Towards unravelling the immunomodulatory mechanisms of S. mansoni & L. major parasites in a mouse model using transgenesis
Schistosoma mansoni and Leishmania major parasites use immunomodulatory strategies to evade the host’s immune response and little is known about the mechanisms by which these two parasites modulate host immune responses. As a result, these parasites develop different subversion mechanisms to escape host immunity and controlling such immunomodulatory pathogens is still not successful. In this project we hypothesized that an understanding of the ability of memory T cells to withstand pathogen manipulation is crucial for the development of effective vaccine strategies to control these pathogens. Therefore, in this study, OVA transgenic S. mansoni and OVA transgenic L. major were generated to use as a tool to measure the degree of immune memory resilience (defined as an ability of the immune memory to withstand pathogen manipulation) of T helper cells on the face of pathogen-mediated immune manipulation in vivo. The specific aims of this thesis are: i) Express recombinant OVA in both S. mansoni and L. major, ii) Demonstrate that OT-II TCR transgenic T cells are able to recognise the OVA expressed by the parasites, iii) Differentiate the OT-II cells into Th1, Th2 and Th17 cells in vitro and demonstrate their functional phenotype, and iv) Investigate whether the parasites expressing OVA are able to influence the cytokine expression profile of the Th1, Th2 and Th17 OT-II memory T cells. To achieve this, first OVA expression by S. mansoni and L. major was confirmed using RT-PCR and western blot. Subsequently, in vitro and in vivo analysis for proliferative responses of OT-II T cells revealed OVA was recognized by OT-II T cells. The proliferated cells also produced cytokine signatures following stimulation with the OVA expressing parasites both in vitro and in vivo. To measure the immune memory resilience of memory T cells against such pathogens, an OT-II mouse model was used as a source of naive OT-II T cells. Hence, Th1, Th2, and Th17 polarised memory cells were generated in vitro and these cells were adoptively transferred to recipient mice to investigate the immune memory resilience in the face of pathogen-mediated manipulation. After transferring memory cells, mice were challenged with OVA-transduced S. mansoni eggs and OVA transfected L. major parasites as well as wild-type controls. After recovery of Th memory cells, their proliferation rate and cytokine signature was analysed using flow cytometry. The in vitro differentiated Th1, Th2 and Th17 memory cells produced cytokines when challenged by OVA-expressing S. mansoni eggs and OVA expressing L. major. Indeed, Th1 memory cells produced significant level of IFN-g and TNF, while, Th2 memory cells produced high level of IL-2, IL-6 and IL-10, similarly, Th17 memory cells produced IL-17 when stimulated with OVA-expressing parasite eggs and OVA expressing L. major. However, Th1, Th2, and Th17 memory T cells didn’t proliferate in response to wild type S. mansoni eggs, and wild type L. major, and when they were left unstimulated. Therefore, the Th phenotypes of the memory T cells remains unaltered in the face of stimulation by two immune manipulative pathogens (S. mansoni and L. major). The ability of memory T cells to remain resilient to manipulation by these two pathogens has important implications for the prospect of developing vaccines against these parasites.
Immune memory, Immunomodulatory pathogen, In vitro, In vivo, , OVA, resilience, Th memory cells.
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