The Immune Synapse: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Measuring the Co-Localization and Dynamics of Mobile Proteins in Live Cells Undergoing Signaling Responses
1 Introduction
2 Materials
2.1 Cell Culture and Transfection
2.2 Dye Conjugation of Labeling Proteins
2.3 Cell Plating and Sample Preparation
2.4 Imaging Reagents
2.5 Microscope and Imaging Software
2.6 Post-Processing and Data Analysis
3 Methods
3.1 Preparation of Fluorescent Fab Fragments
3.2 Cell Preparation
3.3 Imaging
3.4 Post-Processing: Single Molecule Localization
3.5 Post-Processing: Cross-Correlation Analysis
3.6 Post-Processing: Diffusion Analysis
4 Notes
References
Chapter 2: Fluorescence-Based Measurements of Two-Dimensional Affinity in Membrane Interfaces
1 Introduction
2 Materials
2.1 Lipids, Reagents, and Supplies
2.2 Cell Culturing
2.3 Proteins and Labels
2.4 Measurement Cell
2.5 Microscope Setup (TIRF)
3 Methods
3.1 Cell Culturing
3.2 Vesicle Making
3.3 Preparation of Measurement Samples
3.4 SLB Formation
3.5 Functionalizing the SLB with Fluorescently Labeled His-Tagged Proteins
3.6 Adding Cells
3.7 Imaging of Cell Contacts
3.8 Imidazole Addition
3.9 Converting Between Intensity and Protein Density
3.10 Image Analysis and Detection
3.11 Zhu-Golan Analysis
4 Notes
References
Chapter 3: Surfaces for Study of Receptor Dynamics on T Cells
1 Introduction
2 Materials
2.1 Materials for Adhesive Surfaces
2.2 Additional Materials for Activating Surfaces
2.3 Additional Materials for Cell Mimetic Lipid Bilayers
2.4 Imaging T Cell Receptors
3 Methods
3.1 Preparations of Adhesive Surface
3.2 Preparations of Activating and Nonactivating Surface
3.3 Preparations of Cell Mimetic Lipid Bilayers
3.4 Imaging T Cell Surface Receptors
4 Notes
References
Chapter 4: Molecular Dynamics Simulations of Immune Receptors and Ligands
1 Introduction
2 Software
3 Methods
3.1 Protein Structure
3.2 Membrane Embedding
3.3 Equilibration and Simulations
4 Notes
References
5: Investigating Diffusion Dynamics and Interactions with Scanning Fluorescence Correlation Spectroscopy (sFCS)
1 Introduction
2 Materials
2.1 Hardware and Software
2.2 Calibration and Alignment
2.3 Model Membrane Sample Preparation
2.4 Live Cell Samples
3 Methods
3.1 Microscope Calibration Using Point FCS in Solution
3.2 Fluorophore Optimization with FCS Excitation Scans
3.3 Cross-Correlation Controls and Calibrations
3.4 Model Membrane Systems: GUVs
3.5 Model Membrane Systems: SLBs
3.6 Live Cells (Transient Transfection)
3.7 Data Acquisition (sFCS)
3.8 Data Analysis
3.9 Downstream Analysis
3.10 Example Data: Lck Diffusion in T Cells Is Linked to Activatory Signaling
4 Notes
References
Chapter 6: Combined FRET-FLIM and NAD(P)H FLIM to Analyze B Cell Receptor Signaling Induced Metabolic Activity of Germinal Cen...
