Engineering synthetic poly(ethylene) glycol-based hydrogels compatible with injection molding biofabrication.

Hydrogel injection molding is a biofabrication method that is useful for the rapid generation of complex cell-laden hydrogel geometries, with potential utility in biomanufacturing products for tissue engineering applications. Hydrogel injection molding requires that hydrogel polymers have sufficiently delayed crosslinking times to enable injection and molding prior to gelation. In this work, we explore the feasibility of injection molding synthetic poly(ethylene) glycol (PEG)-based hydrogels functionalized with strain promoted azide-alkyne cycloaddition click chemistry functional groups. We evaluate the mechanical properties of a PEG-based hydrogel library, including time to gelation and successful generation of complex geometries via injection molding. We evaluate the binding and retention of adhesive ligand RGD within the library matrices and characterize the viability and function of encapsulated cells. This work demonstrates the feasibility of injection molding synthetic PEG-based hydrogels for tissue engineering applications, with potential utility in the clinic and biomanufacturing.

Differential expression of CCR8 in tumors versus normal tissue allows specific depletion of tumor-infiltrating T regulatory cells by GS-1811, a novel Fc-optimized anti-CCR8 antibody.

The presence of T regulatory (Treg) cells in the tumor microenvironment is associated with poor prognosis and resistance to therapies aimed at reactivating anti-tumor immune responses. Therefore, depletion of tumor-infiltrating Tregs is a potential approach to overcome resistance to immunotherapy. However, identifying Treg-specific targets to drive such selective depletion is challenging. CCR8 has recently emerged as one of these potential targets. Here, we describe GS-1811, a novel therapeutic monoclonal antibody that specifically binds to human CCR8 and is designed to selectively deplete tumor-infiltrating Tregs. We validate previous findings showing restricted expression of CCR8 on tumor Tregs, and precisely quantify CCR8 receptor densities on tumor and normal tissue T cell subsets, demonstrating a window for selective depletion of Tregs in the tumor. Importantly, we show that GS-1811 depleting activity is limited to cells expressing CCR8 at levels comparable to tumor-infiltrating Tregs. Targeting CCR8 in mouse tumor models results in robust anti-tumor efficacy, which is dependent on Treg depleting activity, and synergizes with PD-1 inhibition to promote anti-tumor responses in PD-1 resistant models. Our data support clinical development of GS-1811 to target CCR8 in cancer and drive tumor Treg depletion in order to promote anti-tumor immunity.

A Method for Organoid Transplantation and Whole-Mount Visualization of Post-Engraftment Vascularization.

As the field of organoid development matures, the need to transplant organoids to evaluate and characterize their functionality grows. Decades of research developing islet organoid transplantation for the treatment of type 1 diabetes can contribute substantially to accelerating diverse tissue organoid transplantation. Biomaterials-based organoid delivery methods offer the potential to maximize organoid survival and engraftment. In this protocol, we describe a vasculogenic degradable hydrogel vehicle and a method to deliver organoids to intraperitoneal tissue. Further, we describe a method to fluorescently label and image functional vasculature within the graft as a tool to investigate organoid engraftment.

Immunotherapy via PD-L1-presenting biomaterials leads to long-term islet graft survival.

Antibody-mediated immune checkpoint blockade is a transformative immunotherapy for cancer. These same mechanisms can be repurposed for the control of destructive alloreactive immune responses in the transplantation setting. Here, we implement a synthetic biomaterial platform for the local delivery of a chimeric streptavidin/programmed cell death-1 (SA-PD-L1) protein to direct "reprogramming" of local immune responses to transplanted pancreatic islets. Controlled presentation of SA-PD-L1 on the surface of poly(ethylene glycol) microgels improves local retention of the immunomodulatory agent over 3 weeks in vivo. Furthermore, local induction of allograft acceptance is achieved in a murine model of diabetes only when receiving the SA-PD-L1-presenting biomaterial in combination with a brief rapamycin treatment. Immune characterization revealed an increase in T regulatory and anergic cells after SA-PD-L1-microgel delivery, which was distinct from naïve and biomaterial alone microenvironments. Engineering the local microenvironment via biomaterial delivery of checkpoint proteins has the potential to advance cell-based therapies, avoiding the need for systemic chronic immunosuppression.

