Sylvia M. Kiertscher

Dendritic Cell Vaccination in Glioblastoma Patients Induces Systemic and Intracranial T-cell Responses Modulated by the Local Central Nervous System Tumor Microenvironment

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 μg 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 β2 (TGF-β2). 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-β2 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-β2 expression, may identify a subgroup of glioma patients to target as potential responders in future clinical investigations of dendritic cell–based vaccines.

Microbial Lipopeptides Stimulate Dendritic Cell Maturation Via Toll-Like Receptor 2

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-α, 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-γ 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.

Interferon-alpha and granulocyte-macrophage colony-stimulating factor differentiate peripheral blood monocytes into potent antigen-presenting cells.

The diverse roles of interferon‐α (IFN‐α) in regulating the immune response to infectious agents suggested that it might affect dendritic cell (DC) development. Peripheral blood mononuclear cells cultured with IFN‐α 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 costimulatory molecules, adhesion proteins, and CD40. Elevated DC expression of B7‐2 and HLA‐DR was observed with increasing IFN‐α concentrations up to 5000 U/mL. The effects of IFN‐α on DC immunophenotype were not reversed by adding neutralizing antibodies against interleukin‐4 (IL‐4) or tumor necrosis factor α to the cell cultures or by eliminating lymphocytes from the cultures. The addition of IFN‐α 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‐α 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‐α in the generation of DCs with potent antigen‐presenting capabilities from peripheral blood monocytes. J. Leukoc. Biol. 64: 358–367; 1998.

Activation of Toll-Like Receptor 2 on Human Dendritic Cells Triggers Induction of IL-12, But Not IL-10

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 CD1a+ 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.

Tumors promote altered maturation and early apoptosis of monocyte-derived dendritic cells.

Tumors produce a number of immunosuppressive factors that block the maturation of CD34+ stem cells into dendritic cells (DC). We hypothesized that tumors might also interfere with the maturation and/or function of human monocyte-derived DC. In contrast to stem cells, we found that CD14+ cells responded to tumor culture supernatant (TSN) by increasing expression of APC surface markers, up-regulating nuclear translocation of RelB, and developing allostimulatory activity. Although displaying these characteristics of mature DC, TSN-exposed DC lacked the capacity to produce IL-12, did not acquire full allostimulatory activity, and rapidly underwent apoptosis. The effects of TSN appeared to be specific for maturing DC, and were not reversed by Abs against known DC regulatory factors including IL-10, vascular endothelial growth factor, TGF-β, or PGE2. Supernatants collected from nonmalignant cell sources had no effect on DC maturation. The altered maturation and early apoptosis of monocyte-derived DC may represent another mechanism by which tumors evade immune detection.

HUMAN CD14+ LEUKOCYTES ACQUIRE THE PHENOTYPE AND FUNCTION OF ANTIGEN-PRESENTING DENDRITIC CELLS WHEN CULTURED IN GM-CSF AND IL-4

The combination of granulocyte‐macrophage colony stimulating factor (GM‐CSF) and interleukin‐4 (IL‐4) induces the growth of antigen‐presenting cells (APC) from adherent peripheral blood leukocytes. These cells have been characterized as dendritic cells (DC), yet many questions exist regarding their relationship to other DC populations and the nature of their progenitors. To address these issues, we utilized a combination of immunomagnetic depletion, cell sorting, and cell culture to isolate four distinct APC populations; macrophages expressing high levels of CD14 (CD14bright macrophages), DC produced by culturing adherent cells in GM‐CSF and IL‐4 (cultured DC), and two different subsets of fresh DC that express low levels of CD14 (CD14dim DC). Each population exhibited a unique morphology and a unique profile of cell surface markers. In contrast to macrophages, all three DC populations expressed the DC marker CD83, as well as high levels of MHC molecules and the costimulatory molecules B7‐1 (CD80) and B7‐2 (CD86). In addition, all three DC populations presented soluble tetanus toxin antigen and stimulated T cell proliferation to levels far superior to that of macrophages. Blocking studies demonstrated a costimulatory role for B7‐1, B7‐2, and CD40 in antigen presentation, although B7‐2 expression was the single most important factor. To identify the progenitors of cultured DC, we sorted the adherent fraction of PBMC into discrete subpopulations prior to exposure to GM‐CSF and IL‐4. DC activity derived entirely from CD14+ precursors and was equally demonstrable using either the CD14dim or CD14bright subsets. Although these DC precursors lost expression of CD14 in culture, they maintained most of their other myeloid features. We conclude that human CD14+ leukocytes acquire the phenotype and function of DC when cultured in GM‐CSF and IL‐4.