1 Introduction
2 Materials
2.1 Mice
2.2 Liquids (Sterile)
2.3 Single B Cell Suspensions
2.4 Cell Transfer and Immunization
2.5 Drugs for Anesthesia
2.6 Surgery
2.7 Microscopic Equipment
2.8 Software and Code
3 Methods
3.1 Surgical Preparation of Mice
3.2 System Setup
3.3 Surgical Preparations
3.4 Data Acquisition
3.5 Data Analysis
3.6 Cell Segmentation
3.6.1 Imaris
3.6.2 CellProfiler
4 Notes
References
Chapter 7: Measurement of Molecular Height Using Cell Surface Optical Profilometry (CSOP)
1 Introduction
2 Materials
2.1 Microscopy
2.2 Spherical CSOP Targets
2.3 A Membrane Labeling Reagent
2.4 A Protein Labeling Reagent
2.5 Imaging Buffer
3 Methods
3.1 Preparation of CSOP Samples
3.2 Calibration of the CSOP Imaging System
3.3 Height Measurement
3.4 Image Analysis
4 Notes
References
Chapter 8: Observing Membrane and Cell Adhesion via Reflection Interference Contrast Microscopy
1 Introduction
2 Materials
3 Methods
3.1 Microscope Alignment
3.2 Observation and Recording
3.3 Image Processing and Data Analysis
Box 1
3.4 Dy-RICM Analysis
4 Notes
References
Chapter 9: En-Face Imaging of T Cell-Dendritic Cell Immunological Synapses
1 Introduction
2 Materials and Equipment
2.1 PDMS Device
2.2 Cell Culture
3 Methods
3.1 Photolithography of Wafers
3.2 Production of Soft PDMS Pistons
3.3 Preparation of Bottom Dish with a Glass Slide
3.4 Production of PDMS Micro Pillars
3.5 Preparation of Cells
3.5.1 Isolation and Staining of T Cells
3.5.2 Preparation of Dendritic Cells
3.6 Assembly of Confiner Setup
4 Notes
References
Chapter 10: High- and Super-Resolution Imaging of Cell-Cell Interfaces
1 Introduction
2 Materials
3 Method
3.1 Preparations
3.2 Small Glasses and Chamber Wash and Coating
3.3 Cell Staining (See Note 3)
3.4 Cell-on-Cell Engagement
3.5 Imaging
4 Notes
References
Chapter 11: Separation of Single Core and Multicore Lytic Granules by Subcellular Fractionation and Immunoisolation
1 Introduction
2 Materials
2.1 Primary Murine T Cells
2.2 Cell Homogenization
2.3 Subcellular Fractionation
2.4 Immunoisolation of MCG and SCG
3 Methods
3.1 Cell Homogenization
3.2 Subcellular Fractionation
3.3 Immunoisolation of MCG and SCG
4 Notes
References
Chapter 12: Microvillar Cartography: A Super-Resolution Single-Molecule Imaging Method to Map the Positions of Membrane Protei...
1 Introduction
2 Materials
2.1 Labeling
2.2 Imaging
2.3 Software
2.4 Buffer Preparation
2.5 Microscope
3 Methods
3.1 Labeling of the Cell Membrane and Membrane Proteins (Timing: 1 Day)
3.2 Labeling of Two Different Membrane Proteins for Co-localization Probability (CP) Analysis (Timing: 1 Day)
3.3 Sample Preparation for Microscopy
3.4 Imaging (Timing ~1 h for Imaging of One Cell)
3.4.1 Variable Angle-Total Internal Reflection Microscopy
3.4.2 Stochastic Localization Nanoscopy (SLN)
3.4.3 Analysis (Timing 15-30 min)
4 Notes
References
Chapter 13: T Cell Immunological Synaptosomes: Definition and Isolation
1 Introduction
2 Materials
2.1 Materials for Preparation of T Cell Blasts
2.2 Materials for Bone Marrow-Derived DC (BMDC) Preparation
2.3 Materials for Preparation of Ab-Immobilized CNBr-Activated Sepharose Beads
2.4 Materials for TIS Purification
2.5 Materials for TIS Measurement by Flow Cytometry
2.6 Materials for Evaluation of TIS Activity on DCs
