Antagonistic antibodies to programmed cell death protein 1 (PD1) and its ligand PD-L1 have revolutionized the treatment of multiple metastasized cancers including melanoma and non-small cell lung cancer. Despite these advances, a majority of patients do not achieve durable responses to these therapies. Further insights into the mode of action and potential biomarkers predicting clinical response are therefore warranted. Although the focus has been on biomarker identification in the tumor or peripheral blood, the role of PD-L1 in the tumor draining lymph node (TDLN) has not yet been investigated. As the TDLN is crucial for orchestrating antitumor immune responses, we assessed the role of PD-L1 in the TDLN on survival and anti-tumor immunity. To assess the extent of PD-L1 expression in different tissues during tumor growth and inflammation, we measured PD-L1 levels on multiple cell subsets in the tumor, TDLN and non-TDLN on baseline and following injection of activated dendritic cells (DCs) by multicolor flow cytometry. We exploited the intraperitoneal (i.p.) localization of mesothelioma tumors by injecting a range of anti-PD-L1 antibody concentrations intrapleurally (i.pl). This allowed us to investigate the role of PD-L1 in the TDLN while leaving tumoral PD-L1 intact. When sole targeting of PD-L1 expressed in the TDLN was achieved, we injected advanced tumor-bearing mice with low-dose anti-PD-L1 i.pl and compared the effects on survival and antitumor immune responses to systemic administration of the antibody alone or in combination with DC-induced immune activation. Besides the well-documented expression of PD-L1 by cells in the TME, we detected significant levels of PD-L1 in the TDLN, mainly on macrophages and dendritic cells. Furthermore, surface PD-L1 expression doubled on these cells following adoptive transfer of inflammatory bone-marrow derived DCs. Injecting a near hundredfold lower dose of 2.5µg of anti-PD-L1 antibody i.pl. blocked PD-L1 in the TDLN and prevented translocation of the antibody to other sites. In the advanced disease setting, anti-PD-L1 monotherapy or adoptive DC-transfer only marginally improved survival (median survival of 24 days in untreated mice compared to 25 days for both monotherapies). A single i.pl. injection of low-dose antibody prior to DC-administration was as effective in prolonging survival as compared to repeated high-dose systemic injection of the antibody combined with adoptive DC-transfer (median survival of 35 and 35.5 days, respectively). When investigating the effects on antitumor immune responses, we found the increase in T-cell proliferation to be dependent on systemic anti-PD-L1 administration, whereas activation of T-cells indicated by CD69-positivity, was largely dependent on TDLN-localized PD-L1. Until now, dissecting the spatial roles of PD-L1 in immune regulation has proven difficult. By using a model allowing for separate dosing of PD-L1 blocking antibodies to different anatomic compartments, we identify PD-L1 to be of major importance in the TDLN. Our findings aid in the understanding of how antitumor immunity is regulated and provide a new perspective on biomarker and therapeutic target identification.
Citation Format: Floris F. Dammeijer, Mandy van Gulijk, Melanie M. Lukkes, Menno van Nimwegen, Rudi W. Hendriks, Thorbald T. van Hall, Heleen H. Vroman, Joachim J.G.J.V. Aerts. Specifically targeting PD-L1 in the tumor-draining lymph node unmasks its spatiotemporal role in perturbing antitumor immunity and survival [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A164.