2080

Development of an effective cancer vaccine is challenging because of the lack of defined tumor specific antigens and a tumor induced immuno-suppressive environment. gp96 is an ER resident heat shock protein with tumor rejection properties and is being clinically evaluated as an immunotherapeutic vaccine. Although the clinical anti-tumor activity of gp96 has been established, the exact cellular mechanism for the generation of an immune response has not been characterized. Cellular interactions with gp96 occur through two signaling receptors - Toll like receptors 2 and 4 (TLR2 and TLR4) and two internalization receptors - CD91 and scavenger receptor-A (SR-A). To examine the response of antigen presenting cells (APCs) to a gp96 stimulus, we characterized the interaction of purified gp96 from tumors with a mouse macrophage cell line - RAW264.7. Exogenously added gp96 induced signaling though the MAPK pathway by activating two MAPKKKs - c-Raf and TAK-1 resulting in the activation of the MAPKs - ERK1/2, P38 and JNK. Signaling from TAK-1 also induced the NFκB pathway. These cytoplasmic signaling events culminated in the activation of transcription factors, NFκB, AP-1 and Elk-1. The cellular response to these events was the induction of inflammatory cytokines (TNFα, IL-1β, IL-12 etc.), chemokines (MIP-1α, MCP-1, RANTES, KC etc.) and up-regulation of the co-stimulatory molecule CD86 and MHC-class II. In addition, we demonstrated peptide transfer from internalized gp96 to MHC class I molecules in RAW 264.7 cells using biotinylated VSV-8 complexed with gp96, thus corroborating the cross presentation mechanism of peptides associated with gp96 leading to a specific adaptive immune response. Our studies demonstrate the molecular and cellular mechanisms of gp96 mediated activation of both the innate and adaptive arms of the immune system. Since our experimental model mimics the in vivo scenario of gp96 vaccination, it will be interesting to evaluate if this in vitro mechanism of APC activation translates in a clinical scenario.

99th AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA