Journal: The Journal of Immunology Author Choice

Article Title: LIGHT (TNFSF14) Costimulation Enhances Myeloid Cell Activation and Antitumor Immunity in the Setting of PD-1/PD-L1 and TIGIT Checkpoint Blockade

doi: 10.4049/jimmunol.2101175

Figure Lengend Snippet: Hexameric TIGIT-Fc-LIGHT engages targets with high affinity and activates effector lymphocytes. (A) TIGIT-Fc-LIGHT was synthesized from a single expression vector in mammalian production cells. Shown is a depiction of the hexamer based on Protein Data Bank structures, with dimerization of the central IgG4 Fc domain and trimerization of the TNF-ligand domain. (B) SDS-PAGE Western blot was used to probe all three domains of TIGIT-Fc-LIGHT under nonreducing, 2-ME reduced, and reduced/deglycosylated (PNGaseF) conditions. (C) Individual MSD binding assays were developed to assess binding to each intended recombinant protein target. (D) Cell-based binding assays were developed to assess cell surface receptor binding (using CHOK1 cells engineered to express hPVR and hHVEM, and also A375 cells that express human LTβR). (E) NF-κB/NIK reporter cells were incubated with a recombinant Fc-LIGHT control or TIGIT-Fc-LIGHT (18 nM each), and signaling activity was assessed through luciferase detection. ***p < 0.001, ****p < 0.0001. (F) TIGIT-Fc-LIGHT (or the murine surrogate, both at 10 nM) activation of T cells through IL-2 induction was assessed in human PBMCs or mouse splenocytes cocultured with the superantigen SEB for 3 d. (G) Schematic of a two-cell reporter system in which PVR-expressing CHO-K1 cells are cocultured with Jurkat effector cells expressing TIGIT and DNAM-1. (H) Jurkat effector cells were phenotyped using flow cytometry for the expression of TIGIT, DNAM-1, and HVEM. (I) Jurkat effector and CHO/hPVR reporter cells were cocultured with (all at 150 nM) an IgG4 control, a DNAM-1–blocking Ab, TIGIT-Fc-LIGHT, or TIGIT-Fc-LIGHT preincubated with a LIGHT-blocking Ab for 6 h, and then luciferase signaling activity was assessed using a luminometer.

Article Snippet: AR CPI-acquired resistance BTLA B and T lymphocyte attenuator CPI checkpoint inhibition DcR3 decoy receptor 3 DEG differentially expressed gene h human HVEM herpes virus entry mediator A LTβR lymphotoxin β receptor m mouse MSD Meso Scale Discovery NSCLC non–small cell lung cancer PCA principal component analysis PD-1/L1 PD-1/PD-L1 scRNA-seq single-cell RNA sequencing SEB staphylococcal enterotoxin B SEC size-exclusion chromatography TCGA The Cancer Genome Atlas TIGIT T cell immunoreceptor with Ig and ITIM domains TIL tumor-infiltrating lymphocyte Tn naive T Treg regulatory T cell Tscm T memory stem cell UMAP uniform manifold approximation and projection WT wild-type .

Techniques: Synthesized, Expressing, Plasmid Preparation, SDS Page, Western Blot, Binding Assay, Recombinant, Cell Surface Receptor Assay, Incubation, Control, Activity Assay, Luciferase, Activation Assay, Flow Cytometry, Blocking Assay