Dendritic Cells: Methods and Protocols

Preface
Contents
Contributors
Part I: Overview on Mouse and Human Dendritic Cells
	Chapter 1: Origin, Phenotype, and Function of Mouse Dendritic Cell Subsets
		1 Introduction
		2 Ontogeny of Mouse Dendritic Cells
			2.1 Conventional DC Development
			2.2 Plasmacytoid DC Development
			2.3 Contraction of the DC Family
		3 The Mouse DC Subsets and Their Functional Specialization
			3.1 Conventional DCs
				3.1.1 cDC1s
				3.1.2 cDC2s
			3.2 Plasmacytoid DCs
		4 Conclusion
		References
	Chapter 2: Phenotypes and Functions of Human Dendritic Cell Subsets in the Tumor Microenvironment
		1 Introduction
		2 Identification and Functions of Human DC Subsets
			2.1 Conventional Dendritic Cells
				2.1.1 cDC1
				2.1.2 cDC2 and LC
			2.2 Plasmacytoid Dendritic Cells
			2.3 Mature DC
			2.4 Other DC Subsets
				2.4.1 MoDCs and InfDC
				2.4.2 AS-DC, Axl-DC, or DC5
		3 Role of DC Subsets in Antitumor Immune Response and on the Overall Clinical Impact
			3.1 Sc-RNA seq Studies of Tumor-Associated DC Populations Reveal Five Subsets
			3.2 The Antitumor Function of cDC1: Antigen Cross-Presentation, IFN-λ Secretion, and Cross Talk with NK Cells
			3.3 The Antitumor Properties of Tumor-associated cDC2
			3.4 Inhibition of IFN-I Production by Tumor-Associated pDCs and Their Specific Interaction with Treg
			3.5 Mature DC Have an Antitumor Function and Likely Contribute to TLS Formation
			3.6 MoDC and LC Roles Are Poorly Characterized in Antitumor Immune Responses
		4 In Situ Visualization of Tumor Associated DCs and Prognosis Impact
		5 Conclusion
		References
Part II: In Vivo Study of Dendritic Cells
	Chapter 3: In Vivo Tracking of Dendritic Cell Migration
		1 Introduction
		2 Materials
			2.1 KikGR Mice (See Note 1)
			2.2 Light Source and Protective Glasses
			2.3 Anesthesia, Flow Cytometry, and Other Instruments and Reagents
			2.4 Skin DCs and Lymph Node Cell Purification
		3 Methods
			3.1 Photoconversion of Skin
			3.2 Photoconversion of Proximal Colon or Jejunum and Ileum Excluding the Terminal Ileum
			3.3 Purification of Cells from the Skin
			3.4 Purification of Cells from LN
			3.5 Staining Samples for Flow Cytometric Analysis
			3.6 Flow Cytometric Analysis
		4 Notes
		References
	Chapter 4: In Vivo Analysis of Dendritic Cell Clonality
		1 Introduction
		2 Materials
			2.1 Mice
			2.2 Reagents
			2.3 Equipment
			2.4 Instruments
		3 Methods
			3.1 Tissue Isolation from Clec9aConfetti Mice
			3.2 Tissue Processing for Imaging
			3.3 (Optional) Fluorescence Antibody Staining for cDC Subsets
			3.4 Image Acquisition
		4 Notes
		References
	Chapter 5: Monitoring the Interaction Between Dendritic Cells and T Cells In Vivo with LIPSTIC
		1 Introduction
		2 Materials
			2.1 Mouse Strains and Animal Procedures
			2.2 Isolation and Transfer of Naïve CD4+ T Cells
			2.3 Immunization
			2.4 In Vivo LIPSTIC Labeling and Analysis of LIPSTIC-Labeled Cells by Flow Cytometry
		3 Methods
			3.1 Adoptive Transfer of Ovalbumin-Specific Naïve Cd40lgSrtA/SrtA CD45.1/1 CD4-Cre OT-II CD4+ T Cells into Cd40G5/G5 Hosts (Se...
