Towards Engineering Immunity
Thesis by Lili Yang
In Partial Fulfillment of the Requirements for Degree of Doctor of Philosophy
California Institute of Technology Pasadena, California, USA
2004
(Defended on May 10, 2004)
2004
Lili Yang
All Rights Reserved
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To My Parents
ACKNOWLEDGEMENTS
Before I started to work on my thesis, I thought acknowledgements section would be the easiest one. In reality I found that it was far more difficult than I expected to put together this part of thesis, primarily due to the fact that the bulk of this work would not be completed without the influence of many people in my life. I thank everyone who encouraged me and supported me in many aspects of my life. It is almost impossible to list all names herein and I am sure I have missed some names, for which I apologize.
Foremost, I would like to acknowledge my thesis advisor, David Baltimore, for having guided me into this fascinating area of research, for his constant support and encouragement. I admire his energy, his enthusiasm for science and his good memory.
David allowed me to work on several projects. The opportunities I have been afforded in his group were enormous.
I also would like to thank other members of my committee, Prof. Ellen Rothenberg, Prof. Jose Alberola-Ila, Prof. Pamela Bjorkman, and Prof. Paul Sternberg for their contributions to this work. Their patience and insights have helped move forward much of this thesis work. I appreciate their time dedicated to reviewing my thesis results.
I would like to take this opportunity to thank Prof. Luk Van Parijs at MIT. When I joined David’s laboratory, Luk was a postdoctoral scholar working for David. He acted as my laboratory mentor and helped me initiate my project. He taught me lots of essential experimental skills, ranging from molecular cloning to animal work. His contribution to my scientific career as well as this thesis is enormous.
The 2-year experience at UC riverside was wonderful. The training and knowledge obtained at Walker’s laboratory are very broad and useful. I thank Prof.
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Ameae Walker for her supports and guidance and members in her lab, including Benson, Xiaolei and Georgia.
The experience with the entire Baltimore group was wonderful and unforgettable.
My progress and accomplishments are always associated with their assistance and ideas.
When I first joined the group, Xiaofeng, Wange, Lois, Alex, Matt, Eric, Joe, Mollie had me settle down in the laboratory. I especially appreciate the time I spent working with Mark at our little office. He told me a lot of wonderful and threatening stories about science and Ph.D life. I was always scared by the extremely high citation of his cell paper. I also thank the latter joining Postdocs, including Huatao, Rafael, Konstantin and Shengli for their great time. I certainly enjoyed the company of two other graduate students, Thomas and Jeff. Together, we survived and thrive in the “Postdoc dominant environment”.
Special thanks go to Elissa. She was always very helpful when I tried to find lab stuffs. Her on time ordering and delivery for equipments and reagents kept my experiments moving smoothly.
Eric Santiestevan is a wonderful lab manager. I thank him for critical reading of my manuscripts and thesis and helping me on my English writing. If you can understand my written materials, you should realize his contribution on correction of grammar and careful editing.
I also would like to thank the members of Pepe’s laboratory, especially Harry, Eric, Susannah, Micheline and Gabriela, and members of Ellen’s laboratory, especially Chris, Mary and Shelly. We have wonderful journal club and I have learned lots of immunology from all of you.
I am very fortunate to have a very good group of people at animal facility to take care of my mice, especially Ruben. With his care, my immunodeficient mice, particularly those for bone marrow transfer experiments, have been maintained well, which is crucial for lots of work described in this thesis. Of course Janet and Peggy’s management on animal facility is outstanding, too. I also thank Shirley at Caltech transgenic center for her help on the lentivirus project.
I would like to thank Susan Kovats at City of Hope for allowing me to use her equipments for the proliferation assay described in this thesis.
Life outside the laboratory at Caltech is truly wonderful. Together with Chunhui Mo, Joe Andrieu, Girish N. Aakalu, Pin Wang, Ying Go, we founded Caltech Consulting Club. Knowledge gleaned from this club and experiences obtained from two summer projects with two different companies are precious. I was fortunate to serve as Caltech C president for one term. This experience was wonderful.
