Molecular and cellular control of intrathymic T-Cell development
Authors:   , Kunze-Schumacher Heike 
Published: April 2022

T cell development is a highly dynamic and stepwise process comprimising T lineage commitment, T cell receptor (TCR) gene rearrangements and subsequent selection. From a quantitative point of view, only a few hundred progenitor cells migrate from the bone marrow into the thymus. Developing thymocytes (termed double negative (DN), CD4 CD8 ) can be further divided into DN1 4 cells based on the expression of CD25 and CD44. These developmental events are interspersed by proliferative bursts which ultimately lead to the generation of millions of double positive (DP, CD4+CD8+) thymocytes that then undergo selection. As a consequence, a proportion of naïve T cells evolves to ensure adaptive, but not autoreactive immunity. Previous studies of our lab focused on the quantification of thymus colonization and identified thymus entry to be dependent on expression of the chemokine receptors CCR7 and CCR9 (Krueger et al., 2010; Ziętara et al., 2015). CCR7/9 double knockout (DKO) mice are almost completely devoid of the most immature thymocyte populations (DN1 and DN2), but show near normal DN3 cellularity. Interestingly, a similar defect during early development but a virtually complete recovery of later stages and total thymocyte numbers was also observed in thymi of miR 17~92 deficient mice. Here, a failure of prethymic IL 7 signaling dampens early T cell development (Regelin et al., 2015). For this reason, we hypothesized a tight regulation of thymocyte population size through alterations in the underlying cell cycle kinetics. In this thesis, we employed in vivo single and dual nucleoside pulse labeling combined with determination of DNA replication over time in different WT thymocyte subsets at steady state. Based on this, we assessed alterations in cell cycle kinetics of CCR7/9 and miR 17~92 defcicient mice and identified compensatory mechanisms of thymocytes on the level of cell cycle phase distribution and cell cycle speed. In addition, single cell RNA sequencing helped to obtain information on cell cycle dynamics of early thymocyte subsets, exemplarily shown for WT and CCR7/9 DKO mice. Lastly, we performed cell cycle analyses in a model of endogenous thymic repair upon sublethal total body irradiation which provided insight into intrathymic cell cycle regulation as an adjustable system to re establish normal thymus cellularity. In the second part of the thesis, we addressed the role of miR 21 in the thymus. In various studies, we and others identified miRNAs as key posttranscriptional regulators of the immune system and especially for T cell development (Regelin et al. 2015; Mildner et al. 2017; Li et al. 2007; Ebert et al. 2009; Ziętara et al. 2013; Schaffert et al. 2015). The dynamic expression of miR 21 during T cell development (Neilson et al. 2007; Kirigin et al. 2012; Kuchen et al. 2010) prompted us to hypothesize that miR 21 has a regulatory function in the thymus. A miR 21 knockout mouse model allowed us to study the role of this miRNA for the development of T cells in the thymus and the maintenance of T cells in the periphery. In addition, we performed competitive bone marrow chimera experiments in the context of miR 21 deficiency and overexpression. Further insights were provided by exploring the function of miR 21 in negative selection in vivo as well as in T cell differentiation in coculture experiments in vitro. To unravel implications of miR 21 to regulate cellular stress responses, we assessed the contribution of miR 21 in a model of endogenous regeneration of the thymus after sublethal irradiation. We could not provide evidence for a prominent role for miR 21 during T cell development. Together, our experiments revealed that miR 21 is largely dispensable for physiologic T cell development despite high and dynamic expression in the thymus (Kunze Schumacher et al., 2018). The apparent discrepancy between dynamic expression but lack of a regulatory function in the thymus led us to conclude that miR 21 is rather fine tuning T cell responses than controlling a developmental event.