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Key Points CD161 defines proinflammatory FoxP3+ cells that have classic Treg signatures, yet share effector T-cell properties. CD161+ Treg proinflammatory phenotype is stable upon Treg expansion and thus should be considered in therapeutic strategies using Treg.

Bending et al. establish a new tool, Timer of cell kinetics and activity (Tocky), revealing the temporal dynamics of cellular activation and differentiation in vivo. The tool analyzes the temporal sequence of molecular processes during cellular differentiation and can classify cells based on the frequency they receive signaling events in vivo.

Bacterial-induced intestinal inflammation is crucially dependent on interleukin (IL)-23 and is associated with CD4(+) T helper type 1 (Th1) and Th17 responses. However, the relative contributions of these subsets during the induction and resolution of colitis in T-cell-sufficient hosts remain unknown. We report that Helicobacter hepaticus-induced typhlocolitis in specific pathogen-free IL-10(-/-) mice is associated with elevated frequencies and numbers of large intestinal interferon (IFN)-γ(+) and IFN-γ(+)IL-17A(+) CD4(+) T cells. By assessing histone modifications and transcript levels in IFN-γ(+), IFN-γ(+)IL-17A(+), and IL-17A(+) CD4(+) T cells isolated from the inflamed intestine, we show that Th17 cells are predisposed to upregulate the Th1 program and that they express IL-23R but not IL-12R. Using IL-17A fate-reporter mice, we further demonstrate that H. hepaticus infection gives rise to Th17 cells that extinguish IL-17A secretion and turn on IFN-γ within 10 days post bacterial inoculation. Together, our results suggest that bacterial-induced Th17 cells arising in disease-susceptible hosts contribute to intestinal pathology by switching phenotype, transitioning via an IFN-γ(+)IL-17A(+) stage, to become IFN-γ(+) ex-Th17 cells.

Understanding the mechanisms of cellular differentiation is challenging because differentiation is initiated by signaling pathways that drive temporally dynamic processes, which are difficult to analyse in vivo. We establish a new Tool, Timer-of-cell-kinetics-and-activity (Tocky [toki], time in Japanese). Tocky uses the Fluorescent Timer protein, which spontaneously shifts its emission spectrum from blue-to-red, in combination with computer algorithms to reveal the dynamics of differentiation in vivo. Using a transcriptional target of T cell receptor (TCR)-signaling, we establish Nr4a3-Tocky to follow downstream effects of TCR signaling. Nr4a3-Tocky reveals the temporal sequence of events during regulatory T cell (Treg) differentiation and shows that persistent TCR signals occur during Treg generation. Remarkably, antigen-specific T cells at the site of autoimmune inflammation also show persistent TCR signaling. In addition, by generating Foxp3-Tocky, we reveal the in vivo dynamics of demethylation of the Foxp3 gene. Thus, Tocky is a Tool for cell biologists to address previously inaccessible questions by directly revealing dynamic processes in vivo.SummaryThe authors establish a new Tool, Timer-of-cell-kinetics-and-activity (Tocky) revealing the temporal dynamics of cellular activation and differentiation in vivo. The tool analyses the temporal sequence of molecular processes during cellular differentiation and identifies cells that receive persistent signals in vivo.

Summary Nr4a receptors are activated by T cell receptor (TCR) signaling and play key roles in T cell differentiation. Which TCR signaling pathways regulate Nr4a receptors and their sensitivities to TCR signal strength and duration remains unclear. Using Nr4a1/Nur77-GFP and Nr4a3 -Timer of cell kinetics and activity (Tocky) mice, we elucidate the signaling pathways governing Nr4a receptor expression. We reveal that Nr4a1 – Nr4a3 are Src family kinase dependent. Moreover, Nr4a2 and Nr4a3 are attenuated by calcineurin inhibitors and bind nuclear factor of activated T cells 1 (NFAT1), highlighting a necessary and sufficient role for NFAT1 in the control of Nr4a2 and Nr4a3, but redundancy for Nr4a1 . Nr4a1- GFP is activated by tonic and cognate signals during T cell development, whereas Nr4a3- Tocky requires cognate peptide:major histocompatibility complex (MHC) interactions for expression. Compared to Nr4a3- Tocky, Nr4a1- GFP is approximately 2- to 3-fold more sensitive to TCR signaling and is detectable by shorter periods of TCR signaling. These findings suggest that TCR signal duration may be an underappreciated aspect influencing the developmental fate of T cells in vivo .

ObjectiveGranulocyte–macrophage colony stimulating factor (GM-CSF) is a potent inflammatory mediator that is responsible for recruitment and activation of innate immune cells. Recent data from murine studies have identified Th17 cells as a key source of GM-CSF and suggest that T cell–derived GM-CSF is instrumental in the induction of autoimmune disease. The present study was undertaken to analyze the expression of T cell–derived GM-CSF in the joints of patients with juvenile idiopathic arthritis (JIA) and to investigate the differentiation of Th17 cells and how this relates to GM-CSF+ T helper cells.MethodsSynovial fluid (SF) and peripheral blood (PB) samples from 24 patients with JIA were analyzed, by flow cytometry and reverse transcription–polymerase chain reaction, for expression of GM-CSF and the Th17 marker CD161. A cytokine capture assay was used to purify Th17 cells and test the plasticity of cytokine production in response to interleukin-12 (IL-12) and IL-23.ResultsThe frequency of GM-CSF–producing T helper cells was significantly enriched in SF mononuclear cells compared to PB mononuclear cells from the patients with JIA (24.1% of CD4+ T cells versus 2.9%) and closely correlated with the erythrocyte sedimentation rate (r2 = 0.91, P < 0.001). Synovial GM-CSF+ T cells were predominantly CD161+ and coexpressed interferon-γ (IFNγ), but not IL-17. Culture of Th17 cells in the presence of IL-12 led to rapid up-regulation of GM-CSF and IFNγ, recapitulating the phenotype of GM-CSF–expressing cells within the joint.ConclusionOur results identify a novel outcome of Th17 plasticity in humans that may account for the enrichment of GM-CSF–expressing T cells in the joints of patients with JIA. The association of GM-CSF expression with systemic inflammation highlights the potential role of Th17-related cytokines in the pathology of JIA.

Type one diabetes (T1D) is a complex T cell-mediated autoimmune disease, the defining feature of which is the destruction of the insulin-secreting beta- (β)- cell. Both genetic and environmental factors combine to precipitate disease, and the outcome of the pathological process is dependent on multiple inter-related factors. In this review, the mechanisms behind the initiation and propagation of the autoimmune response are analysed, and the contribution of differing T-helper (T(h)) subsets--in particular T(h)1- and T(h)17-related cytokines--to the disease process are discussed. An argument is then synthesized that proposes that the β-cell's response to stress and inflammation is the critical determinant in predicting disease outcome and that, immunologically, a delicate balance exists between regulation and inflammation at the site of islet infiltration. Strategies for disease intervention, therefore, will not only require the induction of T-cell tolerance by tipping the balance towards regulation but will also need to contain approaches that result in the scavenging of inflammatory mediators, in order to facilitate repair. Ultimately, given that clinical diabetes presents late in the autoimmune process, strategies for β-cell regeneration must now be addressed. There is thus a requirement for an increased, collaborative effort between stem cell biologists and immunologists in order to tailor an optimal therapeutic strategy for the treatment of this debilitating disease.