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Title: Identification of novel immunogenic HLA-DR-restricted peptides from tumour-associated antigens
Author: Rojas, José-Manuel.
Awarding Body: Nottingham Trent University
Current Institution: Nottingham Trent University
Date of Award: 2003
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CD4+ T cells playa central role in antitumour immunity; not only do they provide help for the development of CTL recognising tumour antigens but they can also enhance antitumour responses via indirect cytotoxic mechanisms at the tumour site. Since CD4+ T cells recognise the antigen in the form of pep tides presented on MHC class II molecules, attention has been focused in the recent years on the identification of these peptides derived from tumour antigens. Therefore the aim of this study was to identify novel immunogenic peptides derived from tumour antigens where presentation was restricted to human MHC class II HLA-DRI and/or HLADR4 molecules. The adopted strategy consisted in predicting peptides from the tumour antigens p53, gplOO and bcr-abl(b3a2) using computer-assisted algorithms, and immunisation of HLA-DRI transgenic mice with these peptides in order to assess their immunogenicity. Immunogenic peptides in transgenic mice were then tested in human in in vitro T cell sensitisation assays. To determine peptide immunogenicity in mice, a method was optimised using the reported I-Ak-restricted peptides HEL46-61 and HEL1l 9-132. This model was then successfully established in HLA-DRI transgenic mice with the model peptide HA307- 319. Proliferative responses and IFN-y production were observed when the splenocytes of HLA-DRI transgenic mice were re-presented in vitro with the HA307-319 peptide used in immunisation. Dendritic cells (DC) were shown to be better antigen presenting cells (APC) than syngeneic splenocytes in proliferation assays; thus DC were subsequently used as APC in the all experiments. Further characterisation of DC, generated from bone marrow precursors by culture with GM-CSF, demonstrated that day 8 non-adherent cells matured with LPS were optimal for antigen presentation in this experimental setting. Immunisation of HLA-DRI transgenic mice with predicted peptides from p53, gplOO and bcr-abI(b3a2) resulted in HLA-DRlrestricted responses for two novel p53 peptides (p5363-77 and p53108-122) and two bcrabl peptides (bcr-abIGFK11 and bcr-abIATG1 8). Responses were also observed to two novel gp 100 peptides (gp 1 00194-208 and gp 100566-580). This study demonstrated that HLA-DR-restricted responses to novel peptides can be obtained in HLA-DRI transgenic mice. Furthermore, proliferative responses to p5363-77 in a HLA-DRI + donor, to gpl00566-580 in another HLA-DRl+ donor. and to p53108-122 in two HLA- -1- Abstract DR4+ donors demonstrated that these peptides were also immunogenic in human assays. Collectively, these results indicated that peptide immunisation of HLA-DRI transgenic mice could facilitate the identification of novel immunogenic HLA-DRrestricted pep tides from tumour antigens, that allow us to understand further the role of CD4+ in antitumour immunity and improve cancer immunotherapeutic strategies.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Transgenic mice Medicine Molecular biology Cytology Genetics