T-Helper Cells: Methods and Protocols

243 Pages·2014·5.39 MB·English

by  Ari Waisman

Methods in
Molecular Biology 1193
Ari Waisman
Burkhard Becher Editors
T-Helper Cells
Methods and Protocols
M M B
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hat fi eld, Hertfordshire, AL10 9AB, UK
For further volumes:
http://www.springer.com/series/7651

T-Helper Cells
Methods and Protocols
Edited by
Ari Waisman
Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg
University of Mainz, Mainz, Germany
Burkhard Becher
Institute of Experimental Immunology, University of Zurich, Zurich, Germany
Editors
Ari Waisman Burkhard B echer
Institute for Molecular Medicine Institute of Experimental Immunology
University Medical Center of the University of Zurich
Johannes Gutenberg University of Mainz Zurich , Germany
Mainz, Germany
ISSN 1064-3745 ISSN 1940-6029 (electronic)
ISBN 978-1-4939-1211-7 ISBN 978-1-4939-1212-4 (eBook)
DOI 10.1007/978-1-4939-1212-4
Springer New York Heidelberg Dordrecht London
Library of Congress Control Number: 2014945206
© Springer Science+Business Media New York 2 014
This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is
concerned, specifi cally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction
on microfi lms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation,
computer software, or by similar or dissimilar methodology now known or hereafter developed. Exempted from this
legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifi cally for
the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work.
Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the
Publisher’s location, in its current version, and permission for use must always be obtained from Springer. Permissions
for use may be obtained through RightsLink at the Copyright Clearance Center. Violations are liable to prosecution
under the respective Copyright Law.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not
imply, even in the absence of a specifi c statement, that such names are exempt from the relevant protective laws and
regulations and therefore free for general use.
While the advice and information in this book are believed to be true and accurate at the date of publication, neither
the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be
made. The publisher makes no warranty, express or implied, with respect to the material contained herein.
Printed on acid-free paper
Humana Press is a brand of Springer
Springer is part of Springer Science+Business Media (www.springer.com)
Prefa ce
Ever since the discovery of “cellular antibodies” in the 1960s [1] it became evident that
adaptive immune responses are dependent on antibody-producing B cells and thymus-
derived T cells [2]. After the T cell receptors of mice and men were cloned [3, 4], it became
clear that T cells mastermind cellular and humoral immune responses. In particular CD4 +
T helper (T ) cells are critical for the development of immunological memory and immune
H
regulation.
T cells were later subdivided into T 1 and T 2 cells based on their ability to pro-
H H H
duce distinct sets of cytokines [5] and therefore to specifi cally mediate different immune
responses to cellular vs. extracellular threats. An additional subset of regulatory/sup-
pressor T cells was postulated already in the 1970 [6], but only with the discovery of
H
FoxP3 as a critical transcription factor for their development were Tregs accepted as a
unique and independent member of the growing family of T cell subsets [7–9]. Apart
H
perhaps from Tregs, there appears to be a large degree of plasticity in terms of functional
and phenotypic specialization amongst T cells. Other subsets, such as TGFβ-secreting
H
T 3 cells, T 17 cells (IL-17 producing), T 9 cells (IL-9 producing), and T 22 cells
H H H H
(IL-22 producing), have led to the erroneous assumption that such polarization patterns
are hardwired and irreversible. Instead, it has become clear that these new T cell subsets
are rarely found in vivo (at least in mice) and that their polarization is vastly plastic and
reversible.
To capture the unique features of T cells, which are critical for host defense, but must
H
also be blamed for numerous chronic infl ammatory diseases, novel tools had to be devel-
oped for the isolation of T cells from various tissues and subsequent analysis of their func-
H
tional properties in vitro, ex vivo, and in vivo. Here, we have invited specialists in T cell
H
biology to share their most effi cient and current protocols for the study of this amazingly
versatile and potent cell type.
In this book, we have put together a few “recipes” focusing on working with T cells
of mice and men. In the fi rst four chapters, von Stebut, Greter, Hövelmeyer, Kurts , and col-
leagues describe methods of isolating T cells from various tissues in mice, which permit the
characterization and in vitro culture of these cells immediately ex vivo. The next fi ve chap-
ters by M air, Tosevski, Newell, Ruland, Sparwasser , and their colleagues are dedicated to
protocols for the analysis of T cell function and phenotype using various cutting-edge
technologies. The next three chapters by Zielinski, Segal, Fillatreau , and colleagues
describe methods allowing for the manipulation of T cell function in vitro and in vivo. The
fi nal seven chapters by T aube, Weigmann, Buch, Clausen, Anderton, Maloy, Heikenwälder ,
and their colleagues are dedicated to in vivo models of diseases in which T cells play a
central role in the pathogenesis.