2.7 Regeneration of Ab-Immobilized CNBr-Activated Sepharose Beads
3 Methods
3.1 Preparation of T Cell Blasts
3.2 Preparation of Bone Marrow-Derived DCs (BMDCs)
3.3 Preparation of Ab-Immobilized CNBr-Activated Sepharose Beads
3.4 TIS Purification (Fig. 1)
3.5 TIS Measurement by Flow Cytometry
3.6 Evaluation of TIS Activity on DCs
3.7 Regeneration of Ab-Immobilized CNBr-Activated Sepharose Beads
4 Notes
References
14: Dynamics of Immune Cell Microvilli
1 Introduction
2 Materials
2.1 Microscope and Supplies
2.2 Cell Culture
2.3 Staining and Fixation (see Note 3)
3 Methods
3.1 Cell Culture
3.2 Live Cell Imaging
3.3 Fixed Cell Imaging (Fig. 1e)
3.4 Data Acquisition (Fig. 1f)
3.5 Data Processing (Fig. 1g)
4 Notes
References
Chapter 15: Visualization of Myddosome Assembly in Live Cells
1 Introduction
2 Materials
2.1 Purification and Labeling of His10-IL-1
2.2 Supported Lipid Bilayers Functionalized with IL-1
3 Methods
3.1 Purification and Labeling of IL-1
3.1.1 Labeling HaloTag in His10-Halotag-IL-1β with JF646 Halo Ligand
3.2 Preparing Small Unilamellar Vesicles (SUVs) Suspension for SLB Formation
3.2.1 Formation of SUVs by Freeze-Thaw and Bath Sonication
3.3 Cleaning 96-Well Glass Bottom Plates
3.4 Formation of Supported Lipid Membranes, Labeling SLB with His10-IL1β, Total Internal Reflection Microscopy of SLBs and Ima...
3.4.1 Formation of SLBs in Glass-Bottom 96-Well Plates
3.4.2 Preparing GFP and mScarlet Calibration Wells
3.4.3 Quantification of IL-1 Density and SLB Mobility and Live-Cell Imaging of Myddosome Formation
4 Notes
References
Chapter 16: Detection of Telomere Transfer at Immunological Synapse
1 Introduction
2 Materials
2.1 APCs Purification from Human Primary Peripheral Blood Mononucleate Cells (PBMCs)
2.2 TelC-PNA Probe Live Labeling
2.3 Fluorescence-Activated Vesicle Sorting (FAVS)
2.4 Antigen-Specific Conjugates
2.5 Tzap Overexpression for Alternative Telomere Detection Method
3 Methods
3.1 APCs Purification from Human Primary Peripheral Blood Mononucleate Cells (PBMCs)
3.2 TelC-PNA Probe Live Labeling
3.2.1 Preparation of Glass Beads
3.2.2 APC Telomere Live Labeling
3.2.3 PKH67 Staining and Ionomycin Stimulation
3.3 Fluorescence-Activated Vesicle Sorting (FAVS)
3.4 Antigen-Specific Conjugates
3.4.1 Flowcytometry Analysis to Detect T Cells Acquiring Telomere from APCs
3.4.2 Telomere Immunofluorescence In Situ Hybridization FISH (IF-FISH)
3.5 Tzap Overexpression for Alternative Telomere Detection Method
4 Notes
References
17: Bottom-Up Assembly of Bioinspired, Fully Synthetic Extracellular Vesicles
1 Introduction
2 Materials
2.1 SUV Production
2.2 LUV Production by Emulsification
2.3 Evaluation of Lipid Concentration in LUV Suspension
2.4 Evaluation of Vesicle Concentration and Size by NTA
2.5 Evaluation of Vesicle Size by DLS
2.6 Functionalization of Vesicles with Proteins
3 Methods
3.1 Assembly of synEVs in the Form of SUVs
3.2 Assembly of synEVs in the Form of LUVs by Emulsification
3.3 Evaluation of Lipid Concentration in LUV Suspension
3.4 Evaluation of the Vesicle Concentration and Size by NTA
3.5 Evaluation of the Vesicle Concentration and Size by DLS
3.6 Functionalization of Vesicle Surface with Proteins
4 Notes
References
