Symposium SM02 : Immune Modulatory Materials—From Design to Translational Applications

Spherical nucleic acids (SNAs) are novel nanoparticle architectures that typically consist of a dense shell of oligonucleotides conjugated to a nanoparticle core. The oligonucleotide shell of the SNAs imparts upon the conjugate material unique properties that differ from those of linear nucleic acids, such as rapid cellular uptake and increased resistance to nuclease degradation. SNAs composed of immunomodulatory oligonucleotides and tumor-specific antigens induce the immune system to clear tumors, and are thus promising as cancer vaccines. We have shown that the oligonucleotides on the surface of the SNAs activate the innate immune system through toll-like receptors (TLR) in antigen-presenting cells (APCs). These activated APCs then mature and activate effector T-cells to target and clear tumors expressing the cancer antigen. However, much work is still needed to fully understand how the materials architecture of the SNA (structure), for instance how the antigen and adjuvant are assembled into the SNA surface, affects immune system activation and tumor clearance (function). To this end, we incorporated the peptide antigen into the SNA material via different encapsulation and conjugation schemes. We found that activated T-cells killed five times more tumor cells when the peptide antigen was conjugated to the complementary oligonucleotide compared to when a mixture of linear oligonucleotides and peptide antigens was used. Furthermore, we found that antigen-specific T-cell activation could be increased eight-fold when readily reversible peptide linkers (i.e., disulfides) as opposed to irreversible ones (i.e., thiol-maleimide conjugation) were used. Finally, with these materials, we developed a high-throughput assay, that when coupled with machine learning, could be used to map the immune activation characteristics of libraries of SNA structures with different architectures and hence different properties; we have tested approximately 1,000 SNAs to date.

Introduction: Current paradigms for the treatment of autoimmune diseases (e.g. rheumatoid arthritis [RA]) are woefully inadequate, often missing the mark on desired physiological responses and not targeting the root cause of the disease. Predictably, novel approaches to re-establish immune homeostasis in patients afflicted by autoimmune conditions are now under intense investigation. Notably, we are developing an array of multifunctional, biomaterial-based ‘anti-vaccines’ that can be easily administered to remediate some of the prevalent autoimmune diseases. This talk will focus on different particulate systems currently under development in my lab, which attempt to control critical cellular and humoral mediators that engender conditions such as type 1 diabetes, RA, and autoimmune autism.

Dual microparticle anti-vaccine for conditioning dendritic cells in vivo: The underlying cause of RA is dendritic cell (DC) activation of antigen-specific T cell subsets in the joints, which drive inflammatory responses to the synovial membrane that are typically characterized by inflammatory cell infiltration and over production of pro-inflammatory cytokines by monocytes, macrophages and synovial fibroblasts. The Lewis Lab at UC, Davis is developing a novel, dual microparticle system for in vivo co-delivery of pro-tolerance factors and autoantigens, targeted to DCs. Exogenous conditioning of DCs with certain immuno-modulatory agents has been shown to induce pro-tolerance DC phenotypes that ameliorate RA. We are attempting to build on those successes by using materials to circumvent ex-vivo conditioning, which has been shown to be disadvantageous to full translation into the clinic. Further, we are interested in deciphering the immune effects of synthetic biomaterials on critical immune cells and delineate the properties of biomaterials that influence immune cell polarity.

Biomagnetic Traps for Treatment of Autoimmune Autism: Contemporary molecular biology techniques have helped to shed light on one key trigger of autism – the transfer of maternal autoantibodies against critical proteins in the developing fetal brain during pregnancy, which results in the development of approx. 23% of all autism cases. Further, researchers have pinpointed the specific antigenic determinants recognized by these autism-causing maternal autoantibodies. We seek developing a therapeutic measure for autoimmune autism based on this new knowledge. The immunomodulatory biomaterials lab at UC Davis is currently engineering `biomagnetic traps' – iron oxide-based nanoparticles (NPs) that are surface-conjugated with cognate peptides (epitopes), for the removal of circulating, ASD-causing maternal autoantibodies. Our long-term goal is to develop a modular, nanoparticle platform for autoantibody sequestration and treatment of maternal autoantibody-related (MAR) ASD.

Issues with poor scalability of production have hindered the progression of extracellular vesicle (EV) technology for therapeutic applications. We demonstrate increased cellular production of nano-sized EVs by an order of magnitude compared to naturally occurring EV production by using a method that involves sulfhydryl-blocking. Nanovesicles produced using sulfhydryl-blocking are approximately 50 nm in diameter and can be loaded with therapeutics or engineered via surface modification to achieve desired therapeutic applications. We demonstrate the use of nanovesicles produced by sulfhydryl-blocking for chemotherapeutic delivery of doxorubicin in vivo and for immunotherapy by stimulation of T cells.

