Immunological determinants of immune restoration disease ... · Immunological determinants of immune restoration disease and immune recovery in HIV patients on antiretroviral therapy

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Immunological determinants of immune

restoration disease and immune recovery

in HIV patients on antiretroviral therapy

Dino Bee Aik Tan, B.Sc. (Hons)

This thesis is presented for the degree of

Doctor of Philosophy (Science) of

The University of Western Australia

School of Pathology and Laboratory Medicine

2011

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DECLARATION FOR THESES CONTAINING PUBLISHED WORK AND/OR WORK PREPARED FOR PUBLICATION

The examination of the thesis is an examination of the work of the student. The work must have been substantially conducted by the student during enrolment in the degree. Where the thesis includes work to which others have contributed, the thesis must include a statement that makes the student’s contribution clear to the examiners. This may be in the form of a description of the precise contribution of the student to the work presented for examination and/or a statement of the percentage of the work that was done by the student. In addition, in the case of co-authored publications included in the thesis, each author must give their signed permission for the work to be included. If signatures from all the authors cannot be obtained, the statement detailing the student’s contribution to the work must be signed by the coordinating supervisor. Please sign one of the statements below.

1. This thesis does not contain work that I have published, nor work under review for publication. Student Signature...........................................................................................................................

2. This thesis contains only sole-authored work, some of which has been published and/or prepared for publication under sole authorship. The bibliographical details of the work and where it appears in the thesis are outlined below. Student Signature ..........................................................................................................................

3. This thesis contains published work and/or work prepared for publication, some of which has been co-authored. The bibliographical details of the work and where it appears in the thesis are outlined below. The student must attach to this declaration a statement for each publication that clarifies the contribution of the student to the work. This may be in the form of a description of the precise contributions of the student to the published work and/or a statement of percent contribution by the student. This statement must be signed by all authors. If signatures from all the authors cannot be obtained, the statement detailing the student’s contribution to the published work must be signed by the coordinating supervisor. Student Signature…………………………………………………………………………...………. Coordinating Supervisor Signature.…………………………………………..………..…………

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Thesis Abstract

Antiretroviral therapy (ART) has improved the quality of life for human

immunodeficiency virus (HIV)-infected patients by suppressing viral replication,

allowing reconstitution of the immune system. However, patients initiating ART with

advanced disease may experience immune restoration disease (IRD) or poor recovery of

CD4+ T-cell numbers and/or functions. IRDs are attributed to immune responses that

are immunopathological rather than protective. This thesis addresses possible causes of

IRD and impaired CD4+ T-cell function in HIV patients who were very

immunodeficient (starting ART with <200 cell/µL) but responded virologically to ART.

IRD associated with tuberculosis (TB), cryptococcal meningitis, cytomegalovirus

(CMV) and Varicella Zoster virus (VZV) infections were investigated. Most studies

described here use patients who participated in a study of immune reconstitution after

starting ART at the University of Malaya, Kuala Lumpur, Malaysia.

In Chapter 3, I showed that patients who presented with IRD associated with TB or

cryptococcal meningitis displayed increased interferon-gamma (IFNγ) responses to

antigens from the provoking pathogens, high proportions of activated (HLA-DRhi

) T-

cells and high plasma levels of immunoglobulin (Ig)-G reactive to antigens. IRD was

found to not associate with deficiency of CD4+ T-cells with a regulatory phenotype

(CD25+CD127

lo or CTLA-4

+). These cells have the capacity to suppress immune

responses and were observed to parallel with immune activation regardless of IRD.

In Chapter 4, I found no role for natural killer (NK) cells in the pathogenesis of IRD as

NK cell IFNγ responses remained low in all patients starting ART. NK cell responses

varied between patients with similar IRD, with no consistent peaks preceding diagnosis.

However, NK cell deficiency may promote IRD by impairing the control of the

provoking pathogen before ART as CD16 expression of CD56lo

NK cells was low at

baseline in most IRD patients.

Most IRD events in the cohort were associated with Mycobacterium tuberculosis

(MTB) infection, so the functions of antigen presenting cells and their Toll-like receptor

(TLR)-induced responses were evaluated in patients who experienced TB IRD (Chapter

5). Evaluations of TLR2 (receptor for mycobacterium-derived lipomannan; LM)

expression and LM-induced cytokine responses were included. Some patients who

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developed TB IRD showed increased LM-induced responses around the time of IRD

diagnosis. This involved high expression of TLR2 on both myeloid dendritic cell

(mDC) and monocytes followed by high tumour necrosis factor-alpha (TNFα) and

interleukin (IL)-12p40 production without parallel increases in IL-10 in response to LM.

Thus, inflammatory responses observed during IRD may be caused by hyper-responsive

antigen-presenting cell (APC) promoting over-production of inflammatory cytokine

driving downstream immune responses.

CD4+ T-cell recovery (independent of IRD) was also investigated in this thesis.

Malaysian patients were divided into two groups (Low vs. High) based on the

attainment of more than 200 CD4+ T-cells/µL after 6 months of ART. Purified protein

derivative (PPD) and cytomegalovirus (CMV)-specific IFNγ responses were similar in

the two groups. Higher numbers of CD4+ T-cells after ART was associated with higher

frequencies of IFNα-producing plasmacytoid DC (pDC) and IL-12-producing mDC in

response to TLR7/8 and TLR9 agonists during 3-6 months of ART (Chapter 6).

Recovery of CD4+ T-cells was associated with NK cell subsets and function in the

Malaysian sample set (Chapter 4) and a study of the effect of long-term ART on the

recovery of memory T-cell responses in Australian Caucasian patients (Chapter 7). The

Australian sample set comprised CMV-seropositive patients who had stable virological

responses on 2-5 years of ART and were divided into low and high IFNγ responders

based on CD4+ T-cell responses to CMV. NK IFNγ responses in all patients were lower

than healthy controls in both sample sets and were not associated with CD4+ T-cell

counts. No association was observed between the IFNγ responses of T-cells and NK

cells. However, increased proportions and cytotoxic capacity of CD56hi

NK cells were

observed in the Caucasian patients with low CD4+ T-cell IFNγ responses to CMV

suggesting a compensatory contribution of this NK cell subset.

This work adds to immunological data on the characteristics and causes of IRD as well

as poor CD4+ T-cell recovery on ART. With confirmation in a larger cohort, better

monitoring and treatment strategies for HIV patients starting ART can be developed.

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Table of Contents

THESIS ABSTRACT................................................................................................................................. III

TABLE OF CONTENTS .............................................................................................................................V

ACKNOWLEDGEMENTS .........................................................................................................................X

STATEMENT OF CANDIDATE CONTRIBUTION.......................................................................... XIII

ABBREVIATIONS.................................................................................................................................. XIX

LIST OF FIGURES................................................................................................................................XXII

LIST OF TABLES................................................................................................................................ XXIV

CHAPTER 1 - REVIEW OF THE LITERATURE..................................................................................2

1.1 WHAT IS HIV?.....................................................................................................................................2

1.1.1 Epidemiology............................................................................................................................2

1.1.2 HIV viral structure and life cycle .............................................................................................3

1.1.3 The natural history of HIV infection.........................................................................................4

1.2 THE ADAPTIVE AND INNATE IMMUNE SYSTEMS....................................................................................6

1.2.1 CD4+ T-cells: Regulators of the adaptive immune system .......................................................6

1.2.2 Regulatory T-cells: Suppressors of immune responses ............................................................7

1.2.3 Natural killer cells: Killers of “non-self” cells ........................................................................8

1.2.4 DC and macrophages: Professional antigen presenting cells................................................10

1.2.5 B-cells: Cells of the humoral arm of the immune system........................................................10

1.3 PATHOGENESIS OF HIV INFECTION ....................................................................................................11

1.3.1 Mechanisms of CD4+ T-cell depletion....................................................................................11

1.3.1.1 Virus-mediated killing of infected and uninfected CD4+ T-cells ............................................... 11

1.3.1.2 The role of lymphoid tissues in CD4+ T-cell depletion .............................................................. 12

1.3.1.3 Impaired production of CD4+ T-cells ......................................................................................... 13

1.3.2 Current models of HIV-associated immune activation...........................................................14

1.3.2.1 Microbial translocation .............................................................................................................. 16

1.3.2.2 Elevated Type I IFN response .................................................................................................... 17

1.3.3 HIV viremia increases frequency of T-reg..............................................................................17

1.3.4 HIV immune evasion strategies and NK cell dysfunction.......................................................18

1.3.5 DC and macrophages dysfunction during HIV infection........................................................20

1.4 ANTIRETROVIRAL THERAPY...............................................................................................................21

1.4.1 Basic description and the mode of action...............................................................................21

1.4.2 Outcomes of ART....................................................................................................................22

1.4.2.1 Recovery of CD4+ T-cells on ART are variable among HIV patients........................................ 22

1.4.2.2 Reconstitution of CD4+ T-cell numbers may not parallel restored function............................... 23

1.4.2.3 Immune activation is reduced by ART....................................................................................... 23

1.4.2.4 Outcomes of ART on T-reg, DC and NK cells .......................................................................... 23

1.4.2.5 Adverse clinical outcomes ......................................................................................................... 24

1.5 IMMUNE RESTORATION DISEASE ........................................................................................................26

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1.5.1 Clinical diagnosis...................................................................................................................26

1.5.2 Risk factors for the development of IRD.................................................................................27

1.5.3 Opportunistic infections associated with IRD ........................................................................27

1.5.3.1 Mycobacterium tuberculosis ...................................................................................................... 28

1.5.3.2 Cryptococcus neoformans meningitis ........................................................................................ 34

1.5.3.3 Cytomegalovirus causing retinitis .............................................................................................. 37

1.6 AIMS OF THE THESIS ..........................................................................................................................38

1.6.1 Part 1: Cellular mediators of immune restoration disease ....................................................38

1.6.2 Part 2: Cellular mediators of immune recovery on ART........................................................38

CHAPTER 2 - METHODS AND MATERIALS.....................................................................................40

2.1 IMMUNE RECONSTITUTION STUDY (UMMC, KL, MALAYSIA) ..........................................................40

2.2 SAMPLE COLLECTION, PLASMA HIV RNA LEVEL AND CD4+ T-CELL COUNTS...................................41

2.3 IFNΓ ELISPOT ...................................................................................................................................41

2.4 ELISA ...............................................................................................................................................42

2.5 FLOW CYTOMETRY ............................................................................................................................43

2.6 ANTIGENS/STIMULANTS ....................................................................................................................44

2.6.1 Anti-CD3 ................................................................................................................................44

2.6.2 CMV lysate .............................................................................................................................44

2.6.3 Cryptococcal mannoprotein ...................................................................................................44

2.6.4 K562 cell line..........................................................................................................................45

2.6.5 Lipomannan............................................................................................................................45

2.6.6 Lipopolysaccharide ................................................................................................................45

2.6.7 PHA ........................................................................................................................................45

2.6.8 Tuberculin PPD and ESAT-6 .................................................................................................46

2.6.9 VZV lysate...............................................................................................................................46

CHAPTER 3 - IMMUNOLOGICAL PROFILES OF IMMUNE RESTORATION

DISEASE PRESENTING AS MYCOBACTERIAL LYMPHADENITIS AND

CRYPTOCOCCAL MENINGITIS ...........................................................................................................48

3.1 BACKGROUND ...................................................................................................................................48

3.2 PATIENTS AND METHODS ..................................................................................................................50

3.2.1 Study subjects .........................................................................................................................50

3.2.2 Sample collection and processing ..........................................................................................50

3.2.3 ELISpot assay to quantify the frequency of antigen-specific IFNγ-producing cells ...............50

3.2.4 ELISA to measure pathogen-reactive IgG in plasma .............................................................50

3.2.5 Flow cytometry assay to assess T-cell subsets........................................................................51

3.2.6 Statistical analyses .................................................................................................................51

3.3 RESULTS ............................................................................................................................................52

3.3.1 CD4+ T-cell counts and plasma HIV RNA levels confirm all patients responded to

ART.........................................................................................................................................52

3.3.2 Clinical presentations and microbiological assessment of IRD .............................................52

3.3.3 IFNγ responses to antigens of provoking pathogens peaked at the time of IRD.....................53

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3.3.4 Levels of plasma IgG reactive with Cryptococcus mannoprotein peaked during an

IRD .........................................................................................................................................54

3.3.5 Proportions of activated (HLA-DRhi

) and regulatory CD4+ T-cells were generally

high during an IRD.................................................................................................................55

3.4 DISCUSSION .......................................................................................................................................55

CHAPTER 4 – CHARACTERISTICS OF NK CELLS IN MALAYSIAN HIV

PATIENTS PRESENTING WITH IRD AFTER ART............................................................................65

4.1 BACKGROUND ...................................................................................................................................65

4.2 PATIENTS AND METHODS ...................................................................................................................66

4.2.1 Study subjects .........................................................................................................................66

4.2.2 Sample collection and routine data ........................................................................................67

4.2.3 IFNγ ELISpot assay ................................................................................................................67

4.2.4 Flow cytometry .......................................................................................................................67

4.2.5 Statistical analysis ..................................................................................................................67

4.3 RESULTS ............................................................................................................................................68

4.3.1 Clinical and immunological characteristics of the study population and response

to ART.....................................................................................................................................68

4.3.2 IRD usually parallel IFNγ responses to pathogen antigens ...................................................68

4.3.3 NK IFNγ responses remained impaired after Week 24 in all patients, irrespective

of IRD .....................................................................................................................................69

4.3.4 Proportions of CD56hi

NK cells increased on ART, with a peak in a few IRD

patients ...................................................................................................................................69

4.3.5 Expression of CD16 on CD56lo NK cells was low in most IRD patients at baseline..............70

4.3.6 Perforin levels in CD56hi

NK cells were high in all HIV patients ..........................................70

4.4 DISCUSSION .......................................................................................................................................70

CHAPTER 5 - LONGITUDINAL EVALUATION OF TLR-INDUCED CYTOKINE

RESPONSES BY CIRCULATING DC AND MONOCYTES IN HIV-INFECTED

PATIENTS ON ART AND THEIR ROLE IN THE DEVELOPMENT OF TB IRD ...........................79

5.1 BACKGROUND ...................................................................................................................................79

5.2 PATIENTS AND METHODS ...................................................................................................................80

5.2.1 Study subjects .........................................................................................................................80

5.2.2 Measurement of plasma HIV RNA levels and CD4+ T-cell counts.........................................81

5.2.3 Quantification of IFNγ responses to PPD by ELISpot ...........................................................81

5.2.4 Measurement of CD4+ T-cell activation and TLR2 expression ..............................................81

5.2.5 Quantification of TNFα, IL-12p40 and IL-10 production in response to

lipomannan.............................................................................................................................82

5.2.6 Culture and flow based assay to quantify PPD-specific IFNγ producing CD4+ and

CD8+ T-cells...........................................................................................................................82

5.2.7 Statistical analyses .................................................................................................................82

5.3 RESULTS ............................................................................................................................................83

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5.3.1 Laboratory and clinical features of IRD ................................................................................83

5.3.2 CD4+ T-cell activation and IFNγ responses to PPD parallel the IRD but are seen

in other patients......................................................................................................................83

5.3.3 TB IRD parallels peaks in TLR2 expression on mDC and monocytes in some

patients ...................................................................................................................................84

5.3.4 Elevated TNFα and IL-12p40 responses to lipomannan are restricted to IRD

patients ...................................................................................................................................84

5.3.5 Production of LM-induced cytokines were related to TLR2 expression and immune

activation ................................................................................................................................85

5.4 DISCUSSION .......................................................................................................................................85

CHAPTER 6 - ROBUST IFNΑ AND IL-12 RESPONSES BY DC PARALLEL

EFFICIENT CD4+ T-CELL RECOVERY IN HIV PATIENTS ON ART.............................................93

6.1 BACKGROUND ...................................................................................................................................93

6.2 METHODS ..........................................................................................................................................94

6.2.1 Study subjects .........................................................................................................................94

6.2.2 Sample collection, plasma HIV RNA levels and CD4+ T-cell counts .....................................95

6.2.3 Quantification of CD4+ T-cell activation, IFNα-producing pDC and IL-12-

producing mDC ......................................................................................................................95

6.2.4 Statistical Analyses.................................................................................................................96

6.3 RESULTS ............................................................................................................................................96

6.3.1 Recovery of CD4+ T-cells on ART was adversely affected by low baseline CD4

+ T-

cell counts and high T-cell activation.....................................................................................96

6.3.2 TLR7/8 and TLR9-induced cytokine production in pDC and mDC were correlated

with CD4+ T-cell recovery......................................................................................................96

6.4 DISCUSSION .......................................................................................................................................97

CHAPTER 7 - COULD NATURAL KILLER CELLS COMPENSATE FOR

IMPAIRED CD4+ T-CELL RESPONSES TO CMV IN HIV PATIENTS RESPONDING

TO ANTIRETROVIRAL THERAPY? ...................................................................................................103

7.1 BACKGROUND .................................................................................................................................103

7.2 METHODS ........................................................................................................................................104

7.2.1 Study subjects .......................................................................................................................104

7.2.2 Sample collection and routine data ......................................................................................105

7.2.3 IFNγ ELISpot........................................................................................................................105

7.2.4 Flow cytometry .....................................................................................................................105

7.2.5 RT-PCR ................................................................................................................................105

7.2.6 Statistical Analysis................................................................................................................106

7.3 RESULTS ..........................................................................................................................................106

7.3.1 Proportions of CD56lo

NK cells were lower in HIV Caucasian patients than

controls.................................................................................................................................106

7.3.2 Expression of perforin by CD56hi

NK cells was elevated in HIV patients compared

to healthy controls ................................................................................................................107

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7.3.3 NK cell IFNγ responses were lower in HIV patients than healthy controls .........................107

7.3.4 Levels of mRNA for IL-10 and IL-12 receptors in CD56+ cells do not explain poor

NK cell IFNγ responses in HIV patients ...............................................................................108

7.4 DISCUSSION .....................................................................................................................................108

CHAPTER 8 - CONCLUSIONS AND FUTURE STUDIES................................................................118

8.1 IMMUNE RESTORATION DISEASE: ELEVATION OF INNATE AND ADAPTIVE IMMUNE

RESPONSES.......................................................................................................................................118

8.2 RECOVERY OF CD4+ T-CELLS: ASSOCIATED WITH GOOD DC FUNCTION WITH A POTENTIAL

FOR FUNCTIONAL COMPENSATION BY NK CELLS .............................................................................121

8.3 CONCLUSIONS AND STUDY LIMITATIONS .........................................................................................122

CHAPTER 9 REFERENCES .................................................................................................................124

APPENDIX 1 - HCV IRD STUDY (INDONESIA) .........................................................................................146

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Acknowledgements

Firstly, I would like to give glory and honor to my Lord and Saviour, the Lord Jesus

Christ, whose love and mercy is unconditional and everlasting. Who was crucified,

buried and resurrected from the dead, so that I may have eternal life.

“For God so loved the world that He gave His only begotten Son,

that whoever believes in Him should not perish but have everlasting life”

John 3:16

“For what will it profit a man if he gains the whole world, and loses his own soul?”

Mark 8:36

To my parents: Thank you for your continuous love, support, care and prayers for me.

To my PhD supervisors, Professors Patricia Price and Martyn French (School of

Pathology and Laboratory Medicine, UWA and Department of Clinical Immunology,

RPH): Thank you for all the help, guidance, encouragement, time and effort throughout

my endeavor as a research student. Thank you Tricia for improving my ‘Malaysian’

English, unraveling cryptic sentences and many helpful discussions and Martyn for

critical comments and suggestions that made the presentation of this thesis much better.

I’m truly blessed to have supervisors like you.

To the post-docs: Special thanks to all of you for your advice, input, encouragement,

training and helping me to master the laboratory techniques used in this thesis. Thank

you for welcoming me to the group and I will always treasure the friendship that has

been built over all these years. Thank you Andrew Lim (my flow sifu) for being very

patient with me (and my noobiness) and helping with all my flow cytometry problems.

Thank you also for your constant reminder that there is life outside of the laboratory

such as owning AIs in DOTA, being owned at baddie and fishing for ‘unshamed’ fishes.

Thank you Sonia Fernandez (my ELISpot guru) for being a great teacher and explaining

many things in simple ESL english so that I may understand. Thank you Silvia Lee (my

general lab advisor) for always being available to answer my questions like how do we

heat inactivate FCS, autoclave tips and other lab stuff etc. Thank you Sonia and Silvia

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for lending me great series (Heroes, Dexter, Prison break etc) to help me unwind during

the busy days of labwork.

To the research assistants in MRF level 2 past and present (Steve Roberts, Michael

Scott, Afsana Amini and Ibrahim Fleyfel): Thank you for all your help behind the

scenes. Special mention to Steve Roberts for your excellent Ficoll training, teaching me

how to use the BD FACS Calibur flow machine and helping me pack lab goods for KL.

To Rom Krueger and Kathy Heel (Flow cytometry units, RPH and QEII): Thank you

for the introduction to flow cytometry and guidance on how to use the flow machine

(more importantly how not to wreck the machine).

Thank you to Professor Ian James (Centre for Clinical Immunology and Biomedical

Statistics) for your help with statistical analyses for Chapter 3.

Special thanks to my fellow students (PhD/Honours): Alvina Tiang, Ben Oliver, Caris

Allison, Cheryl Tan, Constance Chew, Edward Chin, Emma De Jong, Jacquita

Suemarni, Joo Tan, Rizal Sumatoh, Sara Tanaskovic, Talia Hammond, Laila Abudulai

and Zayd Aghafar for your encouragement, friendship and company during morning

coffee time, lunch time, afternoon tea time, bubble tea break time etc. To the current

students: I wish you all the best for your studies and may all your statistical analysis

come out significant. To Honours students in the past: Congratulations for graduating

from Honours. I wish you all the best in life wherever you are, even if you have given

up on science ☺

Thank you to the staff members on the MRF building: Liz Doyle and Jane Hall for

passing on my letters or parcels and letting me in whenever I forget my access card.

Thank you to Ray Smith and Gavin Criddle for all the IT support.

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I would also like to acknowledge all the people in University of Malaya Medical Centre,

Kuala Lumpur, Malaysia for their contribution towards this study:

Thank you Professor Adeeba Kamarulzaman, Dr Lian Huat Tan, Margaret Lau and

Clarence Sim for making the study possible. Thank you Dr Sasheela and Dr Sharifah as

well as the other medical officers whom I did not meet for your patient care and helpful

clinical review. Thank you to Professors Kee Peng Ng and Shamala Devi for making

your labs available for sample processing and storage.

To Associate Professor Veera Sekaran, Professor S. Jayaranee, Ms Saraswathi and all

the staff in the Clinical Diagnostic Laboratory and Transfusion Unit, UMMC: Thank

you for your generosity in allowing me to utilize the BD FACS Calibur and FACS

Canto II flow cytometers.

To brother Yean Kong Yong and sister Hong Yien Tan: I’m truly grateful for opening

your home to me and I will never be able to fully repay you for your kindness and

hospitality during my stay in KL. I will never forget the good times that we had during

“ninja night”, “DOTA clicking”, supper with “hollick shake” and “night time kai kai”.

Thank you for introducing me to many great restaurants in KL and feeding me with

your super delicious home cooked food. Not forgetting baby Simon for his company

watching Kung Fu Panda at night, every night!

My gratitudes to Chan Mun Yee, Dr. Kae Shin Sim, Lai Yee Ong, Siew Phooi Teh, Siti

Humaira and Yeat Mei Lee for your friendship and always being there to help when I

needed it. Special mention to Siew Phooi for letting me use her “Oogway che” (tortoise

car) to drive to work and shopping in KL!

Finally thank you all the patients and healthy controls for participating and donating

blood samples for my studies.

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Statement of Candidate Contribution

All work was performed by the author unless otherwise stated in the thesis

……………………………

Dino Tan

(PhD candidate)

…………………………….

Patricia Price

(Co-ordinating supervisor)

……………………………

Martyn French

(Co-supervisor)

xiv

The data presented in Chapter 3 has been published. The bibliographic details and the

contribution of the student to the work are set out below.

Tan DBA, Yong YK, Tan HY, Kamarulzaman A, Tan LH, Lim A, James I, French M,

Price P. Immunological profiles of immune restoration disease presenting as

mycobacterial lymphadenitis and cryptococcal meningitis. HIV Med 2008; 9:307-

316.

Student’s contribution to the work:

Dino Tan undertook laboratory work, data analysis and wrote the manuscript

……………………………

Professor Patricia Price

Co-ordinating supervisor

xv

Data presented in Chapter 4 has been published. The bibliographic details and the


Tan DBA, Yong YK, Tan HY, French M, Kamarulzaman A, Price P. Characteristics

of natural killer cells in Malaysian HIV patients presenting with immune

restoration disease after ART. J AIDS Clinic Res 2010; 1:102.



……………………………



xvi

Data presented in Chapter 5 has been submitted to a journal for publication. The

bibliographic details and the contribution of the student to the work are set out below.

Tan DBA, Lim A, Yong YK, Ponnampalavanar S, Omar S, Kamarulzaman A, French

M, Price P. TLR2-induced cytokine responses may characterize HIV-infected

patients experiencing mycobacterial immune restoration disease. AIDS 2011:Epub

ahead of print.



……………………………



xvii

Data presented in Chapter 6 has been submitted to a journal for publication. The

bibliographic details and the contribution of the student to the work are set out below.