Chapter 18: A DNA Origami-Based Biointerface to Interrogate the Spatial Requirements for Sensitized T-Cell Antigen Recognition
1 Introduction
2 Materials
2.1 Protein Purification
2.2 Microscopy Setup
2.3 Other Components
3 Methods
3.1 Expression of Streptavidin and I-Ek Subunits as Insoluble Inclusion Bodies in E. coli
3.2 Refolding and Purification of Trans dSav
3.3 Refolding I-Ek in Complex with a Placeholder Peptide
3.4 Purification of I-Ek/ANP
3.5 Site-Specific Biotinylation of I-Ek/ANP Using the BirA Biotin Ligase
3.6 Site-Specific Labeling of a Peptide with Maleimide-Conjugated Dyes
3.7 Exchange of the I-Ek-Associated ANP Placeholder Peptide with Site-Specifically Labeled Peptides
3.8 DNA Origami Preparation
3.9 DNA Origami Purification
3.10 DNA Origami Functionalization Strategy Using Divalent Streptavidin
3.11 DNA Origami Quality Control: Gel Electrophoresis
3.12 SLB Preparation
3.13 SLB Functionalization
3.14 Diffusion Analysis of DNA Origami Structures
3.15 Determining the Fraction of DNA Origami Structures Devoid of pMHC
3.16 Determining the Number of pMHCs on a DNA Origami Structure
3.17 Determination of pMHC Surface Density
4 Notes
References
Chapter 19: Leveraging DNA Origami to Study Phagocytosis
1 Introduction
2 Materials
3 Methods
3.1 Prepare Origami Pegboards
3.2 Couple Origamis to Glass and Quantify Relative Origami Concentration
3.3 Generate Supported Lipid Bilayer Coated Beads
3.4 Quantify Origami Conjugation to Supported Lipid Bilayer-Coated Beads
3.5 Phagocytosis Assays
3.6 TIRF Assays
4 Notes
References
Chapter 20: Fabrication of Nanoscale Arrays to Study the Effect of Ligand Arrangement on Inhibitory Signaling in NK Cells
1 Introduction
2 Materials
2.1 Mold Fabrication
2.2 Array Fabrication by Nanoimprint
2.3 Array Fabrication by E-Beam Lithography
2.4 Chemical Functionalization
2.5 Biofunctionalization
2.6 Verification of the Functionalization Site-Specificity by Fluorescent Staining and Microscopy
2.7 NK Cell Stimulation on the Arrays
3 Methods
3.1 Mold Fabrication
3.2 Array Fabrication by Nanoimprint
3.3 Array Fabrication by E-Beam Lithography
3.4 Chemical Functionalization
3.5 Biofunctionalization
3.6 Verification of the Functionalization Site-Specificity by Fluorescent Staining and Microscopy
3.7 NK Cell Degranulation Assay
3.8 NF-κB Activation
3.9 Interferon-γ Secretion by NK Cells
4 Notes
References
Chapter 21: Measurement of Forces for Trans-Endocytosis at Dorsal and Ventral Sides of the Cell
1 Introduction
2 Materials
2.1 TGT
2.2 Micropillar Arrays
2.3 Microfluidics
3 Methods
3.1 Force Measurement Using TGT Substrate
3.1.1 Protein G-ssDNA Conjugation
3.1.2 Hybridization of Protein G-ssDNA and Complementary ssDNA-Biotin
3.1.3 Conjugation of Ligand (CD80-Fc) to the Protein G-TGT
3.1.4 Preparation of TGT-Coated Substrate for Force Measurement
3.1.5 TGT Image Acquisition
3.1.6 TGT Image Analysis
3.2 Micropillar-Based Traction Force Microscopy
3.2.1 Fabrication of Template of Micropillar Arrays
3.2.2 Fabrication of Micropillar Arrays
3.2.3 Coating Micropillar Arrays for Trans-endocytosis Measurement
3.2.4 Coating Micropillar Arrays for Reference Measurement
3.2.5 Micropillar Array Image Acquisition
3.2.6 Micropillar Array Image Analysis
3.3 Microfluidic-Based Force Sensor
3.3.1 Microfluidic Apparatus
