Fundamental Characterization of Antibody Fusion-Single-Chain TNF Recombinant Proteins Directed against Costimulatory TNF Receptors Expressed by T-Lymphocytes.

The costimulatory signal regulated by the members of the tumor necrosis factor receptor (TNFR) superfamily expressed by T cells plays essential roles for T cell responses and has emerged as a promising target for cancer immunotherapy. However, it is unclear how the difference in TNFR costimulation contributes to T cell responses. In this study, to clarify the functional significance of four different TNFRs, OX40, 4-1BB, CD27 and GITR, we prepared corresponding single-chain TNF ligand proteins (scTNFLs) connected to IgG Fc domain with beneficial characteristics, i.e., Fc-scOX40L, Fc-sc4-1BBL, Fc-scCD27L (CD70) and Fc-scGITRL. Without intentional cross-linking, these soluble Fc-scTNFL proteins bound to corresponding TNFRs induced NF-kB signaling and promoted proliferative and cytokine responses in CD4+ and CD8+ T cells with different dose-dependencies in vitro. Mice injected with one of the Fc-scTNFL proteins displayed significantly augmented delayed-type hypersensitivity responses, showing in vivo activity. The results demonstrate that each individual Fc-scTNFL protein provides a critical costimulatory signal and exhibits quantitatively distinct activity toward T cells. Our findings provide important insights into the TNFR costimulation that would be valuable for investigators conducting basic research in cancer immunology and also have implications for T cell-mediated immune regulation by designer TNFL proteins.

OX40 Ligand-Mannose-Binding Lectin Fusion Protein Induces Potent OX40 Cosignaling in CD4+ T Cells.

OX40, a member of the tumor necrosis factor (TNF) receptor superfamily, is induced on activated T cells. Membrane-bound OX40 ligand (OX40L) expressed by activated antigen-presenting cells induces OX40 signaling, which promotes T cell immunity. OX40 agonism would be a potential target for immunotherapy, however, it remains unclear how the activity of OX40 can be successfully controlled by a designer OX40L protein. We prepared a soluble OX40L protein possessing a PA-peptide tag and a collagenous trimerization domain from mannose-binding lectin (MBL), and tested whether PA-MBL-OX40L fusion protein worked as an agonist for OX40. We found that the majority of recombinant PA-MBL-OX40L protein purified from culture supernatants displayed a trimer structure and bound to cell surface OX40 or OX40-Fc fusion protein in a dose-dependent manner. Upon stimulation of CD4+ T cells with TCR/CD3 without CD28, PA-MBL-OX40L displayed significantly increased proliferative and cytokine responses when compared with a benchmark agonistic monoclonal antibody for OX40. Both soluble and immobilized forms of PA-MBL-OX40L induced potent OX40 signaling in CD4+ T cells. Mice administered with PA-MBL-OX40L displayed significantly augmented T cell-mediated delayed-type hypersensitivity responses. Our results suggest that activity of OX40L could be engineered to elicit better T cell responses by rational design of its assembly and architecture.

TNF Receptor-Associated Factor 5 Limits IL-27 Receptor Signaling in CD4+ T Lymphocytes.

TNF receptor-associated factor 5 (TRAF5) restrains early signaling activity of the IL-6 receptor in naive CD4+ T cells by interacting with the shared gp130 chain, although TRAF5 was initially discovered as a cytoplasmic adaptor protein to activate signaling mediated by TNF receptor family molecules. This leads to the question of whether TRAF5 limits signaling via the receptor for IL-27, which is composed of gp130 and WSX-1. The aim of this study is to clarify the role of TRAF5 in IL-27 receptor signaling and to understand the differential role of TRAF5 on cytokine receptor signaling. We found that Traf5 -/- CD4+ T cells displayed significantly higher levels of phosphorylated STAT1 and STAT-regulated genes Socs3 and Tbx21, as early as 1 h after IL-27 exposure when compared with Traf5 +/+ CD4+ T cells. Upon IL-27 and TCR signals, the Traf5 deficiency significantly increased the induction of IL-10 and promoted the proliferation of CD4+ T cells. Traf5 -/- mice injected with IL-27 displayed significantly enhanced delayed-type hypersensitivity responses, demonstrating that TRAF5 works as a negative regulator for IL-27 receptor signaling. In contrast, IL-2 and proliferation mediated by glucocorticoid-induced TNF receptor-related protein (GITR) and TCR signals were significantly decreased in Traf5 -/- CD4+ T cells, confirming that TRAF5 works as a positive regulator for cosignaling via GITR. Collectively, our results demonstrate that TRAF5 reciprocally controls signals mediated by the IL-27 receptor and GITR in CD4+ T cells and suggest that the regulatory activity of TRAF5 in gp130 is distinct from that in TNF receptor family molecules in a T cell.