Dendritic cells (DCs) are considered to be promising adjuvants for inducing immunity to cancer. We used mature, monocyte-derived DCs to elicit resistance to malignant melanoma. The DCs were pulsed with Mage-3A1 tumor peptide and a recall antigen, tetanus toxoid or tuberculin. 11 far advanced stage IV melanoma patients, who were progressive despite standard chemotherapy, received five DC vaccinations at 14-d intervals. The first three vaccinations were administered into the skin, 3 × 106 DCs each subcutaneously and intradermally, followed by two intravenous injections of 6 × 106 and 12 × 106 DCs, respectively. Only minor (less than or equal to grade II) side effects were observed. Immunity to the recall antigen was boosted. Significant expansions of Mage-3A1–specific CD8+ cytotoxic T lymphocyte (CTL) precursors were induced in 8/11 patients. Curiously, these immune responses often declined after the intravenous vaccinations. Regressions of individual metastases (skin, lymph node, lung, and liver) were evident in 6/11 patients. Resolution of skin metastases in two of the patients was accompanied by erythema and CD8+ T cell infiltration, whereas nonregressing lesions lacked CD8+ T cells as well as Mage-3 mRNA expression. This study proves the principle that DC “vaccines” can frequently expand tumor-specific CTLs and elicit regressions even in advanced cancer and, in addition, provides evidence for an active CD8+ CTL–tumor cell interaction in situ as well as escape by lack of tumor antigen expression.

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Dendritic cells form a system of leukocytes specialized to stimulate resting T cells in vivo. Dendritic cells are crucial for the initiation of primary immune responses of both helper and cytotoxic T lymphocytes, and thus act as ‘nature’s adjuvant’. The manifold specializations underlying this in vivo immunostimulatory function are becoming increasingly clear. Methods have been developed to generate large numbers of dendritic cells from hematopoietic precursors in vitro. These techniques now allow molecular studies as well as the use of antigen-charged dendritic cells to vaccinate patients against tumors (e.g. B-cell lymphoma or melanoma) and infection (e.g. HIV-1). Recent data suggest that besides the classical immunostimulatory dendritic cells which belong to the myeloid lineage, there exist regulatory dendritic cells related to the lymphoid lineage. These lymphoid-derived dendritic cells which at least in part express Fas-ligand appear to be involved in the induction of central as well as peripheral tolerance, and in the future might allow a novel approach to induce tolerance in transplantation, autoimmunity, and allergy.

By selective adaptation to glucose-free tissue culture conditions, a gluconeogenic substrain of the opossum kidney (OK) cell line, designated OK_gNg+, was established, analogous to the method recently employed for the isolation of gluconeogenic LLC-PK₁-FBPase+ cells [Gstraunthaler and Handler, Am J Physiol 1987;252:C232-C238]. OK_Gng+ cells express considerable amounts of fructose-1, 6-bisphosphatase activity, and metabolic flux through phosphoenolpyruvate carboxykinase (PEPCK), which is indicative for their gluconeogenic capacity, in contrast to OK wild-type cells. OK_gng+ cells also seem to exhibit a higher degree of differentiation indicated by an increased expression of γ-glutamyltranspeptidase activity. Karyotype analysis revealed that OK_gng+ cells are descendants of OK wild-type cells, exhibiting slight increases in tetraploidy. Remarkable differences between OK_gng+ and LLC-PK₁^-FBPase+ cells have been found in terms of substrate metabolism of the two gluconeogenic renal sub-strains. Cultures were incubated in culture medium containing either Na-L-lactate, Na-pyruvate or lactate/pyruvate mixtures (10:1, 5:5, 1:10) as main gluconeogenic substrates. OKg _gng+ cultures grew better in lactate than in pyruvate, whereas the opposite was observed with LLC-PK₁^-FBPase+ cells. This was further confirmed by the consumption rates of lactate and pyruvate by the substrains. When lactate or lactate/pyruvate were supplied, OKq _gng+ cells consumed lactate at much higher rates as compared to LLC-PK₁^-FBPase+ cultures. When pyruvate was added as the sole gluconeogenic substrate, pyruvate consumption rates by LLC-PK₁^-FBPase+ cells were linear over time, without considerable lactate production. However, pyruvate consumption by OK _gng+ cells was almost completed after 24-48 h, and resulted in an equimolar production and accumulation of lactate in the culture medium, respectively, which was consumed by the cells thereafter. In summary, a novel gluconeogenic strain of OK cells has been established (OK _gng+), as previously shown for gluconeogenic LLC-PK₁^-FBPase+ cells. Both cell strains, however, differ markedly in the metabolism of lactate and pyruvate, and thus obviously in the subcellular localization of PEPCK, in their regulation of gluconeogenesis by the intracellular NADH/NAD ratio, and/or in the rate of mitochondrial substrate fluxes and shuttle activities, respectively.

9002 Background: Emerging data suggests that features of the melanoma tumor microenvironment may determine the clinical outcome to immunotherapies. We recently have observed a gene expression signature that correlated with a favorable clinical outcome in response to an IL-12-based melanoma vaccine. Increased expression of chemokine genes and T cell transcripts, and decreased expression of genes associated with aggressive tumor biology, were observed in the favorable group. To determine whether these patterns were reproducible, gene expression profiling was performed from an independent vaccine clinical trial. Methods: Patients with advanced melanoma were treated with autologous, mature monocyte-derived dendritic cells loaded with a combination of melanoma antigen peptides. Pretreatment biopsies were cryopreserved for RNA extraction and gene expression profiling. Patients were categorized into “long survival” (> 24 months) or “short survival” outcomes. Supervised hierarchical clustering was performed to identify genes differentially expressed in the two outcome groups. Results: RNA that passed quality control was obtained from 17 stage IV patients, 5 with a short survival and 12 with a long survival. 408 genes were differentially at least 2- fold. Consistent with previous observations, tumors from favorable outcome patients expressed higher levels of several T cell-specific genes, including Thy1 and CD28; chemokines, including CCL19, CXCL12, and CXCL14; and other immune genes, including LTβ, IL-1R, IFNαR2, IL27R, CD69, and FcRs. Conversely, tumors from unfavorable outcome patients expressed higher levels of pro- angiogeneic genes, including Flt1; anti-apoptotic genes, including SerpinH1 and Serpine1; and multiple collagens. Conclusions: Our results confirm that a subset of transcripts expressed in melanoma metastases may be useful as a predictive biomarker for response to melanoma vaccines. The categories of genes identified point toward new opportunities for overcoming resistance mechanisms. Future studies should integrate gene expression profiling of pre-treatment biopsies as a stratification or enrichment factor in immunotherapy trials. No significant financial relationships to disclose.