Rowell, Jasmine Elizabeth; (2022) Regulation of the mouse T cell receptor repertoire. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In both mice and humans, as they age, the thymus involutes and decreases in size and function, with the majority being replaced by adipose tissue, and the number of lymphocytes and thymic epithelial cells (TEC) decline. The first aim of this thesis was to characterise the differences in T cell receptor (TCR) repertoires between foetal, young adult and ageing mice by next generation sequencing the recombined TCRa and TCRb gene transcripts expressed in WT thymocytes and peripheral T cells. Wildtype (WT) E18.5 embryos was dissociated and FACS-sorted for CD69- doublepositive (DP), CD69+DP, SP4 (CD3+CD4+) and SP8 (CD3+CD8+) T cells. In adult mice (4 weeks and 1 year old WT mice), the thymocytes were FACS-sorted for CD3- DP, CD3+DP, SP4 and SP8 T cells. Furthermore, the combined lymph nodes and spleen were FACS-sorted for naive CD4 and CD8, effector memory CD4 and CD8, and central memory CD4 and CD8. Analysis of TCR repertoires revealed greater clonality of peripheral effector memory CD4 and CD8 and central memory CD8 aged repertories compared to young adult. Using ecological estimators of diversity, TCR diversity was found to modestly decrease with age, especially in b chain repertoires from CD3+DP and SP8 thymocytes, and in effector memory CD4 and CD8, along with central memory CD8 populations. The diversity of b chain repertoires was more affected than a chain, suggesting that there are age-dependent changes to the rearrangement process of b chains. As mice aged, there was a divergence of sharing in complementary determining region 3 (CDR3) repertoires that could be due to a result of environmental influences to the repertoire. Taken together, the data in this thesis show that the TCR remains highly diverse in aged mice in both thymus and periphery, challenging the idea that thymic rejuvenation is required to produce a good immune response in the elderly. When comparing foetal repertoires to adult repertoires, they were found to be less diverse and contain less information with higher sharing of CDR3 sequences than young adult and aged repertoires. Foetal repertoires were also observed to display biases in TRAV, TRAJ and TRBV gene usage depending on the chromosomal location of the genes. These results point towards the process of rearrangement being more programmed in foetal repertoires. The Hedgehog (Hh) signalling pathway regulates T cell development in the thymus by regulating differentiation, survival and proliferation of the earliest DN thymocytes and reducing TCR signal strength at later stages of development. The second aim of this thesis was to investigate the impact of these changes in TCR signal strength induced by Hh signalling on TCR repertoire selection by next generation sequencing the repertoires from CD3-DP, CD3+DP, SP4 and SP8 thymocyte populations in lckGli2∆N2-transgenic, lck-Gli2∆C2-transgenic, Shh+/- , Gli3+/- , Shhfl/flFoxN1-Cre, and Gli3fl/flFoxN1-Cre compared to wildtype or control mice. The inhibition of Shh signalling in the lck-Gli2∆C2-transgenic mouse significantly reduced the diversity and increased the clonality of the TCR repertoire in the SP4 population and altered the variable joining (VJ) gene usage in comparison to the wildtype. On the other hand, increasing the Hh signal to above wildtype levels in the lck-Gli2∆N2-transgenic increased the diversity of the TCR repertoire in both the CD3- DP population. Heterozygous deletion of Gli3 enriched the TCR diversity and increased sharing of sequences. Conditional deletion of Shh from TEC cells affected the clonality of SP4 cells and VJ gene usage. Taken together, the work in this thesis shows how the TCR repertoire can be altered through changes in TCR signal by modulating Hh signalling.

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