Abstract 2144: Programmed cell death 2 forms coinhibitory microclusters that directly attenuate T cell receptor signaling by recruiting the phosphatase SHP2
Authors: Takehara Tomohiro , Wakamatsu Ei , Machiyama Hiroaki , Soejima Kenzo , Fukunaga Koichi , Yokosuka Tadashi 
Published: September 2020

Abstract
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 an imaging system combined with a planar lipid-bilayer incorporating GPI-anchored major histocompatibility complexes (MHCs) and the other ligands all those are required for T cell activation. We then found the "Microcluster", which is a clustering of receptors and their downstream signaling molecules, functioning as a signalosome for T cell antigen recognition and activation. Here, we imaged the behavior of the PD-1-PD-L2 signalosome recruiting a phosphatase and illustrated the machineries how PD-L2 suppress T cell activation in competition with PD-L1 toward PD-1 binding.
Methods: We purified GPI-anchored murine MHC, adhesion molecule and PD-L2 (mPD-L2) and reconstituted them into lipid-bilayer on a coverslip. Densities of these ligands were finely adjusted as same as those on typical antigen presenting cells (APCs) or tumor cells. Primary CD4+ T cell isolated from moss cytochrome C-specific TCR-Tg mice in Rag2−/- Pdcd1−/− background or T cell hybridoma expressing the same TCR (2D12) were retrovirally transduced with GFP- or HaloTag-tagged proteins (e.g. PD-1 and phosphatases SHP1/2) and imaged these proteins on the lipid-bilayers by confocal microscopy.
Results: We showed PD-1 is translocated to TCRs together forming microclusters and then accumulates at the central region of the immunological synapse in the presence of PD-L2. We also confirmed the rapid (20 seconds after TCR stimulation) and transient recruitment of SHP2, not SHP1, to PD-1 microclusters. PD-1-PD-L2 binding ware biochemically and biologically demonstrated to dephosphorylate the TCR downstream signaling molecules and introduce the reduction of IL-2 production, respectively, correlating with PD-1-PD-L2 microcluster formation. In the presence of both PD-L1 and PD-L2 on a lipid-bilayer, PD-L2 accumulated toward PD-1 microclusters much more stronger than PD-L1 did, suggesting that PD-L2 possesses higher affinity against PD-1 than PD-L1 if they were compared as an each single molecule. We further confirmed more effective suppression of IL-2 production by PD-1-PD-L2 binding.
Conclusions: Our results indicate that the function of PD-L1 and PD-L2 is almost the same in the point of suppressing TCR signaling by the recruitment of SHP2 to PD-1 signalosomes during the initiation of T cell activation. Contrary, PD-L2 more potently occupies PD-1 microclusters than PD-L1. We are now evaluating the effects of anti-PD-1/L1/L2 antibody for TCR signaling.
Citation Format: Tomohiro Takehara, Ei Wakamatsu, Hiroaki Machiyama, Kenzo Soejima, Koichi Fukunaga, Tadashi Yokosuka. Programmed cell death 2 forms coinhibitory microclusters that directly attenuate T cell receptor signaling by recruiting the phosphatase SHP2 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2144.