3.3.2 Preparation for Ligand-Conjugated Particles
3.3.3 Microfluidic Force Sensor Image Acquisition
3.3.4 Microfluidic Force Sensor Image Analysis
4 Notes
References
Chapter 22: Functionalized Lipid Droplets and Microfluidics Approach to Study Immune Cell Polarity In Vitro
1 Introduction
2 Materials
2.1 Microfabrication
2.2 Microfluidic Chip Assembly
2.3 Droplet Formulation and Functionalization
2.4 Microfluidic Pairing to Reconstitute Immune Synapses
3 Methods
3.1 Microfabrication
3.2 Microfluidic Chip Assembly
3.3 Droplet Formulation
3.4 Droplet Functionalization
3.5 Immune Synapse Reconstitution in the Microfluidic Chip
3.6 Final Remarks
4 Notes
References
Chapter 23: Quantifying Immune Cell Force Generation Using Traction Force Microscopy
1 Introduction
2 Materials
2.1 Cover Glass Preparation
2.2 Gel Preparation and Polymerization
2.3 Gel Functionalization
2.4 TFM Acquisition
3 Methods
3.1 Glass Preparation
3.2 Gel Preparation & Polymerization
3.3 Gel Functionalization
3.4 TFM Acquisition
3.5 Data Analysis
4 Notes
References
Chapter 24: Characterizing Biophysical Parameters of Single TCR-pMHC Interactions Using Optical Tweezers
1 Introduction
2 Materials
2.1 PEG Slides
2.2 3500 bp DNA Linker with 20 Base Overhang
2.3 Functionalizing Beads with DNA Linker
2.4 Cleaving Antibody and Crosslinking to 20 Base ssDNA Compliment to Overhang
2.5 Ligating ssDNA-Half 2H11 to Beads with Overhang DNA
2.6 Single-Molecule Slide Preparation
2.7 Optical Tweezers Instrumentation
3 Methods
3.1 PEG Slides
3.2 3500 bp DNA Linker with 20 Base Overhang
3.3 Functionalizing Beads with DNA Linker
3.4 Cleaving Antibody and Crosslinking to 20 Base ssDNA Compliment to Overhang
3.5 Ligating ssDNA-Half 2H11 to Beads with Overhang DNA
3.6 Single-Molecule Slide Preparation
3.7 Single-Molecule Force Spectroscopy Measurement
4 Notes
References
Chapter 25: Isolation of the B Cell Immune Synapse for Proteomic Analysis
1 Introduction
2 Materials
2.1 Preparation of the Beads Coated with Activatory and Non-activatory Ligands
2.2 Primary Mouse B Cell Isolation
2.3 Conjugate Formation
2.4 Synapse Isolation
2.5 In-Gel Digestion
3 Methods
3.1 Coating of Magnetic Beads
3.2 Primary Mouse B Cell Isolation (Sterile Work)
3.3 Conjugate Formation
3.4 Synapse Isolation
3.5 In-Gel Digestion
4 Notes
References
Chapter 26: Analyzing Single Cell Secretions by ``Shadow Imaging´´
1 Introduction
2 Materials
2.1 Disposables
2.2 Equipment
2.3 Reagents
2.4 Biological Materials
3 Methods
3.1 Primary NK Cell Isolation
3.2 Sample Preparation
3.3 Imaging Setup
3.4 Data Analysis
4 Notes
References
Chapter 27: Exploiting the RUSH System to Study Lytic Granule Biogenesis in Cytotoxic T Lymphocytes
1 Introduction
2 Materials
2.1 Cloning of GZMB in a RUSH Construct
2.2 CD8+ Cell Purification from Peripheral Blood and CTL Differentiation
2.3 CTLs Nucleofection with the RUSH-GZMB Construct
2.4 Evaluation of RUSH on Fixed Cells by Confocal Microscopy
2.5 Evaluation of RUSH in Live Cells
3 Methods
3.1 Generation of the RUSH-GZMB Construct
3.1.1 GZMB Amplification by PCR
3.1.2 Insert and Vector Restriction Enzyme Digestion
3.1.3 DNA Ligation and Transformation
3.1.4 Plasmid DNA Preparation and Colony Screening
3.2 CD8+ Cell Purification from Peripheral Blood and CTL Differentiation
