Pagan AJ Dissertation edit2

144 Pages·2012·2.57 MB·English

by  Antonio Pagán

ROLES OF CD28, INDUCIBLE COSTIMULATOR, AND THE INTERLEUKIN-2
RECEPTOR IN HELPER T CELL MEMORY FORMATION

A DISSERTATION
SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL
OF THE UNIVERSITY OF MINNESOTA
BY

ANTONIO JOSÉ PAGÁN

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

MARC K. JENKINS, ADVISOR

July 2012
© ANTONIO JOSÉ PAGÁN 2012
Acknowledgements

I would like to thank my mentor Dr. Marc Jenkins and my Thesis Committee: Dr. Daniel
Mueller, Dr. Stephen Jameson, and Dr. Yoji Shimizu for their guidance throughout my
doctoral training. I am also grateful to my laboratory colleagues for sharing their many
insights and expertise with me, and Dr. Marion Pepper, in particular, for initiating our
collaboration to study helper T cell memory formation and bringing the project to
fruition. Lastly, I would like to thank the faculty, staff, and my classmates in the
Microbiology, Immunology, and Cancer Biology graduate program and my colleagues in
the Center for Immunology for providing an intellectually stimulating and collegial
environment in which to grow as a scientist.
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Dedication

I dedicate this dissertation to my parents. Thank you for instilling in me the joy of
learning and always encouraging me to pursue my dreams.
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Abstract
Numerous studies have suggested that CD28, Inducible Costimulator (ICOS), and
Interleukin-2 receptor (IL-2R) signals shape the size and composition of the helper T cell
memory pool, but the extent to which these signals regulate specific aspects of memory
formation and the precise mechanisms of these processes remain unclear. To address this
question, we used a sensitive peptide:Major Histocompatibility Complex II (p:MHCII)
tetramer and magnetic bead-based enrichment method to track p:MHCII-specific helper T
cells in mice throughout the course of an immune response. We found that CD28, ICOS,
and the IL-2R each control unique aspects of helper T cell memory formation. The major
defect in CD28-deficient T cells was a failure to sustain the proliferation of effector T
cells and not a defect in memory formation per se; CD28-deficient T cells produced
memory cells proportionate to the number of effectors present at the peak of the response.
Our findings suggest that CD28 mediates this process by enhancing TCR-dependent
NFκB signaling independently of the two CD28 cytoplasmic tail sequences previously
implicated in clonal expansion. In contrast, ICOS or IL-2R deficiency did not grossly
reduce the overall size of the effector or memory cell populations. Instead, they
controlled the generation of two distinct subsets of memory T cells from effector cells
that were present several days after infection. T-bethigh T helper type-1 effector memory
(Th1em) cells were generated in an IL-2R-dependent fashion. In contrast, T-betlow CC
chemokine receptor 7 (CCR7)+ CXC cheomokine receptor 5 (CXCR5)+ central memory
T (Tcm) cells were formed in a Bcl6- and ICOS-dependent manner from T follicular
helper (Tfh) effector cells. Thus, CD28, ICOS, and IL-2R regulate distinct aspects of
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helper T cell memory formation.
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Table of Contents
List of Figures...................................................................................................................viii

Chapter 1: Background and Introduction
1.1 Adaptive Immunity...................................................................................1
1.2 Helper T Cell Development......................................................................2
1.3 Primary Helper T Cell Response..............................................................3
1.4 Helper T Cell Memory Formation............................................................5
1.5 CD28 Costimulation in Helper T Cell Priming, Differentiation, and
Memory Formation...................................................................................8
1.6 ICOS Signaling in Helper T Cell Differentiation and in Relation to
CD28 Signaling.......................................................................................15
1.7 IL-2 Signaling in Helper T Cell Immunity.............................................19
1.8 Detection of Numerically Rare Polyclonal Antigen-specific CD4+ T
Cells with p:MHCII Tetramers...............................................................20
1.9 Statement of Thesis.................................................................................21

Chapter 2: CD28 Promotes CD4+ T Cell Clonal Expansion During Infection
Independently of Its PI3K- and Lck-binding Sites.................................22
2.1 Introduction.............................................................................................23
2.2 Materials and Methods............................................................................25
Mice
Listeria monocytogenes Infections
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p:MHCII Tetramer Production
p:MHCII Tetramer Staining and Magnetic Enrichment
Antibodies and Flow Cytometry
DNA staining and Detection
Adoptive Transfer
Bone Marrow Irradiation Chimeras
Peptide Immunization
RelA Translocation Assay
Statistical Analyses
2.3 Results.....................................................................................................30
CD28 Enhances the Expansion of Polyclonal CD4+ T Cells Responding
to an L. monocytogenes-derived pMHCII in vivo

CD28 Is Necessary to Sustain Cell Cycle Activity of pMHCII-specific
CD4+ T Cells In Vivo

The PYAP and Y170F Motifs of CD28 Are Dispensable for CD4+ T
Cell Clonal Expansion in Response to L. monocytogenes Infection

CD28 Deficiency Phenotypically Resembles T Cell-intrinsic NFκB
Signaling Deficiency During Response to L. monocytogenes Infection

CD28 Signaling Increases Antigen-dependent Nuclear Translocation of
RelA In Vivo Independently of the PYAP and YMNM Motifs

2.4 Discussion...............................................................................................46

Chapter 3: Opposing Bcl6 and Interleukin-2 Receptor Signals Generate T Helper-
Type 1 Central and Efector Memory Cells.............................................50
3.1 Introduction.............................................................................................50
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3.2 Materials and Methods............................................................................53
Mice
Generation of r7UP Transgenic Mice
Bone Marrow Irradiation Chimeras
Infections
Tetramer Production
Cell Enrichment and Flow Cytometry
Lymphokine Production
Cell Transfer
Statistical Analyses
3.3 Results.....................................................................................................59
Detection of LLOp:I-Ab-specific CD4+ Memory T Cells
CD4+ Memory T cell Heterogeneity
CXCR5+ Memory Cells are Tcm Cells
Development of Th1em and Tcm Cells
ICOS Signals from B cells Sustain Tcm Cell Differentiation
3.4 Discussion...............................................................................................82

Chapter 4: Germinal Center T Follicular Helper Cells Form Central Memory T
Cells with an Increased Capacity to Produce CXCR5+ Effector Cells...86
4.1 Introduction.............................................................................................87
4.2 Materials and Methods............................................................................88
Mice
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Infections

Tetramer Production

Cell Enrichment and Flow Cytometry

Fluorescence-activated Cell Sorting and Adoptive Transfers
Statistical Analyses

4.3 Results.....................................................................................................91
Detection of Polyclonal GP66:I-Ab-specific CD4+ T Cells

Characterization of CD4+ T Cell Response to LCMV Armstrong
Infection

Secondary Responses by GP66:I-Ab-specific Effector Memory and
Central Memory T Cells

GC-Tfh Cells that Persist into the Memory Phase Lose PD-1 Expression

Secondary Response Potential of Memory Cells Derived from Tfh Cells
or GC-Tfh Cells

4.4 Discussion.............................................................................................104
Chapter 5: Conclusions
5.1 Summary...............................................................................................106
5.2 Therapeutic Implications......................................................................110
References........................................................................................................................112

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