One of the hallmarks of successful immunotherapy is the generation of high avidity cytotoxic T cells which can recognise and respond to very low concentration of antigens. This sensitivity of T cells is usually determined by peptide titration ELISpot assays. Even though these assays are generally useful, they are laborious and sample demanding. The assays become even more difficult if the peptide(s) accountable for the generation of vaccine specific responses are unknown such as whole protein or cell vaccines. Therefore, there is a need to identify markers which can quickly and reliably identify a high avidity T cell response in cancer vaccination settings. To achieve this goal, this study utilised a C57Bl/6J mouse model which could efficiently generate high and low avidity T cell responses, when immunisation was undertaken with two form of vaccines to deliver the target antigens. The antigenic epitopes used for this study were derived from TRP-2 ‘self’ and ovalbumin (OVA) ‘foreign’ antigens. Immunisation of animals with these antigens in a DNA vaccine format induces a high avidity T cell response, in contrast to the response when these are administered in the peptide vaccine format. However, both the immunisations produced same number of peptide specific CD8+ T cells, which was assessed my multimer staining. When these cells were subjected to in vitro stimulations with the target peptides, the functionality of the low avidity T cells was restored whereas the high avidity T cells failed to respond to lower peptide concentrations. This showed the plasticity of antigen specific T cells and their ability to modulate their functionality according to the stimulation they have received. In order to identify markers that are associated with the high avidity T cell responses in vivo, antigen specific CD8+ T cells were isolated from high and low avidity groups using MHC multimer sorting. A global transcriptional profiling was conducted on the mRNA isolated from these cells. Analysis of expression data identified several differentially expressed genes between the groups. Six differentially expressed genes (Granzyme A, Granzyme B, FAS Apoptotic Inhibitory Molecule, Telomerase RNA Component, CD5 Antigen-Like, Spi-C Transcription Factor) were further selected and confirmed using qRT PCR. Expression of three genes were correlated with the microarray gene expression data. Among these, two genes (Granzyme A & B) were further confirmed at the protein level using flow cytometry. Further to this, studies of gene expression activation kinetics of TCR signalling using pentamer sorted cells with anti-CD3/CD28 monoclonal antibody coated microbeads, revealed that ImmunoBody® derived high avidity T cells are more signal competent with rapid up-regulation of genes involving in T cell receptor signalling pathway. Proteomic characterisation of MHC multimer sorted cells using LC-MS profiling identified several proteins uniquely associated with Peptide or ImmunoBody® pentamer positive T cells. Many of these proteins were associated with important T cell functional properties. Further confirmation of these markers and their role in T cell avidity is required, however these studies were limited by the lack of availability of proteins from the low number of peptide-specific cells and antibodies.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:635148 |
| Date | January 2013 |
| Creators | Vadakekolathu, J. |
| Publisher | Nottingham Trent University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://irep.ntu.ac.uk/id/eprint/228/ |
Page generated in 0.0843 seconds