Abstract
Type 1 regulatory T (Tr1) cells are an immunoregulatory CD4 + Foxp3- IL-10 high T cell subset with therapeutic potential for various inflammatory diseases. Retroviral (RV) transduction has been a valuable tool in defining the signaling pathways and transcription factors that regulate Tr1 differentiation and suppressive function. This protocol describes a method for RV transduction of naïve CD4 + T cells differentiating under Tr1 conditions, without the use of reagents such as polybrene or RetroNectin. A major advantage of this protocol over others is that it allows for the role of genes of interest on both differentiation and function of Tr1 cells to be interrogated. This is due to the high efficiency of RV transduction combined with the use of an IL10 GFP /Foxp3 RFP dual reporter mouse model, which enables successfully transduced Tr1 cells to be identified and sorted for functional assays. In addition, this protocol may be utilized for dual/multiple transduction approaches and transduction of other lymphocyte populations, such as CD8 + T cells.