Development of a model system to investigate the effects of haptenation on antigen processing

• Main Author: Charlton, Holly
• Published: University of Leicester 1998
• Subjects: 616.07
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.696519

The objective of this study was to determine whether the haptenation of protein changes qualitatively the peptides produced by antigen processing. This hypothesis suggests that T cells will have no self-tolerance to neo-epitopes exposed by haptenation. Any T cell recognising such an epitope may therefore be involved in the pathogenesis of hapten-induced autoimmunity. The model protein used was the house dust mite antigen, Der p II. To minimise the complexity of processed peptides visualised following antigen processing, the Der p II cDNA was mutated by PCR extension overlap mutagenesis, to replace three tyrosine residues with alanines. The single tyrosine residue of the mutated Der p II fusion protein (Der p II-3Y) was iodinated and Der p II-3Y was haptenated with trinitrophenol (TNP). Iodinated Der p II-3Y +/- TNP was incubated with EBV-transformed B cells. To overcome low antigen uptake by non-specific EBV-transformed B cells, a system developed by C. Watts and A. Lanzavecchia using EBV-transformed B cell clones specific for the C-fragment of tetanus toxin was employed. The C-fragment protein was iodinated throughout and the haptenation studies repeated. The peptide fragmentation patterns seen were unaltered +/- TNP. T cell proliferation offered a more sensitive readout than autoradiography to visualise possible neo-epitope production. Haptenation of Der p II-3Y and C-fragment led to the loss of a proliferation response of specific T cell clones and T cell lines, respectfully. A lower level of C-fragment haptenation caused increased T cell proliferation. To prolong the life of T cells in culture, the caspase inhibitor z-VAD-FMK was investigated for its ability to prevent activation induced cell death (AICD), z-VAD-FMK reduced the level of AICD in PMA-PHA-P and anti-Fas-stimulated Jurkat and C-fragment-specific T cell lines. Der p II-3Y-specific T cell clones appeared to proliferate normally following prior treatment with z-VAD-FMK. Further studies, not only on the effects of haptenation on antigen processing, but also on the uptake, trafficking and efficient presentation of antigen by antigen presenting cells will help in our understanding of the possible role of haptenation in the induction of autoimmune disease.