Regulation Of Programmed Cell Death-1 In CD8 T Cells Open Access

Austin, James Wesley (2014)

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Programmed Death-1 (PD-1) is a transmembrane signaling molecule that negatively regulates effector functions of lymphocytes. Expression and subsequent signaling through PD-1 has been shown to mediate T cell exhaustion, a process that results in the loss of T cell effector function. This dissertation explores the mechanism by which PD-1 is regulated. Using an unbiased, PCR based approach, multiple DNase I hypersensitive sites (DHS) were discovered across the PD-1 locus. DHS, which are indicative of potential regulatory regions, were tested for regulatory activity with two regions showing enhancer function. IL-6 and IL-12, cytokines that activate STAT3 and STAT4 respectively, prolonged and increased PD-1 expression of activated T cells, but cytokine treatment alone was not sufficient to activate PD-1 expression. The transcription factors NFATc1 (T-cell receptor), STAT3 (IL-6), and STAT4 (IL-12) were shown to bind to the enhancer regions in response to their stimuli and were associated with a change in histone modifications at the regions. Both of the regulatory regions interact with the PD-1 promoter, providing a mechanism for their action. NFATc1 and STAT binding is not dependent on each other, although the regulatory function of the region is dependent on both factors being bound. The CCCTC-binding factor (CTCF) bound two locations in the PD-1 locus, flanking the regulatory regions and formed a constitutive regulatory loop, isolating the PD-1 locus from the surrounding chromatin. DNA methylation of the known regulatory regions of PD-1 showed a dynamic pattern that inversely correlated with PD-1 expression during acute infection. In chronic infections with low antigen levels, PD-1 expression is decreased, but without an accompanying increase in DNA methylation. Thus, the inverse correlation of PD-1 expression and DNA methylation was lost in chronic infections. Through this work multiple regulatory mechanisms of PD-1 have been established and provide additional targets for molecular therapies designed to reinvigorate exhausted T cells.

Table of Contents

Chapter 1. Introduction 1

PD-1 Function and its Role in The Immune System 1

Cellular Expression of PD-1 and its Ligands 3

PD-1 Structure and Signaling 5

T Cell Development 7

T Cell Differentiation 10

PD-1 in Chronic Infection 13

PD-1 in Autoimmunity 15

PD-1 in Cancer 17

Modulation of PD-1 and its Ligands by Cytokines 18

Transcription Factors of PD-1 and its Ligands 25

Figure Legends 32

Figures 34

Chapter 2. STAT3, STAT4, NFATc1, and CTCF regulate Pdcd1 through multiple novel regulatory regions in murine T cells 37

Abstract 38

Introduction 38

Materials and Methods 41

Results 45

Discussion 56

Figure Legends 61

Figures 68

Tables 78

Chapter 3. Dynamic DNA Methylation of PD-1 Regulatory Regions 83

Abstract 84

Introduction 84Materials and Methods 88

Results 91

Discussion 97

Figure Legends 101

Figures 104

Tables 110

Chapter 4. Multiple STATs Prolong PD-1 Expression in CD8 T Cells 111

Abstract 112

Introduction 112

Materials and Methods 115

Results 117

Discussion 119

Figure Legends 122

Figures 124

Tables 127

Chapter 5. Discussion 128

Multiple Cis Elements Regulate PD-1 Expression 128

Multiple Factors Effect the DNA Methylation of PD-1 130

Prolonged PD-1 Expression in Response to Cytokine Signaling 133

Additional Potential Regulatory Factors of PD-1 136PD-1 Regulation In Exhausted T Cells 138

Figure Legends 141

Figures 143

References Cited 146

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