Lymph node fibroblastic reticular cells steer immune responses.

Lymph nodes (LNs), where immune responses are initiated, are organized into distinctive compartments by fibroblastic reticular cells (FRCs). FRCs imprint immune responses by supporting LN architecture, recruiting immune cells, coordinating immune cell crosstalk, and presenting antigens. Recent high-resolution transcriptional and histological analyses have enriched our knowledge of LN FRC genetic and spatial heterogeneities. Here, we summarize updated anatomic, phenotypic, and functional identities of FRC subsets, delve into topological and transcriptional remodeling of FRCs in inflammation, and illustrate the crosstalk between FRCs and immune cells. Discussing FRC functions in immunity and tolerance, we highlight state-of-the-art FRC-based therapeutic approaches for maintaining physiological homeostasis, steering protective immunity, inducing transplantation tolerance, and treating diverse immune-related diseases.

Role of lymph node stroma and microenvironment in T cell tolerance.

Lymph nodes (LNs) are at the cross roads of immunity and tolerance. These tissues are compartmentalized into specialized niche areas by lymph node stromal cells (LN SCs). LN SCs shape the LN microenvironment and guide immunological cells into different zones through establishment of a CCL19 and CCL21 gradient. Following local immunological cues, LN SCs modulate activity to support immune cell priming, activation, and fate. This review will present our current understanding of LN SC subsets roles in regulating T cell tolerance. Three major types of LN SC subsets, namely fibroblastic reticular cells, lymphatic endothelial cells, and blood endothelial cells, are discussed. These subsets serve as scaffolds to support and regulate T cell homeostasis. They contribute to tolerance by presenting peripheral tissue antigens to both CD4 and CD8 T cells. The role of LN SCs in regulating T cell migration and tolerance induction is discussed. Looking forward, recent advances in bioengineered materials and approaches to leverage LN SCs to induce T cell tolerance are highlighted, as are current clinical practices that allow for manipulation of the LN microenvironment to induce tolerance. Increased understanding of LN architecture, how different LN SCs integrate immunological cues and shape immune responses, and approaches to induce T cell tolerance will help further combat autoimmune diseases and graft rejection.

Microtubule disruption reduces metastasis more effectively than primary tumor growth.

Clinical cancer imaging focuses on tumor growth rather than metastatic phenotypes. The microtubule-depolymerizing drug, Vinorelbine, reduced the metastatic phenotypes of microtentacles, reattachment and tumor cell clustering more than tumor cell viability. Treating mice with Vinorelbine for only 24 h had no significant effect on primary tumor survival, but median metastatic tumor survival was extended from 8 to 30 weeks. Microtentacle inhibition by Vinorelbine was also detectable within 1 h, using tumor cells isolated from blood samples. As few as 11 tumor cells were sufficient to yield 90% power to detect this 1 h Vinorelbine drug response, demonstrating feasibility with the small number of tumor cells available from patient biopsies. This study establishes a proof-of-concept that targeted microtubule disruption can selectively inhibit metastasis and reveals that existing FDA-approved therapies could have anti-metastatic actions that are currently overlooked when focusing exclusively on tumor growth.

Biophysical Properties of Self-Assembled Immune Signals Impact Signal Processing and the Nature of Regulatory Immune Function.

Outcomes during immunotherapy are impacted not only by the specific therapeutic signals and pharmacodynamics, but also by the biophysical forms in which signals are delivered. This integration is determinative in autoimmunity because the disease is caused by immune dysregulation and inflammation. Unfortunately, the links between nanomaterial design, biophysical properties, and immune regulation are poorly defined. Here we designed cationic peptide antigens with defined charge distributions and then used electrostatics to assemble these peptides into complexes with anionic regulatory cues. We first show complexes induce antigen-specific tolerance during myelin-driven autoimmunity. We next show the affinity between these immune cues is controlled by charge balance and that affinity confers distinct biophysical properties important in immunological processing, including antigen availability. The underlying binding affinities between the self-assembled signals influences inflammatory gene expression in dendritic cells and antigen-specific regulatory outcomes in self-reactive transgenic T cells. This granular understanding of nanomaterial-immune interactions contributes to a more rational immunotherapy design.

