Kurzfassung

The aim of the present project is to characterize the T helper (Th) cell response against KLH in detail. To this end, KLH-specific T cell lines have been established and several T cell clones are currently generated with the aid of a mixture of KLH1 and KLH2 and LPS-activated bone marrow-derived dendritic cells (BMDC) as antigen presenting cells. T cell epitope mapping studies will be performed by using such KLH-specific T cell clones and overlapping KLH peptides as antigen. Alternatively,...The aim of the present project is to characterize the T helper (Th) cell response against KLH in detail. To this end, KLH-specific T cell lines have been established and several T cell clones are currently generated with the aid of a mixture of KLH1 and KLH2 and LPS-activated bone marrow-derived dendritic cells (BMDC) as antigen presenting cells. T cell epitope mapping studies will be performed by using such KLH-specific T cell clones and overlapping KLH peptides as antigen. Alternatively, BMDC or A20 B cell tumor cells will be pulsed with KLH and the MHC-bound peptides extracted (acid wash). These extracts will be fragmented on a C2/C18 reversed phase column and the resulting fractions tested in a Th cell stimulating bioassay. The active fractions will be pooled and rechromatographed under different conditions. Subsequently, these fractions will be again screened with the bioassay. Subsequently peptides from the active fractions will be sequenced using µcap-LC-MS/MS system (Waters). Such KLH-derived T cell epitopes will be used to recruit T cell help for CD8+ T cell and B cell activation. As compared to the whole KLH molecule these peptides should be highly effective since they do not need to be processed. In a further approach the T cell receptor alpha and beta chain of KLH-specific Th clones will be cloned and used to generate a T cell receptor transgenic mouse. This mouse will be used to analyze the helper function of KLH-specific Th cells for CD8+ T cells and B cells. In addition these mice or isolated transgenic Th cells will be used to establish a tumor model. In summary these studies should identify Th epitopes of KLH and elucidate the Th-stimulatory properties of this molecule in vivo. Finally, in this project the outstanding knowledge about the molecular properties of KLH will be combined with the expertise in T cell immunology for detailed analyses of the immune stimulatory function of KLH.» weiterlesen» einklappen