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Almost all melanoma cells express at least one member of the MAGE-A antigen family, making the cytotoxic T cells (CTLs) epitopes with cross-immunizing potential in this family attractive candidates for the broad spectrum of anti-melanoma immunotherapy. In this study, four highly homologous peptides (P264: FLWGPRALA, P264I9: FLWGPRALI, P264V9: FLWGPRALV, and P264H8: FLWGPRAHA) from the MAGE-A antigens were selected by homologous alignment. All four peptides showed high binding affinity and stability to HLA-A*02:01 molecules, and could prime CTL immune responses in human PBMCs and in HLA-A*02:01/K(b) transgenic mice. CTLs elicited by the four epitope peptides could cross-lyse tumor cells expressing the mutual target antigens, except MAGE-A11 which was not tested. However, CTLs induced by P264V9 and P264I9 showed the strongest target cell lysis capabilities, suggesting both peptides may represent the common CTL epitopes shared by the eight MAGE-A antigens, which could induce more potent and broad-spectrum antitumor responses in immunotherapy.

The retinoid-related orphan nuclear receptor gamma (ROR γ) plays critical roles in regulation of development, immunity and metabolism. As transcription factor usually forms a protein complex to function, thus capturing and dissecting of the ROR γ protein complex will be helpful for exploring the mechanisms underlying those functions. After construction of the recombinant tandem affinity purification (TAP) plasmid, pMSCVpuro ROR γ-CTAP(SG), the nuclear localization of ROR γ-CTAP(SG) fusion protein was verified. Following isolation of ROR γ protein complex by TAP strategy, seven candidate interacting proteins were identified. Finally, the heat shock protein 90 (HSP90) and receptor-interacting protein 140 (RIP140) were confirmed to interplay with ROR γ by co-immunoprecipitation. Interference of HSP90 or/and RIP140 genes resulted in dramatically decreased expression of CYP2C8 gene, the ROR γ target gene. Data from this study demonstrate that HSP90 and RIP140 proteins interact with ROR γ protein in a complex format and function as co-activators in the ROR γ-mediated regulatory processes of HepG2 cells.