Cytokine conjugation to enhance T cell therapy.

Adoptive T cell transfer (ACT) therapies suffer from a number of limitations (e.g., poor control of solid tumors), and while combining ACT with cytokine therapy can enhance effectiveness, this also results in significant side effects. Here, we describe a nanotechnology approach to improve the efficacy of ACT therapies by metabolically labeling T cells with unnatural sugar nanoparticles, allowing direct conjugation of antitumor cytokines onto the T cell surface during the manufacturing process. This allows local, concentrated activity of otherwise toxic cytokines. This approach increases T cell infiltration into solid tumors, activates the host immune system toward a Type 1 response, encourages antigen spreading, and improves control of aggressive solid tumors and achieves complete blood cancer regression with otherwise noncurative doses of CAR-T cells. Overall, this method provides an effective and easily integrated approach to the current ACT manufacturing process to increase efficacy in various settings.

STING activation promotes robust immune response and NK cell-mediated tumor regression in glioblastoma models.

Immunotherapy has had a tremendous impact on cancer treatment in the past decade, with hitherto unseen responses at advanced and metastatic stages of the disease. However, the aggressive brain tumor glioblastoma (GBM) is highly immunosuppressive and remains largely refractory to current immunotherapeutic approaches. The stimulator of interferon genes (STING) DNA sensing pathway has emerged as a next-generation immunotherapy target with potent local immune stimulatory properties. Here, we investigated the status of the STING pathway in GBM and the modulation of the brain tumor microenvironment (TME) with the STING agonist ADU-S100. Our data reveal the presence of STING in human GBM specimens, where it stains strongly in the tumor vasculature. We show that human GBM explants can respond to STING agonist treatment by secretion of inflammatory cytokines. In murine GBM models, we show a profound shift in the tumor immune landscape after STING agonist treatment, with massive infiltration of the tumor-bearing hemisphere with innate immune cells including inflammatory macrophages, neutrophils, and natural killer (NK) populations. Treatment of established murine intracranial GL261 and CT-2A tumors by biodegradable ADU-S100-loaded intracranial implants demonstrated a significant increase in survival in both models and long-term survival with immune memory in GL261. Responses to treatment were abolished by NK cell depletion. This study reveals therapeutic potential and deep remodeling of the TME by STING activation in GBM and warrants further examination of STING agonists alone or in combination with other immunotherapies such as cancer vaccines, chimeric antigen receptor T cells, NK therapies, and immune checkpoint blockade.

Mechanical checkpoint regulates monocyte differentiation in fibrotic niches.

Myelofibrosis is a progressive bone marrow malignancy associated with monocytosis, and is believed to promote the pathological remodelling of the extracellular matrix. Here we show that the mechanical properties of myelofibrosis, namely the liquid-to-solid properties (viscoelasticity) of the bone marrow, contribute to aberrant differentiation of monocytes. Human monocytes cultured in stiff, elastic hydrogels show proinflammatory polarization and differentiation towards dendritic cells, as opposed to those cultured in a viscoelastic matrix. This mechanically induced cell differentiation is blocked by inhibiting a myeloid-specific isoform of phosphoinositide 3-kinase, PI3K-γ. We further show that murine bone marrow with myelofibrosis has a significantly increased stiffness and unveil a positive correlation between myelofibrosis grading and viscoelasticity. Treatment with a PI3K-γ inhibitor in vivo reduced frequencies of monocyte and dendritic cell populations in murine bone marrow with myelofibrosis. Moreover, transcriptional changes driven by viscoelasticity are consistent with transcriptional profiles of myeloid cells in other human fibrotic diseases. These results demonstrate that a fibrotic bone marrow niche can physically promote a proinflammatory microenvironment.

Targeting tumor extracellular matrix activates the tumor-draining lymph nodes.

