Down-Regulation Presentation Correlated with Heat Shock Protein-70 Autologous CTLs Is Associated with Inefficient Antigen A Three-Dimensional Tumor Cell Defect in Activating

We described previously a CTL clone able to lyse the autologous carcinoma cell line IGR-Heu after specific recognition of an HLA-A2/ mutated -actinin-4 peptide complex. Here, we used IGR-Heu, cultured either as standard two-dimensional monolayers or as three-dimensional spheroids, to further analyze the influence of target architecture on CTL reactivity. Interestingly, we found that changes in the tumor structure from twoto three-dimensional induced a dramatic decrease in its capacity to activate autologous CTL, as measured by IFNand tumor necrosis factorsecretion. These functional alterations were attributable neither to MHC class I expression nor to tumor antigen (Ag) down-regulation, because IGR-Heu, cultured as twoor three-dimensional, expressed similar levels of HLA-A2 and -actinin-4. More importantly, incubation of three-dimensional cells with synthetic epitope completely restored cytokine release by CTL. This defective Ag presentation correlated with a decrease in heat shock protein (hsp)70 expression by three-dimensional tumors compared with two-dimensional cells. Furthermore, transfection of the tumor cells with hsp70 cDNA completely restored the Ag-presenting potential of spheroids and, therefore, cytokine production by T cells. These data strongly suggest that hsp70 down-regulation in threedimensional cells may result in tumor resistance to the immune response.