Project description:During persistent antigen stimulation, CD8+ cytolytic T cells (CTL) show a gradual decrease in effector function, or “exhaustion”, which impairs the immune response to tumors and infections. Here we show that NFAT, a transcription factor with an established role in T cell activation, in parallel controls a second transcriptional program conferring the characteristic features of CD8+ T cell exhaustion, including upregulation of genes encoding inhibitory cell surface receptors and diminished TCR signaling. Expression of an engineered NFAT1, which induces this negative regulatory program in the absence of the effector program, interferes with the ability of CD8+ T cells to protect against Listeria infection or attenuate tumor growth in vivo. NFAT elicits this second program of gene expression in large part by binding to a subset of the sites occupied by NFAT during a typical effector response, suggesting that a balance between the two pathways determines the outcome of TCR signaling. Determination of NFAT1 binding sites in CD8 T cells in vitro
Project description:During persistent antigen stimulation, CD8+ cytolytic T cells (CTL) show a gradual decrease in effector function, or “exhaustion”, which impairs the immune response to tumors and infections. Here we show that NFAT, a transcription factor with an established role in T cell activation, in parallel controls a second transcriptional program conferring the characteristic features of CD8+ T cell exhaustion, including upregulation of genes encoding inhibitory cell surface receptors and diminished TCR signaling. Expression of an engineered NFAT1, which induces this negative regulatory program in the absence of the effector program, interferes with the ability of CD8+ T cells to protect against Listeria infection or attenuate tumor growth in vivo. NFAT elicits this second program of gene expression in large part by binding to a subset of the sites occupied by NFAT during a typical effector response, suggesting that a balance between the two pathways determines the outcome of TCR signaling. Understanding the role of CA-RIT-NFAT1 in T cells
Project description:Natalizumab is a recombinant monoclonal antibody raised against integrin alpha-4 (CD49d). It is approved for the treatment of patients with multiple sclerosis (MS), a chronic inflammatory autoimmune disease of the CNS. Natalizumab blocks leukocyte extravasation across the blood-brain barrier by inhibiting the molecular interaction between integrin alpha-4/beta-1 heterodimers expressed on leukocytes and VCAM-1 on inflammatory-activated CNS endothelium. Here we investigated whether binding of this adhesion-blocking antibody to T lymphocytes modulated their phenotype by direct induction of intracellular signaling events. Natalizumab induced a mild upregulation of IL-2, IFN-gamma and IL-17 expression in activated primary human CD4+ T cells propagated ex vivo from healthy donors, consistent with a pro-inflammatory costimulatory effect on lymphokine expression. Overall, the relative effect of natalizumab was more pronounced in less than in fully activated T cells. Along with this, natalizumab binding triggered rapid MAPK/ERK phosphorylation. Furthermore, it decreased CD49d surface expression on effector cells within a few hours. Sustained CD49d downregulation could be attributed to integrin internalization and degradation. Importantly, also CD4+ T cells from some MS patients receiving their very first dose of natalizumab produced more IL-2, IFN-gamma and IL-17 already 24 h after infusion. Together these data indicate that in addition to its adhesion-blocking mode of action, natalizumab possesses mild direct signaling capacities, which may support a pro-inflammatory phenotype of peripheral blood T lymphocytes. This might explain why a rebound of disease activity is observed in some MS patients after natalizumab cessation. Human CD4+ T cells from healthy donors were stimulated in the absence or presence of natalizumab for 10 days. To recall the developed program and induce cytokine expression, half of the cells were restimulated for a further 8 h.
