Identification of mTEC precursor cells

Medullary thymic epithelial cells (mTECs) expressing autoimmune regulator (Aire) are critical for preventing the onset of autoimmunity. However, the differentiation program of Aire-expressing mTECs (Aire+ mTECs) is unclear. Here, we describe novel embryonic precursors of Aire+ mTECs. We found the candidate precursors of Aire+ mTECs (pMECs) by monitoring the expression of receptor activator of nuclear factor-κB (RANK), which is required for Aire+ mTEC differentiation. pMECs unexpectedly expressed cortical TEC molecules in addition to the mTEC markers UEA-1 ligand and RANK and differentiated into mTECs in reaggregation thymic organ culture. Introduction of pMECs in the embryonic thymus permitted long-term maintenance of Aire+ mTECs and efficiently suppressed the onset of autoimmunity induced by Aire+ mTEC deficiency. Mechanistically, pMECs differentiated into Aire+ mTECs by tumor necrosis factor receptor-associated factor 6-dependent RANK signaling. Moreover, nonclassical nuclear factor-κB activation triggered by RANK and lymphotoxin-β receptor signaling promoted pMEC induction from progenitors exhibiting lower RANK expression and higher CD24 expression. Thus, our findings identified two novel stages in the differentiation program of Aire+ mTECs

TNF receptor family signaling in the development and functions of medullary thymic epithelial cells

Thymic epithelial cells (TECs) provide the microenvironment required for the development of T cells in the thymus. A unique property of medullary thymic epithelial cells (mTECs) is their expression of a wide range of tissue-restricted self-antigens, critically regulated by the nuclear protein AIRE, which contributes to the selection of the self-tolerant T cell repertoire, thereby suppressing the onset of autoimmune diseases. The TNF receptor family (TNFRF) protein receptor activator of NF-κB (RANK), CD40 and lymphotoxin β receptor (LtβR) regulate the development and functions of mTECs. The engagement of these receptors with their specific ligands results in the activation of the NF-κB family of transcription factors. Two NF-κB activation pathways, the classical and non-classical pathways, promote the development of mature mTECs induced by these receptors. Consistently, TNF receptor-associated factor (TRAF6), the signal transducer of the classical pathway, and NF-κB inducing kinase (NIK), the signal transducer of the non-classical pathway, are essential for the development of mature mTECs. This review summarizes the current understanding of how the signaling by the TNF receptor family controls the development and functions of mTEC

A TCR mechanotransduction signaling loop induces negative selection in the thymus

The T cell antigen receptor (TCR) expressed on thymocytes interacts with self-peptide major histocompatibility complex (pMHC) ligands to signal apoptosis or survival. Here, we found that negative-selection ligands induced thymocytes to exert forces on the TCR and the co-receptor CD8 and formed cooperative TCR–pMHC–CD8 trimolecular ‘catch bonds’, whereas positive-selection ligands induced less sustained thymocyte forces on TCR and CD8 and formed shorter-lived, independent TCR–pMHC and pMHC–CD8 bimolecular ‘slip bonds’. Catch bonds were not intrinsic to either the TCR–pMHC or the pMHC–CD8 arm of the trans (cross-junctional) heterodimer but resulted from coupling of the extracellular pMHC–CD8 interaction to the intracellular interaction of CD8 with TCR–CD3 via associated kinases to form a cis (lateral) heterodimer capable of inside-out signaling. We suggest that the coupled trans–cis heterodimeric interactions form a mechanotransduction loop that reinforces negative-selection signaling that is distinct from positive-selection signaling in the thymus

Mitochondria–Nucleus Shuttling FK506-Binding Protein 51 Interacts with TRAF Proteins and Facilitates the RIG-I-Like Receptor-Mediated Expression of Type I IFN

<div><p>Virus-derived double-stranded RNAs (dsRNAs) are sensed in the cytosol by retinoic acid-inducible gene (RIG)-I-like receptors (RLRs). These induce the expression of type I IFN and proinflammatory cytokines through signaling pathways mediated by the mitochondrial antiviral signaling (MAVS) protein. TNF receptor-associated factor (TRAF) family proteins are reported to facilitate the RLR-dependent expression of type I IFN by interacting with MAVS. However, the precise regulatory mechanisms remain unclear. Here, we show the role of FK506-binding protein 51 (FKBP51) in regulating the dsRNA-dependent expression of type I IFN. The binding of FKBP51 to TRAF6 was first identified by “<i>in vitro</i> virus” selection and was subsequently confirmed with a coimmunoprecipitation assay in HEK293T cells. The TRAF-C domain of TRAF6 is required for its interaction, although FKBP51 does not contain the consensus motif for interaction with the TRAF-C domain. Besides TRAF6, we found that FKBP51 also interacts with TRAF3. The depletion of FKBP51 reduced the expression of type I IFN induced by dsRNA transfection or Newcastle disease virus infection in murine fibroblasts. Consistent with this, the FKBP51 depletion attenuated dsRNA-mediated phosphorylations of IRF3 and JNK and nuclear translocation of RelA. Interestingly, dsRNA stimulation promoted the accumulation of FKBP51 in the mitochondria. Moreover, the overexpression of FKBP51 inhibited RLR-dependent transcriptional activation, suggesting a scaffolding function for FKBP51 in the MAVS-mediated signaling pathway. Overall, we have demonstrated that FKBP51 interacts with TRAF proteins and facilitates the expression of type I IFN induced by cytosolic dsRNA. These findings suggest a novel role for FKBP51 in the innate immune response to viral infection.</p></div

FKBP51 is a possible scaffolding protein in RIG-I-dependent signaling.

