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Human T Cell Response to Substrate Rigidity for Design of Improved Expansion Platform

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Human T Cell Response to Substrate Rigidity for Design of Improved Expansion Platform Human T Cell Response to Substrate Rigidity for Design of Improved Expansion Platform Sarah E. De Leo Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2014 © 2014 Sarah E. De Leo All rights reserved ABSTRACT Human T Cell Response to Substrate Rigidity for Design of Improved Expansion Platform Sarah De Leo Cells have long been known to sense and respond to mechanical stimuli in their environment. In the adoptive immune system particularly, cells are highly special- ized and responsible for detecting and eliminating pathogens from the body. T cell mechanosensing is a relatively new field that explores how force transmission in cell- cell interaction elicits both inter- and intra-cell signaling. Owing to recent advances in genetic manipulation of T cells, it has emerged as new tool in immunotherapy. We recently demonstrated human T cell activation in response to mechanical rigidity of surfaces presenting activating antibodies CD3 and CD28. The work in this dissertation highlights new progress in the basic science of T cell mechanosensing, and the utilization of this knowledge toward the development of a more specialized expansion platform for adoptive immunotherapies. Human T cells are known to trigger more readily on softer PDMS substrates, where Young's Modulus is less than 100 kPa as compared to surfaces of 2 MPa. While the range of effective rigidities has been established, it is important to explore local differ- ences in substrates that may also contribute to these findings. We have isolated the rigidity-dependence of cell-cell interactions apart from material properties to optimize design for a clinical cell expansion platform. Though PDMS is a well understood bio- material and has found extensive use in cellular engineering, a PA gel substrate model allows for rigidity to be tuned more closely across this specific range of rigidities and provides control over ligand density and orientation. These rigidity-based trends will be instrumental in adapting models of mechanobiology to describe T cell activation via the immune synapse. In what is generally accepted as the clinical gold-standard for T cell expansion, rigid (GPa) antibody-coated polystyrene beads provide an increase in the ratio of stim- ulating surface area-per-volume, over standard culture dishes. Herein we describe the development of a soft-material fiber-based system with particular focus on maintaining mechanical properties of PDMS to exploit rigidity-based expansion trends, investigated through atomic force microscopy. This system is designed to ease risks associated with bead-cell separation while preserving a large area-to-volume ratio. Exposing T cells to electrospun mesh of varying rigidities, fiber diameters, and mesh densities over short (3 day) and long (15 day) time periods have allowed for this system's optimization. By capitalizing on the mechanisms by which rigidity mediates cell activation, clinical cell expansion can be improved to provide greater expansion in a single growth period, direct the phenotypic makeup of expanded populations, and treat more patients faster. This technology may even reach some cell populations that are not responsive to current treatments. The aims of this work are focused to identify key material properties that drive the expansion of T cells and optimize them in the design of a rigidity-based cell expansion platform. Contents List of Figuresv List of Tables vii Acknowledgments viii 1 Introduction1 1.1 The advent of personalized medicine...................1 1.2 The immune system functions to fight disease..............2 1.3 Harnessing the immune system through adoptive immunotherapies...7 1.4 Bead-based cell expansion technologies..................8 1.5 Improving ex vivo T cell expansion through mechanobiology...... 12 1.5.1 T cell mechanosensing....................... 12 1.5.2 Adaptation of the motor clutch model may explain rigidity response 15 1.6 Impact and significance........................... 18 2 Polyacrylamide Gels for Delineation of T Cell Response to Surface Rigidity 20 i 2.1 Abstract................................... 20 2.2 Introduction................................. 21 2.3 Materials and Methods........................... 24 2.3.1 Preparation of gel substrates.................... 24 2.3.2 Preparation of antibodies...................... 26 2.3.3 Fluorescence analysis of antibody presentation.......... 26 2.3.4 Atomic force microscopy...................... 27 2.3.5 Cell isolation and culturing.................... 27 2.3.6 Interleukin 2 (IL-2) secretion assay................ 28 2.3.7 Data acquisition and analysis................... 