| Grp170 is an endoplasmic reticulum (ER) resident chaperone that can induce immune responses against the tumor from which it was purified. However, the mechanisms by which grp170 stimulates the immune system is still evasive. To better understand these mechanisms, we targeted grp170 expression by tumor cells to the extracellular microenvironment and showed that this secreted grp170 elicits an immune response in immunocompetent hosts that prevents tumor growth. The murine colon carcinoma cell line, Colon-26, was stably transfected with grp170 cDNA that lacks the ER-retention signal. BALB/c mice challenged with grp170-secreting Colon 26 completely rejected the tumor whereas animals challenged with wild type Colon 26 or mock-transfected Colon-26 developed rapidly progressing tumors. In vivo depletion of lymphocyte subpopulations showed that the tumor rejection phenomenon was the result of an adaptive immune response and is dependent on CD8+ cells. ELISPOT assay revealed an elevated cellular immune response against the AH1 epitope of the Murine leukemia virus (MuLV) envelope protein gp70. Here we propose a novel mechanism of grp170 immune function that involves its binding to full-length protein substrates and cotransporting them to the extracellular compartment. To test for this model the histidine-tagged grp170 was purified from the spent culture media of transfected cells and several proteomic separation techniques (1D-SDS gel electrophoresis, two-dimensional differential gel electrophoresis and two-dimensional liquid chromatography) were applied to resolve the full-length proteins associated with grp170. Subsequently these proteins were identified by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and electrospray ionization tandem mass spectrometry (ESI MS/MS). Secreted grp170 was found associated with tumor antigens such as gp70 of MuLV and large T-antigen. Other identified proteins included components of the intrinsic antigen processing pathway. Together, these results indicate that immunization of animals with grp170-secreting tumor cells results in rejection of the tumor by induction of antigen-specific, CD8-dependent immune responses. Furthermore, the secreted grp170 is able to deliver full-length tumor antigens to the tumor microenvironment, thus making them available for uptake by antigen presenting cells (APCs) to initiate a tumor-specific immune response. This approach has the potential of being exploited as a vaccine against tumors of various histological origins. |