Bladder carcinoma is the most expensive tumor type to treat on a cost-per-patient basis from diagnosis to death. The majority of bladder carcinoma cases are categorized as superficial disease (nonmuscle invasive bladder cancer; NMIBC). Intravesical instillation of live Mycobacterium bovis Bacillus Calmette Guerin (BCG) is the adjuvant therapy of choice for the treatment of NMIBC. Unfortunately, BCG treatment suffers from drawbacks including frequent tumor relapse, resistance, and risk of sepsis. Recently, cyclic di-nucleotides have been reported as a potent immunostimulatory agent that works via stimulation of the STING (STimulator of INterferon Gene) pathway. Clinical translation of cyclic di-nucleotide therapy; however, faces two major challenges. First, the hydrophilic nature of the molecule prevents it from cross the cellular membrane to stimulate the STING pathway via binding with the cytosolic ATP dependent helicase. Second, there is no carrier system capable to deliver the cyclic di-nucleotides in a tumor specific manner. Here we describe a liposomal formulation that has been designed to overcome these two challenges. We have utilized a novel targeting ligand, fibronectin attachment peptide(FAP) to guide the liposomes to the fibronectin (FBN) rich extracellular matrix of the tumor microenvironment. Upon internalization by tumor and macrophagic cells, the pH responsive nature of the liposomes helps to escape the endosome and release the cargo into the cytosol, resulting in the release of a host of cytokines to induce the host innate immune system.

Inducing a strong and specific immune response is the hallmark of a successful vaccine. Nanoparticles have emerged as promising vaccine delivery devices to discover and elicit immune responses, Fine-tuning a nanoparticle vaccine to create an immune response with specific antibody and other cellular responses is influenced by many factors such as shape, size, and composition. Peptide amphiphile micelles are a unique biomaterials platform that can function as a modular vaccine delivery system, enabling control over many of these important factors and delivering payloads more efficiently to draining lymph nodes. In this study, the modular properties of peptide amphiphile micelles are utilized to improve an immune response against a Group A Streptococcus B cell antigen (J8). The hydrophobic/hydrophilic interface of peptide amphiphile micelles enabled the precise entrapment of amphiphilic adjuvants which were found to not alter micelle formation or shape. These heterogeneous micelles significantly enhanced murine antibody responses when compared to animals vaccinated with nonadjuvanted micelles or soluble J8 peptide supplemented with a classical adjuvant. The heterogeneous micelle induced antibodies also showed cross-reactivity with wild-type Group A Streptococcus providing evidence that micelle-induced immune responses are capable of identifying their intended pathogenic targets.

Adoptive cell therapy (ACT) employing antigen-specific T-cells has elicited dramatic clinical responses in leukemia and a subset of melanoma patients. However, strategies to safely and effectively augment T-cell infiltration and function in solid tumors remain of great interest. Our laboratory aims to enhance adoptive T-cell therapy and other cancer immunotherapies through responsive nanoparticle drug delivery. Here we describe a strategy to enhance the tumor-infiltration and function of transferred T-cells by spatiotemporally controlled delivery of immunomodulators. Responsive protein nanogels (NGs) containing large quantities of immunomodulatory drugs are designed and synthesized to release the drugs in response to the reductive environment specific in tumor tissue or on T-cell surface. We show that T-cells increase their cell surface reduction potential upon activation, which we exploit through the design of cell surface-bound NGs that disassemble to release protein cargos in response to this change in the local reductive environment following T-cell receptor (TCR) triggering. The T-cell surface-bound NGs selectively release adjuvant drugs in response to TCR activation, focusing drug release in sites of antigen encounter such as the tumor microenvironment. Using an IL-15 superagonist complex as a candidate adjuvant drug cargo, we demonstrate that relative to systemic administration of free cytokines, NG delivery selectively expands adoptively transferred T-cells 16-fold in tumors, and allows at least 8-fold higher doses of cytokine to be administered without toxicity, leading to substantially increased anti-tumor efficacy and safety. This strategy provides a general approach to augment the function of cell therapies by linking drug release to cell function in vivo.

Herein, we developed a combination strategy of photochemotherapy and immunotherapy by virtue of tumor specific Fenton reactions assisted by near-infrared (NIR) light irradiation, where NIR light upconverted UV/vis light by upconversion nanoparticles (UCNPs) catalyzed the Fenton reaction between the delivered Fe2+ and H2O2 species. First, the UCNPs were coated by tannic acid and crosslinked by ferrous ions. Furthermore, we utilized the ability of GM-CSF in reprogramming anti-inflammatory, pro-tumoral M2 type tumor-associated macrophages to pro-inflammatory, antitumor M1 macrophages, which provided the source of H2O2. In vitro, MB49 cells co-incubated with GMCSF/Man-CS@UCNPs and macrophages showed increased caspase-3 activity and cell inhibition rate. Macrophages exposed to GMCSF/Man-CS@UCNPs displayed increased mRNA associated with pro-inflammatory Th1-type responses. In vivo, GMCSF/Man-CS@UCNPs significantly inhibited growth of subcutaneous and orthortopic tumor in mice. In addition, the combined immunotherapy and photochemodynamic was explored. We found that a burst release of tumor antigen and cytokines stem from cancer cells apoptosis and necrosis post photochemodynamic therapy greatly triggered the immune response in-situ. Furthermore, we investigated the activation of dendritic cells to produce high levels of interferon-gamma, an important cytokine considered as a product of T and natural killer cells. This study is expected to develop a biocompatible, sustainable and highly-efficient anti-tumor treatment strategy, which will pave a new avenue for design of nanomedicine for photochemodynamic therapy and immunotherapy.