<p>(A) Binding of FKBP51 to IRF7 in HEK293T cells. Combinations of Myc-tagged FKBP51 and Flag-tagged TRAF6, MAVS, TBK1, NEMO, IRF7, IRF3, or control were transiently expressed in HEK293T cells. The transfected genes are indicated on the top of the panels. IP and INPUT panels indicate the western blotting analysis of the immunoprecipitated samples and the cell lysates used for immunoprecipitation, respectively. The antibodies used for western blotting are shown on the right of the panels. One representative experiment of two independent experiments is shown. (B) Luciferase activity of HEK293T cells transfected with a combination of plasmids encoding Myc-tagged FKBP51 or control Myc, the Flag-tagged CARD domain of RIG-I (RIG-I-CARD) or control Flag, ISRE-driven luciferase, and β-actin-promoter-driven β-gal. The amount of transfected plasmid encoding FKBP51 or RIG-I-CARD is indicated below the graph. Relative luminescence units (RLU) were normalized to the activity of β-gal. The fold activation was determined as the normalized RLU for each sample relative to those of the sample transfected with the control vector, which indicated the basal activation. Data are the means ± SD of triplicate determinations and are representative of three independent experiments. **P<0.01; Student’s <i>t</i> test with a two-tailed distribution and two-sample equivalent variance parameters. Western blotting analysis of transfected samples using anti-Myc antibody or anti-Flag antibody. Subcellular localization of overexpressed FKBP51 is shown in the bottom panels. (C) Luciferase activity of HEK293T cells transfected with combinations of two plasmids encoding FKBP51 and the CARD domain of MDA-5 (MDA-5-CARD), TBK1, MAVS, IRF7, IRF3 or its control vector. The combinations of the transfected plasmids are indicated. Luciferase activity is shown as the fold induction relative to the basal activation, as in Fig. 6B. Data are the means ± SD of triplicate determinations and are representative of three independent experiments. **P<0.01, *P<0.05; Student’s <i>t</i> test with a two-tailed distribution and two-sample equivalent variance parameters.</p

FKBP51 preferentially accumulates in the mitochondria after cytosolic dsRNA stimulation.

<p>(A) Cytoplasmic accumulation of FKBP51 in MEF cells after stimulation with cytosolic dsRNA. MEF cells were stimulated by Lipofectamine with or without poly I:C. Endogenous FKBP51 was detected with an anti-FKBP51 antibody. The nuclei were visualized by staining with propidium iodide. One representative experiment of three is shown. (B) Cytoplasmic accumulation of FKBP51 in MEF cells after NDV infection. Endogenous FKBP51 was detected with an anti-FKBP51 antibody. The nuclei were visualized by staining with propidium iodide. One representative experiment of three is shown. (C) Subcellular localization of FKBP51 in mitochondria. MEF cells were stimulated by lipofectamine with poly I:C or NDV infection. Endogenous FKBP51 was detected with an anti-FKBP51 antibody. The mitochondria were visualized by immunostaining with anti-TOM20 antibody. One representative experiment of three is shown.</p

A TCR mechanotransduction signaling loop induces negative selection in the thymus

The T cell antigen receptor (TCR) expressed on thymocytes interacts with self-peptide major histocompatibility complex (pMHC) ligands to signal apoptosis or survival. Here, we found that negative-selection ligands induced thymocytes to exert forces on the TCR and the co-receptor CD8 and formed cooperative TCR–pMHC–CD8 trimolecular ‘catch bonds’, whereas positive-selection ligands induced less sustained thymocyte forces on TCR and CD8 and formed shorter-lived, independent TCR–pMHC and pMHC–CD8 bimolecular ‘slip bonds’. Catch bonds were not intrinsic to either the TCR–pMHC or the pMHC–CD8 arm of the trans (cross-junctional) heterodimer but resulted from coupling of the extracellular pMHC–CD8 interaction to the intracellular interaction of CD8 with TCR–CD3 via associated kinases to form a cis (lateral) heterodimer capable of inside-out signaling. We suggest that the coupled trans–cis heterodimeric interactions form a mechanotransduction loop that reinforces negative-selection signaling that is distinct from positive-selection signaling in the thymus