Disruption of the tumor extracellular matrix (ECM) may alter immune cell infiltration into the tumor and antitumor T cell priming in the tumor-draining lymph nodes (tdLNs). Here, we explore how intratumoral enzyme treatment (ET) of B16 melanoma tumors with ECM-depleting enzyme hyaluronidase alters adaptive and innate immune populations, including T cells, DCs, and macrophages, in the tumors and tdLNs. ET increased CD103+ DC abundance in the tdLNs, as well as antigen presentation of a model tumor antigen ovalbumin (OVA), eliciting local OVA-specific CD8+ T cell responses. Delivered in combination with a distant cryogel-based cancer vaccine, ET increased the systemic antigen-specific CD8+ T cell response. By enhancing activity within the tdLN, ET may broadly support immunotherapies in generating tumor-specific immunity.

Nanoparticle Properties Influence Transendothelial Migration of Monocytes.

Nanoparticle-based delivery of therapeutics to the brain has had limited clinical impact due to challenges crossing the blood-brain barrier (BBB). Certain cells, such as monocytes, possess the ability to migrate across the BBB, making them attractive candidates for cell-based brain delivery strategies. In this work, we explore nanoparticle design parameters that impact both monocyte association and monocyte-mediated BBB transport. We use electrohydrodynamic jetting to prepare nanoparticles of varying sizes, compositions, and elasticity to address their impact on uptake by THP-1 monocytes and permeation across the BBB. An in vitro human BBB model is developed using human cerebral microvascular endothelial cells (hCMEC/D3) for the assessment of migration. We compare monocyte uptake of both polymeric and synthetic protein nanoparticles (SPNPs) of various sizes, as well as their effect on cell migration. SPNPs (human serum albumin/HSA or human transferrin/TF) are shown to promote increased monocyte-mediated transport across the BBB over polymeric nanoparticles. TF SPNPs (200 nm) associate readily, with an average uptake of 138 particles/cell. Nanoparticle loading is shown to influence the migration of THP-1 monocytes. The migration of monocytes loaded with 200 nm TF and 200 nm HSA SPNPs was 2.3-fold and 2.1-fold higher than that of an untreated control. RNA-seq analysis after TF SPNP treatment suggests that the upregulation of several migration genes may be implicated in increased monocyte migration (ex. integrin subunits α M and α L). Integrin ß 2 chain combines with either integrin subunit α M chain or integrin subunit α L chain to form macrophage antigen 1 and lymphocyte function-associated antigen 1 integrins. Both products play a pivotal role in the transendothelial migration cascade. Our findings highlight the potential of SPNPs as drug and/or gene delivery platforms for monocyte-mediated BBB transport, especially where conventional polymer nanoparticles are ineffective or otherwise not desirable.

Biomaterials as Local Niches for Immunomodulation.

A major function of the immune system is to detect threat from foreign invaders, tissue damage, or cancer and to mount a counter response that resolves the threat, restores homeostasis, and supplies immunological memory to prevent a second assault. Our increasing understanding of the immune system has opened up numerous avenues for modulating immune responses against infections, cancer, and autoimmunity. However, agents used for immunomodulation have been traditionally administered systemically via bolus injection, leading to unintended consequences by disrupting homeostasis at nontarget sites. Consequently, systemic hyperactivation and hypoactivation can result from bolus administration of immune-activators and immunosuppressants, respectively. Macroscale biomaterial scaffolds can instead be placed at the intended target site to provide both localized, controlled release of immunomodulatory agents and control over local immune cell trafficking and function, potentially maximizing therapeutic efficacy and limiting systemic exposure. These scaffolds have found utility in the area of cancer immunotherapy, especially in situ cancer vaccination where controlled release of factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and the local presentation of tumor antigen and danger signals lead to the recruitment of immature dendritic cells and facilitate their activation and antigen presentation. These cells eventually migrate into secondary lymphoid organs where they prime tumor specific T cells for downstream tumor clearance. Scaffolds can also be used in adoptive T cell therapy to generate large numbers of potent antigen specific T cells or chimeric antigen receptor (CAR) T cells in vitro for subsequent delivery to patients. Macroscale biomaterial scaffolds have also found utility beyond cancer immunotherapy and have been developed to promote immune tolerance by regulatory T cell induction and to expedite tissue regeneration. The design of these macroscale biomaterial scaffolds considers their biocompatibility, biodegradability, mode of delivery, porosity, and kinetics of therapeutic cargo release. Consequently, the numerous approaches that have been developed to fabricate biomaterial scaffolds are aimed at tuning these parameters to achieve the desired therapeutic outcome. This Account will discuss the use of biomaterial scaffolds as niches for immunomodulation and will focus on (1) approaches that have been used to fabricate various biomaterial systems being employed as niches for immunomodulation and (2) how these biomaterial systems have been used to modulate immune responses, specifically in area of cancer immunotherapy, where we will discuss the role of macroscale biomaterial scaffolds for in situ vaccination and in vitro T cell expansion. We will also briefly discuss the utility of biomaterial scaffolds beyond cancer, drawing examples from tolerance and tissue regeneration.

