PO-419 Poly(I:C) prepares glioblastoma cells for anti-PD-L1 therapy via lymphocyte attraction and activation in a TLR3-dependent manner

Abstract

IntroductionGlioblastoma (GBM) still remains one of the deadliest cancer types, emphasising the urgent need for novel treatments. While immunotherapy is generating promising results, combination strategies are warranted to unlock its full potential. Here, we investigated how poly(I:C) affects immunomodulation by GBM cells as well as potential to potentiate immune checkpoint blocking therapy, in particular programmed death ligand (PD-L) blockade.Material and methodsPrimary human GBM cells were cultured from residual tumour tissue obtained from standard surgery of GBM patients. Their phenotype following poly(I:C) treatment was investigated by flow cytometry, immunohistochemistry and multiplex electrochemiluminescence. Mechanistic studies were performed using qRT-PCR, blocking antibodies and chloroquine (inhibitor of Toll-like receptor 3 or TLR3 signalling). Cocultures with peripheral blood mononuclear cells were studied for lymphocyte migration using a transwell assay, and immune activation using flow cytometry and ELISA. Additional PD-L1 or PD-L2 blockade was evaluated using ELISA and CFSE-labelled CD8+ T-cell proliferation.Results and discussionsPoly(IC) stimulated a pro-inflammatory secretome by GBM cells, including type I interferons (IFN), interleukin-15, reduced transforming growth factor-beta, and chemokines CXCL9, CXCL10, CCL4 and CCL5. These treated GBM cells doubled the attraction of CD8+ T cells, and to a lesser extent CD4+ T cells, partly via the CXCR3 and CCR5 ligands, but NK cell migration was not significantly affected. Furthermore, lymphocytes cocultured with poly(I:C)-treated GBM cells showed increased activation (CD69, IFN-gamma) and cytotoxic capacity (CD107a, granzyme B). Poly(I:C) treatment of GBM cells also increased their PD-L1 and PD-L2 expression. Here, poly(I:C) triggered TLR3, resulting in de novo protein, partly via downstream auto-/paracrine IFN-beta. In cocultures, additional blockade of PD-L1, but not PD-L2, strongly invigorated the immune activation. Our results suggest that poly(I:C) modulates GBM cells to prepare the tumour microenvironment for immune checkpoint blocking therapy. Chemokines attract CD8+ T cells to the tumour site, a prerequisite for effective PD-1/PD-L1 blockade, while other cytokines activate the immune cells. Concomitantly, PD-L1 is stimulated on the GBM cells, providing a target for additional PD-L1 blockade, which further propagates the immune activation.ConclusionIn conclusion, our data proposes poly(I:C) to unlock PD-L1 blocking therapy in GBM.