Targeted deletion of PD-1 in myeloid cells induces antitumor immunity.

PD-1, a T cell checkpoint receptor and target of cancer immunotherapy, is also expressed on myeloid cells. The role of myeloid-specific versus T cell-specific PD-1 ablation on antitumor immunity has remained unclear because most studies have used either PD-1-blocking antibodies or complete PD-1 KO mice. We generated a conditional allele, which allowed myeloid-specific (PD-1f/fLysMcre) or T cell-specific (PD-1f/fCD4cre) targeting of Pdcd1 gene. Compared with T cell-specific PD-1 ablation, myeloid cell-specific PD-1 ablation more effectively decreased tumor growth. We found that granulocyte/macrophage progenitors (GMPs), which accumulate during cancer-driven emergency myelopoiesis and give rise to myeloid-derived suppressor cells (MDSCs), express PD-1. In tumor-bearing PD-1f/fLysMcre but not PD-1f/fCD4cre mice, accumulation of GMP and MDSC was prevented, whereas systemic output of effector myeloid cells was increased. Myeloid cell-specific PD-1 ablation induced an increase of T effector memory cells with improved functionality and mediated antitumor protection despite preserved PD-1 expression in T cells. In PD-1-deficient myeloid progenitors, growth factors driving emergency myelopoiesis induced increased metabolic intermediates of glycolysis, pentose phosphate pathway, and TCA cycle but, most prominently, elevated cholesterol. Because cholesterol is required for differentiation of inflammatory macrophages and DC and promotes antigen-presenting function, our findings indicate that metabolic reprogramming of emergency myelopoiesis and differentiation of effector myeloid cells might be a key mechanism of antitumor immunity mediated by PD-1 blockade.

Phosphorylation of PD-1-Y248 is a marker of PD-1-mediated inhibitory function in human T cells.

PD-1 is a target of cancer immunotherapy but responses are limited to a fraction of patients. Identifying patients with T cells subjected to PD-1-mediated inhibition will allow selection of suitable candidates for PD-1-blocking therapy and will improve the therapeutic success. We sought to develop an approach to detect PD-1-mediated inhibitory signaling. The cytoplasmic tail of PD-1 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) encompassing Y223 and an immunoreceptor tyrosine-based switch motif (ITSM) encompassing Y248, which is indispensable for interaction of SHP-2 and delivery of PD-1 inhibitory function. We generated an antibody specific for phosphorylated PD-1-Y248 and examined PD-1pY248+ (pPD-1) expression in human T cells. pPD-1 was upregulated by TCR/CD3 + CD28 stimulation and simultaneous PD-1 ligation. pPD-1+CD8+ T cells were identified in human peripheral blood and had impaired effector function. pPD-1+ T cells were also detected in tumor-draining lymph nodes of tumor bearing mice and in biopsies of patients with glioblastoma multiform. Detection of pPD-1+ T cells might serve as a biomarker for identification of T cells subjected to PD-1-mediated immunosuppression.

Linkage Groups within Thiol-Ene Photoclickable PEG Hydrogels Control In Vivo Stability.

