Zhang, Hegong; (2024) The Role of Bcl6 in T-cell Development and Differentiation. Doctoral thesis (Ph.D), UCL (University College London).

Text Hegong Zhang - 22011162 - PhD Thesis.pdf - Accepted Version Access restricted to UCL open access staff until 1 October 2025. Download (77MB)

Abstract

The transcription factor Bcl6, traditionally recognised as a regulator for follicular B cells and T follicular helper cells, has been recently found to play a pivotal role in early T cell development. It has been revealed that Bcl6 is highly expressed in double positive (CD4+CD8+) thymocytes that are poised for undergoing positive selection. I therefore hypothesised that Bcl6 may be potentially required in positive selection of the T cell repertoire. Here, my data showed that the TCR signalling strength was diminished, and fewer thymocytes were positively selected in the Bcl6cKO mice. To test the hypothesis, I first employed flow cytometry staining to detect phenotype changes associated with TCR signalling strength on a Bcl6 conditional knockout (Bcl6cKO) CD4 Cre mouse model. I observed that the expression of the markers such as CD5, TCRβ, CD3, and CD69 were obviously reduced in the double positive and single positive (CD4+ or CD8+) thymocytes in the Bcl6cKO mice compared to their counterparts, implying a potential role of Bcl6 in regulating TCR signalling strength and facilitating positive selection. Then, I carried out bulk cell RNA sequencing (RNA seq) and functional analysis on the CD69+ double positive and single positive T cells derived from the Bcl6cKO CD4 Cre mouse model, from which several genes associated with regulating TCR signalling were identified as differentially expressed genes (DEGs). Subsequently, given the diminished TCR signalling in the thymocytes in the Bcl6cKO mice and insights gleaned from RNA seq analysis, I further hypothesised that Bcl6 might influence T cell activation. I performed a series of flow cytometry staining experiments to examine peripheral T cells derived from the spleen and lymph nodes, including resting T cells and activated T cells. Observations showed that the percentages of T cell that expressed activation markers CD69 and CD25 were greatly reduced in the Bcl6cKO, suggesting impaired TCR signalling. Following the flow cytometry, I conducted RNA seq for the resting and activated naïve T cells at different time points post-activation. Several DEGs associated with T cell activation, differentiation, and TCR signalling were identified, which corroborated the flow cytometry data. Additionally, I exploited a Nr4a3-Timer transgenic mouse model crossed with Bcl6cKO CD4 Cre mice to further investigate the dynamic changes over time upon activation in TCR signalling of both thymocytes and peripheral T cells. Reduction and delay of TCR 3 signalling and activation were detected. Moreover, differentiation and function of type 1 helper (th1), type 2 helper (th2), type 17 helper T cells (th17), and regulatory T cells (tregs) in the Bcl6 deficient mice were studied. The T-bet and Foxp3 expression was reduced, whereas the Rorc was enhanced. Cytokines such as IFNg, IL4, IL17A, and IL10 all displayed an escalation in the Bcl6cKO mice, suggesting a multifaceted role of Bcl6 in the T cell differentiation and functionality. In addition, I also explored the influence of Bcl6 on γδ T cell development and subset differentiation using another conditional knockout mouse strain—Bcl6 f/f Lck Cre. However, there was no difference observed in the phenotypes associated with γδ T cell development and subset differentiation between the Bcl6cKO mice and the controls. Overall, TCR signalling in thymocytes, splenocytes, and lymph node cells, in both resting and post-activated states, in the Bcl6cKO mice, was found to be reduced compared to the controls. I anticipate the findings from this project to serve as a starting point for developing novel immune-therapeutic strategies for diseases associated with TCR signalling, such as autoimmune disorders and various cancers. Studies for a more detailed mechanism for causing this reduced TCR signalling should follow.

Download activity - last month

Export as CSVXMLJSON

Download activity - last 12 months

Export as CSVJSONXML

Downloads by country - last 12 months

Export as JSONXMLCSV

Archive Staff Only

View Item