Investigating the role of CD4+ and CD8+ T cells in SIV persistence and virus production during antiretroviral therapy Pubblico

Abstract

Human immunodeficiency virus 1 (HIV-1) infection is a global pandemic affecting over 30 million people worldwide. Treatment with antiretroviral therapy (ART) can control virus replication and prolong the life of infected individuals. However, current regimens cannot eradicate the virus and any treatment interruption leads to viral rebound due to a small pool of latently infected cells invisible to both ART and the immune system. This "HIV reservoir" is the major barrier to HIV eradication efforts. In order to better understand the mechanisms and precise cellular subsets involved in HIV reservoir maintenance, we utilized the well-characterized animal model of HIV infection, simian immunodeficiency virus (SIV) infection in non-human primates (NHP). First, we examined the dynamics of a subset of CD4+ memory T-cells with stem cell-like properties, the CD4+ T stem cell memory (CD4+ TSCM), during experimental pathogenic infection of rhesus macaques (RM). We found these long-lived, multipotent memory T-cells were significantly disrupted in pathogenic SIV infection. We found that ART initiation restored some balance to the CD4+ TSCM compartment. Considering their enhanced longevity and susceptibility to infection, CD4+ TSCM are of intense interest as a site of SIV persistence during ART. We found no reduction in the fraction of infected CD4+ TSCM after initiation of ART, confirming a potentially critical role for CD4+ TSCM during SIV persistence. Finally, to further assess the mechanisms of SIV persistence during ART, we performed a CD8+ T-cell depletion study in SIV-infected ART-treated RM. We found that depletion of CD8+ T-cells during continuous ART resulted in a measurable increase in plasma viremia and reconstitution of CD8+ T-cells was associated with re-establishment of viral control. These data suggest a previously underappreciated role for CD8+ T-cells cooperating with ART to suppress viremia in SIV-infected RM. Overall this dissertation provides novel insights into cellular subsets involved in SIV pathogenesis and persistence and demonstrates for the first time a role for CD8+ T- cells in maintaining virus suppression during ART. These results support the design of novel therapeutic strategies that specifically target long-lived CD4+ memory T-cells and enhance the CD8+ T-cell response to HIV/SIV during ART.

Table of Contents

Chapter 1: Introduction

Discovery and origin of HIV. 1

Virology and HIV Life Cycle. 2

Target cells and viral tropism. 4

Clinical Disease Course. 5

Non-human primate models. 7

Immune Response to HIV. 9-20

Innate. 9

Humoral. 11

CD4+ T cells. 13

CD8+ T cells. 15

Immune Dysfunction and Exhaustion. 20

Antiretrovirals: How they changed the course of HIV. 21

Challenges of ART. 24

HIV Persistence. 25-32

Virus replication versus virus production. 25

Latency. 27

The problem of T cell memory. 30

T cell memory Maintenance. 33

Memory T cell subsets and HIV/SIV persistence. 34

Summary. 36

Chapter 2: Divergent CD4+ T memory stem cell dynamics in pathogenic and non-pathogenic SIV infection. 37

Figure 2.1. Identification of CD4+ TSCM- in healthy RM and SM. 53

Figure 2.2. Selective preservation of CD4+ TSCM cells during pathogenic SIV infection of RM. 54

Figure 2.3. Pathogenic SIV infection of RM is associated with significant depletion of CCR5+CD4+ TSCM. 55

Figure 2.4. Pathogenic SIV infection of RM is associated with increased proliferation of CD4+ TSCM that inversely correlates with the level of CD4+ TCM. 56

Figure 2.5. TSCM are unperturbed during non-pathogenic infection of SM. 57

Figure 2.6. Robust levels of CD4+ TSCM infection in vivo are observed in SIV-infected RM but not in SIV-infected SM. 58

Figure 2.S1. Expression of CXCR3, LFA-1, and Bcl-2 in SIV-uninfected RM and SM. 59

