Microbiology & Immunology - Theses
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ItemNo Preview AvailableThe molecular and cellular basis of antigen recognition by CD1a-restricted T cells( 2022)In contrast to conventional T cells that recognise peptide antigens presented by MHC molecules, a group of “unconventional” T cells recognise lipid antigens presented by MHC-like CD1 family members, CD1a, CD1b, CD1c and CD1d. Studies suggest that CD1a-restricted T cells comprise a unique subset in human blood that recognise CD1a-lipid complexes and play a unique functional role in skin immunity. While they comprise a decent proportion of T cells compared to CD1d-restricted, natural killer T (NKT) cells, they remain relatively less well-understood. This thesis describes the phenotypic characterisation of CD1a-restricted T cells in human tissues directly ex vivo. Phenotypic analyses and single cell RNA-sequencing of CD1a-restricted T cells revealed that they are distinct from other CD1-restricted T cells. They did not express typical innate-like markers such as CD161, IL-18R, and PLZF, which are expressed by NKT cells, distinguishing them as a unique population of unconventional T cells. This thesis also elucidates how T cell receptors (TCRs) interact with CD1a-lipid complexes. Profiling the TCR repertoire of CD1a-restricted T cells, demonstrated that while diverse, there is a bias towards TCR variable genes that endow optimal TCR configurations to interact with CD1a and lipid antigens. Experiments with CD1a mutant cell lines revealed that individual TCRs bind at various sites across the entire binding cleft of CD1a, which likely increases the diversity of lipid antigens that can be recognised by CD1a-restricted T cells. Indeed, these T cells were observed to recognise numerous lipid antigens including self-lipids and dideoxymycobactin (DDM), a lipid antigen derived from Mycobacterium tuberculosis, with some CD1a-restricted TCRs even displaying cross-reactivity to lipids with distinct chemical structures. Reagents were developed as tools to study lipid-reactive T cells in macaques, especially for non-human primate models of disease. A suite of CD1 tetramers were generated to isolate CD1-restricted T cells in pig-tailed macaques and for preliminary enumeration and phenotypic analysis of CD1-restricted T cell subsets in macaque tissues. Lastly, tetramers were used to investigate CD1a-restricted T cells in human skin. Populations of lipid-reactive T cells and gd-T cells were isolated for phenotypic analysis and TCR sequencing, thus demonstrating that they may play a role in healthy skin. C12-15 alkyl-benzoate, a common oil in dermatological products, was identified as a novel CD1a antigen, suggesting a role for CD1a-restricted T cells in allergic dermatitis. These studies provide insight into the functional properties of CD1a-restricted T cells and their molecular interactions with CD1a-lipids. Collectively, they represent a step forward in characterising CD1a-restricted T cells and provide a greater understanding of their role in the immune system.
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ItemT cell response to an MHC-II restricted epitope of rodent malaria( 2021)Malaria is caused by different Plasmodium species that can infect a variety of animals including humans and rodents. The life cycle of these parasites is complex and includes a liver stage followed by a blood-stage in their vertebrate hosts. While the host’s immune response against each of these stages is incompletely understood, CD4 T cells are known to play an important role in immunity to Plasmodium infection during both stages. This project aims to examine the specific CD4 T cell response to a novel MHC II-restricted epitope in Plasmodium infection in C57BL/6 mice, and to characterise the protective capacity of these T cells. To this end, we made use of a recently generated TCR transgenic mouse line, termed PbT-II, which responds to a so far unknown Plasmodium derived epitope. In this project, the PbT-II epitope was identified as derived from heat shock protein 90, residues 484 to 496 (Hsp90484-496 or abbreviated DIY). Different priming methods, such as injection of an anti-Clec9A antibody attached to the Hsp90 epitope (aClec9A-DIY), infection with P. berghei ANKA (PbA) infected red blood cells (iRBCs) or immunisation with radiation attenuated PbA sporozoites (RAS), were used to characterise PbT-II memory cell formation. Results revealed the formation of memory PbT-II cells expressing surface markers associated with central memory T cells (TCM), effector memory T cells (TEM) and tissue resident memory T cells (TRM). Given the importance of tissue-resident memory T cells in peripheral immunity, mainly studied in CD8 T cells, we focused our study on the formation and function of CD4 TRM cells in the liver. Parabiosis studies using RAS vaccinated mice confirmed the liver residency of a CD69+ PbT-II cell population. Gene expression profile analysis revealed that these CD4 T cells expressed a core gene signature similar to that of CD8 resident memory T cells. Furthermore, differences in the gene expression profile of PbTII TRM cells generated via different protocols, suggested lineage specific effector mechanisms, such as IL-4 production or perforin expression, for subsets of CD4 TRM cells in the liver. As CD4 T cells can potentially act against both the liver and blood-stage of Plasmodium infection, we sought to investigate the protective potential of PbTII effector and memory cells for both of these stages. While none of the PbT-II priming methods resulted in a reduction of liver parasite burden upon sporozoite infection, mice injected with large numbers of in vitro polarized PbT-II Th1 or Th2 cells showed reduced parasitemia after PbA blood-stage infection. Surprisingly, most of these mice were protected from experimental cerebral malaria (ECM), although they were not able to clear PbA blood-stage infection.