Histatin 5 variant reduces Candida albicans biofilm viability and inhibits biofilm formation.

Candida albicans is a commensal organism and opportunistic pathogen that can form biofilms that colonize surfaces of medical devices, such as implants, catheters, and dentures. Compared to planktonic C. albicans cells, cells in biofilms exhibit increased resistance to treatment. Histatin 5 (Hst-5) is an antimicrobial peptide that is natively secreted by human salivary glands and has strong antifungal activity against C. albicans. However, C. albicans produces secreted aspartic proteases (Saps) that can cleave and inactivate Hst-5, limiting its antifungal properties. We previously showed that residue substitutions K11R and K17R within Hst-5 improve its antifungal activity and prevent proteolytic degradation by Saps when treating planktonic C. albicans. Here, we investigated the use of the K11R-K17R peptide as an alternative therapeutic against C. albicans biofilms by assessing its ability to reduce viability of pre-formed biofilms and to inhibit the formation of biofilms and showed that K11R-K17R had improved activity compared to Hst-5. Based on these results, we incorporated K11R-K17R and Hst-5 into polyelectrolyte multilayer (PEM) surface coatings and demonstrated that films functionalized with K11R-K17R reduced the formation of C. albicans biofilms. Our results demonstrate the therapeutic potential of the K11R-K17R Hst-5 variant in preventing and treating biofilms.

Self-Assembly as a Molecular Strategy to Improve Immunotherapy.

Immunotherapies harness an individual's immune system to battle diseases such as cancer and autoimmunity. During cancer, the immune system often fails to detect and destroy cancerous cells, whereas during autoimmune disease, the immune system mistakenly attacks self-tissue. Immunotherapies can help guide more effective responses in these settings, as evidenced by recent advances with monoclonal antibodies and adoptive cell therapies. However, despite the transformative gains of immunotherapies for patients, many therapies are not curative, work only for a small subset of patients, and lack specificity in distinguishing between healthy and diseased cells, which can cause severe side effects. From this perspective, self-assembled biomaterials are promising technologies that could help address some of the limitations facing immunotherapies. For example, self-assembly allows precision control over the combination and relative concentration of immune cues and directed cargo display densities. These capabilities support selectivity and potency that could decrease off-target effects and enable modular or personalized immunotherapies. The underlying forces driving self-assembly of most systems in aqueous solution result from hydrophobic interactions or charge polarity. In this Account, we highlight how these forces are being used to self-assemble immunotherapies for cancer and autoimmune disease.Hydrophobic interactions can create a range of intricate structures, including peptide nanofibers, nanogels, micelle-like particles, and in vivo assemblies with protein carriers. Certain nanofibers with hydrophobic domains uniquely benefit from the ability to elicit immune responses without additional stimulatory signals. This feature can reduce nonspecific inflammation but may also limit the nanofiber's application because of their inherent stimulatory properties. Micelle-like particles have been developed with the ability to incorporate a range of tumor-specific antigens for immunotherapies in mouse models of cancer. Key observations have revealed that both the total dose of antigen and display density of antigen per particle can impact immune response and efficacy of immunotherapies. These developments are promising benchmarks that could reveal design principles for engineering more specific and personalized immunotherapies.There has also been extensive work to develop platforms using electrostatic interactions to drive assembly of oppositely charged immune signals. These strategies benefit from the ability to tune biophysical interactions between components by altering the ratio of cationic to anionic charge during formulation, or the density of charge. Using a layer-by-layer assembly method, our lab developed hollow capsules composed entirely of immune signals for therapies in cancer and autoimmune disease models. This platform allowed for 100% of the immunotherapy to be composed of immune signals and completely prevents the onset of disease in a mouse model of multiple sclerosis. Layer-by-layer assembly has also been used to coat microneedle patches to target signals to immune cells in the dermal layer. As an alternative to layer-by-layer assembly, one step assembly can be achieved by mixing cationic and anionic components in solution. Additional approaches have created molecular structures that leverage hydrogen bonding for self-assembly. The creativity of engineered self-assembly has led to key insights that could benefit future immunotherapies and revealed aspects that require further study. The challenge now remains to utilize these insights to push development of new immunotherapeutics into clinical settings.

