pH Low Insertion Peptide-Modified Programmed Cell Death-Ligand 1 Potently Suppresses T-Cell Activation Under Acidic Condition

Abstract

Programmed cell death-ligand 1 (PD-L1)/PD-1 axis is critical for maintenance of immune homeostasis by limiting overactivation of effector T-cell responses. The impairment of PD-L1/PD-1 signals play an important role in the pathogenesis of inflammatory diseases, making this pathway an ideal target for novel therapeutics to induce immune tolerance. Given weakly acidic environment as a putative hallmark of inflammation, in this study we designed a new cargo by linking the ectodomain of murine PD-L1 to the N terminus of pHLIPs, a low pH-responding and membrane-insertion peptide, and demonstrated its potent immune-suppressive activity. Specifically, PD-L1-pHLIP spanned the cellular membrane and perfectly recognized its ligand PD-1 in acidic buffer. Immobile PD-L1-pHLIP actively inhibited T-cell proliferation and IFN-γ production. Importantly, soluble PD-L1-pHLIP retained its function to dampen T-cell responses under acidic condition instead of neutral aqueous solution. Overall, these data suggest that PD-L1-pHLIP has potentials to be a novel therapeutic avenue for T-cell-mediated inflammatory diseases.

Acid-driven immune suppression by pHLIP-fused PD-L1 under inflammatory conditions

“…PD-L1-pHLIP inhibit T effector function in pH6.1 rather than pH6.8 buffer Firstly, we determine the inhibitory effects of PD-L1-pHLIP on T cell responses upon TCR stimulation under weakly acidic (pH6.8) and highly acidic (pH6.1) conditions respectively. In consistent with our previous study 10 , soluble PD-L1-pHLIP did not exhibit the suppressive function on lymphocyte proliferation in neutral aqueous solution, whereas actively inhibited lymphocyte expansion in pH6.1 buffer titrated by lactic acid (Fig. 1a).…”

“…Herein, we further determine cell distribution of PD-L1-pHLIP in pH6.8 and 6.1 buffer. In agreement with previous study 10 , no uorescence was visible when PElabeled PD-L1-pHLIP was incubated with anti-CD3/CD28-stimulated lymphocytes in neutral aqueous solutions (data not shown). Unexpectedly, in pH6.8 buffer, uorescence-labeled PD-L1-pHLIP was easily seen on the surface of several types of lymphocytes including NK, dendritic cell, monocyte, macrophage and B cells (Fig.…”

“…Our previous work has demonstrated that PD-L1 ectodomain tethering pHLIP potently dampened immune response to TCR stimulation under acidic conditions in vitro 10 . In this study, we validated its therapeutic effects in vivo using a model of acute intestinal in ammation.…”

“…Our previous study has shown that PD-L1-pHLIP had ability to span across the cellular membrane by conformation changes of pHLIP in response to low pH and retained on the surface of T and B cell lines and primary mouse and human lymphocytes 10 . Herein, we further determine cell distribution of PD-L1-pHLIP in pH6.8 and 6.1 buffer.…”

“…If the environment is acidic, pHLIPs fold and insert across the membrane to form a stable transmembrane helix, thus preferentially locating itself in acidic diseased tissues 8,9 . Recently, we have developed a new cargo by engaging the ectodomain of murine PD-L1 to the N terminus of pHLIPs, and demonstrated its potent immune-suppressive activity under acidic conditions in vitro 10 . Given that acidosis is the hallmark of microenvironment of acute in ammation attributed to augmented glycolysis and lactate e ux in in ltrating immune cells [11][12][13] , we assume that PD-L1-pHLIP may respond to the drop of pH and accumulate in in amed lesions, and thereby deliver negative signals to PD-1-expressing immune cells, ultimately dampen in ammatory responses.…”