I have been very fortunate to gain many lasting friendships over the last five years at Caltech. These friends have been an important part of my graduate school experience not only on a scientific basis but on a personal level as well. I would like to thank the gang like Ying, Qijing, Nan, Aijun, Xun, Xiang, Chunhui, Hongyu, Qiang, Ying, Lan, Huazhang, Xianglei, Weiwei, Jack, Zhigang, Jun, Hui, Wen, Rena, Zhenrong, Suzie, Yen, Joanne, Fanyong, Shuwei, etc, for all the good times.
The research presented in this thesis was funded by NIH Grant.
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ABSTRACT
The aim of engineering immunity is to harness and engineer the immune system to treat infectious diseases and cancer. Towards this goal, accumulating evidence shows that the immune system can be manipulated to achieve the desired and improved functions. In the context of cancer therapy, many strategies have appeared to utilize the principle of immune defense to safely and effectively target tumor cells for destruction.
These strategies fall into two categories: active immunotherapy and passive immunotherapy. Active immunotherapy involves activating the effectors in the host immune system to inhibit cancer cell growth and reject tumors (e.g., cancer vaccination), while passive immunotherapy is a term for directly providing the host with effectors to react against cancer (e.g., adoptive transfer of in vitro expanded antitumor T cells).
We propose a concept of instructive immunotherapy for cancer. This concept is to use a strategy to guide the host in developing in vivo effector cells capable of targeting cancer. This strategy arises from combination of gene therapy, stem cell therapy and immunotherapy to program hematopoietic stem cells (HSCs) to develop into lymphocytes with desired antitumor specificity. Therefore, taking advantage of the longevity and self- renewal of HSCs, life-long supplies of tumor-specific lymphocytes can be generated in vivo, which exceed the current methods of repetitive immunization and adoptive transfer.
To test the feasibility of this approach, I describe in Chapter 2 the procedure of retrovirus-mediated gene transfer of TCR cDNA into RAG1-deficient HSCs. Subsequent transfer of these genetic modified HSCs into RAG1-deficient mice allows the long-term production of functional antigen-specific T cells.
Chapter 3 describes a method to impart anti-tumor specificity to the wild-type mouse T cell repertoire. To achieve this, genes encoding a CD8 T cell receptor with the desired anti-tumor specificity were delivered into wild-type HSCs via a retroviral vector.
When transferred into host mice, these genetically modified HSCs generated a large population of anti-tumor cytotoxic T cells, accounting for more than 20% of peripheral CD8 T cells. These cells displayed a normal response to antigen stimulation and had the ability to generate and maintain long-term memory. Significant tumor rejection was observed in mice containing these T cells, demonstrating feasibility of instructive cancer immunotherapy.
In recognition of the important roles of helper T cells in anti-tumor immunity, Chapter 4 elaborates a two-arm model to augment tumor-specific immune responses. In the experiment, the two arms, both anti-tumor CD4- and CD8 T cells, were generated by HSC gene transfer method. The resultant immune system in mice could not only suppress tumor growth, but could also eradicate large, solid and vascularized tumors.
Coupled with results described in Chapter 3, we demonstrated the great potential of instructive cancer immunotherapy and expanded the scope of engineering immunity.
Successful immunotherapy relies on understanding the molecular mechanisms that control immune responses. For instance, although IL-2 has been approved by FDA to treat renal cancer and melanoma, many results from mice show that the physiological role of IL-2 is complex and unpredictable, hindering the design of better strategies, that would maximize the therapeutic impact of IL-2. I address the role of IL-2 in negative regulatory function and T cell memory in last two chapters, both of which are important for achieve the overall success of immunotherapy and engineering immunity. Chapter 5
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describes the role of IL-2 in maintaining regulatory T cell homeostasis and self-tolerance, and correlates this role with the signaling molecule STAT5. The final chapter (Chapter 6) details the role of IL-2 in generation of CD4 T cell memory.
