Technologies and approaches from the physical sciences and engineering can act as enabling partners with biology to find solutions to difficult problems in medicine. Because the immune system plays a critical role in many diseases, either due to its failure to respond effectively or through the creation of pathology by malfunction, combining engineering tools with biology-driven approaches may hold the key to the discovery and development of novel therapies for many diseases. To this end, we apply our materials-centric engineering background to create new tools for understanding the basic cellular and molecular mechanisms of immunity, and to create new therapeutics.
First, we develop drug delivery materials designed to control the spatial and temporal delivery of cues to the immune system in vitro and in vivo, from antigens and adjuvant molecules to entire cells. Materials allowing precise control over delivery of signals to the immune system (targeting the correct tissue site, controlling entry into the cell, controlling the time evolution of these signals) will provide opportunities for the development of immunotherapies/vaccines and serve as powerful tools for dissecting the biology of the immune system. Second, we have used our ability to build unique model systems (based on the use of synthetic surfaces, particles, and three-dimensional matrices) to gain insight into the basic cell biology of the immune system, particularly as it relates to immune cell trafficking/migration and T cell activation.
Model Systems for the Study of T Cell and Dendritic Cell Biology
Model systems for dissecting T cell activation. T cells are activated when they encounter foreign antigenic peptides displayed on the surface of antigen-presenting cells (APCs). Contact with the APC triggers the assembly of protein receptors and intracellular signaling molecules at the T cell–APC interface, forming a structure known as the immunological synapse (IS). Although the signals delivered at the IS are critical to the regulation of adaptive immunity, dissecting the precise role of the IS and other signals provided to T cells is a major challenge due to the dynamic nature of this cell-cell contact. To address this challenge, we have developed several strategies to interrogate events in T cell activation and allow systematic analysis of the physical and molecular cues governing T cell priming.
In the first approach, we replace the live APC with a synthetic surface displaying patterns of selected APC-derived molecules, substituting the dynamic T cell–APC contact with a T cell–surface contact to control signals delivered to the T cell. To achieve this goal, we first synthesized a class of novel, multifunctional photoresist materials that allow multiple proteins to be immobilized on substrates in desired patterns while exposing these macromolecules only to physiological aqueous conditions. Using this technology, we fabricated patterned substrates (artificial synapse arrays) presenting well-defined physical patterns of APC signals to T cells (mimicking the immunological synapse). We found that surface protein patterns can be used to directly template the organization of T cell surface receptors and intracellular signaling components: T cells form an IS in contact with these surfaces that mirrors the physical pattern of ligands presented to it. We demonstrated that T cells interpret and alter their function in response to even micron-scale changes in the physical pattern of ligand they encounter. The ability to template the physical assembly of the signaling machinery of T cells via a protein pattern presented from a surface opens up opportunities for dissecting the role of synapse structure in T cell recognition.
Although the duration of T cell stimulation and signals governing synapse resolution are critical factors determining the outcome of T cell priming, until now the technical difficulty of studying the late stages of T cell priming has limited our understanding of the end phases of T cell activation. Using our patterned surfaces' model, we have found that T cells exhibit complex dynamics at late times following initial T cell receptor (TCR) triggering: the cells halt migration and form synapses on contact with an activation site presenting a strong TCR stimulus. However, beginning 12–15 hours later, a fraction of cells begin to resolve their synapses, either dividing or "hopping" between activation sites—migrating to several activation sites before finally undergoing cell division. In addition, newly divided daughter cells appear ignorant of TCR ligand, and migrate through activation sites without halting. Such behaviors have been suggested by intravital imaging experiments observing T cell–APC interactions in lymph nodes, but they are difficult to examine mechanistically in vivo.
To complement our protein-patterning strategies in these T cell studies, we have also developed methodologies to study T cell–dendritic cell interactions in model 3D matrices. T cells and DCs interact within highly organized, compartmentalized lymphoid organs during primary immune responses. To explore how the 3D microenvironment of immune cells regulates their interactions, we developed a novel approach to the fabrication of cellular (porous) hydrogel materials (both synthetic and biopolymer gels)—which have a predefined, ordered, 3D porosity—by using colloidal crystals formed from particles tens of microns in diameter as a template for the formation of hydrogel "inverse opals." We have used these scaffolds as a model of the microenvironment within secondary lymphoid tissues where primary immune responses are generated, via the introduction of T cells, DCs, and lymphoid stromal cells.
