Engineering DNA as a molecular tool to investigate T cell mechanical signaling Pubblico
Hu, Yuesong (Fall 2023)
Abstract
The initiation of the T cell immune response occurs when T cell receptors (TCR) recognize foreign antigens presented by target cells. The remarkable sensitivity and specificity of this process have been a subject of wonder for decades. This dissertation sheds new light on this phenomenon by revealing that immunoreceptors on the surface of T cells are subjected to mechanical forces, which play a vital role in promoting signaling during TCR-antigen interactions. Chapter 1 of the dissertation serves as an introduction, discussing T cell recognition, recently proposed mechanosensor model to explain the near-perfect sensitivity, and the utilization of DNA-based force sensors to visualize mechanotransduction. Chapter 2 presents a microparticle force sensor, designed to overcome the limitations of current force measurement tools. This technique enables the study of receptor forces on cellular-sized particle surface with a high-throughput readout using flow cytometry. Chapter 3 introduces a programmable DNA origami-based force sensor. This highly adaptable sensor can be incorporated into supported lipid bilayers and target cell membranes, facilitating the investigation of the magnitude and sources of TCR forces at dynamic physiological membrane junctions. Chapter 4 explores the mechanics of another T cell immunoreceptor, Lymphocyte function-associated antigen 1 (LFA-1), and demonstrates its role in antigen discrimination and cytotoxic degranulation. Finally, Chapter 5 provides a comprehensive summary of the dissertation's findings and discuss potential future research directions in the emerging field of mechanoimmunology.
Table of Contents
Chapter 1. Mechanical regulation of T cell recognition 1
1.1 Introduction 2
1.2 Mechanical forces regulate T cell recognition 3
1.2.1 T cell recognition and TCR signaling 3
1.2.2 TCR-pMHC bond lifetime is crucial to TCR signaling 6
1.2.3 TCR mechanosensor model (conformation change) 11
1.2.3 TCR mechanosensor model (mechanical force) 15
1.3 Tools to investigate cell mechanical forces 17
1.3.1 Summary of current force measurement tools 17
1.3.2 DNA hairpin-based tension sensors 22
1.3.3 DNA duplex-based tension sensors 33
1.4 Aim and Scope of the Dissertation 41
Chapter 2. DNA-based Microparticle Tension Sensors (μTS) for Measuring Cell Mechanics in Non-planar Geometries and for High-throughput Quantification 43
2.1 Abstract 44
2.2 Introduction 44
2.3 Result and discussion 47
2.3.1 Design, synthesis and characterization of μTS 47
2.3.2 Visualizing TCR mechanics in hybrid immunological synapse formed with spherical μTS 48
2.3.3 Mapping platelet adhesion forces by μTS 51
2.3.4 Quantifying integrin forces in high throughput by μTS 54
2.3.5 Mechanopharmacology using the μTS platform: Testing how anti-platelet drugs impact platelet integrin forces 56
2.4 Conclusion 59
2.5 Materials and Methods 61
2.5.1 Materials 61
2.5.2 Methods 63
2.6 Appendix 71
Chapter 3. DNA Origami Tension Sensors to Study TCR Mechanics at Membrane Junctions 85
3.1 Abstract 86
3.2 Introduction 86
3.3 Results 89
3.3.1 Design and characterization of DOTS 89
3.3.2 The dimension of DOTS eliminates intermolecular FRET at high molecular density 90
3.3.3 DOTS detect TCR tension at fluid intermembrane interfaces 92
3.3.4 Actin polymerization and membrane bending contribute to TCR forces 98
3.3.4 Spherical SLB for investigating TCR mechanics in suspension 101
3.3.5 DOTS revealed TCR tension at the physiological T cell-B cell junctions 104
3.4 Conclusions 108
3.5 Materials and Methods 110
3.5.1 Materials 110
3.5.2 Methods 111
3.6 Appendix 121
Chapter 4. LFA-1-ICAM mechanical force regulates T cell recognition and cytotoxic degranulation 156
4.1 Abstract 157
4.2 Introduction 157
4.3 Results and Discussion 159
4.3.1 Measuring LFA-1 force with DNA based tension sensors 159
4.3.2 Mechanical force through the LFA-1/ICAM-1 bond potentiates TCR signaling 160
4.3.3 LFA-1 force potentiates antigen discrimination 163
4.3.4 Degranulation occurs in regions of LFA-1-dependent force exertion 164
4.3.4 LFA-1 force defines cytotoxic degranulation 167
4.5 Materials and Methods 172
4.5.1 Chemicals and oligonucleotides 172
4.5.2 Methods 173
Chapter 5. Summary and Perspective 181
5.1 Summary 182
5.2 Future outlook 185
5.3 Closing Remarks187
6. Bibliography 189
About this Dissertation
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Primary PDF
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Engineering DNA as a molecular tool to investigate T cell mechanical signaling () | 2023-12-07 23:57:11 -0500 |
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Supplemental Files
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Movie A 2.1 (Tension and F-actin signal distribution at the interface between T cell and μTS) | 2023-10-31 11:13:30 -0400 |
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Movie A3.1 (Time lapse video showing origami exclusion from the cell spreading area) | 2023-10-31 11:13:58 -0400 |
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Movie A3.2 (Single molecule experiments showing the spatiotemporal dynamics of DOTS in the immune synapse) | 2023-10-31 11:14:11 -0400 |
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Movie A3.3 (The distribution of F-actin and DOTS at the effector T-cell immune synapse) | 2023-10-31 11:14:49 -0400 |
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Movie A3.4 (3D view of DOTS and tension patterns at the SSLB-T-cell interface) | 2023-10-31 11:15:04 -0400 |
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