The natural selection of resistant bacteria has led to serious antibiotic resistance. Phage therapy has gained increasing attention as an alternative to antibiotics because of high specificity and low inherent toxicity. Importantly, the directed evolution of phage provides an opportunity to overcome the resistance of bacteria. In contrast to chemical compounds, phages can be amplified in bacteria, so the number of phages can increase with the increasing number of host bacteria, effectively suppressing the growth of the bacterial population. However, the use of phage for in vivo anti-bacterial treatments is limited due to the rapid elimination from the body by reticuloendothelial (RES) system clearance. Polyethylene glycol (PEG) and other polymers can be employed, but the steric barrier by the PEG chains can significantly reduce the intrinsic infectivity of phages. Also, any chemical modification is applied to the first generation of phages; i.e., the amplified phages do not have PEG on their surface. In this work, we report the prolonged blood circulation and dramatically increased therapeutic efficacy of lytic phages through the immunological cloaking based on the expression of a self-peptide on the major capsid. The stealth self-peptide was originally suggested through the computational simulations of human CD47 (hCD47) interacting with signal regulatory protein-α (SIRPα) on macrophages and imparting a “do-not-eat-me” signal to avoid phagocytosis (Rodriguez et al., Science, 339, 971). Our results show that the self-peptide expressing T7 phage (Self-T7) suppresses up to 70 % phagocytosis of mouse macrophages compared to the wild-type T7 phage (WT-T7). Real-time in vivo image analysis demonstrates that the Self-T7 exhibits a markedly longer blood circulation compared to the WT-T7. Accordingly, the in vivo anti-bacterial effects of the phage was dramatically increased in a mouse model of E.coli infection of the intestines, as indicated by the high survival rate of mice by the intraperitoneal or intravenous injection of the Self-T7. The WT-T7 and PEG-modified T7 phage showed limited therapeutic effects: the WT-T7 increased the survival time from two days to four days, and the PEG-T7 showed a large variation in the survival rate and no complete recovery. In contrast, all of the mice treated with the Self-T7 were returned to normal conditions and showed no symptoms in 5 days. Plaque assay for the liquid from the infected intestines showed that no single bacteria remained in the mice treated with the Self-T7. The mice treated with WT-T7 and PEG-T7 also the exhibited significantly reduced numbers of plaques compared to non-treated mice, indicating the antibacterial activity of T7 phages. This work demonstrated that the immunological cloaking of lytic phages through genetic expression of a self-peptide is an effective means to improve the in vivo anti-bacterial efficacy of phage through suppressed phagocytosis and prolonged blood circulation.

Although antibody-based immunotherapy is considered to be the most potential clinical translational therapy for tumors due to its low toxicity and high specificity, only bevacizumab was approved by US Food and Drug Administration (FDA) for the treatment of recurrent glioblastoma however showed limited effects. Lack of efficient delivery system compromises antibody therapy for glioblastoma. Extracellular release of monoclonal antibody for them to bind with receptor on the surface of glioblastoma cell membrane to inhibit tumor growth still remains challenging. Here, in our present study, for the first time, high expression of metalloproteinase-2 (MMP-2) in tumor environment was utilized for the extracellular release of antibody. A MMP-2 sensitive nanocapsule based on 2-methacryloyloxy ethyl phosphorylcholine (MPC) was constructed. By simply controlling the content of MPC, the cell uptake of these small sized (~30 nm) antibody carriers was inhibited while effective extracellular release of antibody was realized. Besides, the nanosize and MPC-based shell endow the antibody-loaded nanocapsule with long circulation time, fast permeation ability and high accumulation amount in glioblastoma which are requirements for efficient glioblastoma therapy. Nitozumab was used as the model antibody and was capsulated inside the neutral nanocapsule (~30 nm) by an in situ free radical polymerization method. When the molar ratio of MPC to nitozumab was between 4000 and 5000, the nanocapsules can be degraded enzymatically and meanwhile inhibit the internalization of glioblastoma cells making them capable of extracellular release of nitozumab. Animal experiments proved the long circulation time (half-life is about 50h) of the nanocapsules in blood, fast penetration (about 2h after systemic injection) of the nanocapsules into glioblastoma site and higher accumulation amount of nanocapsules in glioblastoma site. Compared with free nitozumab, nitozumab-loaded nanocapsules can efficiently suppress the glioblastoma of glioblastoma-bearing mice and significantly prolong the survival time of glioblastoma-bearing mice (at day 45, more than 50% mice treated by nitozumab-loaded nanocapsules were survived but all mice died when treated by free nitozumab). Additionally, no significant weight loss of mice treated by nitozumab-loaded nanocapsules was observed during the treatment. The results demonstrated the extracellularly released antibody delivery platform may provide a useful tool for immunotherapy for glioblastoma.