			3.2 Immunization of Cd40G5/G5 Hosts (See Note 8)
			3.3 Injection of SrtA Substrate to Achieve In Vivo LIPSTIC Labeling (See Note 9)
			3.4 Analysis of LIPSTIC-Labeled Cells by Flow Cytometry
		4 Notes
		References
Part III: In Vitro Differentiation of Dendritic Cells
	Chapter 6: In Vitro Generation of Murine Bone Marrow-Derived Dendritic Cells
		1 Introduction
			1.1 GM-CSF-Derived BMDC: A Model for Migratory DCs
			1.2 FLT3L-Derived BMDC: A Model for Conventional DC Subsets
		2 Materials
		3 Methods
			3.1 Bone Marrow Cell Preparation
			3.2 GM-CSF (+/- IL-4) Cultures
			3.3 FLT3L Alone Culture
			3.4 FLT3L + GM-CSF Culture
			3.5 Summary
		4 Notes
		References
	Chapter 7: In Vitro Generation of Murine Dendritic Cells from Hoxb8-Immortalized Hematopoietic Progenitors
		1 Introduction
		2 Materials
		3 Methods
			3.1 Generation and Titration of Hoxb8-ERHBD-Expressing MSCV
				3.1.1 Generation of Hoxb8-ERHBD-Expressing MSCV
				3.1.2 MSCV Titration (See Note 4)
			3.2 Preparation of BM Cells and Generation of Hoxb8-FL Cells
				3.2.1 Preparation of BM Cells
				3.2.2 Infection of BM Cells and Expansion of Hoxb8-FL Cells
				3.2.3 Cell Differentiation and Generation of DC
				3.2.4 CRISPR/Cas9-Mediated Gene Deletion Using Lentiviral Vectors
		4 Notes
		References
	Chapter 8: In Vitro Generation of Murine CD8α+ DEC205+ XCR1+ Cross-Presenting Dendritic Cells from Bone Marrow-Derived Hematop...
		1 Introduction
		2 Materials
			2.1 Cell Lines and Cell Culture
			2.2 Isolation and Culture of Primary BM-Derived Hematopoietic Progenitors
			2.3 Harvesting, Staining, and Flow Cytometric Analysis of Culture-Derived DCs
		3 Methods
			3.1 Generation of Mitomycin C (MMC)-Treated OP9 Cells
			3.2 Production of Flt3L Supernatant for Cell Culture
			3.3 Isolation of Hematopoietic Progenitors from BM
			3.4 Differentiation of Notch DCs from BM Hematopoietic Progenitors
			3.5 Phenotypic Characterization of Culture-Derived DCs
		4 Notes
		References
	Chapter 9: In Vitro Generation of Human Dendritic Cell Subsets from CD34+ Cord Blood Progenitors
		1 Introduction
		2 Materials
		3 Methods
			3.1 MS5 and eMS5 Culture
			3.2 Generation of the Engineered (e) MS5 for the Growth Factor Production by Retroviral Transduction
			3.3 CD34+ Cell Isolation from Cord Blood
			3.4 In Vitro eMSC and CD34+ Cell Coculture
		4 Notes
		References
	Chapter 10: In Vitro Generation of Human Cross-Presenting Type 1 Conventional Dendritic Cells (cDC1s) and Plasmacytoid Dendrit...
		1 Introduction
		2 Materials
			2.1 Cell Lines and Feeder Layer Preparation
			2.2 Expansion of CD34+ CB Cells
			2.3 Cryopreservation and Revival of Expanded CD34+ CB Cells
			2.4 Differentiation of DCs from Expanded CD34+ CB Cells
			2.5 Staining for Flow Cytometry Analysis
		3 Methods
			3.1 Maintenance of Cell Lines and Preparation of the Feeder Layers
			3.2 Expansion of CD34+ CB Cells
			3.3 Cryopreservation of Expanded CD34+ CB Cells
			3.4 Revival of Frozen Expanded CD34+ CB Cells
			3.5 Preparation of the Feeder Layer for CD34+ Cell Coculture
			3.6 Seeding of Expanded CD34+ Cells on Feeder Layer Cells for Differentiation
			3.7 Phenotypic Identification of the DC Populations at the End of the Culture
		4 Notes
		References
	Chapter 11: Culture System Allowing the Simultaneous Differentiation of Human Monocytes into Dendritic Cells and Macrophages U...