Thiol-norbornene (thiol-ene) photoclickable poly(ethylene glycol) (PEG) hydrogels are a versatile biomaterial for cell encapsulation, drug delivery, and regenerative medicine. Numerous in vitro studies with these 4-arm ester-linked PEG-norbornene (PEG-4eNB) hydrogels demonstrate robust cytocompatibility and ability to retain long-term integrity with nondegradable crosslinkers. However, when transplanted in vivo into the subcutaneous or intraperitoneal space, these PEG-4eNB hydrogels with nondegradable crosslinkers rapidly degrade within 24 h. This characteristic limits the usefulness of PEG-4eNB hydrogels in biomedical applications. Replacing the ester linkage with an amide linkage (PEG-4aNB) mitigates this rapid in vivo degradation, and the PEG-4aNB hydrogels maintain long-term in vivo stability for months. Furthermore, when compared to PEG-4eNB, the PEG-4aNB hydrogels demonstrate equivalent mechanical properties, crosslinking kinetics, and high cytocompatibility with rat islets and human mesenchymal stem cells. Thus, the PEG-4aNB hydrogels may be a suitable replacement platform without necessitating critical design changes or sacrificing key properties relevant to the well-established PEG-4eNB hydrogels.

Synthetic poly(ethylene glycol)-based microfluidic islet encapsulation reduces graft volume for delivery to highly vascularized and retrievable transplant site.

Transplant of hydrogel-encapsulated allogeneic islets has been explored to reduce or eliminate the need for chronic systemic immunosuppression by creating a physical barrier that prevents direct antigen presentation. Although successful in rodents, translation of alginate microencapsulation to large animals and humans has been hindered by large capsule sizes (≥500 µm diameter) that result in suboptimal nutrient diffusion in the intraperitoneal space. We developed a microfluidic encapsulation system that generates synthetic poly(ethylene glycol)-based microgels with smaller diameters (310 ± 14 µm) that improve encapsulated islet insulin responsiveness over alginate capsules and allow transplant within vascularized tissue spaces, thereby reducing islet mass requirements and graft volumes. By delivering poly(ethylene glycol)-encapsulated islets to an isolated, retrievable, and highly vascularized site via a vasculogenic delivery vehicle, we demonstrate that a single pancreatic donor syngeneic islet mass exhibits improved long-term function over conventional alginate capsules and close integration with transplant site vasculature. In vivo tracking of bioluminescent allogeneic encapsulated islets in an autoimmune type 1 diabetes murine model showed enhanced cell survival over unencapsulated islets in the absence of chronic systemic immunosuppression. This method demonstrates a translatable alternative to intraperitoneal encapsulated islet transplant.

Local immunomodulation Fas ligand-engineered biomaterials achieves allogeneic islet graft acceptance.

Islet transplantation is a promising therapy for type 1 diabetes. However, chronic immunosuppression to control rejection of allogeneic islets induces morbidities and impairs islet function. T effector cells are responsible for islet allograft rejection and express Fas death receptors following activation, becoming sensitive to Fas-mediated apoptosis. Here, we report that localized immunomodulation using microgels presenting an apoptotic form of the Fas ligand with streptavidin (SA-FasL) results in prolonged survival of allogeneic islet grafts in diabetic mice. A short course of rapamycin treatment boosted the immunomodulatory efficacy of SA-FasL microgels, resulting in acceptance and function of allografts over 200 days. Survivors generated normal systemic responses to donor antigens, implying immune privilege of the graft, and had increased CD4+CD25+FoxP3+ T regulatory cells in the graft and draining lymph nodes. Deletion of T regulatory cells resulted in acute rejection of established islet allografts. This localized immunomodulatory biomaterial-enabled approach may provide an alternative to chronic immunosuppression for clinical islet transplantation.

Design of a vascularized synthetic poly(ethylene glycol) macroencapsulation device for islet transplantation.

