Fig. 3: Pertub-seq reveals cytokine regulation of macrophages at the single-cell level.

a, Scheme of the experimental design for the Hoxb8FL-derived macrophage and the bone-marrow chimera Perturb-seq experiments. Hoxb8FL (top) or LSK (bottom) cells deficient for the receptors of the four essential cytokines driving macrophage polarization in EAE as well as control-edited cells were either transferred as a Hoxb8FL cell pool, as previously described (top), or used to reconstitute the immune system after irradiation in a bone-marrow chimera model (bottom). At the peak of disease, endogenous (not shown on scheme), Hoxb8FL-derived or chimeric macrophages were harvested from the spinal cord and subjected to single-cell transcriptional sequencing. b, UMAP plots of the three EAE macrophage single-cell sequencing datasets after integration. Left, endogenous macrophages from WT EAE; middle, Hoxb8FL-derived macrophages; right, chimeric macrophages. c, Pseudotime across the different monocyte and macrophage clusters in the integrated dataset. Clusters are color coded according to the scale on the right. d, Feature plot of Nos2 and Arg1 expression in the three datasets; expression levels are color coded according to the scales on the right of the plot. e, Density difference plot showing differences in the distribution of myeloid cell subpopulations in the KOs compared to the control cells; density differences are color coded according to the scale on the right.