Abstract: Autophagosomes derived from tumor cells have been proved to induce potent T cell response both in mouse and human. In human in vitro study, dendritic cells(DC)loaded with cytomegalovirus(CMV)pp65 antigen-containing DRibble were capable to efficiently re-stimulate pp65-specific T-cell recall responses from freshly isolated or frozen humanperipheral blood mononuclear cell(PBMC). This study developed more robust assays using in vitro expanded antigen-specific T cells that contained a much higher percentage of antigen-specific T cells. DC cross-presentation efficiency of OX40 and CD80 modified pp65-DRibble was detected by intracellular IFN-γ staining. Compared with Ctrl/pp65 DRibble, the percentage of IFN-γ⁺ in total CD8⁺ T cells andCD4⁺ T cells was improvedwith OX40/pp65 DRibbleand CD80/pp65 DRibble stimulation. In addition, vaccine induced IL-12indendritic cells, whichpolarizes Th cells toward the IFN-γ high Th1 phenotype, evaluated by ELISA inco-culture supernatantwas dramatically higher in OX40/pp65 DRibble and CD80/pp65 DRibblegroups than in Ctrl/pp65 DRibble group. These results have implications for the immuneactivity of OX40 and CD80 modified DRibble and choice for prospective clinical use ofDRibble-based cancer immunotherapy.