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Nanomedicine Center for Mechanobiology Directing the Immune Response

 
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Executive Summary
 

At the present time, there is a major gap in our understanding of molecular complexes in the critical size range between molecules and cells. Both intracellular and extracellular components, in the nanoscale range from about 20 to 800 nm, determine many of the functions that define cell shape, as well as forces exerted by cells on matrices and other cells, that are critical for life. Basically, we have very little knowledge of how cells (about 20 microns in diameter) can create all of the diverse biological forms that range from tiny insects (less than a millimeter long) to huge whales. It is well known that operation of the nanomachinery inside cells is critically dependent on factors in the extracellular environment such as hormones, cytokines, and other cell products. However, beyond single molecule signals from other cells and tissues, we are beginning to understand the importance of the environment surrounding cells that is critical to their function. In other words, the structure, composition and geometry of the extracellular environment influences intracellular molecular function and cell behavior. We have found that immune cells are particularly sensitive to both the chemical and physical nature of their environment. Because of this, we can direct immune cell behavior by manipulating the mechanical environment in which chemical signals are delivered, an underexplored approach. This phenomenon is exemplified by the highly organized immunological synapse formed by T lymphocyte with antigen bearing cells (Grakoui et al, 1999). We are expanding our basic knowledge of mechanical biology in the immune response and applying this new information to improve the manner in which we direct the immune response for cellular immunotherapy (June et al, 2009).
 

Introduction
 

We have little knowledge of how cells, which are about 10 microns in diameter, can create all of the diverse biological forms that range from tiny insects to huge whales. It is well known that operation of the nanomachinery inside cells is critically dependent on factors in the extracellular environment, such as hormones, cytokines, antigens and other cell products. The structure, composition, and geometry of the extracellular environment influence intracellular molecular function and cell behavior (Discher et al, 2005; Meschel et al, 2005; Bershadsky et al, 2006). Mechanical processes affecting cells define the shape of tissues and underlie proper regeneration, repair and immune responses (Vogel and Sheetz, 2006). We have generated evidence that cells of diverse origins use a common set of motile functions to determine the morphology of tissues and protect tissue from infection (Dobereiner et al, 2006; Giannone et al, 2007; Sims et al, 2007). Our research can provide new strategies for treating, cancer, hypertension, cardiovascular diseases, osteoporosis, nerve regeneration, and infectious disease and autoimmuity. For example, we recently discovered that mechanical unfolding of proteins inside and outside the cell transduces force into biochemical signals (Sawada et al, 2006). The unfolding inside the cell exposes new amino acid sequences for phosphorylation, which were not available before the mechanical perturbation. The newly phosphorylated sites cause alterations in cell migration, growth and differentiation. These mechanosensing mechanisms are integrated into the network of molecular interaction that we have defined as the adhesome (Zaidel-Bar et al, 2007; Storrie et al, 2007). We have found that these process act in immune cells as in fibroblastoid cells that form the lymphoid tissue in which immune responses take place. This suggests new approaches to manipulate the immune responses to pathogens in vaccination and strategies to suppress the immune response in autoimmunity.
 

Since so many critical cellular functions are mechanical and occur rapidly, our center is developing new tools to quickly make measurements below the resolution limit of the light microscope and existing force measurement devices. To work at the nanoscale, we began with the knowledge and tools of material science and the growing field of nanotechnology in order to enhance our toolset to specifically manipulate the mechanical properties of biological tissues and make nanomedicine a reality. Our center has embraced the immune response as an initial therapeutic target for the tools of mechanobiology on the nanoscale.
 

NDC Goals
 

Our NDC has a goal of directing the immune response through mechanobiology. Manipulating immune cells through mechanical approach is an understudied area although we have appreciated for many years that immune cells use the same family of major adhesion molecules, the integrins, to regulate close interactions with other cells. We will first introduce out general approach to mechanobiology, which can be applied to many clinical area. Our long-term goal into the center is to have NDC based technology in the preclinical studies and clinical trials in the next 5 years. To achieve these goals we have established a collaboration with the immunotherapy group of Michael Milone and Carl June at the University of Pennsylvania.
 

