Date of Award
2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Program
Biomedical Sciences
Track
Cancer and Developmental Biology
Research Advisor
Yongqiang Feng
Committee
Benjamin Youngblood; Hongbo Chi; Paul Thomas; Yi Lu
Keywords
Developmental age, Precursor cells, Regulatory T cell, Single cell RNA sequencing, Thymocyte differentiation, Timestamp tracing
Abstract
Regulatory T (Treg) cells play a pivotal role in maintaining immune homeostasis and preventing autoimmunity. Like most T cells, Treg development occurs primarily in the thymus, where T-cell precursors undergo a series of developmental stages, including double-negative (DN), double-positive (DP), and single-positive (SP) stages, leading to the formation of distinct mature T cells. To ensure a diverse and self-tolerant T-cell repertoire capable of effectively responding to a wide range of pathogens while preventing autoimmunity, the differentiation process of thymocytes is closely supervised by sequential programs, including the rearrangement of T-cell receptor (TCR) gene segments, β-selection of cells expressing a functional TCRβ chain, positive selection of cells followed by CD4/CD8 lineage specificity, and negative selection or clonal deletion to eliminate autoreactive T cells. The induction of the Treg population is triggered when autoreactive cells escape from negative selection. Despite significant progress in our understanding of thymic T-cell differentiation, investigating how thymic Treg cells differentiate in their native settings remains a key conceptual and technical challenge. One major issue is that previous investigations have been limited by technological constraints. First, studies often rely on predefined markers, which can introduce bias in defining developmental stages. Second, techniques such as adoptive T-cell transfer and in vitro culture of TCR transgenic can induce stress on the cells, emphasizing the necessity of studying T-cell development under native physiological conditions. Third, the thymic T-cell pool is a mix of cells derived at any developmental stages as well as mature T cells recirculating back into thymus. Such heterogeneity complexes the assessment focusing on differentiating thymocytes. To address these challenges, we developed a Rag1CreER timestamping mouse model. This model is based on the CreER-loxp system and enables tracing thymocytes at their earlier stage by tamoxifen-induced Cre recombinase. When combined with Rosa reporter strains, this genetic tool enables inducible labeling of early T cells at the T-cell receptor (TCR) rearrangement stage at any time, allowing temporal tracking of their developmental trajectories. By integrating timestamp tracing with single-cell technology, our deconvolution of thymic cells delineated the dynamic processes of developmental stages, and transcriptome profiles during thymic T-cell differentiation. This method provides a comprehensive and high-resolution view of thymic T-cell differentiation under natural conditions. We identified intermediate CD4 SP thymocytes that precede Treg induction, these cells are featured with Treg-associated gene signatures such as high levels of Tnfrsf9, Tnfrsf4, Ikzf2, Ikzf4 and Il2ra. These cells are induced by TCR signaling through various levels and can differentiate into Treg cells both in vitro and in vivo. We also pulse-chased nascent thymic Treg cells and specified their maturation process. We captured a sharp increase of activation markers such as GITR, CD44, CD25 and CD73 after Foxp3 induction. The acquisition of these markers was further found to occur within the thymus by parabiosis experiment, highlighting a potential transition process of newly derived Treg cells. Further transcriptome and epigenome analysis revealed profound gene expression changes, and demethylation of Treg-specific gene regions is involved in such transition process, thus highlighting a maturation process. Furthermore, we also extended our studies of our timestamp tracing tool. We tracked thymic Treg cells for a longer time and determined the features of recirculating or retained mature thymic Treg cells. These data provide a detailed picture of thymic Treg-cell ontogeny in native conditions. Finally, we applied our timestamping strategy to compare the function difference of T cells derived at neonatal and adult stages and demonstrated that neonatal-derived cells are more active. Thus, the developmental processes of thymic regulatory T cells in native physiological conditions are resolved by a novel genetic tool combined with unbiased approaches, providing a foundation for further exploration.
ORCID
0000-0002-1660-9916
DOI
10.21007/etd.cghs.2024.0679
Recommended Citation
Jiang, Menglin (0000-0002-1660-9916), "On Thymic Regulatory T-Cell Ontogeny" (2024). Theses and Dissertations (ETD). Paper 699. http://dx.doi.org/10.21007/etd.cghs.2024.0679.
https://dc.uthsc.edu/dissertations/699
Declaration of Authorship
