Identification of T-cell specific autoantigens

The mechanisms that lead to the formation of autoantibodies are not understood. Accumulating data suggest that autoantigens might be characterized by post-translational modifications that are part of the natural apoptosis process such as granzyme B cleavage (i.e. topoisomerase I, LA(SS-B), U1-70 kd), caspase cleavage (i.e. topoisomerase I and II), phosphorylation/dephosphorylation (i.e. ribosomal P proteins, LA(SS-B), transglutaminase crosslinking (i.e. histone H2B) and deimination (arginine-citrullin change in fibrin or fibrinogen). T cells, which have been negatively selected or tolerized against native autoantigens, might be primed by modified autoantigens and deliver help to self-reactive B cells. So far, there have been only few reports about autoantigen specific T cells. Since the frequencies of those T cells in the peripheral blood are very low, autoantigen specific T-cell responses are difficult to detect. In our study we are combining the use of monocyte derived dendritic cells (DC) as professional antigen presenting cells with intracellular cytokine staining for the detection of T-cell responses. Monocytes and lymphocytes are purified from freshly isolated PBMC by density gradient centrifugation. Lymphocytes are frozen in liquid nitrogen until usage. Monocytes are differentiated into immature dendritic cells in culture medium containing either 1% FCS or 10% autologous serum and GM-CSF and IL-4. Maturation is induced by addition of GM-CSF, LPS, TNF-α and IL-1-β in the presence or absence of a specific antigen. Mature DC expressing antigen derived peptides are washed and cocultured with thawed autologous lymphocytes. After 4 hours, brefeldin A is added and lymphocytes are intracellulary stained with CD3, CD4, CD69 and TNF-α mAb after o/n culture. We will use this system to identify possible T-cell specific autoantigens by using granzyme B cleaved nuclear extracts and purified modified nuclear antigens.