Figure 1 - Micropatterned costimulation of T-cells

We are approaching the problem of understanding cell mechanical functions by determining first what are the mechanical as well as biochemical bases of cell functions at a sub-micron (nanometer) level. When we know the basic steps and the molecular processes involved, we can hope to intervene to correct or alter cell behavior. It is useful to consider how automobiles work as an analogy to understand how cells work. Similar to the array of cells in the human body, there is a diverse array of automobiles that share many functions, but the individual components involved in a function may be different. A major step forward to controlling operation of a machine is to fully describe each step of the various functions performed. For example, cars have windows that go up and down by a similar mechanism, but the specific parts may be totally different. To describe how turning the window crank causes the window to go down, as in an engineering operations manual, is an important step forward. This requires taking apart the door and identifying which gears, pulleys and other components move, which are stationary, and how they are coupled to accomplish the function. Once this process is described for one car, it will be much easier to understand the mechanism in another even if the components are not identical.

 

We feel that the best way to understand mechanotransduction quantitatively is to develop an integrated approach by examining multiple scales ranging from individual molecules to nanoscale intracellular machinery to whole systems. Biologists, engineers, computer scientists and modelers are all focused on the same questions such as the biophysical and biochemical steps in the formation of the immunological synapse, the immune cell response to substrate compliance, or the assembly of extracellular matrix fibers that underlie the unique structure of lymphoid tissues. Our team has a wide range of expertise including Cell Biophysics and Nanofabrication, Advanced Fluorescence Microscopy and Image Analysis, Immunology and Supported Bilayer Fabrication, Tissue Engineering and Predictive Modeling.

 

Our published results for the first 4 years of the NDC program indicate that many different cells are capable of utilizing similar force-sensing, force-generating and force-bearing systems, suggesting that, for many tasks, cells can use a common tool set and phenotypic differences result from differences in the extent and location of use of one tool versus another. Common tool sets and functional complexes offers the possibility of developing engineering capabilities for intracellular components that could be useful for multiple cells types and tissues which is the part of the promise of Nanomedicine. For example, patterning of antibodies to the T-cell antigen receptor and the innate signal CD28 by microcontact printing can be used to direct T-cell cytokine production (Figure 1). It appears to be a general principle of T-cells from mice an humans that spacing the anti-CD28 signal are far apart in the immunological synapse as possible enhances signaling. This distance is likely related to the recently demonstrated role of actinomyosin force generation in immune cell signaling. Understanding this function in one situation should provide insights into similar functions in other situations. We will develop and test models of mechanotransduction at the cell and molecular level. Nanotechnology developed in our center will enable us to rapidly screen for the optimal matrix rigidity, form, and spacing needed to elicit a given function. Our goal is to customize these technologies to generate sufficient amounts of material for pre-clinical studies in mouse models. We are addressing these questions at the single cell level, at the level of cell-cell interactions with a focus on the immune synapse, and the stem cell like properties of memory T cells that are needed for long-term protection.

 

Intracellular systems sense force and/or nanometer level geometry, transduce the cues into biochemical signals that are then processed over space and time to give mechanoresponses that then cause the cell to move and alter the mechanical cues, producing a new set of signals (Vogel and Sheetz, 2006). The long-term effects of these cycles determine whether cells grow or die, the shape of the organism, and whether many tissue functions are effective. Intricate intracellular protein networks integrate mechanical cues over many length scales. We will need an understanding of how forces regulate signaling pathways and gene expression. For nanomedicine to become a reality, our goal is to fully understand and characterize these physical phenomena. Generating a well-defined, and extensive knowledge base of cellular forces and employing an engineering approach allows us to view the cell as a "complex machine" that can activate highly specialized tool sets to accomplish the necessary tasks at a number of different hierarchical levels.
 