Improving Vaccine and Immunotherapy Design Using Biomaterials.

Polymers, lipids, scaffolds, microneedles, and other biomaterials are rapidly emerging as technologies to improve the efficacy of vaccines against infectious disease and immunotherapies for cancer, autoimmunity, and transplantation. New studies are also providing insight into the interactions between these materials and the immune system. This insight can be exploited for more efficient design of vaccines and immunotherapies. Here, we describe recent advances made possible through the unique features of biomaterials, as well as the important questions for further study.

Leveraging the modularity of biomaterial carriers to tune immune responses.

Biomaterial carriers offer modular features to control the delivery and presentation of vaccines and immunotherapies. This tunability is a distinct capability of biomaterials. Understanding how tunable material features impact immune responses is important to improve vaccine and immunotherapy design, as well as clinical translation. Here we discuss the modularity of biomaterial properties as a means of controlling encounters with immune signals across scales - tissue, cell, molecular, and time - and ultimately, to direct stimulation or regulation of immune function. We highlight these advances using illustrations from recent literature across infectious disease, cancer, and autoimmunity. As the immune engineering field matures, informed design criteria could support more rational biomaterial carriers for vaccination and immunotherapy.

Designing natural and synthetic immune tissues.

Vaccines and immunotherapies have provided enormous improvements for public health, but there are fundamental disconnects between where most studies are performed-in cell culture and animal models-and the ultimate application in humans. Engineering immune tissues and organs, such as bone marrow, thymus, lymph nodes and spleen, could be instrumental in overcoming these hurdles. Fundamentally, designed immune tissues could serve as in vitro tools to more accurately study human immune function and disease, while immune tissues engineered for implantation as next-generation vaccines or immunotherapies could enable direct, on-demand control over generation and regulation of immune function. In this Review, we discuss recent interdisciplinary strategies that are merging materials science and immunology to create engineered immune tissues in vitro and in vivo. We also highlight the hurdles facing these approaches and the need for comparison to existing clinical options, relevant animal models, and other emerging technologies.

In Vivo Intradermal Delivery of Bacteria by Using Microneedle Arrays.

Infectious diseases propagated by arthropod vectors, such as tularemia, are commonly initiated via dermal infection of the skin. However, due to the technical difficulties in achieving accurate and reproducible dermal deposition, intradermal models are less commonly used. To overcome these limitations, we used microneedle arrays (MNAs), which are micron-scale polymeric structures, to temporarily disrupt the barrier function of the skin and deliver a bacterial inoculum directly to the dermis of an animal. MNAs increase reliability by eliminating leakage of the inoculum or blood from the injection site, thereby providing a biologically relevant model for arthropod-initiated disease. Here, we validate the use of MNAs as a means to induce intradermal infection using a murine model of tularemia initiated by Francisella novicida We demonstrate targeted delivery of the MNA bolus to the dermal layer of the skin, which subsequently led to innate immune cell infiltration. Additionally, F. novicida-coated MNAs were used to achieve lethality in a dose-dependent manner in C57BL/6 mice. The immune profile of infected mice mirrored that of established F. novicida infection models, consisting of markedly increased serum levels of interleukin-6 and keratinocyte chemoattractant, splenic T-cell depletion, and an increase in splenic granulocytes, together confirming that MNAs can be used to reproducibly induce tularemia-like pathogenesis in mice. When MNAs were used to immunize mice using an attenuated F. novicida mutant (F. novicida ΔlpxD1), all immunized mice survived a lethal subcutaneous challenge. Thus, MNAs can be used to effectively deliver viable bacteria in vivo and provide a novel avenue to study intradermally induced microbial diseases in animal models.

