Modulating Antigen Availability in Lymphoid Organs to Shape the Humoral Immune Response to Vaccines.

Primary immune responses following vaccination are initiated in draining lymph nodes, where naive T and B cells encounter Ag and undergo coordinated steps of activation. For humoral immunity, the amount of Ag present over time, its localization to follicles and follicular dendritic cells, and the Ag's structural state all play important roles in determining the subsequent immune response. Recent studies have shown that multiple elements of vaccine design can impact Ag availability in lymphoid tissues, including the choice of adjuvant, physical form of the immunogen, and dosing kinetics. These vaccine design elements affect the transport of Ag to lymph nodes, Ag's localization in the tissue, the duration of Ag availability, and the structural integrity of the Ag. In this review, we discuss these findings and their implications for engineering more effective vaccines, particularly for difficult to neutralize pathogens.

Two-dose "extended priming" immunization amplifies humoral immune responses by synchronizing vaccine delivery with the germinal center response.

"Extended priming" immunization regimens that prolong exposure of the immune system to vaccines during the primary immune response have shown promise in enhancing humoral immune responses to a variety of subunit vaccines in preclinical models. We previously showed that escalating-dosing immunization (EDI), where a vaccine is dosed every other day in an increasing pattern over 2 weeks dramatically amplifies humoral immune responses. But such a dosing regimen is impractical for prophylactic vaccines. We hypothesized that simpler dosing regimens might replicate key elements of the immune response triggered by EDI. Here we explored "reduced ED" immunization regimens, assessing the impact of varying the number of injections, dose levels, and dosing intervals during EDI. Using a stabilized HIV Env trimer as a model antigen combined with a potent saponin adjuvant, we found that a two-shot extended-prime regimen consisting of immunization with 20% of a given vaccine dose followed by a second shot with the remaining 80% of the dose 7 days later resulted in increased total GC B cells, 5-10-fold increased frequencies of antigen-specific GC B cells, and 10-fold increases in serum antibody titers compared to single bolus immunization. Computational modeling of the GC response suggested that this enhanced response is mediated by antigen delivered in the second dose being captured more efficiently as immune complexes in follicles, predictions we verified experimentally. Our computational and experimental results also highlight how properly designed reduced ED protocols enhance activation and antigen loading of dendritic cells and activation of T helper cells to amplify humoral responses. These results suggest that a two-shot priming approach can be used to substantially enhance responses to subunit vaccines.

WASP facilitates tumor mechanosensitivity in T lymphocytes.

Cytotoxic T lymphocytes (CTLs) carry out immunosurveillance by scanning target cells of diverse physical properties for the presence of antigens. While the recognition of cognate antigen by the T cell receptor is the primary signal for CTL activation, it has become increasingly clear that the mechanical stiffness of target cells plays an important role in antigen-triggered T cell responses. However, the molecular machinery within CTLs that transduces the mechanical information of tumor cells remains unclear. We find that CTL's mechanosensitive ability requires the activity of the actin-organizing protein Wiskott-Aldrich Syndrome Protein (WASP). WASP activation is modulated by the mechanical properties of antigen-presenting contexts across a wide range of target cell stiffnesses and activated WASP then mediates mechanosensitive activation of early TCR signaling markers in the CTL. Our results provide a molecular link between antigen mechanosensing and CTL immune response and suggest that CTL-intrinsic cytoskeletal organizing principles enable the processing of mechanical information from diverse target cells.

Universal redirection of CAR T cells against solid tumours via membrane-inserted ligands for the CAR.

The effectiveness of chimaeric antigen receptor (CAR) T cell therapies for solid tumours is hindered by difficulties in the selection of an effective target antigen, owing to the heterogeneous expression of tumour antigens and to target antigen expression in healthy tissues. Here we show that T cells with a CAR specific for fluorescein isothiocyanate (FITC) can be directed against solid tumours via the intratumoural administration of a FITC-conjugated lipid-poly(ethylene)-glycol amphiphile that inserts itself into cell membranes. In syngeneic and human tumour xenografts in mice, 'amphiphile tagging' of tumour cells drove tumour regression via the proliferation and accumulation of FITC-specific CAR T cells in the tumours. In syngeneic tumours, the therapy induced the infiltration of host T cells, elicited endogenous tumour-specific T cell priming and led to activity against distal untreated tumours and to protection against tumour rechallenge. Membrane-inserting ligands for specific CARs may facilitate the development of adoptive cell therapies that work independently of antigen expression and of tissue of origin.

