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Title: A study of immune responses to ras transformed tumour cells expressing well-defined antigens
Author: Johnson, Jennifer Linda
ISNI:       0000 0001 3591 2059
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 1991
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In this work tumour cells expressing well-defined antigens were generated to study the importance of interferon modulation of T cell mediated immune responses to foreign antigens. The antigens used were those of a murine leukaemia virus (XLV). DIA clones of this virus were manipulated using standard molecular biology techniques to generate replication-defective viruses expressing individual XLV antigens (env or gag) when transfected into tumour cells. The tumour cell line used in this study (C3H201) was produced in our laboratory by the transformation of murine fibroblasts (C3H10TX) with the Kirsten murine sarcoma virus (MSV) which expresses the ras oncogene. Initially cells transfected with the MLV genes were selected by neomycin resistance and preliminary investigations using these cells suggested that gag is the main T-cell target in this system; the gag expressing cells were more susceptible to MSV/XLV-specific Tc lysis and were less tumourigenic than the env transfected cell lines. However it was found that the cells expressing the neomycin resistance gene alone also elicited an Immune response (increased susceptibility to Tc lysis and less tumourigenic than the untransfected C3H201 cells) thus it was suggested that this selection system was not suitable for cells to be used in immunological studies and an alternative selection system was needed. To overcome the problems observed with neomycin resistance a novel selection system using the major histocompatibility (MHC) antigen D* was developed. The H-2D* gene was transfected into the H-2D* negative cell line C3H201 < H-2* > with the DIA of interest and then the cells expressing the D* antigen were sorted on the FACS following IFI-f treatment and indirect immunofluorescence staining. Experiments to determine the Immunological importance of the antigen of interest were then performed in the FI progeny from a cross between C3H/Ha (H-2*) and C57BL6 (H-2*) mice which recognise the endogenous and acquired KHC antigens of the transfected cell line as self. This selection system thus enabled the examination of the immune response to the proteins of interest without actually interfering with the response they elicit. This system was used to produce gag expressing cells and also a cell line that expresses all the XLV proteins without producing the virus itself (designed to facilitate an examination of the effect of virus replication and subsequent infection on the response to XLV antigens by comparison with a XLV expressing cell line). These cell lines were found to be susceptible to lysis by Xo-MSV/XLV-specific Tc and this lysis was increased by IFI-V treatment (cells transfected with D* alone, DC3H201, showed no such lysis). It is now known that Tc usually recognise antigen in association with class I KHC antigen. Furthermore IFV-y treatment of C3H201 cells has been shown to increases class I KHC antigen expression on these cells. Thus it is proposed that the increased susceptibility to lysis observed for these IFI-t treated cells is due to increased class I KHC antigen expression. These cells also show reduced tumourigenicity in mice in comparison with DC3K201 and C3H201 thus confirming the recognition of gag by the immune system (in the case of DK-gagl and DX- gag2) and furthermore the correlation between the tumourigenicity and susceptibility to Tc lysis of all these cells supports the importance of the Tc element in the immune response to these tumour cells.
Supervisor: Not available Sponsor: Cancer Research UK
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: RC0254 Neoplasms. Tumors. Oncology (including Cancer)