Regulatory T cells (Tregs), either natural or induced, suppress a variety of physiological and pathological immune responses. One of the key issues for understanding Treg function is to determine how ...they suppress other lymphocytes at the molecular level in vivo and in vitro. Here we propose that there may be a key suppressive mechanism that is shared by every forkhead box p3 (Foxp3)+ Treg in vivo and in vitro in mice and humans. When this central mechanism is abrogated, it causes a breach in self-tolerance and immune homeostasis. Other suppressive mechanisms may synergistically operate with this common mechanism depending on the environment and the type of an immune response. Further, Treg-mediated suppression is a multi-step process and impairment or augmentation of each step can alter the ultimate effectiveness of Treg-mediated suppression. These findings will help to design effective ways for controlling immune responses by targeting Treg suppressive functions.
Understanding the mechanisms of cellular differentiation is challenging because differentiation is initiated by signaling pathways that drive temporally dynamic processes, which are difficult to ...analyze in vivo. We establish a new tool, Timer of cell kinetics and activity (Tocky; or 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
-Tocky to follow downstream effects of TCR signaling.
-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
gene. Thus, Tocky is a tool for cell biologists to address previously inaccessible questions by directly revealing dynamic processes in vivo.
Thymus-produced CD4 ⁺ regulatory T (Treg) cells, which specifically express the transcription factor forkhead box p3, are potently immunosuppressive and characteristically possess a self-reactive ...T-cell receptor (TCR) repertoire. To determine the molecular basis of Treg suppressive activity and their self-skewed TCR repertoire formation, we attempted to reconstruct these Treg-specific properties in conventional T (Tconv) cells by genetic manipulation. We show that Tconv cells rendered IL-2 deficient and constitutively expressing transgenic cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) were potently suppressive in vitro when they were preactivated by antigenic stimulation. They also suppressed in vivo inflammatory bowel disease and systemic autoimmunity/inflammation produced by Treg deficiency. In addition, in the thymus, transgenic CTLA-4 expression in developing Tconv cells skewed their TCR repertoire toward higher self-reactivity, whereas CTLA-4 deficiency specifically in developing thymic Treg cells cancelled their physiological TCR self-skewing. The extracellular portion of CTLA-4 was sufficient for the suppression and repertoire shifting. It interfered with CD28 signaling to responder Tconv cells via outcompeting CD28 for binding to CD80 and CD86,or modulating CD80/CD86 expression on antigen-presenting cells. Thus, a triad of IL-2 repression, CTLA-4 expression, and antigenic stimulation is a minimalistic requirement for conferring Treg-like suppressive activity on Tconv cells, in accordance with the function of forkhead box p3 to strongly repress IL-2 and maintain CTLA-4 expression in natural Treg cells. Moreover, CTLA-4 expression is a key element for the formation of a self-reactive TCR repertoire in natural Treg cells. These findings can be exploited to control immune responses by targeting IL-2 and CTLA-4 in Treg and Tconv cells.
Regulatory T cells (Treg) are negative regulators of the immune response; however, it is poorly understood whether and how Foxp3 transcription is induced and regulated in the periphery during T‐cell ...responses. Using Foxp3‐Timer of cell kinetics and activity (Tocky) mice, which report real‐time Foxp3 expression, we show that the flux of new Foxp3 expressors and the rate of Foxp3 transcription are increased during inflammation. These persistent dynamics of Foxp3 transcription determine the effector Treg programme and are dependent on a Foxp3 autoregulatory transcriptional circuit. Persistent Foxp3 transcriptional activity controls the expression of coinhibitory molecules, including CTLA‐4 and effector Treg signature genes. Using RNA‐seq, we identify two groups of surface proteins based on their relationship to the temporal dynamics of Foxp3 transcription, and we show proof of principle for the manipulation of Foxp3 dynamics by immunotherapy: new Foxp3 flux is promoted by anti‐TNFRII antibody, and high‐frequency Foxp3 expressors are targeted by anti‐OX40 antibody. Collectively, our study dissects time‐dependent mechanisms behind Foxp3‐driven T‐cell regulation and establishes the Foxp3‐Tocky system as a tool to investigate the mechanisms behind T‐cell immunotherapies.
Synopsis
Temporally‐persistent Foxp3 expression, established via a Foxp3‐protein dependent autoregulatory transcriptional circuit, drives effector regulatory T‐cell differentiation during inflammation.
Foxp3‐Tocky mice reveal that Foxp3 transcription is highly dynamic in T‐cells in vivo.
During inflammation, both the generation of new Foxp3 expressers and the rate of Foxp3 transcription in Treg increases.
Temporally sustained Foxp3 transcription induces effector Treg differentiation through a Foxp3‐protein driven autoregulatory transcriptional loop.
Foxp3‐Tocky can reveal mechanism of action behind immunotherapies.
A novel mouse model allows to track the kinetic and temporal aspects of Foxp3 expression in regulatory T cells during inflammation, showing that Treg development depends on persistent Foxp3 transcription.
Activation of serum complement triggers Th17 cell-dependent spontaneous autoimmune disease in an animal model. In genetically autoimmune-prone SKG mice, administration of mannan or beta-glucan, both ...of which activate serum complement, evoked Th17 cell-mediated chronic autoimmune arthritis. C5a, a chief component of complement activation produced via all three complement pathways (i.e., lectin, classical, and alternative), stimulated tissue-resident macrophages, but not dendritic cells, to produce inflammatory cytokines including IL-6, in synergy with Toll-like receptor signaling or, notably, granulocyte/macrophage colony-stimulating factor (GM-CSF). GM-CSF secreted by activated T cells indeed enhanced in vitro IL-6 production by C5a-stimulated macrophages. In vivo, C5a receptor (C5aR) deficiency in SKG mice inhibited the differentiation/expansion of Th17 cells after mannan or beta-glucan treatment, and consequently suppressed the development of arthritis. Transfer of SKG T cells induced Th17 cell differentiation/expansion and produced arthritis in C5aR-sufficient recombination activating gene (RAG)-/- mice but not in C5aR-deficient RAG-/- recipients. In vivo macrophage depletion also inhibited disease development in SKG mice. Collectively, the data suggest that complement activation by exogenous or endogenous stimulation can initiate Th17 cell differentiation and expansion in certain autoimmune diseases and presumably in microbial infections. Blockade of C5aR may thus be beneficial for controlling Th17-mediated inflammation and autoimmune disease.
