Panel A: Violin plots show the expression of various genes across different myeloid clusters identified by single-cell RNA sequencing. The x-axis represents different clusters, and the y-axis represents gene expression levels on a log-normalized scale. Panel B: A bar graph displays the results of Gene Set Enrichment Analysis (GSEA) for Gene Ontology Biological Processes (GOBP). The x-axis shows the Normalized Enrichment Score (NES), with positive values in red indicating enrichment in CD1d-KO cells and negative values in blue indicating enrichment in WT c3 Inflam-Mono cells. Panel C: Enrichment plots compare transcriptional signatures of CD1d-KO versus WT c3 Inflam-Mono cells against various signatures. The x-axis represents the enrichment score, and the y-axis lists different signatures. Panel D: A bar graph shows predicted upstream regulators of gene expression changes in CD1d-KO versus WT c3 Inflam-Mono cells, analyzed by Ingenuity Pathway Analysis. The x-axis indicates the activation z-score, with positive values in red and negative values in blue. Panel E: The experimental setup is illustrated at the top, with a representative flow cytometry profile in the middle and quantification of inflammatory monocytes in EO771 tumors from WT and CD1d-KO mice at the bottom. The x-axis shows different cell populations, and the y-axis shows the number of cells per cubic centimeter of tumor. Panel F: A line graph shows tumor growth over time in WT mice orthotopically injected with EO771 cells and treated with IFNAR plus slash minus CD1d or isotype control. The x-axis represents days, and the y-axis represents tumor size in cubic centimeters. Flow cytometry plots and quantification of different myeloid cell populations are also shown, with the x-axis indicating cell markers and the y-axis indicating the percentage of CD45 positive cells.
CD1d controls the transcriptional program and accumulation of inflammatory monocytes. (A) Violin plots showing the expression of the depicted genes in the myeloid clusters identified by scRNA-seq (as in Fig. 3, A and B). (B) GSEAs for GOBP showing top enriched pathways. NES values indicate enrichment (red, positive NES) in CD1d-KO or WT c3_Inflam-Mono cells (blue, negative NES). (C) Enrichment plot for transcriptional signature of CD1d-KO versus WT c3_Inflam-Mono cells compared with the depicted signatures. (D) Predicted upstream regulators of gene expression changes in CD1d-KO versus WT c3_Inflam-Mono cells, by Ingenuity Pathway Analyses. Data show the top cytokine and transcription factor regulators. z-score indicates the predicted activation level, with either positive (red) or negative (blue) values indicating an activated or inhibited regulator, respectively. (E) Experimental setup (top), representative flow cytometry profile (middle), and quantification (bottom) of inflammatory monocytes in EO771 tumors from WT and CD1d-KO mice (n = 4; data are pooled from two independent experiments). Data are shown as the mean ± SEM. (F) WT mice were orthotopically injected with EO771 cells and received αIFNAR ± αCD1d or isotype at the indicated time points (arrows). Data show tumor growth (top left) and weight (bottom left), as well as flow cytometry plots (top right) and quantification (bottom right) of the depicted myeloid cell populations (n = 3–6; data are pooled from three independent experiments). Data are shown as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, unpaired t test (E) or one-way or two-way ANOVA with Tukey’s (F) multiple comparisons. GOBP, Gene Ontology Biological Processes; NES, normalized enrichment score.