Biomaterials constructed from de novo designed peptides and peptidomimetics that assemble into β-sheet rich peptide nanofibers and hydrogels have enormous potential for applications in biology and medicine. Self-assembling peptide biomaterials have been used as multivalent scaffolds for applications in tissue engineering, regenerative medicine, and drug delivery due to their biocompatibility, ease of synthesis, and the rich chemistry with which the primary sequence can be manipulated to impart structure or function. In recent years, supramolecular peptide nanofibers have attracted considerable attention as immune adjuvants for applications in vaccine development and immunotherapy due to their ability to induce strong antibody and cellular responses to conjugated antigens. Unlike traditional depot forming adjuvants (alum or Freund’s), peptide nanofibers are non-inflammatory and do not elicit inflammation at the injection site (recruitment of neutrophils, eosinophils, monocytes, etc.). Several studies have confirmed the efficacy of peptide nanofiber-based vaccines in animal models of infectious and non-infectious diseases; however, the immunological mechanisms that drive the inflammation-free adjuvant potential have not yet been elucidated.

An evolutionarily conserved mechanism that cells use to homeostasis is ‘autophagy’. Autophagy is crucial for the clearance of fibrillar protein aggregates implicated in neurodegenerative diseases such as Alzheimer’s and Huntington’s and a component of innate immunity that is involved in host defense elimination of pathogens. Autophagy has been identified as a route by which cytoplasmic and nuclear antigens are delivered to MHC class II molecules for presentation to CD4+T cells and MHC class I cross-presentation of tumor antigens to CD8+T cells. Here, we report that self-assembling peptide nanofibers bearing CD4+ or CD8+T cell epitopes are processed through mechanisms of autophagy in antigen presenting cells (APCs). Using standard in vitro antigen presentation assays, we confirmed loss and gain of adjuvant function using pharmacological modulators of autophagy and APCs deficient in multiple autophagy proteins. Incorporation of microtubule associated protein 1A/1B-light chain 3 (LC3-II) into the autophagosomal membrane, a key biological marker for autophagy, was confirmed using microscopy. Our findings indicate that autophagy in APCs plays an essential role in the mechanism of adjuvant action of supramolecular peptide nanofibers.

Adoptive cell therapies (ACT) using ex vivo-modified lymphocytes to enhance anti-tumor immunity is rapidly gaining FDA approval for multiple cancer types. ACT has been promising in blood cancers, but poor efficacy persists in many solid tumors. Further, the complex requirements of genetic manipulation and occurrence of immune-related adverse effects remain outstanding challenges for implementing ACT in the clinic. Combining ACT with other therapies such as immune-enhancing drugs can also be limited by inefficient trafficking and activity of drugs to the immunosuppressive tumor microenvironment, and low colocalization of drug with ACT. To address these challenges, we have engineered molecular conjugates using lipid-based tails coupled to TLR agonists (lipo-TLRa) that rapidly and densely insert into the plasma membrane of immune cells. Murine T-cells coated with lipo-CpG-DNA (TLR9a) and Pam2CSK4 (TLR2a) can signal by both autocrine and paracrine mechanisms to enhance therapeutic outcomes while reducing unwanted side effects by tightly colocalizing drugs with cells. Our data showed that lipo-TLRa functionalize immune cell surfaces in a dose and time-dependent manner. Signals loaded for up to 1 h can functionally persist on cell surfaces for multiple days. Lipo-TLRa were shown to 1) activate lipid—inserted cells along with nearby bystander immune cells by co-delivering danger signals, 2) efficiently deliver prolonged signaling to both intracellular and surface TLR receptors, and overcome tumor immunosuppression by 3) recovering proliferative function and 4) enhancing cytokine production in vitro when T-cells were co-cultured with immunosuppressive tumor cells. Further, membrane-inserted lipo-TLRa was equivalent or superior to soluble drug at higher concentrations. These results demonstrate the unique properties and abilities of lipo-TLRa’s to enhance the function of T cells for adoptive transfer.

Dysfunction of the immune system underlies many diseases, and results from certain cancer therapies. However, strategies to effectively accelerate return of immune function and program disease-specific immune responses by manipulating stem cells and immune cells are at an early stage. We are creating biomaterials capable of concentrating, interrogating, and manipulating stem and immune cells ex vivo and in the body by controlling, in space and time, the interaction of the cells with various cues. The utility of this concept in the cancer therapies will be highlighted.