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS iv
ABSTRACT vii
LIST OF TABLES xiv
LIST OF FIGURES xv
CHAPTER
Chapter 1 Introduction to Engineering Immunity 1-63
1 Immunotherapy 2
New Generation Vaccines 2
Target the Negative Immunoregulatory Mechanism 4 Monoclonal Antibodies for Passive Immunotherapy 5
2 Cancer T Cell Therapy 6
Adoptive Immunotherapy 8
Redirecting T Cell Specificity 12
Chimeric Antigen Receptors 15
3 The Unique Role of CD4 T Cells in Antitumor Immunity 18 4 Hematopoietic Stem Cells As Targets for Gene Therapy 22
Mouse Hematopoietic Stem Cells 22
Human Hematopoietic Stem Cells 23
HSC Gene Therapy 26
5 The Role of IL-2 on T Cell Regulatory Function and Memory
31 6 Research Objective and Thesis Outline 34
7 References 46
Chapter 2 Generating Functional Antigen-specific T Cells in Defined Genetic Background
64- 95
1 Introduction 65
2 Materials and Methods 68
Mice 68
MIG-TCR Retroviruses Construction 68 Generation of the THZ Hybridoma Cell Line and
Infection with Retroviruses 68
Bulk LacZ Assay 69
Primary CD4+ T Cell Infection and Stimulation 70 Bone Marrow (BM) Stem Cell Isolation, Infection and
Transfer
70 Immunization of Bone Marrow Recipient Mice 71 In Vitro T Cell Stimulation and Proliferation Assays 71
IL-2 and IFN-γ ELISA 72
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3 Results 72
Functional Expression of the OTII TCR in T Cell Lines and Primary CD4+ T Cells Using Retroviruses
72 Generation of OTII TCR Transgenic T cells in vivo
Following Retrovirus-Based Gene Delivery into Bone Marrow Precursor Cells
74
Normal in vitro and in vivo Antigen Responses of OTII
TCR Transgenic T Cells 76
4 Discussion 77
TCR-Expressing Retroviruses Drive T Cell Development 78 Use of Retroviruses to Study T Cell Development and
Function 79
Therapeutic and Prophylactic Uses of Retrovirus- Generated T Cells
81
5 References 92
Chapter 3 Imparting Anti-tumor Specificity to the Mouse T Cell Repertoire
96- 123
1 Introduction 97
2 Materials and Methods 98
Mice 98
MOT1 Retrovirus 99
Primary T Cell Infection and Stimulation 99 Hematopoietic Stem Cells (HSCs) Isolation, Infection
and Transfer
99 In vitro T cell Stimulation and Functional Assays 100
Antibodies and FACS Analysis 100
T cell Memory Study 101
Tumor Challenge of Mice 101
Dendritic Cell Generation, Antigen Pulsing and Mouse Immunization
102
3 Results and Discussion 103
4 Conclusion 110
5 References and Notes 121
Chapter 4 Construction Of The Two Arms Of The Anti-tumor T Cell Immunity
124
1 Introduction 125
2 Materials and Methods 129
Mice 129
MOT1 and MOT2 Retrovirus 129
Peptides 129
Primary T Cell Infection and Stimulation 130
Hematopoietic Stem Cells (HSCs) Isolation, Infection
and Transfer 130
In vitro T cell Stimulation and Functional Assays 131
Antibodies and FACS Analysis 131
T Cell Memory Study 132
Tumor Challenge of Mice 133
Dendritic Cell Generation, Antigen Pulsing and Mouse
Immunization 133
3 Results 134
Tumor Model 134
Retrovirus Mediates Functional Expression of CD8 and
CD4 T cell Receptors (TCRs) 135
Generation of Monospecific CD8 or CD4 T cells by Retrovirus-Mediated Expression of CD8 or CD4 TCR in RAG1-/- HSCs
136
Comparison of the Transgenic TCR Expression and T cell Development in Mice Receiving Retrovirus- Transduced RAG1-/- HSCs with Those in the Conventional TCR Transgenic Mice
138
Generation of Antigen-Specific CD8 or CD4 T Cells by Retrovirus-Mediated Expression of CD8 or CD4 TCR in Wild-type HSCs
143
Characterization of the CD8 and CD4 T Cells Generated by Viral Transduction of Wild-type HSCs
145 Imparting into Mouse T Cell Repertoire Both Anti-tumor
CD8 Cytotoxic and CD4 Helper T Cell Specificities
148 Test the Concept of Tumor Immunotherapy in E.G7
