As shown in Fig. 1, αDC1s produced significantly higher amounts of the CXCR3 ligands CXCL9/MIG (P = 0.02), CXCL10/IP-10 (P = 0.02) and CXCL11/I-TAC (P = 0.03) (Fig. 1a–c), as compared with PGE2DCs. This chemokine production was not seemingly depressed by the number of contaminating CLL cells LDK378 in the cultures (Fig. 1D). Both

PGE2DCs, as well as αDC1s, showed a mature DC phenotype and morphology (Fig. 2). Importantly, loading with heat-stressed necrotic CLL cells had no significant impact on chemokine production or phenotype. Previously, it has been shown that PGE2DCs generated from healthy blood donors preferentially produced CCL22/MDC and attracted Tregs [17]. In line with this, we could show that monocyte-derived PGE2DCs from patients with CLL produced significantly higher levels of the Th2- and Treg-attracting chemokine CCL22/MDC as compared with αDC1 (P = 0.03). Regarding the production of CCL17/TARC, no statistical significant difference was found (Fig. 3A,B). Once again, tumour cell loading had no significant impact on chemokine production. To examine whether the high production of CXCR3-ligands by αDC1s could be translated into possible recruitment of NK and NKT cells, we used a transwell plate migration assay. Even though there were no differences in total number of recruited lymphocytes, we found that supernatants from tumour-loaded αDC1s induced a substantially higher recruitment of NK (P = 0.04) and NKT (P = 0.04) cells from PBMC in transwell

HIF pathway experiments compared with supernatants from tumour-loaded PGE2DCs (Fig. 4A,B). When reaching the lymph node, antigen-loaded mature DCs undergo an additional activation step, termed ‘licensing’ in response to various stimuli, notably CD40 ligand that is expressed on cognate CD4+ T cells. Signalling through CD40 has multiple effects on DCs, inducing the upregulation of costimulatory molecules and the secretion of cytokines Megestrol Acetate and chemokines. Effective vaccine DCs should optimally mediate a CD4+ T cell-dependent guiding of rare tumour-specific CD8+ T cells to site of antigen-dependent DC–CD4+

T cell interactions by secretion of CCL3/MIP-1α and CCL4/MIP-1β chemokines [20]. We therefore considered whether differentially matured DCs were able to respond to subsequent CD40 ligation (mimicking CD4+ T cell interaction). To optimally mimic the situation in vivo, previously washed mature DCs were cultured in fresh medium for further 24 h (this being an estimation of the time required for the DCs to migrate to a draining lymph node) and subsequently washed before CD40 stimulation by cross-linked soluble CD40L. We found that tumour-loaded αDC1s, produced larger amounts of CCL3 (P = 0.02) and CCL4 (P = 0.04) after CD40 ligation, as compared with PGE2DCs (Fig. 5A,B). Finally, we could show, in accordance with Lee et al. [24], that tumour-loaded αDC1s were superior in producing the Th1-deviating IL-12p70 cytokine compared with PGE2DCs (P = 0.02) after CD40 ligation (Fig. 5C).