Engineering Tertiary Lymphoid Structures For Glioblastoma: A Novel Gene Combination Promotes Therapeutic Tls Formation In An Immune-Competent Mouse Model Of Gbm.

Abstract The absence of conventional lymphatic access to the brain parenchyma restricts the capacity of the peripheral immune system to recognize and elicit durable responses against glioma antigens. In some peripheral solid tumor types and autoimmune diseases such as multiple sclerosis, the spontaneous development of tertiary lymphoid structures (TLS) with varying degrees of organization have been observed in human patients and mice experiencing chronic inflammation. The presence of TLS in the context of cancer is generally associated with improved prognosis, particularly when these TLS are characterized by intratumoral infiltration of CD8+ T-cells and B-cells. We sought to engineer the development of TLS in the brain tumor setting, utilizing our SB28 glioblastoma (GBM) model which is sparsely infiltrated by lymphocytes and characterized by a significant myeloid-derived suppressor cell population. To determine the most critical chemokines and cytokines required to elicit TLS formation in the murine GBM context, we stably transduced SB28 with a combination of TLS-stimulating factors that we identified and injected these cells into the brain parenchyma of syngeneic C57BL/6J mice. A combination of the chemoattractant and lymphoid follicle-stimulating cytokines interleukin (IL)-7, LIGHT (TNFSF14), CCL21, and IL-17 promoted infiltration of CD8+CD3+ T-cells into the tumor and nearby parenchyma, but it also promoted enhanced tumor proliferation. A modified gene combination including IL-7, LIGHT, and CCL21 promoted CD8+ T-cell infiltration and clustering by immunofluorescence analysis. This combination also significantly decreased tumor burden as measured by bioluminescent imaging, resulting in complete tumor rejection in a subset of mice. Finally, modifying this combination to include CXCL13 promoted the infiltration and clustering of B-cells along with the observed CD8+ T-cell infiltration. This combination elicited the formation of vascular structures that stained for PNAd (MECA-79) and LYVE-1, markers for high endothelial venules (HEV) and lymphatic endothelium, respectively. Taken together, these analyses suggest that these factors are sufficient to stimulate the formation of TLS in the GBM setting. Future studies will evaluate a replication competent viral vector based approach for gene delivery and whether TLS formation can promote tumor specific T-cell memory and persistence in GBM. Ultimately, we aim to promote therapeutic TLS in a manner that is highly translational to brain tumor patients and complementary to existing T-cell therapies. Citation Format: Kira Morgan Downey, Bindu Hegde, Zinal Chheda, Jason Zhang, Hideho Okada. Engineering tertiary lymphoid structures for glioblastoma: A novel gene combination promotes therapeutic TLS formation in an immune-competent mouse model of GBM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 74.