Interaction of heat shock protein 70 peptide with NK cells involves the NK receptor CD94.
Full-length Hsp70 protein (Hsp70) and the C-terminal domain of Hsp70 (Hsp70C) both stimulate the cytolytic activity of naive natural killer (NK) cells against Hsp70-positive tumor target cells. Here, we describe the characterization of Hsp70-NK cell interaction with binding studies using the human NK cell line YT. Binding of recombinant Hsp70 protein (Hsp70) and the C-terminal domain of Hsp70 (Hsp70C) to YT cells is demonstrated by immunofluorescence studies. A phenotypic characterization revealed that none of the recently described HSP-receptors (alpha2-macroglobulin receptor CD91, Toll-like receptors 2, 4, 9, CD14) are expressed on YT cells. Only the C-type lectin receptor CD94 is commonly expressed by YT cells and Hsp70 reactive NK cells. A correlation of the cell density-dependent, variable CD94 expression and the binding capacity of Hsp70 was detected. Furthermore, Hsp70 binding could be completely abrogated by preincubation of YT cells with a CD94-specific antibody. Competition assays using either unlabeled Hsp70 protein or an unrelated protein (GST) in 20-fold excess and binding studies with escalating doses of Hsp70 protein provide evidence for a specific and concentration-dependent interaction of Hsp70 with YT cells. In addition to Hsp70 and Hsp70C, a 14-mer Hsp70 peptide termed TKD is known to exhibit comparable stimulatory properties on NK cells. Similar to full-length Hsp70 protein (10 microg/ml-50 microg/ml), a specific binding of this peptide to YT cells was observed at 4 degrees C, at equivalent concentrations (2.0 microg/ml-8.0 microg/ml). Following a 30 min incubation period at 37 degrees C, membrane-bound Hsp70 protein and Hsp70 peptide TKD were completely taken up into the cytoplasm.
Gross
Catharina
C
Department of Hematology and Oncology, University Hospital Regensburg, Franz-Josef Strauss Allee 11, D-93053 Regensburg, Germany.
Hansch
Daniel
D
Gastpar
Robert
R
Multhoff
Gabriele
G
D000906:Antibodies / Q000494:pharmacology; D015703:Antigens, CD / Q000276:immunology / Q000378:metabolism*; D001667:Binding, Competitive; D002452:Cell Count; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005982:Glutathione Transferase / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007694:Killer Cells, Natural / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D037181:Lectins, C-Type / Q000276:immunology / Q000378:metabolism*; D007938:Leukemia / Q000276:immunology / Q000378:metabolism; D051920:NK Cell Lectin-Like Receptor Subfamily D; D009842:Oligopeptides / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000378:metabolism; D013223:Statistics as Topic; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D000906:Antibodies; D015703:Antigens, CD; D018840:HSP70 Heat-Shock Proteins; C497869:KLRD1 protein, human; D037181:Lectins, C-Type; D051920:NK Cell Lectin-Like Receptor Subfamily D; D009842:Oligopeptides; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D005982:Glutathione Transferase