Impact of HIV infection and antiretroviral therapy on Mycobacterium tuberculosis-specific CD4 T cell responses Public

Abstract

HIV is the strongest known risk factor for tuberculosis (TB), largely due to its profound disruption of Mycobacterium tuberculosis (Mtb)-specific CD4 T cell immunity. This dissertation investigates how HIV infection and the timing of antiretroviral therapy (ART) affect the function and transcriptional profile of Mtb-specific CD4 T cells and examines the broader epigenetic and transcriptional landscape of total peripheral CD4 T cells in individuals with and without HIV.

In a unique longitudinal analysis, we profiled Mtb-specific CD4 T cells before and after HIV acquisition using flow cytometry and single-cell RNA sequencing. We observed early and preferential depletion of Mtb-specific effector subsets, including Th1 and Th17 cells, alongside reduced cytokine production and increased representation of TCF7⁺ stem-like cells. Transcriptional dysregulation following HIV infection was marked by enrichment of WNT and hypoxia signaling pathways and downregulation of antigen processing, cytokine signaling, and migration programs. These findings suggest that HIV rapidly impairs the differentiation and function of Mtb-specific CD4 T cells.

In a separate cross-sectional analysis, we performed RNA-seq, ATAC-seq, and reduced representation bisulfite sequencing (RRBS) on total CD4 T cells from people without HIV and people with HIV prior to ART initiation. While transcriptional and chromatin accessibility changes were relatively limited in resting cells, we identified moderate changes in DNA methylation, suggesting early epigenetic reprogramming that may contribute to latent immune dysfunction.

Finally, to evaluate how ART timing shapes Mtb-specific CD4 T cell responses, we functionally profiled these cells in people without HIV and in individuals who started ART early or late after HIV acquisition. HIV infection and delayed ART initiation were associated with diminished frequencies of functional and polyfunctional Mtb-specific CD4 T cells, while early ART preserved effector functionality and subset diversity. Single-cell transcriptomics revealed that delayed ART initiation was linked to transcriptional silencing and skewing toward quiescent states, whereas early ART was associated with greater transcriptional activity and preservation of effector and regulatory subsets.

Together, these findings provide new insights into how HIV alters both the antigen-specific and global CD4 T cell landscape and underscore the critical role of early ART in preserving Mtb-specific immunity. This work offers molecular insights for the heightened risk of TB in people with HIV and informs future strategies for immune restoration in co-infected populations.

Table of Contents

Table of Contents

CHAPTER 1. INTRODUCTION

1.1 Overview

1.2 Epidemiology of Tuberculosis

1.3 Mtb Pathogenesis

1.4 Clinical Spectrum of Mtb Infection

1.5 Diagnosis of Mtb

1.6 Risk Factors for Mtb Infection and Progression

1.7 Mtb Treatment

1.8 Epidemiology of Mtb and HIV Co-infection

1.9 Effect of HIV on TB Progression

1.10 Immune Responses to Mtb

1.11 Mtb-Specific CD4⁺ T Cell Responses in HIV

1.12 Effect of HIV Treatment on Mtb-specific Immune Responses

1.13 Summary

CHAPTER 2: SINGLE-CELL TRANSCRIPTOMICS REVEALS DEPLETION AND DYSREGULATION OF MYCOBACTERIUM TUBERCULOSIS-SPECIFIC TH1 AND TH17 CELLS EARLY AFTER ACQUISITION OF HIV

2.1 Abstract

2.2 Introduction

2.3 Materials and Methods

2.3.1 Study population and sample collection

2.3.2 Ethics Statement

2.3.3 T cell intracellular cytokine staining (ICS) assay and flow cytometry

2.3.4 MIMOSA and COMPASS analysis of flow cytometry data

2.3.5 Activation-induced marker assay and sorting Mtb-specific CD4 T cells

2.3.7 ScRNA-seq data processing and analysis

2.3.8 Statistical Analysis

2.4 Results

2.4.1 Study Participants

2.4.2 Mtb-specific CD4 T cells in PWH demonstrate impaired functionality and polyfunctionality

2.4.3 Single-cell transcriptomics identifies distinct subsets of Mtb-specific CD4 T cells

2.4.4 Mtb-specific Th1 and Th17 cell numbers are diminished following HIV infection

2.4.5 Average expression of effector genes declines after HIV infection

2.4.6 Important pathways are downregulated in Mtb-specific CD4 T cells after HIV infection

2.5 Discussion

2.6 Main Figures

Table 2.1

Figure 2.1

Figure 2.2

Figure 2.3

Figure 2.4

Figure 2.5

2.7 Supplemental Figures

Supplemental Figure 2.1

Supplemental Figure 2.2

Supplemental Figure 2.3

CHAPTER 3. EARLY ART INITIATION FOLLOWING HIV INFECTION PRESERVES THE DIVERSITY AND FUNCTION OF MTB-SPECIFIC CD4 T CELLS

3.1 Abstract

3.2 Introduction

3.3 Methods

3.3.1 Study population and sample collection

3.3.2 Ethics Statement

3.3.3 Total CD4 T cell Sorting

3.3.4 RNA Sequencing and Analysis

3.3.5 ATAC-seq and Analysis

3.3.6 Reduced-Representation Bisulfite Sequencing and Analysis

3.3.7 T cell ICS assay and flow cytometry

3.3.8 COMPASS analysis of flow cytometry data

3.3.9 Activation-induced marker assay and sorting Mtb-specific CD4 T cells

3.3.10 10x Genomics scRNA-seq library preparation

3.3.11 ScRNA-seq data processing and analysis

3.3.12 Statistical Analysis

3.4 Results

3.4.1 Study Participants

3.4.2 HIV infection induces moderate DNA methylation changes with minimal transcriptional or chromatin accessibility alterations in resting CD4 T cells

3.4.3 HIV infection impairs polyfunctional Mtb-specific CD4 T cell responses, with greater dysfunction when ART is initiated more than 1 year after HIV infection

3.4.4 Single cell transcriptional profiling indicates distinct clusters of Mtb-specific CD4 T cells

3.4.5 HIV infection reshapes the transcriptional landscape and subset composition of Mtb-specific CD4 T cells, with early ART preserving effector diversity

3.4.6 Early ART initiation preserves effector and regulatory gene expression in Mtb-specific CD4 T cells

3.4.7 Reduced intensity of effector gene expression within conserved Mtb-specific CD4 T cell clusters

3.5 Discussion

3.6 Main Figures

Table 3.1

Figure 3.1

Figure 3.2 

Table 3.2

Figure 3.3

Figure 3.4

Figure 3.5

Figure 3.6

3.6 Supplemental Figures

Supplemental Figure 3.1

Supplemental Figure 3.2

Supplemental Figure 3.3

CHAPTER 4. DISCUSSION

4.1 Summary

4.2 Implications

4.3 Future Directions

CHAPTER 5. REFERENCES

About this Dissertation

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• Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.

School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Immunology and Molecular Pathogenesis
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Microbiology Health Sciences, Immunology Biology, Virology
Mot-clé	TB-HIV confection Mtb-specific CD4 T cell
Committee Chair / Thesis Advisor	Cheryl Day, Emory University
Committee Members	Eliver Ghosn, Emory University Ann Chahroudi, Emory University Karen Conneely, Emory University Christopher Scharer, Emory University Jacob Kohlmeier, Emory University

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