Membrane-Bound Hsp70 an Activating Ligand for NK Cells

Abstract

A broad variety of tumor cells express Hsp70 on their cell surface. These tumor cells are more sensitive to the lysis mediated by NK cells. Here, it was shown that the 14mer peptide TKD (TKDNNLLGRFELSG, aa450-463) derived from the C-terminus of Hsp70 is the minimal sequence of Hsp70 that has the capacity to activate NK cells. NK cells incubated with low dose IL-2 (100 IU/ml) plus TKD (2 µg/ml) showed an enhanced proliferative capacity and an enhanced cytolytic activity and migratory capacity against Hsp70 membrane-positive tumor cells. Furterhmore, secretion of the cytokines IFN-g and TNF-a was enhanced. TKD stimulated NK cells also showed enhanced intracellular levels of the serine protease granzyme B. Since binding of fulllength Hsp70 protein and TKD to NK cells was specific and concentration-dependent, an involvement of a Hsp70 specific receptor was hypothesized. It was shown that the C-type lectine receptor CD94 is involved in Hsp70/TKD-NK cell interaction: (i) CD94 was upregulated in NK cells after incubation with Hsp70/TKD; (ii) binding of Hsp70 could be inhibited by a CD94 specific antibody; (iii) Hsp70 reactivity correlated with CD94 expression on NK cells; and (iv) Hsp70 reactivity of NK cells could be inhibited by a CD94 specific antibody. Finally, the mechanism of lysis of Hsp70 membrane-positive tumor cells by NK cells could be elucidated. It was shown that the serine protease granzyme B binds to Hsp70/TKD on the cell surface of Hsp70 membrane-positive tumor cells and is specifically taken up by these cells. As demonstrated by different apoptosis assays (Annexin V staining, cytochrome c release, and DAPI staining) granzyme B causes apoptosis specifically in Hsp70 membrane-positive tumor cells, but not in their Hsp70 membrane-negative counterparts.

• Item Type: Thesis (Dissertation, LMU Munich)
• Keywords: Hsp70, TKD, NK cells, CD94, granzyme B
• Dewey Decimal Classification: 600 Natural sciences and mathematics; 600 Natural sciences and mathematics > 570 Life sciences
• Faculties: Faculty of Biology
• Language: English
• Date Accepted: 01. April 2005
• 1. Referee: Weiß, Elisabeth
• Persistent Identifier (URN): urn:nbn:de:bvb:19-35788
• Signature of the printed copy: 0001/UMC 14528