Preface
Contents
Contributors
Part I Overview Chapters
Chapter 1: Alloreactivity
1 Physiological Relevance and History of Alloreactivity
2 T Cell Receptor Recognition of Peptide-Major Histocompatibility Complex Molecules
3 The Phenomenon of Allorecognition
4 Frequencies of Naïve and Memory Alloreactive T Cells
5 The Molecular Basis of Alloreactivity
5.1 The Relative Contribution of Peptide and MHC Molecule to Alloreactivity and the Phenomenon of Polyspecificity Underlying T Cell Alloreactivity
5.2 Alloreactivity as a By-product of the Intrinsic Bias of TCRs to Interact with MHC Molecules
6 Structural Insights into Direct TCR Allorecognition
6.1 The 2C System
6.1.1 Overview of the System
6.1.2 Structural Insights
6.2 The LC13 System
6.2.1 Overview of the System
6.2.2 Structural Insights
6.3 The YAe62 System
6.3.1 Overview of the System
6.3.2 Structural Insights
7 Implications of the Molecular Basis of Alloreactivity in Clinical Transplantation
References
Chapter 2: Antibodies in Transplantation: The Effects of HLA and Non-
1 Clinical Frequency and Relevance of Donor Specific Antibodies
1.1 Frequency and Association with Outcome
1.2 Diagnosis of Antibody-
2 Experimental Techniques to Measure Effects of Antibodies
2.1 In Vitro Techniques
2.1.1 Measurement of HLA Antibody Binding Capacity
2.1.2 Analysis of Intracellular Signaling
2.1.3 Determination of Cell Growth
2.1.4 Measurement of Leukocyte Adherence
2.1.5 Determination of Cytoskeletal Changes and Cell Migration
2.1.6 siRNA and Pharmacological Inhibitors
2.2 In Vivo Models of Antibody-
2.3 Patient Samples
3 Mechanisms of Injury: Fc-Dependent Effects of Antibodies
3.1 Hyperacute and Acute Rejection
3.1.1 Complement Activation
3.1.2 Antibody-
4 Mechanisms of Injury: Target Cell Signaling Induced by HLA I Antibodies
4.1 Survival and Accommodation
4.2 Cell Proliferation
4.2.1 In Vitro Evidence
4.2.2 In Vivo Evidence
4.3 Induction of Secondary Factors
4.4 Leukocyte Recruitment
4.4.1 Infiltration in Antibody-Mediated Rejection
4.4.2 HLA I Antibody-
4.5 Therapies Suggested from Experimental Evidence
5 HLA II Antibodies
5.1 Association with Outcome
5.2 Limitations to Studying HLA II in Model Systems
5.3 Mechanisms
6 Non-HLA Antibodies
6.1 Frequency and Outcome
6.2 Experimental Models
7 Conclusions
References
Chapter 3: Cell Mediated Rejection
1 Tissue Injury
2 Allorecognition and T Cell Activation
2.1 Costimulation
3 T Cell Differentiation
4 B Cell Activation and Function
5 Mechanisms of Graft Destruction
5.1 A Role for the Innate Immune System in Mediating Graft Damage
5.2 Leukocyte Recruitment to the Graft
5.3 Cytotoxic T Cells
6 B Cells and Antibody Mediated Rejection
7 Immunological Memory and Its Impact on Cell Mediated Rejection
References
Chapter 4: Transplantation Tolerance
1 Tolerance Definition
2 Barriers and Limitations to Tolerance
3 Assays for Transplant Tolerance and What They Tell Us About the Immune Response
4 Gene Arrays
5 Urine Biomarkers
6 ImmunKnow ® Assay
7 B Cells
7.1 Urine Biomarkers
7.2 Gene Arrays and PCR
7.3 Flow Cytometry
8 T Cells
8.1 Urine Biomarkers
8.2 Gene Arrays and PCR
8.3 Flow Cytometry
9 Treg
10 Myeloid-Derived Suppressor Cells
11 Immature Dendritic Cells
12 IL-10
12.1 Urine Biomarkers
12.2 Flow Cytometry
13 Transforming Growth Factor Beta
14 IDO-Positive Cells
14.1 Gene Arrays and PCR
14.2 Flow Cytometry
14.3 Protein Arrays and ELISA
14.4 Immunohisto-chemistry
14.5 Other Studies for IDO
15 Chimerism
16 Conclusion
References
Chapter 5: Composite Tissue Transplantation
1 Introduction
2 Functional and Immunological Outcomes
3 Mechanisms and Appearance of Skin Rejection
4 Protocols for Prophylaxis and Treatment of Skin Rejection
