Dendritic cell immunotherapy for melanoma.

Dendritic cells (DC) are the most potent antigen-presenting cells that initiate T cell-mediated immune responses against cancer. It has been almost a decade since the first trial of DC-based cancer immunotherapy was published. Despite the many clinical trials conducted since, few solid conclusions have been reached, and no specific-immunotherapy has routinely demonstrated meaningful anti-tumour responses. Clinical-grade DC can be obtained from three distinct cell populations in the blood - monocytes, CD34(+) progenitors or direct isolation of circulating blood DC. This review discusses the science behind DC-based cancer immunotherapy, with a particular emphasis on the use of monocyte-derived DC in melanoma clinical trials, and the various potential avenues for improvement of patient clinical response rates.

Monocyte-derived DC primed with TLR agonists secrete IL-12p70 in a CD40-dependent manner under hyperthermic conditions.

Fever is an evolutionarily conserved mechanism to improve survival during infection. Previous studies have shown that feverlike temperatures directly enhance the function of murine bone marrow-derived dendritic cells (DCs). In the present study, we examined the response of human monocyte-derived DC to 39.5 degrees C hyperthermia. When primed with toll-like receptor agonists or bacterial extract but not proinflammatory cytokines, hyperthermia specifically enhanced secretion of interleukin (IL)-12p70 by DC, without altering the secretion of IL-10, tumor necrosis factor alpha or IL-1beta. These DC induced significantly higher levels of T-cell proliferation and interferon gamma production in assays of antigen presentation and MLR. Endogenous heat-sock protein 70 colocalized with CD40 in DC exposed to hyperthermic conditions. Recombinant CD40-Fc fusion protein blocked the increase in IL-12p70 secretion by DC primed with bacterial extract and hyperthermia. Thus, DC primed with toll-like receptor-agonists respond to hyperthermia with increased IL-12p70 secretion, mediated by heat-shock protein binding and activation of CD40. The data have important applications for clinical immunotherapy and the mechanism of fever.

IL10 and IL12B polymorphisms each influence IL-12p70 secretion by dendritic cells in response to LPS.

Dendritic cells (DC) are the main producers of the cytokine IL-12p70, through which they play a direct role in the development of IFN-gamma-secreting Th1 cells, costimulation of CTL differentiation and NK-cell activation. In contrast, IL-10, which is also produced by DC, negatively regulates IL-12 production. IL-12p70 production varies widely between individuals, and several polymorphisms in the gene encoding IL-12p40 (IL12B) have been identified that influence susceptibility and severity of infectious, autoimmune and neoplastic disease. Here we show that polymorphisms not only of IL12B, but also in the IL10 promoter, influence IL-12p70 secretion by monocyte-derived DC in response to LPS. Although IL12B promoter homozygotes were prone to making more IL-12p70, presence of the IL10 high genotype restricted IL-12p70 production in these individuals. These observations provide a further genetic control of IL-12p70 regulation and emphasize the complexity of production of this cytokine. They also suggest genotypes that might influence the outcome of DC immunotherapy.

Generation and maturation of dendritic cells for clinical application under serum-free conditions.

Monocyte-derived dendritic cells (MoDCs) in clinical use for cancer immunotherapy are ideally generated in serum-free medium (SFM) with inclusion of a suitable maturation factor toward the end of the incubation period. Three good manfacturing practice (GMP) grade SFMs (AIM-V, X-VIVO 15, and X-VIVO 20) were compared with RPMI-1640, supplemented with 10% fetal bovine serum or 10% human serum. DCs generated for 7 days in SFM were less mature and secreted less interleukin (IL) 12p70 and IL-10 than DCs generated in 10% serum. DC yield was comparable in SFMs, and a greater proportion of cells was viable after maturation. Toll-like receptor (TLR) ligands were compared for their ability to induce cytokine secretion under serum-free conditions in the presence of interferon (IFN) gamma. With the exception of Poly I:C, TLR ligands stimulated high levels of IL-10 secretion. High levels of IL-12p70 were induced by two TLR4-mediated stimuli, lipopolysaccharide and Ribomunyl, a clinical-grade bacterial extract. When T-cell responses were compared in allogeneic mixed leukocyte reaction, DCs stimulated with Ribomunyl induced higher levels of IFNgamma than DCs stimulated with the cytokine cocktail: tumor necrosis factor-alpha, IL-1beta, IL-6, and prostaglandin E2. In the presence of IL-10 neutralizing antibodies, DC IL-12p70 production and T-cell IFNgamma were increased in vitro. Similarly, DCs stimulated with Ribomunyl, IFNgamma, and anti-IL-10 induced high levels of tetanus toxoid-specific T-cell proliferation and IFNgamma secretion. Thus, MoDCs generated in SFM efficiently stimulate T-cell IFNgamma production after maturation in the presence of a clinical-grade TLR4 agonist and IL-10 neutralization.