Abstract 6373: P-selectin glycoprotein ligand-1 modulates the functions of human T cells and macrophages in vitro

Abstract

P-selectin glycoprotein ligand-1 (PSGL-1) is a type I transmembrane protein expressed on the surface of most hematopoietic cells. PSGL-1 can engage multiple ligands (e.g., selectins, VISTA, Siglec-5, versican, CCL19, and CCL21). Apart from being a key adhesion molecule involved in immune cell trafficking, PSGL-1 has been shown to function as a negative immune checkpoint receptor in both T cells and macrophages. PSGL-1 is highly expressed in tumor-infiltrating T cells (TILs) and in tumor-associated macrophages (TAMs) in the tumor microenvironment (TME). PSGL-1 signaling in TILs induces development of T-cell exhaustion and, in TAMs, it promotes immunosuppressive activity of macrophages. In the TME, PSGL-1 is also highly expressed on the surface of myeloid-derived suppressor cells, regulatory T cells, and some types of cancer cells. We hypothesized that PSGL-1 may also act as a ligand to modulate the effector functions of human T cells and macrophages in the TME. In the present study, flow cytometry analysis showed that monocytes and granulocytes had a higher PSGL-1 expression than CD4+ and CD8+ T cells isolated from normal human blood cells. To mimic the cell-expressing PSGL-1, a recombinant human PSGL-1-Fc protein (rhPSGL-1-Fc, R&D Systems) was coated onto 96-well plates overnight. Human peripheral blood mononuclear cells (PBMCs), purified human CD4+ T cells, or CD8+ T cells were cultured in the PSGL-1-coated plates for 3 days in the presence of anti-CD3 antibody. In some experiments, a commercial tool PSGL-1 monoclonal antibody (mAb) with the ability to block PSGL-1/P-selectin interaction was added into the culture system. Cytokine secretion in the culture supernatants was measured using Meso Scale Discovery assays. Similar experiments were also conducted with commercially supplied human M1 and M2 macrophages. The results demonstrated that plate-bound rhPSGL-1-Fc dose-dependently inhibited IFN-γ, IL-2, and TNF-α cytokine production in human PBMCs, CD4+ T cells, and in CD8+ T cells following T-cell receptor stimulation. The tool PSGL-1 mAb was able to partially rescue the suppression of cytokine production. Plate-bound rhPSGL-1-Fc also inhibited TNF-α secretion from human M1 macrophage, and the PSGL-1 mAb significantly enhanced TNF-α production in both human M1 and M2 macrophages treated with plate-bound PSGL-1-Fc. Together, these data indicate that PSGL-1 expressed on immune cells in TME may act as both an inhibitory ligand and receptor to impact on T-cell and macrophage function. Acknowledgements: Thanks to Patrick Mayes and Ricardo Macarron for scientific input and thanks to Qian Wang Yao and Lynn Leffet for technical assistance. Trial Registration: N/A Ethics Approval: N/A Citation Format: Jun Guan, Sundee Dees, Tony Chadderton, Alejandro Amador Arjona. P-selectin glycoprotein ligand-1 modulates the functions of human T cells and macrophages in vitro. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6373.