Figure 2 - Early steps in formation of the immunological synapse
 

Steps In Mechanical Signaling Pathways in the Immune Response- immune synapse formation:
 

  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 mechanobiology 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 (Figure 3). 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 stromal 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 will provide insights needed to direct the immune response during immunotherapy.

 

NDC accomplishments
 

Figure 3 - Effect of MyH9 siRNA on immune synapse formation by primary T-cell

Immune Synapse
 

The movements of the membrane components to form the immune synapse clearly constitute an integral part of the T-cell-antigen recognition process (Figure 3). We have begun to unravel how specific genes control generation of forces that regulate the immune response. In early 2009 we demonstrated for the first time that myosin IIA (MyH9 gene) is important for the formation of the immune synapse. In the absence of myosin II the cells adhere to a larger area and don’t consolidate the T cell receptor and the ICAM-1 into the normal pattern of the immune synapse. The contacts formed in the absence of myosin IIA are motile as indicated by 3 color time lapse image in the left panel.
 

Matrix engineering
 

Lymphoid tissue are formed by fibroblastic reticular cells that ensheath large collagen bundles that serve as scaffolds and conduits for information. We are transferring technologies developed over years of studying stromal cell manipulation of collagen and fibronectin to lymphoid tissue engineering. Cellular remodelling of extracellular matrix fibers is a major determinant of the structure of these secondary lymphoid tissues. We are developing new tools to measure the forces and the details of the fiber movements. In particular, collagen fiber remodeling is critical during the immune response as lymphoid tissue rapidly expand to generate the swollen lymph nodes (“swollen glands”) evident in individuals mounting immune responses. We have learned that matrices age and become increasingly stretched in vitro. This has been learned using fluorescence resonance energy transfer, which operates on the nanoscale. The decrease in FRET reflects the unfolding of fibronectin in the process of fibril formation (Smith et al, 2007). We have begun to apply these methods to matrices produced by cells treated with inflammatory cytokines, signals that are critical for lymphoid tissue development.

 

Figure 4 - Clustered fibronectin receptors withhold force over 6-fold more efficiently than non-clustered ones

Force transduction
 

The use of novel laser tweezer systems has allowed us to distinguish the function of two closely related receptor expressed on stromal and immune cells. A key molecular link between cells and the extracellular matrix is the binding between fibronectin (Fn) and integrins a5b1 and aVb3. However, the roles of these different integrins in establishing adhesion remain unclear. We tested the adhesion strength of Fn-integrin-cytoskeleton linkages by applying physiological nN forces to fibronectin-coated magnetic beads bound to cells. We report that the clustering of fibronectin RGD domains within 40 nm lead in turn to integrin α5β1 clustering, and dramatically increased the ability to sustain force from 0.15 pN/RGD to 1.5 pN/RGD. This force was supported by the α5β1 integrin clusters. Importantly, neither integrin αvβ3 nor talin 1 or 2 had a role in maintaining adhesion strength. Instead, these molecules enabled the connection to the cytoskeleton and reinforcement in response to an applied force. Thus, high matrix forces are primarily supported by clustered α5β1 integrins, while less stable links to αvβ3 integrins initiate mechanotransduction, resulting in reinforcement of integrin-cytoskeleton linkages through talin-dependent bonds.
 

Clustering of Fn domains within 40 nm by fusing Fn domains to a petamerization motif (Figure 4A) led to integrin a5b1 recruitment (not shown), and increased the ability to sustain force by over 6-fold as measured by studies with magnetic tweezers using particles with equivalent amounts of monomeric and multimeric ligands (Figure 4B-D). This force was supported by a5b1 integrin clusters (not shown).
 

