Tumor Immunology: Methods and Protocols (Methods in Molecular Biology, 1393)

✍ Scribed by Attilio Bondanza (editor), Monica Casucci (editor)

• Publisher: Humana
• Year: 2016
• Tongue: English
• Leaves: 166
• Category: Library

✦ Synopsis

This volume explores the still undiscovered secrets of tumor immunology and cancer immunology by discussing the methods and techniques that world-renowned experts in the field use in their laboratories. This book provides a better understanding of the rules governing tumor and cancer immunology, and discusses innovations in the technology of the immunological “smart bullets” (monoclonal antibodies, vaccines, tumor-reactive T cells) used to specifically target cancer cells. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Thorough and cutting-edge, Tumor Immunology: Methods and Protocols contains a wide breadth of subject coverage that any scientist, clinician, or industry professional interested in this field will find valuable.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: ATMPs for Cancer Immunotherapy: A Regulatory Overview
1 Introduction
2 European Regulatory Frame for ATMP
3 Guidance for ATMP Clinical Development
4 Critical Issues for ATMP in Cancer Immunotherapy
5 Conclusion
References
Chapter 2: Natural Antibodies to Tumor-Associated Antigens
1 Introduction
2 Materials
2.1 Sera Collection and Preparation, Cell Line Culture
2.2 Sample Preparation
2.3 Protein Precipitation
2.4 Protein Quantitation by Bradford Protein Assay
2.5 IPG Equilibration and First Dimension
2.6 Second Dimension: SDS-
2.7 Western Blot
2.8 Antibodies
2.9 Colloidal Coomassie Blue Staining
2.10 Image Analysis
3 Methods
3.1 First-Dimension Step: Isoelectric Focusing (IEF)
3.1.1 Sera Collection and Preparation
3.1.2 Cell Line Culture
3.1.3 Sample Solubilization
3.1.4 Protein Precipitation with Acetone
3.1.5 Protein Quantitation by Bradford Protein Assay
3.1.6 IEF
3.2 Second-
3.2.1 IPG Strip Equilibration
3.2.2 Performing SDS-PAGE
3.3 Western Blot
3.3.1 Electrophoretic Transfer
3.3.2 Immunoblot Transferred Proteins
3.4 Staining of Preparative 2D Gel by Colloidal Coomassie Blue Staining
3.5 2D Image Analysis
4 Notes
References
Chapter 3: Generation and Cryopreservation of Clinical Grade Wilms’ Tumor 1 mRNA-Loaded Dendritic Cell Vaccines for Cancer Immunotherapy
1 Introduction
2 Materials
2.1 Generation of  Human Monocyte-­Derived DC
2.2 mRNA Electroporation of Mature DC
2.3 Cryopreservation of Electroporated Mature DC
2.4 Thawing of Electroporated Mature DC and Preparation of the Vaccine
3 Methods
3.1 Generation of Human Monocyte-­Derived DC
3.2 mRNA Electroporation of  Mature DC (See Note 2)
3.3 Cryopreservation of Electroporated Mature DC (See Note 6)
3.4 Thawing of Electroporated Mature DC and Preparation of the Vaccine
4 Notes
References
Chapter 4: Quantitative and Qualitative Analysis of Tumor-Associated CD4+ T Cells
1 Introduction
2 Materials
2.1 Isolation of Peripheral Blood Mononuclear Cells (PBMCs) from  Heparinized Blood
2.2 Isolation of CD4+ T Cells from PBMCs
2.3 Selection of Promiscuous MHC Class II Epitopes within Tumor Ags of  Interest for Synthetic Peptide Synthesis
2.4 Ex Vivo Re-stimulation Assays for Detection and Enumeration of Tumor Ag-Specific CD4+ T Cells
2.5 Immunohisto­chemistry for Expression of GATA-3 (Transcription Factor for Th2) and T-bet (Transcription Factor for Th1)
2.6 Quantification of GATA-3 and T-bet-­Positive Cells
3 Methods
3.1 Isolation of PBMCs from  Heparinized Blood
3.2 Isolation of CD4+ T Cells from PBMCs
3.3 Peptide Synthesis
3.4 Ex Vivo Re-stimulation Assays for Detection and  Enumeration of Tumor Ag-Specific CD4+ T Cells
3.4.1 Short-Term Cultures of CD4+ T Cells for Identification of  Responsive Donors (Ex Vivo Re-stimulation Assay-Type A)
3.4.2 Short-Term Cultures for Frequency Estimation of Ag-Specific CD4+ T Cells (Ex Vivo Re-stimulation Assay-Type B)
3.4.3 Calculation of Peptide-Specific CD4+ T Cell Frequency
3.4.4 Short-Term Cultures for Validation of the Ag Specificity of Expanded CD4+ T Cells (Ex Vivo Re-stimulation Assay-Type C)
