Transcriptional Control And Population Dynamics Of Antiviral Cd8+ T Cell Responses

118 Pages·2014·11.37 MB·English

by  Michael Alexander Paley

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Publicly Accessible Penn Dissertations
2012
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Michael Alexander Paley
University of Pennsylvania, [email protected]
Follow this and additional works at: https://repository.upenn.edu/edissertations
Part of the Allergy and Immunology Commons, Cell Biology Commons, Immunology and Infectious
Disease Commons, Medical Immunology Commons, and the Virology Commons
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Paley, Michael Alexander, "Transcriptional Control and Population Dynamics of Antiviral CD8+ T Cell
Responses" (2012). Publicly Accessible Penn Dissertations. 682.
https://repository.upenn.edu/edissertations/682
This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/682
For more information, please contact [email protected].
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Cytotoxic lymphocytes are central components of cellular immune responses to intracellular pathogens
and malignancy. The transcriptional programs that support proper population dynamics for lifelong
immunity are incompletely understood. Two T-box transcription factors, T-bet and Eomesodermin
+
(Eomes), have critical roles in the development of natural killer cells and the differentiation of CD8 T cells
in response to acutely resolved infections. In both cases, these two factors support distinct but
complementary cellular populations. In this thesis, we first used a recently generated reagent to examine
and separate cellular populations with differential Eomes expression. We found that, while Eomes
+
expression does not identify effector CD8 T cells with enhanced cytotoxic potential, early Eomes
+
expression does correlate with more efficient formation of long-lived, self-renewing central memory CD8
+
T cells. This validated tool was then employed in our investigation into the population dynamics of CD8 T
cell responses during a chronic viral infection. While memory lymphocytes maintain lifelong immunity by
slow self-renewal, chronic infections strain the regenerative capacity of antiviral T lymphocyte
populations, leading to failure in long-term immunity. The cellular and molecular events controlling the
regenerative capacity during chronic infection, however, are unknown. We demonstrate that two distinct
+
states of virus-specific CD8 T cells exist in chronically infected mice and humans. The opposing
+
properties of renewal and differentiation of these CD8 T cell populations are supported by the differential
+
expression of T-box transcription factors, which cooperatively maintain the pool of antiviral CD8 T cells
hi
during chronic viral infection. T-bet cells have low intrinsic turnover but proliferate in response to
hi
persisting antigen, giving rise to Eomes terminal progeny. Genetic elimination of either subset results in
failure to control chronic infection, suggesting that imbalance in differentiation and renewal could
underlie the collapse of immunity in humans with chronic infections. Furthermore, this work
+
demonstrates new roles for T-bet and Eomes in CD8 T cells regarding self-renewal and terminal
differentiation, highlighting how T-box transcription factors can operate in exquisitely context-dependent
manners.
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Dissertation
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Doctor of Philosophy (PhD)
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Immunology
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E. J. Wherry
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CD8 T cells, Cellular Differentiation, Chronic Viral Infection, Population Dynamics, T-bet and
Eomesodermin, Transcription Factors
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Allergy and Immunology | Cell Biology | Immunology and Infectious Disease | Medical Immunology |
Virology
This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/682
TRANSCRIPTIONAL CONTROL AND POPULATION DYNAMICS
OF ANTIVIRAL CD8+ T CELL RESPONSES
Michael A. Paley
A DISSERTATION
in
Immunology
Presented to the Faculties of the University of Pennsylvania
in
Partial Fulfillment of the Requirements for the
Degree of Doctor of Philosophy
2012

Supervisor of Dissertation
_____________________
E. John Wherry
Associate Professor of Microbiology

Graduate Group Chairperson
_______________________
E. John Wherry, Associate Professor of Microbiology

Dissertation Committee
Steven L. Reiner M.D., Professor of Microbiology & Immunology and Pediatrics
Paula M Oliver Ph.D., Assistant Professor of Pathology and Laboratory Medicine
Daniel Douek M.D., MRCP, Ph.D., NIH Faculty
David Artis Ph.D., Associate Professor of Microbiology
TRANSCRIPTIONAL CONTROL AND POPULATION DYNAMICS
OF ANTIVIRAL CD8+ T CELL RESPONSES
COPYRIGHT
2012
Michael Alexander Paley
ACKNOWLEDGMENTS

