Hence, the aim of this study was to determine whether NK cells could play a role in the immune response against HPV infection and related cancers. On tissue samples, we observed an infiltration of NKp46+ NK cells in HPV-associated preneoplastic lesions. In vitro, NK cells displayed a higher cytotoxic activity against HPV+ cells in the presence of HPV-VLPs, by increasing the exocytosis of their cytotoxic granules and 3-deazaneplanocin A research buy by secreting TNF-α and IFN-γ. We also

demonstrated that VLPs rapidly entered into blood NK cells by macropinocytosis, independently of the clathrin and caveolin pathways. Entry of VLPs did not occur into CD16− blood NK cells or into the CD16− NK92 cell line. Moreover, NK92 cells did not degranulate or secrete cytokines in response to VLPs. Finally, the transduction of CD16 into NK92 cells restored VLP entry, degranulation and cytokine production, demonstrating the major role of CD16 in the NK-cell response against HPVs. In order to determine whether NK cells are present in HPV-associated lesions, we stained tissue samples for NKp46, a specific marker of NK cells 12 (Fig. 1). Because more than 85% of HPV-associated cervical lesions occur in the region of the junction between

the endocervix and exocervix 18, we chose these tissues as normal controls. The quantification of NK cells in the epithelia (Fig. 1F) showed a Navitoclax cell line significant infiltration of NKp46+ cells in SILs (Fig. 1C) compared with normal Bay 11-7085 epithelia (Fig. 1A and B), but not in squamous cell carcinoma (SCC) (Fig. 1D) despite the presence of more numerous NK cells in the surrounding stroma (Supporting Information Fig. 1). Interestingly, virus particles have been detected mainly in SILs and not in SCC 19 where the virus is usually integrated into the host genome 20. Our results thus suggest that NK cells could interact with virus particles. In order to determine whether HPV–VLPs could modify the cytotoxic activity of NK cells, we analyzed in vitro the exocytosis of cytotoxic granules of NK cells, negatively selected from blood of healthy donors, in the presence of VLPs

by measuring the expression of lysosomal-associated membrane protein 1 (CD107a) on the NK-cell surface. CD16 engagement has been described to induce degranulation in NK cells 21. Consequently, we used an anti-CD16 mAb as positive control. VLPs significantly increased the number of CD107a+ NK cells after 1 and 6 h of incubation (Fig. 2A and B). We also assessed cytotoxicity of NK cells against CasKi, a HPV+ SCC cell line, and observed a higher cytotoxic activity of NK cells in response to VLPs (Fig. 2C). In addition to their capacity to exhibit cytotoxic activity, NK cells are able to secrete cytokines to promote cell-mediated immune responses. Consequently, we measured NK-cell cytokine production and we noticed a significant increase in TNF-α and IFN-γ after 6 h (Fig. 2D and E) and 24 h (data not shown) of culture in the presence of VLPs.