Δ9-Tetrahydrocannabinol regulates Th1/Th2 cytokine balance in activated human T cells

Human leukocytes express cannabinoid (CB) receptors, suggesting a role for both endogenous ligands and Delta 9-tetrahydrocannabinol (THC) as immune modulators. To evaluate this, human T cells were stimulated with allogeneic dendritic cells (DC) in the presence or absence of THC (0.625-5 microg/ml). THC suppressed T cell proliferation, inhibited the production of interferon-gamma and shifted the balance of T helper 1 (Th1)/T helper 2 (Th2) cytokines. Intracellular cytokine staining demonstrated that THC reduced both the percentage and mean fluorescence intensity of activated T cells capable of producing interferon-gamma, with variable effects on the number of T cells capable of producing interleukin-4. Exposure to THC also decreased steady-state levels of mRNA encoding for Th1 cytokines, while increasing mRNA levels for Th2 cytokines. The CB2 receptor antagonist, SR144528, abrogated the majority of these effects. We conclude that cannabinoids have the potential to regulate the activation and balance of human Th1/Th2 cells by a CB2 receptor-dependent pathway.

Helper-Dependent Adenoviral Vectors Efficiently Express Transgenes in Human Dendritic Cells but Still Stimulate Antiviral Immune Responses

Adenoviral (AdV) vectors can be used to transduce a wide range of human cells and tissues. However, pre-existing immunity to AdV, and enhancement of this immunity after repeated administration, limits their clinical application. This may be especially relevant when vectors are loaded into APCs. Helper-dependent AdV (Hd-AdV), in which viral coding regions are replaced by human stuffer DNA, offers a new approach for limiting antiviral responses. To evaluate their immunogenicity, human dendritic cells (DCs) were infected with either an Hd-AdV or a conventional replication-deficient E1-deleted AdV (E1-AdV) and were evaluated for their capacity to stimulate antiviral T cell responses. Hd-AdV proved to be 50- to 275-fold more effective than E1-AdV at expressing the lacZ transgene in human DCs. PCR demonstrated similar transduction efficiencies, but RT-PCR revealed much higher expression of transgene mRNA after transduction with Hd-AdV. Functionally, DCs transduced with Hd-AdV stimulated the proliferation of autologous T cells to the same level as DCs transduced with E1-AdV. Identical viral-specific T cell responder frequencies were observed and T cells stimulated with either type of AdV-transduced DC lysed viral-infected target cells. Disrupting transcription of vector-based genes had no effect on T cell activation, suggesting that responses against both vectors were directed against preformed components of the viral capsid. We conclude that Hd-AdV vectors can be used to obtain higher transgene expression in human DCs but that they still evoke a vector-related immune response similar to that generated by E1-AdV.

Modifying Adenoviral Vectors for Use as Gene-Based Cancer Vaccines

The past decade has produced significant advances in our understanding of antigen-presenting cells, tumor antigens, and other components of the immune response to cancer. Gene-based vaccination is emerging as one of the more promising approaches for loading dendritic cells (DC) with tumor-associated antigens. In this respect, it is proposed that adenoviral (AdV) vectors can deliver high antigen concentrations, promote effective processing and MHC expression, and stimulate potent cell-mediated immunity. While AdV vectors have performed well in pre-clinical vaccine models, their application to patient care has limitations. The in vivo administration of AdV vectors is associated with both innate and adaptive host responses that result in tissue inflammation and injury, viral neutralization, and premature clearance of AdV-transduced cells. A variety of strategies have been developed to address these limitations. The ideal vaccine would avoid vector-related immune responses, have relative specificity for transducing DC, and induce high levels of transgene expression. This review describes the range of host responses to AdV vaccines, identifies strategies to reduce viral recognition and enhance transgene antigen expression, and suggests future approaches to vector development and administration. There is every reason to believe that safer and more effective forms of AdV-based vaccines can be developed and applied to patient therapy.

Vaccination with helper-dependent adenovirus enhances the generation of transgene-specific CTL

Recombinant adenoviral vectors (AdV) have been used experimentally as vaccines to present antigenic transgenes in vivo. However, administration of first-generation vectors (FG-AdV) is often limited by their induction of antiviral immunity. To address this limitation, helper-dependent vectors (HD-AdV) were developed that lack viral coding regions. While the administration of HD-AdV results in long-term gene expression in vivo, their utility as immunogens has never been examined. Direct vaccination with 108 blue-forming units (BFU) of HD-AdV injected into C57BL/6 mice lead to superior transgene-specific CTL and antibody responses when compared to the same amount of a FG-AdV. The antibody responses to viral antigens were high in response to both the vectors. As a mechanism to reduce viral exposure, dendritic cells (DC) were transduced with HD-AdV in vitro and then used as a cell-based vaccine. DC transduced with HD-AdV expressed higher levels of transgene-specific mRNA and up to 1200-fold higher levels of transgene protein than did DC transduced with a FG-AdV. In addition, HD-AdV-transduced DC stimulated superior transgene-specific CTL responses when administered in vivo, an effect that was further enhanced by maturing the DC with LPS prior to administration. In contrast to direct immunization with HD-AdV, vaccination with HD-AdV-transduced DC was associated with limited antibody responses against the AdV. We conclude that HD-AdV stimulates superior transgene-specific immune responses when compared to a FG-AdV, and that immunization with a DC-based vaccine maintains this efficacy while limiting antiviral reactivity.