Hassall’s corpuscles induce thymic IFNα expression

Hassall’s corpuscles (HCs) are composed of cornifying, terminally differentiated medullary thymic epithelial cells (mTECs) that are developed under the control of Aire. Here, we demonstrated that HC-mTECs show features of cellular senescence and produce inflammatory cytokines and chemokines including CXCL5, thereby recruiting and activating neutrophils to produce IL-23 in the thymic medulla. We further indicated that thymic plasmacytoid dendritic cells (pDCs) expressing IL-23 receptors constitutively produced Ifna, which plays a role in single positive (SP) cell maturation, in an Il23a-dependent manner. Neutrophil depletion with anti-Ly6G antibody injection resulted in a significant decrease of Ifna expression in the thymic pDCs, suggesting that thymic neutrophil activation underlies the Ifna expression in thymic pDCs in steady state conditions. A New Zealand White mouse strain showing HC hyperplasia exhibited greater numbers and activation of thymic neutrophils and pDCs than B6 mice, whereas Aire-deficient B6 mice with defective HC development and SP thymocyte maturation showed significantly compromised numbers and activation of these cells. These results collectively suggested that HC-mTECs with cell-senescence features initiate a unique cell activation cascade including neutrophils and pDCs leading to the constitutive IFNα expression required for SP T-cell maturation in the thymic medulla

Calcium sparks enhance the tissue fluidity within epithelial layers and promote apical extrusion of transformed cells

In vertebrates, newly emerging transformed cells are often apically extruded from epithelial layers through cell competition with surrounding normal epithelial cells. However, the underlying molecular mechanism remains elusive. Here, using phospho-SILAC screening, we show that phosphorylation of AHNAK2 is elevated in normal cells neighboring RasV12 cells soon after the induction of RasV12 expression, which is mediated by calcium-dependent protein kinase C. In addition, transient upsurges of intracellular calcium, which we call calcium sparks, frequently occur in normal cells neighboring RasV12 cells, which are mediated by mechanosensitive calcium channel TRPC1 upon membrane stretching. Calcium sparks then enhance cell movements of both normal and RasV12 cells through phosphorylation of AHNAK2 and promote apical extrusion. Moreover, comparable calcium sparks positively regulate apical extrusion of RasV12-transformed cells in zebrafish larvae as well. Hence, calcium sparks play a crucial role in the elimination of transformed cells at the early phase of cell competition

A human PSMB11 variant affects thymoproteasome processing and CD8+ T cell production

The Psmb11-encoded β5t subunit of the thymoproteasome, which is specifically expressed in cortical thymic epithelial cells (cTECs), is essential for the optimal positive selection of functionally competent CD8+ T cells in mice. Here, we report that a human genomic PSMB11 variation, which is detectable at an appreciable allele frequency in human populations, alters the β5t amino acid sequence that affects the processing of catalytically active β5t proteins. The introduction of this variation in the mouse genome revealed that the heterozygotes showed reduced β5t expression in cTECs and the homozygotes further exhibited reduction in the cellularity of CD8+ T cells. No severe health problems were noticed in many heterozygous and 5 homozygous human individuals. Long-term analysis of health status, particularly in the homozygotes, is expected to improve our understanding of the role of the thymoproteasome-dependent positive selection of CD8+ T cells in humans

Increased c-Myc activity and DNA damage in hematopoietic progenitors precede myeloproliferative disease in Spa-1-deficiency.

Mice deficient for Spa-1 encoding Rap GTPase-activating protein develop myeloproliferative disorder (MPD) of late onset with frequent blast crises. The mechanisms for MPD development as well as the reasons for long latency, however, remain elusive. We demonstrate here that preleukemic, disease-free Spa-1(-/-) mice show reduced steady-state hematopoiesis and attenuated resistance to whole body γ-ray irradiation, which are attributable to the sustained p53 response in hematopoietic progenitor cells (HPCs). Preleukemic Spa-1(-/-) HPCs show c-Myc overexpression with increased p19Arf as well as enhanced γH2AX expression with activation of Atm/Chk pathway. We also show that deregulated Rap signaling in the absence of Spa-1 enhances post-transcriptional c-Myc stability and induces DNA damage in a p38MAPK-dependent manner, leading to p53 activation. Genetic studies indicate that the introduction of p53(+/-) and p53(-/-) mutations in Spa-1(-/-) mice results in the acceleration of typical MPD and rapid development of blastic leukemia, respectively. These results suggest that increased c-Myc expression and DNA damage in HPCs precede MPD development in Spa-1(-/-) mice, and the resulting p53 response functions as a barrier for the onset of MPD and blast crises progression

Medullary Thymic Epithelial Stem Cells Maintain a Functional Thymus to Ensure Lifelong Central T Cell Tolerance

SummaryMedullary thymic epithelial cells (mTECs) are crucial for central T cell self-tolerance. Although progenitors of mTECs have been demonstrated in thymic organogenesis, the mechanism for postnatal mTEC maintenance remains elusive. We demonstrate that implantation of embryonic TECs expressing claudin-3 and claudin-4 (Cld3,4) in a medulla-defective thymic microenvironment restores medulla formation and suppresses multiorgan autoimmunity throughout life. A minor SSEA-1+ fraction within the embryonic Cld3,4hi TECs contained self-renewable clonogenic TECs, capable of preferentially generating mature mTECs in vivo. Adult SSEA-1+Cld3,4hi TECs retained mTEC reconstitution potential, although the activity decreased. The clonogenicity of TECs also declined rapidly after birth in wild-type mice, whereas it persisted in Rag2−/− adult mice with defective thymopoiesis. The results suggest that unipotent mTEC-restricted stem cells that develop in the embryo have the capacity to functionally reconstitute the thymic medulla long-term, thus ensuring lifelong central T cell self-tolerance

Lymphocyte-stromal cell interaction induces IL-7 expression by interferon regulatory factors.

The interaction between lymphocytes and stromal cells plays important roles in coordinated development of early lymphocytes. IL-7 is an essential cytokine for early lymphocyte development produced by stromal cells in the thymus and bone marrow. Although IL-7 is induced by interaction of early lymphocytes and stromal cells, its molecular basis is still unknown. To address this question, we employed co-culture system with an IL-7-dependent pre-B cell line, DW34, and a thymic stromal cell line, TSt-4. Co-culture with DW34 cells enhanced the levels of IL-7 transcripts in TSt-4 cells. Interestingly, the co-culture also induced transcripts of IFN-α and IFN-β but not of IFN-γ. In addition, exogenous IFN-β stimulation increased the levels of IL-7 transcripts in TSt-4 cells. Next, to elucidate the molecular mechanism of IL-7 induction, we analyzed the IL-7 promoter activity by reporter assay. The IL-7 promoter showed specific transcriptional activity in TSt-4 cells. An interferon-stimulated response element (ISRE) in the IL-7 promoter was essential for the induction of IL-7 transcription by both co-culture and IFN-β stimulation. Finally, overexpression of wild-type and dominant-negative forms of interferon regulatory factors (IRFs) activated and repressed, respectively, the IL-7 promoter in TSt-4 cells. Collectively, these results suggested that IRFs activated by lymphocyte adhesion induce IL-7 transcription through ISRE in stromal cells and that type I IFNs may be involved in the activation of IRFs. Thus, this study implied a physiological function of the IFN/IRF signal during lymphocyte development