The use of immunoisolating macrodevices in islet transplantation confers the benefit of safety and translatability by containing transplanted cells within a single retrievable device. To date, there has been limited development and characterization of synthetic poly(ethylene glycol) (PEG)-based hydrogel macrodevices for islet encapsulation and transplantation. Herein, we describe a two-component synthetic PEG hydrogel macrodevice system, designed for islet delivery to an extrahepatic islet transplant site, consisting of a hydrogel core cross-linked with a non-degradable PEG dithiol and a vasculogenic outer layer cross-linked with a proteolytically sensitive peptide to promote degradation and enhance localized vascularization. Synthetic PEG macrodevices exhibited equivalent passive molecular transport to traditional microencapsulation materials (e.g., alginate) and long-term stability in the presence of proteases in vitro and in vivo, out to 14 weeks in rats. Encapsulated islets demonstrated high viability within the device in vitro and the incorporation of RGD adhesive peptides within the islet encapsulating PEG hydrogel improved insulin responsiveness to a glucose challenge. In vivo, the implementation of a vasculogenic, degradable hydrogel layer at the outer interface of the macrodevice enhanced vascular density within the rat omentum transplant site, resulting in improved encapsulated islet viability in a syngeneic diabetic rat model. These results highlight the benefits of the facile PEG platform to provide controlled presentation of islet-supportive ligands, as well as degradable interfaces for the promotion of engraftment and overall graft efficacy.

PEG hydrogel containing calcium-releasing particles and mesenchymal stromal cells promote vessel maturation.

The use of human mesenchymal stromal cells (hMSC) for treating diseased tissues with poor vascularization has received significant attention, but low cell survival has hampered its translation to the clinic. Bioglasses and glass-ceramics have also been suggested as therapeutic agents for stimulating angiogenesis in soft tissues, but these effects need further evaluation in vivo. In this study, calcium-releasing particles and hMSC were combined within a hydrogel to examine their vasculogenic potential in vitro and in vivo. The particles provided sustained calcium release and showed proangiogenic stimulation in a chorioallantoic membrane (CAM) assay. The number of hMSC encapsulated in a degradable RGD-functionalized PEG hydrogel containing particles remained constant over time and IGF-1 release was increased. When implanted in the epidydimal fat pad of immunocompromised mice, this composite material improved cell survival and stimulated vessel formation and maturation. Thus, the combination of hMSC and calcium-releasing glass-ceramics represents a new strategy to achieve vessel stabilization, a key factor in the revascularization of ischemic tissues. STATEMENT OF SIGNIFICANCE: Increasing blood vessel formation in diseased tissues with poor vascularization is a current clinical challenge. Cell therapy using human mesenchymal stem cells has received considerable interest, but low cell survival has hampered its translation to the clinic. Bioglasses and glass-ceramics have been explored as therapeutic agents for stimulating angiogenesis in soft tissues, but these effects need further evaluation in vivo. By incorporating both human mesenchymal stem cells and glass-ceramic particles in an implantable hydrogel, this study provides insights into the vasculogenic potential in soft tissues of the combined strategies. Enhancement of vessel formation and maturation supports further investigation of this strategy.

The adaptor molecule RIAM integrates signaling events critical for integrin-mediated control of immune function and cancer progression.

Lymphocyte activation requires adhesion to antigen-presenting cells. This is a critical event linking innate and adaptive immunity. Lymphocyte adhesion is accomplished through LFA-1, which must be activated by a process referred to as inside-out integrin signaling. Among the few signaling molecules that have been implicated in inside-out integrin activation in hematopoietic cells are the small guanosine triphosphatase (GTPase) Rap1 and its downstream effector Rap1-interacting molecule (RIAM), a multidomain protein that defined the Mig10-RIAM-lamellipodin (MRL) class of adaptor molecules. Through its various domains, RIAM is a critical node of signal integration for activation of T cells, recruits monomeric and polymerized actin to drive actin remodeling and cytoskeletal reorganization, and promotes inside-out integrin signaling in T cells. As a regulator of inside-out integrin activation, RIAM affects multiple functions of innate and adaptive immunity. The effects of RIAM on cytoskeletal reorganization and integrin activation have implications in cell migration and trafficking of cancer cells. We provide an overview of the structure and interactions of RIAM, and we discuss the implications of RIAM functions in innate and adaptive immunity and cancer.

Vasculogenic hydrogel enhances islet survival, engraftment, and function in leading extrahepatic sites.

