2010 Progress Report – Executive Summary
The Nanomedicine Center for Mechanobiology Directing the Immune Response strives to generate useful cellular components for immunotherapy applications. This effort builds on principles that were discovered in the preceding period using fibroblastoid, neural and immune cell systems and the addition of a clinical collaboration with the immunotherapy group of Michael Milone and Carl June at U. Penn focusing on effector and memory T cells. In the past year we have added an additional clinical collaboration with Bruce Blazer at U. Minnesota to study regulatory T cells. The main tool used by the U. Penn and U. Minnesota groups is adoptive immunotherapy- growing billions of memory and effector T cells or regulatory T cells under GMP conditions that are infused into patients to augment or suppress immunity. The control of T cell characteristics is limited and the current focus of our center is to establish conditions that can better control the types of T- cells generated during the production phase to optimize long-term benefit to the patient. The production of memory CD8+ T cells is currently considered the best way to achieve tumor control. In other contexts having a mixture of memory and effector cells in known proportions may be useful and other contexts call for activity of regulatory T cells to suppress immunity. The transfer of the center to NYU has reinforced the immune cell focus while maintaining the unique mechanical biology approach to this problem. We have successfully shifted our focus from general mechanical biology to application of mechanobiology principles to the immune response. As of this progress report, every participating lab has members engaged full time in projects that are directly aligned with the immunotherapy goals.
The key event in T cell activation and differentiation is the formation of an immunological synapse, which integrates adhesion and antigen recognition and takes place in a tissue environment defined by fibroblastic reticular cells that generate a network of ECM fibers that provide a physical scaffold for the immune response. In the past year we have made a number of advances in these areas: 1. Force spectroscopy as a method to follow the stabilization of immunological synapse (35). 2. The role of myosin II in formation of the immunological synapse (38). 3. The polyvalent nature of receptor engagement during mechanotranduction (39). 4. The signaling networks activated in the virological synapse (44). 5. The potential of PKC-θ inhibition to enhance the activity of regulatory T cells both in immunotherapy (22). These published advances are all fully aligned with the goals of the NDC to improve immunotherapy.
We would like to highlight one of our new pathway to medicine projects. Our center discovered that PKC-θ controls immunological synapse stability in CD4+ T cells (3). While we have been engaged in an effort to understand the exact mechanism, we have used this observation as an approach to understand the importance of synapse stability. We found that effector CD4+ T cells function more efficiently when they form stable synapses (45). We then noted that CD4+ Treg, cells that suppress Teff, have a hyperstable synapse. This led to the hypothesis that PKC-θ activity might be attenuated in the Treg synapse. Consistent with this, we found the PKC-θ is sequestered from the Treg synapse and that this pool of PKC-θ can be released by treatment of the Treg with tumor necrosis factor-α. The functional implications were tested with a small molecule inhibitor of PKC-θ, which enhanced the activity of Treg from normal individuals and revived defective Treg from patients with active rheumatoid arthritis (22). We were able to confirm these findings with expanded Treg, cells currently in use for immunotherapy, with clinical collaborator Bruce Blazar. Finally, we demonstrated in a mouse model that Treg treated ex vivo with a PKC-θ inhibitor had significantly improved efficacy in preventing colitis (22). This study opened a new area in nanomedicine for us- the concept that knowledge of nanoscale mechanical biology principles would lead to insight in small molecule drug discovery or therapeutic application of existing pharmaceuticals. Significant future effort will be focused on the full translational application of these findings using both classical pharmacology and nanotechnology approaches within our center.
The strengths of our team are in integrating studies on the effects of biological forces on nanoscale assemblies in cells and the surrounding tissue structures including the extracellular matrix. In addition to the published progress, every member has had a key role in the past year and has made significant progress. The Sheetz lab has made progress on the role of CasL in the immunological synapse and further discovery of additional force sensing modules in model systems. The Kam lab has discovered that Lck behaves in a fundamentally different manner in human and mouse T cells and that laid the groundwork for rigidity and pillar studies. The Hone lab has supervised the scale up for fabrication that has made materials available to all labs in sufficient quantities for mouse studies. The Wind lab has succeeded in generating the long planned active substrates, a new and exciting direction. The Wiggins lab has worked with the Bonneau lab to set up a gene expression analysis process that will help the center determine the outcome of mechanical stimuli and to put this in context of biological responses. The Milone and June groups have made breakthrough observations about mechanical and chemical signals that alter T cell differentiation. The Vogel lab has determined that immune cytokines relax Fn fibrils and determine that T cell can remodel extracellular matrix. The Geiger lab has made progress on the analysis of lymphoid cell motility and the role of PKC-ξ in T cell migration and activation. The Spatz lab has developed methods to use AFM to quantify adhesion forces between immune cells. The Dustin lab has determined that PKC-θ in the distal pole co-localizes with intermediate filaments. Thus, all funded components are contributing to center goals in a synergistic and organized fashion.
Our center has begun to address the hypothesis that we can use mechanical parameters of T cell stimulation to make therapeutically improve T cells. We have very promising results with rigidity in human T cells from the Milone lab. Specifically, T cells grown on softer surfaces have a surface phenotype that is more similar to a memory cells. We have also developed computational methods to start relating the surface phenotype to gene expression profiles that determine functional capacity. We have also made progress in the fabrication pipeline to provide pillar masters and other demanding substrates in large numbers and area. We know that mechanical parameters are linked to specific signaling pathways through molecules like CasL and Talin. We see these parameters as significant modifiers of the chemical signals that are classically studied for immune cell differentiation. The result that putting anti-CD3 and anti-CD28 on a soft surface promotes cells with memory surface markers is very promising. Another unique aspect of our center is that we combine deep expertise in both the immunochemical and mechanical components so that we will be in an excellent position to exploit mechanical parameters to which T cells are responsive to determine the potential for immunotherapy.
The Nanomedicine Center for Mechanobiology Directing the Immune Response has refocused its multidisciplinary efforts on the problem of generating useful T cells for immunotherapy applications- including memory, effector and regulatory cells. The NDC funded effort has been effectively re-targeted in the last year to the immune cell focus with all groups performing T cell based experiments as of this progress report. The director and co-director have coordinated this effort with the assistance of the Program Manager. The director has been able to use the bi-weekly video conference to exert a regular influence on the trainees in all centers and this has helped to control the activity in the center and keep the many project groups on task. The increased fabrication of substrates is meeting the needs of the clinical collaborators such that in vivo experiments are now in progress both in the Dustin (mouse) and Milone (human) labs. Addition of a clinical collaborator focusing on regulatory T cells will enable to center to impact both immune stimulatory and inhibitory therapies. This is a very exciting time in mechanical biology and immunotherapy and our NDC is positioned to bridge these rapidly moving areas to generate synergistic discoveries with therapeutic potential.
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