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Population and single cell RNAseq analysis of CD4+ T cells in FOXP3 mutant mice ( scurfy") and IPEX patients

ABSTRACT: Population and single cell RNAseq analysis of CD4+ T cells in FOXP3 mutant mice ( scurfy") and IPEX patients

PROVIDER: PRJNA707555 | ENA |

REPOSITORIES: ENA

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			Action	DRS
		Other
	SRR13723800_1.fastq.gz	Fastqsanger.gz
	SRR13723800_2.fastq.gz	Fastqsanger.gz
	SRR13723801_1.fastq.gz	Fastqsanger.gz
	SRR13723801_2.fastq.gz	Fastqsanger.gz

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RNAseq analysis of human CD4+ regulatory Tregs and Tconvs in IPEX and healthy donors isolated from peripheral blood.

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Single-cell RNAseq (10x Genomics) analysis of mouse splenic CD4+ T cells in WT and ∆Foxp3 mice and in WT/∆Foxp3 bone marrow chimeras

Project description:Single-cell RNAseq (10x Genomics) analysis of mouse splenic CD4+ T cells in WT and ∆Foxp3 mice and in WT/∆Foxp3 bone marrow chimeras. Mouse CD4+T cells in 21-day-old male WT and ∆Foxp3 mice were isolated from spleen by flow cytometry as DAPI–TCRβ+CD4+ cells for 10x Genomics Single Cell 3′ Reagent Kit (V2 chemistry, one sample per channel). For bone marrow chimera experiment: 7 week-old CD45.2-recipient mice were irradiated with 1000 Rad, reconstituted with 4 million CD3-depleted bone marrow cells: 50% CD45.1 x Foxp3-IRES-GFP (WT, 21d-old male) and 50% Foxp3DeltaEGFPiCre/RFP x ROSA-YFP x CD45.1/2 (scurfy, 21d-old male). 10 weeks later, spleen were harvested and tagged using a different Hashtags fo each mouse. ∆Foxp3 CD4+ T cells were sorted as DAPI–TCRb+CD4+CD45.1+CD45.2+. WT CD4+ T cells were sorted as DAPI–TCRb+CD4+CD45.1+CD45.2–. Control WT DAPI–TCRb+CD4+GFP+ Treg cells and GFP- Tconvs cells were also tagged and sorted. Samples with different hastags were pooled before single cell encapsulation using 10x Genomics Single Cell 3′ Reagent Kit (V3 chemistry).

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Single-cell RNAseq (10x Genomics) analysis of human CD4+ T cells in IPEX patients, healthy donors and heterozygous mothers (blood).

Project description:Single-cell RNAseq (10x Genomics) analysis of human CD4+ T cells in IPEX patients, healthy donors and heterozygous mothers (blood). Human CD4+T cells from IPEX, HD and mothers were isolated from frozen peripheral blood mononuclear cells by flow cytometry as DAPI–CD3+CD4+ cells. In cohort 1, cells from separate donor were encapsulated in separate channel following 10x Genomics Single Cell 3′ Reagent Kit (V2 chemistry). In cohort 2, samples were tagged using a different Hashtags, pooled and encapsulared following 10x Genomics Single Cell 3′ Reagent Kit (V3 chemistry).

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Project description:Analysis of Foxp3(+)epigenetics(-) T cells, Foxp3(-)epigenetics(+) T cells, and Foxp3(+)epigenetics(+) T cells. Results indicate regulatory T cell (Treg) ontogenesis requires two independent processes, expression of the transcription factor Foxp3 and establishment of Treg epigenetic programs induced by T cell receptor (TCR) stimulation. GFP+CD4+ and GFP-CD4+ splenocytes were sorted from DEREG and DEREG/Scurfy mice. These cells were activated with anti-CD3/CD28 antibodies, and then transduced with Foxp3-expressing retrovirus (pGCSamIN, NGFR marker). NGFR+ T cells sorted were subjected to microarray analysis (Affymetrix, mouse genome 430 2.0 array). To normalize the experimental conditions, Tregs (GFP+ T cells from DEREG) and Tconv (GFP- T cells from DEREG) were also activated and transduced with empty vector. Two replicates each.

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Project description:Studies of the monogenic autoimmune disease immunodysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX) have elucidated the essential function of the transcription factor FOXP3 and thymic-derived regulatory T cells (Tregs) in controlling peripheral tolerance. However, the presence and the source of autoreactive T cells in IPEX remain undetermined. Here, we investigated how FOXP3 deficiency affects the T cell receptor (TCR) repertoire and Treg stability in vivo and compared T cell abnormalities in patients with IPEX to those in patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome (APECED). To study Tregs independently of their phenotype and to analyze T cell autoreactivity, we combined Treg-specific demethylation region analyses, single-cell multi-omic profiling, and bulk TCR sequencing. We found that patients with IPEX, unlike patients with APECED, have expanded autoreactive T cells originating from both autoreactive effector T cells (Teffs) and Tregs. In addition, a fraction of the expanded Tregs lost their phenotypic and functional markers including CD25 and often also FOXP3. Functional experiments with CRISPR/Cas9-mediated FOXP3 knock-out Treg and Tregs from patients with IPEX indicated that the patients’ Tregs gain a Th2 skewed Teff-like function, which is consistent with immune dysregulation observed in these patients. Analyses of FOXP3 mutation-carrier mothers and a patient with IPEX after hematopoietic stem cell transplantation, indicated that Tregs expressing non-mutated FOXP3 prevent the accumulation of autoreactive Teffs and unstable Tregs. Collectively, we describe Treg instability and Teff autoreactivity in patients with IPEX. These findings could be directly used for diagnostic and prognostic purposes and for monitoring the effects of immunomodulatory treatments.

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Transcription profiling of regulatory T cells (Treg) and Effector CD4+ T cells in healthy B6 mice and sick mice with scurfy mutation - Foxp3-deficient Treg cells do not revert into conventional Effector CD4+ T cells but constitute a unique cell subset

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Expression data between FOXP3 wild type and 2T>C(mut)

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Project description:Effect of NIK overexpression on Foxp3+CD4+ and Foxp3-CD4+ T cell subsets

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