Tan DBA, Yong YK, Lim A, Tan HY, French M, Kamarulzaman A, Price P. Robust

interferon-α and IL-12 responses by dendritic cells are related to efficient CD4+ T-

cell recovery in HIV patients on ART. Clin Immunol 2011; 139:115-121



……………………………



xviii

Data presented in Chapter 7 has been published. The bibliographic details and the


Tan DBA, Fernandez S, French M, Price P. Could natural killer cells compensate for

impaired CD4+ T-cell responses to CMV in HIV patients responding to

antiretroviral therapy? Clin Immunol 2009; 132:63-70



……………………………



xix

Abbreviations

ADCC Antibody-dependent cell cytotoxicity

AIDS Acquired immunodeficiency syndrome

APC Antigen presenting cell

ART Antiretroviral therapy

ATT Anti-TB therapy

BCG Bacillus Calmette-Guérin

BSA Bovine serum albumin

CD Cluster of differentiation

CMV Cytomegalovirus

CNS Central nervous system

CRP C-reactive protein

CSF Cerebral spinal fluid

CTLA-4 Cytotoxic T-lymphocyte Antigen 4

DC Dendritic cell

DMSO Dimethylsulphoxide

DNA Deoxyribonucleic acid

EDTA Ethylenediaminetetraacetic acid

ELISA Enzyme linked immunosorbent assay

ELISpot Enzyme linked immunospot

ESAT-6 Early secreted antigenic target 6kDa protein

FcγRIIIA Fc-gamma receptor-3A

FCS Fetal calf serum

FITC Fluorescein isothiocyanate

Foxp3 Forkhead box P3 transciption factor

gp Glycoprotein

HBV Hepatitis-B virus

HCV Hepatitis-C virus

HHV Human Herpes virus

HIV Human immunodeficiency virus

HLA Human leukocyte antigen

HPC Hematopoietic progenitor cells

HSV Human Simplex virus

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IFN Interferon

Ig Immunoglobulin

IL Interleukin

INSHI The International Network for the Study of HIV-associated IRD/IRIS

IRD Immune restoration disease

ITAM Immunoreceptor tyrosine-based activation motif

ITIM Immunoreceptor tyrosine-based inhibition motif

KIR Killer Ig-like receptor

KL Kuala Lumpur

LAM Lipoarabinomannan

LAMP Lysosome-associated membrane protein

LBP LPS-binding protein

LIR Leukocyte Ig-like receptor

LM Lipomannan

LPS Lipopolysaccharide

LTα Lymphotoxin-alpha

LTNP Long-term non-progressors

mAb Monoclonal antibody

mDC Myeloid dendritic cell

MHC Major histocompatibility complex

MIC MHC-I chain related molecule

µL Microlitre

mL Millilitre

mRNA Messenger RNA

MTB Mycobacterium tuberculosis

NCR Natural cytotoxicity receptor

Nef HIV negative factor protein

NFκB Nuclear Factor kappa-B

NK cell Natural killer cell

NNRTI Non-nucleoside reverse transcriptase inhibitor

NRTI Nucleoside reverse transcriptase inhibitor

OI Opportunistic infection

PBMC Peripheral blood mononuclear cells

PBS Phosphate buffered saline

PCP/PJP Pneumocytis carinii/jirovecii pneumonia

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PCR Polymerase chain reaction

PE-Cy5 Phycoerythrin-cyanin 5

pDC Plasmacytoid dendritic cell

PE Phycoerythrin

PHA Phytohemagglutinin

PI Protease inhibitor

PMA Phorbol myristate acetate

PPD Purified protein derivative

RNA Ribonucleic acid

RPH Royal Perth Hospital, Australia

rpm Revolutions per minute

RT Reverse transcriptase

SIV Simian immunodeficiency virus

Tat HIV transactivator of transcription protein

TB Tuberculosis

TCR T-cell receptor

TGF Tumour growth factor

Th1 T-helper type 1 immune response



T-reg Regulatory T-cell

TLR Toll-like receptor

TMB Tetramethylbenzidine

TNF Tumour necrosis factor

TNFR Tumour necrosis factor receptor

TRAIL TNF-related apoptosis-inducing ligand

Vpr HIV viral protein R

VZV Varicella Zoster virus

UM University of Malaya

UMMC University of Malaya Medical Centre

UNAIDS The Joint United Nations program on HIV/AIDS

UWA University of Western Australia

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List of figures

Figure 1.1 HIV prevalence (%) in adults (aged 15-49) in Asia, 2007............................3

Figure 1.2 The replication cycle of HIV.........................................................................4

Figure 1.3 Quantitative and qualitative measures of HIV disease progression..............5

Figure 1.4 Diversification of CD4+ T-cell lineages........................................................7

Figure 1.5 Process of DC migration and transmission of HIV in lymphoid

tissues ..........................................................................................................13

Figure 1.6 Cells in the bone marrow susceptible to HIV infection. CMP,

common myeloid progenitor; CLP, common lymphoid progenitor;

HSC, hematopoietic stem cell (Alexaki et al. 2008) ...................................14

Figure 1.7 The potential role of host genetic in events leading to mycobacterial

IRD..............................................................................................................27

Figure 1.8 Architecture of granulomas in separate scenarios.......................................29

Figure 1.9 Multiple strategies in fighting against MTB infection................................30

Figure 1.10 Case definition for paradoxical TB IRIS ....................................................32

Figure 1.11 Case definition for ART-associated TB and provisional case

definition for unmasking TB IRIS ..............................................................33

Figure 1.12 Case definition for paradoxical cryptococcal IRIS .....................................35

Figure 1.13 Case definition for ART-associated cryptococcosis and unmasking

cryptococcal IRIS........................................................................................36

Figure 3.1 Gating strategies for CD4+ T-cell subsets. ..................................................59

Figure 3.2 All patients achieved increased CD4+ T-cells after commencement

of ART ........................................................................................................60

Figure 3.3 IRD patients showed a peak in IFNγ responses to antigens from the

provoking pathogens ...................................................................................61

Figure 3.4 Evaluation of antigen-reactive plasma IgG titres by ELISA ......................62

Figure 3.5 Proportions of activated and regulatory CD4+ T-cells remained

higher in patients than control during first year of ART.............................63

Figure 4.1 ELISpot analysis of IFNγ-producing PBMC ..............................................74

Figure 4.2 Longitudinal evaluation of NK subsets.......................................................75

Figure 4.3 Longitudinal evaluation of CD16 expression on NK subsets .....................76

Figure 4.4 Longitudinal evaluation of perforin levels in NK subsets ..........................77

Figure 5.1 IFNγ responses to PPD are largely mediated by CD4+ T-cells...................88

xxiii

Figure 5.2 IRD is associated with increased activated CD4+ T-cells and IFNγ

responses .....................................................................................................89

Figure 5.3 Increased TLR2 expression during ART was restricted to IRD

patients ........................................................................................................90

Figure 5.4 High TNFα and IL-12p40 responses to LM characterised some

patients who experienced TB IRD ..............................................................91

Figure 6.1 HIV patients who achieved higher numbers of CD4+ T-cells on

ART are characterized by high frequencies of cytokine-producing

DC. ..............................................................................................................99

Figure 6.2 Gating methods for IFNα-producing pDC and IL-12-producing

mDC. .........................................................................................................100

Figure 6.3 Frequencies of cytokine-producing DC were associated with CD4+

T-cell counts at baseline and Week 12......................................................101

Figure 7.1 CD56lo

NK cells produced more IFNγ in response to K562 cells and

expressed more CD16 and perforin than CD56hi

NK cells. ......................113

Figure 7.2 Proportion of CD56lo

NK cell was low in CMV-lo and CMV-hi

patients ......................................................................................................114

Figure 7.3 Low NK cell IFNγ responses were associated with low frequency of

CD56lo

NK cells ........................................................................................115

Figure 7.4 Expression of mRNA for IFNγ, IL-10 receptor and IL-12 receptors

by RT-PCR in purified CD56+ cells..........................................................116

xxiv

List of Tables

Table 1.1 Regional HIV and AIDS statistics, 2008 ......................................................2

Table 1.2 Overview of the Malaysia HIV Epidemic.....................................................3

Table 1.3 Opportunistic infections and malignancies in HIV-infected patients ...........5

Table 1.4 Mechanism of CD4+ T-cell death mediated by HIV proteins.....................12

Table 1.5 Similarities and differences between natural SIV and HIV infection.........15

Table 1.6 Characteristics of NK cell functions in HIV viremic patients ....................19

Table 2.1 ELISpot buffers and solutions.....................................................................42

Table 2.2 ELISA buffers and solutions.......................................................................43

Table 2.3 Flow buffers and solutions ..........................................................................44

Table 3.1 Laboratory diagnosis of cryptococcal and TB IRD ....................................58

Table 4.1 Demographics of study cohort ....................................................................73

Table 5.1 Clinical characteristic of HIV patients presenting with IRD after

ART.............................................................................................................87

Table 7.1 Demographics of study cohort ..................................................................112

1

Chapter 1

Review of the literature

The intent of this literature review:

This thesis focuses on the recovery of cellular and functional immune

competence in human immunodeficiency virus (HIV)-infected patients on

their first year of treatment with antiretroviral therapy (ART) in Malaysia.

Some of these patients experienced immune restoration disease (IRD),

hence the pathogenesis of this disease is investigated.

The literature review will first introduce HIV by reviewing its epidemiology,

life-cycle and natural history, followed by an overview of HIV pathogenesis

and its impact on components of the immune system. Secondly, treatment of

HIV by ART as well as the virological, immunological and clinical

responses will be described. Lastly, adverse outcomes of ART will be

elaborated, focusing on the development of IRD and immune responses to

some pathogens associated with IRD (Mycobacterium tuberculosis,

Cryptococcus neoformans and cytomegalovirus).

Chapter 1 - Literature Review

2

Chapter 1 - Review of the literature

1.1 What is HIV?

Human immunodeficiency virus (HIV) infects cells of the immune system causing

progressive immunodeficiency leading to acquired immunodeficiency syndrome

(AIDS). If left untreated, HIV patients are increasingly prone to opportunistic infections

and malignancies. Common routes of HIV transmission include sharing of needles by

intravenous drug users, sexual contact and vertical (mother to child) transmission.

1.1.1 Epidemiology

The HIV pandemic began in the mid 1970s. By 1980, it had spread to North America,

South America, Europe, Africa and Australia. The Joint United Nations Programme on

HIV/AIDS (UNAIDS) estimates that 33.4 million people are living with HIV

worldwide. A total of 2.7 million new infections and 2 million AIDS-related deaths per

annum were recorded by the end of 2008. To date, Sub-Saharan Africa is the most

affected region, followed by the South and South-East Asia region (Table 1.1).

Table 1.1 Regional HIV and AIDS statistics, 2008

People living

with HIV

New HIV

infections

Adult

prevalence

(15-49) [%]

AIDS

deaths

Sub-Saharan Africa 22.4 million 1.9 million 5.2% 1.4 million

South and South-East Asia 3.8 million 280 000 0.3% 270 000

Latin America 2.0 million 170 000 0.6% 77 000

East Europe & Central Asia 1.5 million 110 000 0.7% 87 000

North America 1.4 million 55 000 0.6% 25 000

East Asia 850 000 75 000 <0.1% 59 000

Western & Central Europe 850 000 30 000 0.3% 13 000

Middle East & North Africa 310 000 35 000 0.2% 20 000

Caribbean 240 000 20 000 1.0% 12 000

Oceania 59 000 3 900 0.3% 2 000

TOTAL 33.4 million 2.7 million 0.8% 2.0 million

Within South-East Asia, Thailand has the highest HIV prevalence in adults (1.4%)

followed by Cambodia (0.8%), Myanmar (0.7%), Vietnam and Malaysia (0.5%) (2007

data; Figure 1.1). The first cases of HIV infection in Malaysia were detected in 1986,


3

with an estimated 87710 HIV infections and 13394 AIDS-related deaths recorded by

2009 (Table 1.2). HIV transmission in Malaysia is predominantly through intra-venous

drug use (70.6%), followed by heterosexual intercourse (16.9%) (Malaysian Ministry of

Health, 2010).

Figure 1.1 HIV prevalence (%) in adults (aged 15-49) in Asia, 2007

(Source: UNAIDS 2008 report on the global AIDS epidemic)

Table 1.2 Overview of the Malaysia HIV Epidemic

Cumulative number of reported HIV infections since 1986 87 710

Cumulative number of reported HIV/AIDS related deaths since 1986 13 394

Women reported with HIV as of Dec 2009 8 091

Children under 13 with HIV as of Dec 2009 870

New HIV infections detected in 2009 3 080

HIV/AIDS related deaths in 2009 805

Number of PLHIV accessing ART 9 962

Estimated adult (aged 15-49 years) HIV prevalence 0.5%

Source: Malaysian Ministry of Health 2010

1.1.2 HIV viral structure and life cycle

HIV is a lentivirus which is able to convert single-stranded viral ribonucleic acid (RNA)

into double-stranded deoxyribonucleic acid (DNA) and integrate into the host’s

genome. HIV has an outer lipid membrane with pin-shaped glycoproteins (trans-


4

membrane gp41 and surface gp120). These bind to CD4 and chemokine receptors

CCR5 or CXCR4 expressed by target cells to mediate HIV entry (Davis et al. 1997).

The HIV inner core contains viral RNA and enzymes. In the host cell, HIV RNA is

converted to pro-viral DNA using the viral enzyme, reverse transcriptase (RT). RT has

no proof reading activity and allows a high rate of mutations. The pro-viral DNA then

integrates into the host’s cell genome using the viral enzyme, integrase, and uses the

host cell’s genetic machinery to replicate and make new virions (Figure 1.2) (Weiss

2001). Genetic diversity and integration of HIV are key strategies for immune evasion

(Johnson et al. 2002).

Figure 1.2 The replication cycle of HIV

(Weiss 2001)

1.1.3 The natural history of HIV infection

The primary or acute phase of HIV infection may last several weeks. Patients may

experience non-specific symptoms such as fever, sweats, malaise, lethargy, headache,

sore throat, general lymphadenopathy and thrombocytopenia (Cooper et al. 1985). HIV

viremia peaks in parallel with rapid depletion of memory CD4+ T-cells in mucosal

tissues (Figure 1.3) (Grossman et al. 2006). The killing of memory CD4+ T-cells is

largely mediated by direct viral infection. Immuno-pathogenesis of HIV infection will

be reviewed in detail in section 1.3.


5

Around the time of sero-conversion, HIV infection progresses into the chronic phase,

which is usually asymptomatic and may last 8-10 years. HIV viremia gradually

increases as peripheral blood CD4+ T-cell counts decrease (Figure 1.3) (Grossman et al.

2006). Depletion of CD4+ T-cell during the chronic phase is mainly caused by HIV-

associated immune activation rather than direct killing (section 1.3.2). As the number of

CD4+ T-cells drop below 200 cells/µL, HIV patients usually present with opportunistic

infections and/or malignancies (Table 1.3). Hence, AIDS is diagnosed and the patients

will die without treatment.

Figure 1.3 Quantitative and qualitative measures of HIV disease progression

(Grossman et al. 2006)

Table 1.3 Opportunistic infections and malignancies in HIV-infected patients

System Infections/Malignancies

Respiratory Pulmonary tuberculosis (TB)

Pneumocystis Carinii/Jirovecii Pneumonia (PCP/PJP)

Gastro-intestinal Oral candidiasis

Cryptosporidiosis

Cytomegalovirus (CMV) gastroenteritis/colitis

Kaposi’s Sarcoma caused by Human Herpes Virus type 8 (HHV-8) infection

Central nervous Cryptococcal meningitis

CMV retinitis/encephalitis

Toxoplasmosis

Skin Varicella Zoster Virus (VZV) infection

Human Simplex Virus (HSV) infection

Kaposi’s Sarcoma caused by Human Herpes Virus type 8 (HHV-8) infection


6

1.2 The adaptive and innate immune systems

Resistance to infectious diseases involves the interplay between the adaptive and innate

immune systems. Adaptive immune responses are centrally regulated by distinct CD4+

T-cell lineages (section 1.2.1). It is antigen-specific and requires initial sensitization by

antigen presenting cells from the innate immune system. Cells of the adaptive immune

system include T-cells and B-cells (section 1.2.5).

The innate immune system is non-specific in terms of the pathogens recognized and

does not require prior sensitization to elicit rapid responses to control an infection and

activate the adaptive responses. Natural killer (NK) cells (section 1.2.3) and dendritic

cells (DC; section 1.2.4) link the innate and adaptive immune responses. NK cells

express activatory and inhibitory receptors (e.g. the KIR family) which help them

distinguish between normal cells and tumour or infected cells. NK cells have cytotoxic

and cytokine-producing function which can activate downstream adaptive responses.

DC expresses pattern recognition receptors such as the toll-like receptors (TLR) which

recognise pathogen-associated molecular patterns on bacteria, fungi and viruses.

Antigenic peptides are processed from engulfed pathogens by DC and presented to T-

cells or B-cells. This activates the adaptive immune responses which are further

regulated by DC-derived cytokines such as interleukin (IL)-12.

1.2.1 CD4+ T-cells: Regulators of the adaptive immune system

Naïve T-cells undergo clonal expansion when they first encounter an antigen and the

clones differentiate into either antigen-specific ‘effector’ or ‘memory’ T-cells. Effector

T-cells are involved in pathogen clearance through killing of infected cells and

production of cytokines to regulate immune responses. Memory T-cells differentiate

into effector T-cells when they re-encounter antigen (Mackay et al. 2001). Maturation

and differentiation of effector CD4+ T-cells may generate a T-helper type 1 (Th1), Th2

or Th17 bias (Figure 1.4). Th1 cells promote cell-mediated responses through the

production of IFNγ and lymphotoxin-alpha (LTα). These cytokines will ‘help’ cytotoxic

CD8+ T-cells, NK cells or macrophages to improve killing of intracellular pathogens or

pathogen-infected cells and hence clear the infections (Kaufmann et al. 1995; Ladel et

al. 1995; Kalams et al. 1998; Egen et al. 2008). Th2 cells promote antibody-mediated

responses by B-cells through the production of IL-4, IL-5 and IL-13 (section 1.2.5).


7

The Th17 CD4+ T-cell lineage is characterized by the production of IL-17 and IL-6. The

differentiation of Th17 cells is promoted by tumour growth factorbeta (TGFβ), IL-6 and

IL-23 (Weaver et al. 2006). Th17 cells are important in clearing extracellular pathogens

such as bacteria and fungi (Ye et al. 2001; Huang et al. 2004; Happel et al. 2005). On

the other hand, IL-17 is also associated with chronic inflammation and plays a major

role in autoimmune diseases by promoting the recruitment of monocytes and

neutrophils (Witowski et al. 2000; Woltman et al. 2000).

Figure 1.4 Diversification of CD4+ T-cell lineages

(Weaver et al. 2006)

1.2.2 Regulatory T-cells: Suppressors of immune responses

Induction of effector T-cell responses is usually accompanied by regulatory T-cells (T-

reg) that are capable of suppressing T-cell responses (Ho et al. 2006). T-reg can

suppress the activation, proliferation and production of cytokines by CD4+ and CD8

+ T-

cells (Kinter et al. 2004), hence maintain T-cell tolerance to self antigens and suppress

immune responses after an infection has been cleared. T-reg can be generally divided

into natural and adaptive or induced T-reg. Natural T-reg develop in the thymus while

induced T-reg develop in the peripheral lymphoid tissues (Weaver et al. 2006). T-reg

may exert their suppressive effect through direct cell contact (natural T-reg) or through

the production of regulatory cytokines such as IL-10 and TGFβ (induced T-reg). They

are generally identified by expression of the transcription factor forkhead box p3

(Foxp3) except for IL-10-producing T-reg (Figure 1.4). Loss of T-reg numbers and/or

functions is usually associated with autoimmune and inflammatory diseases (Weaver et


8

al. 2006). T-reg are also characterized (although not uniquely) by increased expression

of IL-2 receptor alpha chain (CD25; expressed by activated T-cells), cytotoxic T-

lymphocyte-associated antigen-4 (CTLA-4; expressed by other cells with

immunosuppressive activity) and lack of IL-7 receptor (CD127) (Nixon et al. 2005;

Seddiki et al. 2006).

1.2.3 Natural killer cells: Killers of “non-self” cells

NK cells are large granular lymphocytes important in controlling tumours and infections

by viruses, mycobacteria and fungus (Carrington et al. 2006; Lodoen et al. 2006;

Millman et al. 2008; Marr et al. 2009). NK cells lyse tumour cells and pathogen-

infected cells through the release of cytotoxic granules (containing perforin and

granzyme B). NK cells can also produce pro-inflammatory (e.g. tumour necrosis factor-

alpha; TNFα), Th1 (e.g. IFNγ) and Th2 (e.g. IL-13) cytokines (Cooper et al. 2001a;

Caligiuri 2008). Target cell recognition and the activation of NK cells depends on

activatory and inhibitory signals from activatory receptors carrying cytoplasmic

immuno-receptor tyrosine-based activation motifs (ITAM) and inhibitory receptors

contain immuno-receptor tyrosine-based inhibition motifs (ITIM), respectively.

Inhibitory signals are dominant over the activatory signals, to prevent the killing of

‘normal’ major histocompatibility complex (MHC)-1 expressing cells. Cells with

reduced expression of MHC class I molecules due to viral infection or as a characteristic

of certain tumour cells are targeted and killed by NK cells. This mechanism of NK cell

recognition is known as the “missing self” paradigm (Farag et al. 2002). Major NK cell

receptors include the Fc-gamma receptor IIIa (FcγRIIIa; CD16), natural cytotoxicity

receptors (NCR), leukocyte immunoglobulin (Ig)-like receptors (LIR)-1, killer cell Ig-

like receptors (KIR) and C-type lectin superfamily (Moretta et al. 2000; Cooper et al.

2001a; Farag et al. 2002).

CD16 is an activatory receptor which has a low affinity for IgG but can mediate

antibody-dependent cell cytotoxicity (ADCC) (Sun 2003). CD16 ligation with

monoclonal antibody (mAb) or immune complexes activates NK cell proliferation,

cytotoxicity and cytokine production (Warren et al. 1999). In healthy individuals, the

CD56lo

NK subset should have high expression of CD16. Lower expression of CD16 on

CD56lo

NK cells in untreated HIV patients associated with defects in CD16-dependent

signal transduction and release of cytotoxic granules measured as the mobilization of


9

CD107a in response to CD16 cross-linking (Lichtfuss et al. 2011). CD16 is also

expressed by granulocytes, monocytes and DC (Lund-Johansen et al. 1993; Piccioli et

al. 2007).

NCR are activatory receptors expressed exclusively by NK cells and only three have

been identified (NKp46, NKp44 and NKp30) (Moretta et al. 2000). Cellular ligands for

NCR have yet to be characterised, but NCR have been shown to associate with

molecules from various pathogens. NKp46 interacts with haemagglutinins protein of

influenza virus and is required to stimulate NK cell lysis of infected cells (Mandelboim

et al. 2001). NKp46 also binds to vimentin expressed on Mycobacterium tuberculosis

(MTB)-infected monocytes, allowing NK cell recognition and clearing of infected cells

(Garg et al. 2006). NKp44 is shown to bind the cell wall of all species of

mycobacterium and certain species of bacteria (e.g. Pseudomonas aeruginosa). The

human CMV protein pp65 can associate with NKp30 to inhibit NK cell cytotoxicity

(Arnon et al. 2005).

LIR-1 is an inhibitory receptor expressed by NK cells, monocytes, DC, B-cells and T-

cells which recognises a broad range of MHC class-1 molecules (human leukocyten

antigen; HLA-A, -B, -C, -E, -F, -G) (Colonna 1997). The high affinity of LIR-1 for UL-

18 (an human CMV-encoded MHC-I homolog molecule) inhibits the killing of CMV-

infected target cells by LIR-1-expressing NK cells (Prod'homme et al. 2007).

KIR and C-type lectin receptors are expressed by NK cells and a small proportion of

CD8+ T-cells (Henel et al. 2006; Warren et al. 2006; Caligiuri 2008). KIR bind classical

MHC-I molecules (HLA-A, -B or -C) and may be activatory (KIR2DS1 to KIR2DS5

and KIR3DS1) or inhibitory (KIR2DL1-5 and KIR3DL1-3). The C-type lectin receptors

consist of the CD94/NKG2 heterodimer which binds the non-classical MHC-I molecule

HLA-E. CD94/NKG2C heterodimer is an activatory receptor while CD94/NKG2A is

inhibitory. HIV infection causes inverted ratio of the NKG2A/NKG2C expression on

NK cells which distinguish HIV viremic patients from healthy controls and long term

non-progressors (LTNP) (Mela et al. 2005; Brunetta et al. 2010). NKG2D is another

activatory C-type lectin receptor which forms homodimer and recognises MHC-I chain-

related (MIC)-A or MIC-B expressed by cells under stress conditions (Bahram 2000;

Cerwenka et al. 2001).


10

There are two functionally distinct NK cell subsets defined by the levels of CD56

expression. CD56lo

NK cells represent up to 90% of the CD56+ NK cell in blood and

express high levels of CD16, KIR and perforin. This characteristic makes them effective

responders to NK-sensitive target cells and mediators of natural cytotoxicity and

ADCC, but moderate producers of cytokines. In contrast, CD56hi

NK cells express low

levels of CD16, KIR and perforin but have high expression cytokine receptors such as

for IL-2 and IL-12. Hence, they exert weak cytotoxicity but are efficient cytokine

producers in response to exogenous cytokines (e.g. IL-2) and mitogens (e.g. phorbol

myristate acetate plus ionomycin) conferring an immuno-regulatory role (Cooper et al.

2001b; Jacobs et al. 2001; Ferlazzo et al. 2004; Maghazachi 2005; Farag et al. 2006;

Caligiuri 2008). A relatively inert NK cell subset (CD56-CD16

+) exists in low

frequencies in healthy individuals but is expanded in HIV patients (Mavilio et al. 2005).

1.2.4 DC and macrophages: Professional antigen presenting cells

Dendritic cells (DC) and macrophages are professional antigen presenting cells (APC)

that link the innate and adaptive immune systems. They phagocytose pathogens,

preventing the spread of infections. Antigens are then processed and presented to T-

cells via MHC class I (to CD8+ T-cells) or MHC class II (CD4

+ T-cells) resulting in

activation of adaptive immune responses. This process is regulated by production of

cytokines such as Type 1 IFN, IL-12, IL-10 and TNFα (Krutzik et al. 2005; Tosi 2005).

Two major subsets of DC are plasmacytoid DC (pDC) and myeloid DC (mDC). PDC

are characterized by the expression of CD123 (IL-3 receptor) and produce Type 1 IFN

(IFNα/β). MDC is characterized by the expression of CD11c and produce IL-12. The

role of DC and/or macrophages in controlling MTB and HIV infection is discussed in

Chapter 5 and Chapter 6, respectively.

1.2.5 B-cells: Cells of the humoral arm of the immune system

B-cell responses to protein antigens are T-cell dependent. Th2-mediated responses

‘help’ naïve B-cells to proliferate and differentiate into antibody-producing plasma cells

or memory B-cells. Memory B-cells differentiate into plasma cells when they re-

encounter antigen. Optimal expansion, differentiation and antibody class switching

require B-cell receptor triggering by antigen, T-cell help (through CD40 and CD40

ligand interaction) and additional signal by TLR agonists or DC-derived cytokines

(Ruprecht et al. 2006; Lanzavecchia et al. 2009).


11

1.3 Pathogenesis of HIV infection

Proportional and functional changes of T-cells, NK cells, DC and B-cells which occur

during HIV infection may involve direct infection (via CD4 and CCR5), binding of

immuno-modulatory viral proteins (Vpr and Nef) or indirect mechanisms such as HIV-

associated immune activation or inflammation of lymphoid tissues. Overall, HIV

infectionaffects all cells necessary to maintain immune surveillance and effective

immunity against foreign pathogens.

HIV prefentially infects CD4+ T-cells due to their high expression of CD4 and

CCR5/CXCR4 and causes CD4+ T-cell depletion (section 1.3.1). NK cells, DC and

macrophages are also susceptible to HIV infection as they express CD4 and

CCR5/CXCR4 (Sections 1.3.4 and 1.3.5). CD8+ T-cells which cannot be directly

infected by HIV have increased susceptibility to cell death and cellular dysfunction due

to HIV-associated immune activation (Gougeon et al. 1996; Petrovas et al. 2006). B-

cell dysfunctions during HIV infection include abnormal distribution of B-cell subsets,

reduced efficacy of vaccines and lower titres of antibodies. Impaired B-cell responses

may be due to direct interaction with HIV (e.g. via complement receptor and C-type

lectin receptors expressed on B-cell) or indirect effects such as expansion of

hyperactivated B-cell (e.g. increased polyclonal B-cell activation and cell turnover),

increased frequencies of immature B-cells and exhausted B-cells (i.e. enrichment of

HIV-specific response but reduced responses to recall antigens) (Doria-Rose et al. 2009;

Moir et al. 2009).

1.3.1 Mechanisms of CD4+ T-cell depletion

1.3.1.1 Virus-mediated killing of infected and uninfected CD4+ T-cells

Acute HIV infection is characterized by rapid depletion of memory CD4+ T-cells in the

gut mucosa (Douek et al. 2003). HIV preferentially infects memory and effector CD4+

T-cells because of their higher activation state and higher expression of CCR5

(Brenchley et al. 2004). HIV-infected cells are susceptible to apoptosis mediated by Fas

and Fas-ligand (FasL) because HIV induces surface expression these molecules

(Estaquier et al. 1996). HIV proteins such as the envelope glycoprotein (gp120), viral

protein R (Vpr), negative factor (Nef), trans-activator of transcription (Tat) and HIV

protease facilitate CD4+ T-cell apoptosis by mechanisms outlined in Table 1.4. These

proteins may be released into the local environment and cause apoptosis of nearby

uninfected CD4+ T-cells.


12

Continuous HIV replication leads to the build up of unintegrated linear viral DNA,

resulting in cellular toxicity. Continuous budding of HIV virions disrupts the plasma

membrane, leading to cell death (Alimonti et al. 2003). HIV infection disrupts

membrane integrity of the mitochondria which promote activation of apoptotic

pathways. Mitochondrial-mediated apoptosis relates to increased cellular pro-apoptotic

regulator proteins (e.g Bax) and decreased anti-apoptotic regulator proteins (e.g. Bcl-2)

(Petit et al. 2003).

