Sylvia M. Kiertscher

PURPOSE: We previously reported that autologous dendritic cells pulsed with acid-eluted tumor peptides can stimulate T cell-mediated antitumor immune responses against brain tumors in animal models. As a next step in vaccine development, a phase I clinical trial was established to evaluate this strategy for its feasibility, safety, and induction of systemic and intracranial T-cell responses in patients with glioblastoma multiforme. EXPERIMENTAL DESIGN: Twelve patients were enrolled into a multicohort dose-escalation study and treated with 1, 5, or 10 million autologous dendritic cells pulsed with constant amounts (100 mug per injection) of acid-eluted autologous tumor peptides. All patients had histologically proven glioblastoma multiforme. Three biweekly intradermal vaccinations were given; and patients were monitored for adverse events, survival, and immune responses. The follow-up period for this trial was almost 5 years. RESULTS: Dendritic cell vaccinations were not associated with any evidence of dose-limiting toxicity or serious adverse effects. One patient had an objective clinical response documented by magnetic resonance imaging. Six patients developed measurable systemic antitumor CTL responses. However, the induction of systemic effector cells did not necessarily translate into objective clinical responses or increased survival, particularly for patients with actively progressing tumors and/or those with tumors expressing high levels of transforming growth factor beta(2) (TGF-beta(2)). Increased intratumoral infiltration by cytotoxic T cells was detected in four of eight patients who underwent reoperation after vaccination. The magnitude of the T-cell infiltration was inversely correlated with TGF-beta(2) expression within the tumors and positively correlated with clinical survival (P = 0.047). CONCLUSIONS: Together, our results suggest that the absence of bulky, actively progressing tumor, coupled with low TGF-beta(2) expression, may identify a subgroup of glioma patients to target as potential responders in future clinical investigations of dendritic cell-based vaccines.

The ability of dendritic cells (DC) to initiate immune responses in naive T cells is dependent upon a maturation process that allows the cells to develop their potent Ag-presenting capacity. Although immature DC can be derived in vitro by treatment of peripheral blood monocytes with GM-CSF and IL-4, additional signals such as those provided by TNF-alpha, CD40 ligand, or LPS are required for complete maturation and maximum APC function. Because we recently found that microbial lipoproteins can activate monocytes and DC through Toll-like receptor (TLR) 2, we also investigated whether lipoproteins can drive DC maturation. Immature DC were cultured with or without lipoproteins and were monitored for expression of cell surface markers indicative of maturation. Stimulation with lipopeptides increased expression of CD83, MHC class II, CD80, CD86, CD54, and CD58, and decreased CD32 expression and endocytic activity; these lipopeptide-matured DC also displayed enhanced T cell stimulatory capacity in MLR, as measured by T cell proliferation and IFN-gamma secretion. The lipid moiety of the lipopeptide was found to be essential for induction of maturation. Preincubation of maturing DC with an anti-TLR2 blocking Ab before addition of lipopeptide blocked the phenotypic and functional changes associated with DC maturation. These results demonstrate that lipopeptides can stimulate DC maturation via TLR2, providing a mechanism by which products of bacteria can participate in the initiation of an immune response.

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) for the initiation of antigen-specific T-cell activation. DCs may be highly enriched from peripheral blood-adherent leukocytes by short-term (7-day) culture in the presence of interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor. Various methods of gene transfer were studied, including DNA/liposome complexes, electroporation, CaPO4 precipitation, and recombinant adenovirus (AdV) vectors. Low levels of expression were obtained with the physical methods tested. In contrast, AdV vectors expressing luciferase, beta-galactosidase, IL-2, and IL-7 all readily transduced human DCs. Increasing levels of gene expression were observed over a range of multiplicity of infection (MOI) of 10:1 to 10,000:1, with transduction efficiencies exceeding 95% at higher MOI. Although levels of maximal gene expression in DCs were significantly lower than those obtained using human tumor cell lines, IL-2 and IL-7 production of up to 5 x 10(2) ng/10(6) DC were achieved. These results suggest that AdV vectors are a promising vehicle for genetically engineering human DCs.

The diverse roles of interferon-alpha (IFN-alpha) in regulating the immune response to infectious agents suggested that it might affect dendritic cell (DC) development. Peripheral blood mononuclear cells cultured with IFN-alpha and granulocyte-macrophage colony-stimulating factor (GM-CSF) developed a dendritic morphology and expressed high levels of the class I and II human leukocyte antigens (HLA), B7 co-stimulatory molecules, adhesion proteins, and CD40. Elevated DC expression of B7-2 and HLA-DR was observed with increasing IFN-alpha concentrations up to 5000 U/mL. The effects of IFN-alpha on DC immunophenotype were not reversed by adding neutralizing antibodies against interleukin-4 (IL-4) or tumor necrosis factor alpha to the cell cultures or by eliminating lymphocytes from the cultures. The addition of IFN-alpha to cultures containing optimal concentrations of IL-4 and GM-CSF significantly increased the B7-2 and HLA-DR levels above those present on DCs grown in two cytokines. The DCs generated with IFN-alpha and GM-CSF were potent antigen-presenting cells in allogeneic mixed leukocyte reactions. They also were capable of taking up, processing, and presenting tetanus toxin to autologous T lymphocytes. These results demonstrate an important role for IFN-alpha in the generation of DCs with potent antigen-presenting capabilities from peripheral blood monocytes.

Mammalian Toll-like receptors (TLRs) are required for cell activation by bacterial lipoproteins (bLP) and LPS. Stimulation of monocytes with bLP and LPS results in a TLR-dependent induction of immunomodulatory genes leading to the production of pro-inflammatory cytokines. In this paper, we compared the expression and response of TLRs on monocytes and dendritic cells (DC). TLR2, but not TLR4, was detected on peripheral blood monocytes and DC, in lymphoid tissue CD1alpha+ DC as well as on in vitro monocyte-derived DC. Upon stimulation with bLP or LPS, monocytes produced IL-12 and IL-10 at similar levels, whereas monocyte-derived DC produced comparable levels of IL-12, but little IL-10. Greater than 90% of the bLP-induced production of IL-12 was blocked by anti-TLR2 mAb. Thus, DC express TLR2 and activation of this receptor by bLP provides an innate mechanism by which microbial pathogens preferentially activate cell-mediated immunity.