Islet transplantation is a promising alternative therapy for insulin-dependent patients, with the potential to eliminate life-threatening hypoglycemic episodes and secondary complications of long-term diabetes. However, widespread application of this therapy has been limited by inadequate graft function and longevity, in part due to the loss of up to 60% of the graft in the hostile intrahepatic transplant site. We report a proteolytically degradable synthetic hydrogel, functionalized with vasculogenic factors for localized delivery, engineered to deliver islet grafts to extrahepatic transplant sites via in situ gelation under physiological conditions. Hydrogels induced differences in vascularization and innate immune responses among subcutaneous, small bowel mesentery, and epididymal fat pad transplant sites with improved vascularization and reduced inflammation at the epididymal fat pad site. This biomaterial-based strategy improved the survival, engraftment, and function of a single pancreatic donor islet mass graft compared to the current clinical intraportal delivery technique. This biomaterial strategy has the potential to improve clinical outcomes in islet autotransplantation after pancreatectomy and reduce the burden on donor organ availability by maximizing graft survival in clinical islet transplantation for type 1 diabetes patients.

Immunometabolic Regulations Mediated by Coinhibitory Receptors and Their Impact on T Cell Immune Responses.

Host immunity provides wide spectrum protection that serves to eradicate pathogens and cancer cells, while maintaining self-tolerance and immunological homeostasis. Ligation of the T cell receptor (TCR) by antigen activates signaling pathways that coordinately induce aerobic glycolysis, mitochondrial activity, anabolic metabolism, and T effector cell differentiation. Activation of PI3K, Akt, and mTOR triggers the switch to anabolic metabolism by inducing transcription factors such as Myc and HIF1, and the glucose transporter Glut1, which is pivotal for the increase of glucose uptake after T cell activation. Activation of MAPK signaling is required for glucose and glutamine utilization, whereas activation of AMPK is critical for energy balance and metabolic fitness of T effector and memory cells. Coinhibitory receptors target TCR-proximal signaling and generation of second messengers. Imbalanced activation of such signaling pathways leads to diminished rates of aerobic glycolysis and impaired mitochondrial function resulting in defective anabolic metabolism and altered T cell differentiation. The coinhibitory receptors mediate distinct and synergistic effects on the activation of signaling pathways thereby modifying metabolic programs of activated T cells and resulting in altered immune functions. Understanding and therapeutic targeting of metabolic programs impacted by coinhibitory receptors might have significant clinical implications for the treatment of chronic infections, cancer, and autoimmune diseases.

Local release of dexamethasone from macroporous scaffolds accelerates islet transplant engraftment by promotion of anti-inflammatory M2 macrophages.

Transplant-associated inflammatory responses generate an unfavorable microenvironment for tissue engraftment, particularly for cells susceptible to inflammatory stress, such as pancreatic islets. The localized delivery of anti-inflammatory agents, such as glucocorticoids, offers a promising approach to minimize the detrimental side effects associated with systemic delivery; however, the dosage must be carefully tailored to avoid deleterious responses, such as poor engraftment. Herein, we employed a polydimethylsiloxane (PDMS)-based three-dimensional scaffold platform for the local and controlled delivery of dexamethasone (Dex). Incorporation of 0.1% or 0.25% Dex within the scaffold was found to significantly accelerate islet engraftment in a diabetic mouse model, resulting in improved control of blood glucose levels during the early transplant period. Investigation into the mechanism of this impact found that local Dex delivery promotes macrophage polarization towards an anti-inflammatory (M2) phenotype and suppresses inflammatory pathways during the first week post-implantation. Alternatively, higher Dex loadings (0.5% and 1%) significantly delayed islet engraftment and function by impairing host cell migration into the implanted graft. Our results demonstrate the dose-dependent impact of local glucocorticoid delivery on the modulation of inflammatory responses at the implant site in vivo. Outcomes highlight the potential of this platform for generating favorable host responses that improve overall cellular transplant outcomes.

Clinical significance of T cell metabolic reprogramming in cancer.