Table 1.4 Mechanism of CD4+ T-cell death mediated by HIV proteins

HIV proteins Mechanisms References

gp120

• Cross-linking with CD4 molecules without

TCR ligation induces apoptosis

• Increased expression of Fas/FasL

• Increased Bax and decreased Bcl-2

(Marschner et al. 2002)

(Arthos et al. 2002)

(Westendorp et al. 1995)

Tat


• Increased pro-apoptotic Bax and decreased

anti-apoptotic Bcl-2

(Li-Weber et al. 2000)

(Sastry et al. 1996)

Nef


• Causes membrane disorder and lysis of both

CD4+ T-cell and CD4

- T-cells

(Zauli et al. 1999)

(Azad 2000)

Vpr

• Cell cycle arrest

• Mitochondria disruption

• Causes membrane disorder and lysis of both

CD4+ T-cell and CD4

- T-cells

(Watanabe et al. 2000)

(Roumier et al. 2002)

(Azad 2000)

Protease • Inactivate anti-apoptotic Bcl-2

• Activate pro-apoptotic pro-caspase 8

(Strack et al. 1996)

(Nie et al. 2002)

1.3.1.2 The role of lymphoid tissues in CD4+ T-cell depletion

Effective immune responses against infection involve the capture and processing of

antigens by DC in the local tissue, followed by the maturation and migration of DC to

lymph nodes where they activate antigen-specific T-cells (Figure 1.5) (Banchereau et al.

1998; Stoll et al. 2002). DC infected by HIV at mucosal surfaces migrate to lymph

nodes so HIV can then be transmitted to interacting CD4+ T-cells and follicular DC

which reside in lymph nodes (Granelli-Piperno et al. 1999). Follicular DC can serve as a

reservoir for infectious HIV, providing a mechanism for viral persistence and immune

activation (Keele et al. 2008).


13

Lymph nodes from patients with chronic HIV infection accumulate effector T-cells with

markers of immune activation (CD38) and apoptosis (Fas). CD4+ T-cells may be

inappropriately attracted to and retained in lymph nodes leading to lymphadenopathy

(Biancotto et al. 2007). CD4+ T-cells from HIV-infected patients migrate to lymph

nodes at higher rates than in normal individuals (Chen et al. 2002). Thus, the frequency

of circulating CD4+ T-cells decreases more than whole body reserves. HIV infection

also increases collagen deposition in lymph nodes, leading to fibrosis and the disruption

of lymphoid architecture. This affects the ability of the lymphoid tissue to support

homeostasis so the amount of fibrosis correlates inversely with CD4+ T-cell counts in

HIV patients (Schacker 2008).

Figure 1.5 Process of DC migration and transmission of HIV in lymphoid

tissues

(Granelli-Piperno et al. 1999)

1.3.1.3 Impaired production of CD4+ T-cells

Mechanisms of CD4+ T-cell depletion described above affect mature CD4

+ T-cells. HIV

infection is also associated with impaired production and differentiation of CD34+

hematopoietic progenitor cells (HPC) from the bone marrow. HIV-mediated depletion

of CD34+CD4

+ cells may be due to a combination of direct HIV infection or indirect

mechanisms due to impaired ability of accessory cells of the bone marrow (e.g. T-cells,

monocytes, macrophages, micro-vascular endothelial cells and stromal cells) to regulate

the development of hematopoietic cells (Moses et al. 1998; Alexaki et al. 2008).

Furthermore, cells that developed from HPC in the bone marrow can be infected and

serve as HIV reservoirs throughout the body (Figure 1.6).


14

Figure 1.6 Cells in the bone marrow susceptible to HIV infection. CMP,

common myeloid progenitor; CLP, common lymphoid progenitor; HSC,

hematopoietic stem cell (Alexaki et al. 2008)

The production of naïve (new) T-cells from the thymus is critical during early life.

Although thymic function decreases with age, production of naïve T-cells is maintained

in late adulthood. HIV infection causes thymocyte depletion and loss of thymic

epithelium leading to thymus involution (Bonyhadi et al. 1993; McCune 1997).

Expression of T-cell receptor rearrangement excision circles (a marker of thymic

function) was lower in HIV-infected children and adults compared to age-matched

uninfected controls. The thymus also contributes to full immune reconstitution in

children and partial recovery in adults given ART (Douek et al. 1998; Correa et al.

2002; Fernandez et al. 2006a). Amongst HIV patients with a sustained response to

ART, CD4+ T-cell counts are determined by thymus function and immune activation

(Fernandez et al. 2006a; Fernandez et al. 2006b).

1.3.2 Current models of HIV-associated immune activation

HIV infection is also characterised by high levels of immune activaton in contrast to the

non-pathogenic Simian immunodeficiency virus (SIV) infection in its natural host

(sooty mangabeys and African green monkeys). However in new non-adapted host

species (rhesus macaques), SIV infection is pathogenic and progresses to AIDS. The


15

immunological characteristics of HIV infections are similar to non-pathogenic SIV

infection during the acute phase, but differ during the chronic phase (Table 1.5; section

1.1.3). Natural SIV infections involves limited immune activation without progressive

depletion of CD4+ T-cells during the chronic phase (Sodora et al. 2009). Several

mechanisms of HIV-induced immune activation are illuminated by these differences

(section 1.3.2.1 and 1.3.2.2).

Table 1.5 Similarities and differences between natural SIV and HIV infection

Natural SIV infection HIV infection

Acute phase

Virus replication peaks within a few

weeks and soon decline indicating

partial control of virus replication

Virus replication peaks within a few

weeks and soon decline indicating

partial control of virus replication

Elevated virus-specific innate and

adaptive immune responses

Elevated virus-specific innate and


Marked type I IFN response Marked type I IFN response

Depletion of mucosal CD4+ T-cells Depletion of mucosal CD4

+ T-cells

Variable levels of bystander T-cell

activation and apoptosis

High levels of bystander T-cell

activation and apoptosis

Establishment of anti-inflammatory

milieu

Chronic phase

High set point of virus replication

persists despite host’s immune response

High set point of virus replication

persists despite host’s immune response

Virus replication mostly occurring in

short-lived, activated CD4+ T-cells

Virus replication mostly occurring in

short-lived, activated CD4+ T-cells

No progressive depletion of circulating

and mucosal CD4+ T-cells

Progressive depletion of circulating and

mucosal CD4+ T-cells

Resolution of type I IFN responses Persistent type I IFN responses

Limited immune activation and T-cell

apoptosis

Generalized immune activation and

significant T-cell apoptosis

Preservation of mucosal Th17 cells Loss of mucosal Th17 cells

Absence of microbial translocation Microbial translocation is prominent

Clinical outcome

Progression in AIDS is very rare Progression to AIDS in most cases

Vertical transmission is rare Vertical transmission is common


16

The frequency of HIV-infected cells is low in blood and lymphoid tissues (Pantaleo et

al. 1991), so direct HIV infection cannot account for the depletion of CD4+ T-cells. It is

now accepted that HIV-induced immune activation plays a major role in disease

progression. Elevated pro-inflammatory cytokines/chemokines (e.g. IL-1, IL-6, IL-18,

TNFα, CCL5 and CCL2) are found in untreated HIV infection (Breen 2002; Connolly et

al. 2005; Jin et al. 2009). CD4+ T-cells from HIV patients have higher expression of

cellular activation markers (e.g. HLA-DR and CD38) and rates of apoptosis and

proliferation than uninfected control donors (Gougeon et al. 1996; Douek et al. 2001;

Hazenberg et al. 2003). T-cell activation results in the increase of intracellular nuclear

factor kappa B (NFκB) which enhances transcription of integrated virus and the

production of new virions that can infect new targets (Kawakami et al. 1988).

1.3.2.1 Microbial translocation

Pathogenic SIV and HIV infections begin with an early and rapid depletion of memory

CD4+ T-cells in the gastrointestinal mucosa (Sodora et al. 2009). In healthy subjects,

lymphocytes below the mucosal surfaces prevent translocation of microbes and their

products. Damage to mucosal immunity during early HIV infection and subsequent

translocation of products of intestinal flora into the blood may underlie HIV-associated

immune activation (Brenchley et al. 2006a). Rhesus macaques are susceptible to

pathogenic SIV disease, fail to retain and accumulate memory CD4+ T-cells in

respiratory mucosa. Hence SIV or HIV disease progression is associated with an

inability to maintain immune defences on mucosal surfaces (Picker et al. 2004).

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial cell walls

and is an indicator of microbial translocation. Levels of LPS and LPS-binding protein

(LBP) in plasma were higher in patients with chronic HIV infection than in patients

with acute HIV infection or healthy controls. LPS can activate monocytes/macrophages

through TLR4, whilst LBP is produced by gastrointestinal and hepatic epithelial cells in

response to LPS stimulation. Plasma LPS levels were proportional to plasma levels of

soluble CD14 and IFNα as well as the frequency of activated CD8+ T-cells in

circulation. Soluble CD14 is a marker of monocyte or macrophages activation by LPS

stimulation and IFNα is primarily produced by activated plasmacytoid DC (Brenchley et

al. 2006b). T-cells can be activated directly by microbial products or indirectly by

inflammatory cytokines derived from DC and macrophages (Caron et al. 2005). These


17

findings support the role of increased microbial translocation in causing chronic

immune activation in HIV patients.

1.3.2.2 Elevated Type I IFN response

Type I interferon (e.g. IFNα) is produced by pDC and induces Th1 responses. Its

antiviral activity includes HIV (Schmidt et al. 2005). HIV itself can induce the

production of IFNα in pDC through TLR7 and up-regulate the expression of TLR7 to

enhance this response (Heil et al. 2004; Lester et al. 2008). Frequencies of IFNα-

producing cells were higher in the lymphoid tissues from HIV patients than uninfected

controls with the cells localized in the T-cell rich area (Herbeuval et al. 2006). However

the production of IFNα by pDC in lymphoid tissue from natural SIV host was limited,

thus minimising immune activation (Mandl et al. 2008).

High levels of IFNα in lymphoid tissues may be detrimental to CD4+ T-cells. IFNα

induces the expression of TNF-related apoptosis-inducing ligand (TRAIL) by

monocytes and DC (Herbeuval et al. 2005c). Apoptosis follows the binding of TRAIL

to the TRAIL death receptor 5 (DR5) on CD4+ T-cells. Expression of DR5 can be

induced directly by infectious and non-infectious HIV particles (Herbeuval et al.

2005b). Compared to uninfected controls, untreated HIV patients displayed higher

expression of type I IFN-stimulated genes in ex vivo activated (CD25+) CD4

+ T-cells.

These genes include IL-12RB2 and T-bet, both of which promote Th1 effector

differentiation. CD4+ T-cell depletion may reflect preferential differentiation of CD4

+

T-cells into short-lived effector cells under the influence of type I IFN.

1.3.3 HIV viremia increases frequency of T-reg

HIV infection is associated with increased frequency of T-reg (Weiss et al. 2004).

Exposure of T-reg to HIV selectively promotes their survival via a CD4-gp120

dependent pathway. Increased T-reg proportions in circulation is associated with

immune activation and T-reg have the phenotype of activated T-cells (Lim et al. 2007).

HIV also promotes the accumulation of T-reg in the lymphoid tissues with disease

progression and correlates with viral load (Andersson et al. 2005; Nilsson et al. 2006).

In vitro depletion of T-reg from peripheral blood mononuclear cells (PBMC) from HIV

patients increases CD4+ and CD8

+ T-cell responses against HIV and CMV antigens

(Aandahl et al. 2004). Therefore, T-reg may contribute to impaired T-cell responses

observed during HIV infection.


18

1.3.4 HIV immune evasion strategies and NK cell dysfunction

Viral infection of CD4+ T-cells decreases expression of MHC class I molecules which

should allow NK cells to restrict the spread of infection. However, HIV can evade

recognition by NK cells by altering the expression of MHC class I molecules on

infected CD4+ T-cells. For example, HIV-infected T-blasts have decreased HLA-A and

-B, where as HLA-C and -E remained on the surface. Increased HLA-E expression on

target cells inhibits NK cell cytotoxicity while the blocking of HLA-C and -E molecules

improved NK cell mediated ADCC of anti-gp120-coated infected cells (Bonaparte et al.

2004; Ward et al. 2004; Nattermann et al. 2005). HIV viremia is mostly associated with

impaired cytotoxicity and cytokine production by NK cells, but some studies have

shown increased NK cell responses in viremic HIV patients (Table 1.6).

Early loss of CD56hi

NK cells is seen during acute HIV infection, followed by the loss

of CD56lo

NK cells during the chronic phase. Increased proportion of CD56-CD16

+ NK

cells are also evident in patients with chronic HIV infection (Alter et al. 2004; Mavilio

et al. 2005). CD56-CD16

+ NK cells are relatively inert, displaying more inhibitory NK

cell receptors and less natural cytotoxicity receptors (Mavilio et al. 2003; Mavilio et al.

2005). Impaired NK cell function in HIV patients is also attributed to alterations to the

NK cell receptor repertoire (Fauci et al. 2005). For example, expression of CD158a and

CD161 (NK cell receptors that bind HLA-C and HLA-E) were selectively increased in

HIV patients relative to controls. Expression of inhibitory NK cell receptors (e.g

NKG2A and LIR-1) was higher in HIV viremic patients than in controls, whilst

expression of NCR was lower (Parato et al. 2002; Mavilio et al. 2003).


19

Table 1.6 Characteristics of NK cell functions in HIV viremic patients

Target cells/stimulants Assay References

Decreased NK cell function in HIV viremic patients compared to healthy controls

Cytotoxicity against HIV-infected cell lines 51

Cr release assay (Chehimi et al. 2007)

Cytotoxicity against K562 cells 51

Cr release assay (Azzoni et al. 2002;

O'Connor et al. 2007)

Cytotoxicity against K562 cells after IL-2

stimulation 51

Cr release assay (Mavilio et al. 2003)

Degranulation activity after mitogen

stimulation

CD107a

expression

flow cytometry

(Alter et al. 2005)

IFNγ responses after mitogen stimulation Flow cytometry (Alter et al. 2005)

IFNγ and TNFα responses in response to

IL-2 and IL-12 Flow cytometry (Azzoni et al. 2002)

Production of IFNγ and TNFα after IL-2 and

PHA stimulation

ELISA on

supernatants (Mavilio et al. 2003;

Mavilio et al. 2005)

Production of CCL5, MIP-1α and MIP-1β

after IL-2 stimulation

ELISA on

supernatants (Kottilil et al. 2003)

Increased NK cell function in HIV viremic patients compared to healthy controls

Cytotoxicity against K562 cells Nuclear stain (PI)

by flow cytometry (Parato et al. 2002)

Degranulation activity in response to K562

cells

CD107a

expression by flow

cytometry

(Alter et al. 2004;

Alter et al. 2005)

Production of IFNγ in response to K562 cells Flow cytometry (Alter et al. 2005)

CD107a, lysosome-associated membrane protein-1 (LAMP-1) and marker of cell

degranulation; ELISA, enzyme-linked immunosorbent assay; K562 cells, Human

erythromyeloblastoid leukemia cell line. MIP, macrophage inflammatory protein


20

1.3.5 DC and macrophages dysfunction during HIV infection

DC and macrophages are susceptible to HIV infection. Infected DC and macrophages

can be resistant to cytotoxicity or apoptosis and thus become long-term viral reservoirs.

Phagocytosis and antigen presentation by APC are impaired in HIV patients (Aquaro et

al. 2002; Kedzierska et al. 2002; MacDougall et al. 2002). This includes decreased

expression of MHC and co-stimulatory molecules (Andrieu et al. 2001; Brown et al.

2005). DC from HIV infected patients showed impaired maturation, which affects their

migration to secondary lymphoid tissues and antigen presentation (Barron et al. 2003).

Numbers of circulating pDC are lower in HIV patients than healthy controls. They

correlate directly with CD4+ T-cell counts and inversely with plasma HIV RNA levels

(Chehimi et al. 2002; Killian et al. 2006). Production of IFNα by pDC in response to

synthetic TLR7/8 agonists and other viruses including HSV was lower in HIV viremic

patients than healthy controls. Production of IFNα correlated inversely with plasma HIV

RNA levels (Chehimi et al. 2002; Finke et al. 2004; Chehimi et al. 2007; Martinson et

al. 2007), so high levels of IFNα may be detrimental to CD4+

T-cell survival (section

1.3.2.2).

The numbers of mDC are lower in HIV viremic patients than healthy controls (Finke et

al. 2004; Fontaine et al. 2009). Production of IL-12 by mDC in response to TLR4,

TLR7, TLR8 and TLR9 agonists was higher in HIV infected patients than healthy

controls (Martinson et al. 2007; Louis et al. 2010). Increased IL-12 responses in HIV

patient may promote differentiation of CD4+ T-cells into short-lived effector cells

contributing the pathogenesis of HIV (section 1.3.2.2).


21

1.4 Antiretroviral therapy

ART is the current standard of care for HIV infection and has improved the survival rate

and life quality of HIV patients worldwide. Since its introduction in the 1990s, access to

ART has increased even in resource limited countries in Asia and Africa. ART

effectively treats and prevent transmission of HIV, though immune recovery varies

between individuals (section 1.4.2.1). Some patients experience persistent immune

dysfunction or worse clinical outcomes such as IRD (section 1.5).

1.4.1 Basic description and the mode of action

ART suppresses the replication of HIV, allowing recovery of the immune system. HIV

is not completely eradicated by ART as it can integrate in the genome of long-lived

immune cells (e.g. memory T-cells and DC). These cells act as persistent viral

reservoirs, so treatment is life long. Ideally, ART should be started as early as possible

to ensure minimal damage of the immune system and subsequently better immune

recovery. The International AIDS Society-USA panel now recommends the initiation of

ART in symptomatic patients regardless of CD4+ T-cell counts and for asymptomatic

individuals with CD4+ T-cell counts less than or equal to 500 cell/µL. Initiating ART at

a CD4+ T-cell count of 500 cells/µL decreased mortality and generated better outcomes

(Thompson et al. 2010). Most HIV patients in South East Asia initiate ART at a much

lower CD4+ T-cell counts. In 2009, 37% HIV patients in Malaysia with CD4

+ T-cell

counts below 350 cells/µL were still untreated (UNGASS 2010).

Most commonly used drugs in ART are protease inhibitors (PI) or nucleoside/non-

nucleoside reverse transcriptase inhibitors (NRTI/NNRTI). PI disrupt post-translational

processing of viral proteins and enzymes (Randolph et al. 2004). NRTI/NNRTI inhibit

HIV reverse transcriptase, which is important for the conversion of viral RNA to DNA

(De Clercq 1998; Squires 2001). There are newer drug classes such as integrase strand

transfer inhibitors, entry inhibitors and CCR5 antagonists (Greene 2004; Thompson et

al. 2010). Newly developed drugs such as the second generation PI (darunavir) and

integrase inhibitor (raltegravir) are better tolerated among HIV patients compared with

older drugs (Grinsztejn et al. 2007; Ortiz et al. 2008). However, availability of these

drugs is limited by cost in developing countries such as those in South East Asia

including Malaysia. Current treatment in these countries uses older generic drugs (e.g.

stavudine, and zidovudine) which are associated with neuropathy, metabolic

complications, anaemia and other adverse outcomes (section 1.4.2.5).


22

1.4.2 Outcomes of ART

The outcomes of ART can be considered as virological, immunological and clinical

responses. Virological responders are patients who achieved suppression of plasma HIV

RNA to undetectable levels (viral load of <50 copies/ml determined by reverse

transcriptase polymerase chain reaction assay; RT-PCR). Ideally, drug regimens should

be changed until viral suppression is achieved. However in Asian countries, HIV RNA

assays are prohibitively expensive so clinicians often rely on clinical or immunological

markers (e.g. CD4+ T-cell counts). In addition, the choice of second line therapies may

be limited by cost and/or availability. Currently, second line therapies are only partially

subsidised by the Government in Malaysia (UNGASS 2010).

Immunological responses are defined by increases in CD4+ T-cell numbers, which may

or may not include the recovery of immune function. Whilst a favourable clinical

response on ART includes the general improvement of patient’s health and amelioration

of opportunistic infection-associated symptoms, some patients experienced adverse

outcomes such as IRD due to ART-mediated restoration of immune responses that is

immunopathological. IRD will be a focus of this thesis (section 1.5).

1.4.2.1 Recovery of CD4+ T-cells on ART are variable among HIV

patients

Not all HIV patients who have virological responses on ART (i.e. suppressed plasma

HIV RNA levels) achieve optimal restoration of CD4+ T-cell counts (e.g. to over 500

cells/µL after 6 months). The reconstitution of CD4+ T-cells after initiation of ART

occurs in two major phases. Early reconstitution in the first few months mainly involves

redistribution from lymphoid tissue, possibly associated with decreased immune

activation (Autran et al. 1997; Pakker et al. 1998; Bucy et al. 1999). The next phase of

recovery is at a slower rate involving reconstitution of naïve CD4+ T-cells which is

associated with thymus size (Ruiz-Mateos et al. 2004).

Poor recovery of CD4+ T-cell numbers and functions has been associated with late

initiation of ART, low nadir CD4+ T-cell counts (Negredo et al. 2010), low numbers of

naïve CD4+ T-cells (Schacker et al. 2010), high levels of immune activation (Fernandez

et al. 2006b), high rate of apoptosis (Massanella et al. 2010), lymphoid tissue damage

(Krathwohl et al. 2006) and less abundant thymic tissue/activity (Smith et al. 2000; Al-

Harthi et al. 2002; Ruiz-Mateos et al. 2004; Fernandez et al. 2006a).


23

1.4.2.2 Reconstitution of CD4+ T-cell numbers may not parallel

restored function

HIV patients with increased and stable CD4+ T-cell count after ART may have

persistent suboptimal CD4+ T-cell responses. This is clearest in those who had low nadir

CD4+ T-cell counts. Responses to antigens such as tetanus toxoid, CMV, Candida and

MTB antigens were still lower than the median of healthy controls even after more than

a year on ART (Lederman et al. 2003; Keane et al. 2004; Burgess et al. 2006;

Sutherland et al. 2006). Poor antigen-specific CD4+ T-cell responses may reflect limited

recovery of T-cell receptor repertoire on ART (Connors et al. 1997). Numbers and

functions of DC relate to CD4+ T-cell responses after ART (Fernandez et al. 2008), and

hence will be investigated in Chapter 6.

Effector immune responses can be assessed in vitro by the production of cytokines or

cytotoxicity in response to mitogens or antigens from pathogens (e.g. MTB,

Cryptococcus neoformans and CMV). Functional assays used in this thesis utilise IFNγ

ELISpot, flow cytometry and ELISAs. Recovery of CD4+ T-cell function may relate

directly or indirectly to NK cell and DC functions, addressed later in this thesis (Section

8.2)

1.4.2.3 Immune activation is reduced by ART

Spontaneous CD4+ T-cell apoptosis and cellular markers of immune activation (CD38

and HLA-DR) were reduced after ART but remained higher than healthy controls

(Roger et al. 1999; Almeida et al. 2002), see Chapter 3. There is an immediate decrease

in Ki67 expression (marker of cell proliferation) in naïve T-cells after starting ART

(Hazenberg et al. 2003). Patients with effective suppression of viral replication but

persistent immune activation display lower gains of CD4+ T-cells on short-term (median

of 21 months) ART and long-term (median of 103 months) ART (Hunt et al. 2003;

Fernandez et al. 2006b). Decreased LPS levels (mediator of immune activation, see

section 1.3.2.1) are also associated with better CD4+ T-cell recovery on ART

(Brenchley et al. 2006b).

1.4.2.4 Outcomes of ART on T-reg, DC and NK cells

Recovery of T-reg and DC during ART are briefly described here and discussed in

detail in relevant chapters (T-reg; Chapter 3 and DC; Chapter 6). The proportion of

circulating T-reg decreased during ART, but remains higher than healthy controls even


24

when viral suppression is achieved (Weiss et al. 2004; Lim et al. 2007). The numbers of

pDC and mDC increased on ART with increased functional capacity and phenotypic

maturation of circulating DC (Barron et al. 2003).

The number of CD56lo

and CD56hi

NK cells are increased on ART with a slight

decrease in CD56- NK cells (Alter et al. 2005). Data on the restoration of NK cell

functions were inconsistent. In one study, aviremic patients (>24 months on ART)

showed persistently low cytotoxicity against K562 cells (Mavilio et al. 2003), whilst

IFNγ production recovered. In another study, cytotoxicity recovered but IFNγ

production remained low (Azzoni et al. 2002). On the other hand, cytotoxicity against

K562 cells was higher in viremic patients which then normalized when patients became

aviremic after 24 weeks of ART (Parato et al. 2002). Expression of CD107a (a marker

of lysosomal granule exocytosis) and intracellular IFNγ in NK cells stimulated with

K562 cells were higher in viremic patients (regardless of treatment) than healthy

controls. Expression of CD107a and IFNγ were similar in aviremic patients on ART (>6

months) and healthy controls (Alter et al. 2004). The discrepancies may reflect

characteristics of the patients (baseline CD4+ T-cell counts, recovery of CD4

+ T-cell

function), but these parameters are not uniformly reported.

1.4.2.5 Adverse clinical outcomes

HIV drug resistance is a challenging problem especially in patients receiving long-term

ART (Gilks et al. 2006). Lists of mutations associated with ART drug resistance are

regularly updated by the International AIDS Society-USA Drug Resistance Mutations

Group. Criteria for the identification of mutations include (1) in vitro passage

experiments or validation of contribution to resistance by using site-directed

mutagenesis; (2) susceptibility testing of laboratory or clinical isolates; (3) genetic

sequencing of viruses from patients in whom the drug is failing; (4) correlation studies

between genotype at baseline and virologic response in patients exposed to the drug

(Johnson et al. 2009).

Complications of ART are attributed to drug toxicity, drug-drug interactions (e.g. with

anti-TB therapy; ATT) and/or increased inflammation. Phenotypes include changes in

subcutaneous fat, impaired endothelial function, low levels of high-density lipoprotein

cholesterol and increased risk of cardiovascular disease (Dube et al. 2010). The use of

some NRTI has been associated with lipodystrophy and metabolic syndromes, whilst


25

some NNRTI can cause severe skin reactions, hepatotoxicity and psychosis. Stavudine

(a NRTI) and indinavir (a PI) potentiate sensory neuropathy in HIV patients (Pettersen

et al. 2006; Smyth et al. 2007). Susceptibility to neuropathy is affected by

polymorphisms in the TNF gene (Affandi et al. 2008; Cherry et al. 2008), and may

relate to the accumulation of monocytes around peripheral nerves that have been

damaged by HIV or ART (Chew et al, in press).

The role of immune activation in a patients’ outcome on ART was investigated in

patients enrolled in the Strategies for Management of Anti-Retroviral Therapy

(SMART) study with plasma HIV RNA levels below 400 copies/mL. At baseline, they

had higher levels of high sensitivity C-reactive protein (CRP) and IL-6 (markers of

inflammation); D-dimer (marker of coagulation and fibrolysis) and cystatin C (marker

of impaired renal function) compared to the general population (Neuhaus et al. 2010).

Elevations of these markers are associated with mortality and non-AIDS defining

illnesses like cardiovascular and renal diseases (Panichi et al. 2001; Pepys et al. 2006).

HIV patients who experienced new AIDS-related opportunistic infections or mortality

had higher baseline levels of plasma immune activation markers; soluble tumour

necrosis factor receptor (sTNFR) family, soluble CD40 ligand and IL-6 compared to

controls (Kalayjian et al. 2010).

HIV patients who initiate ART when they are very immunodeficient are susceptible to

developing immune reconstitution disorders (French 2007). The most common

disorders are characterised by inflammatory processes due to the restoration of immune

response against pathogen-specific antigens and patients may have atypical presentation

of opportunistic infections such as mycobacterial, cryptococcal or herpes-viral infection.

They are known as immune restoration disease (IRD) or immune reconstitution

inflammatory syndrome (IRIS) (section 1.5).


26

1.5 Immune restoration disease

Around one quarter of ART-treated HIV patients develop atypical presentation of

disease associated with pre-existing pathogens such as MTB, Cryptococcus neoformans

and CMV known as IRD or IRIS. In countries with limited resources, IRD is a major

cause of morbidity in ART-treated HIV patients. Mortality attributed to IRD is less

common and is usually due IRD involving the central nervous system (CNS). IRD is

attributed to pathogen-specific immune responses that are immunopathological rather

than protective (Price et al. 2009; French 2010). However, the immunopathogenic

mechanisms of each IRD remain to be elucidated. It is important to distinguish an IRD

event from a usual presentation of an opportunistic infection or failure of treatment of

an opportunistic treatment so that more appropriate treatment can be administered.

