Bending the Rules: Amplifying PD-L1 Immunoregulatory Function Through Flexible Polyethylene Glycol Synthetic Linkers

Immune checkpoint signaling, such as programmed cell death protein-1 (PD-1), is a key target for immunotherapy due to its role in dampening immune responses. PD-1 signaling in T cells is regulated by complex physicochemical and mechanical cues. However, how these mechanical forces are integrated with biochemical responses remains poorly understood. Our previous work demonstrated that the use of an immobilizing polyethylene glycol (PEG) linker on synthetic microgels for the presentation of a chimeric form of PD-L1, SA-PD-L1, lead to local regulatory responses capable of abrogating allograft rejection in a model of cell-based transplantation. We herein provide evidence that enhanced immune regulating function can be obtained when presentation of SA-PD-L1 is achieved through a longer more flexible PEG chain. Presentation of SA-PD-L1 through a linker of high molecular weight, and thus longer length (10 kDa, 60 nm in length), led to enhance conversion of naive T cells into T regulatory cells (Tregs) in vitro. In addition, using a subcutaneous implant model and protein tethered through three different linker sizes (6, 30, and 60 nm) to the surface of PEG hydrogels, we demonstrated that longer linkers promoted PD-1 immunomodulatory role in vivo through three main functions: (1) augmenting immune cell recruitment at the transplant site; (2) promoting the accumulation of naive Tregs expressing migratory markers; and (3) dampening CD8⁺ cytolytic molecule production while augmenting expression of exhaustion phenotypes locally. Notably, accumulation of Treg cells at the implant site persisted for over 30 days postimplantation, an effect not observed when protein was presented with the shorter version of the linkers (6 and 30 nm). Collectively, these studies reveal a facile approach by which PD-L1 function can be modulated through external tuning of synthetic presenting linkers. Impact statement Recently, there has been a growing interest in immune checkpoint molecules as potential targets for tolerance induction, including programmed cell death protein-1 (PD-1). However, how the mechanics of ligand binding to PD-1 receptor affect downstream activation signaling pathways remains unresolved. By taking advantage of the effect of polyethylene glycol chain length on molecule kinetics in an aqueous solution, we herein show that PD-L1 function can be amplified by adjusting the length of the grafting linker. Our results uncover a potential facile mechanism that can be exploited to advance the role of immune checkpoint ligands, in particular PD-L1, in tolerance induction for immunosuppression-free cell-based therapies.