Project description:We show that Ly49+CD122+ CD8+ Treg have a distinct expression profie compared to conventional Foxp3+ CD4+ Treg and CD49 lo CD122+ CD8 effector like cells. RNA was extracted from sorted cells and sent for sequencing
Project description:Human FOXP3+CD25+CD4+ regulatory T cells (Tregs) play a dominant role in the maintenance of immune homeostasis. Several genes are known to be important for murine Tregs, but for human Tregs the genes and underlying molecular networks controlling the suppressor function still largely remain unclear. We here performed a high-time-resolution dynamic analysis of the transcriptome during the very early phase of human Treg/ CD4+ T-effector cell activation. After constructing a correlation network specific for Tregs based on these dynamic data, we described a strategy to identify key genes by directly analyzing the constructed undirected correlation network. Six out of the top 10 ranked key hubs are known to be important for Treg function or involved in autoimmune diseases. Surprisingly, PLAU (the plasminogen activator urokinase) was among the 4 new key hubs. We here show that PLAU was critical for expression regulation of FOXP3, EOS and several other important Treg genes and the suppressor function of human Tregs. Moreover, we found Plau inhibits murine Treg development and but promotes the suppressive function. Further analysis unveils that PLAU is particularly important for memory Tregs and that PLAU mediates Treg suppressor function via STAT5 and ERK signaling pathways. Our study shows the potential for identifying novel key genes for complex dynamic biological processes using a network strategy based on high-time-resolution data, and highlights a critical role of PLAU in both human and murine Tregs. The construction of a dynamic correlation network of human Tregs provides a useful resource for the understanding of Treg function and human autoimmune diseases. The high-time-resolution time-series transcriptomic data during the very early phase of human Treg/Teff activation could be generally used for further mechanistic analysis of human Treg function. These data could be further used for biological network analysis, dynamic analysis, modeling by experimental researchers, bioinformaticians, computational biologists and systems biologists. We have measured the genome-wide expression of 38,500 genes (probes) by performing a high-time-resolution time-series analysis during the activation process of human regulatory T cells /CD4+ T-effector cells at 19 time points for the first 6h with an equal interval of 20 min. We have also overexpressed the GARP gene in human effector T cells and measured the genome-scale expression for the GARP-overexpressed cells and ThGFP cells at time point 0, 100 and 360min following activation. The stimulation source used in this work is a combination of anti-CD3/-CD28 Dynal beads with IL2 100U/ml.
Project description:Gene expression profiles were compared between regulatory T cells (Treg) and Effector CD4+ T cells in healthy B6 mice and sick mice with scurfy mutation. We used microarrays to elucidate the molecular mechanisms underlying the suppression function of the Treg cells. Experiment Overall Design: To identify candidate genes that might be related to the suppressive activity, the Treg cells expressing functional and mutant Foxp3 transcription factor were compared
Project description:Regulatory T (Treg) cells can facilitate transplant tolerance and attenuate autoimmune- and inflammatory diseases. Therefore, it is clinically relevant to stimulate Treg cell expansion and function in vivo and to create therapeutic Treg cell products in vitro. We report that TNF receptor 2 (TNFR2) is a unique costimulus for naïve, thymus-derived (t)Treg cells from human blood that promotes their differentiation into non-lymphoid tissue (NLT)-resident effector Treg cells, without Th-like polarization. In contrast, CD28 costimulation maintains a lymphoid tissue (LT)-resident Treg cell phenotype. We base this conclusion on transcriptome and proteome analysis of TNFR2- and CD28-costimulated CD4+ Treg cells and conventional T (Tconv) cells, followed by bioinformatic comparison with published transcriptomic Treg cell signatures from NLT and LT in health and disease, including autoimmunity and cancer. These analyses illuminate that TNFR2 costimulation promotes Treg cell capacity for survival, migration, immunosuppression and tissue regeneration. Functional studies confirmed improved migratory ability of TNFR2-costimulated tTreg cells. Flow cytometry validated the presence of the TNFR2-driven Treg cell signature in effector/memory Treg cells from the human placenta as opposed to blood. Thus, TNFR2 can be exploited as driver of NLT-resident Treg cell differentiation for adoptive cell therapy or antibody-based immunomodulation in human disease.
Project description:Gene-edited rats were generated in which EGFP was placed under the transcriptional control of the Foxp3 promoter. EGFP expression by CD4+ and CD8+ T cells allowed to define regulatory T cells (Treg) in both T cell compartments. This Foxp3-EGFP rats constitute a useful model to identify CD4+ and CD8+ natural and induced Treg. Transcriptomic analyses showed similarities but also differences among CD4+ and CD8+ EGFP+ cells, this being the first description of the transcriptomic profile in natural FOXP3+ CD8+ Treg.