I have often heard it takes a village to raise a child. For a budding scientist, that
village is quite large. First, I must thank my two incredible mentors, E. John Wherry and
Steve L. Reiner, for both supporting and challenging me throughout my tenure in their
laboratories. I find I have grown immensely and become much more intellectually limber
after having two great minds approach my data from quite disparate perspectives.
Second, I must thank members of both the Wherry and Reiner laboratories, Scott
Gordon, Maria Ciocca, Levi Rupp, Burton Barnett, Julie Chaix, Douglas Dolfi, Jonathan
Johnnidis, Pamela Odorizzi, Erietta Stelekati, Makoto Kurachi, Vesselin Tomov, Alison
Crawford, Travis Doering, Kathleen Mansfield, Mohammed Ali, Arnob Banerjee, and
John Chang, who created an intellectually stimulating and plain-old-fun work
environment and who were wonderfully tolerant of a fellow lab member that was missing
50% of the time. Third, I am grateful to my Thesis Committee, David Artis, Paula Oliver,
and Daniel Douek, for their valuable advice. Fourth, I wish to thank Bob Doms, Paul
Bates, and the Virology Training Grant for two years of financial support as a graduate
student. Fifth, I am grateful for the guidance provided by the Combined Degree Office
and the Immunology Graduate Group. Sixth, I would like to thank my parents, Robert
Paley and Marianne Steiner, and my sister Jessica Paley, for all the various forms of
support I have received during the many ups and downs in the pursuit of scientific
discovery. Seventh, I must thank my close friends of 24 years, Daniel Abravanel and
Jeffrey Rechler, for their ability to pull me back into the world outside the academic
bubble. And eighth, I must thank my partner in all that I do, Grace Lin, for her patience,
love, unending generosity, and many late nights of companionship in front of the flow
cytometer.
iii
ABSTRACT

TRANSCRIPTIONAL CONTROL AND POPULATION DYNAMICS
OF ANTIVIRAL CD8+ T CELL RESPONSES

Michael A. Paley

E. John Wherry, Ph.D.

Cytotoxic lymphocytes are central components of cellular immune responses to
intracellular pathogens and malignancy. The transcriptional programs that support
proper population dynamics for lifelong immunity are incompletely understood. Two T-
box transcription factors, T-bet and Eomesodermin (Eomes), have critical roles in the
development of natural killer cells and the differentiation of CD8+ T cells in response to
acutely resolved infections. In both cases, these two factors support distinct but
complementary cellular populations. In this thesis, we first used a recently generated
reagent to examine and separate cellular populations with differential Eomes expression.
We found that, while Eomes expression does not identify effector CD8+ T cells with
enhanced cytotoxic potential, early Eomes expression does correlate with more efficient
formation of long-lived, self-renewing central memory CD8+ T cells. This validated tool
was then employed in our investigation into the population dynamics of CD8+ T cell
responses during a chronic viral infection. While memory lymphocytes maintain lifelong
immunity by slow self-renewal, chronic infections strain the regenerative capacity of
antiviral T lymphocyte populations, leading to failure in long-term immunity. The cellular
and molecular events controlling the regenerative capacity during chronic infection,
iv
however, are unknown. We demonstrate that two distinct states of virus-specific CD8+ T
cells exist in chronically infected mice and humans. The opposing properties of renewal
and differentiation of these CD8+ T cell populations are supported by the differential
expression of T-box transcription factors, which cooperatively maintain the pool of
antiviral CD8+ T cells during chronic viral infection. T-bethi cells have low intrinsic
turnover but proliferate in response to persisting antigen, giving rise to Eomeshi terminal
progeny. Genetic elimination of either subset results in failure to control chronic infection,
suggesting that imbalance in differentiation and renewal could underlie the collapse of
immunity in humans with chronic infections. Furthermore, this work demonstrates new
roles for T-bet and Eomes in CD8+ T cells regarding self-renewal and terminal
differentiation, highlighting how T-box transcription factors can operate in exquisitely
context-dependent manners.

v
TABLE OF CONTENTS

ACKNOWLEDGMENTS...........................................................................III

ABSTRACT ..............................................................................................IV

LIST OF FIGURES ...................................................................................IX

CHAPTER 1: INTRODUCTION.................................................................1

1.1 Viral challenges to human health.............................................................................1

1.2 Adaptive immune responses.....................................................................................2

1.3 CD8+ T cell responses................................................................................................3

