Nanotechnology Center for Mechanics in Regenerative Medicine
The Nanotechnology Center for Mechanics in Regenerative Medicine has refocused its effort in the past year on the mechanisms by which nanoscale functional assemblies in immune cells respond to changes in the physical environment to generate useful cellular components for immunotherapy applications. This effort builds on principles that were discovered in the preceding period using fibroblastoid and immune cell systems and the addition of a clinical collaboration with the immunotherapy group of Michael Milone and Carl June at U. Penn. The immune system is critical to tissue regeneration following injury. Chronic viral infections and cancer, the therapeutic focus of the U. Penn. group, are both situations in which immune cells can contribute to eradication of disease and initiation of regenerative processes to restore health of the patient. The main tool used by the U. Penn. group is adoptive immunotherapy- growing billions of CD8+ memory and effector T lymphocyte (Tcells) under GMP conditions that are infused into patients to attack virally infected and tumor cells. 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 this goal. All of the elements of our center have key roles in this new focus on defining conditions to generate memory CD8+ T cells using nanotechnology. 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. 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 demonstrated that 1. Microfabricated substrates control T-cell function (2). 2. Force generating molecular assemblies are required for immunological synapse formation (8). 3. Substrate imposed membrane shape controls signaling networks (19). 4. Nanopatterned adhesion gradients control integrin dependent adhesion (20). 5. Fibroblasts continuously remodel extracellular matrix to increase rigidity (3). These advances can be directly applied to the problem of generating memory T cells from bottom up and top down approaches.
The field of T cell development is currently divided on how memory cells develop and this split is based in part on the use of different experimental models and the unavailability of definitive markers for memory T-cells. Investigators working in acute viral infection models like LCMV tend to favor a model in which memory cells arise from surviving effector cells during the contraction phase of the response (21). An alternative model favored by investigators working with intracellular bacterial infections is that memory cells are generated early in the response and maintained at relatively low, constant numbers throughout the response. This later model has been mechanistically supported by recent findings that stable immunological synapses organize a process of asymmetric cell division leading to one daughter cell that is a memory precursor and one daughter cell that is an effector precursor (6, 7). In this model the axis of asymmetry is perpendicular to the plane of the immunological synapse. The daughter cell proximal to the synapse inherits more LFA-1 and CD8, whereas the distal daughter inherits more PKC-ζ, a polarity network associated kinase (7). It is possible that both of these mechanisms for generation of memory cells can operate in vivo under different conditions. This would not be surprising since biological systems often have built in redundancy. However, the mechanism by which a few effector cells reverse-differentiate to form memory cells is entirely unknown and thus this phenomenom is not useful for our efforts. The mechanism by which memory cells can be produced early in a response by asymmetric cell division is well defined and is based on a stable immunological synapse, a structure that we have already demonstrated that we can manipulate at the nanoscale in multiple studies. Therefore, our center focuses our effort on manipulating the immunological synapse to control asymmetric cell divisions to generate memory and effector precursors that will then be expanded for infusion into patients as pure populations to achieve long term and rapid target eradication, respectively.
Our interdisciplinary team was originally chosen to incorporate all the components needed to undertake studies on mechanotransduction in mesenchymal cells, including immune cells. In this case, the plan is to address the important aspects of the cell biology, mechanical environment, cell signaling networks, bioinformatics and modeling of the process that leads to the production of memory t-cells. We have found that the behavior of tcells is profoundly dependent upon mechanical factors, since the formation of the immune synapse requires myosin II and differentiation is affected by the geometry of the substrate (more recently, even the rigidity appears to matter). We are taking a multi-pronged approach to the problem that ranges from the testing of in vivo derived matrices to the in silico modeling of signaling pathways. There are currently 6 major elements to the effort. 1) At the level closest to in vivo, the Vogel lab is testing the effect of decellularized lymph nodes on the process of immune cell differentiation with defined antigen presenting cells and t-cells. 2) Cellular studies in the Dustin and Sheetz labs are aimed at defining the cellular steps and modular protein machines that are involved in the process of activation, taking clues from both the geometry and the SiRNA screens. 3) Cellular studies in the Kam and Milone labs are testing how to generate large matrix arrays to produce sufficient numbers of activated t-cells. 4) Fabrication of different matrices is critical for our ability to mimic matrix environments in vivo and to control cell-cell interactions with nano-structured surfaces. 5) Screening with SiRNA libraries for the important proteins in mechanical processes have identified several proteins that play a major roll in t-cell differentiation and encourage us to look for other proteins in those pathways. 6) Modeling of the signaling process based upon the quantitative measurements that we have made, can direct experiments to test between different models of the critical elements in the differentiation process. Immune responses take place in secondary lymphoid tissues that are scaffolded by fibroblastic reticular cells. The extracellular matrix provides a dynamic environment for the dendritic and immune cells that clearly enables normal immune function and understanding the important elements of these collagen and fibronectin based matrices is critical. Using single cell approaches will enable us to apply some of the lessons from in vitro studies of fibroblasts and immune cells to the behavior in a more physiological environment. Further, such factors as high local density of ligand, force-dependent stretch and time-dependent oscillations in force influence the types of surfaces that will be prepared to test for improvements in the desired differentiation process. At the application level, we already have improved upon the current methods of production of activated t-cells and are working out methods to reach the needed scale for clinical application that may present new problems. The fabrication groups are presented with a number of challenges to get surfaces for these different applications that include new and challenging capabilities. Already, the screens of factors involved in rigidity sensing have provided new targets that could affect t-cell differentiation. Finally, if we understand this process, we should be able to model it and the current models don’t incorporate the mechanics.
The Nanotechnology Center for Mechanics in Regenerative Medicine has refocused its multidisciplinary efforts on the problem of generating memory T cells for immunotherapy applications. The NDC funded effort of each group has been retasked to execute tasks needed to attain this goal. All groups are working on substrates focused on T-cell problems or working directly with T cells and antigen presenting cells to address these programs. Coordination of this effort is being controlled by the director and co-director of the NDC. Although this provides a major challenge for the group, everyone is excited about the multidisciplinary approach that increases the probability that we can find a way to improve the production of activated immune cells as well as understand the important aspects of the immune cell activation process.