This is not an all-encompassing compendium of all the available tools and technologies
to study T cells, but a relatively broad selection of cutting-edge protocols to enable the reader
to immediately apply them at the bench. If a specifi c subject is missing, we are confi dent
v
vi Preface
that the creative lab scientist will be able to adapt the protocol to suit his/her needs and
to add the newly developed protocol to this ever-growing list of methods to study this
fascinating leukocyte, the helper T cell.
Zürich, Switzerland Burkhard Becher
Mainz, Germany Ari Waisman
References
1. Govaerts A (1960) Cellular antibodies in kidney secretion lead to different functional properties.
homotransplantation. J Immunol 85: 516–522 Annu Rev Immunol 7: 145–173
2. Miller JF, Mitchell GF (1967) The thymus and 6. Gershon RK, Kondo K (1970) Cell interactions
the precursors of antigen reactive cells. Nature in the induction of tolerance: the role of thymic
216:659–663 lymphocytes. Immunology 18:723–737
3. Yanagi Y, Yoshikai Y, Leggett K, Clark SP, 7. Fontenot JD, Gavin MA, Rudensky AY (2003)
Aleksander I, Mak TW (1984) A human T cell- Foxp3 programs the development and function
specifi c cDNA clone encodes a protein having of CD4+CD25+ regulatory T cells. Nat Immunol
extensive homology to immunoglobulin chains. 4:330–336
Nature 308:145–9 8. Hori S, Nomura T, Sakaguchi S (2003) Control
4. Hedrick SM, Cohen DI, Nielsen EA, Davis MM of regulatory T cell development by the
(1984) Isolation of cDNA clones encoding T transcription factor Foxp3. Science 299:
cell-specifi c membrane-associated proteins. Nature 1057–1061
308:149–53 9. Khattri R, Cox T, Yasayko SA, Ramsdell F (2003)
5. Mosmann TR, Coffman RL (1989) TH1 and An essential role for Scurfi n in CD4+CD25+ T
TH2 cells: different patterns of lymphokine regulatory cells. Nat Immunol 4:337–342
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i x
PART I ISOLATION OF T CELLS FROM THE TISSUES
1 Isolation of T Cells from the Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Beate L orenz and Esther von Stebut
2 I solation of Leukocytes from Mouse Central Nervous System . . . . . . . . . . . . . 1 5
Iva Lelios and M elanie G reter
3 I solation of T Cells from the Gut . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1
Sonja Reißig, C hristopher Hackenbruch, and Nadine H övelmeyer
4 T Cell Isolation from Mouse Kidneys . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 7
Isis L udwig-Portugall and Christian K urts
PART II ANALYSIS OF T CELL FUNCTION AND PHENOTYPES
5 Intracellular Staining for Cytokines and Transcription Factors . . . . . . . . . . . . . 3 9
Florian M air and Vinko T osevski
6 Tracking Cells and Monitoring Proliferation . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1
Vinko Tosevski and Florian Mair
7 Mass Cytometry Analysis of Human T Cell Phenotype and Function. . . . . . . . 5 5
Evan W . N ewell and Lai L i Y un
8 D etection of NF-κB Pathway Activation in T Helper Cells. . . . . . . . . . . . . . . . 69
Oliver Gorka, Stefan W anninger, and J ürgen R uland
9 A ssessing the Suppressive Activity of Foxp3+ Regulatory T Cells . . . . . . . . . . . 8 5
Christian T homas M ayer and Tim Sparwasser
10 I n Vitro Generation of Microbe-Specific Human Th17 Cells. . . . . . . . . . . . . . 9 7
Julia M. B raun and Christina E . Z ielinski
11 In Vitro Polarization of T-Helper Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Julie R umble and Benjamin M . S egal
PART III IN VIVO MODELS OF T CELL FUNCTION
12 A Method for the Generation of TCR Retrogenic Mice. . . . . . . . . . . . . . . . . . 1 17
Elisa K ieback, E llen Hilgenberg, and Simon F illatreau