1.3.1 Initiation of antiviral CD8+ T cell responses...........................................................4

1.3.2 Generation of lifelong CD8+ T cell memory after acutely resolved infections........4

1.3.3 Maintenance of lifelong CD8+ T cell memory to acutely resolved infections.........5

1.3.4 Responses to persistent antigen and inflammation: CD8+ T cell exhaustion........6

1.4 T-box transcription factors........................................................................................8

1.4.1 Activity of T-box transcription factors is context-dependent..................................8

1.4.2 T-box factors regulate self-renewal and differentiation..........................................9

1.4.3 T-box factors regulate antiviral immunity.............................................................10

1.4.4 T-bet and Eomes in Natural Killer cells...............................................................10

1.4.5 T-bet and Eomes in CD8+ T cells during acute infection.....................................11

1.4.6 T-bet in CD8+ T cells during chronic infection.....................................................13

1.4.7 Eomes in CD8+ T cells during chronic infection...................................................13

1.5 The Lymphocytic Choriomeningitis Model of Viral Infection...............................14

1.5.1 Acutely resolved LCMV infection.........................................................................14

1.5.2 Chronic LCMV infection.......................................................................................15

CHAPTER 2: FLUORESCENT REPORTER REVEALS INSIGHTS INTO
EOMESODERMIN BIOLOGY IN CYTOTOXIC LYMPHOCYTES...........16

2.1 Introduction:.............................................................................................................16

2.2 Methods.....................................................................................................................18

2.2.1 Mice and Infection:..............................................................................................18

2.2.2 Flow cytometry, division dye labeling, and real-time PCR:..................................19

2.2.3 Cytotoxicity assay:...............................................................................................20

vi
2.3 Results.......................................................................................................................20

2.3.1 Expression of Eomesgfp is largely restricted to cytotoxic lineages and correlates
with Eomes protein expression....................................................................................20

2.3.2 GFP expression quantitatively parallels Eomes protein expression....................23

2.3.3 Eomes expression does not identify the most cytotoxic effector CD8+ T cells....25

2.3.4 Progressive enrichment of Eomes expression in memory CD8+ T cells.............29

2.3.5 Expression of the Eomes locus is reduced in the absence of Eomes protein.....30

2.3.6 Eomesgfp allows for detection of putative intermediates of NK cell development.35

2.4 Discussion................................................................................................................37

CHAPTER 3: A CRITICAL ROLE FOR BALANCING PROGENITOR
AND TERMINAL SUBSETS OF EXHAUSTED CD8+ T CELLS DURING
CHRONIC VIRAL INFECTION................................................................41

3.1 Introduction:.............................................................................................................41

3.2 Methods:....................................................................................................................44

3.2.1 Human Subjects:.................................................................................................44

3.2.2 Mice and Infections:.............................................................................................45

3.2.3 Flow cytometry and real-time PCR:.....................................................................45

3.2.4 Bone Marrow Chimeras.......................................................................................46

3.2.5 BrdU Treatment and Detection............................................................................46

3.2.6 CFSE labeling, temporal deletion and adoptive transfers:..................................46

3.2.7 Statistical Analysis:..............................................................................................47

3.3 Results:.....................................................................................................................47

3.3.1 Eomes expression is enhanced during chronic viral infection.............................47

3.3.2 Eomes expression correlates with features of CD8+ T cell exhaustion...............49

3.3.3 Deletion of Eomes leads to reduced features of CD8+ T cell exhaustion............53

3.3.4 T-bet and Eomes regulate distinct proliferative properties..................................56

3.3.5 T-bethi progenitors give rise to Eomeshi progeny.................................................62

3.3.6 Loss of T-bet or Eomes leads to loss of the CD8+ T cell response and impaired
viral control..................................................................................................................66

3.3.7 Antigen drives the differentiation from T-bethi precursors to Eomeshi progeny....68

3.3.8 Antigen drives the differentiation of T-bethi precursors to Eomeshi progeny during
hepatitis C infection.....................................................................................................68

3.4 Discussion:...............................................................................................................72

CHAPTER 4: CONCLUDING REMARKS AND FUTURE DIRECTIONS77

4.1 Overview of Results.................................................................................................77

4.2 Distinct or identical activity of T-box factors in response to acutely resolved vs
chronic viral infection...................................................................................................78

4.3 Use of a fluorescent reporter to identify transcriptional targets and regulators
of Eomesodermin..........................................................................................................79

4.4 Senescence versus Exhaustion: distinct or overlapping processes?................81

vii