Abstract C023: Tertiary lymphoid structures generate anti-tumor immunity independently of immune responses in secondary lymphoid organs upon STING and lymphotoxin-β receptor activation

Abstract Tertiary lymphoid structures (TLS) are ectopic lymphoid aggregates that recapitulate key organizational and functional features of secondary lymphoid organs (SLO) and are increasingly recognized as critical hubs for orchestrating local anti-tumor immunity. Composed of germinal centers (GC), T-cell zones, follicular dendritic cell (FDC) networks, and high endothelial venules (HEV), TLS form structured intratumoral immune environments. Clinically, TLS presence correlates with improved survival and enhanced responsiveness to immune checkpoint blockade across a wide range of solid tumors. We previously demonstrated that combination therapy with a stimulator of interferon genes (STING) agonist and a lymphotoxin-β receptor (LTβR) agonist induces mature, HEV-positive intratumoral TLS with robust GC activity in the KPC pancreatic cancer model. Most existing studies of TLS have used human FFPE samples, in which TLS function cannot be evaluated independently from the influence of draining lymph nodes, the spleen, and other SLO. Consequently, the exact contribution of TLS to tumor immunity remains unresolved. To directly interrogate the intrinsic anti-tumor capacity of TLS, we generated SLO-null mice by combining lymphotoxin-α deficiency, which is essential for peripheral lymph node development, with splenectomy at 4-5 weeks of age. Using this unique system, we examined whether TLS can be induced therapeutically by the STING/LTβR agonist combination in the complete absence of SLO, whether these TLS exhibit fully functional GC reactions, and whether they are sufficient to mediate durable tumor control, prevent recurrence, and support long-term survival. SLO-null mice bearing syngeneic KPC tumors received a single intratumoral STING agonist injection followed by intraperitoneal LTβR agonist administration twice weekly for four doses. Fourteen days after treatment, tumors displayed mature TLS containing fully developed HEV, BCL6+ GC B cells and BCL6+ follicular helper T cells, CD23+/CD21+ FDC networks, and abundant plasma cells, demonstrating active intratumoral GC responses despite the complete absence of SLO. In tumor recurrence studies, tumors were resected on day 14, and mice were re-challenged 3 weeks later; TLS-inducing therapy in SLO-null mice prevented tumor regrowth and enabled long-term survival comparable to wild-type controls. These results show that functional TLS can develop and act as independent hubs of anti-tumor immunity without conventional immune responses of SLO, suggesting that TLS alone are capable of supporting sufficiently robust immune responses. Citation Format: Yasuhiro Kikuchi, Masanobu Komatsu. Tertiary lymphoid structures generate anti-tumor immunity independently of immune responses in secondary lymphoid organs upon STING and lymphotoxin-β receptor activation abstract. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr C023.