Self-Assembly of Immune Signals Improves Codelivery to Antigen Presenting Cells and Accelerates Signal Internalization, Processing Kinetics, and Immune Activation.

Vaccines and immunotherapies that elicit specific types of immune responses offer transformative potential to tackle disease. The mechanisms governing the processing of immune signals-events that determine the type of response generated-are incredibly complex. Understanding these processes would inform more rational vaccine design by linking carrier properties, processing mechanisms, and relevant timescales to specific impacts on immune response. This goal is pursued using nanostructured materials-termed immune polyelectrolyte multilayers-built entirely from antigens and stimulatory toll-like receptors agonists (TLRas). This simplicity allows isolation and quantification of the rates and mechanisms of intracellular signal processing, and the link to activation of distinct immune pathways. Each vaccine component is internalized in a colocalized manner through energy-dependent caveolae-mediated endocytosis. This process results in trafficking through endosome/lysosome pathways and stimulation of TLRs expressed on endosomes/lysosomes. The maximum rates for these events occur within 4 h, but are detectable in minutes, ultimately driving downstream proimmune functions. Interestingly, these uptake, processing, and activation kinetics are significantly faster for TLRas in particulate form compared with free TLRa. Our findings provide insight into specific mechanisms by which particulate vaccines enhance initiation of immune response, and highlight quantitative strategies to assess other carrier technologies.

Harnessing the lymph node microenvironment.

PURPOSE OF REVIEW: To evaluate role of the lymph node in immune regulation and tolerance in transplantation and recent advances in the delivery of antigen and immune modulatory signals to the lymph node. RECENT FINDINGS: Lymph nodes are a primary site of immune cell priming, activation, and modulation, and changes within the lymph node microenvironment have the potential to induce specific regulation, suppression, and potentially tolerance. Antigen enters the lymph node either from tissues via lymphatics, from blood via high endothelial venules, or directly via injection. Here we review different techniques and materials to deliver antigen to the lymph node including microparticles or nanoparticles, ex-vivo antigen presenting cell manipulation, and use of receptor conjugation for specific intralymph node targeting locations. SUMMARY: The promising results point to powerful techniques to harness the lymph node microenvironment and direct systemic immune regulation. The materials, techniques, and approaches suggest that translational and clinical trials in nonhuman primate and patients may soon be possible.

Engineering Immunological Tolerance Using Quantum Dots to Tune the Density of Self-Antigen Display.

Treatments for autoimmunity - diseases where the immune system mistakenly attacks self-molecules - are not curative and leave patients immunocompromised. New studies aimed at more specific treatments reveal development of inflammation or tolerance is influenced by the form self-antigens are presented. Using a mouse model of multiple sclerosis (MS), we show for the first time that quantum dots (QDs) can be used to generate immunological tolerance by controlling the density of self-antigen on QDs. These assemblies display dense arrangements of myelin self-peptide associated with disease in MS, are uniform in size (<20 nm), and allow direct visualization in immune tissues. Peptide-QDs rapidly concentrate in draining lymph nodes, co-localizing with macrophages expressing scavenger receptors involved in tolerance. Treatment with peptide-QDs reduces disease incidence 10-fold. Strikingly, the degree of tolerance - and the underlying expansion of regulatory T cells - correlates with the density of myelin molecules presented on QDs. A key discovery is that higher numbers of tolerogenic particles displaying lower levels of self-peptide are more effective for inducing tolerance than fewer particles each displaying higher densities of peptide. QDs conjugated with self-antigens could serve as a new platform to induce tolerance, while visualizing QD therapeutics in tolerogenic tissue domains.

Impact of dose, route, and composition on the immunogenicity of immune polyelectrolyte multilayers delivered on gold templates.