5 Unique Biological and Immunological Features of VCA
6 Novel Concepts for Immune Modulation in Reconstructive Transplantation
7 Summary and Conclusion
References
Chapter 6: Transplantation of the Sensitized Patient: Histocompatibility Testing
1 Introduction
2 Identifying the Most Likely Route to Transplantation
3 Prior to Transplantation: Patient Evaluation
3.1 Desensitization
3.2 Kidney Paired Donation
4 Testing After Transplantation
4.1 Non-HLA Antibodies
5 Summary
References
Chapter 7: Thoracic Organ Transplantation: Laboratory Methods
1 Introduction
2 Antibody Screening and Characterization
3 Virtual Crossmatch
4 CPRA
5 CPRA, Desensitization, and Monitoring
6 Non-HLA Antibodies
7 Complement Fixing Antibodies
8 Post-transplant Monitoring
9 AlloMap
10 Immune Monitoring (Cylex ®)
11 Other Potential Biomarkers
12 Conclusions
References
Part II Methods Chapters
Chapter 8: HLA Typing by Sequence-Specific Primers
1 Introduction
2 Materials
3 Methods
3.1 Reaction Components
3.2 Template DNA Requirements
3.3 PCR-SSP Setup
3.4 Electrophoresis
3.5 Interpretation of Results
4 Notes
References
Chapter 9: Human Leukocyte Antigen (HLA) Typing by DNA Sequencing
1 Introduction
1.1 Overview of Methods
1.2 Use of Methods in Clinical Practice
2 Materials
2.1 DNA Preparation
2.2 Polymerase Chain Reaction Co-amplification of Both Alleles at an HLA Class I Locus
2.3 Allele Group-Specific Amplification of HLA Class I Loci, HLA-A and HLA-B
2.4 Allele Group-Specific Polymerase Chain Reaction Amplification of HLA-DRB Loci with Intron Primers
2.5 Polymerase Chain Reaction Amplification of DQB1
2.6 Gel Electrophoresis
2.7 Purification of PCR Amplicons Using AMPure
2.8 DNA Sequencing
2.9 Sequence Analysis
2.10 HLA Allele Isolation by Cloning
3 Methods
3.1 DNA Preparation
3.2 Polymerase Chain Reaction Co-amplification of Both Alleles at an HLA Class I Locus
3.3 Allele Group-Specific Amplification of HLA Class I Loci, HLA-A and HLA-B
3.4 Allele Group-Specific Polymerase Chain Reaction Amplification of HLA-DRB Loci with Intron Primers
3.5 Polymerase Chain Reaction Amplification of DQB1
3.6 Gel Electrophoresis
3.7 Purification of PCR Amplicons Using AMPure
3.8 DNA Sequencing
3.9 Sequence Analysis
3.10 HLA Allele Isolation by Cloning
4 Notes
References
Chapter 10: Next-Generation HLA Sequencing Using the 454 GS FLX System
1 Introduction
1.1 Protocol Overview
1.2 Experience with the System
2 Materials
2.1 Sample Preparation Components
2.2 Genomic PCR Components
2.3 Amplicon Cleanup Components
2.4 Amplicon Quantification Components
2.5 Amplicon Quality Check Components
2.6 Amplicon Dilution and Pooling
2.7 Emulsion PCR Setup and Thermal Cycling
2.8 Emulsion Breaking
2.9 Bead Washing, Enrichment, Counting, and Sequencing Primer Annealing
2.10 Sequencing
2.11 HLA Genotyping
3 Methods
3.1 Sample Preparation
3.2 Genomic PCR
3.3 Amplicon Purification by AMPure
3.4 E-Gel Check for Amplicon Generation and Removal of Primer Dimer
3.5 Choosing the Amplicons for the Final Emulsion PCR Pool
3.6 Quantification by PicoGreen Fluorescence
3.7 Amplicon Dilutions and Pooling for emPCR
3.8 Emulsion PCR, Emulsion Breaking, Sequencing and Genotyping Guidelines
4 Notes
References
Chapter 11: Chimerism Testing by Quantitative PCR Using Indel Markers
1 Introduction
2 Materials
3 Methods
3.1 Cell Separation
3.2 DNA Isolation
3.3 DNA Dilution for Screening Test
3.4 Quantification Test
3.4.1 Informative Marker Selection
3.4.2 Quantification Set-Up
3.4.3 Calculation Number of Wells
3.5 Data Analysis Algorithms
4 Notes
References
Chapter 12: Gene-Specific PCR Typing of Killer Cell Immunoglobulin-
1 Introduction
2 Materials
2.1 Facility
