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Dustin Sheetz Milone Kam Geiger Vogel
Wiggins Wind Hone Spatz Bonneau June

 

 

 

 

 

The Nanomedicine Center for Mechanobiology aims to advance understanding of cellular mechanical biology in order to develop technologies for regenerative medicine and advance immunotherapy (Our Pathway to Medicine).


Theme

To understand and modify fundamental nanoscale force- and geometry-sensing pathways involved in the generation of immunological memory to advance immunotherapy.

 

Our Approach

Immune responses are based on the orchestrated action of differentiated mesenchymal cells that form lymphoid tissues and lymphoid effector cells.  In most immune responses, there are many mechanically dependent steps that are controlled by the nanometer-level force sensing protein modules in cells.  We have recently discovered that lymphocytes utilize many of the same universal motility associated modules as fibroblasts, including force and rigidity sensing mechanisms. This has provided a new appreciation of the role of force sensing in the immune response and we can now apply tools that have been developed through many years of research on force sensing in fibroblasts to the medically critical problem of immunological memory. The Nanotechnology Center for Mechanics in Regenerative Medicine has developed nano-tools and expertise to measure the pattern of force and rigidity sensing.   These tools will enable us to understand both the natural forces that shape the immune response and to manipulate these to generate pure populations of memory cells for immunotherapy applications.

 

Medical Significance of Mechanical Factors

Recent in vivo analysis of immune responses by intravital microscopy have called attention to the intricate mechanical process involved in a) detection of foreign invaders, b) the immune response to cancer and c) the genesis of autoimmune diseases.    In all of these medically important settings a central process is the formation of an immunological synapse- a stable cell-cell junction that performs a number of functions in innate and adaptive immunity.  These functions include the regulation of immune cell growth and differentiation and the killing of infected cells. Recent publications from this center and others have demonstrated that the immunological synapse is built from universal nanoscale modules that function in force sensing by evolutionarily diverse motile cells.  This discovery emphasizes the importance of physical forces in the immune response.  It is also clear that the lymphoid tissues, in which immune responses take place, have a unique extracellular matrix and are populated with contractile mesenchymal cells that define the force environment for immune responses.  An integrated interdisciplinary approach is urgently needed to understand how physical factors are sensed and produce appropriate responses over time to develop the appropriate immune response to pathogens and tumor and block immunopathology to allow normal tissue homeostasis and regeneration.  The read-out of physical factors by cells involves alterations of biochemical signaling responses.   We have described several molecular force transduction mechanisms but we need additional tools (physical and biochemical) to determine which mechanisms are responsible for functions at the cellular level. To measure and generate forces at the specific molecular-scale locations during these functions, a number of nano-structured devices were designed and built by a new high throughput process.  Our previous biochemical studies and those of other labs have identified potential target molecules that are responsible for mechanotransduction in immune cells.  These can be combined with tools for making matrices of different rigidities and strains to screen for the desired effects on specific cell functions in the immune cells.

Steps In Mechanical Signaling Pathways in the Immune Response

1. Adhesion (motility on reticular fibroblasts and immune cell receptor binds)

2. Force Generation (reticular fiber rigidity and actinomyosin contractility)

3. Force Sensing (force exposed substrate domains and conformational changes)

4. Signaling (integrin, costimulatory and antigen receptor signals)

5. Response  (cytokine production, asymmetric cell division, memory, killing)

At the basic science level, a number of important findings have fundamentally altered the way that we approach biomechanical problems.  First, there is a growing consensus that similar mechanical signaling pathways play important if not critical roles in immune cell functions.  We have found that immune cells mechanically pull on ligands and matrices and the mechanical resistance that they encounter plays a critical role in the signal that is generated.  The immune synapse involves the motor-dependent, mechanical sorting of the T cell antigen receptors and other components.  Further, mechanical aspects of the ligands control the T cell response. Another emerging result is that the forces exerted by lymphoid cells will alter the environment.  Immune cells will perturb the presenting cells by pulling on them and mesenchymal cells, which form the superstructure of the lymph node, allow dramatic expansion of lymph nodes during an immune response.  Molecular-level studies show that tyrosine kinase phosphorylation of stretched proteins or protein binding to stretched-exposed sites can provide a primary cue for force-dependent signaling and we have recently translated initial findings in fibroblasts to immune cells, demonstrating a universal aspect of mechanotransduction.  Important proteins in immune cells and stem cells are likely candidates to be stretched.  Thus, the study of the mechanisms of mechanosensing in the lymphoid cells and the specialized fibroblastoid cells that form lymph nodes.            

           

Our Tools               

A critical aspect for the Center is the development of new nanometer level devices for measuring or creating sub-cellular forces. Nanoimprint lithography (NIL) has proven to be a reliable way to fabricate samples in far greater quantities (and far less time) than would be possible by direct-write electron beam lithography.  All members of our nanofabrication team incorporate NIL into their fabrication schemes, and we have developed some new techniques and optimized others, making our NIL fabrication more robust and repeatable.  At multiple scales from sub-10 nm dimensions to 10 micrometers, we can stamp out surfaces with NIL.  In addition, we have developed new techniques for fabricating elastomeric surfaces with multi- and variable rigidity with micron-scale and nanometer-scale precision.  The next generation of devices is being fabricated to enable periodic stretching of the surface to mimic in vivo activity of tissues.  These devices are critical to understanding the roles of geometry and force in cell function and behavior and they are designed to be generally applicable to immune cells and lymphoid tissue reticular fibroblasts.

 

Our Pathway to Medicine        

Our Pathway to Medicine is based on close ties to groups of Michael Milone and Carl June at U. Penn that have pioneered adoptive immunotherapy for treatment of cancer, chronic viral infections and autoimmune diseases.  June’s group developed methods for massive expansion of human T cells using antibody coated magnetic beads, but they cannot always control the types of T cells that are generated and the long term survival of transferred T cells is a challenge that might be overcome by generating long lived memory cells. Preliminary findings with Michael Dustin and Lance Kam show that the spatial patterning of adhesion sites can cause dramatic increases in T cell cytokine production. Milone is leading the effort to extend these approaches for amplification of memory T cells in close collaboration with multiple elements of the center that include bioengineering, fabrication, imaging and modeling.

Integration of nanoscale technologies, combined with cell biology and computational modeling has provided new quantitative insights into mechanical regulation of cell function.  These advances were made possible by the interaction of Center members working toward the common goal of understanding important aspects of cellular mechanics at the nanometer scale.  The tools that have been created are now ready to be applied to the engineering of specialized environments for directing growth, differentiation and fate of immune cells. The Nanotechnology Center for Mechanics in Regenerative Medicine has begun to apply these technologies and has plans for many more applications that are being tested.  Such a coordinated analysis of the physical parameters in conjunction with the biochemical changes has only been possible through such a center, where the many aspects can be approached in parallel and different types of expertise can be applied to the same cell systems simultaneously. Presentation of the signals in physically different patterns changes the cell response and we plan to develop the proper pattern for the appropriate signal generation leading to appropriate supplementation of a patient’s immune response through infusion of regeneration competent memory T cells.