Table Of ContentMethods in
Molecular Biology 1591
George Edward Rainger
Helen M. Mcgettrick Editors
T-Cell
Traffi cking
Methods and Protocols
Second Edition
M M B
ethods in olecular iology
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
For further volumes:
http://www.springer.com/series/7651
T-Cell Trafficking
Methods and Protocols
Second Edition
Edited by
George Edward Rainger
Institute of Cardiovascular Sciences, University of Birmingham, Birmingham, UK
Helen M. Mcgettrick
Institute of Inflammation and Ageing, College of Medicine and Dental Sciences,
University of Birmingham, Birmingham, UK
Editors
George Edward Rainger Helen M. Mcgettrick
Institute of Cardiovascular Sciences Institute of Inflammation and Ageing
University of Birmingham College of Medicine and Dental Sciences
Birmingham, UK University of Birmingham
Birmingham, UK
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-6929-6 ISBN 978-1-4939-6931-9 (eBook)
DOI 10.1007/978-1-4939-6931-9
Library of Congress Control Number: 2017932550
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Preface
Welcome to the second edition of Methods and Protocols for assessing T cell Trafficking,
in the Methods in Molecular Biology series. The trafficking of T cells is relevant in numer-
ous contexts. It occurs during population and maturation of T cells in the thymus and is
required for dissemination of antigen naïve T cells to the secondary lymphatic organs where
immune responses are initiated. Indeed, T cell trafficking within lymph nodes plays an
important role in the maturation of primary and secondary immune responses. Antigen
experienced effector T cells can undertake compartmentalized recirculation during immune
surveillance. In addition they are recruited to tissues during inflammation where they play
important roles in the inflammatory response. Importantly, we now recognize that inap-
propriate or persistent trafficking of T cells into such sites makes a major contribution to
the pathogenesis of immune-mediated inflammatory diseases which have an autoimmune
or chronic inflammatory component.
Thus the trafficking of T cells has both physiological and pathological relevance and
provides some challenging environments in which to make quantitative measurements.
This has seen the development of expertise which goes well beyond the standard laboratory
methodologies which can be supported by commercially available kits and reagents. The
methods in this edition have been developed by experts in T cell trafficking, who have spent
many years perfecting them. Each chapter contains a step-by-step guide to conducting the
assays, with useful hints to avoid common pitfalls. The volume is organized into three sec-
tions. The first addresses homeostatic T cell trafficking during thymic maturation, followed
by the subsequent colonization of and egress from secondary lymphoid organs. The second
addresses T cell trafficking during “normal” inflammatory and immune responses. Lastly,
we include a section on T cell trafficking in disease. Each section is headed by an informa-
tive and accessible introduction written by experts who are actively investigating the regula-
tion of T cell trafficking in these different scenarios. We believe this will ensure that this
book will become an essential point of reference for those new to the field of T cell traffick-
ing, or to those looking to expand their technical capabilities.
We would like to thank all the authors for their invaluable contributions and willingness
to share their expertise. Thanks also to Professor John Walker, the series editor, for guid-
ance in the process of compiling the book.
G. Ed. Rainger is generously supported by the British Heart Foundation of the UK.
Helen M. Mcgettrick is supported by generous funds from Arthritis Research UK.