Low protease activity in B cell follicles promotes retention of intact antigens after immunization.

The structural integrity of vaccine antigens is critical to the generation of protective antibody responses, but the impact of protease activity on vaccination in vivo is poorly understood. We characterized protease activity in lymph nodes and found that antigens were rapidly degraded in the subcapsular sinus, paracortex, and interfollicular regions, whereas low protease activity and antigen degradation rates were detected in the vicinity of follicular dendritic cells (FDCs). Correlated with these findings, immunization regimens designed to target antigen to FDCs led to germinal centers dominantly targeting intact antigen, whereas traditional immunizations led to much weaker responses that equally targeted the intact immunogen and antigen breakdown products. Thus, spatially compartmentalized antigen proteolysis affects humoral immunity and can be exploited.

Deciphering the Mechanics of Cancer Spheroid Growth in 3D Environments through Microfluidics Driven Mechanical Actuation.

Uncontrolled growth of tumor cells is a key contributor to cancer-associated mortalities. Tumor growth is a biomechanical process whereby the cancer cells displace the surrounding matrix that provides mechanical resistance to the growing cells. The process of tumor growth and remodeling is regulated by material properties of both the cancer cells and their surrounding matrix, yet the mechanical interdependency between the two entities is not well understood. Herein, this work develops a microfluidic platform that precisely positions tumor spheroids within a hydrogel and mechanically probes the growing spheroids and surrounding matrix simultaneously. By using hydrostatic pressure to deform the spheroid-laden hydrogel along with confocal imaging and finite element (FE) analysis, this work deduces the material properties of the spheroid and the matrix in situ. For spheroids embedded within soft hydrogels, decreases in the Young's modulus of the matrix are detected at discrete locations accompanied by localized tumor growth. Contrastingly, spheroids within stiff hydrogels do not significantly decrease the Young's modulus of the surrounding matrix, despite exhibiting growth. Spheroids in stiff matrices leverage their high bulk modulus to grow and display a uniform volumetric expansion. Collectively, a quantitative platform is established and new insights into tumor growth within a stiff 3D environment are provided.

Mannose-binding lectin and complement mediate follicular localization and enhanced immunogenicity of diverse protein nanoparticle immunogens.

Nanoparticle (NP) vaccine formulations promote immune responses through multiple mechanisms. We recently reported that mannose-binding lectin (MBL) triggers trafficking of glycosylated HIV Env-immunogen NPs to lymph node follicles. Here, we investigate effects of MBL and complement on NP forms of HIV and other viral antigens. MBL recognition of oligomannose on gp120 nanoparticles significantly increases antigen accumulation in lymph nodes and antigen-specific germinal center (GC) responses. MBL and complement also mediate follicular trafficking and enhance GC responses to influenza, HBV, and HPV particulate antigens. Using model protein nanoparticles bearing titrated levels of glycosylation, we determine that mannose patches at a minimal density of 2.1 × 10-3 mannose patches/nm2 are required to trigger follicular targeting, which increases with increasing glycan density up to at least ∼8.2 × 10-3 patches/nm2. Thus, innate immune recognition of glycans has a significant impact on humoral immunity, and these findings provide a framework for engineering glycan recognition to optimize vaccine efficacy.

A particulate saponin/TLR agonist vaccine adjuvant alters lymph flow and modulates adaptive immunity.

Saponins are potent and safe vaccine adjuvants, but their mechanisms of action remain incompletely understood. Here, we explored the properties of several saponin formulations, including immune-stimulatory complexes (ISCOMs) formed by the self-assembly of saponin and phospholipids in the absence or presence of the Toll-like receptor 4 agonist monophosphoryl lipid A (MPLA). We found that MPLA self-assembles with saponins to form particles physically resembling ISCOMs, which we termed saponin/MPLA nanoparticles (SMNP). Saponin-containing adjuvants exhibited distinctive mechanisms of action, altering lymph flow in a mast cell­dependent manner and promoting antigen entry into draining lymph nodes. SMNP was particularly effective, exhibiting even greater potency than the compositionally related adjuvant AS01B in mice, and primed robust germinal center B cell, TFH, and HIV tier 2 neutralizing antibodies in nonhuman primates. Together, these findings shed new light on mechanisms by which saponin adjuvants act to promote the immune response and suggest that SMNP may be a promising adjuvant in the setting of HIV, SARS-CoV-2, and other pathogens.

Reprogramming NK cells and macrophages via combined antibody and cytokine therapy primes tumors for elimination by checkpoint blockade.

