Browse
Submit Data
Databases
API
Help

Dataset Information

29 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Fast and Efficient Genome Editing of Human FOXP3⁺ Regulatory T Cells.

ABSTRACT: FOXP3⁺ regulatory T cells (Tregs) are central for maintaining peripheral tolerance and immune homeostasis. Because of their immunosuppressive characteristics, Tregs are a potential therapeutic target in various diseases such as autoimmunity, transplantation and infectious diseases like COVID-19. Numerous studies are currently exploring the potential of adoptive Treg therapy in different disease settings and novel genome editing techniques like CRISPR/Cas will likely widen possibilities to strengthen its efficacy. However, robust and expeditious protocols for genome editing of human Tregs are limited. Here, we describe a rapid and effective protocol for reaching high genome editing efficiencies in human Tregs without compromising cell integrity, suitable for potential therapeutic applications. By deletion of IL2RA encoding for IL-2 receptor α-chain (CD25) in Tregs, we demonstrated the applicability of the method for downstream functional assays and highlighted the importance for CD25 for in vitro suppressive function of human Tregs. Moreover, deletion of IL6RA (CD126) in human Tregs elicits cytokine unresponsiveness and thus may prevent IL-6-mediated instability of Tregs, making it an attractive target to potentially boost functionality in settings of adoptive Treg therapies to contain overreaching inflammation or autoimmunity. Thus, our rapid and efficient protocol for genome editing in human Tregs may advance possibilities for Treg-based cellular therapies.

SUBMITTER: Van Zeebroeck L

PROVIDER: S-EPMC8365355 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Activated human Foxp3+ regulatory T cells produce membrane-bound TNF.

Project description:TNF is a multifunctional cytokine that is critical to host defense against pathogens but can also drive the pathophysiology of inflammatory diseases. Inhibition of TNF occasionally causes exacerbation of some autoimmune diseases, suggesting a role for TNF in the regulation of immune homeostasis. Here, we demonstrate that human peripheral blood CD4+CD25+Foxp3+ regulatory T cells (Tregs) express membrane-bound TNF, a potent activator of the type 2 TNF receptor. While the type 1 TNF receptor can cause cell death and is expressed ubiquitously, the type 2 receptor promotes cell growth and its expression is limited mainly to immune and endothelial cells. When autocrine TNF is blocked in an in vitro culture without IL-2, activated Tregs stop proliferating. These data indicate a novel role for TNF as a Treg-derived autocrine growth factor.

| S-EPMC6281785 | biostudies-literature

Efficient Therapeutic Function and Mechanisms of Human Polyclonal CD8+CD103+Foxp3+ Regulatory T Cells on Collagen-Induced Arthritis in Mice.

Project description:ObjectiveTo investigate the potential therapeutic effect in a rheumatoid arthritis model of stable human CD8+ regulatory T cells (hCD8+Tregs) induced by TGF-β1 and rapamycin (RAPA) in vitro.MethodsHuman CD8+T cells were isolated from human peripheral blood mononuclear cells and induced/expanded with TGF-β1 and RAPA along with anti-CD3/28 beads and IL-2 in vitro and harvested as hCD8+Tregs. The phenotypes, suppressive characteristics, and stability of the hCD8+Tregs in an inflammatory microenvironment were examined in vitro. Human CD8+Tregs were transfused into an acollagen-induced arthritis (CIA) mouse model, and their therapeutic effects and related mechanisms were investigated.ResultsHuman CD8+Tregs induced by TGF-β1/RAPA showed high expression of Foxp3 and CD103, exhibited vigorous suppression ability, and were stable in inflammatory microenvironments. In CIA mice, the clinical scores, levels of anti-collagen IgG antibody, and cartilage destruction were significantly reduced after adoptive transfusion with hCD8+Tregs. Moreover, hCD8+Treg treatment significantly reduced the number of Th17 cells, increased the number of CD4+IFN-γ +T cells, and produced self CD4+Foxp3+Tregs in vivo. In an in vitro cell coculture assay, hCD8+Tregs significantly inhibited mouse CD4+ effector T cell proliferation, induced mouse CD4+Foxp3+Treg and CD4+IFN-γ +Th1 cell production, reduced Th17 cell development, and downregulated CD80/86 expression on mature DCs (mDCs).ConclusionTGF-β1/RAPA can induce hCD8+Tregs with stable suppressive characteristics, which could significantly alleviate the severity of CIA based on their stable suppressive ability in an inflammatory microenvironment and further influence the function of other downstream cell subtypes. Human CD8+Tregs might be a therapeutic strategy for rheumatoid arthritis.