		1 Introduction
		2 Materials
			2.1 Blood Monocyte Isolation
				2.1.1 PBMC Purification
				2.1.2 Monocyte Isolation
			2.2 Monocyte Culture
			2.3 Quantification of Differentiated Cells by Flow Cytometry
		3 Methods
			3.1 Blood Monocyte Isolation
				3.1.1 PBMC Purification
				3.1.2 Monocyte Isolation
			3.2 Monocyte Culture
			3.3 Quantification of Differentiated Cells by Flow Cytometry
		4 Notes
		References
	Chapter 12: Clonal Analysis of Human Dendritic Cell Progenitors Using a Stromal Cell Culture
		1 Introduction
		2 Materials
		3 Methods
			3.1 Culture of Stromal Cells (Day 4 to Day 0)
				3.1.1 Passaging Stromal Cells
				3.1.2 Treating Stromal Cells with Mitomycin C
				3.1.3 Plating Mitomycin C-Treated Stromal Cells
			3.2 Purification of CD34+ HSPC from Human Cord Blood (Day 0)
				3.2.1 Isolation of Cord Blood Mononuclear Cells Using Ficoll-Paque (See Note 8)
				3.2.2 Enrichment of CD34+ Cells
			3.3 Culture of Purified Cells in Single-Cell Environment (Day 0-7)
				3.3.1 Single-Cell Sorting and Plating
				3.3.2 Cell Culture Over Time
			3.4 Harvest and Analyze CD34+ Cells´ Output (Day 7-21)
				3.4.1 Read Cells via Flow Cytometry
		4 Notes
		References
Part IV: Enrichment of Dendritic Cells
	Chapter 13: Enrichment of Large Numbers of Splenic Mouse Dendritic Cells After Injection of Flt3L-Producing Tumor Cells
		1 Introduction
		2 Materials
			2.1 Injection of B16-Flt3L Tumor Cells In Vivo
			2.2 Preparation of Single-Cell Suspensions of Splenocytes
			2.3 Depletion of T cells, B cells, and Macrophages
			2.4 Positive Selection of DCs
		3 Methods
			3.1 Implantation of B16-Flt3L Tumors in Mice
			3.2 Preparation of Single-Cell Suspensions of Splenocytes
			3.3 Depletion of T cells, B cells, and Macrophages
			3.4 Positive Selection of DC Cells
				3.4.1 Protocol A (Positive Selection of CD11c+ Cells)
				3.4.2 Protocol B (Positive Selection of CD11c+ mPDCA1+ Cells)
				3.4.3 Magnetic Positive Isolation
				3.4.4 Verification of Cell Purity by Flow Cytometry
		4 Notes
		References
	Chapter 14: Isolation and Identification of Dendritic Cell Subsets from Human and Mouse Tumors
		1 Introduction
		2 Materials
			2.1 Human Tumor Processing
				2.1.1 Mechanical and Enzymatic Tumor Digestion
				2.1.2 DC Enrichment
				2.1.3 Cell Sorting
			2.2 Mouse Tumor Processing
		3 Methods
			3.1 Human Tumor Processing
				3.1.1 Mechanical and Enzymatic Tissue Digestion
				3.1.2 Dendritic Cell Enrichment (See Note 8)
				3.1.3 Cell Staining and Dendritic Cell Sorting
			3.2 Mouse Tumor Processing
				3.2.1 Mouse Mammary Tumor Processing
				3.2.2 Cell Preparation for FACS Analysis of Murine  DCs
		4 Notes
		References
Part V: Functional Characterization of Dendritic Cells
	Chapter 15: Optimized Nonviral Gene Disruption in Primary Murine and Human Myeloid Cells
		1 Introduction
		2 Materials
			2.1 Human Monocyte Preps and Myeloid Cell Culturing
			2.2 Mouse BMDM and BMDC Preps and Culturing
			2.3 Cas9-RNA Complex Formation
			2.4 Monocyte Nucleofection
