Foxp3-expressing CD4+ regulatory T (Treg) cells play key roles in the prevention of autoimmunity and the maintenance of immune homeostasis and represent a major barrier to the induction of robust antitumor immune responses. Thus, a clear understanding of the mechanisms coordinating Treg cell differentiation is crucial for understanding numerous facets of health and disease and for developing approaches to modulate Treg cells for clinical benefit. Here, we discuss current knowledge of the signals that coordinate Treg cell development, the antigen-presenting cell types that direct Treg cell selection, and the nature of endogenous Treg cell ligands, focusing on evidence from studies in mice. We also highlight recent advances in this area and identify key unanswered questions.

to induce inflammatory bone resorption in Dap12 -/-mice. Mechanistically, RBP-J inhibited induction of NFATc1, BLIMP1, and c-FOS by suppressing PLCγ2 expression and activity and downstream calcium-CaMKK-PYK2 signaling via the TGF-β pathway during osteoclastogenesis. Thus, RBP-J deficiency allows RANK or TNFR signaling to induce osteoclast differentiation independently of ITAM-mediated costimulation. These results indicate that RBP-J imposes a requirement for ITAM-mediated costimulation and limits the crosstalk between ITAM and RANK signaling, thereby restraining both pathways in osteoclastogenesis. Results RBP-J deficiency reverses the osteopetrotic bone phenotype of Dap12- Dap12-/-or TKO chimeric mice relieved the osteopetrotic phenotype caused by lack of DAP12 or both DAP12 and FcRγ, respectively. Rbpj ΔM/ΔM Dap12-/-mice exhibited trabecular bone volume, number, thickness, and spacing similar to those of control mice (Figure 1, A Figure 1C). Notably, serum TRAP levels in Rbpj Dap12-/-mice were comparable to those in control mice ( Figure 1C and Fcrg-/-cells leads to attenuation of PLCγ2 activation followed by impaired NFATc1 induction and osteoclast differentiation even in the presence of RANKL (18, 19). Thus, ITAM-mediated costimulatory signals and PLCγ2 activity, which determine downstream calcium signaling, are required for osteoclast differentiation. RANK signaling induces a transient increase in PLCγ2 activity, but does not regulate its expression and is not required for basal PLCγ2 activity in osteoclast precursors. Instead, basal PLCγ2 activity, which has been observed in myeloid cells including osteoclast precursors (17, 21-24), appears to be maintained by tonic ITAM signaling mediated by engagement of ITAM-associated receptors by constitutively expressed ligands (13); consistently, addition of ligands/agonists for ITAMassociated receptors is not required for osteoclast differentiation in vitro. Importantly this basal level of costimulation and PLCγ2 activation together with RANKL is sufficient for NFATc1 induction and osteoclast differentiation (16,18,19). The ligands for FcRγ-and DAP12-associated receptors that mediate tonic signaling are suggested to be expressed by osteoblasts/stromal cells and osteoclast precursors, respectively (18, 24). Thus, cell-cell interaction could provide basal/"tonic" ITAM-mediated PLCγ2/ calcium costimulatory signaling during osteoclastogenesis. There has been great interest in delineating the mechanisms by which ITAM signals crossregulate heterologous receptors, such as TLRs and cytokine receptors (13), but less is known about mechanisms/factors in the cells that fine tune ITAM-mediated PLCγ2/calcium signaling.Recombinant recognition sequence binding protein at the J κ site (RBP-J, also called RBP-J κ , CSL, or CBF1) functions as a central transcription factor that receives input from several signaling pathways, such as the canonical NOTCH pathway, the WNT/ β-catenin pathway, the NF-κB pathway, and the TLR-and TNFsignaling pathways (25-31), to mediate diverse cellula...

Increased osteoclastogenesis is responsible for osteolysis that is a severe consequence of inflammatory diseases associated with bone destruction, such as rheumatoid arthritis (RA) and periodontitis. The mechanisms that limit osteoclastogenesis under inflammatory conditions are largely unknown. We previously identified transcription factor RBP-J as a key negative regulator that restrains TNF-α induced osteoclastogenesis and inflammatory bone resorption. In this study, we tested whether RBP-J suppresses inflammatory osteoclastogenesis by regulating expression of microRNAs (miRNAs) important for this process. Using high throughput sequencing of miRNAs (miRNA-seq), we obtained the first genome-wide profile of miRNA expression induced by TNF-α in mouse bone marrow derived macrophages (BMMs)/osteoclast precursors during inflammatory osteoclastogenesis. We furthermore identified miR-182 as a novel miRNA that promotes inflammatory osteoclastogenesis driven by TNF-α and whose expression is suppressed by RBP-J. Downregulation of miR-182 dramatically suppressed the enhanced osteoclastogenesis program induced by TNF-α in RBP-J-deficient cells. Complementary loss and gain of function approaches showed that miR-182 is a positive regulator of osteoclastogenic transcription factors NFATc1 and Blimp1. Moreover, we identified that direct miR-182 targets Foxo3 and Maml1 play important inhibitory roles in TNF-α mediated osteoclastogenesis. Thus, RBP-J-regulated miR-182 promotes TNF-α induced osteoclastogenesis via inhibition of Foxo3 and Maml1. Suppression of miR-182 by RBP-J serves as an important mechanism that restrains TNF-α induced osteoclastogenesis. Our results provide a novel miRNA mediated mechanism by which RBP-J inhibits osteoclastogenesis and suggest that targeting of the newly described RBP-J-miR-182-Foxo3/Maml1 axis may represent an effective therapeutic approach to suppress inflammatory osteoclastogenesis and bone resorption.

