Novel Tools for Investigating Microglia Cellular Turnover Kinetics and Microglia-CD4 T Cell Interaction

SUMMARY Under homeostatic conditions microglia maintain a tightly-controlled population size, contributing to 5-12% of cellular tissue in the mouse brain, depending on brain region. Following injury and under inflammatory conditions microglia undergo microgliosis, a rapid expansion in number and change in activation state. Microgliosis has been observed in multiple neurological diseases as well as in the long-term degenerative effects of traumatic brain injury and in age-related neuro-inflammation. The mechanisms which govern microglia cellular turnover kinetics to allow tight population control during homeostasis and rapid expansion during microgliosis are yet to be fully eluded. Recent studies have shown that microglia-CD4 T cell interactions are important during brain development for microglia maturation and their subsequent ability to perform important homeostatic functions in the brain. Increased MHCII expression in activated microglia further supports the hypothesis for T cell influence over microglia function during inflammation. Moreover, the change in microglia phenotype in the aged brain is coupled with CD4 T cell infiltration. This circumstantial evidence suggests that CD4 T cells may influence microglia population kinetics during ageing and microgliosis, however formal testing has been limited by a lack of appropriate tools. As microglia are a locally self-renewing population based on foetal progenitors, we cannot use bone marrow chimeras to investigate their competitive cellular kinetics. Instead, we must develop new research tools. Here I characterised and validate a novel chimeric system, which utilises X-inactivation to create mosaic knockout mice to study the cellular mechanisms which underlie short-term microglia population kinetics under both homeostatic and inflammatory conditions. Studies have focused on the role of microglial MHCII expression to elucidate the effect of cognate CD4 T cell interactions during ageing and traumatic brain injury on microglia proliferation, survival and phenotype. A role for conventional CD4 T cells in microgliosis suggests a potential for regulatory T cells (Tregs) to be counteractive and prevent microglia activation. This is supported by data in Yshii, et al. (Nature Immunology, 2022) using a novel adeno-associated virus (AAV)-IL2 delivery system to increase IL2 concentrations locally in the brain to stimulate Treg proliferation. In the context of traumatic brain injury, in preclinical mouse models AAV-IL2 delivery drove a Treg-dependent modification of microglia activity and resulted in smaller lesion size. With the aim of improving the AAV-delivery system as both a research tool and therapeutic, I have characterised a number of computationally designed IL2 muteins modifying production yield and cellular selectivity. Characterisation has revealed a number of muteins which have increased production (potentially reducing the amount of AAV sufficient to elicit a response) and increased cellular selectivity. Delivery of AAV-IL2 in high concentrations increases MHCII on the surface of microglia in a T cell-dependent manner. Thus, IL2 provides another novel way to investigate microglia-T cell interaction and by modifying the IL2 cellular selectivity there is potential to unpick changes in microglia phenotype dependent on specific expansion of T cell subsets. Overall, I have developed several novel tools for the investigation of both competitive cellular kinetics of microglia, as well as for the investigation of the effect of CD4 T cell interaction on microglia phenotype. These tools will aid us in gaining a better understanding of the complex immune environment in the brain and help identify novel therapeutic targets for neuro-inflammatory disease.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2024

Author dc:creator

• Dashwood, Amy

Advisor dc:contributor.advisor

• Liston, Adrian

• neuroimmunology
• immunology
• microglia
• T cell
• IL2
• interleukin 2

Licence

• https://www.repository.cam.ac.uk/bitstreams/b2c7dc98-20e4-4d15-933f-929accf7adda/download
• http://purl.org/NET/rdflicense/allrightsreserved

Author Identifier

0000-0002-4340-377X

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/385561