Programmed Cell Death 2 Forms Coinhibitory Microclusters That Directly Attenuate T Cell Receptor Signaling By Recruiting The Phosphatase Shp2

Background: Anti-PD-1 antibodies have made tremendous therapeutic effects on advanced or recurrent non-small cell lung cancer. However, the expression level of PD-L1 in tumor tissue which is the biomarker in the clinical setting is not necessarily correlated with their efficacy. In recent reports, some kinds of tumors express PD-L2 which is another ligand of PD-1.It is possible that the binding between PD-L2 and PD-1 is contributing to this mechanism. Because we intended to analyze the microstructural basis of immunological synapse, we used a system of a planar bilayer incorporated with glycophosphatidylinositol (GPI)-anchored intercellular adhesion molecule 1 (ICAM-1) and major histocompatibility complex (MHC) class II (I-Ek) loaded with a moth cytochrome c (MCC) peptide. Using our unique imaging analysis, we found the microcluster, where TCR proximal signaling molecules are recruited. In addition, PD-1 colocalizes with TCR microclusters in the presence of PD-L1 and SHP2 recruiting to PD-1 suppresses T cell activity by dephosphorylating the activated signaling molecules at TCR microclusters. Here, we investigated the PD-1-PD-L2 pathway using this dynamicimaging technique to reveal the molecular behavior of PD-1 bounded by PD-L2, not PD-L1. Methods: We established tumor cell line (BHK) highly expressing murine PD-L2 (mPD-L2)-GPI and purified mPD-L2-GPI by affinity column with anti-PD-L2. Primary CD4+ T cell isolated from AND-Tg mice in Rag2-/-Pdcd1-/-background and T cell hybridoma expressing AND-TCR (2D12) were infected with retroviral supernatants of PD-1-GFP or GFP-SHP2. We used planar bilayers of mPD-L2-GPI, mICAM-1 and I-Ekloaded with MCC peptideand observed PD-1-GFP-transfected CD4+ T cell and GFP-SHP2-transfected 2D12 by confocal microscopy. Each GPI protein was reconstituted into the planar bilayer as the same density of these ligands expressed by LPS-stimulated B cells. Results: We showed PD-1 is translocated to TCR microclusters and then accumulates at the central region of the immunological synapse in the presence of PD-L2. We also confirmed SHP2 is immediately but transiently recruited to PD-1-PD-L2 microclusters. Conclusions: Our results indicate that both PD-L1 and PD-L2 suppress TCR signaling by the recruitment of SHP2 at the same signaling clusters for the initiation of T cell activation. Further experiments are ongoing to elucidate the mechanism of the individual effect for cancer immunotherapy by PD-1-, PD-L1- and/or PD-L2 blockade and also of the in vivo effects of each blocking antibody. Citation Format: Tomohiro Takehara, Ei Wakamatsu, Hiroaki Machiyama, Hiroyuki Yasuda, Kenzo Soejima, Tadashi Yokosuka. Programmed cell death 2 forms coinhibitory microclusters that directly attenuate T cell receptor signaling by recruiting phosphatase SHP2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-057.