3.3 CTL Nucleofection with the RUSH-GzmB Construct
3.4 Evaluation of RUSH on Fixed Cells by Confocal Microscopy
3.5 Evaluation of RUSH on Live Cells
4 Notes
References
28: Interactions of Tissue-Resident T Cells
1 Introduction
2 Materials
2.1 Mice
2.2 Preparation of T Cells for Adoptive Transfer
2.3 Models for Generation of Cutaneous CD8+ TRM
2.4 Skin Preparation and Staining Methods for Analysis by Microscopy
3 Methods
3.1 Preparation of CD8+ T Cells for Adoptive Transfer-Naïve CD8+ T Cell Purification
3.2 Preparation of CD8+ T Cells for Adoptive Transfer-Generation of Early Effector CD8+ T Cells
3.3 Models for Generation of Cutaneous CD8+ TRM- Zosteriform Model of HSV Infection
3.4 Models for Generation of Cutaneous CD8+ TRM- OVA Plasmid Tattoo
3.5 Models for Generation of Cutaneous CD8+ TRM- DNFB-Induced Skin Inflammation
3.6 Skin Preparation and Staining Methods for Analysis by Microscopy- Cryosectioning
3.7 Skin Preparation and Staining Methods for Analysis by Microscopy- Skin Whole Mount
3.8 Skin Preparation and Staining Methods for Analysis by Microscopy- Epidermal Sheet
4 Notes
References
Chapter 29: Live Imaging of CAR T Cell Ca2+ Signals in Tumor Slices Using Confocal Microscopy
1 Introduction
2 Materials
2.1 Machines and Equipment
2.2 Buffers and Powders
2.3 Instruments and Tools
2.4 Antibodies
3 Methods
3.1 CAR-T Production and Culture
3.2 Labeling CAR-T and Non-transduced T Cells with Fluo-4
3.3 Obtaining Human Tumor Samples
3.4 Tissue Processing
3.5 Preparation of Agarose Gel
3.6 Embedding of Tumor Samples in Agarose Gel
3.7 Vibratome Slicing of Human Tumor Samples
3.8 Immunostaining of Tumor Slices
3.9 Microscope Preparation
3.10 Imaging of Ca2+ Levels of CAR-T
3.11 Analyzing CAR-T Activation
4 Notes
References
Chapter 30: Measuring CTL Lytic Granule Secretion and Target Cell Membrane Repair by Fluorescent Lipophilic Dye Uptake at the ...
1 Introduction
2 Materials
3 Methods
3.1 Cell Culture
3.2 Procedure for Time-Lapse Microscopy
3.3 Procedure for Flow Cytometry
4 Notes
References
Chapter 31: In Vitro Generation of Human Tolerogenic Monocyte-Derived Dendritic Cells
1 Introduction
2 Materials
2.1 Peripheral Blood Mononuclear Cell (PBMC) Isolation
2.2 Monocyte Isolation
2.3 Generation and Culture of Monocyte-Derived Dendritic Cells (moDC)
3 Methods
3.1 Peripheral Blood Mononuclear Cell (PBMC) Isolation
3.2 Monocyte Isolation
3.3 Generation and Culture of tolDC
4 Notes
References
Chapter 32: Methods of Machine Learning-Based Chimeric Antigen Receptor Immunological Synapse Quality Quantification
1 Introduction
2 Materials
2.1 Recommended Hardware Configuration
2.2 Software Installation
3 Methods
3.1 Immunofluorescence Imaging of CAR IS
3.2 Automated CAR IS Quantification Using Machine Learning
3.3 Alternative Method: Manual Evaluation of CAR IS Using ImageJ
4 Notes
References
Chapter 33: Imaging CAR-T Synapse as a Quality Control for CAR Engineering
1 Introduction
2 Materials
3 Methods
3.1 Lentivirus Production in HEK293T Cells
3.2 Estimation of Virus Titer
3.3 Generation of Primary CAR T Cells
3.4 Culturing Primary CAR T Cell and Raji B-mCherry-CAAX Cells
3.5 Imaging CAR T Cell-Tumor Cell Conjugation and CD45 Exclusion in the Synapse
4 Notes
References
Index