1.5.1 Clinical diagnosis

The International Network for the Study of HIV-associated IRD/IRIS (INSHI) was

established in 2006 to harmonize ongoing and future studies concerning IRD worldwide

utilizing the same clinical definition for the diagnosis of IRD. To date, consensus

clinical case definitions have been developed for TB IRD (Meintjes et al. 2008a) and

cryptococcal IRD (Haddow et al. 2010). Generally, definitions of IRD involved the

following criteria:

1. Patients must have received ART and achieved virological response (>1 log10

decrease in plasma HIV RNA).

2. Clinical deterioration of an infectious or inflammatory condition must be temporally

related to the initiation of ART.

3. Symptoms cannot be explained by expected clinical course of associating pathogen,

drug toxicity, treatment failure and complete non-adherence.

4. Increases in peripheral blood CD4+ T-cell count may be used as a marker of immune

reconstitution when plasma HIV RNA result is lacking. However, this is not

included as a criteria for IRD as:

a) Some cases of IRD occur without an increase in peripheral CD4+ T-cell counts.

b) CD4+ T-cell counts in the peripheral blood may not reflect function or counts

present at the site of an infection.

c) CD4+ T-cells are not the only cellular mediators of IRD. (Possible role of other

immune cells is discussed in section 8.1).


27

1.5.2 Risk factors for the development of IRD

The critical immune pathways differ according to the invading pathogen and are

described later for MTB (section 1.5.3.1), Cryptococcus neoformans (section 1.5.3.2)

and CMV (section 1.5.3.3). Hence, the immunopathological mechanisms and risk

factors will differ based on the type of IRD. This is confirmed by the various genetic

associations observed in IRD which are specific for various provoking agents (Price et

al. 2001a; Price et al. 2002; Affandi et al. 2010). Host genetics may affect IRD through

several mechanisms. Using TB IRD as an example (Figure 1.7), host genetics can affect

different scenarios (orange boxes) which results in the level of mycobacterial antigenic

burden and the immunological characteristics that lead to the development of IRD.

Figure 1.7 The potential role of host genetic in events leading to mycobacterial

IRD

(Price et al. 2009)

1.5.3 Opportunistic infections associated with IRD

If IRD represent pathogen-specific immune responses that are immunopathological,

they should differ with the causative pathogen. There are two possible clinical scenarios

of IRD (paradoxical vs. unmasking) defined for tuberculosis. The distinction may apply

to other infections. Initiation of ART in patients who had an active opportunistic

infection that has been treated may result in IRD appearing as a ‘paradoxical’ relapse of

the infection. IRD may also appear as new presentations of subclinical opportunistic


28

infections ‘unmasked’ by restored immune responses. Generally, HIV patients who

initiate ART with severe immunodeficiency (i.e. lower baseline CD4+ T-cell count)

have the highest risk of developing IRD (French 2009). This is true for the AIDS-

defining infections (e.g. CMV) but not for infections that can occur without HIV disease

(e.g. VZV and HCV).

1.5.3.1 Mycobacterium tuberculosis

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis

(MTB). One third of the world’s population is infected with MTB and it is the leading

cause of HIV-related deaths. Worldwide, almost 14 million people are co-infected with

HIV and MTB (World Health Organization). MTB infection may present as pulmonary

TB (approximately 85% of TB cases) and/or disseminated TB involving other organs

such as the lymph nodes, spleen and CNS (Singh et al. 2003).

Primary MTB infection is acquired through the inhalation of airborne droplets

containing mycobacteria. Following phagocytosis of mycobacteria by alveolar

macrophages, innate responses (production of TNFα and IL-12 by APC) and cell-

mediated responses (notably IFNγ production by CD4+ T-cells) are induced to promote

the recruitment and activation of other macrophages, DC and T-cells to the site of

infection. Mycobacteria engulfed in macrophages can resist killing by preventing

phagosome maturation or by the protective effect of their lipid-rich cell wall (Pieters

2008).

Granulomas are formed to prevent the spread of infection and may appear as non-

caseating or caseating. This process is highly dependent on TNFα and IFNγ production

by macrophages and T-cells which may prevent active disease in infected individuals

but viable MTB may remain dormant inside the granuloma. Maintenance of the

immunological components within and surrounding the granulomas is essential to

prevent re-activation of dormant MTB (Chan et al. 2004; Co et al. 2004; Salgame 2005)

(Figure 1.8a and b). Hence, MTB can be reactivated when the host is immuno-

compromised (e.g. by HIV or old age) (Figure 1.8c).


29

Figure 1.8 Architecture of granulomas in separate scenarios

(a) Non-caseating granulomas consist of macrophages (many contain mycobacteria)

surrounded by lymphocytes and a layer of fibroblasts. Lung damage is usually minimal

in this scenario. (b) Caseating granulomas have an acellular core of necrotic material

containing extracellular bacteria surrounded by macrophages and lymphocytes. They

are usually surrounded by secondary lymphoid follicle-like structures. (c) Reactivating

granulomas are disorganised with enhanced MTB replication which may be caused by

immune cell depletion and dysfunction (Salgame 2005).

The Th1-mediated IL-12-IFNγ axis is important in controlling TB disease by ultimately

improving the killing capacity of macrophages, cytotoxic CD8+ T-cells and NK cells

(Figure 1.9) (Young et al. 2008). Knockout mice lacking IFNγ or IL-12 genes cannot

control mycobacterial growth (Flynn et al. 1993; Cooper et al. 1997; Cooper et al.

2002). Children with non-synonymous mutation in their IFNγ receptor gene are highly

susceptible to mycobacterial infection (Newport et al. 1996). Impaired Th1 responses in

HIV patients increase the risk of TB disease (Corbett et al. 2003). Th17 mediated

immune responses (section 1.2) are also involved in protection against MTB infection

(Khader et al. 2008).


30

Figure 1.9 Multiple strategies in fighting against MTB infection

(Young et al. 2008)

In retrospective and prospective studies, up to 43% of HIV/MTB co-infected patients

develop IRD. Most patients present within the first 2 to 6 weeks of ART (Meintjes et al.

2008a). The INSHI group has defined two categories of TB IRD: paradoxical TB IRIS

and ART-associated TB (Figure 1.10 and Figure 1.11). Patients in the latter group have

not received anti-tubercular therapy before starting ART. ART-associated TB can be

further subdivided into “unmasking” events where subclinical TB become symptomatic

due to ART and “immune reconstitution disease” which refer to severe presentation of

TB associated with exaggerated and overtly inflammatory disease (Lawn et al. 2008).

TB IRD patients may present with TB-related symptoms such as fever, coughing and/or

shortness of breath. TB IRD may involve multiple sites which contain mycobacterial

antigens. IRD in the lymph nodes will cause lymphadenitis and/or lymphadenopathy.

TB IRD patient may also develop cold abscess and accumulation of pus under the skin.

A case report showed the pus contained high levels of infiltrating neutrophils (Meintjes

et al. 2008a). Neurologic TB IRD may present as meningitis, tuberculoma or

radiculomyelopathy with a mortality rate of 13% and 26% of patients experiencing

permanent neurologic disability (Pepper et al. 2009).

Immunological data on TB IRD is limited and is largely associated with the restoration

of Th1 (IFNγ responses) and delayed-type hypersensitivity responses against

mycobacterial antigens (Bourgarit et al. 2006; Meintjes et al. 2008c; Elliott et al.


31

2009). This is addressed in Chapter 3. However, in all these studies, some patients have

mycobacteria-specific IFNγ responses but do not develop TB IRD. Furthermore, clinical

symptoms of mycobacterial IRD can be ameliorated by steroids (Meintjes et al. 2010),

but this did not decrease mycobacteria-specific IFNγ responses (Meintjes et al. 2008c).

Hence, Th1 or IFNγ responses may not be a necessary or sufficient prelude of a TB

IRD. TB IRD may involve a range of other pathologies such as the exaggeration of

Th17 responses promoting neutrophil responses in patients who present with cold

abscess and lymphadenitis. Increased innate immune responses are also evident in TB

IRD which may generate suppurative inflammatory responses through increased levels

of soluble mediators (e.g. CXCL10 and IL-18) that promote cellular recruitment to the

site of IRD (e.g. lymph node) (Oliver et al. 2010). The role of innate responses in IRD

is addressed in Chapter 5.

Patients with shorter interval from the diagnosis/treatment of TB infection to the

initiation of ART had higher risk of developing TB IRD (Lawn et al. 2007). Detectable

lipoarabinomannan (LAM; present in the cell wall of mycobacteria) in urine pre-ART

may predict TB IRD. A South African study on 100 HIV/TB co-infected patients with

suspected TB IRD showed that patients with undiagnosed drug resistance MTB strain

are at greater risk of developing TB IRD (Meintjes et al. 2009). These studies support

the model that increased mycobacterial pathogen load increases the risk of developing

IRD (Meintjes et al. 2008b).


32

(A) Antecedent requirements

Both of the two following requirements must be met:

• Diagnosis of TB: The TB diagnosis was made before starting ART and should fulfill WHO criteria for

diagnosis of smear-positive pulmonary TB, smear-negative pulmonary TB or extrapulmonary TB

• Initial response to TB treatment*: The patient’s condition should have stabilized or improved on

appropriate TB treatment before ART initiation (e.g. cessation of night sweats, fevers, cough or weight

loss).

*Note: this does not apply to patients starting ART within 2 weeks of starting TB treatment since

insufficient time may have elapsed for a clinical response to be reported

(B) Clinical criteria

The onset of TB IRIS manifestations should be within 3 months of ART initiation, reinitiation or regimen

change because of treatment failure.

Of the following, at least one major criterion or two minor clinical criteria are required:

(i) Major criteria

• New or enlarging lymph nodes, cold abscesses, or other focal tissue involvement (e.g. tuberculous arthritis)

• New or worsening radiological features of TB (found by chest radiography, abdominal ultrasonography,

CT or MRI)

• New or worsening CNS TB (e.g. meningitis or focal neurological deficit caused by tuberculoma)

• New or worsening serositis (pleural effusion, ascites, or pericardial effusion)

(ii) Minor criteria

• New or worsening constitutional symptoms (e.g. fever, night sweats or weight loss)

• New or worsening respiratory symptoms (e.g. cough, dyspnoea or stridor)

• New or worsening abdominal pain accompanied by peritonitis, hepatomegaly, splenomegaly or abdominal

adenopathy

(C) Alternative explanations for clinical deterioration must be excluded if possible*

• Failure of TB treatment because of TB drug resistance

• Poor adherence to TB treatment

• Another opportunistic infection or neoplasm (it is particularly important to exclude an alternative diagnosis

in patients with smear-negative pulmonary TB and extrapulmonary TB where the initial TB diagnosis has

not been microbiologically confirmed)

• Drug toxicity or reaction

*Note: it may be difficult or impossible in resource-poor settings to confirm TB drug resistance and to exclude

certain other infections or neoplasia. Cases where alternative diagnoses cannot be fully excluded because of

limited diagnostic capacity should be regarded as “probable paradoxical TB IRIS”. In these probable cases,

should resolution of clinical or radiological findings of the suspected IRIS episode occur without a change in

TB treatment or ART having been made, they could then be reclassified as ”paradoxical TB IRIS” cases.

Figure 1.10 Case definition for paradoxical TB IRIS

Adapted from (Meintjes et al. 2008a)


33

(A) ART-associated TB

The authors propose that ART-associated TB (all cases of TB that are diagnosed during ART) should be

defined as follows:

• Patient is not receiving treatment for TB when ART is initiated

• Active TB is diagnosed after initiation of ART

• The diagnosis of TB should fulfill WHO criteria for smear-positive pulmonary TB, smear-negative

pulmonary TB or extrapulmonary TB

(B) Unmasking TB IRIS (provisional)

The following criteria were proposed to suggest a diagnosis of unmasking TB IRIS:

• Patient is not receiving treatment for TB when ART is initiated and then presents with active TB within 3

months of starting ART

AND one of the following criteria must be met:

• Heightened intensity of clinical manifestations, particularly if there is evidence of a marked inflammatory

component to the presentation. Examples include TB lymphadenitis or TB abscesses with prominent acute

inflammatory features, presentation with pulmonary TB that is complicated by respiratory failure due to

adult respiratory distress syndrome, and those who present with a marked systemic inflammatory syndrome

related to TB

• Once established on TB treatment, a clinical course that is complicated by a paradoxical reaction

Researchers in the field are encouraged not to regard all patients with ART-associated TB as having TB IRIS,

but only those that fit this provisional unmasking TB IRIS case definition.

Figure 1.11 Case definition for ART-associated TB and provisional case

definition for unmasking TB IRIS

Adapted from (Meintjes et al. 2008a)


34

1.5.3.2 Cryptococcus neoformans meningitis

Cryptococcus neoformans is a capsulated, single-celled fungus and is the most common

pathogen causing meningitis in immunocompromised individual. Cryptococcal

meningitis refers to the inflammation of the membrane surrounding the CNS due to

invading fungal cells. The route of transmission is similar to MTB infection, i.e. through

the inhalation of fungal spores. Failure to clear the infection due to immunodeficiency

(e.g. AIDS patients) or infection of the virulent strain in immunocompetent individuals

results in systemic invasion causing cryptococcemia, lymphadenitis and/or meningitis.

Cryptococcal infection has emerged as the second most common cause of death (after

TB) in AIDS patients in Uganda (French et al. 2002).

Formation of polysaccharide capsule and the presence of cell wall-associated melanin

are among the main virulence factors of Cryptococcus neoformans. The capsule can

prevent phagocytosis and have immunosuppressive properties due to the presence of

glucuronoxylomannan (Del Poeta 2004; Monari et al. 2006). Stimulation of mouse

derived DC with encapsulated strain of Cryptococcus neoformans induced lower levels

of IL-12 and TNFα production compared with DC stimulated with the acapsular strain.

This will ultimately affect IFNγ-mediated responses (Lupo et al. 2008).

Th1 responses (IFNγ and IL-12) driven by the production of TNFα in the lung are

important in clearing pulmonary C. neoformans (Herring et al. 2002). Development of

Th1 responses was dependent on CCR2-mediated accumulation of DC in the lung

(Traynor et al. 2000; Osterholzer et al. 2008). Increased production of IL-1β, TNFα,

IFNγ, CCL2, CCL5 and CXCL10 in the brain is also associated with protective immune

responses against C. neoformans infection (Uicker et al. 2005). Cryptococcal

mannoproteins are responsible for recognition by mannose receptors on APC (especially

DC) and inducing T-cell responses (Levitz et al. 2006).

Cryptococcal IRD may present as meningitis, localized inflammatory lesions in the

brain or spinal cord, or lymphadenitis, despite the use of anti-fungal therapy (Figure

1.12 and Figure 1.13) (Skiest et al. 2005; Haddow et al. 2010). It affects 19 to 50% of

HIV patients initiating ART with a prior diagnosis of cryptococcal disease but some

cases may appear as “unmasking” events, i.e. patients who were not diagnosed with

cryptococcal disease prior to ART (Price et al. 2009). Risk factors for cryptococcal IRD

include previous presentation of AIDS-related cryptococcal meningitis, high titres of


35

cryptococcal antigen in the cerebrospinal fluid (CSF) and initiating ART within 30 to 60

days of cryptococcal diagnosis. Patients who experienced cryptococcal meningitis IRD

had higher CSF opening pressures, glucose levels and white blood cells than patients

who presented with AIDS-related cryptococcal meningitis (Shelburne et al. 2005). CSF

or blood may have detectable cryptococcal antigens but are usually culture negative

(Jenny-Avital et al. 2002). It is suggested that cryptococcal IRD is caused by

immunopathological responses to cryptococcal antigens in HIV patients, but this has

never been demonstrated. It is addressed in this thesis in Chapter 3.

(A) Antecedent requirements

• Taking ART

• Cryptococcal disease diagnosed before ART by positive culture or typical clinical features plus positive

India ink staining or antigen detection

• Initial clinical response to antifungal therapy with partial or complete resolution of symptoms or signs,

fever, or other lesions, or reduction in CSF cryptococcal antigen concentration or quantitative culture

(B) Clinical criteria

• Event occurs within 12 months of ART initiation, reintroduction, or regimen switching after previous

failure

• Clinical disease worsening with one of the following inflammatory manifestations of cryptococcosis:

⇒ Meningitis

⇒ Lymphadenopathy

⇒ Intracranial space-occupying lesion or lesions

⇒ Multifocal disease

⇒ Cutaneous or soft-tissue lesions

⇒ Pneumonitis or pulmonary nodules

⇒ Other rarer manifestations (Haddow et al. 2010)

(C) Other explanations for clinical deterioration to be excluded

• Non-adherence or suboptimum antifungal therapy, indicated by an increase in quantitative culture or

antigen titre or any positive cryptococcal culture after 3 months of antifungal therapy

• Alternative infection or malignant disease in the affected site

• Failure of ART excluded if possible (e.g. failure to achieve ≥1 log10 copies/mL decrease in viral load by 8

weeks of ART)

Figure 1.12 Case definition for paradoxical cryptococcal IRIS

Adapted from (Haddow et al. 2010)


36

(A) ART-associated cryptococcosis

• Patient taking ART

• No recognized cryptococcal disease at ART initiation

• Clinical disease worsening caused by cryptococcosis occurs after initiation, re-introduction, or regimen

switch after previous failure (supported by microbiological, histological, or serological evidence)

• Cryptococcal infection characterized by meningitis, CNS complications, skin or soft-tissue lesions,

lymphadenopathy, lung disease, or disseminated disease

(B) Unmasking cryptococcal IRIS (provisional)

• Criteria for ART-associated cryptococcosis are met

• Unusual, exaggerated, or heightened inflammatory manifestations, such as the following:

⇒ Meningitis with CSF WBC >50×106/L or CSF opening pressure >20 cm that is refractory to therapy

⇒ Painful or suppurating lymphadenopathy

⇒ Rapidly expanding CNS lesions, cryptococcomas

⇒ Unusual focal site (i.e. not within the CNS, lung, skin, or lymph nodes)

⇒ Granulomatous inflammation on histology

⇒ Pneumonitis, particularly if cavitating or necrotic

• Event occurs early after ART initiation*

• Failure of ART excluded if possible (e.g. ≥1·0 log10 copies/mL decrease in HIV-1 viral load by 8 weeks

treatment)

*No specific time limit was proposed for unmasking cryptococcal IRIS, pending further research. Typically,

onset within 3 months of starting ART could be assumed to support a diagnosis of IRIS owing to early and

rapid changes in immune function. However, late presentations of cryptococcal IRIS have been reported in

patients with good responses to therapy assessed by CD4+ cell counts.

Figure 1.13 Case definition for ART-associated cryptococcosis and unmasking

cryptococcal IRIS

Adapted from (Haddow et al. 2010)


37

1.5.3.3 Cytomegalovirus causing retinitis

CMV is a member of the herpesvirus family which has the ability to remain latent in the

host. Other members include the HSV type 1 and 2, VZV, Epstein Barr virus (EBV) and

HHV (type 6-8). CMV disease occurs in the setting of severe immunodeficiency (e.g.

AIDS, old-age and immuno-suppressive drugs), leading to blindness, neurological

disorders or death (Gilden et al. 2007). Retinitis is the most common manifestation of

CMV disease in HIV patients in the pre-ART era, affecting up to 40% of American

AIDS patients (Springer et al. 2004). Data from Thailand suggest that 5-25% of all HIV

patients in the developing world can be expected to develop this “blinding disorder” at

some point in their illness. Hence screening for CMV retinitis is recommended for all

HIV patients with CD4+ T-cell counts below 50 cells/µL (Heiden et al. 2007).

The incidence of CMV diseases has decreased since the introduction of ART, but

recurrences of CMV retinitis or uveitis as an IRD (also known as immune recovery

uveitis or retinitis) were observed in a proportion of ART-treated patients (Jacobson et

al. 1997; Casado et al. 1999). Uveitis refers to the inflammation of the uveal tract

comprising the iris, ciliary body and choroid. Detectable plasma CMV viremia at the

time of ART initiation and increased CD4+ T-cell counts were associated with the

development CMV IRD (Casado et al. 1999; Karavellas et al. 2001). CMV IRD

(presenting as retinitis or encephalitis) was associated with nadir CD4+ T-cell below 30

CD4+ T-cells/µL and increased plasma levels of anti-CMV IgG antibodies, soluble

CD30 and IL-6 (Stone et al. 2001; Stone et al. 2002b). Although T-cell activation

(increased soluble CD30) was associated with CMV IRD, CMV-specific CD4+ T-cell

responses were consistently low in patients who develop CMV IRD and in those who

develop AIDS-related CMV disease (Gerna et al. 2001; Hsieh et al. 2001; Price et al.

2001a; Keane et al. 2004). Hence, I proposed the role of NK cell-mediated immune

responses in CMV IRD (Chapter 4).

Low nadir CD4+ T-cell counts correlated with low CMV-specific IFNγ production but

high IFNγ production by NK cells (Price et al. 2008). Increased NK cell activity may

compensate for deficiency in CD4+ T-cell function in ART-treated patients (Chapter 7).

This suggests innate responses mediated by NK cells may be more prominent than T-

cell responses in CMV IRD (Chapter 4). Evidence for the role of NK cells in IRD is

limited, but higher numbers of activating KIRs (section 1.3.4) were observed in patients

who experienced CMV IRD than non-IRD controls (Price et al. 2007).


38

1.6 Aims of the thesis

HIV patients who initiate ART with advanced immunodeficiency and several

opportunistic infections are at highest risk of developing IRD or experiencing

inadequate immune recovery. Based on current literature, it is unclear why some

patients experience these adverse outcomes whilst others do not. This thesis

characterises the immunological profiles of patients with effieicent and uneventful

immune recovery, patients who develop IRD and patients with poor immune recovery.

This is achieved using longitudinal sample sets from HIV patients enrolled into an

immune reconstitution study in Kuala Lumpur, Malaysia (section 2.1) and a study of

West Australian patients with a stable response to ART (Chapter 7). Ultimately, better

understanding of the immunological features of a response to ART may allow us to

identify patients who at risk of adverse outcomes and to optimize their therapy.

1.6.1 Part 1: Cellular mediators of immune restoration disease

The development of IRD is associated with the restoration of pathogen-specific adaptive

responses involving Th1-associated cytokines. Supporting data have mostly involved

patients who developed mycobacterial IRD. Hence, IRD associating with other infection

such as Cryptococcal meningitis and CMV were also investigated. Increased in Th1

responses on ART have been associated with IRD, but careful comparisons with

equivalent control groups have yielded uneven results. Details of other potential

immunopathological mechanisms of IRD are limited and no studies have addressed the

role of innate immune cells in this context. Hence, the role of T-reg, NK cell and

DC/monocytes in IRD are evaluated. The characteristics of NK cells will be described

in patients who presented with TB, Cryptococcal, CMV and VZV IRD compared to

non-IRD controls (Chapter 4). The roles of TLR-mediated DC/monocytes immune

responses are determined in Chapter 5.

1.6.2 Part 2: Cellular mediators of immune recovery on ART

The immunological outcome of ART is routinely assessed using peripheral blood CD4+

T-cell numbers. Here HIV patients who have good or poor recovery of CD4+ T-cells

during 1 year of ART will be identified based on rate of CD4+ increases on ART. This

will then be related to the recovery pathogen-specific T-cell responses. As the functions

of CD4+ T-cells, NK cells and dendritic cells are inter-related, phenotypic and

functional parameters of these cell types will be correlated with the recovery of CD4+ T-

cell counts after ART.

39

Chapter 2

Methods and Materials

This chapter presents general descriptions of the assays and

antigens/stimulants used throughout my thesis. The assays include

ELISpot, ELISA and flow cytometry. Each result chapters will then have

a separate section to describe the cohort of patients used and assays

performed.

Chapter 2 - Methods and Materials

40


2.1 Immune Reconstitution Study (UMMC, KL, Malaysia)

This was a prospective observational cohort study of immune reconstitution and its

consequences in immunodeficient HIV-infected patients beginning ART. Patients were

enrolled at the Infectious Disease Out-patient Clinics in the University Malaya Medical

Centre (UMMC), Kuala Lumpur (KL), Malaysia under the direction of Professor

Adeeba Kamarulzaman. In this clinic, ART was recommended to HIV patients who

were asymptomatic and presented with less than 200 CD4+ T-cells as suggested by the

WHO when the cohort was established in year 2004. Patients who were commencing

ART with less than 200 CD4+ T-cells were invited to join the study.

Blood samples were collected at Weeks 0, 6, 12, 24 and 48 plus an additional sample

during an IRD event from patients. Samples were collected once from healthy controls.

Patients with no evidence of IRD and healthy controls were selected to match with the

IRD patients by sex (male), ethnicity (Chinese) and age. Healthy controls were mostly

hospital staff, residing in Kuala Lumpur and presumed to be HIV-negative. Recruitment

of patients and controls was ongoing since 2006, so the numbers of IRD sample sets

used in Chapters 3 to 6 were changed over time. Institutional ethics approval was

obtained for the study and informed consent was given by all participants.

The inclusion criteria:

1) Age: >18 years old

2) Starting ART with <200 CD4+ T-cells/µL

3) ART naïve at enrolment

The exclusion criteria:

1) Defaulted ART

2) Autoimmune disease (e.g. Type-1 Diabetes, Systemic Lupus Erythematosus etc)

3) Immune cell-related malignancies such as lymphoma

4) Pregnancy

General definition of an IRD:

1) Adherence and a virological response to ART (i.e. decreased viral load)

2) New or worsening symptoms of opportunistic infection

IRD observed in the cohort include TB (n=9), cryptococcal (n=4), CMV (n=3), VZV

(n=4), Kaposi’s sarcoma (n=3) and histoplasmosis (n=1) by the end of 2009.


41

2.2 Sample collection, plasma HIV RNA level and CD4+ T-cell

counts

Blood was collected into ethylene-diamine-tetra-acetic acid (EDTA) treated tubes and

centrifuged. Plasma was collected and stored at -80oC. Peripheral blood mononuclear

cells (PBMC) were then obtained by Ficoll gradient centrifugation and cryopreserved in

liquid nitrogen. The method was standardized carefully between the laboratories in

Perth and KL. Samples were processed by Steve Roberts (Perth), Paul Teoh (KL), Hong

Yien Tan (KL), Siew Phooi Teh (KL) and Yeat Mei Lee (KL). I thank these people for

their contribution to this thesis.

Routine plasma HIV RNA levels and CD4+ T-cell counts were available pre-ART and

at least twice during the first year of ART (i.e. at approximately Week 12-24 and/or

Week 24-48). Plasma HIV-1 RNA was measured using the COBAS Amplicor HIV-1

Monitor Test, v.1.5 (Roche Diagnostics, Indianapolis, In, USA). The cut-off defining an

undetectable viral load was <50 HIV-1 RNA copies/mL. CD4+ T-cell counts were

quantified by flow cytometry.

2.3 IFNγ ELISpot

Enzyme-linked immunospot (ELISpot) is used to quantify antigen-specific IFNγ-

producing cells. Number of spots in a well represents the number of IFNγ-producing

cells. Total numbers of antigen-specific IFNγ-producing cells were calculated by

subtracting the number of spots in unstimulated wells from the number of spots in

antigen-stimulated wells.

Nitrocellulose 96-well plates (Millipore, MA, USA) were coated with 90µl anti-human

IFNγ antibody (15µg/ml; Mabtech, Stockholm, Sweden) diluted in sterile-filtered 0.1 M

bicarbonate buffer (pH 9.5) and incubated overnight at 40C. The plates were washed six

times with 200ul PBS before adding PBMC in RPMI 1640 with 10% fetal calf serum

(FCS) at 1.0 x 105 to 2.0 x 10

5 cells per well and stimulated with antigens for 18-20hrs.

