Differential Immunostimulatory Effects of EGFP and +36 GFP on Immune Cells

Introduction: Green fluorescent protein (GFP) and its variants are pivotal in tracking gene expression across various gene delivery systems.While GFP is typically employed for intracellular reporting, it can be modified to display on cell surfaces for labeling.Previous research indicates GFP might have immunogenic effects, notably enhancing tumor-specific T cell responses.This study explores the immunostimulatory differences between enhanced GFP (EGFP) and the supercharged variant, +36 GFP.Methods: Recombinant EGFP and +36 GFP proteins were generated using an Escherichia coli expression system.Murine bone marrow-derived dendritic cells (BMDCs) were generated using established protocols.Splenocytes were isolated from murine spleens via mechanical disruption and red blood cell lysis.The RAW 264.7 macrophage cell line was cultured in complete DMEM medium.Immune cells were then incubated with varying concentrations of EGFP and +36 GFP, separately, for 48 h.Cytokine levels (IFN-γ, TNF-α, IL-10) were quantified using sandwich ELISA.Results: Purified EGFP and +36 GFP migrated as a single band of ~27 kDa on SDS-PAGE, consistent with their predicted molecular weights.Both EGFP and +36 GFP elicited cytokine production from T cells and macrophages.EGFP significantly increased TNF-α secretion and decreased IL-10 secretion compared to +36 GFP in both T cells and macrophages.Conclusion: Our findings demonstrate that EGFP and +36 GFP differentially stimulate immune cells, suggesting that the selection of GFP variant could influence the immunological outcomes in therapeutic applications.This necessitates careful consideration in vector design, vaccine development, and other biotherapeutic strategies to optimize efficacy and safety through appropriate GFP choice, dosing, and control inclusion.