Transcriptional responses to IFN-α, IFN-γ, and TNF underlie the improved target cell killing in the presence of cGAMP. (A) Overview of bulk RNA sequencing approach; HL-60 and human T cells were cocultured for 8 hours in the presence of AMG 330 ± cGAMP and separated by FACS before subjecting them to RNA sequencing. c-BiTE conditions served as control. (B) Principal component analysis (PCA) applied to the RNA sequencing data collected from the 4 experimental conditions and the 2 indicated cell types (as outlined in panel A; n = 3). (C) Gene set enrichment analysis for the comparison of c-BiTE and AMG 330 + cGAMP in HL-60 and T cells. Normalized enrichment score (NES) is depicted for the indicated hallmark gene sets; the size of the dot represents the adjusted P value for each gene set. (D) Volcano plot showing the gene expression differences between cGAMP treatment and combined AMG 330 + cGAMP treatment in HL-60 cells (left) and AMG 330 treatment and combined AMG 330 + cGAMP treatment in T cells (right). Negative log₁₀-adjusted P values (y-axis) are plotted against the log₂-transformed fold changes in gene expression (x-axis). Significantly upregulated (adjusted P < .05; absolute fold change > 2) genes are shown in blue, with genes associated with an ISG response highlighted in green (left) and genes associated with T-cell activation highlighted in red (right). (E) Heat maps of the top 200 most variable genes in HL-60 and T cells clustered hierarchically across all conditions; relevant genes are highlighted. Color coding corresponds to normalized and log₂-transformed read counts. FACS, fluorescence activated cell sorting; PC1, principal component 1.