Introduction: Type 1 diabetes (T1D) is characterized by a period of β cell dysfunction prior to β cell death. During this time, β cells lack a first-phase insulin response and exhibit an intolerance to glucose. However, the underlying mechanisms of this dysfunction remain unclear. Here, we use live human pancreas tissue slices (LPTS) to investigate the possible mechanisms underlying β cell dysfunction in T1D. Methods: LPTS from organ donors without diabetes (ND, n=13), donors without T1D and positive for one (sAAb+, n=6) or multiple T1D-relavent autoantibodies (mAAb+, n=2), and individuals with short-duration T1D (T1D+, n=9) were studied. Simultaneous time-lapse Ca2+ imaging and CD3+ T-cell tracking were conducted to assess alterations in β-cell function. Additionally, fixed pancreas sections (ND, n=8; sAAb+, n=7; mAAb+, n=7; T1D+, n=6) were probed with a panel of 22 antibodies to investigate mechanisms underlying β-cell dysfunction including insulin, CD3, ATP5B (a mitochondrial ATP synthase subunit), and ATPIF1 (ATP synthase inhibitor). Results: Islets within LPTS from ND and AAb+ donors exhibited typical Ca2+ mobilization in response to high glucose (HG) and potassium chloride (KCl) stimulations. Islets from T1D+ donors had significantly diminished Ca2+ responses to HG compared to ND donors (p<0.05, one-way ANOVA). Low glucose and KCl stimulations between ND, AAb+, and T1D+donors were similar, indicating that the dysfunction occurs in the glucose metabolism pathway. Additionally, there were no significant differences in HG responses in insulitic and non-insulitic islets from T1D+ LPTS with both being dysfunctional. Fixed tissue staining revealed a significant (p<0.0001, two-way ANOVA) decrease in mitochondria in β cells from T1D+ donors. Conclusion: The loss of HG responses in T1D+ β cells independent of the local T cell infiltration, coupled with the decrease in mitochondria, indicates that the dysfunction likely originates in the β cell and contributes to T1D pathogenesis. Disclosure M. Huber: None. A. Widener: None. D. Smurlick: None. H. Hiller: None. M. Beery: None. E. Verney: None. I. Kusmartseva: None. M. Campbell-Thompson: None. M.A. Atkinson: None. C.E. Mathews: None. E. Phelps: Research Support; Immunocore, Ltd, MESO SCALE DIAGNOSTICS, LLC. Funding T32DK108736F31DK130607P01; AI42288 R01DK132387

Introduction & Objective: The recent successful large-scale generation of human stem cell (hPSC)-derived β-like cells (sBC) provides an abundant cell source for replacement therapy efforts to treat patients suffering from autoimmune diabetes. However, even in the presence of systemic immune suppression, transplanted sBC, like primary human islets, are susceptible to allogeneic and recurrent autoimmune attack. Different strategies leveraging genome engineering technologies have been tested to achieve localized immune protection for β-cell grafts. Increased expression of the immune checkpoint inhibitor PD-L1 at the graft site has shown promise in reducing either xenogeneic or allogeneic immune responses in preclinical animal models. However, whether PD-L1 expression can protect sBC grafts from recurring autoimmunity is poorly understood, largely due to the absence of appropriate model systems. Methods: Here we adoptively transferred diabetogenic mouse T cells from NOD mice expressing human HLA class I into immunodeficient NSG mice bearing HLA matched sBC grafts as a novel model of human autoimmunity in vivo. Results: Using this novel autoimmune in vivo model, sBC grafts that were genetically modified to express high levels of PD-L1 exhibit greater survival compared to unmodified control grafts but were also lost within 3 weeks due to robust T cell infiltration. Interestingly, we also observed destruction of HLA class 1 knockout sBC grafts, suggesting a potential and important role for autoreactive CD4+ T cells. We are currently expanding our findings using HLA matched in vitro co-culture assays and performing additional transplantation studies with purified diabetogenic CD8+ T cells. Conclusion: Collectively, our data suggest that PD-L1 expression on sBC alone might not provide sufficient protection from recurring autoimmunity, unlike allogenic responses, and highlights the need for better model systems to interrogate human autoimmunity in a matched manner. Disclosure J. Barra: None. R. Castro-Gutierrez: None. J. Santini-Gonzalez: None. C. Manson: None. A. Widener: None. L. Landry: None. M. Nakayama: None. E. Phelps: Research Support; Immunocore, Ltd, MESO SCALE DIAGNOSTICS, LLC. H.A. Russ: Advisory Panel; Sigilon Therapeutics, Inc. Consultant; Minutia. Advisory Panel; Prellis Biologics. Consultant; Eli Lilly and Company. Funding NIH/NIDDK (R01DK132387); JDRF (2-SRA-2023-1313-S-B); JDRF (2-SRA-2023-1367-S-B); Diabetes Research Connection (grant #49)

This review aims to understand the current progress in immune-instructive granular hydrogels and identify the key features used as immunomodulatory strategies. Published work is systematically reviewed and relevant information about granular hydrogels used throughout these studies is collected. The base polymer, microgel generation technique, polymer crosslinking chemistry, particle size and shape, annealing strategy, granular hydrogel stiffness, pore size and void space, degradability, biomolecule presentation, and drug release are cataloged for each work. Several granular hydrogel parameters used for immune modulation: porosity, architecture, bioactivity, drug release, cell delivery, and modularity, are identified. The authors found in this review that porosity is the most significant factor influencing the innate immune response to granular hydrogels, while incorporated bioactivity is more significant in influencing adaptive immune responses. Here, the authors' findings and summarized results from each section are presented and suggestions are made for future studies to better understand the benefits of using immune-instructive granular hydrogels.