Chapter 3: Initiation of antiretroviral therapy restores CD4+TSCM homeostasis in SIV-infected RM. 60

Figure 3.1. ART effectively reduces viremia and restores CD4+ T cells in PBMC. 79

Figure 3.2. ART partially restores CCR5+CD4+ TSCM in PBMC and lymph node. 80

Figure 3.3. ART reduces the fraction of proliferating CD4+ T cells in PBMC but not in lymph nodes. 81

Figure 3.4. Inverse relationship between proliferating TSCM and frequency of TCM is maintained during SIV-infection and ART treatment. 82

Figure 3.5. Fraction of SIV-infected CD4+TSCM and TCM is stable after ART initiation. 83

Chapter 4: CD8+ lymphocytes are required to maintain virus suppression in SIV-infected macaques treated with ART. 84

Figure 4.1. Treatment of SIV-infected ART-treated RM with the anti-CD8+ antibody MT-807R1 results in CD8+ lymphocyte depletion from peripheral blood, lymph node and rectal mucosa and a variable increase in CD4+ T-cell proliferation. 105

Figure 4.2. CD8+ lymphocyte depletion results in measureable increase in plasma viremia in 13/13 ART-treated SIV-infected macaques. 106

Figure 4.3. CD8+ depletion results in increased SIV-RNA in lymph nodes of ART-treated SIV-infected macaques. 107

Figure 4.4. Ultrasensitive viral load assay confirms dramatic increase in viremia post-CD8+ lymphocyte depletion. 108

Figure 4.5. Pre-depletion levels of SIV infection in CD4+ T-cells predicts post-depletion changes in viral load in ART-treated SIV-infected macaques. 109

Figure 4.6. Virus emerging during CD8+ lymphocyte depletion closely resembles virus during the peak of acute infection. 110

Table 4.1. Identity of mutations in SIVmac239 envelope identified by single genome analysis. 111

Figure 4. S1. MT-807R1 administration also depletes CD8α+ NK cells. 112

Figure 4.S2. Number of pre-depletion SIV-specific CD8+ T cells predicts post depletion viral loads. 113

Figure 4.S3. NK cell reconstitution does not coincide with viral load decline in ART-treated SIV-infected RMs. 114

Figure 4.S4. No significant changes in total cell-associated SIV-DNA in sorted memory CD4+ T cell subsets of SIV-infected ART-treated RM before and after CD8+ depletion. 115

Table 4.S1. Antiretroviral drug regimen and length of treatment for SIV-infected ART-treated RM. 116

Table 4.S2. Magnitude of virus suppression during ART, prior to CD8+ lymphocyte depletion is >99.9% in all animals where at least 1 undetectable time point was achieved. 117

Table 4.S3. Length of the periods 1-3 used for statistical analysis as described in Figure 2 for each SIV-infected ART-treated RM. 118

Table 4.S4. No correlation between the frequency of proliferating (Ki67+) CD4+ T-cells and plasma viral load at weeks 1 and 3 post-CD8+ depletion. 119

Chapter 5: Discussion

HIV Cure: Where have we been?. 121

Role of CD4+ memory T cell subsets in HIV/SIV pathogenesis. 121

Figure 5.1. Model for differentiation and self-renewal capacity of memory T cells. 123

Why ART is not enough. 124-127

Immune activation. 124

Viral Reservoir. 125

Moving Forward: Functional Cure. 127

Early Treatment. 127

Role of CD8+ T cells. 129

Kick and Kill. 132

Checkpoint Blockade. 134

Summary. 137

References. 139

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Health Sciences, Immunology
Parola chiave	SIV antiretroviral therapy CD8+ T cell depletion T cells
Committee Chair / Thesis Advisor	Silvestri, Guido, Emory University
Committee Members	Chahroudi, Ann, Emory University Altman, John D, Emory University Kohlmeier, Jacob, Emory University Amara, Rama Rao, Emory University

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