Hypoxia-induced NETs establish a self-reinforcing immunosuppressive circuit that can be targeted to enhance immunotherapy in DLBCL

The efficacy of immune checkpoint inhibitors (ICI) in diffuse large B-cell lymphoma (DLBCL) is often limited by the immunosuppressive tumor microenvironment (TME), which comprises diverse cellular and non-cellular components. Among these, neutrophil extracellular traps (NETs) have emerged as potential immunomodulators that may contribute to immune suppression; however, their functional significance and regulatory mechanisms in DLBCL remain elusive. NETs levels were evaluated in DLBCL patient specimens and their effects on clinical outcomes were analyzed. Using a syngeneic murine lymphoma model, we assessed the therapeutic effect of DNase I-mediated NET degradation, both alone and in combination with anti-PD-L1 antibody. Mechanistic investigations included in vivo macrophage depletion, transcriptomic profiling, and in vitro assays under normoxic and hypoxic conditions. NETs were significantly elevated in DLBCL tissues and were correlated with advanced disease stage, elevated PD-L1 expression, and poor survival. Deoxyribonuclease I (DNase I) treatment potently inhibited tumor growth and exhibited marked synergy with PD-L1 blockade. This combination therapy robustly reduced immunosuppressive macrophages and myeloid-derived suppressor cells while enhancing CD8 + T cell infiltration and function. Mechanistically, NETs promoted T cell exhaustion primarily through macrophage-dependent pathways and affected by the oxygen tension: under normoxia, NETs drove macrophages into a dysfunctional phenotype, whereas under hypoxia, they were associated with PI3K-Akt pathway to drive macrophages toward an immunosuppressive phenotype. Furthermore, hypoxia promoted NET formation via the HIF-1α/IL-8 axis, establishing a self-reinforcing immunosuppressive circuit. Our study identifies NETs as pivotal regulators of immunosuppression in DLBCL and demonstrates that targeting NETs with DNase I effectively remodels the TME and enhances ICI efficacy, providing a compelling rationale for clinical translation of this combinatorial approach.