| الوصف: |
T cell development and differentiation are dependent on T cell receptor (TCR) signalling. This project investigates the events downstream of TCR-signalling in CD4+T cell development and differentiation by studying the transcriptional dynamics of two key genes in individual cells: an immediate early transcribed gene upon TCR engagement (Nr4a3) and a more lately transcribed gene following TCR signalling (Foxp3). Because there were no technologies to study those transcriptional dynamics in vivo in individual cells, a new reporter technology (a fluorescent timer protein called Timer) was used. The first part of this project establishes two new transgenic mouse strains expressing Timer fluorescent protein, Nr4a3Timer and Foxp3Timer. Several independent reporter lines were assessed by their Timer expression, and selected. The second part studies the dynamics of Timer protein expression in the selected transgenic strains. An analysis framework was established to reveal the dynamics of expression of the reported genes (Nr4a3 and Foxp3) by using the expression and colour maturation of Timer. The last part of the project was dedicated to addressing biological questions using our new fluorescent reporter strains. First, I used the Timer technology to investigate the temporal dynamics of Nr4a3, CD25 and Foxp3 expression during neonatal thymocyte development upon TCR signalling. This information placed CD25 expression as an early event downstream of TCR signalling, while Foxp3 expression appeared later on, with or without coexpression of CD25. Second, the investigation of the mechanisms downstream of TCR-signalling in peripheral CD4+ T cells in vitro showed the modulatory role of IL-6 and TGFβ on Nr4a3 expression. Finally, investigation of the role of Nr4a3 overexpression in T cell apoptosis showed that Nr4a3 proapoptotic effects are context- dependent, depending on the cytokine environment. This project constitutes the first application of a fluorescent timer reporter system to the field of T cell immunology. It also provides a better understanding of the role of Nr4a3 in TCR-mediated events in T cell differentiation and activation. |
