The role of regulatory T cells in stroke recovery

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Malone, Kyle
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University College Cork
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Background: Acute ischaemic stroke is a major cause of mortality worldwide. Despite the search for new therapies, tissue plasminogen activator remains the only FDA-approved medication. The immune system is involved in all stages of stroke, from risk factors to pathogenesis and tissue repair. The presence of a lymphocyte subpopulation termed regulatory T cells (Tregs) appears to correlate with improved disease outcome. However, the impact of stroke on Tregs, and the contribution of these cells to stroke recovery remains under debate. Likewise, attempts at developing Treg-targeted immunotherapies have been hampered by high cost, toxicity, and the stability of expanded Tregs. The aim of this thesis was to explore the role Tregs play in ischaemic stroke recovery and evaluate the neuroprotective and immunomodulatory effects of two potential Treg-targeted stroke immunotherapies, namely fingolimod and recombinant pregnancy-specific glycoprotein-1 (rPSG1-Fc). Methods: The effect of fingolimod on Tregs in a mouse model of permanent brain ischaemia was first investigated using a combination of flow cytometry and immunohistochemistry. Next, the impact of fingolimod on Treg suppressive function was characterised via Treg suppression assay. Following this, the immunomodulatory and neuroprotective properties of rPSG1-Fc in permanent brain ischaemia were determined. Finally, the changes in Treg frequency and function in the peripheral blood of mild stroke patients were quantified. Results: Fingolimod increased peripheral Treg frequency in the post-ischaemic mouse. Fingolimod augmented brain infiltration of FoxP3+ T cells, possibly via CCR8 signalling. Fingolimod also enhanced the secretion of IFN-γ, IL-17, and IL-10 from CD4+ T cells. Likewise, fingolimod promoted both suppressive and effector T cell function. rPSG1-Fc improved neurobehavioural recovery in mice post-brain ischaemia, possibly via increased CD4+IL-10+ T cells. Finally, an increased Treg frequency and an increased expression of functional markers of suppression (CTLA-4, PD-1) was observed in stroke patients at 24 hours post-ischaemia, and specifically among proliferating Tregs. However, by 7 days, the expression of PD-1/CTLA-4 among proliferating Treg frequency had returned to baseline. Conclusions: This thesis has made a number of novel insights. A positive impact of fingolimod on both Treg frequency and function post-ischaemia was revealed. The observed dual effect of fingolimod on regulatory and pro-inflammatory T cell function may explain why the drug fails to consistently improve experimental stroke outcome. The immunomodulatory and neuroprotective effects of rPSG1-Fc post-stroke were also characterised for the first time. Finally, the impact of clinical stroke on Treg frequency and phenotype was comprehensively quantified. Overall, this thesis shows Tregs may not play a major role in the early stages of recovery of mild stroke, but therapies manipulating them can still promote functional recovery in a mouse model. It provides a basis for further study on Tregs in ischaemic stroke. It also illustrates rigorous methods by which researchers should test future Treg-targeted stroke immunotherapies.
Tregs , Regulatory T cells , Fingolimod , Stroke , Brain ischaemia
Malone, K. 2022. The role of regulatory T cells in stroke recovery. PhD Thesis, University College Cork.