T-cells express a5b1 integrins and thus the comparison of T-cell responses on substrates with multimeric or an equal total density of monomeric Fn will lead to a better understanding of requirements for integrin signaling in T-cells. MHC-peptide tetramers have been widely used in cell binding, but not in functional analysis. The manner in which these tetrahedral reagents bind to cells is not known. The system described here is much better defined because all 5 arms can bind a cell. We will construct MHC-peptide pentamers and compare these to the monomers that we currently use in a variety of surface and force configurations. This system will provide another controlled variable to define the optimal conditions for generating of memory T-cells.

 

Pathway to Medicine

Future Clinical Applications
 

There are three major therapeutic areas in which we will focus our effort- 1) directing memory CD8+ T-cell production, 2) directing helper and regulatory T cells differentiation and 3) optimizing the retargeting of T cells with chimeric antigen receptors by determining how these are integrated into the immune synapse. These topics were chosen in response to needs of the clinicial collaborators (Fig 5).
 

Figure 5 - A schematic of how basic mechanobiology, immune synapse analysis and clinical application are related in the center

Memory T cells are stem cells that maintain a particular antigen specificity in a state of readiness for years or decades. Following maturation in the thymus, T-cells enter a series of differentiation choices, leading to production of many classes of cells, including memory, regulatory, and effector as well as numerous subtypes. Memory cells exhibit extended life-spans which can include long periods without or with very slow cell division, combined with the ability to respond to appropriate stimulus with rapid proliferation and further differentiation, and thus act as stem cells in the context of the immune system. We will attempt to utilize the ability of T cells to undergo asymmetric cell division as a deterministic pathway to generation of memory and effector cell precursors (Chang et al, 2007). We will use the asymmetric division process and novel on chip sorting systems to collect effector and memory cell precursors and then further expand these to generate specifically programmed effector cells and memory cells that can be transferred in appropriate ratios for short and long term protection, respectively. The ability to control the differentiation process ex vivo, including the use of beads or other engineered surfaces to direct cells, has wide impact on a range of immunotherapy approaches.

 

There are many new tools to measure and generate forces at the submicron level, to organize molecules at the nanometer level, and to simulate the effect of force on protein structure. We have plans for additional tools to measure new parameters that are biologically relevant; these tools are conceptually feasible but still require significant development. In addition, we plan to extend our well-established techniques to new dimensions and new systems. For example, we have implemented devices for measuring submicron 2-D forces and have plans for modifying those devices to enable 3-D force measurement (aided by magnetic beads and wires as well as molecular force probes integrated into force-bearing elements). These tools will play a critical role in optimization of chimeric antigen receptors for redirecting immune cells to the correct cellular targets (tumors or infected cells). The tools that we are developing are flexible, and we are committed to helping other labs apply them to their specific systems. Further, the software that we are generating has general application to other cell systems and we will make that available. Finally, the modeling of the systems will be done in a format that can be easily applied to other systems. Since we are only addressing a small fraction of the force-dependent effects, we expect that there will be many more analogous systems where the technologies that we develop will find application.

 

References

Background Reading
 

Bershadsky, A., M. Kozlov, and B. Geiger. 2006. Adhesion-mediated mechanosensitivity: a time to experiment, and a time to theorize. Curr Opin Cell Biol. 18:472-81.
 

Chang, J. T., V. R. Palanivel, I. Kinjyo, F. Schambach, A. M. Intlekofer, A. Banerjee, S. A. Longworth, K. E. Vinup, P. Mrass, J. Oliaro, N. Killeen, J. S. Orange, S. M. Russell, W. Weninger, and S. L. Reiner. 2007. Asymmetric T lymphocyte division in the initiation of adaptive immune responses. Science 315:1687-1691.
 

Discher, D.E., P. Janmey, and Y.L. Wang. 2005. Tissue cells feel and respond to the stiffness of their substrate. Science. 310:1139-43.

Dobereiner, H. G., B. J. Dubin-Thaler, J. M. Hofman, H. S. Xenias, T. N. Sims, G. Giannone, M. L. Dustin, C. H. Wiggins, and M. P. Sheetz. 2006. Lateral membrane waves constitute a universal dynamic pattern of motile cells. Phys Rev Lett 97:038102.
 

