Fig 2: Exhausted monocytes express elevated levels of CD38 and exhibit compromised mitochondria respiration. BMDMs were stimulated with PBS or high dose LPS (100 ng/mL) for 5 days. (A) CD38 MFIs on Day 1,3 and 5 were analyzed with flow cytometry. The relative fold changes in MFIs of CD38 comparing LPS-treated vs PBS-treated monocytes were determined and plotted in the graphs. n=3. (B) NAD+ levels in monocytes treated with either PBS or high dose LPS were measured on Day 5 using a fluorescence-based assay. n=4. (C) OCR/ug was determined by Seahorse assay. n=3. (D, E) The basal and maximum OCR/ug levels were quantified. n≥9. (F) Intracellular ROS levels (n=3) were determined using flow cytometry. The data are representative of at least three independent experiments, and error bars represent means ± SEM. ****p < 0.0001, Student’s t test.

Fig 3: Treatment with hSFRP2 mAb in mice with metastatic osteosarcoma decreases CD38 protein levels in splenocytes. Splenocytes isolated from mice with RF577 OS lung metastases treated with IgG1 control (n = 5) versus hSFRP2 mAb (n = 5) were lysed and prepared for Western blot analysis probing for CD38. Int. ratio: intensity ratios listed below CD38 Western blot were calculated after normalization to both actin and the reference sample (indicated by 1.00). Overall, CD38 protein levels were reduced in splenocytes from mice treated with hSFRP2 mAb by 82%, compared to the control group (p = 0.004).

Fig 4: TRAM is required for the generation exhausted monocytes. BMDMs from WT and TRAM KO mice were treated with PBS or high dose LPS (100 ng/mL) for 5 days. (A) Ly6C+ CD11b+, Ly6C++ CD11b+ and Ly6C- CD11b+ populations in the cell cultures were analyzed with flow cytometry. (B–D) The frequencies of indicated populations were quantified, n=3. (E–H) The MFIs of CD38 (E), PD-L1 (F), CD86 (G) and MHC-II (H) in WT and TRAM KO monocytes were determined with flow cytometry. (I) Intracellular ROS levels in WT and TRAM KO monocytes were determined with flow cytometry, n=3. (J, K) Mitochondrial OCR/ug levels in WT (J) and TRAM KO (K) monocytes were detected with Seahorse assay, n=5. (L–O) The basal and maximum OCR/ug levels in WT and TRAM KO were compared (n≥14). The data are representative of at least three independent experiments, and error bars represent means ± SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: non-significant, (B–I) one-way ANOVA and (L–O) Student’s t test.

Fig 5: Exhausted monocytes exhibit sustained activation of STAT1. (A–E) BMDMs were stimulated with PBS or high dose LPS (100 ng/mL) for 5 days. The levels of phosphorylated STAT1 and total STAT1 were determined by Western blot, n=3. (A). The relative levels of pSTAT1 (B) and STAT1 (C) were normalized to β-actin. The level of KLF-4 was determined by Western blot, n=3 (D), and the relative level was normalized to β-actin (E). (F–J) BMDMs were pre-treated with STAT1 inhibitor (Fludarabine 10 uM) on Day 0 for 2 hours, followed by stimulation with PBS or high dose LPS (100 ng/mL). The levels of pSTAT1 and STAT1 were determined using Western blot, n=3 (F) and flow cytometry, n=3 (G). The expressions of CD38 (H), PD-L1 (I) and CD86 (J) were analyzed with flow cytometry, n=3. The data are representative of at least three independent experiments, and error bars represent means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, (B, C, E) Student’s t test and (G–J) one-way ANOVA.