Distinct Foxp3 enhancer elements coordinate development, maintenance, and function of regulatory T cells.

The transcription factor Foxp3 plays crucial roles for Treg cell development and function. Conserved non-coding sequences (CNSs) at the Foxp3 locus control Foxp3 transcription, but how they developmentally contribute to Treg cell lineage specification remains obscure. Here, we show that among Foxp3 CNSs, the promoter-upstream CNS0 and the intergenic CNS3, which bind distinct transcription factors, were activated at early stages of thymocyte differentiation prior to Foxp3 promoter activation, with sequential genomic looping bridging these regions and the promoter. While deletion of either CNS0 or CNS3 partially compromised thymic Treg cell generation, deletion of both completely abrogated the generation and impaired the stability of Foxp3 expression in residual Treg cells. As a result, CNS0 and CNS3 double-deleted mice succumbed to lethal systemic autoimmunity and inflammation. Thus, hierarchical and coordinated activation of Foxp3 CNS0 and CNS3 initiates and stabilizes Foxp3 gene expression, thereby crucially controlling Treg cell development, maintenance, and consequently immunological self-tolerance.

Epigenetic conversion of conventional T cells into regulatory T cells by CD28 signal deprivation.

Foxp3-expressing regulatory T cells (Tregs) can be generated in vitro by antigenic stimulation of conventional T cells (Tconvs) in the presence of TGF-ß and IL-2. However, unlike Foxp3+ naturally occurring Tregs, such in vitro induced Tregs (iTregs) are functionally unstable mainly because of incomplete Treg-type epigenetic changes at Treg signature genes such as Foxp3 Here we show that deprivation of CD28 costimulatory signal at an early stage of iTreg generation is able to establish Treg-specific DNA hypomethylation at Treg signature genes. It was achieved, for example, by TCR/TGF-ß/IL-2 stimulation of CD28-deficient Tconvs or CD28-intact Tconvs without anti-CD28 agonistic mAb or with CD80/CD86-blocked or -deficient antigen-presenting cells. The signal abrogation could induce Treg-type hypomethylation in memory/effector as well as naive Tconvs, while hindering Tconv differentiation into effector T cells. Among various cytokines and signal activators/inhibitors, TNF-α and PKC agonists inhibited the hypomethylation. Furthermore, CD28 signal deprivation significantly reduced c-Rel expression in iTregs; and the specific genomic perturbation of a NF-κB binding motif at the Foxp3 CNS2 locus enhanced the locus-specific DNA hypomethylation even in CD28 signaling-intact iTregs. In addition, in vitro maintenance of such epigenome-installed iTregs with IL-2 alone, without additional TGF-ß or antigenic stimulation, enabled their expansion and stabilization of Treg-specific DNA hypomethylation. These iTregs indeed stably expressed Foxp3 after in vivo transfer and effectively suppressed antigen-specific immune responses. Taken together, inhibition of the CD28-PKC-NF-κB signaling pathway in iTreg generation enables de novo acquisition of Treg-specific DNA hypomethylation at Treg signature genes and abundant production of functionally stable antigen-specific iTregs for therapeutic purposes.

Scalable, multimodal profiling of chromatin accessibility, gene expression and protein levels in single cells.

Recent technological advances have enabled massively parallel chromatin profiling with scATAC-seq (single-cell assay for transposase accessible chromatin by sequencing). Here we present ATAC with select antigen profiling by sequencing (ASAP-seq), a tool to simultaneously profile accessible chromatin and protein levels. Our approach pairs sparse scATAC-seq data with robust detection of hundreds of cell surface and intracellular protein markers and optional capture of mitochondrial DNA for clonal tracking, capturing three distinct modalities in single cells. ASAP-seq uses a bridging approach that repurposes antibody:oligonucleotide conjugates designed for existing technologies that pair protein measurements with single-cell RNA sequencing. Together with DOGMA-seq, an adaptation of CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing) for measuring gene activity across the central dogma of gene regulation, we demonstrate the utility of systematic multi-omic profiling by revealing coordinated and distinct changes in chromatin, RNA and surface proteins during native hematopoietic differentiation and peripheral blood mononuclear cell stimulation and as a combinatorial decoder and reporter of multiplexed perturbations in primary T cells.