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Epigenetic features such as DNA accessibility dictate transcriptional regulation in a cell type- and cell state- specific manner, and mapping this in health vs. disease in clinically relevant material is opening the door to new mechanistic insights and new targets for therapy. Assay for Transposase Accessible Chromatin Sequencing (ATAC-seq) allows chromatin accessibility profiling from low cell input, making it tractable on rare cell populations, such as regulatory T (Treg) cells. However, little is known about the compatibility of the assay with cryopreserved rare cell populations. Here we demonstrate the robustness of an ATAC-seq protocol comparing primary Treg cells recovered from fresh or cryopreserved PBMC samples, in the steady state and in response to stimulation. We extend this method to explore the feasibility of conducting simultaneous quantitation of chromatin accessibility and transcriptome from a single aliquot of 50,000 cryopreserved Treg cells. Profiling of chromatin accessibility and gene expression in parallel within the same pool of cells controls for cellular heterogeneity and is particularly beneficial when constrained by limited input material. Overall, we observed a high correlation of accessibility patterns and transcription factor dynamics between fresh and cryopreserved samples. Furthermore, highly similar transcriptomic profiles were obtained from whole cells and from the supernatants recovered from ATAC-seq reactions. We highlight the feasibility of applying these techniques to profile the epigenomic landscape of cells recovered from cryopreservation biorepositories. [ABSTRACT FROM AUTHOR]

There has been much interest in the ability of regulatory T cells (Treg) to switch function in vivo , either as a result of genetic risk of disease or in response to environmental and metabolic cues. The relationship between levels of FOXP3 and functional fitness plays a significant part in this plasticity. There is an emerging role for Treg in tissue repair that may be less dependent on FOXP3, and the molecular mechanisms underpinning this are not fully understood. As a result of detailed, high-resolution functional genomics, the gene regulatory networks and key functional mediators of Treg phenotype downstream of FOXP3 have been mapped, enabling a mechanistic insight into Treg function. This transcription factor-driven programming of T-cell function to generate Treg requires the switching on and off of key genes that form part of the Treg gene regulatory network and raises the possibility that this is reversible. It is plausible that subtle shifts in expression levels of specific genes, including transcription factors and non-coding RNAs, change the regulation of the Treg gene network. The subtle skewing of gene expression initiates changes in function, with the potential to promote chronic disease and/or to license appropriate inflammatory responses. In the case of autoimmunity, there is an underlying genetic risk, and the interplay of genetic and environmental cues is complex and impacts gene regulation networks frequently involving promoters and enhancers, the regulatory elements that control gene expression levels and responsiveness. These promoter–enhancer interactions can operate over long distances and are highly cell type specific. In autoimmunity, the genetic risk can result in changes in these enhancer/promoter interactions, and this mainly impacts genes which are expressed in T cells and hence impacts Treg/conventional T-cell (Tconv) function. Genetic risk may cause the subtle alterations to the responsiveness of gene regulatory networks which are controlled by or control FOXP3 and its target genes, and the application of assays of the 3D organization of chromatin, enabling the connection of non-coding regulatory regions to the genes they control, is revealing the direct impact of environmental/metabolic/genetic risk on T-cell function and is providing mechanistic insight into susceptibility to inflammatory and autoimmune conditions. [ABSTRACT FROM AUTHOR]

Abstract: The peptidase inhibitor PI16 was shown previously by microarray analysis to be over-expressed by CD4-positive/CD25-positive Treg compared with CD4-positive/CD25-negative Th cells. Using a monoclonal antibody to the human PI16 protein, we found that PI16-positive Treg have a memory (CD45RO-positive) phenotype and express higher levels of FOXP3 than PI16-negative Treg. PI16-positive Treg are functional in suppressor assays in vitro with potency similar to PI16-negative Treg. Further phenotyping of the PI16-positive Treg revealed that the chemokine receptors CCR4 and CCR6 are expressed by more of the PI16-positive/CD45RO-positive Treg compared with PI16-negative/CD45RO-positive Treg or Th cells. PI16-positive Treg showed enhanced in vitro migration towards the inflammatory chemokines CCL17 and CCL20, suggesting they can migrate to sites of inflammation. We conclude that PI16 identifies a novel distinct subset of functional memory Treg which can migrate to sites of inflammation and regulate the pro-inflammatory response at those sites. [Copyright &y& Elsevier]

Abstract: The epithelial tight junction forms a barrier to paracellular solute movement. In this study we show that the heterotrimeric G-protein Gα13 regulates the epithelial tight junction barrier. We generated MDCKII kidney epithelial cell lines in which the expression of an active Gα13 mutant (Gα13Q226L) could be induced. We demonstrated that Gα13Q226L expression increased paracellular permeability and caused the disruption and redistribution of proteins comprising the tight junction and the adherens junction away from sites of cell contact and the appearance of basal stress fibers. The effects on the junctional proteins and the actin cytoskeleton were abrogated by the Rho kinase inhibitor Y27632 but not by the Src kinase inhibitor PP2. The Gα13 mediated increase in permeability was also Src kinase independent but was partly dependent on Rho kinase signalling. Our data establish a link between Gα13, Rho kinase signaling and epithelial barrier function and not only demonstrate that Gα13 regulates epithelial apical junction properties but that it does so via signaling pathways that are distinct from the closely related protein Gα12. [Copyright &y& Elsevier]