Driven by methodological success in identifying reliable lineage markers, regulatory T cells have quickly been recognized as the most numerous subset of immune regulators in the body with critical functions in a wide array of immune responses. In Regulatory T Cells: Methods and Protocols, experts in the field to offer a collection of current techniques to advance the study of regulatory T cells, including the use of the IL-2 receptor alpha chain and other markers, as well as the more recently desirable use of the transcription factor FoxP3. Divided into three sections, the book covers, in equal measure, in vitro, in vivo, and human studies. Written in the highly successful Methods in Molecular Biology series format, chapters include brief introductions to their respective subjects, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known methodological pitfalls. Authoritative and cutting-edge, Regulatory T Cells: Methods and Protocols distills the most vital current techniques through several years of optimization and standardization in order to allow reliable and reproducible use by both young and experienced cellular and molecular immunologists.

Contributors ix
Part I Introduction
1 Regulatory T Cells: History and Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Shimon Sakaguchi
Part II In vitro
2 In Vitro Treg Suppression Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Lauren W. Collison and Dario A.A. Vignali
3 Generation of T Cell Hybridomas from Naturally
Occurring FoxP3+ Regulatory T Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Nagendra Singh, Rafal Pacholczyk, Makio Iwashima,
and Leszek Ignatowicz
4 In Vitro and In Vivo Analyses of Regulatory T Cell
Suppression of CD8+ T Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Kim J. Hasenkrug and Lara M. Myers
5 Flow Cytometric Profiling of Mature and Developing
Regulatory T Cells in the Thymus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Donald M. Simons and Andrew J. Caton
6 ChIP-on-Chip for FoxP3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Ye Zheng
7 Live Imaging of Dendritic Cell Treg Cell Interactions . . . . . . . . . . . . . . . . . . . . . 83
Milka Sarris and Alexander G. Betz
Part III In vivo
8 Genetic Tools for Analysis of FoxP3+ Regulatory
T Cells In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Nadia M. Jeremiah and Adrian Liston
9 In Vivo Treg Suppression Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Creg J. Workman, Lauren W. Collison, Maria Bettini,
Meenu R. Pillai, Jerold E. Rehg, and Dario A.A. Vignali
10 In Vivo Depletion of FoxP3+ Tregs Using the DEREG Mouse Model . . . . . . . . . 157
Katharina Lahl and Tim Sparwasser
11 Antigen-Specific Induction of Regulatory T Cells In Vivo and In Vitro . . . . . . . . 173
Carolin Daniel, Hidde Ploegh, and Harald von Boehmer
12 In Vitro Expansion of Alloantigen-Specific Regulatory T Cells
and Their Use in Prevention of Allograft Rejection . . . . . . . . . . . . . . . . . . . . . . . 187
Clémence Nouzé, Lise Pasquet, and Joost P.M. van Meerwijk
viii Contents
Part IV Human
13 Analysis of Human FOXP3+ Treg Cells Phenotype and Function . . . . . . . . . . . . . 199
Eva d'Hennezel and Ciriaco A. Piccirillo
14 Depletion of Human Regulatory T Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
Amy C. Hobeika, Michael A. Morse, Takuya Osada,
Sharon Peplinski, H. Kim Lyerly, and Timothy M. Clay
15 Assessment of Suppressive Capacity by Human Regulatory
T Cells Using a Reproducible, Bi-Directional CFSE-Based In Vitro Assay . . . . . . 233
Anya Schneider and Jane H. Buckner
16 Measurement of Proliferation and Disappearance of Regulatory
T Cells in Human Studies Using Deuterium-Labeled Glucose . . . . . . . . . . . . . . . 243
Milica Vukmanovic-Stejic, Yan Zhang, Arne N. Akbar
and Derek C. Macallan
17 Flow Cytometric Detection of Human Regulatory T Cells . . . . . . . . . . . . . . . . . . 263
Barbara Fazekas de St Groth, Erhua Zhu, Suzanne Asad
and Loretta Lee
Index .