FIGURE 3 from PSGL-1 Blockade Induces Classical Activation of Human Tumor-associated Macrophages

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posted on 2023-10-26, 14:20 authored by Kevin Kauffman, Denise Manfra, Dominika Nowakowska, Mohammad Zafari, Phuong A. Nguyen, Ryan Phennicie, Elisabeth H. Vollmann, Brian O'Nuallain, Sara Basinski, Veronica Komoroski, Kate Rooney, Elizabeth K. Culyba, Joseph Wahle, Carola Ries, Michael Brehm, Steve Sazinsky, Igor Feldman, Tatiana I. Novobrantseva

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).

Funding

Verseau Therapeutics

History

ARTICLE ABSTRACT

The immune suppressive microenvironment is a major culprit for difficult-to-treat solid cancers. Particularly, inhibitory tumor-associated macrophages (TAM) define the resistant nature of the tumor milieu. To define tumor-enabling mechanisms of TAMs, we analyzed molecular clinical datasets correlating cell surface receptors with the TAM infiltrate. Though P-selectin glycoprotein ligand-1 (PSGL-1) is found on other immune cells and functions as an adhesion molecule, PSGL-1 is highly expressed on TAMs across multiple tumor types. siRNA-mediated knockdown and antibody-mediated inhibition revealed a role for PSGL-1 in maintaining an immune suppressed macrophage state. PSGL-1 knockdown or inhibition enhanced proinflammatory mediator release across assays and donors in vitro. In several syngeneic mouse models, PSGL-1 blockade alone and in combination with PD-1 blockade reduced tumor growth. Using a humanized tumor model, we observed the proinflammatory TAM switch following treatment with an anti-PSGL-1 antibody. In ex vivo patient-derived tumor cultures, a PSGL-1 blocking antibody increased expression of macrophage-derived proinflammatory cytokines, as well as IFNγ, indicative of T-cell activation. Our data demonstrate that PSGL-1 blockade reprograms TAMs, offering a new therapeutic avenue to patients not responding to T-cell immunotherapies, as well as patients with tumors devoid of T cells. This work is a significant and actionable advance, as it offers a novel approach to treating patients with cancer who do not respond to T-cell checkpoint inhibitors, as well as to patients with tumors lacking T-cell infiltration. We expect that this mechanism will be applicable in multiple indications characterized by infiltration of TAMs.