Generation of an MHC class II based tumour vaccine in a murine breast carcinoma model.
Doctoral thesis, UCL (University College London).
This study involved the conversion of class II MHC negative tumour cells, via transduction with the potent class II MHC transcription factor, Class II Transactivator (CIITA) to enhance in de novo expression of MHC class II molecules. CIITA-transfected tumour cells may act as surrogate antigen presenting cells, processing and presenting endogenous tumour specific antigen in an MHC class II restricted manner to CD4+ T cells. CIITA has the major advantage over other cancer immunotherapy strategies, in that CIITA transfection of tumour cells evokes expression of all three MHC class II isotype; avoiding the need to tissue-type the recipient, a limit of previous MHC class I based strategies. MHC class I restricted strategies have proved largely unsuccessful in the past, due to weak CTL responses which have been unable to control tumour growth in vaccinated patients; thought to be due to the fact that MHC class II restricted CD4+ T cell help is required of optimal induction of both humoral and cellular immune responses. Although, CIITA-transfected tumour cells had been shown to be immunogenic, previous in vivo studies indicate that CIITA gene transfection failed to induce tumour rejection in syngeneic mice, due to up-regulation of invariant chain (Ii). Invariant chain is hypothesised to bind to the antigenic peptide-binding cleft of nascent Class II MHC molecules, thus preventing the binding of endogenous tumour antigens. The realisation of this work involved setting various techniques such as cell culture, RNA and protein extraction, RT-PCR, Western blotting, transfection, RNA interference, flow cytometry, immunofluorescence, immune -histochemistry, isolation of CD4+ and CD8+ T cells from lymph nodes, T cell proliferation, cytotoxic T cell and cytokine assays, as well as in vivo studies. Murine 4T1 mammary carcinoma cell line, a poorly immunogenic but highly tumourigenic tumour cell line, which closely mimics metastatic spread of human stage IV breast cancers in BALB/c mice, was genetically modified by transfection with CIITA. This resulted in up-regulation of MHC class II and co-expression of invariant chain, both at the mRNA and protein level. MHC class I expression was also slightly increased after this transfection. The novel technique of RNA interference was employed in this study to inhibit Ii chain expression, previously thought to render MHC class II based vaccine ineffective. However, CIITA-transfected 4T1 tumour cells were rejected in syngeneic BALB/c mice or induced slower tumour without the need to inhibit invariant chain activity. Tumour growth kinetics was found to be dependent on tumour load. Mice rejecting CIITA-transfected cells were found to be resistant to re-challenge with wild-type 4T1 tumour cells, indicating immunological memory. This rejection was also found to be tumour specific. T and B cells were implicated in tumour rejection in studies with BALB/c mice, and immunohistological studies demonstrated that mainly also confirmed by T cell proliferation and CD4+ and CD8+ T cells, as well as dendritic cells were involved in the anti-tumour immune response. The role of CD4+ and CD8+ T cells was also assessed via in vitro studies and this immune response was found to be mediated by Th1 cytokines. Traditional therapies for metastatic tumours, such as surgery, radiation or chemotherapy are invasive, often have quite drastic side-effects and are frequently not effective. However, these results open the possibility of utilising CIITA as a biological tool, utilising the body's own immune system to eradicate or prevent cancer.
|Title:||Generation of an MHC class II based tumour vaccine in a murine breast carcinoma model|
|Additional information:||Permission for digitisation not received|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Brain Sciences > Institute of Ophthalmology|
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