Conversion of normal cells to cancer is accompanied with changes in their metabolism. During this conversion, cell metabolism undergoes a shift from oxidative phosphorylation to aerobic glycolysis, also known as Warburg effect, which is a hallmark for cancer cell metabolism. In cancer cells, glycolysis functions in parallel with the TCA cycle and other metabolic pathways to enhance biosynthetic processes and thus support proliferation and growth. Similar metabolic features are observed in T cells during activation but, in contrast to cancer, metabolic transitions in T cells are part of a physiological process. Currently, there is intense interest in understanding the cause and effect relationship between metabolic reprogramming and T cell differentiation. After the recent success of cancer immunotherapy, the crosstalk between immune system and cancer has come to the forefront of clinical and basic research. One of the key goals is to delineate how metabolic alterations of cancer influence metabolism-regulated function and differentiation of tumor resident T cells and how such effects might be altered by immunotherapy. Here, we review the unique metabolic features of cancer, the implications of cancer metabolism on T cell metabolic reprogramming during antigen encounters, and the translational prospective of harnessing metabolism in cancer and T cells for cancer therapy.

Silencing the Transcriptional Repressor, ZCT1, Illustrates the Tight Regulation of Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus Hairy Roots.

The Catharanthus roseus plant is the source of many valuable terpenoid indole alkaloids (TIAs), including the anticancer compounds vinblastine and vincristine. Transcription factors (TFs) are promising metabolic engineering targets due to their ability to regulate multiple biosynthetic pathway genes. To increase TIA biosynthesis, we elicited the TIA transcriptional activators (ORCAs and other unidentified TFs) with the plant hormone, methyl jasmonate (MJ), while simultaneously silencing the expression of the transcriptional repressor ZCT1. To silence ZCT1, we developed transgenic hairy root cultures of C. roseus that expressed an estrogen-inducible Zct1 hairpin for activating RNA interference. The presence of 17ß-estradiol (5µM) effectively depleted Zct1 in hairy root cultures elicited with MJ dosages that either optimize or inhibit TIA production (250 or 1000µM). However, silencing Zct1 was not sufficient to increase TIA production or the expression of the TIA biosynthetic genes (G10h, Tdc, and Str), illustrating the tight regulation of TIA biosynthesis. The repression of the TIA biosynthetic genes at the inhibitory MJ dosage does not appear to be solely regulated by ZCT1. For instance, while Zct1 and Zct2 levels decreased through activating the Zct1 hairpin, Zct3 levels remained elevated. Since ZCT repressors have redundant yet distinct functions, silencing all three ZCTs may be necessary to relieve their repression of alkaloid biosynthesis.

Controlled Release of Dexamethasone from Organosilicone Constructs for Local Modulation of Inflammation in Islet Transplantation.

Inflammation is a significant detriment to the engraftment of cells and tissues, particularly for islet transplantation, where a low tolerance for the inflammatory milieu results in significant graft loss. Local treatment with anti-inflammatories, such as glucocorticoids, provides the benefits of site-targeted delivery with minimization of the broad side effects associated with systemic delivery. Polydimethylsiloxane (PDMS) is a flexible platform that is capable of providing sustained delivery of hydrophobic drugs. Here, we evaluated the capacity of PDMS constructs loaded with the anti-inflammatory glucocorticoid dexamethasone (Dex) to locally mitigate inflammation in islet grafts. Dex-PDMS constructs, fabricated in rod or disk geometries, demonstrated prolonged and sustained release at therapeutically relevant levels. In vitro, Dex-PDMS constructs inhibited endotoxin-induced human monocyte and macrophage activation, but they did not impair islet viability or function. Dex-PDMS rods, co-transplanted with islet-seeded scaffolds in a murine model, demonstrated suppression of host inflammatory responses during early- and late-phase engraftment, without significantly altering islet graft potency. The facile nature of these glucocorticoid-doped PDMS constructs allows for the optimization of targeted dose delivery with wide applicability in cell and tissue transplantation.