IL‐2‐induced Jak1 activation, which is likely to be involved in the proper regulation of T‐cell activation and development.

RANK signaling induces interferon-stimulated genes in the fetal thymic stroma

Medullary thymic epithelial cells (mTECs) are essential for thymic negative selection to prevent autoimmunity. Previous studies show that mTEC development is dependent on the signal transducers TRAF6 and NIK. However, the downstream target genes of signals controlled by these molecules remain unknown. We performed a microarray analysis on mRNAs down-regulated by deficiencies in TRAF6 or functional NIK in an in vitro organ culture of fetal thymic stromata (2DG-FTOC). An in silico analysis of transcription factor binding sites in plausible promoter regions of differentially expressed genes suggests that STAT1 is involved in TRAF6- and NIK-dependent gene expression. Indeed, the signal of RANK, a TNF receptor family member that activates TRAF6 and NIK, induces the activation of STAT1 in 2DG-FTOC. Moreover, RANK signaling induces the up-regulation of interferon (IFN)-stimulated gene (ISG) expression, suggesting that the RANKL-dependent activation of STAT1 up-regulates ISG expression. The RANKL-dependent expression levels of ISGs were reduced but not completely abolished in interferon α receptor 1-deficient (Ifnar1(-/-)) 2DG-FTOC. Our data suggest that RANK signaling induces ISG expression in both type I interferon-independent and interferon-dependent mechanisms.