| S-EPMC6399536 | biostudies-literature

Signaling Through gp130 Compromises Suppressive Function in Human FOXP3+ Regulatory T Cells.

Project description:The CD4+FOXP3+ regulatory T cell (Treg) subset is an indispensable mediator of immune tolerance. While high and stable expression of the transcription factor FOXP3 is considered a hallmark feature of Treg cells, our previous studies have demonstrated that the human FOXP3+ subset is functionally heterogeneous, whereby a sizeable proportion of FOXP3+ cells in healthy individuals have a diminished capacity to suppress the proliferation and cytokine production of responder cells. Notably, these non-suppressive cells are indistinguishable from suppressive Treg cells using conventional markers of human Treg. Here we investigate potential factors that underlie loss of suppressive function in human Treg cells. We show that high expression of the IL-6 family cytokine receptor subunit gp130 identifies Treg cells with reduced suppressive capacity ex vivo and in primary FOXP3+ clones. We further show that two gp130-signaling cytokines, IL-6 and IL-27, impair the suppressive capacity of human Treg cells. Finally, we show that gp130 signaling reduces the expression of the transcription factor Helios, whose expression is essential for stable Treg function. These results highlight the role of gp130 in regulating human Treg function, and suggest that modulation of gp130 signaling may serve as a potential avenue for the therapeutic manipulation of human Treg function.

| S-EPMC6657659 | biostudies-literature

Human LAP+GARP+FOXP3+ regulatory T cells attenuate xenogeneic graft versus host disease.

Project description:Adoptive transfer of regulatory T cells (FOXP3+ Tregs) has been developed as a potential curative immune therapy to prevent and treat autoimmune and graft-versus-host diseases (GVHD). A major limitation that has hindered the use of Treg immunotherapy in humans is the difficulty of consistently isolating and obtaining highly purified Tregs after ex vivo expansion. Methods: We isolated bona fide Tregs from expansion cultures based on their selective surface expression of latency-associated peptide (LAP). The TCR V? diversity and intracellular cytokine production of Tregs were determined by flow cytometer. The TSDR methylation was determined by epigenetic human FOXP3 qPCR Assay. Their in vitro and in vivo potency was confirmed with suppression assay and humanized xenogeneic GVHD (xGVHD) murine model, respectively. Results: LAP+ repurification results in >90% LAP+FOXP3+ Tregs, leaving behind FOXP3- and FOXP3+ nonTregs within the LAP- population. After 4-week expansion, the LAP+ Tregs were >1 billion cells, highly suppressive and anergic in vitro, >90% demethylated in the TSDR and able to maintain TCR V? diversity. In the xGVHD model, exogenous CD25-PBMC administered alone results in a median survival of 32 days. The co-transfer of LAP+ Tregs increased median survival to 47 days, while the LAP parent (CD25+) and LAP- nonTregs had median survival of 39 and 31 days, respectively. Conclusions: These preclinical data together provide evidence that LAP+ Tregs are highly purified with fully suppressive function for cell therapy. This population results in a more effective and safer product for immunotherapy to treat GVHD and provides the necessary preclinical data for transition into a clinical trial with LAP+ Tregs to prevent or treat GVHD and other autoimmune diseases.

| S-EPMC6531299 | biostudies-literature

Cpf1 enables fast and efficient genome editing in Aspergilli.