3.5 Immunohisto­chemistry for  Expression of GATA-3 (Transcription Factor for Th2) and T-bet (Transcription Factor for Th1)
3.6 Quantification of GATA-3 and T-bet-­Positive Cells
4 Notes
References
Chapter 5: Detection and Functional Analysis of Tumor-Derived LXR Ligands
1 Introduction
2 Materials
2.1 Tumor-­Conditioned Medium Testing by Luciferase-­Based LXR Activation Assay
2.1.1 HeK 293 Seeding
2.1.2 HeK 293 Transfection
2.1.3 HeK 293 Lysate Preparation and  Luciferase Assay
2.1.4 β-Galactosidase  Assay
2.2 Tumor-­Conditioned Medium Testing by ABCG1 mRNA Expression in Maturing Dendritic Cells
2.2.1 Isolation of PBMCs from Buffy Coat of Peripheral Blood
2.2.2 Differentiation of Dendritic Cells from  Peripheral Blood Monocytes
2.2.3 Conditioning of Maturing Dendritic Cells with Tumor-­Derived  Medium
2.2.4 Total RNA Extraction
2.2.5 cDNA Synthesis
2.2.6 QPCR Analysis to Evaluate LXR Activation
3 Methods
3.1 Tumor-­Conditioned Medium Testing by Luciferase-­Based LXR Activation Assay
3.1.1 HeK 293 Seeding
3.1.2 HeK 293 Transfection
3.1.3 HeK 293 Lysate Preparation
3.1.4 Luciferase Assay
3.1.5 β-Galactosidase  Assay
3.2 Tumor-­Conditioned Medium Testing by ABCG1 mRNA Expression in Maturing Dendritic Cells
3.2.1 Isolation of PBMCs from Buffy Coat of  Peripheral Blood
3.2.2 Differentiation of Dendritic Cells from  Peripheral Blood Monocytes
3.2.3 Conditioning of Maturing Dendritic Cells with Tumor-­Conditioned  Medium
3.2.4 Total RNA Extraction
3.2.5 cDNA Synthesis
3.2.6 QPCR analysis to evaluate LXR activation
4 Notes
References
Chapter 6: In Vitro Generation of Antigen-Specific T Cells from Induced Pluripotent Stem Cells of Antigen-Specific T Cell Origin
1 Introduction
2 Materials
2.1 Reprogramming T Cells into Pluripotent Stem Cells
2.2 Differentiation of Hematopoietic Stem/Progenitor Cells from T-iPS Cells [8]
2.3 Differentiation of Antigen-­Specific Cytotoxic T Lymphocytes
2.4 Characterization of Re-differentiated T Cells by Flowcytometry
3 Methods
3.1 Reprogramming of Antigen-­Specific T Cells
3.2 Induction of Hematopoietic Stem/Progenitor Cells from T-iPSCs [8]
3.3 Induction of Antigen-­Specific CD8 T Cells
3.4 Characterization of Antigen-­Specific CD8 T Lymphopoiesis by Flow Cytometry
4 Notes
References
Chapter 7: Chimeric Antigen Receptors for Cancer Immunotherapy
1 Introduction
2 Materials
2.1 Molecular Cloning of a CAR Molecule
2.2 Production of Retroviral Supernatant
2.3 Generation of T Lymphocytes Expressing a CAR
2.4 Phenotypic Analysis
2.5 Cytotoxicity Assay
2.6 Co-culture Assay
2.7 Carboxyflu orescein Diacetate Succinimidyl Ester (CFSE) Assay
2.8 Cytokine Release
2.9 Tumor Cell Inoculation Preparation
2.10 T-Cell Infusion Preparation
2.11 In Vivo Imaging
3 Methods
3.1 Molecular Cloning of a CAR Molecule
3.2 Production of Retroviral Supernatant
3.3 Generation of CAR+ T Cells from Human PBMCs
3.4 Evaluating Transduction Efficiency
3.5 Cytotoxicity Assay
3.6 Co-culture Assay
3.7 CFSE Assay
3.8 Tumor Cell Inoculation Preparation
3.9 T-Cell Infusion Preparation
3.10 In Vivo Imaging
4 Notes
References
Chapter 8: Cancer and Chemokines
1 Introduction
1.1 Chemokines and Tumor-­Associated Leukocytes
1.2 Chemokines and Metastasis
2 Materials
2.1 Chemokines Quantification in Organ Lysates
2.2 Tumor Disaggregation for Leukocyte Staining
2.3 Lung Histology
2.4 Determination of Lung Metastasis in  Vivo by Luminescent Imaging of Luciferase-­Transfected Tumor Cells
3 Methods
3.1 Chemokines Quantification in Serum and Organ Lysate
3.2 Tumor Disaggregation for Leukocyte Staining
3.3 Histology
3.4 Determination of Lung Metastasis in  Vivo by Luminescent Imaging of Luciferase-­Transfected Tumor Cells
4 Notes
References
Chapter 9: T Cells as Antigen Carriers for Anti-tumor Vaccination
1 Introduction
2 Materials (see Note 1)
2.1 Isolation of PBMCs from Buffy Coat of Peripheral Blood or from  Lymphocyte Apheresis
2.2 Transduction and Selection of T Lymphocytes
3 Methods
