Lymphocytes A Practical Approach

by Rowland-Jones, Sarah L.; McMichael, Andrew J.

• ISBN13: 9780199638161
• ISBN10: 0199638160
• Edition: 2nd
• Format: Paperback
• Copyright: 2000-03-16
• Publisher: Oxford University Press

Summary

Cellular immunology is a rapidly moving field in which recent advances have made significant contributions to our understanding of the immune response to infection and malignancy. These in turn, have given rise to new therapeutic opportunities in areas such as vaccines and immunotheraphy. Many investigators have been discourages by the complicated protocols involved in cellular immunological studies, as illustrated, by the meticulous care required for the generation of antigen-specific T-cells. Lymphocytes: A Practical Approach (second edition) contains straight-forward protocols for well- established procedures in the study of lymphocytes including preparation and identification of lymphocytes, immortalization, cell and organ culture, and quantification assays. It also covers the recent technological advances which have revolutionised the field, such as the use of the Interferon-gamma ELISpot assay and peptide-HLA tetrameric assays to quantify antigen-specifidc T-cells directly from peripheral blood, without the need for in vitro culture, and molecular methods for accurate HLA typing.

Author Biography

Charles R. M. Bangham: Department of Immunology, Imperial College School of Medicine at St Mary's, Norfolk Place, London, UK. Judy M. Bastin: Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, UK. Simon C. Biddolph: Department of Paediatric Pathology, Womens' Centre, John Radcliffe Hospital, Headington, Oxford, UK. Marion H. Brown: MRC Cellular Immunology Unit, Sir William Dunn School of Pathology, South Parks Road, Oxford, UK. Michael Browning: Department of Microbiology and Immunology, University of Leicester, Medical Sciences Building, University Road, Leicester, UK. Helmut Fickenscher: Institut fur Klinische und Molekulare Virologie, Friedrich-Alexander-Universitat Erlangen-Nurnberg,, Germany. Y. Furukawa: Department of Immunology, Imperial College School of Medicine at St Mary's, Norfolk Place, London, UK. Kevin C. Gatter: University Department of Cellular Sciences, Level 4, John Radcliffe Hospital, Headington, Oxford, UK. Andrew Carmichael: Department of Medicine, Level 5, Addenbrooke's Hospital, Cambridge, UK. P. Gorak-Stolinska: Department of Immunology, GKT School of Medicine and Dentistry, London, UK. Pete Krausa: Molecular Genetics Division, Forensic Analytical, Hayward, CA, USA. Deirdre E. J. Mcloughlin: Department of Anatomy, University of Birmingham Medical School, UK. Edgar Meinl: Max Planck-Institut fur Neurobiologie, Martinsried, Germany. Kingston H. G. Mills: Infection and Immunity Group, Department of Biology, National University of Ireland, Maynooth, County Kildare Grahams. Ogg: Molecular Human Immunology Unit, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, UK John J. T. Owen: Department of Anatomy, University of Birmingham Medical School, Edgbaston, Birmingham, B15 2TT, UK. Magdalena Plebanski: The Austin Research Institute, Victoria, Australia. Sarah L. Rowland-Jones: Molecular Human Immunology Unit, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK. Antony Symons: The R. W. Johnson Pharmaceutical Research Institute, San Diego, CA, USA. Rusung Tan: Department of Pathology and Laboratory Medicine, BC's Childrens' Hospital, Vancouver, BC, Canada. M. Vukmanovic-Stejic: Department of Immunology, GKT School of Medicine and Dentistry, London, UK. Anna Vyakarnam: Department of Immunology, GKT School of Medicine and Dentistry, London, UK. B. Vyas: Department of Immunology, GKT School of Medicine and Dentistry, London, UK. D. Wallace: Edward Jenner Institute for Vaccine Studies, Compton, Berkshire, UK.

