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.

Delayed graft rejection in autoimmune islet transplantation via biomaterial immunotherapy.

The induction of operational immune tolerance is a major goal in beta-cell replacement strategies for the treatment of type 1 diabetes. Our group previously reported long-term efficacy via biomaterial-mediated programmed death ligand 1 (PD-L1) immunotherapy in islet allografts in nonautoimmune models. In this study, we evaluated autoimmune recurrence and allograft rejection during islet transplantation in spontaneous nonobese diabetic (NOD) mice. Graft survival and metabolic function were significantly prolonged over 60 days in recipients of syngeneic islets receiving the biomaterial-delivered immunotherapy, but not in control animals. The biomaterial-mediated PD-L1 immunotherapy resulted in delayed allograft rejection in diabetic NOD mice compared with controls. Discrimination between responders and nonresponders was attributed to the enriched presence of CD206+ program death 1+ macrophages and exhausted signatures in the cytotoxic T cell compartment in the local graft microenvironment. Notably, draining lymph nodes had similar remodeling in innate and adaptive immune cell populations. This work establishes that our biomaterial platform for PD-L1 delivery can modulate immune responses to transplanted islets in diabetic NOD mice and, thus, can provide a platform for the development of immunologic strategies to curb the allo- and autoimmune processes in beta-cell transplant recipients.

Enteroendocrine Cells Protect the Stem Cell Niche by Regulating Crypt Metabolism in Response to Nutrients.

BACKGROUND & AIMS: The intestinal stem cell niche is exquisitely sensitive to changes in diet, with high-fat diet, caloric restriction, and fasting resulting in altered crypt metabolism and intestinal stem cell function. Unlike cells on the villus, cells in the crypt are not immediately exposed to the dynamically changing contents of the lumen. We hypothesized that enteroendocrine cells (EECs), which sense environmental cues and in response release hormones and metabolites, are essential for relaying the luminal and nutritional status of the animal to cells deep in the crypt. METHODS: We used the tamoxifen-inducible VillinCreERT2 mouse model to deplete EECs (Neurog3fl/fl) from adult intestinal epithelium and we generated human intestinal organoids from wild-type and NEUROGENIN 3 (NEUROG3)-null human pluripotent stem cells. We used indirect calorimetry, 1H-Nuclear Magnetic Resonance (NMR) metabolomics, mitochondrial live imaging, and the Seahorse bioanalyzer (Agilent Technologies) to assess metabolism. Intestinal stem cell activity was measured by proliferation and enteroid-forming capacity. Transcriptional changes were assessed using 10x Genomics single-cell sequencing. RESULTS: Loss of EECs resulted in increased energy expenditure in mice, an abundance of active mitochondria, and a shift of crypt metabolism to fatty acid oxidation. Crypts from mouse and human intestinal organoids lacking EECs displayed increased intestinal stem cell activity and failed to activate phosphorylation of downstream target S6 kinase ribosomal protein, a marker for activity of the master metabolic regulator mammalian target of rapamycin (mTOR). These phenotypes were similar to those observed when control mice were deprived of nutrients. CONCLUSIONS: EECs are essential regulators of crypt metabolism. Depletion of EECs recapitulated a fasting metabolic phenotype despite normal levels of ingested nutrients. These data suggest that EECs are required to relay nutritional information to the stem cell niche and are essential regulators of intestinal metabolism.