			2.5 Flow Cytometric Analysis of Target Gene Disruption
		3 Methods
			3.1 Isolation of Human Monocytes
				3.1.1 Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs)
				3.1.2 Isolation of Human Monocytes
			3.2 Murine Bone Marrow Isolation and BMDM Culture
				3.2.1 Isolate Bone Marrow from Tibias and Femurs of Mice
				3.2.2 BMDM Culture
			3.3 Cas9-RNP Complex Formation
			3.4 Monocyte Nucleofection
				3.4.1 Preparing Cells and Reagents for Electroporation
				3.4.2 Electroporation of Cas9-RNP Complexes Produced in Subheading 3.3
			3.5 Human Myeloid Cell Culturing Post Nucleofection
				3.5.1 Human Monocyte-Derived Dendritic Cell Culturing
				3.5.2 Human Monocyte-Derived Macrophage Culturing
			3.6 Mouse BMDC and BMDM Culturing Post Nucleofection
				3.6.1 BMDC Culturing
				3.6.2 BMDM Culturing
			3.7 Flow Cytometry Analysis of Knockout Frequency
		4 Notes
		References
	Chapter 16: Characterization of Dendritic Cell Metabolism by Flow Cytometry
		1 Introduction
		2 Materials
			2.1 Cellular Systems
			2.2 Flow Cytometry Reagents and Equipment
			2.3 Intracellular Staining for Metabolic Phosphorylated Proteins
			2.4 Metabolic Dyes
		3 Methods
			3.1 Intracellular Staining of Phosphorylated Metabolic Proteins
				3.1.1 Fixation
					Fixation Prior to Tissue Processing
					Fixation of Single-Cell Suspensions After Tissue or Blood Processing
					Fixation of In Vitro-Cultured DCs
				3.1.2 Staining Procedure
			3.2 Glucose and Lipid Metabolism
				3.2.1 Staining Procedure
			3.3 Mitochondrial Metabolism
				3.3.1 Staining Procedure
			3.4 ROS Staining
				3.4.1 Staining Procedure
			3.5 Metabolic Panel Tested on Cytek Spectral Flow Cytometer
		4 Notes
		References
	Chapter 17: In Vivo and In Vitro Assay to Address Dendritic Cell Antigen Cross-Presenting Capacity
		1 Introduction
		2 Materials
			2.1 Reagents and Plasticware
			2.2 Gamma Irradiation Source
			2.3 Transgenic  Mice
		3 Methods
			3.1 In Vitro Assay to Address DC Cross-Presenting Capacity
				3.1.1 Preparation of Single-Cell Suspensions
				3.1.2 Preparation of Labeled OT-I Cells
				3.1.3 Antigen (OVA) Loading of Cells by Osmotic Shock
				3.1.4 Cross-Priming of OT-I by  DCs
			3.2 In Vivo Assay to Address DC Cross-Presenting Capacity
				3.2.1 Preparation of OT-I Cells at Day 0
				3.2.2 Preparation of Labeled OT-I Cells at Day 0
				3.2.3 Injections of Labeled OT-I Cells at Day 0
				3.2.4 Preparation of Antigen-Loaded Cells at Day 1
				3.2.5 Injection of Antigen-Loaded Cells at Day 1
		4 Notes
		References
	Chapter 18: Assays to Detect Cross-Dressing by Dendritic Cells In Vivo and In Vitro
		1 Introduction
		2 Materials
			2.1 In Vitro Assay for Cross-Dressing
			2.2 In Vivo Assay for Cross-Dressing
		3 Methods
			3.1 Assays to Study Cross-Dressing In Vitro (See Fig. 1)
				3.1.1 Culture of Bone Marrow-Derived Dendritic Cells (BMDCs)
				3.1.2 Culture of EL4 and EG7 Cells and Pulsing of EG7 Cells with SIINFEKL
				3.1.3 Detection of Cross-Dressing of BALB/c BMDCs with SIINFEKL-Kb Complexes