Project description:Through their functional diversification, CD4+ T cells play key roles in both driving and constraining immune-mediated pathology. Transcription factors are critical in the generation and maintenance of cellular diversity and negative regulators antagonistic to alternate fates often act in conjunction with positive regulators to stabilize lineage specification1. Polymorphisms within the locus encoding a transcription factor BACH2 are associated with diverse immune-mediated diseases including asthma2, multiple sclerosis3, Crohn¹s disease4-5, coeliac disease6, vitiligo7 and type 1 diabetes8. A role for Bach2 in maintaining immune homeostasis, however, has not been established. Here, we define Bach2 as a broad regulator of immune activation that stabilizes immunoregulatory capacity while repressing the differentiation programmes of multiple effector lineages in CD4+ T cells. Bach2 was required for efficient formation of regulatory (Treg) cells and consequently for suppression of lethal inflammation in a manner that was Treg cell dependent. Assessment of the genome-wide function of Bach2, however, revealed that it represses genes associated with effector cell differentiation. Consequently, its absence during Treg polarization resulted in inappropriate diversion to effector lineages. In addition, Bach2 constrained full effector differentiation within Th1, Th2 and Th17 cell lineages. These findings identify Bach2 as a key regulator of CD4+ T-cell differentiation that prevents inflammatory disease by controlling the balance between tolerance and immunity. The role of Bach2t to regulate immune homeostasis was investigated by mapping DNA binding profiles of Bach2 in iTreg condition. The function of Bach2 was also evaluated by comparing transcriptome in WT and Bach2-deficient iTreg cells and further comparison was done with transcriptome in naive, Th1, Th2, and Th17 conditions.
Project description:Gene expression profiles were compared between regulatory T cells (Treg) and Effector CD4+ T cells in healthy B6 mice and sick mice with scurfy mutation. We used microarrays to elucidate the molecular mechanisms underlying the suppression function of the Treg cells. Keywords: Cell type comparison
Project description:Though T cell expansion and effector differentiation are triggered and, perhaps, maintained by antigen, the proliferative behaviors of CD4+ and CD8+ T cells responding to timed antigen presentation have rarely been compared side by side. Proliferation and effector differentiation of TCR transgenic and polyclonal T cells were analyzed following transient and continuous TCR signals. We found CD4+ T cell proliferation to be dependent on prolonged antigen presence, whereas CD8+ T cells were able to divide and differentiate into effector cells in the absence of it. We excluded CD4+ T cell proliferation to be abrogated by coinhibitory signals or the lack of inflammatory stimuli and found that autonomous proliferation of CD8* T cells was independent of any MHC class I signals. Gene expression analyses illustrated differences in global gene transcription between the two subsets following stimulation periods of different lengths. These T cell data reflect the MHC class difference in that class II but not class I molecules were stabilized on activated DCs in vivo, suggesting a coevolution of MHC molecules and their respective T cell subsets. Samples 1-12: Analysis on day 2. Purified CD4+ AND-TCR transgenic cells and CD8+ OT1-TCR transgenic cells were separately stimulated with anti-CD3 and anti-CD28 antibodies. 48 hours later, the cells were sorted again to a purity of >99 %. Extracted total RNA was amplified twice and hybridized on Affymetrix Mouse 430A2 microarrays. First, we analysed the changes of the CD4+ and CD8+ T cells after stimulation. Second, we compared the differences of the changes between the two cell types after stimulation. For each of the four groups (CD4+ and CD8+, stimulated and unstimulated), we analysed three independent biological replicates. Samples 13-28: Analysis on day 5. AND and OT1 TCR-transgenic T cells were prepared as described before, but transferred into mice that do not or do present their respective antigens. 72 hours later, the cells were FACS-sorted twice to >99 % purity, directly into Trizol. For each of the six groups (CD4+ and CD8+, unstimulated, transient (2 days) and continuous (5 days) stimulation), three independent biological replicates were analyzed, except for CD4+ unstimulated and CD4+ transient, with two replicates each.
Project description:Most people infected by EBV acquire specific immunity, which then controls latent infection throughout their life. Immune surveillance of EBV-infected cells by cytotoxic CD4+ T cells has been recognized; however, the molecular mechanism of generating cytotoxic effector T cells of the CD4+ subset remains poorly understood. Here we compared phenotypic features and the transcriptome of EBV-specific effector-memory CD4+ T cells and CD8+ T cells in mice and found that both T cell types show cytotoxicity and, to our surprise, widely similar gene expression patterns relating to cytotoxicity. Similar to cytotoxic CD8+ T cells, EBV-specific cytotoxic CD4+ T cells from human peripheral blood expressed T-bet, Granzyme B, and Perforin and upregulated the degranulation marker, CD107a, immediately after restimulation. Furthermore, T-bet expression in cytotoxic CD4+ T cells was highly correlated with Granzyme B and Perforin expression at the protein level. Thus, differentiation of EBV-specific cyto toxic CD4+ T cells is possibly controlled by mechanisms shared by cytotoxic CD8+ T cells. T-bet-mediated transcriptional regulation may explain the similarity of cytotoxic effector differentiation between CD4+ T cells and CD8+ T cells, implicating that this differentiation pathway may be directed by environmental input rather than T cell subset.
Project description:In this screen we compared cDNA from rTreg (AICD resistent TReg cells) against cDNA derived from all CD4+CD25hi Treg for further molecular characterization of rTreg Experiment Overall Design: Single comparison of rTReg vs. TReg. We pooled rTreg-cDNA derived from FACS-sorted CD4+CD25hi Treg of eight healthy blood donors and performed gene chip microarray analysis.