Table of Contents

List of Contributors	xvii
Abbreviations	xix
Preparation of lymphocytes and identification of lymphocyte subpopulations	1	(26)
Magdalena Plebanski
Introduction	1	(1)
Sources of lymphoid and professional antigen-presenting cells	1	(2)
Methods of purification	3	(11)
Lymphoid organs	3	(7)
Body fluids	10	(4)
Principles of lymphocyte handling and culture	14	(4)
Short- and long-term storage (freezing)	14	(2)
Principles of long-and short-term culture	16	(2)
Methods of subfractionation for lymphocytes and antigen-presenting cells	18	(9)
Physical properties	19	(1)
Cell-surface markers	20	(6)
Acknowledgement	26	(1)
Immunohistochemistry of lymphoid organs	27	(28)
Simon C. Biddolph
Kevin C. Gatter
Introduction	27	(1)
Preparation of lymphoid cells and tissues for immunohistochemistry	27	(12)
Slide adhesive	27	(1)
Choice of preparation	28	(1)
Transport of specimen	29	(1)
Cell smear and cytocentrifuge preparations	29	(2)
Cell imprints (`dabs' or `touch preparations')	31	(1)
Frozen sections (`cryostat sections')	31	(1)
Paraffin sections	32	(7)
Immunohistochemical staining	39	(16)
Immunohistochemical methods	39	(2)
Labels	41	(4)
Considerations in the choice of method and label	45	(4)
Multiple antibody methods	49	(3)
Quality control	52	(1)
The distribution of cell types in lymphoid tissue	53	(1)
Acknowledgements	53	(1)
References	53	(2)
Viral Transformation of lymphocytes	55	(20)
Edgar Meinl
Helmut Fickenscher
Introduction	55	(1)
Transformation of T lymphocytes	56	(13)
Growth transformation of human T cells by herpesvirus saimiri	56	(12)
Immortalization of human T cells by human T-cell leukaemia virus type 1	68	(1)
B-cell immortalization	69	(6)
EBV- transformation of human B cells	69	(1)
Transformation of macaque B cells with herpesvirus papio	70	(1)
Acknowledgements	71	(1)
References	71	(4)
B- and T-cell hybridomas	75	(20)
Judy M. Bastin
Introduction	75	(1)
Methods for generating B-and T-cell hybridomas and the principles of hybridoma culture	75	(13)
Tissue culture	76	(1)
Materials	76	(1)
Selection procedures	77	(1)
Immunizations	77	(2)
Fusion partners	79	(1)
Fusion protocol	79	(2)
Growth of hybridomas	81	(1)
Screening assays	81	(1)
Cloning hybridomas	82	(1)
Cryopreserving and thawing cells	83	(1)
Large-scale production of hybridomas	84	(1)
Purification	84	(1)
Human hybridomas	85	(1)
Antibody production by chemical and genetic engineering	86	(1)
Disadvantages of hybridomas	87	(1)
Conclusions	87	(1)
Applications of hybridoma technology	88	(7)
B-cell hybridomas	88	(2)
T-cell hybridomas	90	(1)
References	91	(4)
Murine T-cell culture	95	(40)
Kingston H. G. Mills
Introduction	95	(4)
T-cell subtypes and their role in protective immunity	95	(1)
Strategies for the induction of distinct T-cell subtypes	96	(2)
Techniques for detecting T-cell responses	98	(1)
Tissue culture conditions, growth factors, and cell viability	99	(4)
Culture medium and serum	99	(2)
Cytokines and growth factors for culturing T cells	101	(1)
Viable cell count	102	(1)
Preparation of T cells and APCs	103	(8)
Preparation of cells from lymphoid and non-lymphoid organs	103	(3)
Purification of mononuclear cells and removal of dead cells	106	(1)
Purification of T cells, B cells, and T- cell subpopulations	107	(3)
Preparation of macrophages and APCs	110	(1)
Generation and detection of antigen-specific CD4= T cells	111	(16)
In vivo priming of CD4+ T cells	111	(1)
Generation of CD4+ T- cell lines and clones	112	(3)
Antigen-specific T-cell proliferation	115	(4)
Detection of Th1/Th0/Th2 responses by cytokine production	119	(5)
Assay of helper function of T cells and antibody production by B Cells	124	(3)
Generation and detection of antigen-specific CD8+CTLs	127	(3)
In vivo priming of CD8+ CTLs	127	(1)
Generation of CD8+ T- cell lines and clones	127	(1)
Cytotoxic T -cell assay	128	(2)
MHC restriction analysis	130	(5)
Acknowledgements	131	(1)
References	132	(3)
Human CD4 culture	135	(26)
Anna Vyakarnam
B. Vyas
M. Vukmanovic- Stejic
P. Gorak-Stolinska
D. Wallace
A. Noble
D. M. Kemeny
Introduction	135	(1)
General apparatus, media, and reagents for human CD4 culture	136	(1)
Apparatus	136	(1)
Media	137	(1)
Reagents	137	(1)
The isolation and culture of CD4 T- cell subsets from peripheral blood	137	(10)
Principle	140	(1)
The generation of CD4+ Th1, Th2, and Th0 effectors	140	(2)
Generation of antigen-specific and random CD4 Clones	142	(5)
Assays for CD4 function	147	(14)
Principle of proliferation assay	147	(2)
Activation-induced cell death	149	(2)
Measuring cytokine production in culture supernatants by ELISA	151	(2)
Enumerating cytokine-producing cells by flow cytometry: intracytoplasmic cytokine staining	153	(5)
References	158	(3)
Human cytotoxic T- lymphocyte (CTL) studies	161	(18)
Sarah L. Rowland-Jones
Introduction	161	(1)
General principles of CTL culture	162	(1)