				3.1.4 Labeling of OT-I Cells with CFSE
				3.1.5 FACS Sorting of BMDCs After Co-Culture with Tumor Cells
				3.1.6 Functional Validation of Cross-Dressing by Co-Culturing Sorted BMDCs with CFSE-Labeled OT-I Cells
			3.2 Assays to Study Cross-Dressing In Vivo (See Fig. 2)
				3.2.1 Injection of SIINFEKL-Pulsed EL4 Tumor Cells into H2Kbm1 Mice
				3.2.2 Preparation of Single-Cell Suspension from Draining Lymph Nodes
				3.2.3 Staining of the Dendritic Cells in the Single-Cell Suspension
				3.2.4 Sorting and Co-Culture of DCs with CFSE-Labeled OT-I Cells
		4 Notes
		References
	Chapter 19: Assessing the Ability of Human Dendritic Cells to Stimulate Naive CD4+ and CD8+ T Cells
		1 Introduction
		2 Materials
			2.1 Polarization of Blood Naive CD4+ T Cells
				2.1.1 Blood Naive CD4+ T Cell Isolation
				2.1.2 Staining CD4+ T Cells with a Proliferation Dye
				2.1.3 CD4+ T Cells and DC Co-Culture
				2.1.4 Detection of Cytokines by Intracellular Flow Cytometry
			2.2 Differentiation of Blood Naive CD8+ T Cells into Effector Cells
				2.2.1 Blood Naive CD8+ T Cell Isolation
				2.2.2 Staining CD8+ T Cells with a Proliferation Dye
				2.2.3 T Cells and DC Co-Culture
				2.2.4 Detection of Effector Molecules by Intracellular Flow Cytometry
		3 Methods
			3.1 Polarization of Blood Naive CD4+ T Cells
				3.1.1 Blood Naive CD4+ T Cell Isolation
				3.1.2 Staining of Naive CD4+ T Cells with a Proliferation Dye
				3.1.3 Allogeneic Co-Culture with DC
				3.1.4 Detection of Cytokines by Intracellular Flow Cytometry
			3.2 Differentiation of Blood Naive CD8+ T Cells into Effector Cells
				3.2.1 Blood Naive T Cell Isolation
				3.2.2 Staining of Naive CD8+ T Cells with a Proliferation Dye
				3.2.3 Allogeneic Co-Culture with DC
				3.2.4 Detection of Effector Molecules by Intracellular Flow Cytometry
		4 Notes
		References
	Chapter 20: In Vitro and In Vivo Assays to Evaluate Dendritic Cell Phagocytic Capacity
		1 Introduction
		2 Materials
			2.1 In Vitro Generation of Bone-Marrow-Derived Dendritic Cells (BMDCs)
			2.2 In Vitro Generation of Conventional Dendritic Cells Type 1 (cDC1)
			2.3 Ex Vivo Purification of Conventional Dendritic Cells Type 1 (cDC1)
			2.4 Phagocytosis Assay
		3 Methods
			3.1 Generating BM-DCs and cDC1
				3.1.1 Day 0: Collection of Bone Marrow from the Femur and Tibia
				3.1.2 Generation of BMDCs
				3.1.3 Generation of cDC1
					Preparation of the OP9-DL1 Feeder Cell  Line
					Differentiation of BM Cells into cDC1
			3.2 Ex Vivo cDC1 Purified from the Spleen
			3.3 Preparation of NH2 Beads
			3.4 Phagocytosis Assay
				3.4.1 In Vitro Phagocytosis Assay
				3.4.2 Ex Vivo Phagocytosis Assay
		4 Notes
		References
	Chapter 21: Tracking Plasmacytoid Dendritic Cell Response to Physical Contact with Infected Cells
		1 Introduction
		2 Materials
			2.1 Isolation of pDCs from Peripheral Blood Mononuclear Cells (PBMCs)
			2.2 Biological Reagents: Infected Cells
			2.3 Enzyme-Linked Immunosorbent Assay (ELISA)