13 Mouse Models of Allergic Airway Disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Helen M eyer-Martin, S ebastian Reuter, and C hristian T aube
14 I nduction of Colitis in Mice (T-Cell Transfer Model) . . . . . . . . . . . . . . . . . . . 1 43
Benno W eigmann
vii
viii Contents
15 Manipulation of T Cell Function and Conditional Gene
Targeting in T Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 53
Anna Ś ledzińska, L ynsey Fairbairn, and Thorsten Buch
16 Aldara-Induced Psoriasis-Like Skin Inflammation: Isolation
and Characterization of Cutaneous Dendritic Cells
and Innate Lymphocytes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
C. T. Wohn, S. P antelyushin, J . L . O ber-Blöbaum, and B. E. Clausen
17 Induction of Passive EAE Using Myelin-Reactive CD4+ T Cells . . . . . . . . . . . 1 87
Rhoanne C. McPherson, H elen E. Cambrook, R ichard A . O’Connor,
and Stephen M . A nderton
18 E xperimental Mouse Models of T Cell-Dependent
Inflammatory Bowel Disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
George X. S ong-Zhao and K evin J. Maloy
19 Analysis of Chromosomal Aberrations in Murine HCC. . . . . . . . . . . . . . . . . . 2 13
Kristian Unger and M athias H eikenwälder
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 27
Contributors
STEPHEN M. A NDERTON • MRC Centre for Infl ammation Research and Centre
for Multiple Sclerosis Research, Queen’s Medical Research Institute , U niversity
of Edinburgh , E dinburgh, UK
BURKHARD BECHER • Institute of Experimental Immunology , U niversity of Zürich ,
Zürich, S witzerland
JULIA M. BRAUN • Cellular Immunoregulation, Department of Dermatology
and Allergology, Charité-Universitätsmedizin , B erlin, Germany
THORSTEN B UCH • Institute for Medical Microbiology, Immunology and Hygiene,
Technische Universität München , Munich, Germany
HELEN E. C AMBROOK • MRC Centre for Infl ammation Research and Centre
for Multiple Sclerosis Research, Queen’s Medical Research Institute , U niversity
of Edinburgh , E dinburgh, UK
B. E . C LAUSEN • Institute for Molecular Medicine, University Medical Center ,
Johannes Gutenberg-University , Mainz, G ermany
LYNSEY FAIRBAIRN • Institute for Medical Microbiology, Immunology and Hygiene,
Technische Universität München , Munich, G ermany
SIMON FILLATREAU • Deutsches Rheuma-ForschungsZentrum, a Leibniz Institute ,
Berlin , G ermany
OLIVER GORKA • Institut für Klinische Chemie und Pathobiochemie, Klinikum rechts der
Isar, T echnische Universität München , Munich, G ermany
MELANIE GRETER • Institute of Experimental Immunology , University of Zürich ,
Zürich, Switzerland
CHRISTOPHER HACKENBRUCH • Institute for Molecular Medicine, University Medical
Center , J ohannes Gutenberg-University , Mainz, Germany
MATHIAS HEIKENWÄLDER • Institute of Virology , T echnische Universität München,
Helmholtz Zentrum München für Gesundheit und Umwelt (HMGU) , Munich, G ermany
ELLEN HILGENBERG • Deutsches Rheuma-ForschungsZentrum, a Leibniz Institute ,
Berlin, G ermany
NADINE HÖVELMEYER • Institute for Molecular Medicine, University Medical Center ,
Johannes Gutenberg-University , M ainz, Germany
ELISA KIEBACK • Max Delbrueck Center for Molecular Medicine , B erlin, Germany
CHRISTIAN K URTS • Institute of Experimental Immunology , University of Bonn ,
Bonn, G ermany
IVA LELIOS • Institute of Experimental Immunology , U niversity of Zürich ,
Zürich, S witzerland
BEATE LORENZ • Department of Dermatology, University Medical Center, J ohannes
Gutenberg University , M ainz , Germany
ISIS L UDWIG-PORTUGALL • Institute of Experimental Immunology ,
Rheinische Friedrich-Wilhelms-University of Bonn , B onn , G ermany
FLORIAN MAIR • Institute of Experimental Immunology , U niversity of Zürich ,
Zürich, S witzerland
ix