T cell immunity in liver disease Open Access

Abstract

The liver critically participates in a multitude of metabolic functions required for human life. Due to shared blood flow with the gastrointestinal (GI) tract, the liver is perpetually bombarded with antigen rich intestinal blood, oxidative stress, and metabolites, yet maintains order amidst this antigenic chaos. Fibrosis, reversible liver scarring, is one of several mechanisms contributing to preservation of the liver microenvironment. Fibrosis is thought to shunt insults from healthy tissue such that the liver may function unscathed. Once the insult is resolved, fibrosis reverses as healthy tissue replaces scar tissue.

Central to the process fibrosis are activated hepatic stellate cells (HSCs), which upon activation initiate the liver wound healing process. Activated HSC-derived all-trans retinoic acid (ATRA) induces Foxp3 expression on CD4+ T cells. These "regulatory" T cells (Tregs) protect the liver from immune mediated injury by CD8+ T cells and other effectors. While beneficial for preservation of the liver microenvironment, these Tregs are implicated in suppressing antiviral responses and permitting chronic hepatotropic infections such as hepatitis C (HCV). Despite dampened CD8+ T cell immunity, chronic liver disease can manifest systemically as antibody mediated disorders due to aberrant B cell functions. These opposing yet co-existent features suggest that fibrosis-driven alterations of intrahepatic CD4+ T cell-help yield extrahepatic consequences. Surprisingly, we found that a subpopulation of fibrosis-elicited Tregs expressing CD40L and therefore represents one mechanism driving the extrahepatic sequelae of chronic liver disease.

Given the peripheral consequences of liver-specific events, it is entirely plausible that peripheral events can influence the liver. Persistent liver injury results in destruction of the liver architecture; this is associated with digestive perturbations such as defective shuttling of bile. Blockages in bile flow, cholestasis, has been linked to intestinal abnormalities, translocation of microbes to the periphery and imbalances in commensal flora. In the clinic, transient improvement in liver functional tests have been reported following antibiotic treatment of patients suffering from choleostatic liver disorders. Although these strategies are not curative, the outcomes suggest that events in GI tract have the potential to augment intrahepatic processes. Here, we found that microbial translocation from the GI tract to the liver augments disease pathology through interaction with γδ-T cells, a non-conventional lymphocyte population selectively enriched and expanded in the liver.

The studies carried out in this dissertation propose that the events in the liver have extrahepatic consequences. Additionally, this work begins to address the effects of extrahepatic events on intrahepatic processes. Overall, deviations associated with chronic liver disease emphasize the liver's remarkable capacity to maintain a balance between tolerance and inflammatory responses while simultaneously inundated with panoply of antigenic stimuli.

Table of Contents

Table of Contents

Chapter 1: Consequences of intrahepatic environmental cues on T cell functions: a comprehensive review of the literature 1

Introduction: Meet the liver 2

I. Liver-specific signals differentially instruct CD4+ T cell responses 7

CD4+ T cell priming by liver-resident APCs 8

II. Hepatic CD8+ T cell responses 14

Intrahepatic CD8+ T cell priming 14

Memory responses, activated T cell sink, and immune-mediated injury 16

III. Non-conventional T cell populations 20

gd-T cell subsets 20

NK and NKT subsets 22

IV. Liver transplantation highlights intrinsic management of T cell activation 24

V. Concluding remarks and therapeutic implications 26

Chapter 2: CD4+ Foxp3+ T cells promote aberrant IgG production and maintain CD8+ T cell suppression during chronic liver disease 31

I. Abstract 32

II. Introduction 33

III. Materials and Methods 35

IV. Results 42

Aberrant IgG production during hepatic fibrosis requires CD4+ T cells 42

Fibrosis-elicited CD4+Foxp3+ T cells do not suppress B cell activity 45

Fibrosis-elicited CD4+Foxp3+ T cells suppress CD8+ T cell responses 48

Fibrotic liver CD4+ Foxp3+ T cells up-regulate CD40L, CD69 and PD-1 Expression 50

CD40L expression is localized to CD4+Foxp3+CXCR5-ICOS- T cells 52

In vivo CD40L blockade during liver injury attenuates IgG aberrations 54

Accumulation of CD4+Foxp3+CD40L+ T cells in explanted HCV patients livers 56

Aberrant IgG production correlated with frequency of CD4+Foxp3+ T cells in explanted HCV patient livers 58

V. Discussion 60

VI. Supplemental Data 64

Chapter 3: Gut dysbiosis propels pathogenic IL-17A+ Vγ6Jγ1-γδ T cells during chronic liver disease 73

I. Abstract 74

II. Introduction 75

III. Materials and Methods 78

IV. Results 83

Intrahepatic gd T Cells are a predominant source of IL-17a during chronic choleostatic hepatic fibrogenesis 83

Liver-specific alterations to the gd T Cell compartment during choleostatic liver disease 86

Fibrosis drives expansion of Vγ6Jγ1-γδ T cells 88

Increased gut permeability and dysbiosis favor microbial translocation and enrichment in fibrotic liver 91

Intrahepatic γδ T cells produce IL-17 in response to translocated gut microbiota 95

Administration of Mdr2-/- liver-derived Lactobacillus induces IL-17 responses in WT mice 97