Each well had a final volume of 200ul. After the incubation, plates were washed six

times with 200ul PBS.


42

Biotinylated anti-human IFNγ detection antibody (7-B6-1 biotin; Mabtech, Stockholm,

Sweden) was diluted to 1ug/ml in filtered 0.5% FCS/PBS, added to each well

(100ul/well) and incubated for 2 hours at room temperature. Plates were then washed

six times with 200ul PBS. Streptavidin horseradish peroxidase (BD Biosciences

Pharmingen, CA, USA) was diluted 1 in 1000 in filtered 0.5% FCS/PBS, added to the

wells (100ul/well) and incubated for 1 hour at room temperature. Plates were then

washed six times with 200ul PBS. 100ul of filtered TMB substrate (Mabtech,

Stockholm, Sweden) was added to each well and left for 1 minute for color

development. The reaction was stopped by washing the plate with distilled water four

times. After the plate was dry, the spots were counted using the AID ELISpot Reader

v2.9 software (Autoimmun Diagnostika GmbH, Strassberg, Germany)

Table 2.1 ELISpot buffers and solutions

Buffers and Solutions Source Concentration/ Volume

Coating buffer: Dissolved in 1L dH20

Na2CO3 1.59g

NaHCO3

AnalaR, BDH Chemical Ltd.,

Poole, England 2.55g

Wash buffer:

1X PBS Sigma-Aldrich, Inc.,

St. Louis, Mo, USA 1 tablet per 200mL dH2O

Culture medium:

RPMI Invitrogen Australia Pty. Ltd.,

Victoria, Australia

FCS Invitrogen Australia Pty. Ltd.

10% FCS/RPMI

2.4 ELISA

Enzyme-linked immunosorbent assay (ELISA) was used to measure the concentration

of proteins (antibodies or cytokines) in plasma samples or culture supernatants. The

optical density generated is proportional to the concentration of the protein of interest

and can be quantified in comparison to a standard of known concentration. ELISA

methods employed in Chapter 3 and Chapter 5 are described in detail in section 3.2.4

and 5.2.4 respectively.


43

Table 2.2 ELISA buffers and solutions


Coating buffer: Dissolved in 1L dH20

Na2CO3 1.59g

NaHCO3 AnalaR, BDH Chemical

2.55g

Wash buffer:

1X PBS 1 tablet per 200ml distilled

H2O

Tween-20

Sigma-Aldrich

0.05% in 1X PBS

Reagent diluent:

BSA Sigma-Aldrich 1% BSA in 1X PBS

Phosphate/Citrate buffer

Stop solution 2N H2SO4

TMB tablets Sigma-Aldrich

Dissolve one tablet in

10mL of phosphate/citrate

buffer

2.5 Flow cytometry

Flow cytometry can be used to measure the physical (e.g. size and granularity) and

chemical characteristics of cells. Using fluorochrome-conjugated monoclonal

antibodies, cellular protein expression (e.g. lineage markers) or function markers (e.g.

cytokines and cytotoxic molecules) can be assessed. Cells are passed through the

“sensing point” in a single line through hydrodynamic focussing. Optical signals are

then detected and converted to digital signals which can be viewed on a computer.

In this thesis, all samples utilized are frozen PBMC. Hence after PBMC were thawed in

RPMI and cell count performed, PBMC were washed in flow buffer and stained with

monoclonal conjugated antibodies against cellular surface markers for 15 minutes at

room temperature. If required, staining of intracellular markers were performed using

kits or reagents, according to manufacturer’s protocols. These involve fixation and

permeabilisation of cells. All staining was performed in the dark. After staining, cells

were resuspended in 200µL flow buffer, fixed with a drop of 1% paraformaldehyde and

kept at 4oC before data acquisition. Data were acquired using a BD FACSCalibur or a

FACS Canto II flow cytometer (BD Biosciences, San Jose, CA, USA) and analysed

using the FlowJo program v. 5.7.2 (Tree Star, Ashland, OR, USA).


44

Table 2.3 Flow buffers and solutions


Flow Buffer:

1 x PBS 1 tablet/200ml distilled

H2O

NaN3 2 g/L

BSA

Sigma-Aldrich

10 g/L

Flow Fixative:

Paraformaldehyde BDH Chemical Ltd. 1 % in 1X Flow Buffer

2.6 Antigens/Stimulants

2.6.1 Anti-CD3

Anti-CD3 (Mabtech, NSW, Australia) is a mouse monoclonal antibody which binds the

ε chain of human T-cell receptor (TCR; CD3) and thus activate all T-cells. It was stored

at 4oC and used at a concentration of 10ng/ml. In ELISpot, anti-CD3 was used to

stimulate 5 x 104 and 2.5 x 10

4 PBMC per well.

2.6.2 CMV lysate

Human CMV strain (AD169) was kindly provided by Dr Hazel Freeland (Department

of Microbiology, Royal Perth Hospital; RPH). CMV was propagated in human

fibroblasts infected at a high multiplicity. After 7 days, the cells were harvested and

sonicated in 0.1 M glycine buffer (pH 9.5), aliquoted and stored at -80oC. CMV lysate

was used at an optimal concentration, previously selected by antigen titration using

ELISpot assays of healthy CMV-seropositive donors (Keane et al. 2000).

2.6.3 Cryptococcal mannoprotein

Cryptococcal mannoprotein served as critical cryptococcal antigens to stimulate T-cell

responses. It is recognise by mannose receptors expressed by APC, particularly DC

(Levitz et al. 2006). Mannoprotein preparation of Cryptococcus neoformans, (acapsular

strain Cap67) was kindly provided by Dr Stuart M. Levitz (University of Massachusetts

Medical School, Worcester, MA, USA). Details of antigen isolation can be found in

(Mansour et al. 2002). Antigens were stored at -80oC and used at a concentration of

50ug/ml.


45

2.6.4 K562 cell line

K562 erythroleukemia cell line was derived from a patient with chronic myeloid

leukemia and is sensitive to NK cell cytotoxicity due to very low expression of HLA

class I and class II (Ziegler et al. 1981). K562 cells were kindly provided by Dr

Campbell Witt (Department of Clinical Immunology, Royal Perth Hospital, WA,

Australia) and cryo-preserved in liquid nitrogen for long-term storage. A week prior to

use, K562 cells were thawed in plain RPMI, washed and grown in 5% FCS/RPMI. The

cells were then split 1:10 every 3 days. The cells were washed in RPMI a day prior to

using for an ELISpot assay. Before adding to PBMC, K562 cells were washed in RPMI,

cell counts performed using Trypan Blue to exclude dead cells and cells were

resuspended in culture medium (10% FCS/RPMI). The Effector (PBMC) to Target

(K562 cell) ratio of 10:1 yielded the highest responses and was selected in preliminary

experiments for presentation.

2.6.5 Lipomannan

Lipomannans (LM) are lipoglycans composed of a mannan core and a glycosyl-

phosphoinositol anchor which are present in the cell wall of all mycobacteria species.

LM induce strong inflammatory activity regardless of which Mycobacterium species it

is isolated from (Dao et al. 2004). Immune responses to LM are TLR2 and MyD88

dependent (Quesniaux et al. 2004). LM derived from Mycobacterium smegmatis were

obtained from Invivogen (San Diego, CA, USA) and used at a concentration of

10µg/mL.

2.6.6 Lipopolysaccharide

Lipopolysaccharide (LPS) is present in the outer membrane of gram negative bacteria

including Escherichia coli and Salmonella. It is recognized by TLR4, LBP and CD14

expressed on monocytes and myeloid dendritic cells (Kinter et al. 2004). LPS derived

from E. coli were obtained from Sigma-Aldrich Inc. (St. Louis, Mo, USA) stored at 4oC

and used at a concentration of 1 µg/mL.

2.6.7 PHA

Phytohaemagglutinin (PHA; Invitrogen) is a mitogenic lectin molecule extracted from

the red kidney bean Phaseolus vulgaris. PHA is capable of activating T-cells by binding

to cell membrane glycoproteins including the TCR complex (Kay 1991). It was stored at

-20oC and used at a concentration of 0.5% v/v.


46

2.6.8 Tuberculin PPD and ESAT-6

Tuberculin is a purified protein derivative (PPD) prepared from a culture of seven

selected strains of MTB which were then heat-sterilized and filtered (Magnusson et al.

1958). Tuberculin PPD is commonly used in Mantoux skin test to diagnose previous

infection with MTB or vaccination with Bacille Calmette-Guerin (BCG). A positive

reaction does not indicate an immune response specifically to MTB, as there is cross

reactivity with M. bovis, M. africanum, M. microtii and non-tuberculous mycobacteria.

ESAT-6 (early secreted antigenic target, 6kDa) is a small family of proteins secreted

specifically by MTB which can be used to stimulate MTB-specific T-cell responses

(Berthet et al. 1998; Brodin et al. 2004). Unlike PPD, ESAT-6 is a protein specifically

secreted by M. tuberculosis, but not in other mycobacteria such as the vaccine strains of

M. bovis BCG (Harboe et al. 1996). In active tuberculosis, there is strong recognition

and response to ESAT-6 by T-cells measured by proliferation and IFN-γ production

(Mustafa et al 1998). Both PPD and ESAT-6 were obtained from Statens Serum Institut,

(Copenhagen, Denmark) stored at -80oC and used at concentration of 10ug/ml.

2.6.9 VZV lysate

The VARIVAXTM

vaccine (Oka/Merck strain) was obtained from Merck, Sharp and

Dohme, South Granville, BSW, Australia and cultured in MRC-5 cells. VZV lysate was

prepared as in section 2.6.2 and optimal concentration were determined by IFNγ

ELISpot using PBMC from sero-positive healthy controls.

47

Chapter 3

Immunological profiles of immune restoration

disease presenting as mycobacterial

lymphadenitis and cryptococcal meningitis

Data from this chapter has been published. There are minor modifications

reflecting further work has been carried out since 2008.

Immunological profiles of immune restoration disease presenting as

mycobacterial lymphadenitis and cryptococcal meningitis

Tan DBA, Yong YK, Tan HY, Kamarulzaman A, Tan LH, Lim A, James I,

French M, Price P. HIV Med 2008; 9:307-316

The authors thank Professors Kee Peng Ng and Shamala Devi for laboratory facilities,

Dr Stuart M. Levitz for the kind provision of cryptococcal mannoprotein and the

patients and controls who contributed to this study. The project was supported by

Infectious Diseases Research Fund, UMMC and UWA. This is publication 2007-33

(Clinical Immunology and Immunogenetics, RPH, Australia).

Parts of the data in this chapter were also presented in this publication:

Proportions of circulating T-cells with a regulatory cell phenotype

increase with HIV-associated immune activation and remain high on

antiretroviral therapy

Lim A, Tan D, Price P, Kamarulzaman A, Tan HY, James I, French MA.

AIDS 2007;21:1525-1534

Chapter 3 - Immunological profiles of immune restoration disease

48

Chapter 3 - Immunological profiles of immune restoration

disease presenting as mycobacterial lymphadenitis and

cryptococcal meningitis

A proportion of HIV patients may develop IRD after starting ART. Immunological

characteristics of IRD were investigated in a cohort of HIV patients beginning therapy

in UMMC, KL, Malaysia where samples were collected from patients at weeks 0, 6, 12,

24 and (in some cases) 48 of ART. When this study was performed, samples were

available from five patients experiencing IRD (2 with cryptococcal and 3 with MTB

disease), eight non-IRD controls who had begun ART with CD4+ T-cell counts of <100

cells/µL and 17 healthy controls. IFNγ-producing PBMC were quantified by ELISpot

after stimulation with PPD, ESAT-6, Cryptococcus neoformans or CMV antigens.

Plasma IgG reactive with these antigens were assessed by ELISA. Proportions of

activated (HLA-DRhi

) and regulatory (CD25+CD127

lo and CTLA-4

+) CD4

+ T-cells

were quantitated by flow cytometry.

TB IRD patients displayed elevated IFNγ responses and/or plasma IgG to PPD, but

none responded to ESAT-6. Cryptococcal IRD occurred in patients with low baseline

CD4+ T-cell counts and involved clear IFNγ and antibody responses to cryptococcal

antigen. This is the first report of the immunology of cryptococcal IRD. Proportions of

activated and regulatory CD4+ T-cells declined on ART, but remained higher in patients

than healthy controls. At the time of IRD, proportions of activated CD4+ T-cells and

regulatory CD4+ T-cells were generally elevated relative to other patients. Cryptococcal

and TB IRD generally coincide with peaks in the proportion of activated T-cells,

pathogen-specific IFNγ responses and reactive plasma IgG, but did not reflect a paucity

of regulatory CD4+ T-cells.

3.1 Background

Up to 10-40% of patients beginning ART with advanced immunodeficiency experience

IRD associated with common opportunistic pathogens. The timing of these events

coincides with increases in CD4+ T-cell counts on ART, suggesting that restored

immune responses can be immunopathological rather than protective (section 1.5).

However, few longitudinal sample sets have been available to characterize these

responses.


49

(Bourgarit et al. 2006) described dramatic IFNγ responses by cells from seven patients

who developed paradoxical TB IRIS (section 1.5.3). PPD-induced Th1 responses from

cultured PBMC were elevated at the time of IRD, suggesting broad-based immune

activation. Th1-related cytokines that were elevated included IL-2, IL-12, IFNγ, and

CXCL10, Other pro-inflammatory cytokines/chemokines affected were TNFα, IL-6, IL-

1β, IL-10, CCL5 and CCL2. However, PBMC from these patients did not respond to

secreted mycobacterial proteins such as ESAT-6 and 85B protein (Bourgarit et al.

2006). These antigens are only released by viable bacteria, so paradoxical IRD may be

induced by residual antigens from treated infections. This requires confirmation.

Elevated humoral immune responses have been associated with IRD. Patients who

developed CMV retinitis as an IRD has a steady increase in plasma IgG reactive to

CMV antigens in the first year of ART (Stone et al. 2002b). Similarly, HIV and

hepatitis-C virus (HCV) co-infected patients who developed hepatotoxicity and clinical

hepatitis after ART had increased HCV core-specific IgG (Stone et al. 2002a). Serum

antibodies against several mycobacterial antigens (notably PGL-Tb1) mark

symptomatic TB (Lyashchenko et al. 1998; Simonney et al. 2007), but have not been

addressed in an IRD. Levels of pathogen-specific antibodies before and during ART

may help predict and/or diagnose IRD, or shed light on pathogenic mechanisms.

In patients with no previous diagnosis of cryptococcal infection, presentations of

cryptococcal disease in the context of increased CD4+ T-cell counts and decreased

plasma HIV RNA on ART are defined as ‘unmasking’ IRD. They usually present as

central nervous system disease (meningitis or focal enhancing mass lesions) early after

ART or as lymphadenitis within 15 months on ART (Jenny-Avital et al. 2002;

Shelburne et al. 2005; Skiest et al. 2005). Histological examination of lymph nodes may

show granulomatous inflammation, necrosis or suppuration (section 1.5.3.2).

Natural T-reg can suppress the activation, proliferation and production of cytokines by

CD4+ and CD8

+ T-cells (section 1.3.3). Depletion of T-reg from PBMC of HIV-infected

patients increased HIV- and CMV-specific responses by CD4+ and CD8

+ T-cells

(Aandahl et al. 2004). Hence, a deficiency in T-reg may promote the elevated T-cell

responses characteristic of an IRD. However, proportions of T-reg and activated CD4+

T-cells are correlated during the first year of ART (Lim et al. 2007), so T-reg

populations may increase after an IRD as a consequence of immune activation.


50

In this study, T-reg were identified as CD4+CD25

+CD127

lo and CD4

+CTLA-4

+ T-cells.

Low expression of IL-7 receptor (CD127) with co-expression of CD25 identifies CD4+

T-reg expressing the transcription factor FoxP3 (Liu et al. 2006; Seddiki et al. 2006).

These markers differentiate T-reg from activated T-cells, which may also express

CD25. CD4+ T-cells expressing the inhibitory receptor CTLA-4 were assayed because

they influence down-regulation of effector T-cell activation (Read et al. 2000).

3.2 Patients and Methods

3.2.1 Study subjects

Five Chinese male HIV patients with cryptococcal (n=2) or TB (n=3) IRD were

identified from the cohort study of immune reconstitution in UMMC (section 2.1).

Patients with no evidence of IRD (non-IRD; n=8) were selected to match with the IRD

patients by sex (male), ethnicity (Chinese) and age (p=0.22). Healthy Chinese male

donors (n=17; younger than HIV patients, p=0.01) who were mostly hospital staff,

residing in Kuala Lumpur and presumed to be HIV-negative were included as controls.

Non-IRD patients and healthy controls had no known history of cryptococcal disease,

but several patients had a history of MTB and vaccination with BCG is standard

practice in Malaysia.

3.2.2 Sample collection and processing

Refer to section 2.1.

3.2.3 ELISpot assay to quantify the frequency of antigen-specific

IFNγ-producing cells

Refer to section 2.3. PBMC (2 x105 and 1 x 10

5 cells) were stimulated with PPD,

ESAT-6 (section 2.6.8), CMV lysate (section 2.6.2) or Cryptoccus neoformans

mannoprotein (section 2.6.3). ELISpot data were expressed as number of IFNγ-

producing cells per 200,000 PBMC.

3.2.4 ELISA to measure pathogen-reactive IgG in plasma

Half-volume 96-well plates were coated with 50µL/well PPD (section 2.6.8), CMV

lysate (section 2.6.2) or C. neoformans mannoprotein (section 2.6.3) overnight at 4oC.

Wells were then washed and blocked with 100µL/well 5% bovine serum albumin


51

(BSA)/PBS for 2 hours at room temperature. Wells were then washed before adding

samples and standards (50 µL/well) diluted in 1% BSA/PBS for 2 hours at room

temperature. Pooled plasma samples from CMV, Cryptococcal and PPD-seropositive

individuals were used as standards and assigned an arbitrary value of 100units/mL of

antigen-specific IgG. Wells were then washed and incubated with anti-human IgG

peroxidase conjugate (Jackson Immunoresearch Labs, Inc. West Grove, PA, USA) for 2

hours at room temperature. Wells were then washed and developed with 50µL/well

TMB substrate for 15 mins. The reaction was stopped by adding 25µL/well stop

solution and absorbance was read at 450 nm. ELISA buffers are described in section

2.4. Coefficients of variance were below 15%.

3.2.5 Flow cytometry assay to assess T-cell subsets

5 x 105 PBMC were resuspended in 100µL flow buffer and surface-stained for 15

minutes at room temperature with CD4-PECy5, CD25-FITC, CD127-PE and/or HLA-

DR-PE (Coulter Immunotech, Marseille, France). CTLA-4-PE required intracellular

staining achieved using Intraprep reagents (Coulter) applied according to the

manufacturer’s protocol. All staining was performed in the dark. After staining, cells

were resuspended in 400 µL buffer, fixed with a drop of 1% paraformaldehyde and kept

at 4oC before acquisition. 50,000-100,000 events were acquired using a BD

FACSCalibur flow cytometer and analysed using the FlowJo program v. 5.7.2 (Tree

Star, Ashland, OR, USA). Figure 3.1 show gating strategies for each cell population.

3.2.6 Statistical analyses

Mann-Whitney U-Tests were used to compare groups of individuals. No longitudinal

analyses were applied to the IRD patients because the groups were too small.

Longitudinal data from the eight non-IRD patients were analysed on a log10 scale using

continuous piecewise-linear mixed models with a change point at week 15 of ART. The

slope estimates and p-values of the plots relate to these two segments, testing for

divergence from a slope of zero. Statistically significant differences are shown in

figures. Analyses were carried out in S-Plus 7.0 for Windows (Insightful Corporation,

Seattle, WA, USA). P-values below 0.05 were considered to be statistically significant.


52

3.3 Results

3.3.1 CD4+ T-cell counts and plasma HIV RNA levels confirm all

patients responded to ART

All HIV patients (IRD and non-IRD) had plasma HIV RNA levels >100,000 copies/ml

prior to ART and <100 copies/ml after 8-27 weeks on ART. All patients had lower

proportions of CD4+ T-cells (expressed as % of lymphocytes) prior to ART compared to

healthy controls. Proportions of CD4+ T-cells in all HIV patients increased during the

year of study [12% (4.1-20%) at the last sample collection], but remained lower than

healthy controls [33% (20-41%), p < 0.001]. The increase in proportions of CD4+ T

cells was significant in non-IRD patients over the first 15 weeks on ART (slope = 0.02,

p = 0.002). A similar increase was observed in the IRD patients (Figure 3.2).

3.3.2 Clinical presentations and microbiological assessment of IRD

The five cases of IRD presented as atypical opportunistic infections after ART (French

et al. 2004). Two patients experienced ‘unmasking’ cryptococcal IRD and three patients

experienced ‘paradoxical’ TB IRIS. The clinical presentations of IRD and the diagnostic

tests performed are summarised in Table 3.1.

One cryptococcal IRD patient (denoted C1) presented with meningitis 2 weeks after

beginning ART, while the other (denoted C2) presented with cryptococcal meningitis

and cryptococcaemia after 3 weeks on ART. In both patients, symptoms began about 1

week before diagnosis, which was based on examination of CSF. Cryptococcal antigen

was titred by latex agglutination and Cryptococcus neoformans was cultured from the

CSF. Cryptococcus neoformans was isolated from blood in C2 only. Patients were

treated with amphotericin B and fluconazole. Symptoms resolved in C1 with no further

investigations. In C2, a second lumbar puncture had elevated opening pressure and

lower titre of cryptococcal antigen in the CSF. There was no fungal growth in CSF or

blood cultures at that time.

All TB IRD patients had presented with active TB infection 4-7 weeks before starting

ART and received ATT. Paradoxical TB IRIS was diagnosed at 3 weeks (T1), 12 weeks

(T2) and 14 weeks (T3) after commencing ART. All three patients presented with fever

and one or more enlarged lymph nodes 1-2 weeks before diagnosis, which was based on

exacerbations of cervical lymphadenitis, lymphadenopathy and lymph nodes evolving

into abscesses or cold abscess enlargement. T1 had a worsening left parotid abscess.


53

3.3.3 IFNγγγγ responses to antigens of provoking pathogens peaked

at the time of IRD

Samples were collected on 2-6 (median 5) occasions from each patient over a period of

14-53 (median 47) weeks after commencement of ART, with the exception of T1 who

was recruited at week 3 of ART. Antigen-specific IFNγ responses were measured to

assess the hypothesis that IRD is caused by increased cell-mediated responses to

antigens of opportunistic pathogens. The upper limit of the ELISpot assay was 2,000

spots per 200,000 cells. The median and interquartile ranges of ELISpot counts recorded

in healthy control donors (n=17) are presented for comparison.

IFNγ responses to C. neoformans antigens peaked at the time of IRD in C1 and C2

(Figure 3.3a). These responses were approximately ten-fold higher than those seen in

any of the non-IRD patients or healthy controls. After the IRD episodes, responses

declined over the year of study.

Two TB IRD patients (T1, T2) exhibited strong IFNγ responses to PPD which remained

high throughout the study (Figure 3.3b). T3 exhibited a small increase in his PPD

response on ART (13 to 443 spots per 200,000 cells) which peaked prior to the

presentation of IRD. The low response may reflect his low baseline proportions of CD4+

T-cells (data not shown). Tuberculin skin tests performed on T2 and T3 were recorded

as Mantoux negative during their IRD episode, but the clinic considered values below

10mm to be negative.

IFNγ responses to PPD in non-IRD patients increased significantly within 15 weeks of

ART (Figure 3.3b) with no significant change thereafter. One non-IRD patient had peak

IFNγ responses above 400 spots per 200,000 PBMC with no signs of TB disease pre-

and post-ART. This is above the median for healthy controls (120 spots). His Mantoux

tests were recorded as negative at baseline and after 24 weeks on ART. However, he

had higher proportions of CD4+ T-cells before ART than all other patients, so a robust

protective response to mycobacterial antigen was suspected. In other non-IRD patients,

PPD responses increased to levels similar to healthy controls during the first year of

ART.

IFNγ responses to ESAT-6 antigen in TB IRD patients (Figure 3.3c) did not follow the

pattern seen with PPD or reach the median response of healthy controls. The


54

cryptococcal IRD patients (C1 and C2) displayed increased responses to ESAT-6 on

ART, whilst there was no significant increase in responses of non-IRD patients.

CMV was included as a control antigen because no patients experienced CMV IRD.

IFNγ responses to CMV increased in IRD patients, reaching levels similar to controls

within the first year on ART. CMV IFNγ responses increased in non-IRD patients after

15 weeks on ART with no significant change thereafter. C1, C2 and two non-IRD

patients had low IFNγ responses to CMV before and after ART (Figure 3.3d).

The results demonstrate IFNγ responses to the initiating antigens pre-ART and during

IRD. This was compared with plasma IgG antibody levels reactive to PPD and C.

neoformans as an alternative marker of these IRD. CMV was again included as a

control antigen.

3.3.4 Levels of plasma IgG reactive with Cryptococcus

mannoprotein peaked during an IRD

Levels of plasma IgG antibodies reactive with C. neoformans, PPD and CMV were

measured in longitudinal samples from IRD and non-IRD patients. Results are presented

in arbitrary units (Figure 3.4). This allows comparisons between patients but not

between antigens.

Levels of antibodies reactive with cryptococcal antigens peaked at the time of IRD. C2

had higher levels of antibody (Figure 3.4a) and higher antigen titres (Table 3.1) than C1,

despite having a lower IFNγ response. Two TB IRD patients (T2 and T3) had

intermediate and stable levels of anti-cryptococcal IgG. T1 and all non-IRD patients

retained low levels throughout.

TB IRD patients (T2 and T3) had higher anti-PPD antibody levels than non-IRD

patients (Figure 3.4b), but levels were low in T1. Anti-CMV antibody levels began low

in C1, C2 and T3, but increased thereafter. No significant change was observed in non-

IRD patients (Figure 3.4c).

The results show increased IFNγ or IgG responses to the initiating antigen in all five

IRD patients, with concurrent increases in responses to CMV, PPD or ESAT-6 in

several patients who did not experience IRD associated with these pathogens. This


55

could reflect non-specific immune activation and/or expansion of memory T-cells

present at the start of ART. Hence proportions of activated and regulatory CD4+ T-cells

were investigated in our longitudinal sample sets.

3.3.5 Proportions of activated (HLA-DRhi) and regulatory CD4+ T-

cells were generally high during an IRD

High expression of HLA-DR was used to mark CD4+ T-cell activation. Proportions of

activated CD4+ T-cells were higher in patients at baseline, compared to healthy controls

[median (range), 40% (19-72%) vs. 4.9% (1.4-12%), p < 0.001]. Levels declined

significantly after 15 weeks on ART in non-IRD patients, but peaked at the time of IRD

in four of the five IRD patients and remained high (Figure 3.5a). Hence immune

activation is a general feature of IRD.

Patients had higher proportions of CD25+CD127

loCD4

+ T-cells at baseline than healthy

controls [13% (1.3-34%) vs. 6.3% (3.8-8.6%), p = 0.002]. Baseline proportions of

CTLA-4+ CD4

+ T-cells were also higher in patients than controls [17% (2.7-30%) vs.

2.9% (1.4-4.8%), p < 0.001]. There was no significant change in proportions of

CD25+CD127

lo CD4

+ T-cells in non-IRD patients within the first year of ART (Figure

3.5b). However, proportions of CTLA-4+ CD4

+ T-cells in non-IRD patients declined

after 15 weeks on ART (Figure 3.5c). Proportions of T-cells defined by both sets of

markers remained higher in patients than controls throughout.

In cryptococcal IRD patients (C1 and C2), proportions of regulatory T-cells peaked at

the time of IRD and were higher than levels in non-IRD patients. However, TB IRD

patients had levels similar to the non-IRD patients. The low numbers precluded

statistical analyses, but it is clear that IRD does not reflect low proportions of regulatory

T-cells.

3.4 Discussion

This is the first data associating cryptococcal IRD with elevated IFNγ and humoral

responses to cryptococcal antigen. Cryptococcal and TB IRD were associated with T-

cell activation but not a deficiency of regulatory T-cells. Whilst non-IRD patients and

healthy controls were not selected by prior cryptococcal or mycobacterial diseases, all

subjects were recruited locally to achieve a similar spectrum of exposure.