2.2 Materials for PCR Amplification
2.3 Materials for Gel Electrophoresis
3 Methods
3.1 PCR Amplification Procedure
3.2 Gel Electrophoresis Procedure
3.3 Interpretation of Gel Results
3.4 KIR Genotyping Data Analysis
4 Notes
References
Chapter 13: Complement-Dependent Cytotoxicity Crossmatch
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment and Supplies
3 Methods
3.1 Preparation of Patient Serum
3.2 DTT Treatment of Patient/Recipient Sera
3.3 Preparation of Donor Cells from Whole Blood
3.4 Preparation of Donor Cells from Spleen
3.5 Preparation of Donor Cells from Lymph Nodes
3.6 Cell Counting and Viability Determination
3.7 Isolation of T and B Cells by Immunomagnetic Selection
3.8 Standard Complement-Dependent Cytotoxic Crossmatch (CDC)
3.9 Antihuman Globulin Enhanced Cytotoxicity Crossmatch (AHG-CDC)
3.10 Scoring and Interpretation of Results of the Complement-Dependent Cytotoxicity Assay (Both Standard CDC and AHG-CDC)
3.11 Evaluation of Complement
3.12 Evaluation of Antihuman Globulin (AHG)
4 Notes
References
Chapter 14: Lymphocyte Crossmatching by Flow Cytometry
1 Introduction
2 Materials
2.1 Specimen Requirements
2.2 Testing Components
2.3 Equipment and Supplies
2.4 Control Serum
3 Methods
3.1 Initial Serum/ Cell Incubations
3.2 Primary Serum/Cell Incubation
3.3 Addition of Anti-human IgG and Monoclonal Antibodies
3.4 Data Acquisition and Analysis
4 Notes
References
Chapter 15: Solid Phase Assay Measuring C4d Deposition to Determine Complement Fixation by HLA-Specific Antibodies
1 Introduction
2 Materials
2.1 Luminex ® X-Map Microspheres
2.2 Equipment
2.3 Antibodies and Conjugates
3 Methods
4 Notes
References
Chapter 16: C1q Assay for the Detection of Complement Fixing Antibody to HLA Antigens
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment and Supplies
3 Methods
3.1 Serum Preparation
3.2 Testing
3.3 Analysis and Interpretation
4 Notes
References
Chapter 17: Tetramer Staining for the Detection of HLA-Specific B cells
1 Introduction
2 Materials
2.1 Materials and Equipment
2.2 Reagents
3 Methods
3.1 Isolation of Peripheral Blood Mononuclear Cells (PBMC)
3.2 B Cell Enrichment with Untouched Human B Cell Kit
3.3 Tetramer Staining
3.4 Flow Cytometric Analysis
4 Notes
References
Chapter 18: A Flow Cytometric Crossmatch Test Using Endothelial Precursor Cells Isolated from Peripheral Blood
1 Introduction
2 Materials and Equipment
2.1 Specimens
2.2 Reagents Included in XM-ONE ® Kit (Absorber, Stockholm Sweden)
2.3 Additional Reagents (All Stored at 2–8 °C Unless Indicated)
2.4 Equipment
2.5 Additional Supplies
3 Methods
3.1 EPC Isolation
3.2 Serum Preparation and Incubation
3.3 Fluorochrome-Conjugated Antibody Incubation
3.4 Acquisition and Analysis
4 Notes
References
Chapter 19: The Detection of Antibodies to the Angiotensin II-Type 1 Receptor in Transplantation
1 Introduction
2 Materials
2.1 Sample Collection and Storage
2.2 Preparation of Reagents and Samples
3 Methods
3.1 Assay Procedure
3.2 Calculation of Results
4 Notes
References
Chapter 20: Detection of Antibodies to Self-Antigens (K-alpha 1 Tubulin, Collagen I, II, IV, and V, Myosin, and Vimentin) by Enzyme-Linked Immunosorbent Assay (ELISA)
1 Introduction
2 Materials
2.1 Antigens for Coating 96-Well Plates
2.2 Blocking and Loading Serum Samples
2.3 Secondary Ab Incubation and Detection
2.4 Data Analysis
3 Methods
3.1 Coating Plates
3.2 Blocking and Loading Serum Samples
3.3 Secondary Ab Incubation and Detection
3.4 Data Analysis and Sample Calculation
4 Notes
References
Chapter 21: Cylex ImmuKnow Cell Function Assay
1 Introduction
2 Materials
2.1 Specimen
2.2 Reagents
2.3 Equipment
3 Methods
3.1 Assay Procedure Part 1: Cell Stimulation