Birmingham, UK George Edward Rainger
Helen M. Mcgettrick
v
Contents
Preface.......................................................... v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 Introduction to Homeostatic Migration ............................. 1
Mark C. Coles
2 Analysis of Thymocyte Migration, Cellular Interactions, and Activation
by Multiphoton Fluorescence Microscopy of Live Thymic Slices ........... 9
Jessica N. Lancaster and Lauren I.R. Ehrlich
3 Visualizing and Tracking T Cell Motility In Vivo....................... 27
Robert A. Benson, James M. Brewer, and Paul Garside
4 Graph Theory-Based Analysis of the Lymph Node Fibroblastic Reticular
Cell Network ................................................. 43
Mario Novkovic, Lucas Onder, Gennady Bocharov,
and Burkhard Ludewig
5 Visualizing Endogenous Effector T Cell Egress from the Lymph Nodes ..... 59
Manisha Menon, Alexandre P. Benechet, and Kamal M. Khanna
6 Introduction: T Cell Trafficking in Inflammation and Immunity........... 73
Myriam Chimen, Bonita H.R. Apta, and Helen M. Mcgettrick
7 Leukocyte Adhesion Under Hemodynamic Flow Conditions.............. 85
Charlotte Lawson, Marlene Rose, and Sabine Wolf
8 Endocrine Regulation of Lymphocyte Trafficking In Vitro ............... 101
Bonita H.R. Apta, Myriam Chimen, and Helen M. Mcgettrick
9 Mesenchymal Stromal Cells as Active Regulators of Lymphocyte
Recruitment to Blood Vascular Endothelial Cells....................... 121
Helen M. Mcgettrick, Lewis S.C. Ward, George Edward Rainger,
and Gerard B. Nash
10 Monitoring RhoGTPase Activity in Leukocytes Using Classic
“Pull-Down” Assays ............................................ 143
Marouan Zarrouk, David Killock, Izajur Rahman, Jessica Davies,
and Aleksandar Ivetić
11 Utilizing Lentiviral Gene Transfer in Primary Endothelial Cells
to Assess Lymphocyte-Endothelial Interactions........................ 155
Jasmeet S. Reyat, Michael G. Tomlinson, and Peter J. Noy
12 Introduction to Lymphocyte Trafficking in Disease..................... 169
Patricia F. Lalor and Elizabeth A. Hepburn
13 Using Ex Vivo Liver Organ Cultures to Measure Lymphocyte Trafficking.... 177
Benjamin G. Wiggins, Zania Stamataki, and Patricia F. Lalor
vii
viii Contents
14 In Vitro and Ex Vivo Models to Study T Cell Migration Through
the Human Liver Parenchyma..................................... 195
Benjamin G. Wiggins, Konstantinos Aliazis, Scott P. Davies,
Gideon Hirschfield, Patricia F. Lalor, Gary Reynolds, and Zania Stamataki
15 Monitoring Migration of Activated T Cells to Antigen-Rich
Non-lymphoid Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
Eleanor Jayne Ward, Hongmei Fu, and Federica Marelli-Berg
16 Tissue Digestion for Stromal Cell and Leukocyte Isolation ............... 225
Saba Nayar, Joana Campos, Nathalie Steinthal, and Francesca Barone
17 T Cell Response in the Lung Following Influenza Virus Infection.......... 235
Robert A. Benson, Jennifer C. Lawton, and Megan K.L. MacLeod
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
Contributors
Konstantinos aliazis • Centre for Liver Research, Institute for Immunology and
Immunotherapy, University of Birmingham, Birmingham, UK
Bonita H.R. apta • Institute of Cardiovascular Sciences, College of Medicine and Dental
Sciences, University of Birmingham, Birmingham, UK
FRancesca BaRone • Centre for Translational Inflammation Research, Institute of
Inflammation and Ageing, College of Medical & Dental Sciences, University of
Birmingham Research Laboratories, Queen Elizabeth Hospital, Birmingham, UK
alexandRe p. BenecHet • Division of Immunology, Transplantation and Infectious
Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy
RoBeRt a. Benson • Centre for Immunobiology, Institute of Infection, Immunity and
Inflammation, The University of Glasgow, Glasgow, UK
FedeRica MaRelli-BeRg • William Harvey Research Institute—Heart Centre,
Barts and the London School of Medicine and Dentistry, Queen Mary University
of London, London, UK
gennady BocHaRov • Institute of Numerical Mathematics, Russian Academy of Sciences,
Moscow, Russian Federation
JaMes M. BReweR • Institute of Infection, Immunity and Inflammation, College of
Medical, Veterinary and Life Sciences, Glasgow Biomedical Research Centre,
University of Glasgow, Glasgow, UK
Joana caMpos • Centre for Translational Inflammation Research, Institute of
Inflammation and Ageing, College of Medical & Dental Sciences, University of
Birmingham Research Laboratories, Queen Elizabeth Hospital, Birmingham, UK
MyRiaM cHiMen • Institute of Cardiovascular Sciences, College of Medicine and Dental
Sciences, University of Birmingham, Birmingham, UK
MaRK c. coles • Department of Biology, Centre for Immunology and Infection, University
