Role of Interferon-Gamma (IFN-γ) Signaling in Immune Checkpoint Inhibitor-Associated Thrombosis: Tissue Factor Upregulation and Proinflammatory Cytokine Signature

Cancer immunotherapy with immune checkpoint inhibitor (ICI) is associated with venous thrombosis. In a CT26 colorectal murine tumor model, we observed that cancer cell-derived tissue factor (TF) contributes to development of ICI-associated thrombosis (IAT), as demonstrated by reduced IVC thrombus size in mice bearing CRISPR/Cas9-mediated TF-deleted cancer cells. ICI-treated mice also exhibited elevated levels of neutrophil extracellular traps compared to IgG-treated controls. The mechanisms underlying TF induction and neutrophil activation in IAT are still unclear.

To obtain mechanistic insights, we assessed the dynamics of cytokines in the tumor microenvironment (TME) and circulation, and the signaling pathways responsible for cytokine-induced TF upregulation in cancer cells and extracellular vesicles (EV). Cytokine expression in tumor and plasma from mice treated with IgG and ICI (anti-PD-1+anti-CTLA-4) was assessed using a cytokine array (proteome profiler) with densitometric analysis and also quantified with a Meso Scale Discovery (MSD) U-PLEX assay. Spearman's correlation analysis assessed cytokine correlations between tumor and plasma. T cell involvement was evaluated by depleting T cells with anti-CD4 and anti-CD8 antibodies before ICI treatment. IFN-γ effects on TF expression and TF⁺EV release by CT26 cancer cells were analyzed using pharmacological inhibition (JAK1/2 inhibitor) and genetic silencing (siRNA targeting IRF-1 and Rab27a).

Cytokine profiling in ICI-treated mice tumors showed significant increases in IFN-γ (2-fold) and its responsive chemokines, monokine induced by IFN-γ (MIG, 1.8-fold) and interferon-inducible T cell alpha chemoattractant (I-TAC, 8.8-fold), implying T cell recruitment. Elevated tumor necrosis factor (TNF-α, 6.8-fold) and B lymphocyte chemoattractant (BLC, 2.9-fold) were also observed, along with macrophage inflammatory protein-1beta (MIP-1β, 5.4-fold) and regulated on activation, normal T cell expressed and secreted (RANTES, 3.4-fold). Increased plasma keratinocyte-derived chemokine (KC, 4.7-fold), stromal cell-derived factor-1 (SDF-1, 2-fold), and monocyte chemotactic protein-5 (MCP-5, 3.4-fold) indicated myeloid cell activation. The U-PLEX multiplex assay confirmed significant increases in eight tumor cytokines from ICI-treated mice: IFN-γ (3.8-fold), TNF-α (1.6-fold), BLC (2.8-fold), MIP-1β (1.8-fold), RANTES (2.1-fold), SDF-1 (1.9-fold), IL-6 (1.7-fold), and GM-CSF (1.9-fold). The higher sensitivity of MSD platform detected increased circulating levels of IFN-γ (2.6-fold, 1.62 vs 0.63 pg/ml), TNF-α (1.6-fold, 11.98 vs 8.16 pg/ml), IL-6 (1.6-fold, 352.02 vs 218.88 pg/ml), MCP-5 (1.5-fold, 413.03 vs 276.45 pg/ml), and MIP-2 (1.7-fold, 134.18 vs 79.71 pg/ml), in addition to KC (1.7-fold, 177.14 vs 105.76 pg/ml) and MCP-1 (2.1-fold, 115.34 vs 55.79 pg/ml). Elevated KC and MIP-2 (murine IL-8 homologues) align with our previous finding that higher IL-8 pretreatment levels in ICI-treated cancer patients correlate with thrombosis, suggesting endothelial activation. Spearman's correlation analysis showed significant positive correlations in four cytokines between tumor and plasma of ICI-treated mice: IFN-γ (r=0.6520, p=0.0216), TNF-α (r=0.7170, p=0.0026), MIP-1β (r-0.6547, p=0.0081), and RANTES (r=0.7178, p=0.0008), indicating systemic reflection of local tumor inflammatory responses. IFN-γ correlated strongly with MCP-1 and MIP-1β in circulation (r>0.5, p>0.05). T cell depletion significantly reduced MIP-1β, RANTES, and IFN-γ levels (129 vs 60 pg/ml, p=0.0091). In vitro studies with CT26 cancer cells demonstrated that IFN-γ upregulates TF via the JAK1/2-STAT1 pathway and increases TF⁺EV release via IRF1-Rab27a axis, confirmed by siRNA targeting IRF1 and Rab27a.

IFN-γ released by ICI-activated T cells may upregulate TF expression and TF⁺EV secretion in cancer cells, and stimulate monocytes and macrophages in the TME to secrete TNF-α, MIP-1β, RANTES. Elevated circulating IFN-γ (and/or TNF-α) can activate endothelial cells to release of cytokines such as KC and MIP-2 (for neutrophils) and MCP-1 and MCP-5 (for monocytes). Thus, IFN-γ induces TF in the TME and modulates a prothrombotic state in circulation by mediating proinflammatory cytokine release. Understanding these cytokine networks and correlations will provide insight into mechanisms of IAT.