Project description:Background: Chronic rhinosinusitis with nasal polyposis (CRSwNP) in western countries is characterized by eosinophilia, IgE production and Th2 cytokine expression. Type 2 innate lymphoid cells (ILC2) from polyps produce IL-5 and IL-13 in response to IL-25 and IL-33 although the relevance of this axis to local mucosal T cell responses is unknown. Objective: To investigate the role of the IL-25/IL-33 axis in local mucosal T cell responses in CRSwNP. Methods: Polyp tissue and blood were obtained from patients undergoing nasal polypectomy. Control nasal biopsies and blood were obtained from healthy volunteers. Tissue was cultured in a short-term explant model. T cell surface phenotype/intracellular cytokines were assessed by flow cytometry. TCR Vβ analysis was performed with the immunoSEQ assay. Microarrays were performed for gene expression analysis. Results: Using nasal polyp tissue, numerous IL-25 receptor (IL-17RB) positive polarized Th2 cells were identified which were absent in the healthy nasal mucosa and periphery. IL-17RB+CD4+ polyp Th2 cells co-expressed ST2 (IL-33 receptor) and responded to IL-25 and IL-33 with enhanced IL-5 and IL-13 production. Within IL-17RB+CD4+ T cells several identical TCR Vβ CDR3 sequences were identified in different subjects suggesting clonal expansion driven by a common antigen. Abundant IL-17 producing T cells were observed in healthy nasal mucosal and polyp populations with Th17-related genes the most overexpressed compared to peripheral blood T cells. Conclusion: IL-25 and IL-33 may interact locally with IL-17RB+ST2+ polyp T cells to augment Th2 responses in CRSwNP. A local Th17 response may be the default signature in healthy nasal mucosal immune homeostasis. Three biological replicates. T-helper cells were isolated nasal polyps by explant culture from patients with chronic rhinosinusitis. Cells were then sorted based upon expression of IL17RB by flow cytometric sorting. Resting and activated IL-17RB+ve cells were compared with resting and activated IL-17RB-ve cells.
Project description:The protease activity of the paracaspase MALT1 plays an important role in antigen receptor-mediated lymphocyte activation by controlling the activity of the transcription factor NF-kB and is thus essential for the expression of inflammatory target genes. MALT1 is not only present in cells of the hematopoietic lineage, but is ubiquitously expressed. Here we report that Zymosan or S. aureus stimulation induced MALT1 protease activity in human primary keratinocytes. Human primary keratinocytes were treated for 8 h with solvent control (DMSO), PMA/Ionomycin (P/I) or P/I with MALT1-inhibitor LVSR-fmk. Three biological replicates of each stimualtion were analyzed for gene expression profiles.
Project description:we used genome-wide transcriptome analysis to profile the mRNA, long noncoding RNA (lncRNA), and microRNA (miRNA) expression of B10 cells, an antigen-specific Cd1dhiCd5+Cd19hiIl10 competent regulatory B cell. Potential key upstream regulators (including transcription factors, cytokines, trans-membrane receptors, and kinases) for Breg biogenesis and function were identified. B10+ B cells (Cd1dhiCd5+Cd19hiIl10+) and B10- cells (Cd1d-Cd5-Cd19hiIl10-) from mouse splenic B cell were sorted for RNA preparation. Two independent repeats were prepared for RNA-seq
Project description:The complex relationship between Th1 and Th17 cells is incompletely understood. The transcription factor T-bet is best known as the master regulator of Th1 lineage commitment. However, attention is now focused on the repression of alternate T cell subsets mediated by T-bet, particularly the Th17 lineage. Specifically it has recently been suggested that pathogenic Th17 cells express T-bet and are dependent on IL-23. However, T-bet has previously been shown to be a negative regulator of Th17 cells. We have taken an unbiased approach to determine the functional impact of T-bet on Th17 lineage commitment. Genome-wide analysis of functional T-bet binding sites provides an improved understanding of the transcriptional regulation mediated by T-bet, and suggests novel mechanisms by which T-bet regulates T helper cell differentiation. Specifically, we show that T-bet negatively regulates Th17 lineage commitment via direct repression of the transcription factor interferon regulatory factor-4 (IRF4). An in vivo analysis of the pathogenicity of T-bet deficient T cells demonstrated that Th17 responses were augmented in the absence of T-bet, and we have defined a critical temporal window for T-bet function. The interaction of the two key transcription factors T-bet and IRF4 during the determination of T cell fate choice significantly advances our understanding of the mechanisms underlying the development of pathogenic T cells. ChIP-seq analysis of T-bet in WT and Tbet -/- mice.