Dendritic cell (DC) vaccines have been demonstrated to elicit immunological responses in numerous cancer immunotherapy trials. even further potentiated by addition of the TLR4 agonist LPS. Concurrently, upregulation of the anti-inflammatory cytokine IL-10 was modest. Most importantly, DC matured with IFN, R848, and poly I:C had the ability to activate IFN production in allogeneic T cells and this was further enhanced by adding LPS to the cocktail. Furthermore, epitope-specific stimulation of TCR-transduced T cells by peptide- or whole tumor lysate-loaded DC was efficiently stimulated only by DC matured in the full maturation cocktail containing IFN and the three TLR ligands R848, poly I:C, and LPS. We suggest that this cocktail is used for future clinical trials of anti-cancer DC vaccines. Electronic supplementary material The online version of this article (doi:10.1007/s00262-017-2029-4) contains supplementary material, which is available to authorized users. Keywords: Cancer, Dendritic cell-vaccine, IFN, R848, Poly I:C, LPS Introduction Efforts to vaccinate cancer patients XL-888 with preparations of naturally occurring DC from blood has shown promising clinical results [1, 2]. However, these primary DC are rare and therefore the majority of DC trials have been based on DC derived from monocytes ex vivo (Mo-DC). Monocytes are abundant in blood and large quantities of Mo-DC can be easily generated in culture for most individuals. The FDA-approved DC-based vaccine (Sipuleucel-T, Provenge, Dendreon) consists of a leukapheresis product that has been enriched for DC precursors and loaded with a recombinant fusion protein of prostatic acid phosphatase (PAP) and GM-CSF. This cell product was shown to prolong the overall survival in asymptomatic or minimally symptomatic, metastatic, hormone-refractory prostate cancer patients by 4.1?months [3]. Unfortunately, the numerous other trials on Mo-DC vaccination conducted in different malignancies have had limited clinical success even though immunological responses were commonly reported in response to both unmutated tumor-associated antigens and mutated neoepitopes [4C7]. Thus, there is a need for more efficient XL-888 Mo-DC vaccines and/or to combine Mo-DC vaccines with other therapies in cancer patients. The in vitro generation of iDC from monocytes is most commonly stimulated by culturing in the presence of IL-4 and GM-CSF, but for triggering subsequent differentiation into mDC there are numerous different protocols. For an efficient DC-based cancer vaccine, generated mDC have to be able to home to lymphoid tissue and, once there, efficiently stimulate tumor-specific Th1-type CD4+ T cells and CD8+ CTL that are able to eliminate tumor cells. To achieve this, mDC should express the lymph node homing receptor CCR7, high levels of co-stimulatory molecules such as CD80, CD83, and CD86 and produce high levels of the Th1-skewing and CTL-stimulatory cytokine IL-12 [8C10]. For many years the gold standard for a maturation cocktail of DC vaccines contained TNF, IL-1, IL-6, XL-888 and prostaglandin E2 (PGE2) [11]. However, these DC were shown to produce very little IL-12p70, which was attributed to the presence of PGE2 in the cocktail [12]. Thus, alternate cocktails without PGE2 were suggested. Most of these involve the addition of molecules containing pathogen- and/or damage-associated molecular patterns (PAMP and DAMP, respectively), which bind to pattern recognition receptors (PRR), such as TLR expressed by the DC. The TLR expression differs depending on the DC subtype and maturation stage. Monocyte-derived iDC have been reported to express several TLR, including TLR3, 4, and 8, receptors for double-stranded RNA (dsRNA), LPS, and single-stranded RNA (ssRNA), respectively [13C17]. In line with this, maturation cocktails containing a mixture of TNF, IL1, IFN, IFN, and the TLR3 agonist poly I:C [9, 18], IFN and poly I:C [19], IFN and the TLR4 agonists LPS or its derivative monophosphoryl lipid A (MPLA) [19C23] or IFN and the TLR7/8 agonist R848 GRS [19, 21] generated mDC with improved IL-12p70 production while still expressing CCR7. Notably, the addition of IFN [9, 19, 24, 25] or combinations of more than one TLR agonist [24, 26, 27], (poly I:C and LPS, poly I:C and R848, LPS and R848) or both [28] (IFN, poly I:C, and R848 in combination with CD40L) potentiated the IL-12p70 production. Furthermore, the addition of multiple TLR.