Next generation biomaterials will be capable of communicating with the biological microenvironment in ways that are similar to real cells and tissues. A primary way that this is accomplished in situ through secretion and spatiotemporal organization of soluble proteins that can lead to the homing and/or differentiation of specialized endogenous cells involved in regulation of the immune system. New technology has been made available to rationally program biomaterials in silico to produce such spatiotemporal control over release. This talk will present several examples of the programming of biomaterials to mimic the release and organization of natural proteins to achieve site-specific homing and control over the behavior of endogenous regulatory T cells toward treatments for diseases involving destructive inflammation including tissue transplantation, dry eye disease, periodontal disease, and contact dermatitis.

Clinical islet transplantation, the intrahepatic infusion of allogeneic islets, has the potential to provide physiological blood glucose control for insulin-dependent diabetics. The success of clinical islet transplantation, however, is significant hindered by the strong inflammatory and immunological responses to the transplant, in spite of systemic immunosuppression. To address these challenges, our laboratory has focused on engineering biomaterials that serve to modulate immunological responses at the implant site. This can be achieved by designing biomaterials that serve to: immuno-camouflage the transplant via encapsulation; generate a local immunosuppressive graft site via local drug delivery; and/or present surface-mediated tolerogenic signals to peripheral immune cells. A summary of these approaches to extend the duration of foreign cellular grafts via local modulation of host immune cells towards desirable tolerogenic phenotypes will be presented herein.

Monocytes and macrophages play a critical role in tissue development, homeostasis, and injury repair. These innate immune cells participate in guiding vascular remodeling, stimulation of local stem and progenitor cells, and structural repair of tissues such as muscle and bone. Therefore, there is a great interest in harnessing this powerful endogenous cell source for therapeutic regeneration through immunoregenerative biomaterial engineering. These materials seek to harness specific subpopulations of monocytes/macrophages to promote repair by influencing their recruitment, positioning, differentiation, and function within a damaged tissue. Monocyte and macrophage phenotypes span a continuum of inflammatory (M1) to anti-inflammatory or pro-regenerative cells (M2), and their heterogeneous functions are highly dependent on microenvironmental cues within the injury niche. Increasing evidence suggests that division of labor among subpopulations of monocytes and macrophages could allow for harnessing regenerative functions over inflammatory functions of myeloid cells; however, the complex balance between necessary functions of inflammatory versus regenerative myeloid cells remains to be fully elucidated. Historically, biomaterial-based therapies for promoting tissue regeneration were designed to minimize the host inflammatory response; although, recent appreciation for the roles that innate immune cells play in tissue repair and material integration has shifted this paradigm. A number of opportunities exist to exploit known signaling systems of specific populations of monocytes/macrophages to promote repair and to better understand the biological and pathological roles of myeloid cells. This review seeks to outline the characteristics of distinct populations of monocytes and macrophages, identify the role of these cells within diverse tissue injury niches, and offer design criteria for immunoregenerative biomaterials given the intrinsic inflammatory response to their implantation.

Toll-like receptors (TLR) play a critical role in host defence by initiating sterile inflammatory responses to endogenous damage-associated molecular patterns (DAMPs), which are released following tissue injury. Upon ligand binding, TLRs induce inflammation via NF-κB activation and the expression of pro-inflammatory cytokines, including TNF-α and IL-6. Tissue injury accompanying biomaterial implantation likely results in the release of DAMPs. However, the role of DAMP-TLR signaling in host responses to solid polymer implants in unclear. The aim of our research is to understand the molecular mechanisms through which the innate immune system responds to materials, in order to identify potential molecular targets for modulating cell-material interactions. Our current focus is examining the molecular mechanism through which biomaterial-adsorbed DAMPs activate macrophages and mediate the inflammatory biomaterial microenvironment.
In this study, we used cell lysate generated from NIH3T3 fibroblasts via freeze-thaw cycling as a complex, in vitro source of DAMPs. Lysate or fetal bovine serum (FBS) was adsorbed to polydimethylsiloxane (PDMS), poly(methyl methacrylate) (PMMA) surfaces or tissue culture polystyrene (TCPS) surfaces for 30 min and rinsed with PBS. RAW-Blue™ (Invivogen) macrophages, an NF-κB reporter cell line, were seeded on the different polymer surfaces for 20 hours and the NF-κB activity was measured indirectly using an inducible-secreted embryonic alkaline phosphatase (SEAP) activity assay. The concentration of IL-6 and TNF-α in the cell supernatant was measured by ELISA. TLR2 and TLR4 signalling were inhibited using a TLR2 neutralizing antibody and TLR4 inhibitor, CLI-095. PAM3CSK4 and LPS were used as positive controls of TLR2 and TLR4 signalling, respectively.
Macrophages cultured on lysate-adsorbed PMMA, PDMS and TCPS surfaces had a greater than 6-fold increase in NF-κB activity, compared to FBS-adsorbed surfaces and the negative control (p < 0.001). Lysate-adsorbed surfaces also potently induced the secretion of TNF-α and IL-6, compared to the negative control (p < 0.001). The increased NF-κB activity and cytokine secretion on lysate-adsorbed surfaces were strongly attenuated by TLR2 neutralizing antibodies, whereas inhibiting TLR4 resulted in a modest reduction in NF-κB activity.
These data show that lysate-derived DAMPs adsorbed to polymer surfaces potently NF-κB transcription factors, to a greater extent than serum protein; DAMP-adsorbed polymer surfaces activated NF-κB primarily through TLR2; and exposure of RAW-Blue macrophages to DAMP-adsorbed surfaces strongly induced the production of pro-inflammatory cytokines in a TLR2-dependent manner. These results suggest that DAMPs, in their adsorbed conformation, are capable of inducing a potent pro-inflammatory response in macrophages through TLRs, and that TLR pathways should be investigated as potential therapeutic targets for modulating biomaterial host responses.