56

All cases of TB IRD presented as a worsening of TB lymphadenitis and enlarged

abscesses, fitting the definition of a ‘paradoxical’ IRD. TB IRD have been associated

with Th1 and pro-inflammatory responses to PPD (Bourgarit et al. 2006). Here, patients

T1 and T2 had large increases in CD4+ T-cell counts and much higher IFNγ responses

to PPD than were seen in non-IRD patients. IFNγ responses remained high after the

IRD episode. Patient T3 had persistently low CD4+ T-cell counts with a moderate IFNγ

response to PPD which declined rapidly after his IRD. This was seen in one non-IRD

patient who had no evidence of active MTB infection before or during ART. Since only

T2 and T3 had clear IgG responses to PPD before and during therapy, the three patients

present distinct immunological profiles.

No TB IRD patients responded to the MTB-specific protein ESAT-6. This has been

reported (Bourgarit et al. 2006) and suggests that ‘paradoxical’ IRD can reflect

responses to non-viable bacteria. Mycobacteria could not be cultured from T1 before

ART or at the time of his IRD (Table 3.1, panel 2). T2 was not tested but T3 was

culture-positive before his IRD. Hence the failure of T3 to respond to ESAT-6 may

reflect persistent immunodeficiency as his CD4+ T-cells counts remained low and his

responses to PPD were poor. This demonstrates two scenarios where tuberculosis-

diagnosis tests based on ESAT-6 would miss acute responses associated with IRD.

Diagnoses of cryptococcal IRD were based on the overt presentation of cryptococcosis

(antigen and culturable organisms in CSF) in the context of a virological and

immunological response to ART, meeting the definition of ‘incident’ or ‘unmasking’

IRD. Here, I demonstrated increased IFNγ responses and IgG responses to cryptococcal

antigens at the time of the IRD. Antibody levels were higher in the patient with a

disseminated infection and elevated serum antigen levels (C2), but both patients showed

a clear response. No cases of paradoxical cryptococcal IRD occurred in our cohort, so

no conclusion can be made about this important condition.

No individuals who had prior cryptococcal disease were available for comparative

studies. Five HIV patients treated for cryptococcal infection prior to successful ART

displayed no response to cryptococcal antigen (assessed by expression of CD69 and

TNFα) and only one patient displayed a small response to the antigen (Aberg et al.

2002). These authors did not discuss IRD, but no patients relapsed after withdrawal of

anti-fungal therapy.


57

In general, levels of activated (HLA-DRhi

) CD4+ T-cells in non-IRD patients declined

on ART but remained above levels in healthy controls. Elevated cellular and plasma

immune activation markers associate with development of mycobacterial IRD (Pires et

al. 2005) and persistent elevation is described after viral IRD (Price et al. 2001b; Stone

et al. 2001; Almeida et al. 2002). Here proportions of HLA-DRhi

CD4+ T-cells peaked

at the time of IRD. However four non-IRD patients also showed transient increases at

week 6 and proportions of these cells at baseline did not predict IRD as proportions

were similar between IRD and non-IRD patients. Activation markers may rise as a

consequence of IRD, rather than being a cause.

I showed for the first time that increased IFNγ and IgG responses and high proportions

of activated (HLA-DRhi

) CD4+ Tcells at the time of IRD do not reflect reduced

proportions of CD4+ T-cells with regulatory phenotypes (CD25

+CD127

lo and CTLA-

4+). However, the regulatory functions were not investigated. HIV infection changes the

tissue distribution of T-reg (Andersson et al. 2005), so the proportions of regulatory T-

cells in PBMC may not reflect proportions at the site of IRD (lymph nodes in TB IRD

patients or meninges in cryptococcal IRD patients). Further studies of the

immunological profiles of IRD are warranted.


58

Table 3.1 Laboratory diagnosis of cryptococcal and TB IRD

Patient C1 C2 C2

Clinical Presentation Cryptococcal meningitis on ART (IRD)

Time of assay Wk +2

(IRD)

Wk +3

(IRD)

Wk +5

(Recovery

phase)

C. neoformans culture (blood) Neg Pos Neg

Cryptococcal Ag titre (serum) 1:128 1:512 Not done

C. neoformans culture (CSF) Pos Pos Neg

Cryptococcal Ag titre (CSF) 1:32 1:16 1:4

Patient T1 T2 T3

Initial presentation Disseminated TB before IRD

Mycobacterial culture and AFB

(at presentation) Pos (Wk -7) Pos (Wk -6) Pos (Wk -4)


(on anti-TB therapy) Neg (Wk -4) Not done Neg (Wk +3)

Clinical presentation at IRD Increased lymphadenitis on ART

Time of IRD Wk +2 Wk +12 Wk +12


(after IRD)

Neg

(Wk +4 & +8)

AFB pos only

(Wk +12) Not done

Times are shown as the week (Wk) before (-) or after (+) the initiation of ART.

Mycobacterial culture and acid fast bacilli (AFB) were assessed in pus from affected

lymph nodes


59

(A)

FS

C

SSC

CD

4

SSC

CD

25

CD127

CD

4

CD152

CD

4

HLA-DR

A) Lymphocytes

B) CD4+ lymphocytes

C) Regulatory CD4+ T-cells (CD25+CD127lo)

D) Suppressor CD4+ T-cells (CD152+)

E) Activated CD4+ T-cells (HLA-DR+)

(B) (C)

(D) (E)

Figure 3.1 Gating strategies for CD4+ T-cell subsets.


60

Figure 3.2 All patients achieved increased CD4+ T-cells after commencement of

ART

Proportions of CD4+ T-cells in IRD (left panel) and non-IRD (right panel) HIV-infected

patients increased within the first year of ART but remained lower than healthy

controls.


61

Figure 3.3 IRD patients showed a peak in IFNγ responses to antigens from the

provoking pathogens


62

Figure 3.4 Evaluation of antigen-reactive plasma IgG titres by ELISA

Plasma IgG antibody titres in IRD (left panels) and non-IRD (right panels) patients.

Cryptococcal IRD patients have highest plasma IgG reactive to Cryptococcus

neoformans antigens (a) during IRD. TB IRD patients (T2 and T3) have higher anti-

PPD (b) and anti-Cryptococcus neoformans antigens (a) plasma IgG than non-IRD

patients. All IRD patients have increased anti-CMV plasma IgG (c) after commencing

ART. Significant decrease in IgG reactive to PPD (b) was seen in non-IRD patients

within 15 weeks on ART.


63

Figure 3.5 Proportions of activated and regulatory CD4+ T-cells remained

higher in patients than control during first year of ART

Proportions of activated (HLA-DRhi) (a) and regulatory CD25

+CD127

lo (b) or CTLA-4

+ (c)

CD4+ T-cells in IRD (left panels) and non-IRD (right panels) patients remained higher than

healthy controls in the first year of ART. Proportions of activated CD4+ T-cells (a) and CTLA-

4+ CD4

+ T-cells (c) significantly decreased in non-IRD patients after 15 weeks on ART.

Proportions of CD25+CD127

lo CD4

+ T-cells (b) did not change within the first year of ART.

64

Chapter 4

Characteristics of NK cells in Malaysian HIV

patients presenting with IRD after ART

Data from this chapter has been published.

Characteristics of natural killer cells in Malaysian HIV patients

presenting with immune restoration disease after ART

Tan DBA, Yong YK, Tan HY, French M, Kamarulzaman A, Price P. J AIDS

Clinic Res 2010; 1:102

The authors thank Professor Kee Peng Ng for laboratory facilities, Drs Sharifah Omar

and Sasheela Ponnampalavanar for clinical reviews of the patients, Yeat Mei Lee for

technical assistance and the patients and controls who contributed to this study. The

project was supported by Infectious Diseases Research Fund, UMMC and the National

Health and Medical Research council (Grant 513755). This is publication 2010-01

(Clinical Immunology and Immunogenetics, Royal Perth Hospital).

Chapter 4 - NK cell profiles during IRD

65

Chapter 4 – Characteristics of NK cells in Malaysian HIV

patients presenting with IRD after ART

NK cell function was investigated in Malaysian HIV patients beginning ART with

advanced immunodeficiency. Some patients experienced IRD presenting as

exacerbations of pre-existing infections. Whilst most IRD are attributed to IFNγ

produced by T-cells (Chapter 3), NK cells may also contribute. Blood leukocytes were

collected prospectively from ~100 HIV patients over 1 year on ART, plus 36 healthy

controls. Eleven patients who experienced an IRD and 14 matched controls were

assayed. Cells producing IFNγ were quantified by ELISpot after stimulation with K562

cells (an NK cell target) or antigens from pathogens associated with the IRD. NK cell

subsets, CD16 and perforin expression were determined by flow cytometry.

NK cell IFNγ responses to K562 cells were lower in HIV patients at baseline, improved

by Week 24 (p<0.01), but remained lower than uninfected controls (p<0.05).

Proportions of CD56hi

NK cells increased (p<0.01) above controls at Week 24. Perforin

expression on these cells was higher than controls at baseline (p<0.01), but

subsequently declined on ART. Proportions of CD56lo

NK cells were similar in patients

and controls throughout. CD16 expression on these cells was lower than controls

(p<0.05) at baseline, and then increased on ART. IRD patients showed lower CD16

expression on CD56lo

NK cells than non-IRD patients before treatment (p<0.05). NK

cell profiles were restored on ART, but NK cell IFNγ production remained low. Low

CD16 expression on CD56lo

NK cells may mark a predisposition for an IRD.

4.1 Background

NK cells are important in controlling tumors and pathogen-infected cells (section 1.2.3).

Effects of untreated HIV disease on NK cells include depletion of the major CD56lo

and

CD56hi

NK subsets, expansion of a dysfunctional CD56-CD16

+ NK subset, functional

abnormalities and perturbation of NK cell receptor repertoire (section 1.3.4). ART

increased the frequencies of CD56lo

and CD56hi

NK cells but decreased CD56-CD16

+

NK cells. Studies of the restoration of NK cell functions after ART yielded inconsistent

results (section 1.4.2.4). Overall, recovery of NK cells is partial even in patients with

good virological responses on long-term ART.


66

I found that NK cell IFNγ responses and proportions of CD56lo

NK cells are lower in

Caucasian Australian HIV patients on long-term ART (2-5 years) than in healthy

controls (Chapter 7). No previous studies of NK cells in HIV disease had considered

Asian populations. This is important because Asian patients are typically younger and

affected by more opportunistic infections than Caucasians (Ruxrungtham et al. 2004;

Vermund et al. 2007). Moreover genetic profiles pertinent to NK cells vary with

ethnicity (Lee et al. 2008).

IRD associated with MTB and Cryptococcus neoformans parallel robust IFNγ responses

that are evident in vitro following stimulation of blood leukocytes with relevant antigens

(section 1.5.3.1 and 1.5.3.2). These are attributed to CD4+ T-cells but

monocytes/dendritic cells and NK cells may contribute if stimulated via pattern

recognition receptors (PRR). Less is known about the immuno-pathogenesis of IRD

associated with CMV or VZV. Caucasian HIV patients with CMV IRD had higher

numbers of activating KIR genes than non-IRD patients or healthy controls (Price et al.

2007). High numbers of activated NK cells were found in the cerebral spinal fluid

(CSF) of a patient with VZV IRD presenting as transverse myelitis (Clark et al. 2004).

Together, these results suggest NK cells may contribute to several IRD.

In this chapter, I present the first report of the recovery of NK cell function in Asian

HIV patients beginning ART with advanced immunodeficiency and multiple

opportunistic infections. Some patients from the cohort presented with IRD associated

with MTB, cryptococcal, CMV and VZV. These patients are followed individually.

4.2 Patients and methods


Eleven male HIV patients with IRD [MTB (n=6), MTB/CMV retinitis (n=1),

cryptococcal meningitis (n=1), cryptococcal meningitis/Kaposi’s sarcoma (KS; n=1),

CMV retinitis (n=1), dermatomal VZV (n=1)] were identified from our study of

immune reconstitution in KL as described in section 2.1. Approximately 100 patients

had been recruited when this study group was compiled. Fourteen patients with no

evidence of IRD were selected for comparison. These were matched with the IRD

patients by sex (male) and ethnicity (Chinese and Malay). All patients received 2

nucleoside analog reverse transcriptase inhibitors (3TC with d4T or AZT) and 1 non-


67

nucleoside reverse transcriptase inhibitors (EFV or NVP). Healthy Chinese and Malay

donors (n=36) residing in Kuala Lumpur were included as controls.

Non-IRD patients and healthy controls had no known history of cryptococcal, CMV and

VZV disease, but several patients had a history of MTB and vaccination with BCG is

standard practice in Malaysia. HIV patients were slightly older than the healthy controls

(p=0.02), but the IRD and non-IRD groups were similar in age (p=0.35) (Table 4.1).

4.2.2 Sample collection and routine data

Refer to section 2.1.

4.2.3 IFNγγγγ ELISpot assay

IFNγ producing PBMC were assayed by ELISpot as described in section 2.3. PBMC

were stimulated for 20 hours with K562 cells (section 2.6.4), PPD (section 2.6.8),

Cryptococcus neoformans mannoprotein (section 2.6.3), CMV (section 2.6.2) and VZV

lysate (section 2.6.9).

4.2.4 Flow cytometry

PBMC (5 x 105 cells) were surface stained with CD3-APC, CD16-APC-H7 and CD56-

PE (BD Pharmingen, San Jose, CA, USA). Cells were processed using

Cytofix/CytopermTM

Kits (BD Pharmingen) according to the manufacturer’s protocol

for intracytoplasmic staining with Perforin-FITC (BD Pharmingen). 50,000-100,000

events were acquired on a FACSCanto II cytometer (BD Pharmingen). Gating of NK

cell subsets is described in Figure 7.1.

4.2.5 Statistical analysis

Mann-Whitney tests were used to compare groups of individuals. As Week 48 samples

were not available for some patients, Wilcoxon matched-pair tests were used to

compare data from Week 0 and the closest sample to Week 24 to evaluate changes

amongst IRD or non-IRD patients during ART. Spearman’s test was used to evaluate

non-parametric correlation coefficients. For all comparisons, p-values below 0.05 were

considered to be statistically significant.


68

4.3 Results

4.3.1 Clinical and immunological characteristics of the study

population and response to ART

The study comprised IRD patients, non-IRD patients and healthy controls (Table 4.1).

Nadir CD4+ T-cell counts were <110 cells/µL in all patients and were similar in IRD

and non-IRD patients (p=0.40). All patients had increased CD4+ T-cell counts [by Week

11-37; p<0.0001] and frequencies [by Week 13-32; p<0.005] after starting ART. Levels

of plasma HIV RNA were >100,000 copies/mL in all patients pre-ART and were

suppressed to <50-437 copies/ml over 9-39 weeks of ART. HIV RNA levels were

similar in IRD and non-IRD patients (Table 4.1).

All cases of IRD presented as atypical opportunistic infections or inflammatory disease

after ART (section 1.5). Four patients experienced ‘paradoxical’ TB IRIS defined by

initiation of treatment for active MTB disease before ART, followed by lymphadenitis,

lymphadenopathy, lymph nodes evolving into abscesses or cold abscess enlargement on

ART. Three patients with no history of MTB infection pre-ART developed ‘unmasking’

TB IRD, including one who also experienced CMV retinitis as an IRD. Two patients

experienced ‘unmasking’ cryptococcal IRD presenting as cryptococcal meningitis,

including one with coincident worsening of KS. Two patients presented with CMV

retinitis as an IRD (one also experienced TB IRD) and one patient with dermatomal

VZV infection after ART.

4.3.2 IRD usually parallel IFNγ responses to pathogen antigens

Most IRD patients showed increased IFNγ production against antigens from the

provoking pathogens at the time of IRD and for up to 48 weeks. Levels were above the

median value of non-IRD patients and healthy controls (Figure 4.1A, B, D). This was

evident with 5/7 MTB, 2/2 cryptococcal and 1/1 VZV IRD patient. The patient who

experienced both CMV and TB IRD showed a moderate increase in IFNγ responses to

CMV during his IRD, whilst the other CMV IRD patient had persistently low responses

(<10 spots per 200,000 PBMC; Figure 4.1C)


69

4.3.3 NK IFNγγγγ responses remained impaired after Week 24 in all

patients, irrespective of IRD

NK cell IFNγ responses were deficient in IRD and non-IRD patients at baseline

compared to healthy controls (p<0.001; Figure 4.1G). These increased significantly by

Week 24 (p<0.01) but remained lower than healthy controls (p<0.05) after 48 weeks

(Figure 4.1E and F). No significant differences were observed between IRD and non-

IRD patients at Week 0 or Week 24 of ART. NK cell IFNγ responses remained low in

most IRD patients at the time of IRD. One TB IRD patient showed a peak in IFNγ

production during his IRD. Two IRD patients with CMV retinitis (+/- TB IRD) and one

other TB IRD patient showed an increased NK cell IFNγ soon after their IRD was

diagnosed (Figure 4.1E). However, several non-IRD patients also displayed elevated

NK IFNγ responses (Figure 4.1F).

4.3.4 Proportions of CD56hi NK cells increased on ART, with a peak

in a few IRD patients

CD3- lymphocytes were distinguished by expression of CD56 (Figure 7.1), creating NK

subsets that can be quantified as proportions of lymphocytes. CD56lo

NK cells are

naturally more cytotoxic and express higher levels of CD16 than CD56hi

NK cells.

CD56hi

NK cells are less cytotoxic, express low levels of CD16 and are more able to

make cytokines (e.g. IFNγ and TNFα). I found that NK IFNγ responses correlated with

proportions of CD56lo

NK cells in Australian HIV patients after >2 years of ART.

Intracellular cytokine staining assay confirmed that CD56lo

NK cells produced IFNγ in

response to K562 cells (Chapter 7).

Here, proportions of CD56lo

NK cells were similar across all groups of patients and

were relatively stable during one year of ART irrespective of an IRD (Figure 4.2A, B

and C). NK IFNγ responses correlated with proportions of CD56lo

NK cells in

Malaysian controls (r=0.35, p=0.05), but not in Malaysian HIV patients at Week 0 or

Week 24 (r=0.13, p=0.56 and r=0.19, p=0.38 respectively).

Proportions of CD56hi

NK cells increased in IRD and non-IRD patients from Week 0 to

Week 24 of ART (Figure 4.2D, E and F, p<0.01). IRD patients had slightly more

CD56hi

NK cells at Week 24 than healthy controls (Figure 4.2D and F). This pattern

held in several patients to Week 48. The two cryptococcal IRD patients showed


70

increased proportions of CD56hi

NK cells coinciding with peaks in cryptococcal IFNγ

responses during their IRD (Figure 4.1B). The two patients with CMV retinitis (+/- TB

IRD) also showed increased proportions of CD56hi

NK cells on ART above other

patients. The CMV/TB IRD patient had higher IFNγ responses to CMV (Figure 4.1C)

and proportions of CD56hi

NK cells (Figure 4.2D, left panel).

4.3.5 Expression of CD16 on CD56lo NK cells was low in most IRD

patients at baseline


NK cells was lower at baseline in IRD (p<0.001) and non-

IRD patients (p<0.05) compared to healthy controls (Figure 4.3A, B and C) and

increased to levels seen in controls by Week 24 (Figure 4.3C). CD16 expression

displayed equivalent trends when assessed as Mean Fluorescence Intensity (p<0.05;

data not shown). Baseline CD16 expression was lower in IRD patients than non-IRD

patients (p<0.05; Figure 4.3C). An examination of the individual plots (Figure 4.3A)

shows that the low baseline values in patients with IRD associated with MTB (n=3),

CMV (n=1) and cryptococcal meningitis/KS (n=1). These IRD occurred whilst CD16

expression was low. In contrast, CD16 expression on CD56hi

NK cells was similar in all

groups and did not reflect individual IRD events (Figure 4.3D, E and F).

4.3.6 Perforin levels in CD56hi NK cells were high in all HIV patients

Intracellular perforin was assessed in seven IRD patients (5 MTB, 1 MTB/CMV and 1

CMV IRD), 10 non-IRD patients and 23 healthy controls. Perforin expression in CD56lo

NK cells was not deficient in HIV patients at baseline and remained high to Week 48

with no difference between IRD and non-IRD patients (Figure 4.4A, B and C). Perforin

expression on CD56hi

NK cells was higher at baseline in IRD and non-IRD patients

(p<0.01) compared to healthy controls, with a decline by Week 24. Expression

remained similar in IRD and non-IRD patients (Figure 4.4D, E and F).

4.4 Discussion

I established that defects in NK cell IFNγ responses in Asian HIV patients persist for at

least 1 year on ART. The K562 cell line only presents ligands for NK cell activatory

receptors (Garcia-Penarrubia et al. 2002), so the data reflect maximum NK cell

responses – responses to other targets may be even lower. NK cell IFNγ responses to

K562 cells also remained low in Australian (Caucasian) HIV patients on long-term ART


71

(2-5 years) (Chapter 7). Defects in NK cell IFNγ responses increase risk of tumours and

infections (Carrington et al. 2006; Lodoen et al. 2006; Millman et al. 2008; Marr et al.

2009). This may explain the high and rising incidence of non-AIDS-related

malignancies seen in patients with optimal stable responses to ART (Crum-Cianflone et

al. 2009).

NK cell IFNγ responses were limited by proportions of CD56lo

NK cells in Australian

patients on long-term ART (2-5 years) (Chapter 7). Here no correlations were observed

with the proportions of CD56lo

NK cells or CD56hi

NK cells. Expansion of

dysfunctional CD56-CD16

+ NK cells in HIV viremic patients has been linked to

impaired function of the total NK cell population. Compared to CD56+ NK cells, CD56

-

CD16+ NK cells have poorer cytotoxic function and secrete lower levels of IFNγ and

TNFα (Mavilio et al. 2005). However, I did not quantify these cells as our antibody

panel did not allow us to gate out CD16+ monocytes.

Perforin expression on CD56lo

NK cells was not deficient in the Malaysian HIV

patients, suggesting that the cytotoxic potential of their NK cells is not impaired.

Functional assessments of cytotoxic NK cells are warranted as other studies have

demonstrated impaired NK cell cytotoxicity in HIV viremic and aviremic patients (De

Maria et al. 2003; Mavilio et al. 2003; Fogli et al. 2004). Defects affecting the

degranulation pathway or receptor recognition may limit NK function, despite normal or

elevated levels of perforin. Higher expression of perforin in CD56hi

NK cells from HIV

patients sampled at baseline (Figure 4.3D) may reflect immune activation as CD56hi

NK

cells can increase expression of perforin after stimulation with IL-2 (Ferlazzo et al.

2004).

Mechanisms whereby NK cells may be protective or pathological in an IRD. Studies of

mycobacterial and cryptococcal IRD suggest that antigen load is a risk factor for

developing IRD (de Boer et al. 2003; Shelburne et al. 2005; Lawn et al. 2008), so NK

cells may minimize the risk of developing IRD through their capacity to control

mycobacterial, cryptococcal, CMV or VZV infections (Ma et al. 2004; Vankayalapati et

al. 2005; Lodoen et al. 2006). Alternatively, NK cell responses restored on ART may

contribute to the elevated cytokine responses implicated in IRD pathogenesis (Chapter

3). Compartmentalization of NK cell activation to the central nervous system was

observed in a patient with neurological VZV IRD (Clark et al. 2004).


72

Here, NK IFNγ responses only increased before the IRD in one patient (TB IRD; Figure

4.1E), so NK cells are unlikely to contribute cytokines that mediate IRD. No significant

changes were observed with the proportion of CD56lo

NK cells in circulation during the

development of IRD. However, proportions of CD56hi

NK cells did increase in the two

IRD patients who presented with cryptococcal meningitis (represented by green lines),

coinciding with peaks in cryptococcal IFNγ responses during their IRD at Weeks 2 and

3. These patients were not distinguishable by other parameters such as nadir CD4+ T-

cell count, rate of CD4+

T-cell recovery or NK IFNγ responses (data not shown), so this

warrants further investigation as a mechanism of IRD affecting a secluded site (such as

the central nervous system).

CD16 (FcγR3a) is an activatory receptor which can mediate antibody-dependent cell

cytotoxicity (ADCC) (Sun 2003). Here expression of CD16 on CD56lo

NK cells

normalized by Week 24 of ART, which may reflect normal capacity to elicit ADCC.


NK cells was particularly low in IRD patients before ART

(Figure 4.3A). Impaired CD16 expression may affect the recognition, activation and

ADCC function of NK cells against antibody-coated pathogens (Miller et al. 1990;

Orange et al. 2002). This may limit antigen clearance before ART, increasing the risk of

IRD. ADCC should be addressed directly in further studies, as the response of NK cells

toward K562 cells does not depend on CD16.

In conclusion, partial restoration of NK cell profiles is observed with recovery of CD16


NK cells and decreased expression of perforin on CD56hi

NK

cells to levels of healthy controls after approximately 6 months of ART. However, NK

cell IFNγ responses remained impaired compared to healthy controls. No evidence of a

role for NK cells in the pathogenesis of IRD was found as responses varied between

patients with similar IRD, with no consistent peaks preceding diagnosis. However, NK

deficiency may promote IRD by impairing antigen clearance before ART as CD16

expression of CD56lo

NK cells was low at baseline in most IRD patients. This warrants

independent replication. Pathogen-infected target cells (e.g. MTB-infected monocytes

or CMV-infected fibroblasts) should be used to investigate responses of NK cells during

specific IRD. NK genotypes (e.g. KIR) should also be considered. This work is being

undertaken by another PhD student in our laboratory.


73

Table 4.1 Demographics of study cohort

IRD patientsa

Non-IRD

patients

Healthy

Controls

N 11 14 36

Age

(years) 44 (34-56) 41 (33-64) 36 (23-71)

b

CD4+ T-cells/µl blood

[Nadir, Week 0] 22 (0-58) 27 (0-104) NA

CD4+ T-cells/µl blood)

[Week 11-37] 142 (78-307) 176 (54-338) NA

%CD4+ T-cells of

lymphocytes [Week 0] 2.0 (0.5-9.4) 3.9 (0.2-10.8) 35.2 (20.0-46.4)

c

%CD4+ T-cells of

lymphocytes [Week 13-32] 10.2 (4.1-15.7) 8.6 (2.8-20.5) 35.2 (20.0-46.4)

c

Values are presented as median (range), NA = not available

a All parameters assessed were similar to non-IRD patients

b Lower than all HIV patients (p < 0.05)

c Higher than all HIV patients (p < 0.05)


74

Figure 4.1 ELISpot analysis of IFNγ-producing PBMC


75

Figure 4.2 Longitudinal evaluation of NK subsets


76

Figure 4.3 Longitudinal evaluation of CD16 expression on NK subsets


77

Figure 4.4 Longitudinal evaluation of perforin levels in NK subsets

78

Chapter 5

TLR2-induced cytokine responses by DC and

monocytes may characterize HIV-infected

patients experiencing mycobacterial IRD

Data from this chapter has been published.

TLR2-induced cytokine responses may characterize HIV-infected patients

experiencing mycobacterial immune restoration disease

Tan DBA, Lim A, Yong YK, Ponnampalavanar S, Omar S, Kamarulzaman

A, French MA, Price P. AIDS 2011: Epub ahead of print

The authors thank Professor Kee Peng Ng for laboratory facilities, Dr Jeffrey Martinson

(Rush University Medical Centre), Maxwell Pistilli and Professor Luis Montaner (The

Wistar Institute) for expert technical advice, Sonia Fernandez (UWA) for helpful

discussions, Hong Yien Tan and Yeat Mei Lee (UMMC) for technical assistance and all

patients and controls who contributed to this study.

