Membrane-Bound Hsp70 an Activating Ligand for NK Cells

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.