Fig. 5: STAT1 regulates PD-L1 expression in Cluster 0 cells and influences T-cell activation status.

a Dual plot shows expression of Ccr1 and Cd274 in immune cells. b Heatmap shows the marker genes of Cluster 0 and compares their expression between Ccr1⁺ cells, Cluster 0, and other myeloid cell subsets. c Gating strategy to identify FACS sorted CD11b^high CCR1^high, CD11b^high CCR1^low myeloid cells, and CFSE levels on CD8⁺ T cells in co-culture. d Box plots show percentage of proliferating CD4 and CD8 T cells in co-culture with T-cell activation beads with or without myeloid cells. e Biaxial plots (left) show the gating strategy to identify the PD-1 levels on CFSE diluted and proliferating T cells. Box plots (right) show the ratio of PD-1^high proliferating and non-proliferating CD4 and CD8 T cells. f Volcano plot shows DGE on Cluster 0 cells when MCT-treated. g Scatter plot shows the potential gene targets (NES > 3 and p < 0.05 DEG between MCT and Vehicle treated) in Cluster 0 specific STAT1 targetome. h Violin plots show expression of Cd274, Map3k8, Ifitm3, Cd164, Gna13, and H2-D1 genes by Cluster 0 cells (each dot is a cell and cells are from n = 3 biological repeats pooled together). i Biaxial plots show the levels of pStat1 in CD11b⁺ cells in different transfection conditions. j Box plot shows the percentage of pStat1⁺ PD-L1⁺ CD11b⁺ viable cells after shRNA STAT1 transfection relative to pLKO transfection (n = 5 biological repeats). k Representative IF images of Iba1 and PD-L1 stained tumors treated with either Vehicle, MCT, MCT + STAT1 inhibitor, or MCT + STAT1 inhibitor + PD-1 inhibitor (left). Box plot (right) shows the quantified Iba1+ PD-L1 + cells of all the Iba1+ cells (n = 3 to 5 biological repeats). l Histograms show flow cytometry gating strategy and box plots show quantification of PD-L1⁺ CD11b⁺ cells of all the CD45⁺ immune cells in dual-therapy (MCT + PD-1 inhibitor) and tri-therapy (MCT + PD-1 inhibitor + STAT1 inhibitor) treated TNBC tumors (n = 4 biological repeats). a, b, f, g n = 3 biological replicates. c–l In box and violin plots, error bar = Mean ± SEM, center = Means (Diamonds) and Medians (Line), bottom and top boundaries of the box = 25 and 75th percentiles of the data, (whiskers) minima and maxima = lowest and highest data points, and two-sided T-test p-values. Also see associated Fig. S5.