2013 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. Our clinical collaboration with the immunotherapy group of Michael Milone at U. Penn and Carl June at the Abramson Cancer Center provides a clinical setting to test the utility of the culture systems developed by all NDC groups. The main tool used by the Abramson Cancer Center group is adoptive immunotherapy- growing billions of memory and effector T cells under cGMP conditions that are infused into patients to augment and focus immunity. We continue to focus on the application of mechanobiology principles to the immune response, fully working on projects that are directly aligned with the immunotherapy goals.
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 and number of T cells generated during the production phase to optimize benefit to the patient. The production of expanded populations capable of further proliferation after transfer and the development of memory cells correlates with the best in vivo results for tumor immunotherapy in animal models and patients. We are using mechanics to tune signal transduction pathway to balance rapid expansion in vitro while maintaining the capacity to replicate further in vivo. We are using immunology and systems biology tools to evaluate these complex phenotypes using in vivo effector and memory T cells from effective immune responses as a template. The key initiating event in T cell activation and differentiation is the formation of an immunological synapse, which integrates adhesion and antigen recognition. The tissue environment defined by fibroblastic reticular cells that generate a network of ECM fibers provide a physical scaffold for immunological synapse formation and differentiation. Engineering systems that provide more physiological mechanical cues may restore the balance between replication and replicative reserve. In the past year we have made a number of advances in these areas:
1. We showed that rigidity of the substrate used for immobilization of T cells activating ligands controls activation and proliferation of human T cells (1),
2. Using mouse T cells we also showed that their activation through CD3 and CD28 is sensitive to the mechanic
al properties of a substrate presenting ligands to these receptors (2).
3. We have developed an overall step-by-step protocol to integrate response to cell environment with molecular networks that can be used by a wider research community (3).
4. We have continued to develop the technology to generate T cell culture substrates with single molecules patterned at a nanoscale (4) and to visualize biomolecular interactions at the immune synapse with single molecule resolution (5). An important development of the center in the past year has been the expansion of its focus not only at the quantitative level (T cell expansion) but also at the qualitative level, aiming at understanding and manipulating the phenotype of human T cells in culture.
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 and extracellular matrix. Having the optimization of the ex vivo generation of T cells for adoptive therapy, in addition to the published progress, every member has continued to have a key role in the past year adding to the center’s progress. The majority of the groups at our center are now collaborating to apply and understand the effects of substrate rigidity on T cell activation and proliferation (Milone, Kam, Dustin, Hone, Wind, Geiger, Vogel, Bonneau, Sheetz and Hone Groups). The Sheetz, Dustin Kam, Geiger and Vogel labs are defining the nanoscale molecular mechanisms that account for effects of substrate rigidity on T cell activation and differentiation. The Bonneau, Dustin, June and Milone groups are collaborating on gene expression studies to link ex vivo expansion to key characteristics of in vivo memory. The Dustin, Sheetz, Wind, Dunlop, Wiggins, Geiger and Spatz groups are using single molecule imaging and patterning to explore fundamental requirements for T Cell Antigen Receptor and Chimeric Antigen Receptor ligands. The Hone, Wind, Vogel, Sheetz, Kam and Dunlop groups are currently testing a number of secondary candidates. The June group is preparing for the clinical trial by establishing control conditions and the technology needed to “bar code” the cellular products from the nano-engineered and standard of care culture systems. These activities will generate the data needed for key critical decision points regarding the lead and secondary candidates on our translational path.
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 the successful expansion of anergic T cells from patients with hematopoietic malignancies like CLL and ALL. T cell anergy limits the number of patients who can be treated and we anticipate increasing with number by ~30% by improving the culture system. 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 PI’s have coordinated this effort with the assistance of the Scientific Program Manager. The bi-weekly video conference continue to be an extremely useful platform to discuss progress by each group on a monthly basis (Round-robin format), where trainees from all groups give a short presentation on a monthly basis (to allow for sufficient time to discuss results and provide feedback, and have been helpful to exert a regular influence on the trainees in all centers - this has helped to control the activity in the center and keep the 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. Conversely, the Dustin group has been key in providing the knowledge to fabrication/chemistry groups on T cell isolation, culture and assays, as well as generating and providing essential protein reagents. The clinical collaborator labs – Milone and June – have been providing some of the T cells - donor cells – to complement human T cell analysis throughout the center. 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.
- O’Connor RS, Hao X, Shen K, Bashour K, Akimova T, Hancock WW, Kam LC and Milone MC. Substrate rigidity regulates human T cell activation and proliferation. 2012. J Immunol.189(3):1330-9.
- Judokusumo, E, Tabdanov, E, Kumari, S, Dustin, ML, and Kam, LC Mechanosensing in T Lymphocyte Activation. Biophysical Journal - Letters, 2012, 102:L5-7.
- Poultney CS, Greenfield A, Bonneau R. Integrated inference and analysis of regulatory networks from multi-level measurements. Methods Cell Biol. 2012; 110:19-56.
- Palma, M, Abramson, JJ, Gorodetsky, AA, Penzo, E, Gonzalez, RL, Jr., Sheetz, MP, Nuckolls, C, Hone, J, and Wind, SJ (2011).
- Palma, M, Abramson, JJ, Gorodetsky, AA, Penzo, E, Gonzalez, RL, Jr., Sheetz, MP, Nuckolls, C, Hone, J, and Wind, SJ (2011b). Selective biomolecular nanoarrays for parallel single-molecule investigations. J Am Chem Soc 133, 7656-7659.