Giannone, G., B.J. Dubin-Thaler, O. Rossier, Y. Cai, O. Chaga, G. Jiang, W. Beaver, H.G. Dobereiner, Y. Freund, G. Borisy, and M.P. Sheetz. 2007. Lamellipodial actin mechanically links Myosin activity with adhesion-site formation. Cell. 128:561-75.
 

Grakoui, A., S. K. Bromley, C. Sumen, M. M. Davis, A. S. Shaw, P. M. Allen, and M. L. Dustin. 1999. The immunological synapse: A molecular machine controlling T cell activation. Science 285:221-227.
 

June, C. H., B. R. Blazar, and J. L. Riley. 2009. Engineering lymphocyte subsets: tools, trials and tribulations. Nat Rev Immunol 9:704-716.

Meshel, A.S., Q. Wei, R.S. Adelstein, and M.P. Sheetz. 2005. Basic mechanism of three-dimensional collagen fibre transport by fibroblasts. Nat Cell Biol. 7:157-64.
 

Sims, T.N., T.J. Soos, H.S. Xenias, B. Dubin-Thaler, J.M. Hofman, J.C. Waite, T.O. Cameron, V.K. Thomas, R. Varma, C.H. Wiggins, M.P. Sheetz, D.R. Littman, and M.L. Dustin. 2007. Opposing effects of PKCtheta and WASp on symmetry breaking and relocation of the immunological synapse. Cell. 129:773-85.

Smith ML, Gourdon D, Little WC, Kubow KE, Eguiluz RA, Luna-Morris S, and V. Vogel. 2007. Force-Induced Unfolding of Fibronectin in the Extracellular Matrix of Living Cells. PLoS Biol. 2;5(10):e268.
 

Storrie H, Guler MO, Abu-Amara SN, Volberg T, Rao M, Geiger B, and SI. Stupp. 2007. Supramolecular crafting of cell adhesion. Biomaterials. 28(31):4608-18.
 

Vogel, V., and M. Sheetz. 2006. Local force and geometry sensing regulate cell functions. Nat Rev Mol Cell Biol. 7:265-75.
 

Zaidel-Bar, R., S. Itzkovitz, A. Ma'ayan, R. Iyengar, and B. Geiger. 2007. Functional atlas of the integrin adhesome. Nat Cell Biol. 9:858-67.
 