Subcutaneous biodegradable scaffolds for restimulating the antitumour activity of pre-administered CAR-T cells.

The efficacy of adoptive T-cell therapies based on chimaeric antigen receptors (CARs) is limited by the poor proliferation and persistence of the engineered T cells. Here we show that a subcutaneously injected biodegradable scaffold that facilitates the infiltration and egress of specific T-cell subpopulations, which forms a microenvironment mimicking features of physiological T-cell activation, enhances the antitumour activity of pre-administered CAR-T cells. CAR-T-cell expansion, differentiation and cytotoxicity were driven by the scaffold's incorporation of co-stimulatory bound ligands and soluble molecules, and depended on the types of co-stimulatory molecules and the context in which they were presented. In mice with aggressive lymphoma, a single, local injection of the scaffold following non-curative CAR-T-cell dosing led to more persistent memory-like T cells and extended animal survival. Injectable biomaterials with optimized ligand presentation may boost the therapeutic performance of CAR-T-cell therapies.

Surface-Functionalized Microgels as Artificial Antigen-Presenting Cells to Regulate Expansion of T Cells.

Artificial antigen-presenting cells (aAPCs) are currently used to manufacture T cells for adoptive therapy in cancer treatment, but a readily tunable and modular system can enable both rapid T cell expansion and control over T cell phenotype. Here, it is shown that microgels with tailored surface biochemical properties can serve as aAPCs to mediate T cell activation and expansion. Surface functionalization of microgels is achieved via layer-by-layer coating using oppositely charged polymers, forming a thin but dense polymer layer on the surface. This facile and versatile approach is compatible with a variety of coating polymers and allows efficient and flexible surface-specific conjugation of defined peptides or proteins. The authors demonstrate that tethering appropriate stimulatory ligands on the microgel surface efficiently activates T cells for polyclonal and antigen-specific expansion. The expansion, phenotype, and functional outcome of primary mouse and human T cells can be regulated by modulating the concentration, ratio, and distribution of stimulatory ligands presented on microgel surfaces as well as the stiffness and viscoelasticity of the microgels.

Neutrophils bearing adhesive polymer micropatches as a drug-free cancer immunotherapy.

Tumour-associated neutrophils can exert antitumour effects but can also assume a pro-tumoural phenotype in the immunosuppressive tumour microenvironment. Here we show that neutrophils can be polarized towards the antitumour phenotype by discoidal polymer micrometric 'patches' that adhere to the neutrophils' surfaces without being internalized. Intravenously administered micropatch-loaded neutrophils accumulated in the spleen and in tumour-draining lymph nodes, and activated splenic natural killer cells and T cells, increasing the accumulation of dendritic cells and natural killer cells. In mice bearing subcutaneous B16F10 tumours or orthotopic 4T1 tumours, intravenous injection of the micropatch-loaded neutrophils led to robust systemic immune responses, a reduction in tumour burden and improvements in survival rates. Micropatch-activated neutrophils combined with the checkpoint inhibitor anti-cytotoxic T-lymphocyte-associated protein 4 resulted in strong inhibition of the growth of B16F10 tumours, and in complete tumour regression in one-third of the treated mice. Micropatch-loaded neutrophils could provide a potent, scalable and drug-free approach for neutrophil-based cancer immunotherapy.

T Cell Development and Function.

