Table Of ContentMethods in
Molecular Biology 1585
Ritobrata Goswami Editor
Th9 Cells
Methods and Protocols
M M B
ethods in olecular iology
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Th9 Cells
Methods and Protocols
Edited by
Ritobrata Goswami
School of Bio Science, Sir JC Bose Laboratory Complex,
Indian Institute of Technology, Kharagpur, West Bengal, India
Editor
Ritobrata Goswami
School of Bio Science
Sir JC Bose Laboratory Complex
Indian Institute of Technology
Kharagpur, West Bengal, India
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-6876-3 ISBN 978-1-4939-6877-0 (eBook)
DOI 10.1007/978-1-4939-6877-0
Library of Congress Control Number: 2017937939
© Springer Science+Business Media LLC 2017
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Preface
T helper cells that play an important role in adaptive immune response have a new member,
Th9 cells. Th9 cells secrete IL-9, a pleiotropic cytokine having biological effects on distinct
cell types. It has been more than 25 years since the cloning of IL-9. IL-9 can be produced
by various cell types including long-term T cell lines and mast cells; however, T helper cells
produce copious amounts of IL-9 in the presence of IL-4 and TGF-β. This discovery has
propelled the study of Th9 cells with enthusiasm.
Over the last eight years, several studies have tried to optimize the conditions for the
production of Th9 cells, transcriptional regulation of Th9 cells, and the in vivo function of
Th9 cells. One of the goals of this book is to present comprehensive laboratory protocols
that have been used to generate Th9 cells both in vitro and in vivo. Th9 cells have been
ascribed to be involved in several diseases having both beneficial and detrimental roles. In
this book techniques used to study the role of Th9 cells in various inflammatory diseases
models have been described. This includes allergic inflammation model, parasite model,
tumor model, and EAE and IBD model. This book will use the knowledge of expert scien-
tists in the field to provide the reader with the laboratory techniques used to generate Th9
cells for specific downstream events.
West Bengal, India Ritobrata Goswami
v
Contents
Preface.......................................................... v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 Th9 Cells: New Member of T Helper Cell Family...................... 1
Ritobrata Goswami
2 IL-9: Function, Sources, and Detection.............................. 21
Wilmer Gerardo Rojas-Zuleta and Elizabeth Sanchez
3 IL-9 Signaling Pathway: An Update ................................ 37
Dijendra Nath Roy and Ritobrata Goswami
4 A Method to In Vitro Differentiate Th9 Cells from Mouse
Naïve CD4+ T Cells ............................................ 51
Duy Pham
5 T Cell Receptor and Co-Stimulatory Signals for Th9 Generation........... 59
Françoise Meylan and Julio Gomez-Rodriguez
6 Polarizing Cytokines for Human Th9 Cell Differentiation................ 73
Prabhakar Putheti
7 Determining the Frequencies of Th9 Cells from Whole Blood............. 83
Anuradha Rajamanickam and Subash Babu
8 IL-9 Production by Nonconventional T helper Cells.................... 93
Silvia C.P. Almeida and Luis Graca
9 Prediction and Validation of Transcription Factors Binding Sites
in the Il9 Locus ............................................... 111
William Orent and Wassim Elyaman
10 Flow Cytometric Assessment of STAT Molecules in Th9 Cells............. 127
Lucien P. Garo, Vanessa Beynon, and Gopal Murugaiyan
11 Transcription Factors Downstream of IL-4 and TGF-β Signals:
Analysis by Quantitative PCR, Western Blot, and Flow Cytometry ......... 141
Atsushi Sugimoto, Ryoji Kawakami, and Norihisa Mikami
12 Retroviral Transduction and Reporter Assay: Transcription
Factor Cooperation in Th9 Cell Development......................... 155
Rukhsana Jabeen
13 Transcription Factor Binding Studies in CD4+ T Cells:
siRNA Transfection, Chromatin Immunoprecipitation,
and Liquid Luminescent DNA Precipitation Assay...................... 167
Etienne Humblin, François Ghiringhelli, and Frédérique Végran
14 Defining Epigenetic Regulation of the Interleukin-9 Gene
by Chromatin Immunoprecipitation ................................ 179
Alla Skapenko and Hendrik Schulze-Koops
vii
viii Contents
15 Allergic Inflammation and Atopic Disease: Role of Th9 Cells ............. 189
Pornpimon Angkasekwinai
16 Characterization of Th9 Cells in the Development of EAE and IBD ........ 201