The immune response to implanted materials remains a critical challenge for the development of biomaterials used in medical devices and regenerative medicine. Understanding this response and designing better biomaterials requires a multidisciplinary approach involving materials engineering and immunology. The goal of our work is to understand how material properties regulate the function of immune cells, particularly macrophages, versatile regulators of the innate immune system that are involved in inflammation, wound healing, and tissue regeneration. Using cell micropatterning and surface topography, we have previously found that geometry of adhesion, or cell shape, plays a critical role in regulating their polarization towards pro-inflammatory versus pro-healing states. More recently, we have examined how the composition and architecture of three-dimensional extracellular matrices influence macrophage adhesion and function. In particular, we found that fibrin matrices are protective and inhibit macrophage inflammatory activation. Current work is focused on understanding mechanochemical signaling pathways involved, and leveraging these findings to design new materials to encourage macrophage-mediated wound healing.

A major challenge in engineering biomaterials for regenerative medicine is achieving sufficient vascularization to support tissue survival and integration in vivo. Functional blood vessel networks must not only form within the tissue (angiogenesis), but also connect with the existing host vasculature (anastomosis) to achieve graft perfusion upon implantation. Macrophages, the primary cell of the innate immune system, strongly affect the outcome of implanted biomaterials and have been implicated in both angiogenesis and anastomosis. However, the role of macrophage phenotype in these processes is unclear. Previously, we have shown that macrophages activated with pro-inflammatory stimuli interferon-gamma (IFNg) and lipopolysaccharide (LPS) (M1) secrete factors involved in the early stages of angiogenesis, whereas macrophages activated with interleukin-4 (IL4) and IL13 (M2a) secrete factors involved in the stabilization of newly sprouted vessels¹. The purpose of this study was to delineate the contribution of macrophage phenotype to biomaterial vascularization, using in vitro-polarized macrophages in a previously developed 3D model of in vitro blood vessel network formation².

Human adipose microvascular endothelial cells expressing tdTomato together with human adipose-derived mesenchymal stem cells were pre-seeded on porous Gelfoam® scaffolds (Pfizer, New York, NY) to generate self-assembled vascular networks that can be used to examine vascularization dynamics in vitro. PMA-activated, GFP-expressing THP1-derived M0, M1, or M2a macrophages were added to the pre-vascularized constructs on days 3 or 6 of vessel growth, and changes in network development were monitored over 14 days using confocal microscopy. Images were analyzed in 2D and 3D using Angiotool³ and Matlab software, respectively, in terms of vessel density, length, extent of branching and number of endpoints. Statistical analysis was completed in GraphPad Prism 7.0 using analysis of variance with Tukey’s post-hoc analysis (n ≥ 3). Image analysis revealed a significant (p < 0.05) increase in vessel sprouting by M1 macrophages, while both M1 and M2a phenotypes increased vessel connections 1 day post-seeding relative to control constructs without macrophages. However, phenotype-specific changes in network morphology were indistinguishable after 72 hours, despite qualitative differences in M0, M1, and M2a morphology and localization. Interestingly, all macrophage-seeded constructs exhibited significant vessel regression independent of phenotype after 4 days in vitro; this regression was not observed in control constructs. Ongoing work aims to characterize the temporal changes in gene expression of macrophages and endothelial cells during angiogenesis. Ultimately, this work will aid in designing immunomodulatory biomaterials that promote vascularization and integration.

1. Spiller KL et al. Biomaterials 2014; 35.
2. Freiman A et al. Stem Cell Res Ther 2016; 7.
3. Zudaire E et al. PLoS One 2011; 6.