Regulation of immune cell migration. Chemokines are host proteins that regulate immune cell migration; in response to pathogens, chemokines direct dendritic cells (DCs; key initiators of adaptive immunity) and their precursors to sites of infection. We developed microspheres that release chemokines at predetermined rates, allowing gradients of chemokine to be created with tailored characteristics; each microsphere can act as a point source of attractant. These microparticles avidly chemoattract DCs, and loading of these attracting microspheres with antigen or other immunostimulatory molecules allows selective delivery of these compounds to attracted cells. We seek to use this system to determine how the recruitment of unique DC and DC precursor cells by different chemokine signals alters immune responses in vivo. These materials may also form the basis of improved vaccines that mimic the cellular recruitment processes normally triggered during infections. In parallel with the development of this model, we carried out basic studies on chemokine signaling in T cells and demonstrated a role for homeostatic chemokines in regulating T cell and B cell motility.
New Materials for Vaccines and Immunotherapy
A key goal of our laboratory is to advance methods for delivery of antigens and/or immunostimulatory cues to immune cells, using soluble polymers, microparticles, or nanoparticles to create improved vaccines and immunotherapies. We propose that by controlling the temporal sequence, physical form, and quantity of antigen, adjuvant, or immunostimulatory signals delivered to immune cells we can create vaccines and immunotherapies that will provide solutions to the major challenges posed by cancer, HIV, and other infectious diseases.
Delivery of macromolecules into cells. Materials that efficiently deliver macromolecules to the cytosol of cells could be used to create a variety of powerful therapeutics, including anticancer vaccines or immunotherapies based on short interfering RNA (siRNA) or gene delivery. We developed an approach based on monodisperse nanoparticles with a core-shell structure to deliver macromolecules to the cytosol of immune cells: particles with a pH-responsive "proton sponge" core and cell-friendly hydrogel shell were synthesized. When cells internalize these particles, absorption of protons by the particle core and coincident buildup of counterions in the endosome lead to disruption of the confining endosomal/phagosomal membrane and thereby escape of the particles and associated drug cargo (e.g., proteins, siRNA) to the cytosol. By sequestering the hydrophobic proton sponge component of the particles within the core, we achieved delivery without overt cytotoxicity.
Complementary to this approach, in collaboration with Francesco Stellacci's lab (Massachusetts Institute of Technology), we explored the fate of organic ligand monolayer-protected nanoparticles in cells and discovered an unexpected role for the surface organization of chemical groups on amphiphilic particles in regulating the ability of nanoparticles to pass through cell membranes. We are exploring the application of these particles for delivery of siRNA for cancer therapy and for enhanced protein vaccines. In parallel, we are seeking to devise thin films and particles that can allow these concepts to be translated to noninvasive immunotherapy/vaccine delivery through the skin or through intranasal or oral routes.
Matrices for local immunotherapy. A number of promising approaches to the treatment of cancer and infectious disease are based on cell therapy, the injection of immune cells into patients as a biological therapy. We hypothesized that biomaterials could be designed to maximize the potency of such therapies, by allowing the microenvironment where cells are delivered to be controlled and manipulated. To this end, we developed "self-gelling" alginate hydrogels, which begin as a liquid solution but which crosslink (via calcium-loaded microspheres mixed with the gel) within minutes of injection; we load these gels with DCs, key initiators of immune responses, along with cofactors (e.g., immunomodulatory cytokines) that are sequestered and slowly released from the gel into the local tissue. We have demonstrated that such matrix-based delivery of DCs can dramatically alter the resulting immune response; DCs injected in these gels attract more than 100-fold more T cells to their site than the same cells injected as a free cell suspension, via the synergistic binding of DC-derived factors by the polysaccharide matrix. We have used this gel-based system to codeliver DCs with the immunoregulatory cytokines to local tumor sites. In preliminary studies, DC- and cytokine-carrying gels dramatically suppressed tumor growth in the aggressive B16 mouse model of melanoma.
