Visualization of RelB expression and activation at the single-cell level during dendritic cell maturation inRelb-Venusknock-in mice

RelB is activated by the non-canonical NF-κB pathway, which is crucial for immunity by establishing lymphoid organogenesis and B-cell and dendritic cell (DC) maturation. To elucidate the mechanism of the RelB-mediated immune cell maturation, a precise understanding of the relationship between cell maturation and RelB expression and activation at the single-cell level is required. Therefore, we generated knock-in mice expressing a fusion protein between RelB and fluorescent protein (RelB-Venus) from the Relb locus. The Relb Venus/Venus mice developed without any abnormalities observed in the Relb–/– mice, allowing us to monitor RelB-Venus expression and nuclear localization as RelB expression and activation. Relb Venus/Venus DC analyses revealed that DCs consist of RelB – , RelB low and RelB high populations. The RelB high population, which included mature DCs with projections, displayed RelB nuclear localization, whereas RelB in the RelB low population was in the cytoplasm. Although both the RelB low and RelB – populations barely showed projections, MHC II and co-stimulatory molecule expression were higher in the RelB low than in the RelB – splenic conventional DCs. Taken together, our results identify the RelB low population as a possible novel intermediate maturation stage of cDCs and the Relb Venus/Venus mice as a useful tool to analyse the dynamic regulation of the non-canonical NF-κB pathway.