Regulatory T Cell Development.

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.

A local regulatory T cell feedback circuit maintains immune homeostasis by pruning self-activated T cells.

A fraction of mature T cells can be activated by peripheral self-antigens, potentially eliciting host autoimmunity. We investigated homeostatic control of self-activated T cells within unperturbed tissue environments by combining high-resolution multiplexed and volumetric imaging with computational modeling. In lymph nodes, self-activated T cells produced interleukin (IL)-2, which enhanced local regulatory T cell (Treg) proliferation and inhibitory functionality. The resulting micro-domains reciprocally constrained inputs required for damaging effector responses, including CD28 co-stimulation and IL-2 signaling, constituting a negative feedback circuit. Due to these local constraints, self-activated T cells underwent transient clonal expansion, followed by rapid death ("pruning"). Computational simulations and experimental manipulations revealed the feedback machinery's quantitative limits: modest reductions in Treg micro-domain density or functionality produced non-linear breakdowns in control, enabling self-activated T cells to subvert pruning. This fine-tuned, paracrine feedback process not only enforces immune homeostasis but also establishes a sharp boundary between autoimmune and host-protective T cell responses.

The endogenous repertoire harbors self-reactive CD4+ T cell clones that adopt a follicular helper T cell-like phenotype at steady state.

The T cell repertoire of healthy mice and humans harbors self-reactive CD4+ conventional T (Tconv) cells capable of inducing autoimmunity. Using T cell receptor profiling paired with in vivo clonal analysis of T cell differentiation, we identified Tconv cell clones that are recurrently enriched in non-lymphoid organs following ablation of Foxp3+ regulatory T (Treg) cells. A subset of these clones was highly proliferative in the lymphoid organs at steady state and exhibited overt reactivity to self-ligands displayed by dendritic cells, yet were not purged by clonal deletion. These clones spontaneously adopted numerous hallmarks of follicular helper T (TFH) cells, including expression of Bcl6 and PD-1, exhibited an elevated propensity to localize within B cell follicles at steady state, and produced interferon-γ in non-lymphoid organs following sustained Treg cell depletion. Our work identifies a naturally occurring population of self-reactive TFH-like cells and delineates a previously unappreciated fate for self-specific Tconv cells.

Eomes identifies thymic precursors of self-specific memory-phenotype CD8+ T cells.

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 showed upregulation of the transcription factor Eomes during maturation in the thymus, prior to induction of the full memory phenotype, which is 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, which suggests potential roles in antitumor immunity and the response to immunotherapy.

RBP-J-Regulated miR-182 Promotes TNF-α-Induced Osteoclastogenesis.

Increased osteoclastogenesis is responsible for osteolysis, which is a severe consequence of inflammatory diseases associated with bone destruction, such as rheumatoid arthritis 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 the expression of microRNAs (miRNAs) important for this process. Using high-throughput sequencing of miRNAs, we obtained the first, to our knowledge, genome-wide profile of miRNA expression induced by TNF-α in mouse bone marrow-derived macrophages/osteoclast precursors during inflammatory osteoclastogenesis. Furthermore, we 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 B lymphocyte-induced maturation protein-1. 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.

Altered selection on a single self-ligand promotes susceptibility to organ-specific T cell infiltration.

For the large array of self-peptide/MHC class II (pMHC-II) complexes displayed in the body, it is unclear whether CD4+ T cell tolerance must be imparted for each individual complex or whether pMHC-II-nonspecific bystander mechanisms are sufficient to confer tolerance by acting broadly on T cells reactive to multiple self-pMHC-II ligands. Here, via reconstitution of T cell-deficient mice, we demonstrate that altered T cell selection on a single prostate-specific self-pMHC-II ligand renders recipient mice susceptible to prostate-specific T cell infiltration. Mechanistically, this self-pMHC-II complex is required for directing antigen-specific cells into the Foxp3+ regulatory T cell lineage but does not induce clonal deletion to a measurable extent. Thus, our data demonstrate that polyclonal T reg cells are unable to functionally compensate for a breach in tolerance to a single self-pMHC-II complex in this setting, revealing vulnerabilities in antigen-nonspecific bystander mechanisms of immune tolerance.

RBP-J imposes a requirement for ITAM-mediated costimulation of osteoclastogenesis.

Osteoclastogenesis requires activation of RANK signaling as well as costimulatory signals from immunoreceptor tyrosine-based activation motif-containing (ITAM-containing) receptors/adaptors, predominantly tyrosine kinase-binding proteins DAP12 and FcRγ, in osteoclast precursors. It is not well understood how costimulatory signals are regulated and integrated with RANK signaling. Here, we found that osteopetrotic bone phenotypes in mice lacking DAP12 or DAP12 and FcRγ are mediated by the transcription factor RBP-J, as deletion of Rbpj in these mice substantially rescued the defects of bone remodeling. Using a TNF-α-induced model of inflammatory bone resorption, we determined that RBP-J deficiency enables TNF-α to induce osteoclast formation and bone resorption in DAP12-deficient animals. Thus, RBP-J imposes a requirement for ITAM-mediated costimulation of RANKL or TNF-α-induced osteoclastogenesis. Mechanistically, RBP-J suppressed induction of key osteoclastogenic factors NFATc1, BLIMP1, and c-FOS by inhibiting ITAM-mediated expression and function of PLCγ2 and activation of downstream calcium-CaMKK/PYK2 signaling. Moreover, RBP-J suppressed Plcg2 expression and downstream calcium oscillations indirectly by a TGF-ß/PLCγ2/calcium axis. Together, our findings indicate that RBP-J suppresses ITAM-mediated costimulation, thereby limiting crosstalk between ITAM and RANK/TNFR signaling and allowing fine tuning of osteoclastogenesis during bone homeostasis and under inflammatory conditions. Furthermore, these data suggest that environmental cues that regulate RBP-J expression/function potentially modulate the requirement for costimulatory signaling for osteoclast differentiation and bone remodeling.

Morphological differences of primary cilia between human induced pluripotent stem cells and their parental somatic cells.

Human induced pluripotent stem cell (hiPSC) reprogramming possesses enormous potential in stem cell research and disease modeling. Chemical and mechanical signaling has been implicated in the maintenance of pluripotency of hiPSCs, as well as their differentiation pathways toward various lineages. Primary cilia have been shown to play a critical role in mechanochemical signaling across a wide spectrum of cell types. The functions of primary cilia in hiPSCs and their characteristic changes during the reprogramming process remain largely vague. This work focused on understanding how reprogramming affects the mechanical characteristics of primary cilia. Using immunofluorescence imaging assays, we validated the presence of primary cilia on reprogrammed cells. These reprogrammed cells had high expression levels of pluripotency markers, Nanog and Cripto, shown by quantitative polymerase chain reaction assays. We also found high expression of hedgehog signaling proteins Patched1 (Ptch1), Smoothened (Smo), Gli1, and Gli2 in reprogrammed cells. Stimulation of the hedgehog pathway resulted in the concerted movement of Ptch1 out of the cilia and Smo into the cilia, implying that the cilia on iPSCs contain functioning hedgehog machinery. The mean length of primary cilia in reprogrammed cells was shorter than those of parental human fibroblasts. Morphometric analyses revealed that reprogramming resulted in an increase in the curvature of primary cilia from ∼0.015 to 0.064 µm(-1), indicating an underlying approximately fourfold decrease in their rigidity, and a decrease in length of primary cilia from ∼2.38 to ∼1.45 µm. Furthermore, reprogramming resulted in fewer primary cilia displaying kinked geometries.

Superresolution STED microscopy reveals differential localization in primary cilia.

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.