Nanocarrier administration has primarily been restricted to intermittent bolus injections with limited available options for sustained delivery in vivo. Here, we demonstrate that the cylinder-to-sphere transitions of self-assembled filomicelle (FM) scaffolds can be employed for sustained delivery of monodisperse micellar nanocarriers with improved bioresorptive capacity, modularity for customization, and biocompatibility. These controllable transitions in nanostructure morphology were achieved using oxidation-sensitive poly(ethylene glycol)-bl-poly(propylene sulfide) block copolymer (BCP), which promoted spherical micelle formation in response to changes in surface tension under mild oxidative conditions. Cylinder-to-sphere transitions were characterized in 2D by cryogenic electron microscopy and in 3D by cryo-electron tomography. Modular assembly of FMs from diverse BCP chemistries allowed customization for in situ gelation into porous hydrogel scaffolds following subcutaneous injection into mice. Upon photo- or physiological oxidation, molecular payloads within FMs transferred to intact micellar vehicles during the morphological transition, which was verified in vivo by flow cytometry. FMs composed of multiple distinct BCP fluorescent conjugates permitted multimodal analysis of the scaffold’s non-inflammatory bioresorption and micellar delivery to immune cell populations for up to three months. Mice receiving in situ crosslinked FM-scaffolds exhibited significantly greater uptake within the draining lymph nodes than those receiving injections of non-crosslinked FM or free form fluorescent dye. Specifically, macrophages and both immature and mature dendritic cells exhibited a discernible increase in micelle fluorescence in comparison to free form FM and dye controls. Differences in cell uptake reflected the distinct release rates observed when comparing the slower crosslinked scaffolds and the more rapidly dispersing free form FMs. These scaffolds provide a highly efficient mechanism of bioresorption wherein all components participate in retention and transport of therapeutics, presenting previously unexplored mechanisms for controlled delivery of immunotheranostic nanocarriers for sustained targeting and imaging of lymph node resident immune cell populations.

Antibodies are routinely used as therapeutic agents to fight a wide range of disorders including asthma, blood cancers, breast cancer, arthritis, and transplant rejection. Humoral immunity against infections depends on the germinal center (GC) differentiation process in the B cell follicles of secondary lymphoid organs, such as spleen and lymph nodes. In GCs, B cells rapidly proliferate and somatically mutated high-affinity antibody secreting cells, i.e. plasma cells, are generated from naïve B cells in response to T cell-dependent antigen. To date, the scientific community has relied on animal models to generate high-affinity antibodies and discover fundamental knowledge of GC immunology. Yet we are far from understanding the extracellular and intracellular factors that contribute to the exuberant pace of the GC reaction and conversion to antibody secreting cells (ASCs). Such information would be crucial for developing tranlational therapeutics, understanding the basic cellular nature of humoral immunity, as well as the biology of malignant lymphomas that arise from GC B cells and manifest their highly proliferative phenotype. A major impediment to answering such immunological questions is that GC cells in a mouse model is highly heterogenous and mouse models with silenced epigenetic genes do not develop GCs. Infusion of epigenetic inhibitors cannot enable these studies because GCs are heterogeneous with varying cell cycle phases. Therefore, there is a need for tractable, ex vivo platform that can induce bona fide GC reaction and synchronize nearly all cells in the same GC phase. Here we report the development of hydrogel and nanomaterials based ex vivo immune organoids [1, 2] that recapitulate the complex immunobiology of wild type and transgenic knockout mice, and use this immune tissue model to show that Enhancer of zeste homolog 2 (EZH2) histone methytransferase mediates GC formation through repression of cyclin-dependent kinase inhibitor CDKN1A (p21^Cip1). We next describe development of a designer immune organoid, where hydrogels model T cell-like selection process to generate high affinity antibody secreting cells.


[1] A. Purwada, A. Singh, Immuno-engineered Organoids for Regulating the Kinetics of B cell Development and Antibody Production, Nature Protocols 12 (2017) 168-182.

[2] W. Beguelin, M.A. Rivas, M.T. Calvo Fernandez, M. Teater, A. Purwada, D. Redmond, H. Shen, M.F. Challman, O. Elemento, A. Singh, A.M. Melnick, EZH2 enables germinal centre formation through epigenetic silencing of CDKN1A and an Rb-E2F1 feedback loop, Nature Communications 8(1) (2017) 877.

Advances in immuno-biomaterials are providing increasing control of immunity both in vitro and in vivo. Organoids and 3D cultures begin to mimic functions such as antibody production, while biomaterials-based immunotherapies modulate immune responses in animals, often acting directly in the lymph node. A critical element of the immune system that is challenging to incorporate into the design of such materials is tissue-level spatial organization. Cells and extracellular matrix elements in the lymph node, spleen, and thymus are all highly organized. It is thought that spatial proximity, coupled with diffusion and interstitial flow, ensures that secreted signals such as cytokines and chemokines arrive at their target cell in the time required for immunity. Therefore, methods are needed to control the spatial patterning of 3D cultures and to test the role of spatially targeted biomaterials-based therapies.

