Catheptic antigen processing

For an antigen to be recognized by T cells, it must first be processed into short peptide fragments and presented by an MHC molecule at the cell surface. In this thesis, the roles of cathepsins B, D and E in MHC class II antigen processing are investigated in human B cells. A vector-based antisense approach is adopted using EBV-transformed B cell presentation of tetanus toxoid to human T cell clones as a model system. A series of vectors are made containing a portion of each cathepsin gene in a sense or antisense orientation under the control of a metallothionein promoter. Stable transfection of these constructs produces a panel of EBV-cell lines which can be induced to express anti-cathepsin or control RNA. Specific reductions in cathepsin protein levels and activity is monitored in these lines using a variety of biochemical techniques. The effect of such reductions on antigen processing capacity is investigated via tetanus toxoid presentation to T cell clones. The antigen processing roles of cathepsins B, D and E are also investigated by treating B cells with short antisense oligonucleotides. Human B cells expressing the murine MHC II molecule I-Ak are pre-treated with phosphorothioate oligonucleotides and then challenged to present hen egg lysosyme (HEL) to murine T cell hybridomas. The effect of soluble cathepsin inhibitors on antigen processing is also investigated for both described antigen presentation systems, and a comparison of the three investigative approaches is made. Finally, it is shown that transcription of the cathepsin E gene is upregulated upon B cell activation (polyclonal activation of ex vivo tonsillar B cells and PMA-activation of an EBV-transformed B cell line). It is also shown that cells that do not usually present antigen (chondrosarcoma and HeLa cells) can be induced to express cathepsin E when treated with IFNγ. Mutant HeLa cells are used to unravel the potential roles of CTIIA and JAK1 in the transcriptional control mechanism