Stimulation with LPS+IFN- upregulated more genes than IL-4 stimulation when compared to unstimulated cells (1141 and 380 genes, respectively), with only 95 of these genes being induced by both conditions (Fig.?1b and Supplementary Data?1). variable unfavorable cross-regulation between some LPS?+?IFN–specific and IL-4-specific genes results in cell-to-cell heterogeneity in transcription. Interestingly, unfavorable cross-regulation leads to mutually exclusive expression of the T-cell-polarizing cytokine genes and versus the IL-4-associated factors and in single co-stimulated macrophages, and single-cell secretion measurements show that these specialized functions are maintained for at least 48?h. This study suggests that increasing functional diversity in the population is one strategy macrophages use to respond to conflicting environmental cues. and and and and Chil3l3 at 24?h (Supplementary Fig.?1b). In contrast, LPS+IFN–stimulated TNF and were not significantly inhibited at any dose combination. Thus, we concluded that this dose combination might reveal interesting behaviors in individual cells. For sc-RNAseq, we profiled ~1500 single cells per condition to ensure high-quality data with low doublet rates and high sequencing depth per cell (see Methods section). Our final data set had an average of 30,262 unique reads per cell and 4076 genes detected per cell. Stimulation with LPS+IFN- upregulated more genes than IL-4 stimulation when compared to unstimulated cells (1141 and 380 genes, respectively), with only 95 of these genes being induced by both conditions (Fig.?1b and Supplementary Data?1). Interestingly, both LPS+IFN- and IL-4 stimulation caused the RA190 apparent transcriptional downregulation of thousands of genes normally expressed in unstimulated cells, with the number of downregulated genes substantially outnumbering the number of upregulated genes (Fig.?1b). We performed dimensionality reduction using uniform manifold approximation and projection (UMAP)24,25 on the full transcriptional signature to visualize how cells mapped across treatments. We observed that almost all co-stimulated macrophages clustered separately from those stimulated with only one cue and from the unstimulated control population, suggesting that co-stimulation induced a distinct global transcriptional state (Fig.?1c). This result also exhibited that most macrophages were able to respond to both stimuli. This is consistent with the observation that macrophages co-stimulated with LPS+IFN- and IL-4 displayed robust Stat1 and Stat6 phosphorylation downstream of the IFN–receptor or IL-4-receptor, respectively, indicating that a majority of macrophages respond to both signals (Supplementary Fig.?1c). Although the transcriptional state of co-stimulated macrophages was distinct from cells treated with LPS+IFN- or IL-4 alone, we observed cell separation within the co-stimulation cluster. For example, we observed RA190 heterogeneous expression of canonical genes associated with LPS+IFN- stimulation, and and (Fig.?1d), suggesting that there RA190 is cell-to-cell variability in the extent of each individual cells response to LPS+IFN- versus IL-4 RA190 stimulation. To further explore this possibility, we measured the Spearman correlation between genes uniquely upregulated by either LPS+IFN- or IL-4 alone across all single cells to identify which genes are co-expressed or mutually inhibited across single cells. As expected, genes RA190 upregulated by LPS+IFN- alone were more likely to exhibit positive correlations with other LPS+IFN–induced genes, and either no correlation or negative correlation with IL-4-induced genes (Fig.?1e). Similarly, genes upregulated by IL-4 alone were more likely to be positively correlated with other IL-4-induced genes, and uncorrelated or negatively correlated with LPS+IFN–induced genes. Co-stimulated cells generally exhibited weak positive correlations within the LPS+IFN–induced genes and the IL-4-induced genes, while also exhibiting weak unfavorable correlations between some genes across the two programs (Fig.?1e and Supplementary Fig.?1d). This suggests that on average macrophages expressing core genes of one program FAD exhibited slightly reduced expression of core genes of the other program. Of note, not all correlations between LPS+IFN–induced genes and IL-4-induced genes were unfavorable, indicating that some LPS+IFN–induced genes are co-expressed with IL-4-induced genes. However, the strongest unfavorable correlations were between genes induced by the two opposing stimuli, suggesting that single.