Investigation of IGF1R/IRS1 Signalling Mechanisms in B Cell Development

Abstract

The adaptive immune system declines as we age. This includes reduction in B cell number and function, contributing to increased vulnerability to infection in the elderly. Current evidence is consistent with age-associated defects emerging during early B cell development in the bone marrow (BM). Here, B cells develop by transition through distinct progenitor stages as the immunoglobulin heavy and light chain variable (V), diversity (D) and joining (J) genes are differentially rearranged in VDJ recombination. This assembles a complete B cell receptor (BCR) and is the primary step in producing a diverse BCR repertoire; an essential requirement for a robust adaptive immune response. The roles of interleukin-7 receptor, pre-BCR and chemokine receptor type 4 signalling are well established in this process. There is now growing evidence supporting a role for Insulin-like Growth Factor 1 Receptor (IGF1R) signalling in progenitor B cell development. Studies report marked reduction of B cell progenitor numbers and antibody diversity in aged mice and humans. The underlying mechanisms and causes are unclear. Genome-wide analysis of aged mouse BM B cell progenitors revealed that the major defect was multi-layered transcriptional downregulation of components of IGF1R signalling. This was most significant for the IGF1R adaptor protein, Insulin Receptor Substrate 1 (IRS1), and suggests multi-faceted dysregulation of IGF1R/IRS1 signalling in ageing B cell progenitors. IGF1R signalling functions in cellular growth and proliferation in numerous mammalian tissues, but its role in normal B cell development is poorly understood. Its impairment in ageing progenitor B cells, coincident with reduced cell numbers and antibody diversity, points to a key role of IGF1R signalling in B cell development. I therefore hypothesise a non-redundant role for IGF1R/IRS1 signalling in progenitor B cell development in the BM, and therefore that this signalling is critical to support the generation of adaptive immune cells. In this thesis, I aim to characterise the impact of IGF1R and IRS1 deletion in B cells during their development in the BM. To achieve this, I established two progenitor B cell-specific conditional knockout (KO) reporter mouse models. The first targets IRS1, to interrogate intracellular signalling events downstream of the IGF1R. The second targets the IGF1R, to investigate its response to the extracellular environment. Since IRS1 was the most significantly downregulated component of IGF1R signalling in ageing progenitor B cells, the hypothesis for a role of IRS1 in BM B cell development was tested first. Flow cytometry analysis of the IRS1 KO BM indicated a block at the pro- to pre-B cell progenitor transition. The model was then employed in functional assays to investigate the underlying mechanisms of this dysregulation. A mixed BM chimera setup was developed to evaluate the intrinsic ability of IRS1 KO B cells to progress through development in competition with cells without defect. Optimisation of culture methods for in vitro IRS1 KO B cells facilitated interrogation of the impact of IRS1 deletion on the pro- to pre-B transition in vitro. VDJ-seq analysis of the BCR repertoire provided insight into the putative role of IRS1 signalling in VDJ recombination and repertoire selection. Finally, the IGF1R KO was analysed to investigate the impact of IGF1R deletion during B cell development. Findings from this research establish a key role for IGF1R/IRS1 signalling in B cell development and further our understanding of the impact of its impairment in ageing. This study also provides opportunities for developing therapeutic approaches for healthy ageing; a pertinent issue for today’s ageing population.