Mouse Tumor Model: Suppression of syngenic tumor growth by imparting anti-tumor specificities to mouse T cell repertoire
149
Testing the Concept of Tumor Immunotherapy in E.G7 Mouse Tumor Model: Eradication of the established solid tumor by reversal of the functional tumor tolerance via construction of the two arms of anti-tumor T cell immunity
153
4 Discussion 155
5 References 183
Chapter 5 IL-2 Signals Maintain CD25+CD4+ Regulatory T Cell Homeostasis and Self-Tolerance
188- 219
1 Introduction 189
2 Materials and Methods 192
Mice 192
Abs and Flow Cytometry 193
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T Reg Purification and Functional Assays 193 Generation of Inducible STAT5 Transgenic Mice 194
Statistical Analysis 195
3 Results 196
IL-2 Signals Maintain CD25+CD4+ T Reg Homeostasis and Self-Tolerance
196 Bcl-2 Is Not a Target of IL-2 Signals that Establish
Normal CD25+CD4+ T Reg Numbers in the Periphery and Maintain Self-Tolerance
197
CD25+CD4+ T Reg Numbers and Function Are Defective in STAT5-Deficient Mice, Leading to Deregulated Lymphoid Homeostasis
198
STAT5 Activation Increases CD25+CD4+ T Reg Numbers in the Absence of IL-2
200
4 Discussion 201
5 References 216
Chapter 6 IL-2 Regulates CD4 T Cell Memory Generation 220- 270
1 Introduction 221
2 Materials and Methods 224
Mice 224
FUGW, FUIGW-AKT* and FUIGW-STAT5*
Lentiviruses 225
Peptides 225
In vitro OT2 T cell Stimulation and Functional Assays 225 Naïve OT2 CD4 T Cell Infection and Stimulation 226
Antibodies and FACS Analysis 226
T Cell Memory Study 227
3 Results 228
Generation and Characterization of Naïve IL-2 Deficient CD4 T-cells
228 An IL-2 Signal Supports CD4 T-cell Memory Generation
But Is Not Required for Memory Maintenance 231 Development of a Lentiviral Gene Delivery Method as a
Tool to Study IL-2 Signaling in CD4 T-cells
234 Activation of the AKT Signaling Pathway During
Primary Response Cannot Rescue CD4 T-cell Memory Failure Caused by IL-2 Deficiency
238
Activation of the STAT5 Signaling Pathway During Primary Response Is Able to Rescue CD4 T-cell Memory Generation Caused by IL-2 Deficiency
240
4. Discussion 242
5 References 263
LIST OF TABLES
Page
Table 1-1 History of immunotherapy 37
Table 1-2 Mechanisms that account for failure of efficient immune responses
against tumors 38
Table 5-1 CD25+CD4+ regulator T cell numbers in mouse strains with defects in IL-2 and key IL-2 signaling molecules
205
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LIST OF FIGURES
Page
Figure 1-1 A schematic presentation of adoptive T cell transfer therapy for patients with cancer
39 Figure 1-2 Engineering redirected antitumor specificity into mature T cells
from PBL via retrovirus-mediated gene transfer of TCR cDNA genes
40
Figure 1-3 Engineering mature T cells from PBL with antitumor specificity by retrovirus-mediated gene transfer of chimeric antigen receptor
41 Figure 1-4 Cartoon structure of natural antigen receptors and chimera antigen
receptors
42 Figure 1-5 The schematic presentation of the model for CD4 T cells to
orchestrate multiple effector arms of antitumor immunity 43 Figure 1-6 The schematic presentation of a model for multipotent HSCs to
differentiate into different cells in hematopoietic system
44 Figure 1-7 The schematic representation of bone marrow transplantation of
genetic modified HSCs into lethal or sub-lethal irradiated mice to regenerate the hematopoietic system
45
Figure 2-1 Functional expression of the OTII TCR in a T cell hybridoma line and primary CD4+ T cells using retroviruses
83 Figure 2-2 Schematic of our strategy to generate OTII monoclonal T cells in
vivo using retroviruses 85
