RNA-seq of expanded human CRISPR/HDR-mediated FOXP3-KO naive Tregs, Cas9 naive Tregs, and conventional T cells
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA735242 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR14736800_1.fastq.gz | Fastqsanger.gz | |||
| SRR14736800_2.fastq.gz | Fastqsanger.gz | |||
| SRR14736801_1.fastq.gz | Fastqsanger.gz | |||
| SRR14736801_2.fastq.gz | Fastqsanger.gz | |||
| SRR14736802_1.fastq.gz | Fastqsanger.gz |
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Project description:Treg cell therapy is a promising curative approach for a variety of immune-mediated conditions. CRISPR-based genome editing allows precise insertion of transgenes through homology-directed repair, but its use in human Tregs has been limited. We report an optimized protocol for CRISPR-mediated gene knock-in in human Tregs with high-yield expansion. To establish a benchmark of human Treg dysfunction, we target the master transcription factor FOXP3 in naive and memory Tregs. Although FOXP3-ablated Tregs upregulate cytokine expression, effects on suppressive capacity in vitro manifest slowly and primarily in memory Tregs. Moreover, FOXP3-ablated Tregs retain their characteristic protein, transcriptional, and DNA methylation profile. Instead, FOXP3 maintains DNA methylation at regions enriched for AP-1 binding sites. Thus, while FOXP3 is important for human Treg development, it has a limited role in maintaining mature Treg identity. Optimized gene knock-in with human Tregs will enable mechanistic studies and the development of tailored, next-generation Treg cell therapies.
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Project description:Treg cell therapy is a promising curative approach for a variety of immune-mediated conditions. CRISPR-based genome editing allows precise insertion of transgenes through homology-directed repair, but its use in human Tregs has been limited. We report an optimized protocol for CRISPR-mediated gene knock-in in human Tregs with high-yield expansion. To establish a benchmark of human Treg dysfunction, we target the master transcription factor FOXP3 in naive and memory Tregs. Although FOXP3-ablated Tregs upregulate cytokine expression, effects on suppressive capacity in vitro manifest slowly and primarily in memory Tregs. Moreover, FOXP3-ablated Tregs retain their characteristic protein, transcriptional, and DNA methylation profile. Instead, FOXP3 maintains DNA methylation at regions enriched for AP-1 binding sites. Thus, while FOXP3 is important for human Treg development, it has a limited role in maintaining mature Treg identity. Optimized gene knock-in with human Tregs will enable mechanistic studies and the development of tailored, next-generation Treg cell therapies.
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Project description:Genome-wide DNA methylation profile of expanded human CRISPR/HDR-mediated FOXP3-KO naive Tregs, Cas9 naive Tregs, and conventional T cells
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2022-02-25 | E-MTAB-11442 | biostudies-arrayexpress
Project description:Tregs and Tcons were isolated by flow cytometry from the spleen of naïve mice and from the spleen and central nervous system (brain + spinal cord) (CNS) of mice at the peak of experimental autoimmune encephalomyelitis (EAE) induced by immunization with MOG(35-55). Samples at EAE peak were collected at day 16-18 after immunization. Purity was >99.5%. Foxp3.eGFP mice (provided by A. Rudensky) were used to identify Tregs. A bin channel excluded CD11b+ cells during the sort. Mice were 6-10 weeks of age at the time of immunization. The goal was to identify genes differentially expressed between Tregs and Tcons from naive mice, and genes differentially expressed by these cells in the inflamed tissue (central nervous system) at the peak of the disease.
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Project description:Objectives. Immune homeostasis in the intestinal tract is tightly controlled by FOXP3+ regulatory T cells (Tregs), with loss of this Treg-mediated control linked to development of chronic conditions, such as inflammatory bowel disease (IBD). As a mechanism of immune evasion, several species of intestinal parasites strengthen intestinal Treg activity, leading to the notion that parasite-derived products could be harnessed and used as an immunoregulatory therapy for IBD. It has been previously demonstrated that the parasite Heligmosomoides polygyrus secretes a molecule (Hp-TGM) which mimics the ability of TGF-β to induce FOXP3 expression in CD4+ T cells. Our aim was to investigate whether Hp-TGM could induce human FOXP3+ Tregs as a potential therapeutic approach. Methods. Human CD4+ T cells from healthy volunteers were expanded in the presence of Hp-TGM or mammalian TGF-β. The induction of Tregs was measured by flow cytometric detection of FOXP3 and other Treg markers, such as CTLA-4 and CD25. Epigenetic changes were detected using ChIP-Seq and pyrosequencing of FOXP3. Treg phenotype stability was assessed following inflammatory cytokine challenge and Treg function was assessed by cellular co-culture suppression assays and secreted cytokines measured by cytometric bead array. Results. Hp-TGM efficiently induced FOXP3 expression (>60%), in addition to another Treg functional marker CTLA-4. Hp-TGM caused epigenetic modification of the FOXP3 loci to a greater extent than did TGF-β. Hp-TGM-induced Tregs also had superior suppressive function compared to TGF--induced Tregs, and retained their phenotype following exposure to inflammatory cytokines. Hp-TGM also induced a Treg phenotype in in vivo differentiated Th17 cells, indicating its potential to re-program memory cells to enhance immune tolerance. Conclusion. These data indicate Hp-TGM has the potential to be used to generate stable human FOXP3+ Tregs to treat IBD.
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