| Development of an effective cancer vaccine is challenging because of the lack of defined tumor specific antigens and a tumor induced immuno-suppressive environment. Initial attempts at cancer immunotherapy led to the understanding that effective tumor regression requires the activation of an innate immune response and the induction of a specific lymphocyte response against tumor antigens. 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, the interaction of purified gp96 from tumors with a murine macrophage cell line - RAW 264.7, and murine bone marrow derived macrophages (BMMs), was characterized. 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, G-CSF, KC etc.) and up-regulation of the co-stimulatory molecule CD86 and MHC-class II, thus demonstrating the activation of an innate immune response. As a pre-requisite for the induction of an adaptive immune response, transfer of peptides from gp96 to MHC class I molecules was demonstrated in RAW 264.7 cells by using biotinylated VSV-8 complexed with gp96, thus corroborating the cross presentation mechanism of peptides associated with gp96 leading to a specific lymphocyte response. Moreover, examination of the molecular mechanism of peptide elution from gp96 revealed that monomerization of gp96 under acidic conditions leads to the release of its associated peptides, which can then be routed into the antigen cross presentation pathways. These studies therefore demonstrate the molecular and cellular mechanisms of gp96 mediated activation of both the innate and adaptive arms of the immune system, which is essential for effective tumor remission. |
