Fig 2: SFK-mediated phenotypical and functional neutrophil exhaustion. (a) The expression of Fyn and phosphorylated Src at Tyr416 on WT neutrophils stimulated with PBS or LPS (100 ng/ml) in the presence of Dasatinib (100 nM) for 24 h (n = 3). (b) The total STAT1 and activated STAT1 at Tyr701 on WT neutrophils treated with PBS or LPS (100 ng/ml) in the presence of Dasatinib (100 nM) for 24 h (n = 3). (c,d) The levels of PD-L1, ICAM1, SIRPα, CD11a, CD11b, CD29 (c), CXCR2, and CD62L (d) on WT neutrophils treated with PBS or LPS (100 ng/ml) in the presence of Dasatinib (100 nM) for 24 h (n = 3). (e) The swarming size of PBS- or LPS-stimulated (100 ng/ml; 24 h, with or without 100 nM Dasatinib) WT neutrophils co-incubated with polystyrene beads (diameter = 30 µm) for 5, 15, 30, and 45 min (n = 10; each data point is derived from separate fields under the light microscope with ×400 magnification). All n-numbers represent data derived from individual cell cultures with data plotted as mean ± SD unless otherwise stated. Neutrophil to bead ratio in co-incubation was 40:1. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05 using Student’s t-test (a–e). L, LPS; D, Dasatinib.

Fig 3: Expression of ANKLE1 leads to STAT1 activation, mitochondria degradation, DNA damage, and Epithelial to Mesenchymal transition.a Gene Set Enrichment Analysis (GSEA) of RNA-seq results for ANKLE1 overexpressing cells versus control cells identifies hallmarks of STAT1 activation, mitochondria degradation, DNA damage, and Epithelial to Mesenchymal transition. b Gene ontology analysis for differentially expressed genes (Fig. S5C) after 3-days post-transfection of ANKLE1 cells versus control cells show enrichment for STAT1 activation and mitochondria degradation. c ANKLE1 cuts nuclear DNA in non-apoptotic cells, as shown by DNA Pulse Field Gel Electrophoresis (PFGE) from non-apoptotic cells that overexpress ANKLE1 (24 h post-transfection). d ANKLE1 leads to STAT1 activation after 24 h of ANKLE1-transfection, as shown by western blots probed for P-STAT1, STAT1, P-STAT3,P-STAT5, P-IκB and α-Actin of three independent replicates per condition. The mean and standard deviations for the control and ANKLE1 are shown below the blot. The p-value for differential phosphorylation as measured by a two-tailed T-test is 0.008.

Fig 4: Ifngr1 and CD40 signaling non-redundantly drive anti-tumor TAM fate(A) NicheNet analysis from scRNA-seq data from Figure 4 using CD4 T cells as donor cells and MHCIIhi macrophages as acceptor cells. See also Figure S10.(B) Stat1 staining and MFI in MHCIIhi and MHCIIlo intratumoral macrophages at day 7. Data are mean ± SEM; n = 4 mice per group; ****p < 0.0001; Student’s t test.(C) Tumor weights from WT or Ifngr1−/− mice; n = 4–8 mice per group; ****p < 0.0001; Student’s t test for each time point.(D) Proportion of MHCIIhi or MHCIIlo macrophages from WT or Ifngr1−/− mice. Data are mean ± SEM; n = 4–8 mice per group; ****p < 0.0001; Student’s t test for each time point.(E) Proportion of Arg1+ macrophages on day 7. Data are mean ± SEM; ****p < 0.0001; Student’s t test.(F) Proportion of MHCIIloCD206+FRβ+ macrophages from WT or Ifngr1−/− mice. Data are mean ± SEM; n = 4–8 mice per group; ****p < 0.0001; Student’s t test for each time point.(G) PD-L1 MFI gated on total macrophages from WT or Ifngr1−/− mice. Data are mean ± SEM; **p < 0.005; Student’s t test for each time point.(H) Normalized expression of selected genes from bulk RNA sequencing from sorted live MHCIIhi or MHCIIlo tumor macrophages from WT and Ifngr1−/− mice at day 14. See also Figure S11.(I) Gene set enrichment analysis (GSEA) of the top 300 differentially expressed genes from sorted cells in (H).(J) Orthotopic KPC2a tumor weights at day 7 from WT control mice, WT mice treated with CD40L blockade (αCD40L), or Cd40−/− mice. *p < 0.05; one way ANOVA with Tukey’s post test. See also Figure S12.(K) Proportion of MHCIIhi or MHCIIlo macrophages. Each dot is an independent mouse. Data are mean ± SEM; n = 4–5 mice per group; ***p < 0.001; one-way ANOVA with Tukey’s posttest.(L) Proportion of Arg1+ macrophages. Each dot is an independent mouse. Data are mean ± SEM; n = 4–5 mice per group; ***p < 0.001; one-way ANOVA with Tukey’s posttest.(M) Proportion of MHCIIloCD206+FRβ+ macrophages. Each dot is an independent mouse. Data are mean ± SEM; n = 4–5 mice per group; ***p < 0.001; one-way ANOVA with Tukey’s posttest. See also Figure S13.

Fig 5: PPP inhibition modulates glycogen metabolism and the immune response signaling axis UDPG-Stat1-Irg1-itaconate of macrophages(A) Total glycogen amount in J774A.1 macrophages under compound-mediated PPP inhibition and shRNA-mediated knockdown of 6Pgd and Tkt.(B) Western blot analysis of protein expression of hypothesized connecting pathway in J774A.1 macrophages under shRNA-mediated knockdown of 6Pgd and Tkt compared to empty vector control. Mean expression displayed in bar graph analysis and one representative western blot example.(C) Scheme of working hypothesis of PPP metabolism modulating immune response.(D) Amount of 6pgd product ribulose-5-phosphate and Tkt product sedoheptulose-7-phosphate under shRNA-mediated inhibition of 6pgd and Tkt in J774A1 macrophages.(E) Metabolomic analysis of tricarboxylic acid cycle and citrate metabolism with display of enzyme expression of key enzymes under shRNA-mediated PPP knockdown of 6Pgd and Tkt compared to empty vector control J774A.1 macrophages. Amount of metabolites displayed in box and whiskers. Change in enzyme expression displayed in bar graphs. Genes: succinate dehydrogenase (Sdh), ATP citrate lyase (Acly), immunoregulatory gene 1 (Irg1 = Acod1).(F and G) Cytokine expression under 6Pgd inhibition by 6-aminonicotinamide or physcion and Tkt inhibition by oxythiamine in J774A.1 macrophages. (F) IL-6 expression compared to untreated control. (G) IL-10 expression compared to untreated control.(H) ADCP assay of bone marrow-derived macrophages of Irg1+/+ wild-type mice and Irg1−/− knockout mice. Macrophages differentiated out of femoral bone marrow with M-CSF. In (A), (B), and (F–H), bar plots are shown as mean ± SEM; in (D and E) metabolite amount is shown as minimum to maximum and protein expression is shown as calculated −Log2 fold change of control and knockdown macrophages. Technical replicates (A) n = 15, (B) n = 3–6, (D) n = 3, (E) n = 3, (F and G) n = 9–18, (H) n = 35; biological replicates (A) n = 3, (B) n = 3–6, (D) n = 3, (E) n = 3, (F and G) n = 3–6, (H) n = 7. p values were calculated in (A, B, D, and E) using one-way ANOVA, protein expression in (E) using student’s t test, and in (F–H) using unpaired t test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. See also Figures S4 and S5 and Tables S6 and S7.