Generation of MHC II Streptamers for gluten-specific CD4+ T cells
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- Master's theses (KBM) 
Coeliac disease is a chronic inflammatory disorder of the small intestine due to inappropriate immune responses to gluten. Disease-associated HLA-DQ2.5 molecules are complexed with deamidated gluten epitopes displayed on antigen-presenting cells and presented to CD4+ T cells. The investigation of T cells and their interaction with MHC molecules has revolutionised the research of cell-mediated immunology by the multimer technology. This technology was developed by Altman et al. in 1996. Gluten-specific CD4+ T cells have been detected and identified by a similar MHC II tetramer technology. Soluble recombinant HLADQ2.5 molecules with covalently linked epitope peptides are expressed in the baculovirus expression vector system and purified in their folded state. Biotinylated MHC II reagents are multimerised on streptavidin prior to T-cell staining in flow cytometry. A reversible multimer technology based on the tetramer technology and the Streptag:streptavidin interaction has been established for MHC class I molecules and CD8+ T cells. Basic principle of the streptamer technology is that multimerised MHC molecules on streptavidin (Streptactin) are left on the T-cell surface as monomers in the presence of dbiotin. D-biotin binds to streptavidin and leads to dissocation. Hence, the interaction of the Streptag and Streptactin is reversible. This technology is also basis for a recently developed koff-rate assay that measures the dissociation of monomeric MHC molecules from TCR. To date, there are no publications on the so-called Streptamer technology applied on MHC class II molecules and antigen-specific CD4+ T cells. Here, we have generated recombinant, soluble HLA-DQ2.5 molecules fused with the affinity peptide StreptagII. Based on the well-established recombinant DQA and DQB gene sequences in the Sollid lab, the BirA site was replaced with a tandem repeat of the StreptagII sequence (StreptagIII) and a dye conjugation site at the C-terminus of the β chain. Dye-conjugated MHC II can therefore be observed in the absence of the Streptactin backbone. The new MHC II multimer reagents (Streptamers) were quality checked prior to flow cytometry. They were tested in flow cytometry to verify specific staining to gluten-reactive CD4+ T-cell clones (TCCs). Two specific HLA-DQ2.5-Streptag constructs were made. One contruct demonstrated positive staining of the majority of the cognate DQ2.5-reactive TCCs while the other construct showed variable staining signals. Possible interference with the dye conjugation was considered. Subsequently, TCCs that showed high staining with the dyeconjugated Streptamers were tested in the human koff -rate assay in Munich. The performance of the koff-rate assay did not give any useful data, but problems were addressed and investigated in order to improve the MHC II reagents. These variable findings remain to be elucidated and must be further investigated. The Streptamer-positive TCCs remain to be retested in the koff-rate assay.