28 2.4 Results.................................... 29 2.4.1 Polyacrylamide gels with bis-acrylamide crosslinker limited in rigidity spectrum.......................... 29 2.4.2 Hertzian analytical model employed for AFM indentation studies 30 2.4.3 Polyacrylamide gels with piperazine-diacrylamide crosslinker can be made from 100 kPa to 1 MPa................. 35 2.4.4 Human CD4+ T cells activate across rigidity range....... 42 2.5 Discussion.................................. 45 3 Electrospun Mesh Platform for Enhanced T Cell Expansion 47 3.1 Abstract................................... 47 3.2 Introduction................................. 48 3.2.1 Electrospun mesh.......................... 51 3.3 Materials and Methods........................... 57 ii 3.3.1 Atomic force microscopy...................... 57 3.3.2 Surface preparation......................... 57 3.3.3 Confocal microscopy........................ 58 3.3.4 Cell culture............................. 58 3.3.5 Assessment of initial cell-mesh binding.............. 59 3.3.6 Long-term cell expansions in conjunction with CFSE assays.. 59 3.3.7 Flow cytometry........................... 61 3.3.8 Statistical analysis......................... 61 3.4 Results.................................... 61 3.4.1 Characterization of rigidity and protein adhesion on electrospun mesh................................. 61 3.4.2 T cell penetration into and interaction with mesh topology... 65 3.4.3 T cell stimulation and expansion on electrospun mesh at short and long time points........................ 67 3.5 Discussion.................................. 74 4 Conclusions and Future Directions 78 5 Perspective on Personalized Medicine in a Changing Healthcare Land- scape 82 5.1 The Current Healthcare Climate...................... 82 5.2 The Role of Personalized Medicine.................... 84 5.2.1 Reserach and Development..................... 87 5.2.2 The FDA's Regulatory Strategies................. 89 iii 5.2.3 Economic and Ethical Considerations............... 92 5.3 Patients and Advocates Guide Medical Research Efforts......... 96 Bibliography 98 Appendix 113 A 113 A.1 Electrospinning mesh............................ 113 A.2 Mesh characterization........................... 114 iv List of Figures 1.1 Schematic of T Cell Activation......................5 1.2 Adoptive Immunotherapy Method..................... 10 1.3 PDMS Bead Coating Methodology for T Cell Activation........ 11 1.4 Motor-Clutch Model............................ 17 2.1 Atomic Force Microscope System..................... 32 2.2 Chemical Structure of Polyacrylamide Crosslinkers........... 36 2.3 Human CD4+ T Cell Cytokine Secretion on Rigidity-Dependent Poly- acrylamide Gels............................... 44 3.1 Schematic of Electrospinning........................ 52 3.2 SEM images of PMDS-PCL mesh..................... 55 3.3 Schematic of AFM Tip Interaction with Substrate............ 63 3.4 AFM Indentation Measurements...................... 64 3.5 Confocal Images of Mesh and Cell Interaction.............. 66 3.6 Human CD4-CD8 T Cell Activation and Proliferation Study Comparing Antibody Linkers.............................. 69 v 3.7 Human CD4-CD8 T Cell Activation and Proliferation Study Comparing Unaligned and Aligned Fibers with Varying Diameters......... 71 3.8 Human CD4-CD8 T Cell Activation and Proliferation Study with PCL Controls................................... 73 5.1 Integration of molecular, clinical, and genetic factors can be assessed by personalized diagnostics and impact therapeutic options [Bottinger, 2007]. 85 5.2 Probablility of drug success to market by stage of develpment [Arrow- smith, 2012]................................. 90 vi List of Tables 2.1 Polyacrylamide gel compositions with bis-acrylamide crosslinker.... 30 2.2 AFM indentation measurements...................... 38 2.3 PDA-polyacrylamide gels.......................... 39 2.4 Antibody presentation on PA gels..................... 41 2.5 Streptavidin Acrylamide Concentrations................. 41 2.6 Streptavidin Acrylamide Concentrations................. 42 3.1 Morphological data of mesh........................ 56 vii Acknowledgments I would like to thank Professor Kam, whose dedication to science is inspiring and commitment to his graduate students is invaluable. I would not have completed this work without his selfless time and patient guidance. I am also appreciative of members of the MBL group past and present who have been a sounding board for ideas and a source of constant support and encouragement, particularly Keenan, Haoqian, Alex, Debjit, Geri, Susie, Ed, Lester and Helen. I would like to thank our collaborators, without whom this work would not have been possible; special thanks to Dr. Lu for her advice and Danielle for her work in mesh fabrication. Thanks to Corina for her assistance with the AFM.
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