However, DC killing by autologous NK cells in vivo has not been directly demonstrated to date and the potential relevance of this lysis during a physiological immune response remains to be evaluated. A number of in vivo models have provided evidence that NK cell recognition of MHC class I-deficient target cells results in an enhanced generation of CTLs against tumors [21,35]. In these experimental models, activated NK cells produce cytokines that, in turn, appear to first promote DC activation and subsequently a protective CTL response against parental tumors. This prompted us to investigate whether, during a protective immune response against tumors, activated NK cells might also select a more immunogenic subset of DCs. We show here that DC editing occurs in vivo and that this phenomenon plays a key role for tumor- specific CTL development and mice survival in a murine model of tumor vaccination. Results and Discussion Activation of NK Cells in Peripheral Tissues Results in a Perforin-dependent Decrease of DC Content in Draining Lymph Nodes Mice were inoculated s.c. with YAC-1, a MHC-devoid cell line, as an NK cell target able to induce NK cell activation. After 36 h, DCs derived from both draining and controlateral LN were analyzed. As shown in figure 1, both the percentage and the absolute number of CD11c+ DCs were dramatically decreased in draining LNs compared to controlateral LNs (p = 0.0029 for the percentage and p = 0.007 for absolute number). In vivo depletion of NK cells by injecting anti-asialo-GM1 monoclonal antibodies (mAbs) reverted this phenomenon, confirming the major role played by NK cells. In draining LNs from NK cell-depleted mice, both the percentage and the absolute number of CD11c+ DCs were comparable with controlateral LNs, indicating that NK cells should be involved in the decrease of DCs. Anti-asialo GM1 mAb treatment led to a decrease of at least 80% of NK cells (Figure 1,B). These results suggested that the decline in DC number observed in draining LN was NK cell-dependent and apparently consequent to NK cell activation upon recognition of MHC-devoid cells. One possible explanation for the observed reduction of DC cell numbers upon NK cell activation is the release of specific cytokines, by NK cells, able to influence the survival of DCs or their ability to migrate from the periphery to the LN. Alterna- tively, NK cells may affect the number of DCs in draining LN by direct lysis. To elucidate the mechanism underlying the decline in DC numbers, we repeated the same experiment in perforin knockout (pfn2/2) mice, since perforin is an essential molecule for NK cell cytotoxicity. In the pfn2/2 mice, there were no differences in the number and percentage of CD11c+ DCs found in draining and controlateral LNs (not shown). These data suggest that perforin- dependent cytotoxicity might represent a pathway in the NK cell- dependent decrease of LN DCs. NK Cell Activation Selects Lymph Node DC with Higher T Cell Activating Capability NK cells have been shown to kill immature DCs in vitro [13,15]. One interpretation for the specific killing of healthy autologous cells by NK cells was that NK cells might act to control the quality of DCs undergoing maturation [26]. This hypothesis implies that NK cells would prevent the survival of less immunogenic immature DCs, which would induce inappropriate, low affinity T-cell priming, eventually resulting in a state of tolerization. The final outcome of this DC editing process mediated by NK cells In Vivo DC Editing by NK Cells might therefore be the selection of more immunogenic DCs, due to the removal of DCs that would fail to mediate optimal T cell priming. In order to verify whether this mechanism is functionally relevant in vivo, we tested whether DCs persistent in the draining LN after NK cell editing were phenotipically and functionally more immunogenic. Groups of mice that were either NK cell depleted or not were inoculated s.c. with YAC-1 cells. After 36 h DCs from the draining and controlateral lymph nodes were analyzed for the expression of co-stimulatory molecules and maturation markers such as CD40, CD80, CD83, CD86 by flow cytometry and for IL12p35, IL-12p40 and IL23p19 mRNA by real time PCR. In addition, we assessed whether DCs persistent in draining LN upon NK cell editing presented higher T cell activating capability. Splenocytes from C57BL/6 mice were stimulated with DCs sorted from lymph nodes of BALB/c mice injected with YAC-1 cells 24 h before lymph node removal. Six days later, the rate of proliferation was evaluated as loss of CFSE dye. Although significant differences for the analyzed surface molecules or cytokines were not discernable (not shown), allogeneic T cell proliferation was significantly decreased when DCs from NK cell-depleted mice were used as stimulus (Figure 2). These results indicate that NK cell-mediated decrease of DC number represents a functionally relevant in vivo mechanism by which the more immunogenic DCs are efficiently selected. Indeed, following peripheral NK cell activation, DCs contained in the draining LNs were reduced in number but endowed with more potent T cell activating properties. The Editing of DCs by NK Cells Promotes Antigen Specific T Cell Expansion and a More Protective Immune Response During Cancer Cell Vaccination Our data show that in vivo activation of NK cells results in a perforin-dependent decrease of LN dendritic cells, which is associated with the presence of more immunogenic DCs in draining LN. We therefore investigated whether it results in a more protective adaptive immune response in a cancer cell vaccination model. Groups of mice, which had either undergone NK cell depletion or mock treatment, were injected s.c. with the MHC-devoid cells (i.e. YAC-1 cells) and TS/A cells, a NK cell- resistant mammary adenocarcinoma tumor cell line. As controls, groups of mice were injected with either TS/A or YAC-1 cells alone. After three weeks, spleens were collected and cells were restimulated in culture with TS/A tumor cells or the AH1 peptide, which is an immunodominant MHC class I- restricted epitope of TS/A gp70env antigen [36,37]. Splenocytes from mice that had been injected with TS/A plus YAC-1 cells showed significantly higher numbers of IFN-c producing cells upon stimulation as compared to NK cell depleted mice or control mice (p,0.05 and ,0.01) (Figure 3,A). These data indicate that lysis of DCs by activated NK cells supports the expansion of antigen-specific T lymphocytes in vivo. No increase in IFN-c producing cells was observed when cultures were restimulated by YAC-1 cells, indicating that the increase in IFN-c producing cells was not associated with NK cell activation. In addition, restimulation by either TS/A or their immunodominant MHC class I-restricted epitope AH1 induced a similar increase of IFN-c producing splenocytes, indicating that they were mainly represented by MHC-class I-restricted cytotoxic T lymphocytes (CTL). To elucidate whether NK cell help for CTL expansion was in fact associated to NK cell-mediated DC lysis, we set the same experiment using groups of pfn2/2 mice, which lack NK cell cytolytic activity. Spleen cells from pfn2/2 mice injected with TS/ A plus the MHC-negative cells YAC-1 contained a significantly PLoS ONE | www.plosone.org 2 June 2012 | Volume 7 | Issue 6 | e39170