Figure 2. Peripheral activation of NK cells results in the selection of lymph node DCs with higher T cell activating capabilites. Draining lymph node DCs of mice injected subcutane- In Vivo DC Editing by NK Cells ously with MHCnegcells were sorted and cultured in the presence of allogeneic splenocytes previously labeled with CFSE. A: Splenocytes were cultured alone (No stimulus) or with 10% of highly purified lymph node DCs from mice NK cell depleted (10% DC draining LN + anti-asialo GM1) or control (10% DC draining LN) before administration of MHC- negative target cells. CFSE dilution of the splenocytes at 6 days of culture is shown. NK cell depletion compromises the induction of proliferation by LN DCs. Data are representative of four independent experiments summarized in panel B: & = 10% DC draining LN; m = 10% DC draining LN + anti-asialo GM1. * = p,0,02. doi:10.1371/journal.pone.0039170.g002 activation of NK cells induced by MHC-negative cells may promote a more protective immune response during cancer cell vaccination. Concluding Remarks We have here shown that NK cells can elicit DC editing in vivo. NK cells, upon activation by MHC class I devoid cells, acquire the ability to select the most immunogenic myeloid DCs in draining LNs via a perforin-dependent mechanism. Although our current data do not constitute a formal proof of direct NK cell-mediated cytotoxicity against DCs, the abrogation of the phenomenon in pfn2/2 mice might indicate a direct DC lysis by NK cells. Remarkably, this putative killing leads to the selection of more immunogenic DCs, characterized by a higher capacity to induce proliferation of allogeneic T cells. Thus, in addition to a direct stimulation of DCs mediated by NK cell-released cytokines, NK cells can contribute to T cell activation by selecting the most immunogenic DCs. The identification of the type of NK cells responsible for the editing process, as well as the sites where DC killing allegedly occurs, still remains to be identified. In this context, it is possible that this event might take place in the periphery, where NK cells are equipped with a higher cytolytic activity as compared to those detected in lymph nodes. On the other hand, we cannot exclude that, under particular conditions (for example a cytokine storm), lymph node NK cells might switch their functional phenotype and acquire higher cytolytic potential, as previously proposed [38]. An alternative possibility is that peripheral NK cells displaying high cytolytic activity could migrate to lymph nodes following activation and acquisition of appropriate chemokine receptors, as suggested by recent reports [39–41]. The interaction between activated NK cells and DCs is also relevant for establishing a protective immune response. In a model of anti-cancer vaccination, administration of tumor cells together with NK activating MHC-devoid cells, boosted the expansion of tumor-specific CTLs resulting in enhanced survival of mice upon challenge with a lethal dose of tumor cells. Depletion of NK cells impaired this tumor-specific T cell response as well as its protective role against tumor challenge. Taken together, these results indicate that, in vivo, NK cells can edit DCs for the optimal generation of adaptive immune responses by an efficient selection of the more immunogenic DCs. Finally, our data also suggest that cancer cell vaccines could be improved by strategies aimed at NK cell activation, such as the use of NK-sensitive cancer cells. Materials and Methods Cell Lines, Animal Model and Experimental Conditions The TS/A mammary adenocarcinoma murine cell line [36] (kindly provided by P.L. Lollini, University of Bologna, Bologna, Italy) was cultured in DMEM /10% FCS (Cambrex, Charles City, IA, USA). The YAC-1 (ATCC TIB-160) [42] was instead cultured in RPMI 1640/10% FCS (Cambrex, Charles City, IA, USA). 5–8 PLoS ONE | www.plosone.org 4 June 2012 | Volume 7 | Issue 6 | e39170