Recent center publications
 

  1. Antia, M., Baneyx, G., Kubow, K.E., and Vogel, V. (2008). Fibronectin in aging extracellular matrix fibrils is progressively unfolded by cells and elicits an enhanced rigidity response. Faraday Discuss 139, 229-249; discussion 309-225, 419-220. PMCID: PMC2613444
  2. Arnold, M., Hirschfeld-Warneken, V.C., Lohmuller, T., Heil, P., Blummel, J., Cavalcanti-Adam, E.A., Lopez-Garcia, M., Walther, P., Kessler, H., Geiger, B., and Spatz, J.P. (2008). Induction of cell polarization and migration by a gradient of nanoscale variations in adhesive ligand spacing. Nano Lett 8, 2063-2069. NIHMSID pending author submission. NIHMSID: 132670
  3. Dubin-Thaler, B.J., Hofman, J.M., Cai, Y., Xenias, H., Spielman, I., Shneidman, A.V., David, L.A., Dobereiner, H.G., Wiggins, C.H., and Sheetz, M.P. (2008). Quantification of cell edge velocities and traction forces reveals distinct motility modules during cell spreading. PLoS ONE 3, e3735. PMCID: PMC2581916
  4. Hirschfeld-Warneken, V.C., Arnold, M., Cavalcanti-Adam, A., Lopez-Garcia, M., Kessler, H., and Spatz, J.P. (2008). Cell adhesion and polarisation on molecularly defined spacing gradient surfaces of cyclic RGDfK peptide patches. Eur J Cell Biol 87, 743-750. PMCID: PMC2564985.
  5. Hofman, J.M., and Wiggins, C.H. (2008). Bayesian approach to network modularity. Phys Rev Lett 100, 258701. NIHMSID: 128814
  6. Ilani, T., Vasiliver-Shamis, G., Vardhana, S., Bretscher, A., and Dustin, M.L. (2009). TCR signaling and immunological syanpse stability requires myosin IIA. Nat. Immunol. 10, 531-539. NIHMSID: 128700
  7. Little, W.C., Smith, M.L., Ebneter, U., and Vogel, V. (2008). Assay to mechanically tune and optically probe fibrillar fibronectin conformations from fully relaxed to breakage. Matrix Biol 27, 451-461. NIHMSID: 129907
  8. Neves, S.R., Tsokas, P., Sarkar, A., Grace, E.A., Rangamani, P., Taubenfeld, S.M., Alberini, C.M., Schaff, J.C., Blitzer, R.D., Moraru, II, and Iyengar, R. (2008). Cell shape and negative links in regulatory motifs together control spatial information flow in signaling networks. Cell 133, 666-680. NIHMSID: 131814
  9. Selhuber-Unkel, C., Lopez-Garcia, M., Kessler, H., and Spatz, J.P. (2008). Cooperativity in adhesion cluster formation during initial cell adhesion. Biophys J 95, 5424-5431. PMC Journal - In Process.
  10. Schvartzman, M., A. Mathur, J. Hone, C. Jahnes, and S. J. Wind. 2008. Plasma fluorination of carbon-based materials for imprint and molding lithographic applications. Appl Phys Lett 93:153105. PMCID: PMC2684694
  11. Shen, K., Thomas, V.K., Dustin, M.L., and Kam, L.C. (2008). Micropatterning of costimulatory ligands enhances CD4+ T cell function. Proc Natl Acad Sci U S A 105, 7791-7796. PMCID: PMC2409411
  12. Tsai, J., Sun, E., Gao, Y., Hone, J.C., and Kam, L.C. (2008). Non-Brownian diffusion of membrane molecules in nanopatterned supported lipid bilayers. Nano Lett 8, 425-430. NIHMSID: 128710
  13. Walczak, A.M., Mugler, A., and Wiggins, C.H. (2009). A stochastic spectral analysis of transcriptional regulatory cascades. Proc Natl Acad Sci U S A 106, 6529-6534. PMC Journal - In Process.
  14. Wolfenson, H., Lubelski, A., Regev, T., Klafter, J., Henis, Y.I., and Geiger, B. (2009). A role for the juxtamembrane cytoplasm in the molecular dynamics of focal adhesions. PLoS ONE 4, e4304. PMCID: PMC2627934
  15. Wolfram, T., Spatz, J.P., and Burgess, R.W. (2008). Cell adhesion to agrin presented as a nanopatterned substrate is consistent with an interaction with the extracellular matrix and not transmembrane adhesion molecules. BMC Cell Biol 9, 64. PMCID: PMC2612657
  16. Zhang, X., Jiang, G., Cai, Y., Monkley, S.J., Critchley, D.R., and Sheetz, M.P. (2008). Talin depletion reveals independence of initial cell spreading from integrin activation and traction. Nat Cell Biol 10, 1062-1068. NIHMSID: 128826
  17. Moore, S.W., N. Biais, and M.P. Sheetz. 2009. Traction on immobilized netrin-1 is sufficient to reorient axons. Science 325:166. PMC Journal - In Process.
  18. Schvartzman, M., K. Nguyen, M. Palma, J. Abramson, J. Sable, J. Hone, M. P. Sheetz, and S. J. Wind. 2009. Fabrication of nanoscale bioarrays for the study of cytoskeletal protein binding interactions using nanoimprint lithography. Journal of Vacuum Science and Technology B: Microelectronics and Nanometer Structures 27:61-65. PMCID: PMC2748349
     

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