Biomaterial vaccines offer new capabilities that can be exploited for both infectious disease and cancer. We recently developed a novel vaccine platform based on self-assembly of immune signals into immune polyelectrolyte multilayers (iPEMs). These iPEM vaccines are electrostatically assembled from peptide antigens and nucleic acid-based toll-like receptor agonists (TLRas) that serve as molecular adjuvants. Gold nanoparticles (AuNPs) coated with iPEMs stimulate effector cytokine secretion in vitro and expand antigen-specific T cells in mice. Here we investigated how the dose, injection route, and choice of molecular adjuvant impacts the ability of iPEMs to generate T cell immunity and anti-tumor response in mice. Three injection routes-intradermal, subcutaneous, and intramuscular-and three iPEM dosing levels were employed. Intradermal injection induced the most potent antigen-specific T cell responses and, for all routes, the level of response was dose-dependent. We further discovered that these vaccines generate durable memory, indicated by potent, antigen-specific CD8+ T cell recall responses in mice challenged with vaccine 49 days after a prime-boost immunization regimen. In a common exogenous antigen melanoma model, iPEM vaccines slowed or stopped tumor growth more effectively than equivalent ad-mixed formulations. Further, iPEMs containing CpG-a TLR9a-were more potent compared with iPEMs containing polyIC, a TLR3a. These findings demonstrate the ability of iPEMs to enhance response to several different classes of vaccine cargos, supporting iPEMs as a simple vaccine platform that mimics attractive features of other nanoparticles using immune signals that can be self-assembled or coated on substrates. Biotechnol. Bioeng. 2017;114: 423-431. © 2016 Wiley Periodicals, Inc.

Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia.

Acetyl coenzyme A (AcCoA) is the central biosynthetic precursor for fatty-acid synthesis and protein acetylation. In the conventional view of mammalian cell metabolism, AcCoA is primarily generated from glucose-derived pyruvate through the citrate shuttle and ATP citrate lyase in the cytosol. However, proliferating cells that exhibit aerobic glycolysis and those exposed to hypoxia convert glucose to lactate at near-stoichiometric levels, directing glucose carbon away from the tricarboxylic acid cycle and fatty-acid synthesis. Although glutamine is consumed at levels exceeding that required for nitrogen biosynthesis, the regulation and use of glutamine metabolism in hypoxic cells is not well understood. Here we show that human cells use reductive metabolism of α-ketoglutarate to synthesize AcCoA for lipid synthesis. This isocitrate dehydrogenase-1 (IDH1)-dependent pathway is active in most cell lines under normal culture conditions, but cells grown under hypoxia rely almost exclusively on the reductive carboxylation of glutamine-derived α-ketoglutarate for de novo lipogenesis. Furthermore, renal cell lines deficient in the von Hippel-Lindau tumour suppressor protein preferentially use reductive glutamine metabolism for lipid biosynthesis even at normal oxygen levels. These results identify a critical role for oxygen in regulating carbon use to produce AcCoA and support lipid synthesis in mammalian cells.

In situ engineering of the lymph node microenvironment via intranodal injection of adjuvant-releasing polymer particles.

Recent studies have demonstrated a simple, potentially universal strategy to enhance vaccine potency, via intralymph node (i.LN) injection. To date, intranodal immunization studies have focused on the delivery of unadjuvanted vaccines (e.g., naked DNA, peptide, or protein). We hypothesized that combining i.LN vaccination with controlled release biomaterials permitting sustained dosing of molecular adjuvants to the local tissue microenvironment would further enhance this promising vaccination strategy. To test this idea, we encapsulated the Toll-like receptor-3 ligand poly(inosinic:cytidylic acid) (polyIC) in biodegradable poly(lactide-co-glycolide) microparticles (MPs) designed to remain extracellular and release polyIC in the LN over several days. Intranodal injection of MPs increased persistence of polyIC in LNs compared to the same dose of soluble polyIC or polyIC formulated in nanoparticles, leading to increased accumulation of Toll-like receptor agonist in LN-resident antigen presenting cells and more enduring dendritic cell activation. Intralymph node injection of ovalbumin mixed with polyIC-releasing MPs enhanced the humoral response and expanded ovalbumin-specific T cells to frequencies as high as 18% among all CD8(+) cells following a single injection (8.2-fold greater than the same vaccine given i.m.), a response that could not be matched by antigen mixed with polyIC-loaded nanoparticles or a 10-fold greater dose of soluble polyIC. Thus, i.LN immunization with slow release-formulated adjuvants may be a broadly applicable strategy to enhance therapeutic or prophylactic vaccines.