T cells play critical roles in the immune system, including in responses to cancer, autoimmunity, and tissue regeneration. T cells arise from common lymphoid progenitors (CLPs) that differentiate from hematopoietic stem cells in the bone marrow. CLPs then traffic to the thymus, where they undergo thymopoiesis through a number of selection steps, resulting in mature single positive naive CD4 helper or CD8 cytotoxic T cells. Naive T cells are home to secondary lymphoid organs like lymph nodes and are primed by antigen-presenting cells, which scavenge for both foreign and self-antigens. Effector T cell function is multifaceted, including direct target cell lysis and secretion of cytokines, which regulate the functions of other immune cells (refer to "Graphical Abstract"). This review will discuss T cell development and function, from the development of lymphoid progenitors in the bone marrow to principles that govern T cell effector function and dysfunction, specifically within the context of cancer.

Enhancing CAR-T cell functionality in a patient-specific manner.

Patient responses to autologous CD19 chimeric antigen receptor (CAR) T-cell therapies are limited by insufficient and inconsistent cellular functionality. Here, we show that controlling the precise level of stimulation during T-cell activation to accommodate individual differences in the donor cells will dictate the functional attributes of CAR-T cell products. The functionality of CAR-T cell products, consisting of a diverse set of blood samples derived from healthy donors, acute lymphoblastic leukemia (ALL), and chronic lymphocytic lymphoma (CLL) patient samples, representing a range of patient health status, is tested upon culturing on artificial antigen-presenting cell scaffolds to deliver T-cell stimulatory ligands (anti-CD3/anti-CD28) at highly defined densities. A clear relationship is observed between the dose of stimulation, the phenotype of the T-cell blood sample prior to T-cell activation, and the functionality of the resulting CAR-T cell products. We present a model, based on this dataset, that predicts the precise stimulation needed to manufacture a desired CAR-T cell product, given the input T-cell attributes in the initial blood sample. These findings demonstrate a simple approach to enhance CAR-T functionality by personalizing the level of stimulation during T-cell activation to enable flexible manufacturing of more consistent and potent CAR-T cells.

Adoptive T cell transfer and host antigen-presenting cell recruitment with cryogel scaffolds promotes long-term protection against solid tumors.

Although adoptive T cell therapy provides the T cell pool needed for immediate tumor debulking, the infused T cells generally have a narrow repertoire for antigen recognition and limited ability for long-term protection. Here, we present a hydrogel that locally delivers adoptively transferred T cells to the tumor site while recruiting and activating host antigen-presenting cells with GMCSF or FLT3L and CpG, respectively. T cells alone loaded into these localized cell depots provided significantly better control of subcutaneous B16-F10 tumors than T cells delivered through direct peritumoral injection or intravenous infusion. T cell delivery combined with biomaterial-driven accumulation and activation of host immune cells prolonged the activation of the delivered T cells, minimized host T cell exhaustion, and enabled long-term tumor control. These findings highlight how this integrated approach provide both immediate tumor debulking and long-term protection against solid tumors, including against tumor antigen escape.

Generation of functionally distinct T-cell populations by altering the viscoelasticity of their extracellular matrix.

The efficacy of adoptive T-cell therapies largely depends on the generation of T-cell populations that provide rapid effector function and long-term protective immunity. Yet it is becoming clearer that the phenotypes and functions of T cells are inherently linked to their localization in tissues. Here we show that functionally distinct T-cell populations can be generated from T cells that received the same stimulation by altering the viscoelasticity of their surrounding extracellular matrix (ECM). By using a model ECM based on a norbornene-modified collagen type I whose viscoelasticity can be adjusted independently from its bulk stiffness by varying the degree of covalent crosslinking via a bioorthogonal click reaction with tetrazine moieties, we show that ECM viscoelasticity regulates T-cell phenotype and function via the activator-protein-1 signalling pathway, a critical regulator of T-cell activation and fate. Our observations are consistent with the tissue-dependent gene-expression profiles of T cells isolated from mechanically distinct tissues from patients with cancer or fibrosis, and suggest that matrix viscoelasticity could be leveraged when generating T-cell products for therapeutic applications.

Nonsurgical treatment reduces tendon inflammation and elevates tendon markers in early healing.

