SUMMARY Title: Age-associated Changes in the Germinal Centre Response upon Vaccination Name: Alyssa Silva Cayetano

Vaccination efficacy is reduced in older individuals due to diminished immune system function with advancing age. The germinal centre (GC) response plays a crucial role in generating protective immunity upon vaccination through the production of memory B cells and long-lived antibody-secreting plasma cells. However, the GC response deteriorates with age contributing to poor humoral immunity after vaccination and therefore to increased susceptibility to infections in older individuals. I aimed to assess the age-dependent cellular changes of the GC response using a combination of in vivo and in silico tools to determine the key mechanisms driving the GC deterioration with age. After immunisation, aged (24-month old) BALB/c and C57BL/6 mice had a decrease in frequency of GC B cells and an accumulation of T follicular helper (Tfh) cells compared to adult (2-month old) mice. This indicated a reduction in the magnitude of the GC response with age. The quality of the GC was also impaired as aged mice had lower serum titres of high-affinity antigen-specific antibodies. Confocal imaging revealed that the structure of GCs is altered with age. The GC is segregated into two specialised compartments: the dark zone (DZ) where B cells proliferate and the light zone (LZ) which contains the follicular dendritic cell (FDC) network and Tfh cells that facilitate the selection of high affinity GC B cells. I found that the size of the FDC network is significantly reduced and that Tfh cells are overrepresented in the DZ of the GC in aged mice.

I hypothesised a B cell intrinsic defect was responsible for the decrease in GC magnitude in aged mice. However, adoptive transfer of SWHEL B cells from 24-month old mice refuted this hypothesis. To understand which cellular factor(s) may be causing the poor GC response in aged mice I used in silico modelling of the GC. This revealed that a combination of a reduced FDC network and an aberrant localisation of Tfh cells in the DZ is responsible for the poor GC response in aged mice. This altered localisation of Tfh cells corresponded with a higher expression of CXCR4 on Tfh cells from aged mice and increased sensitivity to CXCL12. I investigated the role of CXCR4 on Tfh function by adoptive transfer of CXCR4 deficient CD4+ T cells. Upon immunisation, these cells were capable of differentiating into phenotypically intact Tfh cells and were restricted to the LZ area of the GC. This finding suggests CXCR4 expression by Tfh cells is required for DZ localisation and overexpression of CXCR4 in aged Tfh cells may have deleterious effects on the GC response by driving Tfh cell localisation towards the DZ. Together, I have shown that the age-dependent GC deterioration is a multifactorial process, likely driven by a reduction in FDC network size and aberrant Tfh cell localisation, and results in poor protective immunity after vaccination.