Chapter 5 - TLR2-induced responses in TB IRD

79

Chapter 5 - Longitudinal evaluation of TLR-induced cytokine

responses by circulating DC and monocytes in HIV-infected

patients on ART and their role in the development of TB IRD

Most HIV patients who experience TB IRD display elevated IFNγ responses against

mycobacterial antigens, but these can occur without an IRD. Recognition of

mycobacteria-associated molecular patterns through TLR on DC and monocytes

induces cytokine production. TLR-induced responses in IRD are evaluated in this

chapter. PBMC were collected at Weeks 0, 6, 12, 24 and 48 after ART from five

patients who experienced TB IRD and nine matched non-IRD patients. Samples were

collected once from fifteen healthy controls. IFNγ by PBMC stimulated with PPD was

assessed by ELISpot. TLR2 expression on mDC and monocytes was assessed by flow

cytometry. TNFα, IL-12p40 and IL-10 were measured by ELISA in 24 hour cultures of

PBMC with lipomannan (LM; mycobacteria-derived TLR2 agonist).

TLR2 expression on mDC and monocytes was higher in patients than controls at

baseline (p<0.005). TLR2 expression decreased to normal levels on mDC by Week 12,

but remained higher on monocytes at Week 24 (p=0.02). At Week 24, IRD patients

showed higher IFNγ responses to PPD (p=0.02), TLR2 expression on monocytes

(p=0.006) and LM-induced TNFα production (p=0.016) than non-IRD patients. LM-

induced TNFα and IL-12p40 responses paralleled TB IRD in the patients with high

TLR2 expression. IL10 levels did not associate with IRD. TLR2-induced pro-

inflammatory cytokines by DC or monocytes may contribute to the pathogenesis of

mycobacterial IRD.

5.1 Background

Presentations of TB IRD (paradoxical TB-IRIS or ART-associated TB) have been

associated with the increased T-cell responses (e.g. IFNγ) to mycobacterial antigens

(section 1.5.3.1; Chapter 3) and increased plasma levels of soluble mediators of innate

responses, such as IL-18 and CXCL-10, relative to non-IRD patients (Oliver et al.

2010). Post mortem examination of a HIV patient who experienced fatal ART-

associated TB with bronchiolitis obliterans organizing pneumonia showed a large

infiltrate of macrophages but few T-cells (Lawn et al. 2009). Hence, evaluation of the

role of innate immune cells in the development of TB IRD is needed.


80

Effective innate immune responses against MTB infection involve a balance between

pro-inflammatory cytokines (e.g. TNFα, IL-12) and regulatory cytokines (e.g. IL-10) by

DC and monocytes/macrophages (Fulton et al. 1998; Giacomini et al. 2001; Pereira et

al. 2004; Jamil et al. 2007). TLR2 is critical for the production of TNFα and IL-12 by

mouse DC and macrophages in vitro, as production was lower in MTB-infected TLR2-

deficient mice (Reiling et al. 2002; Drennan et al. 2004; Bafica et al. 2005; Holscher et

al. 2008; Drage et al. 2009).

TLR2 recognizes mycobacterial cell wall constituents such as lipomannan (LM) and

lipoarabinomannan (LAM). Mycobacterial LM induces IL-12 production in

macrophages. LAM from pathogenic mycobacterial species like MTB inhibits IL-12

production, while LAM from non-pathogenic species lacks this activity (Briken et al.

2004; Dao et al. 2004), so IL-12 may contribute to disease. Stimulation with LM

activates human and mouse DC and macrophages via TLR2 pathways to produce TNFα,

IL-12p40 and IL-10 (Song et al. 2003; Briken et al. 2004; Jang et al. 2004; Quesniaux

et al. 2004; Elass et al. 2008). IL-10 is involved in immune tolerance and inhibits

inflammatory processes (Moore et al. 2001; O'Garra et al. 2009), including the

production of TNFα and IL-12 by human DC and monocytes/macrophages (Fulton et al.

1998; Demangel et al. 2002; Higgins et al. 2009).

No studies have assessed cytokine responses to LM or the role of TLR in TB IRD. In

this chapter, the role of TLR2-mediated innate immune responses in TB IRD is

evaluated for the first time in a detailed longitudinal study of a small series of patients

(TB IRD, n=5 vs. non-IRD, n=9) beginning ART with extreme immunodeficiency and

numerous co-infections, including TB, candidasis and Pneumocystis jirovecii

pneumonia (PJP).

5.2 Patients and methods


Five male HIV patients who expecienced TB IRD were identified from our study of

immune reconstitution in KL (section 2.1). Nine patients with no evidence of IRD were

selected for comparison. These were matched with the IRD patients by sex (male) and

ethnicity (Chinese). Healthy Chinese donors (n=15) residing in Kuala Lumpur were

included as controls [Age median (range): Patients, 41 (33-52) vs. Controls 38 (23-71)


81

respectively, p=0.29]. Vaccination with BCG is standard practice in Malaysia and

received by all subjects.

Blood was collected into EDTA tubes from patients after approximately 0, 6, 12, 24 and

48 weeks on ART, and once from control donors. PBMC were obtained by Ficoll

gradient centrifugation and cryopreserved in liquid nitrogen. Institutional ethics

committee approval was obtained and informed consent was given by all participants.

5.2.2 Measurement of plasma HIV RNA levels and CD4+ T-cell

counts

Refer to section 2.2. Plasma HIV RNA levels and absolute CD4+ T-cell counts were

determined before treatment and on one or two occasions during the first year of ART

[median (range): Week 14 (7-19) and/or Week 33 (22-47)] by flow cytometry in routine

clinical laboratories at UMMC. Plasma HIV-1 RNA was measured using the COBAS

Amplicor HIV-1 Monitor Test, v1.5 (Roche Diagnostics, Indianapolis, IN, USA) with a

cut-off of <50 copies/mL.

5.2.3 Quantification of IFNγγγγ responses to PPD by ELISpot

Refer to section 2.3. IFNγ-producing PBMC were quantified by ELISpot, after 20 hours

stimulation with MTB-derived PPD (10 µg/mL; Statens Serum Institute, Copenhagen,

Denmark).

5.2.4 Measurement of CD4+ T-cell activation and TLR2 expression

PBMC were thawed into RPMI and washed with 1% BSA/PBS. 0.5 x 106 cells were

surface stained with CD3-APC, CD4-PerCP-Cy5.5, HLA-DR-PE, and CD38-FITC (BD

Biosciences, San José, CA, USA) to measure frequencies of activated [HLA-

DRhi

CD38+] CD4

+ T-cells. 1 x 10

6 cells were surface stained with CD4-PerCP-Cy5.5,

CD8-APC-H7, CD14-PE-Cy7, CD19-PE-Cy7 (BD Biosciences), TLR2-FITC

(eBioscience, San Diego, CA, USA) and BDCA-1-APC (Miltenyi Biotec, Bergisch

Gladbach, Germany) to measure TLR2 expression on mDC and monocytes, expressed

as mean fluorescence intensity (MFI). All staining was performed at room temperature

in the dark. 100,000 events were acquired for the CD4+ T-cell activation tubes and

300,000-500,000 events were acquired for the TLR2 expression tubes using a BD

FACSCanto II cytometer and analyzed using FlowJo software version 7.2.2 (Tree Star,

Ashland, OR, USA).


82

5.2.5 Quantification of TNFα, IL-12p40 and IL-10 production in

response to lipomannan

PBMC were cultured in RPMI 1640 with 10% fetal calf serum (FCS) at 5 x 105 cells per

well in 24-well plates and stimulated with LM-derived from M. smegmatis (10µg/mL;

InvivoGen, San Diego, CA, USA). Supernatants were collected after 24 hours and

concentrations of TNFα, IL-10 (R&D Systems, Minneapolis, MN, USA) and IL-12p40

(BD Biosciences) were measured by ELISA.

5.2.6 Culture and flow based assay to quantify PPD-specific IFNγ

producing CD4+ and CD8+ T-cells

Thawed PBMC were resuspended at 1 x 106 cells/mL in 10% FCS/RPMI. 500 µL

PBMC were stimulated PPD (section 2.6.8) for 20 hours at 37oC with 5% CO2.

Following incubation, Brefeldin-A (BFA; BD Biosciences) was added and PBMC were

incubated for a further 4 hours at 37oC with 5% CO2. PBMC were then washed with 2

mL cold FB and blocked with 10 µL FcR blocking reagent (Miltenyi) for 20 minutes at

4oC. PBMC were then washed with 1 mL FB, transferred to labelled flow tubes and

surface stained for 15 minutes at room temperature with CD4-PerCP-Cy5.5 and CD8-

APC-Cy7 (BD Biosciences). IFNγ-FITC (BD Biosciences) was stained intracellularly

and processed using the BD Cytofix/Cytoperm kit. All staining was performed in the

dark. After staining procedures, cells were resuspended in 250 µL FB, fixed with a drop

of 1% paraformaldehyde in FB and kept at 4oC before acquisition. 150,000-200,000

events were acquired using a BD FACS Canto II flow cytometer and analysed using the

FlowJo program v. 5.7.2 (Tree Star, Ashland, OR, USA). Figure 5.1 shows gating

strategies for each cytokine-producing cell population.

5.2.7 Statistical analyses

Mann-Whitney tests were used to compare groups of individuals. Spearman’s test was

used to calculate the significance of non-parametric correlation coefficients. For all

comparisons, p-values below 0.05 were considered to be statistically significant.


83

5.3 Results

5.3.1 Laboratory and clinical features of IRD

Nadir CD4+ T-cells counts were similar in IRD and non-IRD patients [median (range):

14 (0-36) vs. 27 (0-53) cells/µL respectively, p=0.23]. After 11-39 weeks of ART, all

patients achieved suppressed plasma HIV RNA (<50-437 copies/mL) and increased

CD4+ T-cell counts. At this time, CD4

+ T-cell counts were similar in IRD and non-IRD

patients [median (range): 131 (78-262) vs. 174 (13-275) cells/µL respectively, p=0.90].

One TB IRD (T2) showed a rapid increase in frequencies of CD4+ T-cells, while other

patients showed steady increases over one year on ART (Figure 5.2A).

All cases of TB IRD (patient T1-T5) presented as TB or TB-IRIS after ART (Table

5.1). Patients T1, T2 and T3 experienced paradoxical TB-IRIS, whilst patients T4 and

T5 developed ART-associated TB. Patient T3 developed a second TB IRD episode (TB

lymphadenitis) at Week 97. MTB resistant to rifampicin was found at this time which

was the same strain of mycobacteria isolated pre-ART (i.e. not a novel resistant strain).

PBMC were collected at this time and also at Week 81. All TB IRD patients received

anti-TB treatment, after the IRD was diagnosed.

5.3.2 CD4+ T-cell activation and IFNγ responses to PPD parallel the

IRD but are seen in other patients

As the cohort is small, patients are described individually. IRD patients are represented

with a consistent colour code (Table 5.1), whilst non-IRD patients are represented by

black lines. Three TB IRD patients (T2, T3 and T5) had a peak in CD4+ T-cell

activation above non-IRD patients at IRD diagnosis, while T4 had a large increase after

his IRD (Figure 5.2B). Patient T3 also had an increase in CD4+ T-cell activation during

his second IRD episode (3.5% at Week 81 to 19% at Week 97).

The paradoxical TB IRIS patients (T1, T2 and T3) displayed high IFNγ responses to

PPD at or before IRD diagnosis. During his second IRD, T3 retained IFNγ responses to

PPD similar to the median of healthy controls (66 spots per 200,000 cells). T4 displayed

a large increase after his IRD was diagnosed. T5 had low/moderate responses

throughout (21-54 spots per 200,000 cells). Responses of TB IRD patients were higher

than non-IRD patients at Week 24 (p=0.02) and marginally higher at Weeks 0 and 12

(p=0.07).


84

5.3.3 TB IRD parallels peaks in TLR2 expression on mDC and

monocytes in some patients

All patients had higher TLR2 expression on mDC and monocytes than healthy controls

before ART (p<0.001; Figure 5.3A and B). TLR2 expression on mDC was marginally

above healthy controls at Week 12 and Week 24 (p=0.09), whilst expression on

monocytes remained higher than controls at Week 12 (p=0.004) and Week 24

(p=0.024). At Week 24, TLR2 expression on monocytes was higher in IRD than non-

IRD patients (p=0.006).

Patients T2 and T5 displayed clear peaks in TLR2 expression on mDC and monocytes

during their IRD. Patient T4 had increased TLR2 expression after his diagnosis. These

peaks were above levels seen in non-IRD patients or controls. Patient T1 and T3 had

similar TLR2 expression on mDC and monocytes at all times compared to non-IRD

patients and controls. However, patient T3 displayed increased TLR2 expression on

mDC (MFI of 2035 to 2461) and monocytes (MFI of 5195 to 9743) during his second

IRD. This is higher than non-IRD patients at Week 48 and healthy controls.

5.3.4 Elevated TNFα and IL-12p40 responses to lipomannan are

restricted to IRD patients

Production of TNFα, IL-12p40 and IL-10 in response to LM was similar in patients and

controls at Weeks 0 and 24. At Week 12, IL-12p40 responses were higher in patients

than controls and higher in IRD patients than non-IRD patients. TNFα responses were

higher in IRD patients than non-IRD patients at Weeks 12 (p=0.03) and 24 (p=0.03).

Strikingly high TNFα responses were evident in the three TB IRD patients (T2, T4 and

T5) who had high TLR2 expression on mDC and monocytes (Figure 5.4A). Levels of

IL-12p40 were also high in these patients (Figure 5.4B), whilst their IL-10 responses

were low (Figure 5.4C). Marking his second IRD, T3 displayed increased TNFα (2740

to 3600 pg/mL), IL-12p40 (140 to 675 pg/mL) and possibly IL-10 (2085 to 2355

pg/mL) from Week 81 to Week 97. At Week 97, these levels were higher than all non-

IRD patients (at Week 48) and healthy controls.


85

5.3.5 Production of LM-induced cytokines were related to TLR2

expression and immune activation

When all patients were combined (n=14), TLR2 expression on mDC was proportional

to the production of TNFα at Week 12 and 24 (r=0.80, p=0.002 and r=0.79, p=0.002

respectively), IL-12p40 at Week 12 (r=0.80, p=0.002) and IL-10 at Week 24 (r=0.69,

p=0.01). TLR2 expression on monocytes showed no associations with LM-induced

cytokines at Week 12, but was proportional to the production of TNFα (r=0.80,

p=0.002) and IL-12p40 (r=0.60, p=0.04) at Week 24. Hence, LM-induced cytokine

responses are related to TLR2 expression.

5.4 Discussion

TB IRD are associated with an over-production of IFNγ based on data from Malaysia

(Chapter 3), France and Cambodia (Bourgarit et al. 2006; Elliott et al. 2009). However

many patients experience a rise in IFNγ without an IRD. Symptoms of mycobacterial

IRD are ameliorated by steroids, but IFNγ responses to PPD did not decline in parallel

(Meintjes et al. 2008c; Skolimowska et al. 2009; Meintjes et al. 2010). Hence IFNγ

responses may not cause IRD (Barber et al. 2010). Furthermore, data from Cambodia

showed more prominent Mtb-specific IFNγ responses in ART-associated TB cases

(Elliott et al. 2009), with higher plasma levels of IL-18 but lower CXCL-10 compared

to paradoxical TB-IRIS cases (Oliver et al. 2010). This suggests that IRD with different

clinical manifestations may reflect different immunopathogenic mechanisms including

defects in innate immune responses.

Most TB IRD patients described here sustained high CD4+ T-cell activation on ART

compared to non-IRD patients which showed the declining trends after commencing

ART (Chapter 3). TLR2 expression on mDC and monocytes was higher in all patients

than controls pre-ART and generally decreased on ART, suggesting an association with

immune activation. Immune activation in HIV patients reflects levels of circulating

microbial products from gastrointestinal tract (Brenchley et al. 2006b). Microbial

products containing TLR2 agonists could maintain TLR2 expression and TLR-2

induced responses. Here, TLR2 expression was proportional to LM-induced cytokine

responses (e.g. TNFα).


86

TNFα and IL-12p40 (but not IL-10) responses to LM were notably high in the four IRD

patients (including T3’s second IRD) where there was high TLR2 expression on mDC

and monocytes. Production of TNFα and IL-12p40 without equivalent regulatory

cytokines (e.g. IL-10) may facilitate TB IRD. The findings implicate LM-induced pro-

inflammatory cytokines in some cases of paradoxical TB-IRIS (T2 and T3’s second

IRD) and ART-associated TB (T4, T5), but did not distinguish these clinical scenarios

of TB IRD. TLR7/8 or TLR9-induced IFNα and IL-12 responses in this cohort showed

no association with IRD. However, efficient DC responses (IFNα and IL-12) via

TLR7/8 and TLR9 correlated with better recovery of CD4+ T-cells (Chapter 6).

Innate responses to MTB infection may also involve IL-1 and IL-18 (produced by

activated macrophages). IL-1 receptor and TLR knockout mice establish a role for IL-1

that is independent of TLR (Reiling et al. 2008). Future studies should address whether

TB IRD patients with no evidence of heightened TLR2 activity have increased IL-1 or

IL-18 responses. IL-1 has not been investigated but increased levels of plasma IL-18

were described in TB IRD patients from Cambodia (Oliver et al. 2010).

In conclusion, TLR2-mediated cytokine responses were observed in some cases of TB

IRD. These were characterized by higher expression of TLR2 on both mDC and

monocytes, and higher TNFα and IL-12p40 production without a parallel increase in IL-

10 in response to mycobacteria-derived TLR2 ligands. These data provide us with

further information on the immunopathogenesis of TB IRD. The findings should be

confirmed in a larger cohort of patients distinguished by their clinical presentations.


87

Table 5.1 Clinical characteristics of HIV patients presenting with TB IRD

after ART and non-IRD patients

Chapter 5 - TLR-induced responses in TB IRD

88

Figure 5.1 IFNγ responses to PPD are largely mediated by CD4+ T-cells


89

Figure 5.2 IRD is associated with increased activated CD4+ T-cells and IFNγ

responses

(A) Proportions of CD4+ T-cells and (B) activated CD4

+ T-cells were quantified by flow

cytometry. (C) PBMC producing IFNγ after stimulation with PPD was enumerated by

ELISpot. IRD patients are represented with different colored lines as described in Table

1 with open circles indicating the time of TB IRD diagnosis (left panel). Matched non-

IRD patients are represented by black lines (right panel). Healthy controls are

represented as crosses (horizontal lines indicate median values).


90

Figure 5.3 Increased TLR2 expression during ART was restricted to IRD

patients

TLR2 expression on myeloid DC (A) or on monocytes (B) are presented as mean

fluorescence intensity. IRD patients are represented with different coloured lines as

described in Table 1 with open circles indicating the time of TB IRD diagnosis (left

panel). Matched non-IRD patients are represented by black lines (right panel). Healthy

controls are represented as crosses (horizontal lines indicate median values).


91

Figure 5.4 High TNFα and IL-12p40 responses to LM characterised some

patients who experienced TB IRD

Concentrations of TNFα (A), IL-12p40 (B) and IL-10 (C) in culture supernatants after

24 hours stimulation of PBMC with mycobacterial lipomannan. IRD patients are

represented with different coloured lines as described in Table 1 with open circles

indicating the time of TB IRD diagnosis (left panel). Matched non-IRD patients are

represented by black lines (right panel). Healthy controls are represented as crosses

(horizontal lines indicate median values).

92

Chapter 6

Robust IFNα and IL-12 responses by DC parallel

efficient CD4+ T-cell recovery in HIV patients on

ART

Data from this chapter has been published

Robust interferon-α and IL-12 responses by dendritic cells are related to

efficient CD4+ T-cell recovery in HIV patients on ART

Tan DBA, Yong YK, Lim A, Tan HY, Kamarulzaman A, French M, Price P.

Clin Immunol 2011; 115-121.

The authors would like to thank Professor Kee Peng Ng for laboratory facilities, Dr

Sharifah Omar and Dr Sasheela Ponnampalavanar (UMMC) for clinical reviews of the

patients, Jeffrey Martinson (Rush University Medical Centre), Maxwell Pistilli and Luis

Montaner (The Wistar Institute) for expert technical advice, Sonia Fernandez (UWA)

for helpful discussions, Yeat Mei Lee for technical assistance and all patients and

controls who contributed to this study. The project was supported by the University of

Malaya Research Grant and the National Health and Medical Research Council (Grant

513755).

Chapter 6 - Dendritic cell function on ART

93

Chapter 6 - Robust IFNα and IL-12 responses by DC parallel

efficient CD4+ T-cell recovery in HIV patients on ART

Amongst HIV patients with successful virological responses to ART, poor CD4+ T-cell

recovery is associated with low nadir CD4+ T-cell counts and persistent immune

activation. These factors might be influenced by DC function. IFNα-producing

plasmacytoid DC and IL-12-producing myeloid DC were quantified by flow cytometry

after stimulation with agonists to TLR7/8 (CL075) or TLR9 (CpG-ODN). These were

compared between Malaysian HIV patients who achieved CD4+ T-cell counts above or

below 200cells/µL after 6 months on ART (High vs. Low groups).

High Group patients had more DC producing interferon-α or IL-12 at Weeks 6 and 12

on ART than Low Group patients. The frequencies of cytokine-producing DC at Week

12 were directly correlated with CD4+ T-cell counts at baseline and at Week 12.

Patients with good recovery of CD4+ T-cells had robust TLR-mediated interferon-α

responses by plasmacytoid DC and IL-12 responses by myeloid DC during early ART

(1-3 months).

6.1 Background

Successful ART suppresses HIV replication, allowing the recovery of CD4+ T-cell

numbers. However, some patients with a virological response to ART experience slow

or impaired recovery of CD4+ T-cells. This is more common in patients with low nadir

CD4+ T-cell counts, high levels of immune activation and a high rate of CD4

+ T-cell

apoptosis (Fernandez et al. 2006b; Massanella et al. 2010; Negredo et al. 2010).

Immune activation and CD4+ T-cell apoptosis may be mediated by IFNα (Herbeuval et

al. 2007; Sedaghat et al. 2008). Single-stranded RNA (ssRNA) viruses including HIV

can induce the production of IFNα in pDC and IL-12 in mDC through TLR7 and TLR8,

respectively (Heil et al. 2004). HIV ssRNA up-regulates expression of these TLRs and

enhances TLR-mediated responses, so DC in HIV patients may be hyper-sensitized to

TLR-mediated activation (Lester et al. 2008).

IFNα may promote activation-induced CD4+ T-cell depletion during HIV infection

through increased expression of tumor necrosis factor (TNF)-related apoptosis-inducing

ligand (TRAIL) by monocytes and DC (Herbeuval et al. 2005a; Herbeuval et al. 2007).


94

Apoptosis follows the binding of TRAIL to the TRAIL death receptor 5 (DR5) on CD4+

T-cells. Expression of DR5 can be directly induced by infectious and non-infectious

HIV particles (Herbeuval et al. 2007). CD4+ T-cell depletion may also occur as a result

of preferential differentiation of CD4+ T-cells into short-lived effectors under the

influence of type I IFN (IFNα and/or IFNβ) (Sedaghat et al. 2008).

Despite the potential for IFNα to impair CD4+ T-cell survival, HIV patients who

exhibited high TLR-induced IFNα production had higher CD4+ T-cell counts on ART

(Sachdeva et al. 2008). HIV patients had lower production of IFNα than controls in

response to synthetic TLR7/8 agonists and to other viruses such as Influenza and Herpes

Simplex Virus (HSV), whilst the production of IL-12 by mDC was higher in HIV

patients (Chehimi et al. 2002; Finke et al. 2004; Chehimi et al. 2007; Martinson et al.

2007). ART increased pDC numbers but they remained lower than controls, whereas

mDC numbers normalized (Finke et al. 2004; Chehimi et al. 2007). IFNα responses to

TLR agonists were higher at week 52 than at baseline but remained lower than controls

(Chehimi et al. 2007). There are few studies of earlier time points on therapy and no

longitudinal studies of IL-12 responses to illuminate whether poor production of these

cytokines predicts immune recovery. No studies of non-Caucasian populations were

found or those that involve patients with advanced immunodeficiency or from resource-

constrained countries.

I performed a longitudinal study of a small group of Malaysian Chinese HIV patients

who had acquired immunodeficiency syndrome (AIDS) and began ART with very low

CD4+ T-cell counts (below 53 CD4

+ T-cells/µL). Following stimulation with agonists to

TLR7/8 or TLR9, the frequencies of IFNα-producing pDC and IL-12-producing mDC

were compared between patients who achieved CD4+ T-cell counts above or below

200/µL after 6 months on ART.

6.2 Methods


Fourteen Chinese male HIV patients were selected from the study of immune

reconstitution after ART in UMMC (section 2.1) including IRD patients described in

section 5.2.1. Patients were divided based on attainment of a CD4+ T-cell count above

or below 200/µL after a minimum of 6 months (26-63 weeks) on ART (n=7 in each


95

group; Figure 6.1A), as the median CD4+ T-cell count was 206 cells/µL at this time. All

patients had been diagnosed with AIDS and experienced one or more opportunistic

infections pre-ART, including oral candidiasis (n=11), Pneumocystis jirovecii

pneumonitis (n=7) or active TB disease (n=5). Local Chinese male healthy controls

(n=15) were included and matched by age [median and range: 41 (33-44) for Low

Group; 40 (33-52) for High Group; 38 (23-71) for controls]. Institutional ethics

approval was obtained from the University of Malaya and informed consent was given

by all participants.

6.2.2 Sample collection, plasma HIV RNA levels and CD4+ T-cell

counts

Refer to section 2.1

6.2.3 Quantification of CD4+ T-cell activation, IFNα-producing pDC

and IL-12-producing mDC

CD4+ T-cell activation (co-expression of HLA-DR and CD38) was quantified by flow

cytometry (section 5.2.4). Frequencies of IFNα-producing pDC and IL-12-producing

mDC were determined by flow cytometry following stimulaton of PBMC for 6 hours

with CL075 (a synthetic agonist of TLR7 and TLR8; 10 µg/ml) and Class A CpG 2336

(a synthetic agonist of TLR9; 5 µM) (InvivoGen) in RPMI 1640 with 10% FCS at 1 x

106 cells/ml. The protein transport inhibitor, Brefeldin-A (5 µg/ml; BD Biosciences)

was added 1 hour after the addition of CL075. Cells were then washed with 1%

BSA/PBS and blocked with FcγR blocking reagent (Miltenyi Biotec). Cells were then

surface stained with Lin-1-FITC (CD3, CD14, CD16, CD19, CD20 and CD56 cocktail),

CD123-PerCP-Cy5.5, HLA-DR-APC-H7 (BD Biosciences) and CD11c-PECy7

(eBioscience) and intracellular stained with IFNα-AF647 and IL-12-PE (BD

Biosciences). Cells were processed using the BD Cytofix/CytopermTM

Kit according to

the manufacturer’s protocol for intracytoplasmic staining. All staining was performed at

room temperature in the dark. 300,000-500,000 events were acquired using a BD

FACSCanto II cytometer and analyzed using FlowJo software version 7.2.2 (Tree Star).

Gating methods are shown in Figure 6.2 and results were expressed as frequencies of

IFNα-producing pDC and IL-12-producing mDC.


96

6.2.4 Statistical Analyses

Data were analyzed with Prism software v5.02 (GraphPad Inc., La Jolla, CA, USA).

Mann-Whitney tests were used to compare between the Low Group, High Group and

healthy controls. Spearman’s tests were used to calculate non-parametric correlation

coefficients. For both tests, P-values less than 0.05 were considered to be statistically

significant.

6.3 Results

6.3.1 Recovery of CD4+ T-cells on ART was adversely affected by

low baseline CD4+ T-cell counts and high T-cell activation.

All patients had plasma HIV RNA levels >100,000 copies/mL before ART and

suppressed their plasma HIV RNA (<50-437 copies/mL) over 11-39 weeks of ART. All

patients also started ART with less than 53 CD4+ T-cells/µL which were then divided

according to their recovery of CD4+ T-cells on ART (Low and High Groups). After 2

years on ART (Week 97;77-118), median (range) of CD4+ T-cell counts were 122 (81-

239) cells/µL in Low Group patients and 464 (200-880) cells/µL in High Group

patients. The most recent data showed four High Group patients and no Low Group

patients achieved >500 CD4+ T-cells/µL after 200 (97-245) weeks on ART.