Here, we report the development of a microfluidic method for chemical stimulation of engineered 3D cultures and live tissue sections on the regional scale. A microfluidic chip was built with 1 – 10 parallel channels running beneath a tissue culture chamber. Each channel connected to a single vertical port, providing focal stimulation to a hydrogel slab or tissue sample in the culture chamber. This system enabled precise control over quantity delivered and timing of delivery to the sample, as demonstrated by delivering fluorescent dextrans with 200 – 300 micron resolution into a live slice of murine lymph node. This resolution was sufficient to target functional regions such as a B cell follicle or the T cell zone. The method was compatible with live fluorescent imaging, making it possible to deliver fluorescently-labeled bioactive proteins into live tissue in order to measure diffusion coefficients and tissue tortuosity. More recently, we have redesigned the device to make the port mobile beneath the tissue, so that the region of stimulation could be chosen on-demand and even moved during the experiment by the user. We anticipate that microfluidic local delivery will be used to create spatially patterned protein distributions in engineered 3D cultures, as well as to test the effects of immuno-modulating materials in specific regions of the lymph node. Combining engineered biomaterials with the fluidic and spatial control of microfluidics promises a new level of insight and control over the spatiotemporal dynamics of inflammation and immunity.

A robust experimental model of a lymph node (LN) is imperative for the better understanding of mechanistic processes underlying immunity in health and disease. A 3D cell culture on a microfluidic chip is an ideal middle-ground to mimic in vivo responses while providing the advantages of an in vitro cell culture. Such a model must include the necessary cell-cell and cell-matrix interactions required to sufficiently generate immune responses, and should also integrate both supportive biomaterials and fluidic control.
The LN is comprised of a compartmentalized architecture; in a simplified view, it has three main regions: the sub-capsular sinus, the B cell follicles, and the deep para-cortex (both T and Dendritic cells zone). To replicate this structure, we generated a micro-patterned 3D cell culture of primary murine splenocytes inside of a microfluidic housing, creating the first 3D patterned LN-on-chip. The platform was designed to be customizable, allowing control over cellular distribution, matrix composition in different regions, and allows both incoming and interstitial fluid flow. The LN-on-chip was composed of two microfluidics “afferent lymphatic” channels, a central culture chamber, and one microfluidic “efferent lymphatic” channel. Inside the culture chamber, the hydrogel was excluded from the peripheral area through the use of micro-fabricated posts, creating a LN “sinus.” We used UV-patterning through a photomask to pattern a human-sized LN (10 mm diameter, 100 μm thick), composed of two to six 400-µm diameter “B cell follicles,” surrounded by a “para-cortex” region containing a distinct population of cells. A hydrogel matrix, 8% gelatin methacrylate cross-linked with 0.1% and lithium phenyl-2,4,6-trimethylbenzoylphosphinate, was selected to match the mechanical properties of healthy lymph nodes. We found that the patterning process retained high cell viability and basic cellular function such as cell migration. We also achieved the same fluid flow velocities as observed in vivo for incoming (5 - 6 μm/s) and interstitial (0.10 - 1 μm/s) fluid flows.
In summary, we describe a novel LN-on-chip that contains the cell types, spatial patterning, and fluid flow rates required to reproduce simple inflammatory and adaptive immune responses. In the future, this modular platform will be used to directly test hypotheses of how spatial structure and fluid flow patterns affect efficacy of an immune response.

Nitric oxide (NO) is a therapeutic implicated for the treatment of a variety of pathologies that afflict lymphatic tissues, ranging from cardiovascular and infectious diseases to cancer. Existing technologies available for NO therapy, however, provide poor bioactivity within lymphatic tissues. In this work, we address this technology gap with a NO encapsulation and delivery strategy leveraging the formation of S-nitrosothiols on lymphatic-targeting Pluronic-stabilized, poly(propylene sulfide)-core nanoparticles (SNO-NP). In an in vivo lymphatic delivery murine model, we evaluated the lymphatic versus systemic delivery of NO resulting from intradermal administration of SNO-NP, examined signs of toxicity systemically as well as localized to the site of injection, and benchmarked against a commonly used commercially available small molecule S-nitrosothiol NO donor. We found SNO-NP to facilitate the controlled and sustained delivery of NO to LN, in contrast to a small molecule NO donor. As a result, over 72 hr post injection, SNO-NP increased the delivery of SNO to LN by two orders of magnitude. Treatment also resulted in dramatic increases in the abundance of LN-resident cells despite no apparent LN cell death associated with treament nor signs of systemic toxicity after administration. Since NO has shown in vitro/in vivo promise in lymphatic-related cancer therapy and infectious disease applications but has been unable to progress due to delivery-related challenges, with further development this NO delivery technology has the potential to advance such NO-based therapeutic appraoches through enhancement of lymphatic targeting.