Project description:BackgroundCRISPR technology has revolutionized fungal genetic engineering by increasing the speed and complexity of the experiments that can be performed. Moreover, the efficiency of the system often allows genetic engineering to be introduced in non-model species. The efficiency of CRISPR gene editing is due to the formation of specific DNA double-strand breaks made by RNA guided nucleases. In filamentous fungi, only Cas9 has so far been used as the CRISPR nuclease. Since, gene editing with Cas9 is limited by its 5'-NGG-3' protospacer adjacent motif (PAM) sequence, it is important to introduce RNA guided nucleases that depend on other PAM sequences in order to be able to target a larger repertoire of genomic sites. Cpf1 from Lachnospiraceae bacterium employs a PAM sequence composed of 5'-TTTN-3' and therefore serves as an attractive option towards this goal.ResultsIn this study we showed that Lb_cpf1 codon optimized for Aspergillus nidulans can be used for CRISPR based gene editing in filamentous fungi. We have developed a vector-based setup for Cpf1-mediated CRISPR experiments and showed that it works efficiently at different loci in A. nidulans and in A. niger. Specifically, we used our setup to demonstrate that Cpf1 is able to catalyze oligonucleotide-mediated genomic site-directed mutagenesis and marker-free gene targeting.ConclusionsIn this paper we introduce Cpf1 as a new tool in the fungal CRISPR toolbox. Our experiments demonstrate that Cpf1 can be efficiently used in Aspergilli for gene editing thereby expanding the range of genomic DNA sequences that can be targeted by CRISPR technologies.

| S-EPMC6492335 | biostudies-literature

Efficient genome editing in erythroid cells unveils novel MYB target genes and regulatory functions.

Project description:Targeted genome editing holds great promise in biology. However, efficient genome modification, including gene knock-in (KI), remains an unattained goal in multiple cell types and loci due to poor transfection efficiencies and low target genes expression, impeding the positive selection of recombined cells. Here, we describe a genome editing approach to achieve efficient gene targeting using hard to transfect erythroid cell lines. We demonstrate robust fluorescent protein KI efficiency in low expressed transcription factor (TF) genes (e.g., Myb or Zeb1). We further show the ability to target two independent loci in individual cells, exemplified by MYB-GFP and NuMA-Cherry double KI, allowing multicolor labeling of regulatory factors at physiological endogenous levels. Our KI tagging approach allowed us to perform genome-wide TF analysis at increased signal-to-noise ratios, and highlighted previously unidentified MYB target genes and pathways. Overall, we establish a versatile CRISPR-Cas9-based platform, offering attractive opportunities for the dissection of the erythroid differentiation process.

| S-EPMC10475484 | biostudies-literature

Baculoviral delivery of CRISPR/Cas9 facilitates efficient genome editing in human cells.

Project description:The CRISPR/Cas9 system is a highly effective tool for genome editing. Key to robust genome editing is the efficient delivery of the CRISPR/Cas9 machinery. Viral delivery systems are efficient vehicles for the transduction of foreign genes but commonly used viral vectors suffer from a limited capacity in the genetic information they can carry. Baculovirus however is capable of carrying large exogenous DNA fragments. Here we investigate the use of baculoviral vectors as a delivery vehicle for CRISPR/Cas9 based genome-editing tools. We demonstrate transduction of a panel of cell lines with Cas9 and an sgRNA sequence, which results in efficient knockout of all four targeted subunits of the chromosomal passenger complex (CPC). We further show that introduction of a homology directed repair template into the same CRISPR/Cas9 baculovirus facilitates introduction of specific point mutations and endogenous gene tags. Tagging of the CPC recruitment factor Haspin with the fluorescent reporter YFP allowed us to study its native localization as well as recruitment to the cohesin subunit Pds5B.

| S-EPMC5480884 | biostudies-literature

Transcriptome profiling of human FoxP3+ regulatory T cells.