3.2 Assay Part 2: CD4 Cell Selection and ATP Release
3.3 Assay Part 3: ATP Measurement
3.4 Calibration
3.5 Result Interpretation
3.6 Calculation of Results
3.7 Reporting Results
3.8 Interpretation of PHA Stimulated Sample Results
3.9 Quality Control
4 Notes
References
Chapter 22: Detection of Intracellular Cytokines
1 Introduction
2 Materials
2.1 Activation Components
2.2 Fluorescence-
2.3 Reagents
2.4 Equipment
3 Methods
3.1 Sample Collection for Assays
3.2 Activation of Cells
3.3 Staining Procedures
3.3.1 PBMC Cultures
3.3.2 Whole Blood
3.4 Data Analysis
4 Notes
References
Chapter 23: Tolerogenic Dendritic Cells and Induction of T Suppressor Cells in Transplant Recipients
1 Introduction
2 Materials
2.1 Flow Cytometry
2.2 Cell Sorting Using Magnetic Beads
2.3 Cell Culture Assays
3 Methods
3.1 Induction of Tolerogenic Phenotypes in APC by CD8+ CD28− T Cells from the Peripheral Blood of Transplant Recipients
3.2 Proliferation Assay
3.3 Measurement of the ILT3/ILT4 Inducing Capacity of Recipient Sera
4 Notes
4.1 Magnetic Sorting of CD4, CD8, CD14 Cells by Negative Selection
4.2 Depletion of Cell Subsets from Magnetically Sorted T cells
4.3 RNA Extraction Using the RNEasy Kit (Qiagen)
4.4 cDNA Preparation
4.5 Real-Time PCR
References
Chapter 24: Discovery and Customized Validation of Antibody Targets by Protein Arrays and Indirect ELISA
1 Introduction
2 Materials
2.1 Protein Arrays (ProtoArray ®, Life Technologies, Carlsbad, CA)
2.2 ELISA Assays
3 Methods
3.1 Serum and Plasma Processing and Sample Preparation
3.1.1 Serum Processing
3.1.2 Plasma Processing
3.1.3 Sample Preparation (Serum or Plasma)
3.2 ProtoArray
3.2.1 Blocking and Detecting
3.2.2 Drying of Slides and Scanning
3.2.3 Data Generation
3.2.4 Data Analysis (Using Prospector Analyzer ®, Life Technologies)
3.2.5 Data Analysis Using Significant Analysis of Microarrays (SAM)
3.3 Conventional ELISA Method (A Summary of Indirect ELISA is Presented in Fig. 1b)
3.4 MSD ELISA
4 Notes
4.1 Protein Arrays
4.2 ELISA
4.2.1 Conventional ELISA Method
4.2.2 MSD ELISA Method
References
Chapter 25: RNA Purification and Expression Analysis Using Microarrays and RNA Deep Sequencing
1 Introduction
2 Materials
2.1 RNA Purification from Various Samples
2.1.1 RNA Extraction from Cells and Tissue
2.1.2 RNA Extraction from Blood PaxGene Tubes
2.2 Global Gene Expression Profiling Using Affymetrix DNA Microarrays: Gene 1.1 ST Array Plates
2.3 Target Hybridization and Processing for Gene 1.1 ST Peg Arrays on the Affymetrix GeneTitan Instrument
2.4 Global Gene Expression Profiling Using RNA Deep Sequencing (RNAseq) on an Illumina HiSEQ2000
3 Methods
3.1 RNA Extraction from Cells and Tissues
3.1.1 Trizol Hybrid Protocol
3.1.2 RNA Extraction from Blood PaxGene Tubes Using the QiaCube Robot (Fig. 1)
Additional Considerations for RNA Purification
3.2 Global Gene Expression Profiling Using Affymetrix DNA Microarrays: Gene 1.1 ST Array Plates
3.2.1 Ambion WT Expression Kit
3.2.2 Fragmentation and Labeling of the cDNA generated from the WT Expression Kit
3.2.3 NuGEN Ovation Pico WTA System V2
3.2.4 Target Hybridization and Processing for Gene 1.1 ST Peg Arrays on the Affymetrix GeneTitan Instrument
3.2.5 Preparation of GeneTitan Instrument
Additional Considerations for Global Gene Expression Profiling Using Affymetrix DNA Microarrays
3.3 Global Gene Expression Profiling Using RNA Deep Sequencing (RNAseq) on an Illumina HiSEQ2000
3.3.1 Additional Considerations for Global Gene Expression Profiling Using RNA Deep Sequencing (RNAseq)
4 Notes
4.1 RNA Extraction from Cells and Tissues
4.2 Global Gene Expression Profiling Using Affymetrix DNA Microarrays
References
INDEX