Operative treatment is assumed to provide superior outcomes to nonoperative (conservative) treatment following Achilles tendon rupture, however, this remains controversial. This study explores the effect of surgical repair on Achilles tendon healing. Rat Achilles tendons (n = 101) were bluntly transected and were randomized into groups receiving repair or non-repair treatments. By 1 week after injury, repaired tendons had inferior mechanical properties, which continued to 3- and 6-week post-injury, evidenced by decreased dynamic modulus and failure stress. Transcriptomics analysis revealed >7000 differentially expressed genes between repaired and non-repaired tendons after 1-week post-injury. While repaired tendons showed enriched inflammatory gene signatures, non-repaired tendons showed increased tenogenic, myogenic, and mechanosensitive gene signatures, with >200-fold enrichment in Tnmd expression. Analysis of gastrocnemius muscle revealed elevated MMP activity in tendons receiving repair treatment, despite no differences in muscle fiber morphology. Transcriptional regulation analysis highlighted that the highest expressed transcription factors in repaired tendons were associated with inflammation (Nfκb, SpI1, RelA, and Stat1), whereas non-repaired tendons expressed markers associated with tissue development and mechano-activation (Smarca1, Bnc2, Znf521, Fbn1, and Gli3). Taken together, these data highlight distinct differences in healing mechanism occurring immediately following injury and provide insights for new therapies to further augment tendons receiving repaired and non-repaired treatments.

Relative Quantification of siRNA Strand Loading into Ago2 for Design of Highly Active siRNAs.

In RNA interference (RNAi), silencing is achieved through the interaction of double-stranded small interfering RNAs (siRNAs) with essential RNAi pathway proteins, including Argonaute 2 (Ago2). Based on these interactions, one strand of the siRNA is loaded into Ago2 forming the active RNA-induced silencing complex (RISC). Optimal siRNAs maximize RISC activity against the intended target and minimize off-target silencing. To achieve the desired activity and specificity, selection of the appropriate siRNA strand for loading into Ago2 is essential. Here, we provide a protocol to quantify the relative loading of individual siRNA strands into Ago2, one factor in determining the capacity of a siRNA to achieve silencing activity and target specificity.

Terminal Duplex Stability and Nucleotide Identity Differentially Control siRNA Loading and Activity in RNA Interference.

Efficient short interfering RNA (siRNA)-mediated gene silencing requires selection of a sequence that is complementary to the intended target and possesses sequence and structural features that encourage favorable functional interactions with the RNA interference (RNAi) pathway proteins. In this study, we investigated how terminal sequence and structural characteristics of siRNAs contribute to siRNA strand loading and silencing activity and how these characteristics ultimately result in a functionally asymmetric duplex in cultured HeLa cells. Our results reiterate that the most important characteristic in determining siRNA activity is the 5' terminal nucleotide identity. Our findings further suggest that siRNA loading is controlled principally by the hybridization stability of the 5' terminus (Nucleotides: 1-2) of each siRNA strand, independent of the opposing terminus. Postloading, RNA-induced silencing complex (RISC)-specific activity was found to be improved by lower hybridization stability in the 5' terminus (Nucleotides: 3-4) of the loaded siRNA strand and greater hybridization stability toward the 3' terminus (Nucleotides: 17-18). Concomitantly, specific recognition of the 5' terminal nucleotide sequence by human Argonaute 2 (Ago2) improves RISC half-life. These findings indicate that careful selection of siRNA sequences can maximize both the loading and the specific activity of the intended guide strand.

Dextran functionalization enhances nanoparticle-mediated siRNA delivery and silencing.

Understanding the endocytosis and intracellular trafficking of short interfering RNA (siRNA) delivery vehicle complexes remains a critical bottleneck in designing siRNA delivery vehicles for highly active RNA interference (RNAi)-based therapeutics. In this study, we show that dextran functionalization of silica nanoparticles enhanced uptake and intracellular delivery of siRNAs in cultured cells. Using pharmacological inhibitors for endocytotic pathways, we determined that our complexes are endocytosed via a previously unreported mechanism for siRNA delivery in which dextran initiates scavenger receptor-mediated endocytosis through a clathrin/caveolin-independent process. Our findings suggest that siRNA delivery efficiency could be enhanced by incorporating dextran into existing delivery platforms to activate scavenger receptor activity across a variety of target cell types.