The maturation phases of dendritic cells (DCs) are pivotal determinants in their capacity to either promote immune tolerance or incite inflammatory responses (Li and Shi, 2015). Immature dendritic cells (imDCs), characterized by their lower expression of MHC II and co-stimulatory molecules, are capable of inducing T cell tolerance. In contrast, mature DCs, which exhibit high expression levels of these molecules, can trigger inflammatory T cell responses (Ravindranath et al., 2021). Earlier studies have illustrated the potential of imDCs in cell therapy, either to rectify immune imbalances or to foster immune tolerance (Fu et al., 1996, Mahnke et al., 2002, Lutz et al., 2000). Nonetheless, imDCs exhibit an unstable phenotype, and they may undergo activation and maturation in response to inflammatory stimuli, either in vivo or in vitro, thereby posing a risk of initiating immune reactions (Morelli and Thomson, 2007). Current research indicates that the resistance of imDCs to danger signals can be enhanced by regulating the expression of specific genes, and such cells with stable tolerogenic functions are known as tolerogenic dendritic cells (tolDCs) (Iberg and Hawiger, 2020). TolDCs have been recognized for their significant potential in preventing transplant rejections, making them a focal point in transplant immunology research.

Using recombinant adenovirus (rAd) vectors for dendritic cell gene modification is an efficient method, offering higher stability and transduction efficiency compared to non-viral interference strategies such as siRNA or electroporation (Tan et al., 2005). In various studies employing adenoviral vectors for imDCs transduction, discrepancies have been reported: some studies indicate a marked alteration in imDCs' phenotype, while others demonstrate the retention of their immature state alongside adequate transduction efficiency (Okada et al., 2003, Lin et al., 2019, Youlin et al., 2010, Cai et al., 2010, Tuettenberg et al., 2010, Knippertz et al., 2009). ImDCs, known for their efficient immune recognition, possess an abundance of receptors for danger signals. Their phenotype and functions are prone to alterations under the influence of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) (Morelli et al., 2000). Research on tolerogenic cells requires phenotypically stable imDCs. Presently, there is an absence of ample evidence to evaluate the extent to which variations in adenovirus transduction outcomes might affect the induction process of tolDCs.

In our study, aimed at elucidating the causes behind the disparities in imDCs transduction outcomes, we set the adenovirus transduction conditions to align with commonly used parameters. We discovered pronounced differences in imDCs' phenotypes when using various culture mediums like optiMEM and RPMI and different MOI levels, such as 50 and 100. Notably, while certain adenoviral transduction conditions altered imDCs' phenotype in this study, they did not significantly impact their capability to activate CD4+ and CD8+ T cells. A distinctive feature of our research is the examination of the in vivo tolerogenic functions induced by adenovirus-transfected imDCs. In a mouse heart transplantation model, we noted that imDCs, which exhibited substantial phenotypic changes under in vitro conditions, led to an increased ratio of TH1/TH17 cells in vivo, potentially linked to significant alterations in their cytokine secretion patterns. Our findings have significant implications for improving the application of adenovirus transduction technology in research on tolerogenic cells.