Figure 2-3 Development of functional wild type and IL-2-deficient OTII monoclonal T cells derived from retrovirally-transduced hematopoietic precursor cells
87
Figure 2-4 In vivo responses of wild type and IL-2-deficient OTII monoclonal T cells upon immunization with cognate antigen
89- 90 Figure 3-1 Imparting a desired antigen specificity to the mouse T cell
repertoire by retrovirus-mediated expression of TCR cDNAs in hematopoietic stem cells (HSCs)
112
Figure 3-2 Functional expression of the OT1 TCR in primary CD8 T cells using MOT1 retroviruses
114 Figure 3-3 Characterization of the OT1 T cells generated by viral transduction
of wild-type B6 HSCs 115
Figure 3-4 Suppression of syngenic tumor growth by imparting anti-tumor specificity to the mouse T cell repertoire via viral tranduction of HSCs
117
Figure 3-5 Comparison of TCR expression in OT1 T cells from B6/MOT1 mice with those from OT1 Tg mice
119
Figure 4-1 Retrovirus mediated functional expression of OT1 CD8 and OT2 CD4 T cell receptors (TCRs)
159
Figure 4-2 Generation of monospecific CD8 or CD4 T cells by retrovirus- mediated expression of CD8 or CD4 TCR cDNAs in RAG1-/- hematopoietic stem cells (HSCs)
161- 162 Figure 4-3 Comparison of the transgenic TCR expression and T cell
development in mice receiving retrovirus-transduced RAG1-/- HSCs with those in the conventional TCR transgenic mice
164- 165 Figure 4-4 Generation of antigen-specific CD8 or CD4 T cells by retrovirus-
mediated expression of CD8 or CD4 TCR cDNAs in wild-type HSCs
168- 169 Figure 4-5 Characterization of the CD8 and CD4 T cells generated by
retrovirus-mediated expression of CD8 or CD4 TCR cDNAs in wild-type HSCs
171- 174 Figure 4-6 Imparting to mouse T cell repertoire both anti-tumor CD8 cytotoxic
and CD4 helper T cell specificities
177 Figure 4-7 Suppression of syngenic tumor growth by imparting anti-tumor
CD8 cytotoxic or/and CD4 helper T cell specificity/specificities to mouse T cell repertoire
179
Figure 4-8 Reversal of tumor tolerance and eradication of the established solid tumors by constructing both arms of the anti-tumor T cell immunity
181
Figure 5-1 Jak3 is required to establish normal CD25+CD4+ T reg numbers in the periphery and to prevent the accumulation of activated CD4+ T cells
206
Figure 5-2 IL-2 and STAT5, but not Bcl-2, are required to establish normal CD25+CD4+ T reg numbers in the periphery and to prevent the accumulation of activated CD4+ T cells
208
Figure 5-3 Tg expression of Bcl-2 does not rescue CD25+CD4+ T reg numbers or prevent the accumulation of activated CD4+ T cells and the development of splenomegaly in IL-2-deficient mice
210
Figure 5-4 Adoptive transfer of CD25+CD4+ T regs prevents the development of autoimmunity in STAT5-deficient mice 212 Figure 5-5 Transient activation of STAT5 increases CD25+CD4+ T reg
numbers in IL-2-deficient mice
214
Figure 6-1 Generation and activation of OT2 CD4 T cells with IL-2 deficiency 249 Figure 6-2 Analysis of IL-2 requirement on CD4 T cell memory generation 251 Figure 6-3 Evaluation of the potential of using FUGW lentiviral vector to
genetically modify naïve T cells 253
Figure 6-4 Potential of lentivirus-mediated gene transfer for study of IL-2 signaling
255 Figure 6-5 Evaluation of possibility of activation of AKT signaling pathway to
rescue CD4 memory failure caused by IL-2 deficiency
257 Figure 6-6 Evaluation of possibility of activation of STAT5 signaling pathway
to rescue CD4 memory failure caused by IL-2 deficiency
259 Figure 6-7 Summarized results from study of the role of IL-2 in CD4 T cell
memory
261
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Figure 6-8 Schematic representation of the proposed “Paths to STAT5”
memory model. 262
