A novel principle for immunomodulator delivery with hematopoietic cells as vehicles Focus on soluble TNF-receptor gene expression, targeting to secretory lysosomes and regulated secretion

Detta är en avhandling från Divison of Hematology and Transfusion Medicine, Lund University

Sammanfattning: The goal of this research is to use hematopoietic cells as vehicles for targeting transgenic immunomodulators to the inflammatory sites. The work presented was focused on the anti-inflammatory soluble TNF receptor. A transmembrane soluble TNF receptor (sTNFR1) construct was expressed in hematopoietic cell lines. ER export was facilitated by addition of a transmembrane (tm) sequence, and constitutive secretion was overcome by incorporating a tyrosine-base cytosolic sorting signal (Y) taken from CD63. This signal directed sTNFR1-tm-Y from the trans-Golgi network (TGN) to secretory lysosomes, followed by generation of membrane-free soluble sTNFR1. The secretion of sTNFR1 could be triggered by a calcium-ionophore or by crosslinking of the IgE receptors on mast cells leading to degranulation. In contrast, sTNFR1-tm-Y-egfp in which the sorting signal (Y) was hidden by egfp was, at least in part, transported from TGN to the plasma membrane. This was followed by endocytosis and possible secretory lysosome targeting. PKC-activation with phorbol ester induced ectodomain shedding of sTNFR1 from sTNFR1-tm-Y-egfp. In addition to cell lines, the soluble TNF receptor construct sTNFR1-tm-Y was also successfully expressed in normal murine hematopoietic progenitor cells. During granulopoietic differentiation sTNFR1-tm-Y targeting was achieved to primary granules followed by proteolytic generation of sTNFR1. Finally, our work has shown that an immunomodulatory agent such as soluble TNF receptor can be synthesized with a native conformation, be targeted to a storage compartment, and become secreted upon stimulation.