Antibody-mediated blockade of the γδ TCR attenuates fibrosis 99

V. Discussion 101

VI. Supplemental Data 105

Chapter 4: Discussion 114

I. When a "Treg" is not really a "Treg": intrahepatic environmental cues can tip the balance between tolerance and immune responses 115

II. Gut-Liver Axis is disrupted during liver diseases: implications on pathologic outcomes 118

III. The liver does not exist in a vacuum 121

Appendices 124

Appendix 1: References 124

Appendix 2: Reprint licensure information for "CD4+ Foxp3+ T cells promote aberrant IgG production and maintain CD8+ T cell suppression during chronic liver disease" courtesy of John Wiley and Sons and Copyright Clearance Center 143

Appendix 3: Unpublished supplemental data figures 144

List of Tables and Figures

Chapter 2: CD4+ Foxp3+ T cells promote aberrant IgG production and maintain CD8+ T cell suppression during chronic liver disease 31

Figure 2-1: Aberrant IgG-production during hepatic fibrosis requires CD4+ T cells 44

Figure 2-2: Fibrosis-elicited CD4+Foxp3+T cells do not suppress B cell activity 47
Figure 2-3: Fibrotic liver CD4+Foxp3+ T cells suppress CD8+ T cell response 49
Figure 2-4: Fibrotic liver CD4+Foxp3+ T cells up-regulate CD69, PD-1 and CD40L 53
Figure 2-5: CD40L is primarily expressed on CXCR5 and ICOS double negative CD4+Foxp3+ T cells 53
Figure 2-6: In vivo blockade of CD40L attenuates Ig-mediated manifestations of liver fibrosis 55
Figure 2-7: Phenotypic analysis of CD4+Foxp3+ T cells in explanted liver from HCV patients 57
Figure 2-8: Aberrant IgG production correlates with frequency of CD4+Foxp3+ T cells in explanted HCV patient livers 59

Chapter 2: Supplemental Data 64

Figure S2-1: Antibody mediated depletion of CD4+ T cells in C57BL6/J treated with CCl4 64

Figure S2-2: CCl4 treatment primarily alters the hepatic B cell compartment 65
Figure S2-3: Increased B cell activation and survival during liver fibrosis 66
Figure S2-4: CD25 negative CD4+ T cell interaction with B cells is not influenced by fibrosis 67
Figure S2-5: Phenotypic analysis of splenic CD4+Foxp3+ T cells 68

Figure S2-6: Intrahepatic accumulation of CD4+Foxp3+ T cells and incidence of spontaneous IgG production are independent of HCV viral load 70
Table S2-1: Fibrosis Lesion Scoring analysis Rubric and Findings 71
Table S2-2: Clinical Characteristics of HCV patients in this study 72

Table S2-3: Clinical characteristics of non-fibrotic control subjects in this study 72

Chapter 3: Gut dysbiosis propels pathogenic IL-17A+ Vγ6Jγ1-γδ T cells during chronic liver disease 73

Figure 3-1: Intrahepatic γδ -T cells are a predominant source of IL-17A during chronic liver disease 85

Figure 3-2: Fibrotic liver γδ-T cell compartment is altered in favor of IL-17 producing subsets 87
Figure 3-3: Fibrosis drives expansion of IL-17A+ invariant Vγ6Jγ1 γδ -T cells 90
Figure 3-4: Increased gut permeability and dysbiosis favor microbial translocation and enrichment of lactobacillus in fibrotic liver 93
Figure 3-5: Intrahepatic γδ -T cells produce IL-17 in response to translocated gut microbiota 96
Figure 3-6: Administration of Lactobacillus can induce hepatic inflammation and increase serum IL-17 in wild-type FVB/N mice98
Figure 3-7: Antibody-mediated blockade of the γδ TCR attenuates hepatic fibrosis 100

Chapter 3: Supplemental Data 105

Figure S3-1: Neutralization of IL-17A attenuates hepatic inflammation in Mdr2-/- mice 105

Figure S3-2: In vivo administration of anti- γδ TCR (Clone UC7-13D5) preferentially targets IL-17 producing subsets 106
Figure S3-3: Accumulation of intrahepatic in vivo bound anti-TCR + corresponds with increased serum IL-17A levels in Mdr2^-/- mice 107
Figure S3-4: Gut microbial composition is altered in 8-wk-old Mdr2-/- mice 108
Figure S3-5: Identification of culture isolates 109

Figure S3-6: Increased IL-17A in FVB/N mice inoculated with Lactobacillus 110
Figure S3-7: Blockade of TCR results in reduced intrahepatic expansion of IL-17+ population and corresponds to reduced neutrophilic infiltration 111
Figure S3-8: Fibrosis scores in control Mdr2-/- vs. anti-IL-17A treated and anti-TCR treated mice 112

Table S3-1: Fibrosis scoring criteria and representative findings 113

Chapter 4: Discussion 114

Figure 4-1: The fibrotic liver microenvironment promotes aberrant CD4+ T cell functions 117

Appendix 3: Unpublished supplemental data figures 144

Figure A3-1: ANA-assay of naive splenic B cell supernatants co-cultured with the indicated total CD4+ T cell source. 144

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, General Health Sciences, Immunology
Keyword	Tregs Immunology Liver disease Gamma delta T cells T cells
Committee Chair / Thesis Advisor	Grakoui, Arash, Emory University
Committee Members	Speck, Sam, Emory University Ford, Mandy L, Emory University Evavold, Brian, Emory University Suthar, Mehul S, Emory University

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