Investigating MHC class II antigen presentation in HLA- DP4 and HLA-DR4 humanised melanoma cell lines

Major histocompatibility complex class II (MHC-II) molecules play an essential role in the adaptive immune response by presenting antigens to CD4+ T-lymphocytes (T-cells). MHC II is normally expressed on professional antigen presentation cells (APCs) and can be induced by interferon-gamma (IFNγ ) in most cells, including melanoma. This can be exploited to develop vaccines for melanoma. Immunisation with tumour-specific antigens or neoantigens can result in the uptake of these antigens by APCs, which activate CD4+ T-cells. Activated CD4+ T-cells then secrete IFNγ , which induces MHC II expression in melanoma. Tumour- specific antigens and neoantigens bound to MHC II are presented to activated CD4+ T-cells, which can then directly kill the tumour cells. This thesis has used biochemical and proteomic techniques to characterise and elucidate MHC II antigen presentation by HLA-DP4 and HLA- DR4 in HLA-humanised B16F10 melanoma cell lines. Immunofluorescence microscopy imaging and biochemical assays were used to characterise the expression of HLA-DP4 and HLA-DR4 in humanised B16F10 melanoma. Mass spectrometry was used to investigate the proteome, interactome and peptidome of HLA-DP4 and HLA-DR4 in melanoma cell lines. Findings revealed the similarities and differences in proteomics changes and interacting proteins of HLA-DP4 and HLA-DR4, providing a better understanding of MHC II antigen presentation in melanoma. Tumour antigens and neoantigens were identified and were shown to elicit IFNγ response in in vivo immunisation studies. In conclusion, this thesis investigated and categorised the expression and cell biology that drives the processing and presentation of antigens/neoantigens by HLA-DP and HLA-DR in constitutive and inducible HLA humanised melanoma cell lines.