Analysis and modelling of immune cell behaviour in lymph nodes based on multi-photon imaging

This thesis presents quantitative studies of T cell and dendritic cell (DC) behaviour in mouse lymph nodes (LNs) in the naive state and following immunisation. These processes are of importance and interest in basic immunology, and better understanding could improve both diagnostic capacity and therapeutic manipulations, potentially helping in producing more effective vaccines or developing treatments for autoimmune diseases. The problem is also interesting conceptually as it is relevant to other fields where 3D movement of objects is tracked with a discrete scanning interval. A general immunology introduction is presented in chapter 1. In chapter 2, I apply quantitative methods to multi-photon imaging data to measure how T cells and DCs are spatially arranged in LNs. This has been previously studied to describe differences between the naive and immunised state and as an indicator of the magnitude of the immune response in LNs, but previous analyses have been generally descriptive. The quantitative analysis shows that some of the previous conclusions may have been premature. In chapter 3, I use Bayesian state-space models to test some hypotheses about the mode of T cell search for DCs. A two-state mode of movement where T cells can be classified as either interacting to a DC or freely migrating is supported over a model where T cells would home in on DCs at distance through for example the action of chemokines. In chapter 4, I study whether T cell migration is linked to the geometric structure of the fibroblast reticular network (FRC). I find support for the hypothesis that the movement is constrained to the fibroblast reticular cell (FRC) network over an alternative 'random walk with persistence time' model where cells would move randomly, with a short-term persistence driven by a hypothetical T cell intrinsic 'clock'. I also present unexpected results on the FRC network geometry. Finally, a quantitative method is presented for addressing some measurement biases inherent to multi-photon imaging. In all three chapters, novel findings are made, and the methods developed have the potential for further use to address important problems in the field. In chapter 5, I present a summary and synthesis of results from chapters 3-4 and a more speculative discussion of these results and potential future directions.

The pathophysiology of CADASIL: studies in a Scottish cohort

Since identification that mutations in NOTCH3 are responsible for cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) in the early 1990s, there has been extensive characterisation of the clinical and radiological features of the disease. However therapeutic interventions remain elusive, partly due to a limited understanding of the vascular pathophysiology and how it leads to the development of strokes, cognitive decline and disability. The apparent rarity and heterogenous natural history of CADASIL potentially make conducting any longitudinal or therapeutic trials difficult. The role of disease biomarkers is therefore of some interest. This thesis focuses on vascular function in CADASIL and how it may relate to clinical and radiological markers of disease. Establishing the prevalence of CADASIL in the West of Scotland was important to assess the impact of the disease, and how feasible a trial would be. A mutation prevalence of 10.7 per 100,000 was demonstrated, suggesting significant under diagnosis of the disease across much of Scotland. Cerebral hypoperfusion is thought to be important in CADASIL, and it has been shown that vascular abnormalities precede the development of brain pathology in mouse models. Investigation of vascular function in patients, both in the brain and systemically, requires less invasive measures. Arterial spin labelling magnetic resonance imaging (MRI) and transcranial Doppler ultrasound (TCD) can both be used to obtain non-invasive and quantifiable indices of vascular function. Monitoring patients with MRI whilst they receive different concentrations of inspired oxygen and carbon dioxide can provide information on brain function, and I reviewed the practicalities of this technique in order to guide the design of the studies in this thesis. 22 CADASIL patients were recruited to a longitudinal study. Testing included peripheral vascular assessment, assessment of disability, neurological dysfunction, mood and cognition. A CO2 reactivity challenge during both TCD and arterial spin labelling MRI, and detailed MRI sequences were obtained. I was able to demonstrate that vasoreactivity was associated with the number of lacunes and brain atrophy, as were carotid intima-media thickness, vessel stiffness, and age. Patients with greater disability, higher depressive symptoms and poorer processing speed showed a tendency to worse cerebral vasoreactivity but numbers were small. This observation suggests vasoreactivity may have potential as a therapeutic target, or a biomarker. I then wished to establish if arterial spin labelling MRI was useful for assessing change in cerebral blood flow in CADASIL patients. Cortical grey matter showed the highest blood flow, mean (SD), 55 (10) ml/100g/min and blood flow was significantly lower within hyperintensities (19 (4) ml/100g/min; p <0.001). Over one year, blood flow in both grey matter (mean -7 (10) %; p = 0.028) and deep white matter (-8 (13) %; p = 0.036) declined significantly. Cerebrovascular reactivity did not change over one year. I then investigated whether baseline vascular markers were able to predict change in radiological or neuropsychological measures of disease. Changes in brain volume, lacunes, microbleeds and normalised subcortical hyperintensity volume (increase of 0.8%) were shown over one year. Baseline vascular parameters were not able to predict these changes, or those in neuropsychological testing. NOTCH3 is found throughout the body and a systemic vasculopathy has been seen particularly affecting resistance vessels. Gluteal biopsies were obtained from 20 CADASIL patients, and ex vivo myography investigated the response to vasoactive agents. Evidence of impairment in both vasodilation and vasoconstriction was shown. The addition of antioxidants improved endothelium-dependent relaxation, indicating a role for oxidative stress in CADASIL pathology. Myography measures were not related to in vivo measures in the sub-group of patients who had taken part in both studies. The small vessels affected in CADASIL are unable to be imaged by conventional MR imaging so I aimed to establish which vessels might be responsible for lacunes with use of a microangiographic template overlaid onto brain images registered to a standard brain template. This showed most lacunes are small and associated with tertiary arterioles. On the basis of this thesis, it is concluded that vascular dysfunction plays an important role in the pathophysiology of CADASIL, and further assessment of vascular measures in longitudinal studies is needed. Arterial spin labelling MRI should be used as it is a reliable, non-invasive modality that can measure change over one year. Furthermore conventional cardiovascular risk factor prevention should be undertaken in CADASIL patients to delay the deleterious effects of the disease.