Engineering metabolism to modulate immunity.

Metabolic programming and reprogramming have emerged as pivotal mechanisms for altering immune cell function. Thus, immunometabolism has become an attractive target area for treatment of immune-mediated disorders. Nonetheless, many hurdles to delivering metabolic cues persist. In this review, we consider how biomaterials are poised to transform manipulation of immune cell metabolism through integrated control of metabolic configurations to affect outcomes in autoimmunity, regeneration, transplant, and cancer. We emphasize the features of nanoparticles and other biomaterials that permit delivery of metabolic cues to the intracellular compartment of immune cells, or strategies for altering signals in the extracellular space. We then provide perspectives on the potential for reciprocal regulation of immunometabolism by the physical properties of materials themselves. Lastly, opportunities for clinical translation are highlighted. This discussion contributes to our understanding of immunometabolism, biomaterials-based strategies for altering metabolic configurations in immune cells, and emerging concepts in this evolving field.

Advancing immunotherapy using biomaterials to control tissue, cellular, and molecular level immune signaling in skin.

Immunotherapies have been transformative in many areas, including cancer treatments, allergies, and autoimmune diseases. However, significant challenges persist in extending the reach of these technologies to new indications and patients. Some of the major hurdles include narrow applicability to patient groups, transient efficacy, high cost burdens, poor immunogenicity, and side effects or off-target toxicity that results from lack of disease-specificity and inefficient delivery. Thus, there is a significant need for strategies that control immune responses generated by immunotherapies while targeting infection, cancer, allergy, and autoimmunity. Being the outermost barrier of the body and the first line of host defense, the skin presents a unique immunological interface to achieve these goals. The skin contains a high concentration of specialized immune cells, such as antigen-presenting cells and tissue-resident memory T cells. These cells feature diverse and potent combinations of immune receptors, providing access to cellular and molecular level control to modulate immune responses. Thus, skin provides accessible tissue, cellular, and molecular level controls that can be harnessed to improve immunotherapies. Biomaterial platforms - microneedles, nano- and micro-particles, scaffolds, and other technologies - are uniquely capable of modulating the specialized immunological niche in skin by targeting these distinct biological levels of control. This review highlights recent pre-clinical and clinical advances in biomaterial-based approaches to target and modulate immune signaling in the skin at the tissue, cellular, and molecular levels for immunotherapeutic applications. We begin by discussing skin cytoarchitecture and resident immune cells to establish the biological rationale for skin-targeting immunotherapies. This is followed by a critical presentation of biomaterial-based pre-clinical and clinical studies aimed at controlling the immune response in the skin for immunotherapy and therapeutic vaccine applications in cancer, allergy, and autoimmunity.

Extracellular vesicles as carriers of mRNA: Opportunities and challenges in diagnosis and treatment.

Extracellular vesicles (EVs) are produced by all cells in the body. These biological nanoparticles facilitate cellular communication through the transport of diverse cargoes, including small molecules, proteins, and nucleic acids. mRNA cargoes have gained particular interest given their role in the translation of functional proteins. As a biomarker platform, EVs can be found in nearly all biofluids-blood, mucus, urine, cerebrospinal fluid, and saliva-providing real-time insight into parent cell and tissue function. mRNAs carried by EVs are protected from degradation, resulting in improved detection compared to free mRNA, and recent work demonstrates promising results in using these mRNA cargoes as biomarkers for cancer, neurological diseases, infectious diseases, and gynecologic and obstetric outcomes. Furthermore, given the innate cargo carrying, targeting, and barrier crossing abilities of EVs, these structures have been proposed as therapeutic carriers of mRNA. Recent advances demonstrate methods for loading mRNAs into EVs for a range of disease indications. Here, we review recent studies using EVs and their mRNA cargoes as diagnostics and therapeutics. We discuss challenges associated with EVs in diagnostic and therapeutic applications and highlight opportunities for future development.