Publication: Towards a surrogate system to express human lipid binding TCRs

What makes a protein an allergen, and in particular a food allergen, is not well understood. Previously we reported that natural nut lipids were necessary for sensitization and that Natural Killer T Cells (NKTs) must play a critical role in the development of Brazil nut allergic responses. One of the major bottlenecks in the further studies of the interaction of nut natural lipids with the cells of the immune system is the lack of well-characterized lipid responsive human cell lines. In the present study, we engineered human TCRs sequences TRAV10 (clone J3N.5T) and TRBV25 (clone BM2a.t) responsive to GalCer into a stable murine iNKT hybridoma cell line. This system has shown to be problematic as far as expression of new and functional human TCR sequences is concerned. Here we show that the expression of human TCR sequences has been achieved either driven by a viral bidirectional promoter or a polycistronic construct using plasmids or a lentivirus system using either murine or human stable DC cell lines as lipid presenting cells and a stable T cell line as a surrogate system. Further, we show that the commercial human Jurkat cell line containing an inducible secreted luciferase reporter construct regulated by human NFAT binding sites was functional and can be used for a transient expression of human TCRs in a lipid screening program. We also show that transfection efficiencies were improved using the lentivirus polycistronic constructs containing the P2A sequence in a TCR αβ/γδ null cell line (Jurkat 76). These results suggest that indeed the mis-pairing of the endogenous α/β TCR during ER folding in the presence of the new human TCR sequences could be impairing the functionality of the TCR lipid receptors. This will help towards a surrogate system to express functional human lipid binding TCR sequences. These are important first steps in the establishment of cell specific lipid responsive libraries for the study of natural lipid substances.

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