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.

Investigating the role of target cell availability in the pathogenesis of feline immunodeficiency virus infection

Feline immunodeficiency virus (FIV) is a naturally occurring lentivirus of domestic cats, which shares many similarities with its human counterpart, human immunodeficiency virus (HIV). FIV infects its main target cell, the CD4+ T lymphocyte, via interactions with its primary receptor CD134 (an activation marker expressed on activated CD4+ T lymphocytes), and, the chemokine receptor CXCR4. According to the different ways in which FIV isolates interact with CD134, FIV may be categorised into two groups. The first group contains strains that tend to dominate during the earlier phase of infection, such as GL8 and CPG41. These strains are characterized by their requirement for an additional interaction with the second cysteine rich domain (CRD2) of the CD134 molecule and are classified as “CRD2-dependent” strains. The second group, on the other hand, contains either laboratory-adapted isolates or isolates that emerge after several years of infection, such as PPR or the GL8 variants that emerged in cats 6 years post experimental infection and were studied in this thesis. These isolates are designated “CRD2-independent” as they can infect target cells without interacting with CRD2 of the CD134 molecule. This study provides the first evidence that FIV compartmentalisation is related to FIV-CD134 usage and the tissue availability of CD134+ target cells. In tissue compartments containing high levels of CD134+ cells such as peripheral blood and lymph nodes, CRD2-dependent viruses predominated, whereas CRD2-independent viruses predominated in compartments with fewer CD134+ cells, such as the thymus. The dynamics of CD4+CD134+ T lymphocytes at different stages of FIV infection were also described. The levels of CD4+CD134+ T lymphocytes, which were very high in the early phase, gradually decreased in the later phase of infection. The dynamics of CD4+CD134+ T lymphocyte numbers appeared to correlate with FIV tropism switching, as more CRD2-independent viruses were isolated from cats in the late phase of infection. Moreover, it was observed that pseudotypes bearing Envs of CRD2-dependent variants infected CD134+ target cells more efficiently than pseudotypes bearing Envs of CRD2-independent variants, confirming the selective advantage of CRD2-dependent variants in environments with high levels of CD134+ target cells. In conclusion, this study demonstrated that target cell types and numbers, as well as their dynamics, play important roles in the selection and expansion of FIV variants within the viral quasispecies. Improved understanding of the roles of target cells in FIV transmission and pathogenesis will provide important information required for the development of an improved, more successful protective FIV vaccine and will provide insight into the development of effective vaccines against other lentiviral infections such as HIV.