In vitro activity: Resiquimod activates immune cells and induces proliferation of wild-type splenocytes via the Toll-like receptor 7 (TLR7)-MyD88-dependent signaling pathway. Resiquimod also modulates dendritic cells to augment cytomegalovirus- and HIV-1-specific T cell responses. Resiquimod induces the differentiation of myeloid-derived suppressor cells into macrophages and dendritic cells, and may improve cancer immunotherapy by reducing immunosuppressive MDSCs.

Kinase Assay: For luciferase assay, FG-9307 cells are transfected with the firefly NF-κB-specific luciferase reporter vector pNFκB-Met-Luc2. Transfection efficiency is monitored by co-transfection with the pSEAP2 control vector, which constitutively expresses the human secreted enhanced alkaline phosphatase (SEAP). Then the cells are treated with Resiquimod (R848, 1 μg/mL), CQ (10 μM), CQ plus R848 or PBS and incubated at 22°C for 24 h. The culture medium of the transfectants is then analyzed for luciferase activity and SEAP activity using Luciferase Assay Kit and the Great EscAPe(TM) SEAP Chemiluminescence Detection Kit, respectively. The assay is performed three times.

Cell Assay: Unlike LPS-induced activation, which was MyD88-independent, Resiquimod-induced activation of NF-κB was completely dependent on MyD88. It was implied that Resiquimod activated macrophages via TLRs.