Project description:The major goal of this study was to perform an in depth characterization of the "gene signature" of human FoxP3(+) T regulatory cells (Tregs). Highly purified Tregs and T conventional cells (Tconvs) from multiple healthy donors (HD), either freshly explanted or activated in vitro, were analyzed via RNA sequencing (RNA-seq) and gene expression changes validated using the nCounter system. Additionally, we analyzed microRNA (miRNA) expression using TaqMan low-density arrays. Our results confirm previous studies demonstrating selective gene expression of FoxP3, IKZF2, and CTLA4 in Tregs. Notably, a number of yet uncharacterized genes (RTKN2, LAYN, UTS2, CSF2RB, TRIB1, F5, CECAM4, CD70, ENC1 and NKG7) were identified and validated as being differentially expressed in human Tregs. We further characterize the functional roles of RTKN2 and LAYN by analyzing their roles in vitro human Treg suppression assays by knocking them down in Tregs and overexpressing them in Tconvs. In order to facilitate a better understanding of the human Treg gene expression signature, we have generated from our results a hypothetical interactome of genes and miRNAs in Tregs and Tconvs.

| S-EPMC4761514 | biostudies-literature

PRKAG2.2 is essential for FoxA1+ regulatory T cell differentiation and metabolic rewiring distinct from FoxP3+ regulatory T cells.

Project description:Forkhead box A1 (FoxA1)+ regulatory T cells (Tregs) exhibit distinct characteristics from FoxP3+ Tregs while equally effective in exerting anti-inflammatory properties. The role of FoxP3+ Tregs in vivo has been challenged, motivating a better understanding of other Tregs in modulating hyperactive immune responses. FoxA1+ Tregs are generated on activation of the transcription factor FoxA1 by interferon-β (IFNβ), an anti-inflammatory cytokine. T cell activation, expansion, and function hinge on metabolic adaptability. We demonstrated that IFNβ promotes a metabolic rearrangement of FoxA1+ Tregs by enhancing oxidative phosphorylation and mitochondria clearance by mitophagy. In response to IFNβ, FoxA1 induces a specific transcription variant of adenosine 5'-monophosphate-activated protein kinase (AMPK) γ2 subunit, PRKAG2.2. This leads to the activation of AMPK signaling, thereby enhancing mitochondrial respiration and mitophagy by ULK1-BNIP3. This IFNβ-FoxA1-PRKAG2.2-BNIP3 axis is pivotal for their suppressive function. The involvement of PRKAG2.2 in FoxA1+ Treg, not FoxP3+ Treg differentiation, underscores the metabolic differences between Treg populations and suggests potential therapeutic targets for autoimmune diseases.

| S-EPMC10732530 | biostudies-literature

Efficient genome editing of differentiated renal epithelial cells.

Project description:Recent advances in genome editing technologies have enabled the rapid and precise manipulation of genomes, including the targeted introduction, alteration, and removal of genomic sequences. However, respective methods have been described mainly in non-differentiated or haploid cell types. Genome editing of well-differentiated renal epithelial cells has been hampered by a range of technological issues, including optimal design, efficient expression of multiple genome editing constructs, attainable mutation rates, and best screening strategies. Here, we present an easily implementable workflow for the rapid generation of targeted heterozygous and homozygous genomic sequence alterations in renal cells using transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeat (CRISPR) system. We demonstrate the versatility of established protocols by generating novel cellular models for studying autosomal dominant polycystic kidney disease (ADPKD). Furthermore, we show that cell culture-validated genetic modifications can be readily applied to mouse embryonic stem cells (mESCs) for the generation of corresponding mouse models. The described procedure for efficient genome editing can be applied to any cell type to study physiological and pathophysiological functions in the context of precisely engineered genotypes.

| S-EPMC5222933 | biostudies-literature