of York, North Yorkshire, UK
scott p. davies • Centre for Liver Research, Institute for Immunology and
Immunotherapy, University of Birmingham, Birmingham, UK
Jessica davies • Cytoskeleton/Membrane Signalling Research Group, Cardiovascular
Division, King’s College London, London, UK
lauRen i.R. eHRlicH • Department of Molecular Biosciences, Institute for Cellular
and Molecular Biology, The University of Texas at Austin, Austin, TX, USA
HongMei Fu • William Harvey Research Institute—Heart Centre, Barts and the London
School of Medicine and Dentistry, Queen Mary University of London, London, UK
paul gaRside • Institute of Infection, Immunity and Inflammation, College of Medical,
Veterinary and Life Sciences, Glasgow Biomedical Research Centre, Wellcome Trust
Centre for Molecular Parasitology, Glasgow, UK
elizaBetH a. HepBuRn • Department of Cellular Pathology, Cheltenham General
Hospital, Cheltenham, UK
gideon HiRscHField • Centre for Liver Research, Institute for Immunology and
Immunotherapy, University of Birmingham, Birmingham, UK
ix
x Contributors
aleKsandaR ivetić • Cytoskeleton/Membrane Signalling Research Group, Cardiovascular
Division, King’s College London, London, UK
KaMal M. KHanna • Department of Immunology, University of Connecticut Health,
Farmington, CT, USA
david KillocK • Cytoskeleton/Membrane Signalling Research Group, Cardiovascular
Division, King’s College London, London, UK
patRicia F. laloR • Centre for Liver Research, Immunity and Immunotherapy, Institute
of Biomedical Research, University of Birmingham, Birmingham, UK
Jessica n. lancasteR • Department of Molecular Biosciences, Institute for Cellular
and Molecular Biology, The University of Texas at Austin, Austin, TX, USA
cHaRlotte lawson • Comparative Biomedical Sciences, Royal Veterinary College,
London, UK
JenniFeR c. lawton • Centre for Immunobiology, Institute of Infection, Immunity
and Inflammation, The University of Glasgow, Glasgow, UK
BuRKHaRd ludewig • Institute of Immunobiology, Kantonsspital St . Gallen, St . Gallen,
Switzerland
Megan K.l. MacLeod • Centre for Immunobiology, Institute of Infection, Immunity
and Inflammation, The University of Glasgow, Glasgow, UK
Helen M. McgettRicK • Institute of Inflammation and Ageing, College of Medicine
and Dental Sciences, University of Birmingham, Birmingham, UK
ManisHa Menon • Department of Immunology , University of Connecticut Health,
Farmington, CT, USA
geRaRd B. nasH • Institute of Cardiovascular Sciences, University of Birmingham,
Birmingham, UK
saBa nayaR • Centre for Translational Inflammation Research, Institute of Inflammation
and Ageing, College of Medical & Dental Sciences, University of Birmingham Research
Laboratories, Queen Elizabeth Hospital, Birmingham, UK
MaRio novKovic • Institute of Immunobiology, Kantonsspital St . Gallen, St . Gallen,
Switzerland
peteR J. noy • School of Biosciences, College of Life and Environmental Sciences, University
of Birmingham, Birmingham, UK
lucas ondeR • Institute of Immunobiology, Kantonsspital St . Gallen, St . Gallen,
Switzerland
izaJuR RaHMan • Cytoskeleton/Membrane Signalling Research Group, Cardiovascular
Division, King’s College London, London, UK
geoRge edwaRd RaingeR • Institute of Cardiovascular Sciences, University of
Birmingham, Birmingham, UK
JasMeet s. Reyat • School of Biosciences, College of Life and Environmental Sciences,
University of Birmingham, Birmingham, UK
gaRy Reynolds • Centre for Liver Research, Institute for Immunology and
Immunotherapy, University of Birmingham, Birmingham, UK
MaRlene Rose • Harefield Hospital, Imperial College, London, UK
zania staMataKi • Centre for Liver Research, Immunity and Immunotherapy, Institute
of Biomedical Research, University of Birmingham, Birmingham, UK
natHalie steintHal • Centre for Translational Inflammation Research, Institute of
Inflammation and Ageing, College of Medical & Dental Sciences, University of
Birmingham Research Laboratories, Queen Elizabeth Hospital, Birmingham, UK
Contributors xi
MicHael g. toMlinson • School of Biosciences, College of Life and Environmental Sciences,
University of Birmingham, Birmingham, UK
eleanoR Jayne waRd • William Harvey Research Institute—Heart Centre, Barts and the
London School of Medicine and Dentistry, Queen Mary University of London, London, UK
lewis s.c. waRd • Institute of Inflammation and Ageing, University of Birmingham,
Birmingham, UK
BenJaMin g. wiggins • Centre for Liver Research, Immunity and Immunotherapy,
Institute of Biomedical Research, University of Birmingham, Birmingham, UK
saBine wolF • Comparative Biomedical Sciences, Royal Veterinary College, London, UK
MaRouan zaRRouK • Cytoskeleton/Membrane Signalling Research Group, Cardiovascular
Division, King’s College London, London, UK