Sakshi Malik, Valerie Dardalhon, and Amit Awasthi
17 B16 Lung Melanoma Model to Study the Role of Th9 Cells in Cancer ...... 217
Alka Dwivedi, Sushant Kumar, and Rahul Purwar
18 Th9 Cells and Parasitic Inflammation: Use of Nippostrongylus
and Schistosoma Models.......................................... 223
Miguel Enrique Serrano Pinto and Paula Licona-Limón
19 Isolation and Purification of Th9 Cells for the Study
of Inflammatory Diseases in Research and Clinical Settings ............... 247
Patricia Keating and James X. Hartmann
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
Contributors
Silvia C.P. almeida • Faculdade de Medicina, Instituto de Medicina Molecular,
Universidade de Lisboa, Lisbon, Portugal; Instituto Gulbenkian de Ciencia, Oeiras,
Portugal
PornPimon angkaSekwinai • Department of Medical Technology, Faculty of Allied Health
Sciences, Thammasat University, Pathumthani, Thailand; Graduate Program, Faculty of
Allied Health Sciences, Thammasat University, Pathumthani, Thailand
amit awaSthi • Center for Human Microbial Ecology (CHME), Translational Health
Science & Technology Institute (THTI), Faridabad, Haryana, India
SubaSh babu • National Institutes of Health - International Center for Excellence
in Research, National Institute of Research in Tuberculosis (Formerly Tuberculosis
Research Center), Chennai, India
vaneSSa beynon • Ann Romney Center for Neurologic Diseases, Brigham and Women’s
Hospital and Harvard Medical School, Boston, MA, USA
valerie dardalhon • Institut de Génétique Moléculaire de Montpellier, Centre National
de la Recherche Scientifique UMR5535, Université de Montpellier, Montpellier, France
alka dwivedi • Department of Bioscience and Bioengineering, Indian Institute
of Technology Bombay (IIT Bombay), Mumbai, Maharashtra, India
waSSim elyaman • Ann Romney Center for Neurologic Diseases, Brigham and Women’s
Hospital and Harvard Medical School, Boston, MA, USA; Program in Translational
Neurogenomics and Neuroimmunology, Department of Neurology, Brigham and
Women’s Hospital, Harvard Medical School, Broad Institute at Harvard University and
MIT, Boston, MA, USA
luCien P. garo • Ann Romney Center for Neurologic Diseases, Brigham and Women’s
Hospital and Harvard Medical School, Boston, MA, USA
FrançoiS ghiringhelli • Université Bourgogne Franche-Comté, Dijon, France;
Centre de Recherche INSERM LNC-UMR1231, Dijon, France; Plateforme de Transfert
en Biologie Cancérologique, Centre GF Leclerc, Dijon, France
Julio gomez-rodriguez • Genetic Disease Research Branch, National Human Genome
Research Institute, National Institutes of Health, Bethesda, MD, USA
ritobrata goSwami • School of Bio Science, Sir JC Bose Laboratory Complex, Indian
Institute of Technology, Kharagpur, West Bengal, India
luiS graCa • Faculdade de Medicina, Instituto de Medicina Molecular, Universidade de
Lisboa, Lisbon, Portugal; Instituto Gulbenkian de Ciencia, Oeiras, Portugal
JameS X. hartmann • Florida Atlantic University, Boca Raton, FL, USA
etienne humblin • Université Bourgogne Franche-Comté, Dijon, France;
Centre de Recherche INSERM LNC-UMR1231, Dijon, France
rukhSana Jabeen • HB Wells Center for Pediatric Research, Indiana School of Medicine,
Indianapolis, IN, USA
ryoJi kawakami • Department of Experimental Immunology, Immunology Frontier
Research Center, Osaka University, Osaka, Japan
PatriCia keating • Florida Atlantic University, Boca Raton, FL, USA
ix
x Contributors
SuShant kumar • Department of Bioscience and Bioengineering, Indian Institute
of Technology Bombay (IIT Bombay), Mumbai, Maharashtra, India
Paula liCona-limón • Departamento de Biología Celular y del Desarrollo, Instituto de
Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, Mexico
SakShi malik • Center for Human Microbial Ecology (CHME), Translational Health
Science & Technology Institute (THTI), Faridabad, Haryana, India
FrançoiSe meylan • Immunoregulation Section, Autoimmunity Branch, National
Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of
Health, Bethesda, MD, USA
norihiSa mikami • Department of Experimental Immunology, Immunology Frontier
Research Center, Osaka University, Osaka, Japan
goPal murugaiyan • Ann Romney Center for Neurologic Diseases, Brigham and Women’s
Hospital and Harvard Medical School, Boston, MA, USA
william orent • Ann Romney Center for Neurologic Diseases, Brigham and Women’s
