IFN-alpha promotes definitive maturation of dendritic cells generated by short-term culture of monocytes with GM-CSF and IL-4.

Dendritic cells (DC) generated in vitro have to be viable and phenotypically mature to be capable of inducing T cell-mediated immunity after in vivo administration. To facilitate optimization of DC-based vaccination protocols, we investigated whether the cytokine environment and the mode of activation affect maturation and survival of DC derived from monocytes by a short-term protocol. Monocytes cultured for 24 h with granulocyte macrophage-colony stimulating factor and interleukin-4 were stimulated with proinflammatory mediators for another 36 h to generate mature DC. Additional activation with CD40 ligand and interferon (IFN)-gamma increased viability of DC and promoted definitive maturation as defined by maintenance of a mature phenotype after withdrawal of cytokines. Addition of IFN-alpha to DC cultures prior to stimulation further enhanced definitive maturation: IFN-alpha-primed DC expressed high levels of costimulatory molecules and CC chemokine receptor 7 (CCR7) up to 5 days after cytokine withdrawal. Compared with unprimed DC, IFN-alpha-primed DC displayed equal capacity to migrate upon CCR7 ligation and to prime antigen-specific T helper cell as well as cytolytic T cell responses. In conclusion, we show that optimal maturation and survival of monocyte-derived DC require multiple activation signals. Furthermore, we identified a novel role for IFN-alpha in DC development: IFN-alpha priming of monocytes promotes definitive maturation of DC upon activation.

FastDC derived from human monocytes within 48 h effectively prime tumor antigen-specific cytotoxic T cells.

Previously, we have shown that dendritic cells (DCs) with full T-cell stimulatory capacity can be derived from human monocytes after 48 h of in vitro culture (FastDC). Compared to a standard 7-day protocol, this new strategy not only reduces the time span and the amount of recombinant cytokines required, but may also resemble DC development in vivo more closely. Using a melanoma antigen model, we show here that FastDC prime CTL responses against tumor antigens as effectively as standard monocyte-derived DCs (moDCs). FastDC and moDCs derived from monocytes of HLA-A2(+) donors were loaded with the melanoma-associated, HLA-A(*)0201-restricted peptide Melan-A and cocultured with autologous CD3(+) T cells. After two weekly restimulations with freshly prepared, peptide-loaded FastDC or moDCs, binding of CD8(+) T cells to fluorescently labeled MHC-I/Melan-A-peptide complexes and intracellular cytokine staining revealed that the two DC preparations had an equal capacity to prime Melan-A-specific, IFN-gamma producing CD8(+) T cells. CTLs derived from cocultures with FastDC lysed Melan-A-loaded T2 cells even more effectively than CTLs primed by moDCs. Comparative analysis also revealed that FastDC possess an equal capacity to migrate in response to the chemokine receptor CCR-7 ligand 6Ckine. Importantly, DCs can be generated with higher yield and purity using the FastDC-protocol. The reliability and efficacy of this new strategy for DC development from monocytes may facilitate clinical investigation of DC-based tumor immunotherapy.

Chemosensitization of pancreatic carcinoma cells to enhance T cell-mediated cytotoxicity induced by tumor lysate-pulsed dendritic cells.

Dendritic cells (DCs) can induce cytotoxic T-cell (CTL) responses against tumor antigens in vitro and in vivo, yet few cancer patients experience tumor regression after DC-based vaccination. Combination with other treatment modalities, such as radiation or pharmacologic anticancer agents, may reduce tumor cell resistance against immune responses. The authors tested whether treatment with gemcitabine or cyclooxygenase-2 (COX-2) inhibitors increases the sensitivity of pancreatic carcinoma cells to CTL-mediated killing. Monocyte-derived DCs of HLA-A2+ donors were loaded with lysate from the HLA-A2+ pancreatic carcinoma cell line Panc-1 and co-cultured with autologous CD3+ T cells. ELISPOT and cytotoxicity assays performed after two rounds of in vitro stimulation confirmed induction of a tumor-specific CTL response. Changes in the magnitude and the effector mechanism of the CTL response were analyzed after treatment of Panc-1 cells with gemcitabine and COX-2 inhibitors. Compared with gemcitabine, COX-2 inhibitors more effectively sensitized Panc-1 cells to CTL-mediated killing and showed less inhibition of T-cell activation by DCs in vitro. Using anti-CD95 blocking antibody, the authors showed that the increase in CTL-mediated tumor cell killing observed after treatment with COX-2 inhibitors is dependent on CD95/CD95 ligand interaction. Increased apoptosis of Panc-1 cells treated with COX-2 inhibitor was also observed after incubation with agonistic anti-CD95 antibody. Sensitization of cancer cells to CD95-dependent killing by CTLs represents a novel mechanism of action for COX-2 inhibitors and provides a rationale for their concomitant use with immunotherapeutic strategies such as DC-based vaccination.

Development of a new protocol for 2-day generation of mature dendritic cells from human monocytes.

We developed a new 2-day protocol for the generation of dendritic cells (DCs) from human monocytes in vitro. First, we demonstrated that 24 hours of culture with GM-CSF and IL-4 are sufficient to generate immature DCs capable of antigen uptake. We then compared two different strategies for DC maturation: proinflammatory mediators were either added together with GM-CSF and IL-4 from the beginning of cell culture or added after 24 hours of differentiation with GM-CSF and IL-4. After 48 hours of total culture period, expression of activation markers was more pronounced in cells generated by the 2-step differentiation and activation method. Our new protocol for 2-day DC differentiation reduces labor, cost and time and also reliably renders high numbers of mature and viable DCs.

Mature dendritic cells derived from human monocytes within 48 hours: a novel strategy for dendritic cell differentiation from blood precursors.

It is widely believed that generation of mature dendritic cells (DCs) with full T cell stimulatory capacity from human monocytes in vitro requires 5-7 days of differentiation with GM-CSF and IL-4, followed by 2-3 days of activation. Here, we report a new strategy for differentiation and maturation of monocyte-derived DCs within only 48 h of in vitro culture. Monocytes acquire immature DC characteristics by day 2 of culture with GM-CSF and IL-4; they down-regulate CD14, increase dextran uptake, and respond to the inflammatory chemokine macrophage inflammatory protein-1alpha. To accelerate DC development and maturation, monocytes were incubated for 24 h with GM-CSF and IL-4, followed by activation with proinflammatory mediators for another 24 h (FastDC). FastDC expressed mature DC surface markers as well as chemokine receptor 7 and secreted IL-12 (p70) upon CD40 ligation in the presence of IFN-gamma. The increase in intracellular calcium in response to 6Ckine showed that chemokine receptor 7 expression was functional. When FastDC were compared with mature monocyte-derived DCs generated by a standard 7-day protocol, they were equally potent in inducing Ag-specific T cell proliferation and IFN-gamma production as well as in priming autologous naive T cells using tetanus toxoid as a model Ag. These findings indicate that FastDC are as effective as monocyte-derived DCs in stimulating primary, Ag-specific, Th 1-type immune responses. Generation of FastDC not only reduces labor, cost, and time required for in vitro DC development, but may also represent a model more closely resembling DC differentiation from monocytes in vivo.