Dataset Information

Tracking Regulatory T Cell Development in the Thymus Using Single-Cell RNA Sequencing/TCR Sequencing.

ABSTRACT: Recent studies have demonstrated that regulatory T cells (T_regs) develop in the thymus via two pathways involving distinct T_reg progenitors (T_regP): CD25⁺FOXP3^- (CD25⁺ T_regP) and CD25^-FOXP3^lo (FOXP3^lo T_regP) T_reg progenitors. To examine this process in more detail, we carried out single-cell RNA sequencing (scRNA-Seq) and TCR-Seq on sorted murine CD4⁺CD8⁺ double-positive (DP) thymocytes, CD4⁺ single-positive (CD4SP) thymocytes, CD25⁺FOXP3^-CD73^- T_regP, CD25^-FOXP3^loCD73^- T_regP, newly generated mature CD25⁺FOXP3⁺CD73^- T_regs, and FOXP3⁺CD73⁺ recirculating/long-term resident T_regs (RT-T_regs). Sorted populations were individually hashtagged and then combined into one scRNA-Seq/TCR-Seq library before sequencing and subsequent analysis. We found that both CD25⁺ T_regP and FOXP3^lo T_regP arise via an initial agonist-activated state that gives rise to a second transitional stage before differentiating into mature T_regs Using both scRNA-Seq and bulk RNA-Seq on sorted thymocyte subsets, we demonstrate that CD25⁺ T_regP are significantly enriched for Il2 production, suggesting that they are the major source of IL-2 needed to convert T_regP into mature T_regs Using TCR-Seq, we found that several TCRs were clearly biased in favor of the conventional or T_reg lineages, but that a large fraction of TCRs were found in both these lineages. Finally, we found that RT-T_regs in the thymus are not monomorphic but are composed of multiple distinct subsets and that these RT-T_regs express the most diverse TCR repertoire of all CD4SP thymocytes. Thus, our studies define multiple stages of T_reg differentiation within the murine thymus and serve as a resource for future studies on CD4⁺ thymocyte development and T_reg differentiation.

SUBMITTER: Owen DL

PROVIDER: S-EPMC9529998 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Publications

Tracking Regulatory T Cell Development in the Thymus Using Single-Cell RNA Sequencing/TCR Sequencing.

Owen David L DL La Rue Rebecca S RS Munro Sarah A SA Farrar Michael A MA

Journal of immunology (Baltimore, Md. : 1950) 20220829 7

Recent studies have demonstrated that regulatory T cells (Tregs) develop in the thymus via two pathways involving distinct Treg progenitors (TregP): CD25+FOXP3- (CD25+ TregP) and CD25-FOXP3lo (FOXP3lo TregP) Treg progenitors. To examine this process in more detail, we carried out single-cell RNA sequencing (scRNA-Seq) and TCR-Seq on sorted murine CD4+CD8< ...[more]

PMID: 36038290

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Project description:Cell-type-specific tools facilitate the identification and functional characterization of distinct cell types, which underly the complexity of neuronal circuits. A large collection of existing genetic tools in Drosophila relies on enhancer activity to label different subsets of cells. These enhancer-based GAL4 lines often fail to show a predicable expression pattern to reflect the expression of nearby gene(s), partly due to an incomplete capture of the full gene regulatory elements. While genetic intersectional technique such as the split-GAL4 system further improve cell-type-specificity, it requires significant time and resource to generate and screen through combinations of enhancer expression patterns. In addition, since existing enhancer-based split-GAL4 lines that show cell-type-specific labeling in adult are not necessarily active nor specific in early development, there is a relative lack of tools for the study of neural development. Here, we use an existing single-cell RNA sequencing (scRNAseq) dataset to select gene pairs and provide an efficient pipeline to generate cell-type-specific split-GAL4 lines based on the native genetic regulatory elements. These gene-specific split-GAL4 lines can be generated from a large collection of coding intronic MiMIC/CRIMIC lines either by embryo injection or in vivo cassette swapping crosses and/or CRISPR knock-in at the N or C terminal of the gene. We use the developing Drosophila visual system as a model to demonstrate the high prediction power of scRNAseq-guided gene specific split-GAL4 lines in targeting known cell types. The toolkit allows efficient cluster annotation in scRNAseq datasets but also the identification of novel cell types. Lastly, the gene-specific split-GAL4 lines are broadly applicable to Drosophila tissues. Our work opens new avenues for generating cell-type-specific tools for the targeted manipulation of distinct cell types throughout development and represents a valuable resource to the fly research community.Significance statementUnderstanding the functional role of individual cell types in the nervous systems has remained a major challenge for neuroscience researchers, partly due to incomplete identification and characterization of underlying cell types. To study the development of individual cell types and their functional roles in health and disease, experimental access to a specific cell type is often a prerequisite. Here, we establish an experimental pipeline to generate gene-specific split-GAL4 guided by single-cell RNA sequencing datasets. These lines show high accuracy for labeling targeted cell types from early developmental stages to adulthood and can be applied to any tissues in Drosophila. The collection of gene-speicifc-split-GAL4 will provide a valuable resource to the entire fly research community.

Dataset Information

Tracking Regulatory T Cell Development in the Thymus Using Single-Cell RNA Sequencing/TCR Sequencing.

Publications

Tracking Regulatory T Cell Development in the Thymus Using Single-Cell RNA Sequencing/TCR Sequencing.

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