Treatments aiming to augment immune checkpoint blockade (ICB) in cancer often focus on T cell immunity, but innate immune cells may have important roles to play. Here, we demonstrate a single-dose combination treatment (termed AIP) using a pan-tumor-targeting antibody surrogate, half-life-extended interleukin-2 (IL-2), and anti-programmed cell death 1 (PD-1), which primes tumors to respond to subsequent ICB and promotes rejection of large established tumors in mice. Natural killer (NK) cells and macrophages activated by AIP treatment underwent transcriptional reprogramming; rapidly killed cancer cells; governed the recruitment of cross-presenting dendritic cells (DCs) and other leukocytes; and induced normalization of the tumor vasculature, facilitating further immune infiltration. Thus, innate cell-activating therapies can initiate critical steps leading to a self-sustaining cycle of T cell priming driven by ICB.

Controlling timing and location in vaccines.

Vaccines are one of the most powerful technologies supporting public health. The adaptive immune response induced by immunization arises following appropriate activation and differentiation of T and B cells in lymph nodes. Among many parameters impacting the resulting immune response, the presence of antigen and inflammatory cues for an appropriate temporal duration within the lymph nodes, and further within appropriate subcompartments of the lymph nodes- the right timing and location- play a critical role in shaping cellular and humoral immunity. Here we review recent advances in our understanding of how vaccine kinetics and biodistribution impact adaptive immunity, and the underlying immunological mechanisms that govern these responses. We discuss emerging approaches to engineer these properties for future vaccines, with a focus on subunit vaccines.

An Engineered Tumor-on-a-Chip Device with Breast Cancer-Immune Cell Interactions for Assessing T-cell Recruitment.

Recruitment of immune cells to a tumor is determined by the complex interplay between cellular and noncellular components of the tumor microenvironment. Ex vivo platforms that enable identification of key components that promote immune cell recruitment to the tumor could advance the field significantly. Herein, we describe the development of a perfusable multicellular tumor-on-a-chip platform involving different cell populations. Cancer cells, monocytes, and endothelial cells were spatially confined within a gelatin hydrogel in a controlled manner by using 3D photopatterning. The migration of the encapsulated endothelial cells against a chemokine gradient created an endothelial layer around the constructs. Using this platform, we examined the effect of cancer cell-monocyte interaction on T-cell recruitment, where T cells were dispersed within the perfused media and allowed to infiltrate. The hypoxic environment in the spheroid cultures recruited more T cells compared with dispersed cancer cells. Moreover, the addition of monocytes to the cancer cells improved T-cell recruitment. The differences in T-cell recruitment were associated with differences in chemokine secretion including chemokines influencing the permeability of the endothelial barrier. This proof-of-concept study shows how integration of microfabrication, microfluidics, and 3D cell culture systems could be used for the development of tumor-on-a-chip platforms involving heterotypic cells and their application in studying recruitment of cells by the tumor-associated microenvironment. SIGNIFICANCE: This study describes how tumor-on-chip platforms could be designed to create a heterogeneous mix of cells and noncellular components to study the effect of the tumor microenvironment on immune cell recruitment.

Three-Dimensional Monolayer Stress Microscopy.

Many biological processes involve the collective generation and transmission of mechanical stresses across cell monolayers. In these processes, the monolayer undergoes lateral deformation and bending because of the tangential and normal components of the cell-generated stresses. Monolayer stress microscopy (MSM) methods have been developed to measure the intracellular stress distribution in cell monolayers. However, current methods assume plane monolayer geometry and neglect the contribution of bending to the intracellular stresses. This work introduces a three-dimensional (3D) MSM method that calculates monolayer stress from measurements of the 3D traction stresses exerted by the cells on a flexible substrate. The calculation is carried out by imposing equilibrium of forces and moments in the monolayer, subject to external loads given by the 3D traction stresses. The equilibrium equations are solved numerically, and the algorithm is validated for synthetic loads with known analytical solutions. We present 3D-MSM measurements of monolayer stress in micropatterned islands of endothelial cells of different sizes and shapes. These data indicate that intracellular stresses caused by lateral deformation emerge collectively over long distances; they increase with the distance from the island edge until they reach a constant value that is independent of island size. On the other hand, bending-induced intracellular stresses are more concentrated spatially and remain confined to within one to two cell lengths of bending sites. The magnitude of these bending stresses is highest at the edges of the cell islands, where they can exceed the intracellular stresses caused by lateral deformations. Our data from nonpatterned monolayers suggests that biomechanical perturbations far away from monolayer edges also cause significant localized alterations in bending tension. The localized effect of bending-induced stresses may be important in processes like cellular extravasation, which are accompanied by significant normal deflections of a cell monolayer (i.e., the endothelium) and require localized changes in monolayer permeability.