			2.4 Flow Cytometry Analysis for the Profiling of pDC Activation
			2.5 Imaging Combined with Flow Cytometry Analysis of the Frequency of pDC/Infected Cell Conjugates (Image Stream X Technology)
			2.6 Antiviral Activity of pDCs Defined by Flow Cytometry Analysis of Viral Spread
			2.7 Imaging of the Cell-Cell Contacts by Confocal Microscopy
			2.8 Live Imaging of the Cell Contacts by Spinning-Disk Confocal Microscopy
		3 Methods
			3.1 Isolation of PBMCs from the Blood of Healthy Adult Human Volunteers
			3.2 Isolation of pDCs from PBMCs
			3.3 Infection of the Cells and Coculture with Isolated pDCs for the Analysis of Cytokine Production
			3.4 Flow Cytometry Analysis of the Activation Profile of pDCs Induced by Contact with Infected Cells
			3.5 Quantification of pDC/Infected Cell Conjugates and Its Molecular Regulation by a Technology Combining Imaging and Flow Cyt...
			3.6 Antiviral Activity of pDCs Defined by Flow Cytometry
			3.7 Analysis of Cell-Cell Contacts at Subcellular Level by Immunofluorescence and Confocal Microscopy Analysis
			3.8 Imaging of the Cell-Cell Contacts at Single-Cell Level by Spinning-Disk Confocal Microscopy
		4 Notes
		References
	Untitled
Part VI: Omic Approaches to Study Dendritic Cells
	Chapter 22: Harnessing Single-Cell RNA Sequencing to Identify Dendritic Cell Types, Characterize Their Biological States, and ...
		1 Introduction
		2 Materials
			2.1 List of Files to Download
			2.2 Download the Input Files
			2.3 Download the Files for the Signature Generation
			2.4 Download the .R Scripts to Run Single-Cell CMAP
			2.5 Download the Docker File
			2.6 Programs and Packages Used in This Workflow
		3 Methods
			3.1 Running the Seurat Workflow on All the Cells from the Original Data File
			3.2 Running the Seurat Workflow on the Selected cDC1s
			3.3 Trajectory Inference Using Monocle
			3.4 Trajectory Inference Based on Velocity Analysis
			3.5 Analysis of Mature cDC1s
			3.6 Analyzing the Transcriptional Differences between Clusters of Interest
		4 Notes
		References
	Chapter 23: Characterization of Developmental Trajectories of Dendritic Cell Hematopoiesis Through Single-Cell RNA Sequencing ...
		1 Introduction
		2 Materials
			2.1 Mouse Bone Marrow Processing
			2.2 Datasets
			2.3 Software for Processing and Analyses of scRNA-seq Data
			2.4 Packages for Trajectory Analyses of scRNA-seq Bone Marrow Data
		3 Methods
			3.1 Processing of Mouse Bone Marrow
			3.2 Enrichment of Bone Marrow Progenitors
			3.3 Staining of Mouse Bone Marrow Progenitors for Cell Sorting
				3.3.1 If Progenitor Cells Were Not Enriched
				3.3.2 If Progenitor Cells Were Enriched
			3.4 ScRNA-seq Analyses of DC Progenitor Trajectories
				3.4.1 Analyses of scRNA-seq Data Using the Seurat Pipeline
				3.4.2 Analyses of Developmental Trajectories Using mpath
				3.4.3 Employing cMap to Identify Gene Enrichment for pDC Versus cDC Lineage Commitment
				3.4.4 Analyses of Developmental Trajectories Using Slingshot
		4 Notes
		References
Index