Unprimed mice harbor a substantial population of "memory-phenotype" CD8 + T cells (CD8-MP cells) that exhibit hallmarks of activation and innate-like functional properties. Due to the lack of faithful markers to distinguish CD8-MP cells from bona fide CD8 + memory T cells, the developmental origins and antigen specificities of CD8-MP cells remain incompletely defined. Using deep T cell antigen receptor (TCR) sequencing, we found that the TCRs expressed by CD8-MP cells are highly recurrent and distinct from the TCRs expressed by naive-phenotype CD8 + T cells. CD8-MP clones exhibited reactivity to widely expressed self-ligands. T cell precursors expressing CD8-MP TCRs upregulated the transcription factor Eomes during maturation in the thymus, prior to induction of the full memory phenotype, suggestive of a unique program triggered by recognition of self-ligands. Moreover, CD8-MP cells infiltrate oncogene-driven prostate tumors and express high densities of PD-1, suggesting a potential role in anti-tumor immunity and response to immunotherapy.

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Inflammatory bone resorption mediated by osteoclasts is a major cause of morbidity and disability in many inflammatory disorders, including rheumatoid arthritis (RA). The mechanisms that regulate osteoclastogenesis and bone resorption in inflammatory settings are complex and have not been well elucidated. In this study, we identify the immunoregulator Def6 as a novel inhibitor of osteoclastogenesis in both physiological and inflammatory conditions. Def6 deficiency in Def6−/− mice enhanced the sensitivity of osteoclast precursors to the physiological osteoclastogenic inducer RANKL, and Def6−/− osteoclasts formed actin rings. Furthermore, Def6 deficiency markedly increased TNF-α-induced osteoclastogenesis both in vitro and in vivo and enhanced bone resorption in an inflammatory osteolysis mouse model. TNF-α serum levels negatively correlated with Def6 expression levels in osteoclast precursors obtained from RA patients and the osteoclastogenic capacity of the osteoclast precursors was significantly inversely correlated with their Def6 expression levels, indicating that Def6 functions as an inhibitor of excessive osteoclast formation and bone destruction in RA. Mechanistically, Def6 suppressed osteoclastogenesis and the expression of key osteoclastogenic factors NFATc1, Blimp1 and c-Fos by regulating an endogenous IFN-β mediated autocrine feedback loop. The Def6-dependent pathway may represent a novel therapeutic target to prevent pathological bone destruction.

The primary cilium is an organelle that serves as a signaling center of the cell and is involved in the cAMP, Wnt, and hedgehog signaling pathways. Adenylyl cyclase type III (ACIII) is enriched in primary cilia and acts as a marker that is involved in cAMP signaling, while also playing an important role in regulating ciliogenesis and sensory functions. Ciliary function relies on the transportation of molecules between the primary cilium and the cell, which is facilitated by intraflagellar transport (IFT). The detailed localization and interactions of these important proteins remain unclear due to the limited resolution of conventional microscopy. We conducted superresolution imaging of immunostained ACIII and IFT88 in human fibroblasts using stimulated emission depletion (STED) microscopy. Instead of a homogeneous distribution along a primary cilium, our STED images revealed that ACIII formed a periodic punctate pattern with a roughly equal spacing between groups of puncta. Superresolution imaging of IFT88, an important protein of the IFT complexes, demonstrated two novel distinct distribution patterns at the basal end: a triangle of three puncta with similar fluorescence intensities, and a Y-shaped configuration of a bright punctum connected to two branches. We also performed STED imaging of IFT88 in mouse inner medullary collecting duct cells and mouse embryonic fibroblasts. The similar three-puncta and Y-shape patterns were observed in these cells, suggesting that these distribution patterns are common among primary cilia of different cell types. Our results demonstrate the ability of superresolution STED microscopy to reveal novel structural characteristics in primary cilia.

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