Tissue culture reagents and conditions	162	(1)
What persuades memory CTLs to grow in vitro?	163	(1)
Generation of antiviral CTL	163	(6)
Influenza-specific CTLs	164	(1)
HIV-specific CTLs	165	(1)
Using peptides to stimulate CTLs	166	(1)
Generating virus-specific CTL clones	167	(2)
Measuring CTL activity	169	(7)
The CTL lysis assay	169	(3)
Measuring the antigen-specific release of cytokines by CTLs	172	(3)
How do different methods of quantifying CTLs compare?	175	(1)
Conclusions	176	(3)
References	176	(3)
Limiting dilution analysis for the quantitation of antigen-specific T cells	179	(18)
Andrew Carmichael
Theoretical considerations	179	(4)
Quantitation at the clonal level using limiting dilution analysis (LDA)	179	(1)
The Poisson distribution	180	(3)
Advantages of LDA in addition of quantitation	183	(1)
Limitations of LDA	183	(1)
Methodology	183	(7)
Responder cells	184	(3)
Stimulator cells	187	(3)
Calculation and interpretation of results	190	(7)
Calculation of cytotoxic activity in individual microcultures	190	(1)
Classification of positive and negative wells	190	(1)
Estimation of precursor frequency	191	(3)
Interpretation	194	(1)
References	194	(3)
HLA---peptide tetrameric complexes	197	(12)
Graham S. Ogg
Introduction	197	(1)
Cloning of modified HLA heavy chain	198	(1)
Protein expression	199	(1)
Principle	199	(1)
Comments	200	(1)
Inclusion body purification	200	(1)
Principle	200	(1)
Refolding by dilution	201	(1)
Principle	201	(1)
Comments	202	(1)
Enzymatic biotinylation	202	(1)
Principle	202	(1)
Comments	203	(1)
Purification of refolded protein	203	(1)
Principle	203	(1)
Elisa	204	(2)
Principle	204	(2)
Comments	206	(1)
Formation of tetramers	206	(1)
Principle	206	(1)
Comments	206	(1)
Flow cytometry	207	(1)
Principle	207	(1)
Comments	207	(1)
Conclusions	207	(2)
References	208	(1)
Expansion of human cytotoxic T lymphocytes for immunotherapy	209	(12)
Rusung Tan
Introduction	209	(2)
Adoptive transfer of CTLs in humans	210	(1)
Peptide recognition by CTLs	211	(2)
Peptide binding motifs	211	(2)
Establishing CTL lines	213	(8)
Establishing CTL clones	213	(2)
Expansion of CTL clones	215	(2)
Ensuring the safety of CTL infusions	217	(1)
References	218	(3)
HLA typing methods	221	(52)
Pete Krausa
Michael Browning
Introduction	221	(2)
The HLA system	223	(7)
Polymorphism in relation to structure and function	224	(2)
Nomenclature	226	(1)
HLA in populations	227	(3)
Methods of HLA typing	230	(1)
Serological HLA typing	230	(9)
Preparation of components	232	(5)
The micro-lymphocytotoxicity assay	237	(1)
Discussion on serology	238	(1)
Molecular biological approaches to tissue typing	239	(1)
The PCR reaction	240	(3)
Reagent composition	240	(2)
The PCR	242	(1)
Detection by oligonucleotide probing (PCR-SSOP)	243	(6)
PCR-SSOP protocols	245	(3)
PCR-SSOP discussion	248	(1)
HLA typing by PCR-SSP	249	(9)
Five-locus determination PCR-SSP---phototyping	249	(7)
High-resolution of PCR-SSP typing	256	(1)
PCR-SSP discussion	257	(1)
Sequence-based typing	258	(6)
SBT strategies	259	(2)
HLA SBT protocol	261	(3)
Discussion	264	(9)
Key references	267	(1)
Commercial suppliers	267	(1)
References	268	(5)
Biochemical characterization of lymphocyte surface antigens	273	(26)
Antony Symons
Marion H. Brown
Introduction	273	(1)
Immunoprecipitation	274	(11)
Introduction	274	(1)
Labelling cellular antigens	274	(6)
Solubilization of membrane proteins	280	(1)
Immunoprecipitation	281	(4)
SDS-polyacrylamide gel electrophoresis (PAGE)	285	(4)
Introduction	285	(3)
Detection of proteins	288	(1)
Immunoblotting	289	(7)
Introduction	289	(1)
Preparation of protein samples	290	(1)
Blotting proteins	291	(3)
Detection of antigen	294	(2)
Strategies for the identification of new molecule	296	(3)
Affinity chromatography	296	(1)
Ligand hunting	297	(1)
References	297	(2)
Measurement of cells undergoing apoptosis	299	(16)
Y. Furukawa
C. R. W. Bangham
Introduction	299	(4)
Definition of apoptosis	299	(1)
Importance of apoptosis in lymphocyte biology	299	(1)
Recognition of apoptotic cells by phagocytes	300	(1)
Recent advances in our understanding of apoptosis	300	(3)
Methods available to assay apoptosis	303	(12)
DNA laddering	305	(1)
Analysis of light scatter by flow cytometry (size and granularity)	305	(1)
Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL)	305	(3)
Annexin V	308	(1)
CMXRos and related dyes	309	(4)
References	313	(2)
Thymic organ culture	315	(28)
John J. T. Owen
Deirdre. E. J. McLoughlin
Introduction	315	(1)
Microdissection and organ culture of the murine fetal thymus	316	(6)
The production of alymphoid thymus lobes	321	(1)
Newborn thymic slice culture	322	(2)
Reaggregate thymus organ culture	324	(7)
References	329	(2)
Appendices
A1 List of suppliers	331	(6)
A2 Commonly used lymphoma lines	337	(6)
Edgar Meinl
Helmut Fickenscher
Index	343