Abstract
Effective immune-based therapies against the plasma cell malignancy, multiple myeloma (MM), are currently lacking. Identification of novel antigens (Ag) on the surface of MM cells to use as cellular targets for the destruction of cancer cells by the body’s immune system has been of great interest. We and others have demonstrated that CD40 stimulation of MM cells results in marked upregulation of membrane bound proteins such as Ku86. Using CD40 triggered MM cells as immunogens and hybridoma technology; we generated a monoclonal antibody (mAb), 6D11, that recognizes a CD40 induced cell membrane Ag on MM cells. This Ag is detectable on the surface of MM cells using indirect immunofluorescence flow cytometric analysis. Moreover, in Western immunoblotting assays, 6D11 mAb reacts with a 94 kDa protein, which is strongly associated with a 78 kDa protein. Using high performance liquid chromatography and protein microsequencing, we confirm that these proteins are the heat shock proteins (HSP), glucose-regulated peptide 94 (GRP94) and GRP78, respectively. These data were confirmed using co-immunprecipitation experiments. Furthermore, we demonstrate through indirect immunofluorescence flow cytometric analysis and quantitative real time reverse transcription polymerase chain reaction (RQ-PCR) that CD40 ligand (CD40L) stimulation of MM cells results in rapid upregulation of both GRP94 and GRP78. Since HSPs have been shown to play a role in both Ag presentation, as well as the intracellular transport of cellular Ags, it is tempting to speculate that cell membrane expression of tumor-specific peptides could also be induced via CD40 triggering. Accordingly, CD40 induced cell membrane HSP expression resulted in increased antigenicity as determined by increased co-stimulatory molecule expression on Ag presenting cells (APC) and by increased immunoreactivity in mixed lymphocyte reactions (MLR). This suggests that CD40 induced HSP expression may indeed result in increased recognition of MM cancer by the immune system. Our study therefore supports the development of CD40-based targeted cell therapies against MM.
Functions of Heat Shock Proteins in Pathways of the Innate and Adaptive Immune System