3.1 Isolation of PBMCs from Buffy Coat of Peripheral Blood or from  Lymphocyte Apheresis
3.2 Transduction and Selection of T Lymphocytes
3.2.1 Cells Thawing (Day 0)
3.2.2 First round  of Transduction (Day 2)
3.2.3 Second round  of Transduction (Day 3)
3.2.4 First Step of Selection (Day 4)
3.2.5 Second Step of Selection (Day 7)
3.2.6 Expansion of the Transduced Cells (Day 10)
3.2.7 Freezing of the Transduced Cells (Day 14)
4 Notes
References
Chapter 10: Exploiting Secreted Luciferases to Monitor Tumor Progression In Vivo
1 Introduction
2 Materials
2.1 Generation and Production of the Lentiviral Vector Carrying the  LUCIA-­luciferase Gene
2.2 Tumor Cell Transduction, FACS Analysis and Sorting
2.3 In Vitro and In Vivo LUCIA-­luciferase Assay
2.4 In Vivo Imaging
3 Methods
3.1 Generation and Production of a Lentiviral Construct Carrying the LUCIA-­luciferase Gene
3.2 Tumor Cell Transduction, FACS Analysis and Sorting (see Fig. 1)
3.3 In Vitro LUCIA-­luciferase Assay (see Fig. 1)
3.4 In Vivo LUCIA-­luciferase Blood Assay for Monitoring Tumor Growth (see Fig. 2)
3.5 In Vivo Imaging with LUCIA-­luciferase (see Note 7)
4 Notes
References
Chapter 11: Efficient RNA Interference of Primary Leukemic Cells for Loss-of-Function Studies in Xenograft Mouse Models
1 Introduction
1.1 RNA Interference: An Overview
1.2 Efficient Lentiviral Transduction with shRNAs
2 Materials
3 Methods
3.1 ShRNA Lentiviral Vector Design and Construction
3.1.1 Identification of the siRNA Target Sequence and Oligo Design
3.1.2 Cloning of the shRNA into the Backbone
3.1.3 Lentiviral Vector Production
3.2 Stable Transduction of Cell Lines: Validation and Toxicities
3.2.1 Transduction of AML and MM Cell Lines
3.2.2 shRNA-Mediated Knockdown Validation: mRNA Analysis
3.2.3 shRNA-Mediated Knockdown Validation: Protein Analysis
3.2.4 ShRNA Knockdown Toxicities
3.3 Stable Transduction of AML Primary Blasts with shRNA-­Encoding Lentiviral Vector
3.3.1 Prepare AML Cells
3.3.2 Transduce AML Primary Blasts
3.4 Xenograft Model
3.4.1 Engraftment of Silenced-AML Primary Blasts in Immunodeficient Mice
3.4.2 Follow the Engraftment
3.4.3 At Sacrifice: Bone Marrow and Spleen Analysis
3.4.4 FACS Analysis
4 Notes
References
Chapter 12: Modeling Human Graft-Versus-Host Disease in Immunocompromised Mice
1 Introduction
2 Materials
2.1 Isolation of Human PBMCs
2.2 Infusion of Human PBMCs in Sublethally Irradiated NSG Mice
2.3 Monitoring and Sacrifice of NSG Mice
3 Methods
3.1 Isolation of Human PBMCs
3.2 Infusion of Human PBMCs in Sublethally Irradiated NSG Mice (See Notes 2 and 3)
3.3 Monitoring and Sacrifice of NSG Mice (See Note 7)
4 Notes
References
Chapter 13: ICOS Expression as Immunologic Marker in Immune Activating Monoclonal Antibodies
1 Introduction
2 Materials
2.1 Isolation and Cryopreservation of Lymphocytes
2.2 Immunofluore­scence Analysis of ICOS Expression
3 Methods
3.1 Lymphocytes Isolation (see Note 1)
3.2 Analysis of ICOS Expression on Lymphocytes
4 Notes
References
Chapter 14: Identifying NK Alloreactive Donors for Haploidentical Hematopoietic Stem Cell Transplantation
1 Introduction
2 Materials and Methods
2.1 Evaluation of Donor and Recipient by Four-Digit HLA Typing
2.2 KIR Genotyping and Phenotyping
2.3 Functional Assessment of Alloreactive NK Cell Repertoires
3 Conclusion
References
Chapter 15: Human Microtumors Generated in 3D: Novel Tools for Integrated In Situ Studies of Cancer Immunotherapies
1 Introduction
2 Materials
3 Methods
3.1 Generation of Microtumors
3.2 Treatment of Microtumors
3.3 Monitoring of Microtumor Growth and Cytokine Release
3.3.1 Monitoring of Microtumor Growth
3.3.2 Monitoring of Cytokine Release by mHag CTLs
3.4 Embedding of the Microtumors in Agar and Paraffin
3.4.1 Embedding of the  Microtumors in Agar
3.4.2 Embedding of the Microtumors in Paraffin
3.5 Histological and Immunohisto­chemical Analysis of Microtumors
3.5.1 Histological Analysis of Microtumors
3.5.2 Immunohisto­chemical Analysis of Microtumors
3.5.3 Application
3.6 Conclusion
4 Notes
References
ERRATUM TO
Index