OBJECTIVE: Patients with type 1 diabetes and microalbuminuria are at increased risk of cardiovascular disease (CVD). Abnormalities in vascular progenitor cells, which participate in vascular repair, ...may be implicated in this susceptibility. RESEARCH DESIGN AND METHODS: We studied the number and function of vascular progenitor cells in 22 type 1 diabetic patients with history of microalbuminuria (MA⁺) and 22 type 1 diabetic patients without history of microalbuminuria (MA⁻), of similar age, diabetes duration, glycemic control, renal function, and no history of CVD. RESULTS: MA⁺ patients had lower circulating CD34⁺ and CD34⁺/CD133⁺ cell numbers compared with MA⁻ patients (P < 0.006). In in vitro functional assays, MA⁺ patients had a significantly lower number of colony-forming units and impaired vascular endothelial growth factor (VEGF)-A-mediated tube formation, when compared with MA⁻ patients (P < 0.01). CONCLUSIONS: In type 1 diabetic patients with microalbuminuria, a marker of microvascular injury and a risk factor for CVD, circulating vascular progenitor cell number is reduced and function is impaired.
CTLA-4 Control over Foxp3⁺ Regulatory T Cell Function Wing, Kajsa; Onishi, Yasushi; Prieto-Martin, Paz ...
Science (American Association for the Advancement of Science),
10/2008, Letnik:
322, Številka:
5899
Journal Article
Recenzirano
Naturally occurring Foxp3⁺CD4⁺ regulatory T cells (Tregs) are essential for maintaining immunological self-tolerance and immune homeostasis. Here, we show that a specific deficiency of cytotoxic T ...lymphocyte antigen 4 (CTLA-4) in Tregs results in spontaneous development of systemic lymphoproliferation, fatal T cell-mediated autoimmune disease, and hyperproduction of immunoglobulin E in mice, and it also produces potent tumor immunity. Treg-specific CTLA-4 deficiency impairs in vivo and in vitro suppressive function of Tregs--in particular, Treg-mediated down-regulation of CD80 and CD86 expression on dendritic cells. Thus, natural Tregs may critically require CTLA-4 to suppress immune responses by affecting the potency of antigen-presenting cells to activate other T cells.
Currently, molecular diagnosis of haemophilia A and B (HA and HB) highlights the excess risk-inhibitor development associated with specific mutations, and enables carrier testing of female relatives ...and prenatal or preimplantation genetic diagnosis. Molecular testing for HA also helps distinguish it from von Willebrand disease (VWD). Next-generation sequencing (NGS) allows simultaneous investigation of several complete genes, even though they may span very extensive regions. This study aimed to evaluate the usefulness of a molecular algorithm employing an NGS approach for sequencing the complete F8, F9 and VWF genes. The proposed algorithm includes the detection of inversions of introns 1 and 22, an NGS custom panel (the entire F8, F9 and VWF genes), and multiplex ligation-dependent probe amplification (MLPA) analysis. A total of 102 samples (97 FVIII- and FIX-deficient patients, and five female carriers) were studied. IVS-22 screening identified 11 out of 20 severe HA patients and one female carrier. IVS-1 analysis did not reveal any alterations. The NGS approach gave positive results in 88 cases, allowing the differential diagnosis of mild/moderate HA and VWD in eight cases. MLPA confirmed one large exon deletion. Only one case did have no pathogenic variants. The proposed algorithm had an overall success rate of 99 %. In conclusion, our evaluation demonstrates that this algorithm can reliably identify pathogenic variants and diagnose patients with HA, HB or VWD.
Hemato-oncologic patients with chemotherapy-induced thrombocytopenia are one of the populations receiving platelet transfusions. The general practice with these patients is to give prophylactic ...platelet transfusions when platelet counts fall below 10×109PLT/L. However, in more than 40% of these patients, platelet transfusion does not prevent bleeding. The reason of the low efficacy of platelet transfusion in the context of chemotherapy patients is not entirely understood.
We therefore aimed at immunophenotyping the expression of platelet surface and activation markers and thrombopoietin levels from hemato-oncologic patients before and after transfusion. A more detailed follow-up was performed in three patients that underwent autologous bone marrow transplantation.
As previously reported, basal platelet activation was observed in hemato-oncologic patients. Based on flow cytometry parameters, i.e. the percentage of positivity and mean fluorescence intensity (MFI) distribution, our data provide an additional interpretation of platelet acquired qualitative changes in the hemato-oncologic patient. From our results we propose: first, the underlying activation of platelets in the hemato-oncologic patient is accompanied by loss of expression of the platelet receptors that are susceptible to protease-mediated shedding; second, soon after transfusion, the newly circulating donor platelets show additional activation, which may result in subsequent platelet receptor recycling and potential accelerated clearance of these activated platelets.
In conclusion, the immunophenotype of circulating platelets changes after prophylactic platelet transfusion. Next to platelet count increment, exploration of this immunophenotype might help to explain transfusion refractory bleeding in hemato-oncologic patients. Eventually this may lead to personalization and improvement of the present platelet transfusion support regime.
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