Skeletal muscle-on-a-chip: an in vitro model to evaluate tissue formation and injury.

Engineered skeletal muscle tissues can be used for in vitro studies that require physiologically relevant models of native tissues. Herein, we describe the development of a three-dimensional (3D) skeletal muscle tissue that recapitulates the architectural and structural complexities of muscle within a microfluidic device. Using a 3D photo-patterning approach, we spatially confined a cell-laden gelatin network around two bio-inert hydrogel pillars, which induce uniaxial alignment of the cells and serve as anchoring sites for the encapsulated cells and muscle tissues as they form and mature. We have characterized the tissue morphology and strain profile during differentiation of the cells and skeletal muscle tissue formation by using a combination of fluorescence microscopy and computational tools. The time-dependent strain profile suggests the existence of individual cells within the gelatin matrix, which differentiated to form a multinucleated skeletal muscle tissue bundle as a function of culture time. We have also developed a method to calculate the passive tension generated by the engineered muscle tissue bundles suspended between two pillars. Finally, as a proof-of-concept we have examined the applicability of the skeletal muscle-on-chip system as a screening platform and in vitro muscle injury model. We studied the dose-dependent effect of cardiotoxin on the engineered muscle tissue architecture and its subsequent effect on the passive tension. This simple yet effective tool can be appealing for studies that necessitate the analysis of skeletal muscle structure and function, including preclinical drug discovery and development.

Chemotaxis-driven assembly of endothelial barrier in a tumor-on-a-chip platform.

The integration of three-dimensional micropatterning with microfluidics provides a unique opportunity to create perfusable tissue constructs in vitro. Herein, we have used this approach to create a tumor-on-a-chip with an endothelial barrier. Specifically, we photopatterned a mixture of endothelial cells and cancer spheroids within a gelatin methacrylate (GelMA) hydrogel inside a microfluidic device. The differential motility of endothelial and cancer cells in response to a controlled morphogen gradient across the cell-laden network drove the migration of endothelial cells to the periphery while maintaining the cancer cells within the interior of the hydrogel. The resultant endothelial cell layer forming cell-cell contact via VE-cadherin junctions was found to encompass the entire GelMA hydrogel structure. Furthermore, we have also examined the potential of such a tumor-on-a-chip system as a drug screening platform using doxorubicin, a model cancer drug.

3D cardiac µtissues within a microfluidic device with real-time contractile stress readout.

We present the development of three-dimensional (3D) cardiac microtissues within a microfluidic device with the ability to quantify real-time contractile stress measurements in situ. Using a 3D patterning technology that allows for the precise spatial distribution of cells within the device, we created an array of 3D cardiac microtissues from neonatal mouse cardiomyocytes. We integrated the 3D micropatterning technology with microfluidics to achieve perfused cell-laden structures. The cells were encapsulated within a degradable gelatin methacrylate hydrogel, which was sandwiched between two polyacrylamide hydrogels. The polyacrylamide hydrogels were used as "stress sensors" to acquire the contractile stresses generated by the beating cardiac cells. The cardiac-specific response of the engineered 3D system was examined by exposing it to epinephrine, an adrenergic neurotransmitter known to increase the magnitude and frequency of cardiac contractions. In response to exogenous epinephrine the engineered cardiac tissues exhibited an increased beating frequency and stress magnitude. Such cost-effective and easy-to-adapt 3D cardiac systems with real-time functional readout could be an attractive technological platform for drug discovery and development.

Embedded 3D Photopatterning of Hydrogels with Diverse and Complex Architectures for Tissue Engineering and Disease Models.

Techniques that can create three-dimensional (3D) structures to provide architectural support for cells have a significant impact in generating complex and hierarchically organized tissues/organs. In recent times, a number of technologies, including photopatterning, have been developed to create such intricate 3D structures. In this study, we describe an easy-to-implement photopatterning approach, involving a conventional fluorescent microscope and a simple photomask, to encapsulate cells within spatially defined 3D structures. We have demonstrated the ease and the versatility of this approach by creating simple to complex as well as multilayered structures. We have extended this photopatterning approach to incorporate and spatially organize multiple cell types, thereby establishing coculture systems. Such cost-effective and easy-to-use approaches can greatly advance tissue engineering strategies.

