SulTlnlaryImmunization with defined tumor antigens is currently limited to a small number of cancers where candidates for tumor rejection antigens have been identified. In this study we investigated whether pulsing dendritic cells (DC) with tumor-derived P,.NA is an effective way to induce CTL and tumor immunity. DC pulsed with in vitro synthesized chicken ovalbumin (OVA) P, NA were more effective than OVA peptide-pulsed DC in stimulating primary, OVA-specific CTL responses in vitro. DC pulsed with unfractionated P, NA (total or polyA +) from OVA-expressing tumor cells were as effective as DC pulsed with OVA peptide at stimulating CTL responses. Induction of OVA-specific CTL was abrogated when polyA + P,.NA from OVA-expressing cells was treated with an OVA-specific antisense oligodeoxynucleotide and P,.Nase H, showing that sensitization of DC was indeed mediated by OVA P,.NA. Mice vaccinated with DC pulsed with P, NA from OVA-expressing tumor cells were protected against a challenge with OVA-expressing tumor cells. In the poorly immunogenic, highly metastatic, B16/F10.9 tumor model a dramatic reduction in lung metastases was observed in mice vaccinated with DC pulsed with tumor-derived P, NA (total or polyA +, but not polyA-P,.NA). The finding that RNA transcribed in vitro from cDNA cloned in a bacterial plasmid was highly effective in sensitizing DC shows that amplification of the antigenic content from a small number of tumor cells is feasible, thus expanding the potential use of P,.NA-pulsed DCbased vaccines for patients bearing very small, possibly microscopic, tumors.

Dendritic cells (DC) generated from the peripheral blood mononuclear cells of healthy individuals or from cancer patients transfected with carcinoembryonic antigen (CEA) mRNA stimulate a potent CD8+ cytotoxic T lymphocyte (CTL) response in vitro. DCs are effectively sensitized with RNA in the absence of reagents commonly used to facilitate mammalian cell transfection. RNA encoding a chimeric CEA/LAMP-1 lysosomal targeting signal enhances the induction of CEA-specific CD4+ T cells, providing a strategy to induce T-help that may be necessary to generate and/or maintain an optimal CD8+ CTL response in vivo. CEA RNA-transfected DCs also serve as effective targets in cytotoxicity assays, thus providing a general method for inducing, as well as measuring, CEA-specific CTL responses across a broad spectrum of HLA haplotypes.

Despite recent advances, many cancers remain refractory to available immunotherapeutic strategies. Emerging evidence indicates that the tolerization of local dendritic cells (DCs) within the tumor microenvironment promotes immune evasion. Here, we have described a mechanism by which melanomas establish a site of immune privilege via a paracrine Wnt5a-β-catenin-peroxisome proliferator-activated receptor-γ (PPAR-γ) signaling pathway that drives fatty acid oxidation (FAO) in DCs by upregulating the expression of the carnitine palmitoyltransferase-1A (CPT1A) fatty acid transporter. This FAO shift increased the protoporphyrin IX prosthetic group of indoleamine 2,3-dioxgenase-1 (IDO) while suppressing interleukin(IL)-6 and IL-12 cytokine expression, culminating in enhanced IDO activity and the generation of regulatory T cells. We demonstrated that blockade of this pathway augmented anti-melanoma immunity, enhanced the activity of anti-PD-1 antibody immunotherapy, and suppressed disease progression in a transgenic melanoma model. This work implicates a role for tumor-mediated metabolic reprogramming of local DCs in immune evasion and immunotherapy resistance.