Unknown,Transcriptomics,Genomics,Proteomics

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Development of a humanized mouse model of graft-versus-host disease to assess human regulatory T cell function

ABSTRACT: We developed a humanized mouse model of graft-versus-host disease in which Treg injected two days after PBMC persist in the blood of infused mice and can be characterized. This model provides in vivo assessment of manipulated Treg. We used that model to evaluate the impact of ex vivo priming of Treg with TNF-alpha on their ability to prevent xenogeneic GVHD. Indeed, it had been demonstrated that Treg function can be enhanced through stimulation of TNFR2. scRNAseq analysis was performed to assess the impact of TNF priming on Treg transcriptomics. Peripheral blood mononuclear cells were isolated from the blood of three healthy donors. Treg were isolated through immuno-magnetic selection then cultured for 24 hours with IL-2 at 10 ng/mL with or without TNF at 100 ng/mL. After 24 hours, cells were collected and washed. We performed cell multiplexing to label samples individually, then pooled them for single cell RNA sequencing. For each donor (named donors 3, 4 and 7), half cells were in the control group (no TNF) and half cells were in the experimental group (with TNF). 10x Genomics 3’ Single Cell Gene Expression with CellPlex multiplexing (Set A). Cells were labeled with Cell Multiplexing Oligos prior to pooling and library preparation.

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Homo sapiens

SUBMITTER: Charline BEGUIN

PROVIDER: E-MTAB-15881 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Hematopoiesis in embryonic and adult life is the process of producing blood cells. In mammalian embryos, hematopoiesis occurs in three consecutive overlapping waves (Neo et al. 2021; Dzierzak and Bigas 2018) and is regulated by transcription factors (TFs) and signaling molecules. In this study, we investigated the function of three relatively poorly studied TFs in early embryonic hematopoietic development at single-cell resolution: Activating transcription factor 3 (Atf3), Zinc finger protein 711 (Zfp711), and B cell CLL/lymphoma 6, member B (Bcl6b) respectively. We observed that these three TFs are upregulated early in development when hematopoietic and endothelial lineages separate from cardiac and other mesodermal lineages. To study the roles of these TFs in a rapidly changing system with diverse cell types and small cell populations during early developmental stages, we employed multiplexed single-cell RNA sequencing (scRNA-seq) of TF knockouts (KO) of in vitro differentiating mouse embryonic stem cells (mESCs) and also capturing changes with Flow Cytometric Analysis (FCA). This approach offered a valuable method to dissect the functions of these TFs in lineage induction, specification, and separation, providing access to sufficient numbers of various progenitor cells. We adapted available multiplexing technology for single-cell RNA sequencing (scRNA-seq) -multiplexing knockouts (KO) and Control conditions with biological replicates-, accompanied by Flow Cytometric Analysis (FCA) to study the role of three TFs in early embryonic hematopoietic development. Using this adaptation, the depth and coverage of this study can be placed between large-scale multiplexed CRISPR-based perturbation studies covering multiple candidate genes together (Datlinger et al. 2017; Jaitin et al. 2016; Dixit et al. 2016; Adamson et al. 2016) and single gene perturbation studies, which focus on the in-depth function/role of a particular gene with multiple experimental procedures (Harland et al. 2021). With adapted methodology, we studied the role of the three TF genes at once in a cost-efficient manner in one experiment by including three biological replicates to minimize false positive results, to capture a sufficient number of cells to detect changes in low abundance cell types, to minimize the creation of potential batch effects (e.g., each replicates creates a separate library) and prevent the loss of biological information during computational integration steps by skipping computational integration step. Following the scRNA-seq analysis, our findings are compared with publicly available datasets to categorize our findings. This categorized information can be used as a launching pad for future in-depth follow-up studies investigating the roles of these three TFs in hematoendothelial development.

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Development of a humanized mouse model of graft-versus-host disease to assess human regulatory T cell function

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