3D traction stresses activate protease-dependent invasion of cancer cells.

Cell invasion and migration that occurs, for example, in cancer metastasis is rooted in the ability of cells to navigate through varying levels of physical constraint exerted by the extracellular matrix. Cancer cells can invade matrices in either a protease-independent or a protease-dependent manner. An emerging critical component that influences the mode of cell invasion is the traction stresses generated by the cells in response to the physicostructural properties of the extracellular matrix. In this study, we have developed a reference-free quantitative assay for measuring three-dimensional (3D) traction stresses generated by cells during the initial stages of invasion into matrices exerting varying levels of mechanical resistance. Our results show that as cells encounter higher mechanical resistance, a larger fraction of them shift to protease-mediated invasion, and this process begins at lower values of cell invasion depth. On the other hand, the compressive stress generated by the cells at the onset of protease-mediated invasion is found to be independent of matrix stiffness, suggesting that 3D traction stress is a key factor in triggering protease-mediated cancer cell invasion. At low 3D compressive traction stresses, cells utilize bleb formation to indent the matrix in a protease independent manner. However, at higher stress values, cells utilize invadopodia-like structures to mediate protease-dependent invasion into the 3D matrix. The critical value of compressive traction stress at the transition from a protease-independent to a protease-dependent mode of invasion is found to be ∼165 Pa.

Fusarisetins: Structure-function studies on a novel class of cell migration inhibitors.

Herein, we report the effects of fusarisetin A on cell morphology focusing in particular on actin and microtubules dynamics. We also report the synthesis and structure-function studies of a designed library of synthetic fusarisetins in cell-based assays.

Engineering the cell-material interface for controlling stem cell adhesion, migration, and differentiation.

The effective utilization of stem cells in regenerative medicine critically relies upon our understanding of the intricate interactions between cells and their extracellular environment. While bulk mechanical and chemical properties of the matrix have been shown to influence various cellular functions, the role of matrix interfacial properties on stem cell behavior is unclear. Here, we report the striking effect of matrix interfacial hydrophobicity on stem cell adhesion, motility, cytoskeletal organization, and differentiation. This is achieved through the development of tunable, synthetic matrices with control over their hydrophobicity without altering the chemical and mechanical properties of the matrix. The observed cellular responses are explained in terms of hydrophobicity-driven conformational changes of the pendant side chains at the interface leading to differential binding of proteins. These results demonstrate that the hydrophobicity of the extracellular matrix could play a considerably larger role in dictating cellular behaviors than previously anticipated. Additionally, these tunable matrices, which introduce a new control feature for regulating various cellular functions offer a platform for studying proliferation and differentiation of stem cells in a controlled manner and would have applications in regenerative medicine.

Osteoarthritic chondrocyte-secreted morphogens induce chondrogenic differentiation of human mesenchymal stem cells.

OBJECTIVE: The potential of stem cells to repair compromised cartilage tissue, such as in osteoarthritis (OA), depends strongly on how transplanted cells respond to factors secreted from the residing OA chondrocytes. This study was undertaken to determine the effect of morphogenetic signals from OA chondrocytes on chondrogenic differentiation of human mesenchymal stem cells (MSCs). METHODS: The effect of OA chondrocyte-secreted morphogens on chondrogenic differentiation of human MSCs was evaluated using a coculture system involving both primary and passaged OA chondrocytes. The findings were compared against findings for human MSCs cultured in OA chondrocyte-conditioned medium. Gene expression analysis, biochemical assays, and immunofluorescence staining were used to characterize the chondrogenic differentiation of human MSCs. Mass spectrometry analysis was used to identify the soluble factors. Numerical analysis was carried out to model the concentration profile of soluble factors within the human MSC-laden hydrogels. RESULTS: The human MSCs cocultured with primary OA chondrocytes underwent chondrogenic differentiation even in the absence of growth factors; however, the same effect could not be mimicked using OA chondrocyte-conditioned medium or expanded cells. Additionally, the cocultured environment down-regulated hypertrophic differentiation of human MSCs. Mass spectrometry analysis demonstrated cell-cell communication and chondrocyte phenotype-dependent effects on cell-secreted morphogens. CONCLUSION: The experimental findings, along with the results of the numerical analysis, suggest a crucial role of soluble morphogens and their local concentrations in the differentiation pattern of human MSCs in a 3-dimensional environment. The concept of using a small number of chondrocytes to promote chondrogenic differentiation of human MSCs while preventing their hypertrophic differentiation could be of great importance in formulating effective stem cell-based cartilage repair.