Figure 3|. TBK1 inhibition remodels the tumour immune microenvironment.

a, Uniform Manifold Approximation and Projection (UMAP) of all immune cells (n=53,637) with 25 unique populations identified among CD45⁺-enriched immune cells from single cell RNA sequencing on tumour-infiltrating leukocytes from B16-ova tumours from Control (Vehicle/IgG, n=3), αPD-1 (Vehicle/anti-PD-1, n=4), TBK1i (TBK1i/IgG n=4), and αPD-1 + TBK1i (TBK1i/anti-PD-1, n=4) treated tumours. b, Downsampled cell density projections by condition. c, UMAP of 43,068 cells and 19 unique populations identified among sub-clustered myeloid cells. d, Downsampled myeloid sub-cluster cell density projections by condition. e, Bar plots of proportional changes by unique cluster of myeloid sub-cluster immune cells by treatment. Means (bars) and individual values (circles) +/− s.e.m (error bars) are shown (n = 4 biologically independent samples, except for control, n = 3; multiple unpaired t-test, **P < 0.01; ***P < 0.001; **** P <.0001). f, Top enriched or decreased Hallmark gene signatures in the myeloid sub-cluster determined by GSEA Prerank on differentially expressed genes calculated by a logistic regression by condition. g, Mountain plots showing enrichment scores for the TNFα Signaling Via NFκB Hallmark gene set and Inflammatory Response gene set in the myeloid sub-cluster by condition. h-i, downsampled UMAP of all immune cells showing (h) Tnf gene expression and (i) Il1a gene expression by condition. j, gene expression (qRT-PCR) of Tnf and Il1a in bone marrow-derived macrophages (BMDMs) pre-treated with TBK1i (1 μM) for 24 hours prior to 2-hour stimulation with LPS (20 ng/mL) plus IFNγ (20ng/mL) versus PBS control. Means (bars) and individual values (open circles) are shown (n=4 biological replicates; 2-way ANOVA with Sidak’s multiple comparisons test, **P < 0.01; ***P < 0.001; **** P <.0001).