We compared fluorescence in CD56bright CD16− versus CD56dim CD16+ NK cells and observed a higher fluorescence in this latter subpopulation (Fig. 6D). Moreover, using a co-immunoprecipitation assay, we observed a direct interaction between CD16 and VLPs

since we detected the presence of L1 from VLPs only when viral particles and CD16 were immunoprecipitated with anti-CD16 antibody (Fig. 6E). We used normal mice IgG and an antibody against an unrelated protein (EGF receptor, EGFR) as negative controls. Finally, we confirmed the role of CD16 by blocking the LYNX-VLP binding and internalization with a pre-incubation of NK cells with blocking anti-CD16 mAb (Fig. 6E). Similarly, this mAb also inhibited VLP entry into NK92 HIF-1 cancer CD16+ cells (data not shown). FITC-dextran uptake assays selleck chemical showed that VLP internalization is mediated by macropinocytosis in NK92 CD16+ cells (Fig. 6F) (viability of NK92 in the presence of drugs is shown in Supporting Information Fig. 3B). In contrast, the presence of VLPs did not change FITC-dextran uptake by NK92 CD16− cells (Supporting Information Fig. 6). In order to determine the role of CD16 in NK-cell function in the presence of VLPs, we compared the cytotoxic activity of CD16+ and CD16− NK92 cells. As opposed to NK92 CD16+ cells, NK92 CD16− cells were not able to degranulate in the presence of VLPs although

these cells increased their cytotoxic granule release in the presence of PMA/ionomycin which is the most common and potent stimulator of NK-cell cytotoxic function (Fig. 7A). Similarly, VLPs induced an increased killing of CasKi cells by NK92 CD16+ cells (Fig. 7B) but not by NK92 CD16− cells (Fig. 7C). We also observed higher cytokine production, both of IFN-γ (Fig. 7D) and TNF-α (Fig. 7E), in the presence of VLPs only in NK92 CD16+ Cyclin-dependent kinase 3 culture supernatant. Understanding the interactions between HPVs and immune cells is important in order to dissect the mechanisms responsible for the viral clearance observed in the majority of patients with SIL 8. Moreover, the immune response against HPV induced by HPV–VLP vaccination is poorly characterized. In this

study, we demonstrated that NK cells recognize, internalize and respond to VLPs by cytotoxic granule exocytosis and cytokine production. In cervical tissue samples, we observed that NK cells infiltrate mainly HPV-associated preneoplastic lesions where HPV particles are produced, but less SCC where the expression of L1 protein is not detected 19. These findings confirm previous data using a less specific marker for NK cells, CD56, and showing an increased number of CD56+ cells in HPV-related preneoplastic lesions 29, 30. Moreover, NK cells may also interact with VLPs used as a prophylactic anti-HPV vaccine 6, since the adjuvant present in the vaccine induces local inflammation 31, and since infiltration of NK cells has been observed in inflamed tissues 32.