Genetically encoded calcium indicators (GECIs) and high-resolution confocal microscopy enable dynamic visualization of calcium signals in cells and tissues. Two-dimensional and 3D biocompatible materials mimic the mechanical microenvironments of tumor and healthy tissues in a programmable manner. Cancer xenograft models and ex vivo functional imaging of tumor slices reveal physiologically relevant functions of calcium dynamics in tumors at different progression stages. Integration of these powerful techniques allows us to quantify, diagnose, model, and understand cancer pathobiology. Here, we describe detailed materials and methods used to establish this integrated interrogation platform, from generating transduced cancer cell lines that stably express CaViar (GCaMP5G + QuasAr2) to in vitro and ex vivo calcium imaging of the cells in 2D/3D hydrogels and tumor tissues. These tools open the possibility for detailed explorations of mechano-electro-chemical network dynamics in living systems.

Inter-particle secondary crosslinks allow microporous annealed particle (MAP) hydrogels to be formed. Methods to introduce secondary crosslinking networks in MAP hydrogels include particle jamming, annealing with covalent bonds, and reversible noncovalent interactions. Here, we investigate the effect of two different approaches to secondary crosslinking of polyethylene glycol (PEG) microgels via reversible guest-host interactions. We generated a dual-particle MAP-PEG hydrogel using two species of PEG microgels, one functionalized with the guest molecule, adamantane, and the other with the host molecule, β-cyclodextrin (Inter-MAP-PEG). In a different approach, a mono-particle MAP-PEG hydrogel was generated using one species of microgel functionalized with both guest and host molecules (Intra-MAP-PEG). The Intra-MAP-PEG formed a homogenous distribution due to the single type of microgels used. We then compared the mechanical properties of these two types of MAP-PEG hydrogels and found that Intra-MAP-PEG resulted in significantly softer gels with lower yield stress. We investigated the effect of intra-particle guest-host interactions through titrated weight percentage and the concentration of functional groups added to the hydrogel. We found that there was an ideal concentration of guest-host molecules that enables intra- and inter-particle guest-host interactions with sufficient covalent crosslinking. Based on these studies, Intra-MAP-PEG provides a homogeneous guest-host hydrogel that is shear-thinning with reversible secondary crosslinking.

Microporous annealed particle (MAP) hydrogels have emerged as a versatile biomaterial platform for regenerative medicine. MAP hydrogels have been used for the delivery of cells and organoids but often require annealing post injection by an external source. We engineered an injectable, self-annealing MAP hydrogel with reversible interparticle linkages based on guest-host functionalized polyethylene glycol maleimide (PEG-MAL) microgels. We evaluated the effect of guest-host linkages on different types of microgels fabricated by either batch emulsion or mechanical fragmentation methods. Batch emulsion generated small spherical microgels with controllable 10-100 μm diameters and mechanical fragmentation generated irregular microgels with larger diameters (100-200 μm). Spherical microgels (15 μm) showed self-healing behavior and completely recovered from high strain while fragmented microgels (133 μm) did not recover. Guest-host interactions significantly contributed to the mechanical properties of spherical microgels but had no effect on fragmented microgels. Spherical microgels were superior to the fragmented microgels for co-injection of immune cells and pancreatic islets due to their lower force of injection, demonstrating more homogeneously distributed cells and greater cell viability after injection. Based on these studies, the spherical guest-host MAP hydrogels provide a controllable, injectable scaffold for engineered microenvironments and cell delivery applications.

We report the development of a polyethylene glycol (PEG) hydrogel scaffold that provides the advantages of conventional bulk PEG hydrogels for engineering cellular microenvironments and allows for rapid cell migration. PEG microgels were used to assemble a densely packed granular system with an intrinsic interstitium-like negative space. In this material, guest-host molecular interactions provide reversible non-covalent linkages between discrete PEG microgel particles to form a cohesive bulk material. In guest-host chemistry, different guest molecules reversibly and non-covalently interact with their cyclic host molecules. Two species of PEG microgels were made, each with one functional group at the end of the four arm PEG-MAL functionalized using thiol click chemistry. The first was functionalized with the host molecule β-cyclodextrin, a cyclic oligosaccharide of repeating d-glucose units, and the other functionalized with the guest molecule adamantane. These two species provide a reversible guest-host interaction between microgel particles when mixed, generating an interlinked network with a percolated interstitium. We showed that this granular configuration, unlike conventional bulk PEG hydrogels, enabled the rapid migration of THP-1 monocyte cells. The guest-host microgels also exhibited shear-thinning behavior, providing a unique advantage over current bulk PEG hydrogels.

The goal of this study was to perform in situ electrochemical polymerization of poly(3,4-ethylenedioxythiophene) (PEDOT) in peripheral nerves to create a soft, precisely located injectable conductive polymer electrode for bi-directional communication. Intraneural PEDOT polymerization was performed to target both outer and inner fascicles via custom fabricated 3D printed cuff electrodes and monomer injection strategies using a combination electrode-cannula system. Electrochemistry, histology, and laser light sheet microscopy revealed the presence of PEDOT at specified locations inside of peripheral nerve. This work demonstrates the potential for using in situ PEDOT electrodeposition as an injectable electrode for recording and stimulation of peripheral nerves.