FIGURE 3 from PSGL-1 Blockade Induces Classical Activation of Human Tumor-associated Macrophages
Anti-PSGL-1 repolarizes macrophages and leads to a proinflammatory response. A, Fold change of M2 markers CD163, CD206, and CD16 measured by flow cytometry on LPS stimulated M2c macrophages treated with anti-PSGL-1 relative to IgG4 isotype control (n = 4 donors). B, Fold change of secreted proteins TNFα, IL6, and GMCSF measured by Luminex in the supernatants from LPS stimulated M2c macrophages treated with anti-PSGL-1 relative to IgG4 isotype control (n = 4 donors). See Supplementary Fig. S5 for absolute levels in pg/mL for reference. For both A and B, antibodies were added at various concentrations to M2c on day 7 of the polarization process for 30 minutes followed by the addition of 100 ng/mL LPS for 24 hours. C, Fold change of secreted proinflammatory proteins IL2, TNFα, and IFNγ measured in the supernatants from a multicellular PBMC-SEB assay treated with anti-PSGL-1 relative to the IgG4 isotype control treatment. PBMCs were sequentially treated with anti-PSGL-1 at 10 ug/mL for 30 minutes followed by SEB for 3 days. On day 3, the supernatants were collected, and secreted proteins were measured in triplicate by a Luminex custom 10-plex. (n = 6 donors). D, Fold change of secreted cytokines from an MLR assay. Monocytes were treated with anti-PSGL-1 throughout the 9-day differentiation and polarization of M0 macrophages and then coincubated with allogeneic T cells for 4 days. On day 13, Luminex was used to measure secreted mediators in the supernatants (D) and flow cytometry was used to measure T cells proliferation and activation (E). Significance was measured using mixed-effect models by concentration and treatment for each marker using Graph Prism software in A and B. Significance was measured using Kolmogorov–Smirnov test for each analyte using Graph Prism software in C, D, and E (given the large variance of fold changes across samples for the same analyte, the nonparametric test was more sensitive than the t test for most comparisons; however, the t test produced statistically significant results for all the comparisons as well).