A probability value of P < 0·05

was considered statistically significant. We first established the immunostimulatory capacity of FLT3L in our model. To this end, mice pretreated with PBS or FLT3L were immunized Z-VAD-FMK order s.c. with irradiated EL-4mOVA cells and OVA257–264 specific CD8+ T cell responses in spleens were determined 7 days later by intracellular cytokine staining upon stimulation with OVA257–264 or with control peptide. As expected, FLT3L-treated mice showed a greater induction of OVA257–264-specific IFNγ-producing CD8+ T cells compared to PBS-treated mice (Fig. 1a and b). FLT3L-treated, but not PBS-treated, mice were protected from EL-4-mOVA challenge 35 days after the initial immunization (Fig. 1c). This protection was CD8+ T cell-dependent, as antibody-mediated GSK-3 inhibitor review depletion of CD8+ T cells before tumour challenge resulted in tumour growth comparable to that observed in naive mice (data not shown). As FLT3L has been shown to increase NK cell numbers and their activation status [44,45], we determined if NK cells played a role in the increased CD8+ T cell priming in FLT3L-treated mice. Temporary elimination of NK T cells by antibody depletion prior to immunization did not affect the magnitude of the antigen-specific T cell response or survival upon tumour challenge in PBS- and FLT3L-treated mice. Moreover, NK T cell depletion after immunization (but before tumour challenge) GNAT2 did not affect

the FLT3L-mediated protection from tumour outgrowth, demonstrating that both the protection to tumour growth and increased OVA257–264-specific CD8+ T cell response in FLT3L-treated mice was NK T cell-independent (Fig. 1d, and data not shown). As FLT3L treatment has been shown

to expand DCs in the spleen and secondary lymphoid organs [34], we next analysed the effect of FLT3L treatment on frequency of total DC, the frequency of different DC subsets (CD11b DCs, CD11c+CD11b+PDCA-1-CD8α-; CD8 DCs, CD11c+CD11b-PDCA-1-CD8α+; pDC, CD11c+CD11b-PDCA-1+CD8α-; mcDC, CD11c+CD11b-PDCA-1-CD8α- (Fig. 2a) and their functional capacity. Importantly, not only the absolute number of DC but also the distribution of different DC populations within the CD11+ population changed dramatically upon FLT3L treatment (Fig. 2b). While total CD11b DCs expanded ∼ twofold (2·2 ± 0·3) upon FLT3L treatment, CD8 DCs, mcDC and pDC expanded ∼ ninefold (9·6 ± 2·3-, 9·2 ± 1·6- and 8·3 ± 1·1-fold, respectively). Interestingly, FLT3L treatment did not affect the functional profile of the DC supsets. The expression levels of major histocompatibility complex (MHC) I/II or co-stimulatory molecules [CD40, CD54, CD80, CD86, CD274 programmed cell death ligand 1 (PD-L1), CD273 (PD-L2)] were comparable with the corresponding DC populations from PBS-treated mice (data not shown). In addition, the cytokine induction by DCs upon interaction with apoptotic cells was also unaltered (Fig. 2c).