Hospital and Harvard Medical School, Boston, MA, USA; Program in Translational
Neurogenomics and Neuroimmunology, Department of Neurology, Brigham and
Women’s Hospital, Harvard Medical School, Broad Institute at Harvard University and
MIT, Boston, MA, USA
duy Pham • Department of Pathology, University of Alabama at Birmingham,
Birmingham, AL, USA
miguel enrique Serrano Pinto • Departamento de Biología Celular y del Desarrollo,
Instituto de Fisiología Celular, Universidad Nacional Autónoma de México,
Mexico City, Mexico
rahul Purwar • Department of Bioscience and Bioengineering, Indian Institute
of Technology Bombay (IIT Bombay), Mumbai, Maharashtra, India
Prabhakar Putheti • Department of Medicine, Weill-Cornell Medical College, New York,
NY, USA
anuradha raJamaniCkam • National Institutes of Health - International Center for
Excellence in Research, National Institute of Research in Tuberculosis (Formerly
Tuberculosis Research Center), Chennai, India
wilmer gerardo roJaS-zuleta • Department of Rheumatology, Universidad de
Antioquia, Medellín, Colombia
diJendra nath roy • Department of Bioengineering, National Institute of Technology,
Jirania, NIT-Agartala, Tripura, India
elizabeth SanChez • Department of Physiology, Universidad Nacional de Colombia,
Bogotá, Colombia
hendrik SChulze-kooPS • Division of Rheumatology and Clinical Immunology,
Department of Medicine IV, University of Munich, Munich, Germany
alla SkaPenko • Division of Rheumatology and Clinical Immunology, Department of
Medicine IV, University of Munich, Munich, Germany
atSuShi Sugimoto • Department of Experimental Immunology, Immunology Frontier
Research Center, Osaka University, Osaka, Japan
Frédérique végran • Université Bourgogne Franche-Comté, Dijon, France; Centre de
Recherche INSERM LNC-UMR1231, Dijon, France; Plateforme de Transfert en
Biologie Cancérologique, Centre GF Leclerc, Dijon, France
Chapter 1
Th9 Cells: New Member of T Helper Cell Family
Ritobrata Goswami
Abstract
T Helper cells (CD4+ T cells) constitute one of the key arms of adaptive immune responses. Differentiation
of naïve CD4+ T cells into multiple subsets ensure a proper protection against multitude of pathogens in
immunosufficient individual. After differentiation, T helper cells secrete specific cytokines that are critical to
provide immunity against various pathogens. The recently discovered Th9 cells secrete the pleiotropic cyto-
kine, IL-9. Although IL-9 was cloned more than 25 years ago and characterized as a Th2 cell-specific cyto-
kine, not many studies were carried out to define the function of IL-9. This cytokine has been demonstrated
to act on multiple cell types as a growth factor. After the discovery of Th9 cells as an abundant source of
IL-9, renewed focus has been generated. In this chapter, I discuss the biology and development of IL-9-
secreting Th9 cells. Furthermore, I highlight the role of Th9 cells and IL-9 in health and diseases.
Key words Th9 cells, IL-9, Transcription factors, Epigenetic modification, Allergic inflammation,
Autoimmune disorder, Tumor immunity, Helminthic infection
1 Introduction
An adaptive immune response begins when a naïve CD4+ T cell
interacts with an antigen presenting cell with a nonself peptide in
the context of class II MHC molecule. Following this interaction,
the naïve CD4+ T cell differentiates into distinct T helper subsets.
Differentiation into distinct T helper subset would depend on cyto-
kines present in the microenvironment and each of these subsets
would express their signature cytokines. The newest member of the
ever growing T helper subset is the interleukin-9 (henceforth to be
known as IL-9) secreting T helper cells, also known as Th9 cells. T
helper cells are characterized by their distinct functions. Th1 cells
are responsible to fight against intracellular pathogens, Th2
cells provide immunity against extracellular parasites, while Th17
cells mediate immunity against fungal infections and extracellular
bacteria. Even though IL-9 was cloned almost three decades back,
we have started unraveling the factors that control the expression
and function of the gene recently. The cytokine microenvironment
leading to the production of IL-9 by mouse CD4+ T cells was first
Ritobrata Goswami (ed.), Th9 Cells: Methods and Protocols, Methods in Molecular Biology, vol. 1585,
DOI 10.1007/978-1-4939-6877-0_1, © Springer Science+Business Media LLC 2017
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