Objective: Previously, our group has shown that mild thermal stress (39.5^oC, 6 hours) can significantly enhance direct lysis of tumor cells by NK cells compared to hypothermic (33^oC) or heat-shock (42^oC) temperatures. Our purpose is to identify the mechanisms underlying thermal enhancement of NK cell mediated tumor cytotoxicity.

Methods: Human NK cells were purified from the peripheral blood of healthy donors with gradient centrifugation followed by magnetic bead capture of non-NK cells. NK cells and Colo205 human colon adenocarcinoma cells were incubated at various temperatures for 6 hours in vitro before cytotoxicity assays.

Results: Enhanced NK cytotoxicity required the incubation of both NK cells and Colo205 cells at elevated temperatures and was dependent on the interaction of NK cell activating receptor NKG2D and its stress-induced ligand, MICA. We were unable to observe thermal enhancement of NK cell cytotoxicity when syngeneic human PBMC were used as targets, or when the IL-2 dependent human NK cell lines NKL or NK92MI, or IL-2 activated NK cells were used as effectors. Thermal stress controls MICA transcription, leading to the upregulation of MICA on Colo205 cell surface. The presence of a consensus hsf-1 binding site in the MICA promoter led us to investigate the role of hsf-1 on thermal regulation of MICA expression. Inhibition of hsf-1 with Quercetin resulted in reversal of thermal enhancement of MICA surface expression as well as NK mediated cytotoxicity. Preliminary data with siRNA against hsf-1 support these inhibition studies. Consistent with this, we found that hsf-1 binding to its consensus sequence from MICA promoter is enhanced at elevated temperatures. On the NK cell side, we observed an increased clustering of NKG2D on NK cells with elevated temperatures, suggesting the potential for enhanced efficiency of activating signals through aggregation of signaling molecules.

Conclusion: Mild thermal stress enhances NK cytotoxic activity by upregulating MICA expression on target cells through activation of transcription factor hsf-1. Further, changes on the NK cell (i.e., clustering of NKG2D receptors for MICA) may lower the activation threshold of NK cells. These studies should help to identify strategies that use heat to enhance NK mediated tumor cell killing.