Baseline CD4+ T-cell counts were lower in Low than High Group patients [5 (0-36) vs.

46 (14-53) cells/µL, respectively; Figure 6.1A], whilst CD4+ T-cell activation at

baseline was higher in Low Group patients (Figure 6.1B). CD4+ T-cell activation at

Weeks 6, 12 and 24 were inversely correlated with baseline CD4+ T-cell counts (r = -

0.56 to -0.68, p = 0.01 to 0.05). Similarly, CD4+ T-cell counts recorded at Weeks 12 and

24 were inversely correlated with baseline CD4+ T-cell activation (r = -0.71, p = 0.006

and r = -0.60, p = 0.03, respectively).

6.3.2 TLR7/8 and TLR9-induced cytokine production in pDC and

mDC were correlated with CD4+ T-cell recovery

The frequencies of IFNα-producing pDC and IL-12-producing mDC induced by CL075

were directly related at all time-points (r=0.71-0.87, p<0.005). The frequency of IFNα-

producing pDC at Weeks 6 and 12 was higher in High Group patients than Low Group

patients (Figure 6.1C). At the same time-points, the frequency of IL-12-producing mDC

was higher in High Group patients than Low Group patients and healthy controls


97

(Figure 6.1D). The frequency of IFNα-producing pDC induced by CpG-ODN was

higher in High Group than Low Group patients at Week 6 [median (range): 4.1% (0.75-

27) vs. 0.52% (0-7.3), p=0.04], but was similar between groups at other time-points

(data not shown).

The frequencies of IFNα- and IL-12-producing DC in response to CL075 at Week 12

were directly correlated with CD4+ T-cell counts at Week 12 (Figure 6.3A), weakly

associated with CD4+ T-cell counts at baseline (Figure 6.3B), and inversely correlated

with CD4+ T-cell activation at baseline (Figure 6.3C). IFNα/IL-12 responses to CL075

and IFNα responses to CpG-ODN at baseline were not correlated with CD4+ T-cell

counts at Weeks 12 and 24 (r = 0.15-0.44; p = 0.12-0.61), and hence were not predictive

of CD4+ T-cell recovery.

6.4 Discussion

In most patients, ART-mediated suppression of HIV replication leads to immune

reconstitution measured by increased CD4+ T-cell counts. However, DC should be

considered in the definition of immune reconstitution as they regulate innate and

adaptive immune responses and generate cytokines that can influence CD4+ T-cell

recovery. I focussed on patients with advanced immunodeficiency and associate good

recovery of CD4+ T-cells (>200/µL after 6 months of ART) with robust TLR-mediated

IFNα responses by pDC and IL-12 responses by mDC during early ART (1-3 months).

It should be noted that these patients were assessed in an Asian clinic and many had

AIDS-related opportunistic infections. Our findings support previous studies associating

good recovery of CD4+ T-cells with higher TLR7- and TLR9-induced IFNα production

by pDC in samples collected after 3-6 years of ART (Sachdeva et al. 2008). An

association between the recovery of CD4+ T-cells and TLR8-induced IL-12 responses

by mDC has not been addressed previously.

Improved cytokine responses by DC may assist CD4+ T-cell homeostasis. IFNα and IL-

12 synergistically induce IL-2 secretion by CD4+ central memory T-cells (Davis et al.

2008). This is important for maintaining T-cell homeostasis and preventing CD95 (Fas)-

mediated apoptosis of CD4+

T-cells from HIV patients, though this effect was stronger

in CD4+ T-cells from healthy controls (Estaquier et al. 1996; Rodriguez et al. 2006). IL-

12 can prevent TCR-induced or Fas-mediated apoptosis of CD4+

T-cells from HIV


98

patients (Estaquier et al. 1995; Clerici et al. 1996). An alternative explanation for this

data is that immune activation in patients with low CD4+ T-cell counts increases the

activation of DC in vivo and hence limits the increase since seen after in vitro

stimulation. However, no cytokine production was detectable without in vitro

stimulation of DC from any donor.

Herbeuval et al. suggest a negative role for IFNα as it promotes the expression of the

death ligand (TRAIL) which promote CD4+ T-cell apoptosis upon ligation to DR5

(Herbeuval et al. 2007). The expression of DR5 and Fas correlated inversely with CD4+

T-cell counts in HIV patients (Sousa et al. 2002; Herbeuval et al. 2005b). Expression of

TRAIL, DR5 and Fas mRNA in circulating CD4+ T-cells decreased in HIV patients

who responded to ART and had >300 CD4+ T-cells (Herbeuval et al. 2009). Increased

TRAIL proteins in plasma and mRNA expression of DR5 was associated with reduction

in CD4+ T-cells after ART (Herbeuval et al. 2005b). However, in our patients

production of IFNα by pDC was directly (not inversely) related to CD4+ T-cell

recovery.

Rather than placing DC as the cause or effect of impaired recovery of CD4+ T-cells

during ART, it is also possible that they are influenced by a common factor. It is known

that during HIV infection, CD4+ T-cells and DC accumulate in lymphoid tissues (e.g.

lymph nodes) and display high rates of apoptosis (Chen et al. 2002; Dillon et al. 2008).

Fibrosis and damage to the architecture of lymph nodes during untreated HIV infection

were correlated with depletion of CD4+ T-cells (mostly within the naïve population) and

predicts poor recovery of peripheral T-cells later on ART (Schacker et al. 2006;

Schacker 2008). The recovery of peripheral DC able to produce cytokines may also be

limited by damage to the lymphoid tissues during untreated HIV infection.

In conclusion, better recovery of CD4+ T-cells is associated with higher frequencies of

IFNα-producing pDC and IL-12-producing mDC in response to TLR7/8 and TLR9

agonists during the first few months of ART. TLR-induced cytokine responses by DC

may therefore be informative as markers of functional immune reconstitution. CD4+ T-

cell counts and frequencies of cytokine-producing DC during ART are associated with

low baseline CD4+ T-cell counts and high immune activation. Therefore, robust TLR-

induced IFNα and IL-12 responses by DC may be influenced by factors that regulate

recovery of circulating CD4+ T-cells.


99

Figure 6.1 HIV patients who achieved higher numbers of CD4+ T-cells on ART

are characterized by high frequencies of cytokine-producing DC.

Patients were divided based on attainment of a CD4+ T-cell count above or below 200/µL (A).

Longitudinal evaluation of ex vivo CD4+ T-cell activation (B) and the frequencies of IFNα-

producing pDC (C) and IL-12-producing mDC (D) upon stimulation with agonists to TLR7/8

(CL075). The right panel shows cross-sectional comparisons of CD4+ T-cell counts, CD4

+ T-

cell activation and the frequency of cytokine-producing DC between Low Group patients, High

Group patients and healthy controls. Horizontal lines in each group represent median.


100

Figure 6.2 Gating methods for IFNα-producing pDC and IL-12-producing

mDC.

LIN-1-HLA-DR

+ cells (B) were first gated from PBMC population (A). PDC were

identified by expression of HLA-DR and CD123 (C), and subsequently analysed for

production of IFNα (E) MDC were identified by expression of HLA-DR and CD11c

(D), and subsequently analysed for production of IL-12 (F).


101

Figure 6.3 Frequencies of cytokine-producing DC were associated with CD4+ T-

cell counts at baseline and Week 12.

Associations between the frequency of IFNα-producing pDC (left panel) and IL-12-

producing mDC (right panel) in response to CL075 at Week 12 with CD4+ T-cell counts

at Week 12 (A), CD4+ T-cell counts at baseline (B) and CD4

+ T-cell activation at

baseline (C).

102

Chapter 7

Could Natural Killer cells compensate for

impaired CD4+ T-cell responses to CMV in HIV

patients responding to antiretroviral therapy?

Data from this chapter has been published

Could natural killer cell compensate impaired CD4+ T-cell functions in

HIV patients after long-term ART?

Tan DBA, Fernandez S, Tan HY, French M, Price P. Clin Immunol 2009;

132:63-70.

The authors thank the patients and staff of Royal Perth Hospital who donated blood for

this study. The project was supported by NHMRC (Australia) Grant 404028.

Chapter 7 - NK cells in HIV patients responding to ART

103

Chapter 7 - Could Natural Killer cells compensate for impaired

CD4+ T-cell responses to CMV in HIV patients responding to

antiretroviral therapy?

NK cell subsets and functions was evaluated in Australian HIV patients who were

immunodeficient before they began ART, but had virological response to treatment that

was stable over several years. This can be regarded as the best outcome for patients

beginning ART with advanced immunodeficiency in the developing world. NK subsets

were studied by flow cytometry and cytokine receptor mRNA was quantitated in

purified CD56+ cells. Data were correlated with CD4

+ T-cell counts and IFNγ responses

to CMV. NK cell IFNγ responses to K562 cells and proportions of CD56lo

NK cells

were correlated in patients (p<0.001) and both were lower than in controls (p<0.001 and

p=0.008, respectively), so all patients had poor NK cell function. Proportions of CD56hi

NK cells correlated inversely with current CD4+ T-cell counts (p=0.006) and perforin

expression in CD56hi

NK cells was higher in patients than controls (p<0.05). Hence

increased proportions and cytolytic function of CD56hi

NK cells may partially

compensate for CD4+ T-cell deficiency. NK cell IFNγ responses correlated inversely

with expression of IL-10 and IL-12 receptor mRNA. Expression of these transcripts is

reduced by exposure to the cytokines, which may reflect immune activation in

immunodeficient patients.

7.1 Background

Subsets of NK cells are defined as CD56lo

and CD56hi

. CD56lo

NK cells have high

expression of CD16, KIR and perforin which makes them respond better to NK-

sensitive target cells and mediate natural cytotoxicity and ADCC effectively. However,

on a per cell basis, CD56lo

NK cells secrete cytokines at lower levels compared to

CD56hi

NK cells. CD56hi

NK cells have important immuno-regulatory roles as they are

efficient cytokine producers (eg. IFNγ and TNFα) following cytokine and mitogen

stimulation (Section 1.2.3).

HIV infection causes the loss of CD56lo

and CD56hi

NK cell subsets but increase in the

functionally defective CD56-CD16

+ NK cells (Section 1.3.4). These changes are

partially restored after >6 months of successful ART (Sondergaard et al. 1999; Goodier


104

et al. 2003; Alter et al. 2005; Fauci et al. 2005; Chehimi et al. 2007). The baseline

CD4+ T-cell count was not defined in these studies and may affect the recovery of NK

cells on ART.

Abnormalities of NK cell function by untreated HIV infection have been reviewed

(Table 1.6; section 1.3.3.2) and different views on the effects of ART on restoration of

NK cell function have been highlighted (section 1.4.2.4). In Chapter 4, I have shown

that IFNγ responses by NK cells remained lower in HIV patients than healthy controls

after 1 year of ART but CD16 expression on CD56lo

NK cells recovered to normal

levels. Opposing results from different studies probably reflect characteristics of their

patients, such as baseline CD4+ T-cell counts and recovery of CD4

+ T-cell function.

However, these parameters are not uniformly reported.

Following ART, the recovery of NK cell IFNγ responses might parallel the recovery of

CD4+ T-cell IFNγ responses. Alternatively NK cells may compensate for inadequate

CD4+ T-cell function. To address this, I did a cross-sectional study of NK cell numbers

and function in HIV patients with good recovery of CD4+ T-cell numbers on ART who

were stratified by CMV-specific IFNγ CD4+ T-cell responses. I measured the

frequencies of NK subsets, FcγRIII (CD16) and perforin expression, as well as IFNγ

responses to K562 cells. To elucidate mechanisms underlying the observed

deficiencies, mRNA for IFNγ, IL-10R1, IL-12Rβ1 and IL-12Rβ2 were measured in NK

cells enriched from PBMC using CD56-coated magnetic beads.

7.2 Methods


Eighteen male CMV-seropositive, HIV patients attending clinics at Royal Perth

Hospital (Perth, Western Australia) and nine age-matched, male, CMV-seropositive

healthy volunteers were selected for a study of the effect of innate immunity (dendritic

cells and NK cells) on the recovery of a memory T-cell response. The effects of DC

were published by a colleague, Dr Sonia Fernandez (Fernandez et al. 2008). All patients

had started ART with <50 CD4+ T-cells/µl and maintained undetectable plasma HIV

RNA (<50 copies/ml) for more than 12 months after at least 17 months on treatment.

HIV patients were divided into low (n=8) and high (n=10) IFNγ responders based on

CD4+ T-cell responses to CMV assessed by ELISpot (denoted CMV-lo and CMV-hi,


105

respectively; Table 7.1). All patients had been assayed approximately 19 months

previously. Responses were stable, with all patients remaining in the same group. This

enabled us to use samples collected earlier or later from five patients for mRNA studies,

as the original samples were exhausted. Institutional ethics approval was obtained for

the study and informed consent was given by all participants.

7.2.2 Sample collection and routine data

Whole blood was collected into lithium heparin tubes. PBMC were obtained by Ficoll

gradient centrifugation and separated using magnetic bead technology or cryopreserved

in liquid nitrogen. Plasma HIV-1 RNA was measured using the COBAS Amplicor HIV-

1 Monitor Test, v1.5 (Roche Diagnostics, Indianapolis, IN, USA). CD4+ T-cell counts

were performed by routine flow cytometric methods.

7.2.3 IFNγγγγ ELISpot

Refer to section 2.3. PBMC were stimulated with CMV lysate (section 2.6.2) or K562

cells (section 2.6.4).

7.2.4 Flow cytometry

PBMC (5 x 105 cells) were surface stained with the following monoclonal antibodies:

CD3-APC, CD16-PECy5 and CD56-PE (Coulter Immunotech, Marseille, France). For

the staining of intracellular perforin, cells were permeablised using FACSlyse and

incubated with Perforin-FITC (BD Pharmingen, San Jose, CA, USA). Data were

acquired on the same day using a BD FACSCanto cytometer for 4-colour protocols.

50,000-100,000 events were recorded per tube and analyzed using the FlowJo program

v5.7.2 (Tree Star, Ashland, OR, USA). Gating strategies are shown in Figure 7.1.

7.2.5 RT-PCR

CD56+ cells were purified from fresh PBMC using conjugated magnetic bead kits

(Miltenyi Biotec, Bergisch Gladbach, Germany) by Dr Sonia Fernandez (Fernandez et

al. 2008). Two samples from each group were excluded because the purity was <70%.

For the remainder, the median purity was 80% (range 70-93) based on the phenotype

CD3-CD56

+. Most contaminants were CD56

+CD8

+ T-cells (data not shown). Real-time

PCR were used to quantify mRNA for β-actin, IFNγ, IL-10R1, IL-12Rβ1 and IL-12Rβ2.

The PCR protocol comprised 95oC for 300 sec, followed by 40 cycles of denaturation

(95oC for 10 sec), annealing (62

oC for β-actin and IL-12Rβ2, 68

oC for IL-12Rβ1 for 15


106

sec) and extension (72oC for 25 sec). Primer sequences for β-actin were 5’-

GATGACCCAGATCATGTTTGA-3’ and 5’-GACTCCATGCCCAGGAAGGAA-3’,

for IL-12Rβ1 were 5’-CTTCCAGAAGGCTGTCAAG-3’ and 5’-

GTATGGTGGCAGATGCCTG-3’ and for IL-12Rβ2 were 5’-

GGATGCTCATTGGCATTTAT-3’ and 5’-CAGGCCAGTTTGCAGACAA-3’

(Parrello et al. 2000). IFNγ and IL-10R1 mRNA levels were determined using

Quantitect®

sequence specific probes and Hs_IL10RA_SG_1 Quantitect®

Primer Assay

(Qiagen, CA, USA). Real-time PCR was performed on a RotorgeneTM

(Corbett,

Sydney, Australia). Quantitation utilized standard curves generated from amplification

of serial 10-fold dilution of pooled PCR products and results were expressed relative to

β-actin mRNA. The lower limit of detection was a ratio of 0.00001.

7.2.6 Statistical Analysis

Mann-Whitney tests were used to compare groups of individuals. Spearman’s test was

used to calculate the significance of non-parametric correlation coefficients. For all

comparisons, p-values below 0.05 were considered to be statistically significant.

7.3 Results

7.3.1 Proportions of CD56loNK cells were lower in HIV Caucasian

patients than controls

Patients with a stable virological response to ART were divided into CMV-lo and

CMV-hi groups based on IFNγ responses to CMV. The groups had similar durations of

treatment and current CD4+ T-cell counts, but the CMV-lo group had a lower median

nadir CD4+ T-cell count (Table 7.1). Proportions of NK cell subsets were calculated as a

percentage of lymphocytes and correlated with the parameters indicated. NK cells were

defined as CD3- lymphocytes which were CD56

-CD16

+, CD56

lo or CD56

hi.

Patients from CMV-lo and CMV-hi groups had a lower proportion of CD56lo

NK cells

than controls (p<0.03; Figure 7.2A), so the major NK cell population was deficient in

all HIV patients after long term ART (1.4 - 9.2 years) with prolonged viral suppression.

The proportions of CD56hi

and CD56-CD16

+ NK cells were similar in all groups.

Patient cohorts were then combined and proportions of each subset of NK cells were

correlated with CD4+ T-cell IFNγ responses to CMV and current or nadir CD4

+ T-cell


107

counts. Proportions of CD56hi

NK cells correlated inversely with current CD4+ T-cell

counts (r = -0.62, p = 0.006). Hence CD56hi

cells may compensate for CD4+ T-cell

deficiency.

7.3.2 Expression of perforin by CD56hi NK cells was elevated in HIV

patients compared to healthy controls

Perforin and CD16 were analyzed in CD56lo

and CD56hi

NK cells expressed as a

percentage of each NK subset and as mean fluorescence intensity (MFI). CD56lo

NK

cells have high expression of perforin and CD16 while CD56hi

NK cells have low or no

expression of perforin and CD16 (Figure 7.1A & B). Hence, perforin and CD16

expression was higher in CD56lo

NK cells than CD56hi

NK cells in all subjects (Figure

7.2B & C).

CD16 expression by CD56lo

and CD56hi

NK cells was similar in all groups (Figure

7.2C). When the patient groups were combined, expression of CD16 by CD56lo

NK

cells correlated with NK IFNγ responses (r=0.55, p=0.028) and CMV-specific IFNγ

CD4+ T-cell responses (r=0.48, p=0.043).

Perforin expression by CD56lo

NK cells was similar across all groups (Figure 7.2B &

C). Perforin expression by CD56hi

NK cells analyzed by MFI was higher in CMV-lo

and CMV-hi patients than healthy controls (p=0.015 & p=0.006 respectively). When the

CMV-lo and CMV-hi groups were combined, expression of perforin by CD56hi

NK

cells was higher in patients than controls (p=0.048 by percentages, p=0.003 by MFI).

Perforin expression by CD56hi

NK cells is increased by IL-2 in vitro (Ferlazzo et al.

2004) and peripheral fibroblasts administered as targets (Chan et al. 2007), so persistent

immune activation may explain the observed elevation.

7.3.3 NK cell IFNγγγγ responses were lower in HIV patients than

healthy controls

NK cell IFNγ responses were lower in the CMV-lo (p = 0.003) and CMV-hi (p = 0.002)

groups than healthy controls (Figure 7.3A). Amongst patients, NK cell IFNγ responses

did not correlate with CD4+ T-cell IFNγ responses to CMV (r = 0.21, p = 0.44), nadir

CD4+ T-cell counts (r = -0.34, p = 0.20) or current CD4

+ T-cell count (r = 0.41, p =

0.11). However they correlated directly with the proportion of CD56lo

NK cells (r =


108

0.92, p < 0.001; Figure 7.3B) and with expression of CD16 in this NK subset (r=0.55,

p=0.028; data not shown). Therefore, NK cell IFNγ responses to K562 cells are

attributed to CD56lo

NK cells. I confirmed this using samples from four controls and

two HIV patients assessed by flow cytometry. CD56lo

NK cells were the major

producers of IFNγ after stimulation with K562 cells (Figure 7.1).

7.3.4 Levels of mRNA for IL-10 and IL-12 receptors in CD56+ cells

do not explain poor NK cell IFNγγγγ responses in HIV patients

Impaired NK cell IFNγ responses in HIV patients may reflect low expression of

receptors for IL-10 or IL-12 since these cytokines are important in NK cell activation.

This was assessed in cells purified using CD56-coated magnetic beads. IFNγ mRNA

was assessed in parallel. This and expression of perforin and CD16 reflect NK cell

activity without in vitro stimulation.

Expression of mRNA for IFNγ, IL-10R1, IL-12Rβ1 and IL-12Rβ2 was similar in

CMV-lo, CMV-hi and healthy control groups (Figure 7.4A). Hence, low expression of

these transcripts does not explain the poor NK cell IFNγ responses of patients.

Expression of the receptors did not correlate with expression of IFNγ mRNA by

unstimulated CD56+ NK cells in patients (Figure 7.4B), so their expression does not

limit NK responses. However, several inverse correlations were observed. Co-

expression of perforin and CD16 correlated inversely with the expression of mRNA for

IL-10R1 (r = -0.56, p = 0.016) and IL-12Rβ1 (r = -0.63, p = 0.005) but not IL-12Rβ2 (r

= -0.22, p = 0.46). NK cell IFNγ responses correlated inversely with the expression of

mRNA for IL-10R1 (r = -0.76, p = 0.002), IL-12Rβ1 (r = -0.74, p = 0.004) and IL-

12Rβ2 (r = -0.64, p = 0.024) in the combined patient group (Figure 7.4B).

7.4 Discussion

Here, I established that there are persistent defects in NK IFNγ responses to a pan-NK

target (K562 cells) in HIV patients who had undergone long term ART (median of 8.5

years) compared to healthy controls. NK cell IFNγ responses in patients were attributed

to lower proportions of the major CD56lo

NK cell subset but not correlated with the

CD4+ T-cell IFNγ responses. As reviewed in (Lodoen et al. 2006), NK cell production

of IFNγ is important in controlling various viruses, parasites and bacteria infections. For

example, the inhibition of human CMV viral replication is dependent on IFN-β


109

production by infected cells induced by NK cells IFNγ (Iversen et al. 2005). Therefore,

HIV patients with stable responses to ART but poor NK function may remain at risk of

other infections.

The expression of CD16 and perforin were not deficient in our HIV patients. This

means the cytotoxicity and ADCC capabilities of NK cells were normal after long-term

ART. Provided that the NK receptors (eg KIR and NCR) required to recognize

malignant cells are intact, restored NK cell cytotoxicity could contribute to the less risk

of developing cancer observed in HIV patients on ART (Cheung et al. 2005; Spano et

al. 2008).

Expression of CD16 on CD56lo

NK cells correlates directly with NK cell IFNγ

responses in patients and CMV-specific CD4+ T-cell IFNγ responses This probably

reflects the activation or functional capacity of CD56lo

NK cells rather than the direct

role of CD16 in IFNγ production by NK cells or CD4+ T-cells as CD16 is not important

in the interaction of NK cells and K562 cells (Robertson et al. 1990; Vivier et al. 1991;

Garcia-Penarrubia et al. 2002).

Here, the proportion of CD56hi

NK cells correlated inversely with current CD4+ T-cell

counts. CD56hi

NK cells can acquire the phenotype of CD56lo

NK cells and increase

their perforin and CD16 expression upon stimulation (Ferlazzo et al. 2004; Chan et al.

2007). The cytotoxic capability of NK cells highly depends on the perforin content of

their granules (Jacobs et al. 2001). The expression of perforin by CD56hi

NK cells was

elevated in patients compared to healthy controls. Hence, increased proportion and

cytotoxic capacity of CD56hi

NK cells may reflect the ability of this NK subset to

compensate for poor CD4+ T-cell recovery in HIV patients on ART.

Proportions of CD56lo

NK cells and IFNγ responses to a pan-NK target (K562 cells)

were significantly lower in HIV patients than controls and were not correlated with the

CD4+ T-cell IFNγ responses. NK cell IFNγ responses in patients were attributed to

CD56lo

NK cells. Our data shows a persistent defect in IFNγ responses of CD56lo

NK

cells, but makes it unlikely that CD4+ T-cell IFNγ responses are limited by NK cell

IFNγ responses or vice versa. IL-10 production by NK cells can suppress antigen-

specific CD4+ T-cell proliferation and IFNγ production (Deniz et al. 2008), so higher


110

IL-10 secretion by NK cells could affect CD4+ T-cell responses. Here IL-10 mRNA was

not detectable in unstimulated CD56+ cells from the HIV patients (data not shown), but

IL-10 production by stimulated NK cells was not assessed.

Deficient NK cell IFNγ responses in HIV patients responding to ART may be caused by

low levels of cytokines important in the activation of NK cells. IL-10 and IL-12 can

stimulate the proliferation, cytotoxicity and IFNγ production of human CD56+ NK cells

in the presence of IL-2 (Carson et al. 1995; Cooper et al. 2001a). Expression of IL-10

mRNA by unstimulated PBMC declines following a virological response to ART

(Imami et al. 1999). IL-10 production was also lower in PHA-stimulated PBMC from

aviremic patients than healthy controls (Stylianou et al. 2000). However the source of

IL-10 affecting NK cells and the effects of prolonged ART on its release are unclear.

IL-10 and IL-12 derived from dendritic cells (DC) or monocytes warrant consideration

as factors limiting IFNγ production by the patients’ NK cells. Production of IL-10 and

IL-12 by myeloid DC was impaired in viremic, but not aviremic HIV patients. This

correlated with impaired activation and production of IFNγ by NK cells (Mavilio et al.

2006). Moreover IL-12 mRNA levels were not lower in patients than controls here , or

in our earlier study which included cells stimulated with PMA (Lee et al. 2004), so

there is no evidence that IL-12 is limiting in patients responding to ART.

NK cell activation and IFNγ production may also be affected by IFNα secreted by

plasmacytoid DC (Tomescu et al. 2007). Aviremic patients in the present study were

assessed for proportions of accessory cells and levels of cytokine mRNA in each

purified fraction (Fernandez et al. 2008). Proportions of plasmacytoid DC (and not other

populations) were directly correlated with CD4+ T-cell IFNγ responses to CMV.

However, proportions of DC and levels of IL-10, IL-12 and IFNα mRNA in DC and

monocytes did not correlate with NK cell IFNγ responses here (data not shown).

Here expression of IL-10R1, IL-12Rβ1 and IL-12Rβ2 mRNA was similar in both

patient groups and controls, so low receptor expression does not explain the low NK

cell IFNγ responses in patients. However, a significant inverse correlation was observed

between levels of all three transcripts with induced NK IFNγ responses and the co-

expression of perforin and CD16 in the combined patient group (Fig. 4B). This suggest


111

low IL-10R1, IL-12Rβ1 and IL-12Rβ2 mRNA may reflect chronic activation of NK

cells. This level of activation may increase NK cell IFNγ responses but not to levels of

healthy controls.

Chronic activation can differentially affect the levels of cytokine receptor mRNA and

protein. For example; IL-2 reduces levels of IL-10R mRNA but not the binding of

labelled IL-10 to NK cells. The authors concluded that expression of just 90 IL-10

receptors per cell yields optimal stimulation, so high levels of IL-10R1 mRNA are

redundant (Carson et al. 1995). Chronic activation of NK cells by IL-2 is unlikely in

HIV patients. Indeed levels of IL-2 mRNA in T-cell fractions from our patients did not

correlate with NK IFNγ responses or cytokine mRNA levels in NK cells (data not

shown).

Rather chronic immune activation in HIV patients may reflect bacterial products leaking

from a gut damaged during periods of extreme immunodeficiency (Brenchley et al.

2006b). NK cells express Toll-like receptors (TLR) and can be directly activated by

bacterial products (Chalifour et al. 2004). Alternatively, pDC activated via TLR may

activate NK cells via IFNα (Tomescu et al. 2007). In our cohort, nadir CD4+ T-cell

count inversely correlated with NK cell IFNγ responses and level of immune activation

of CD4+ T-cells were significantly higher than controls (p=0.01), so the degree of

previous immunodeficiency and residual immune activation affect NK cell function on

ART (Price et al. 2008). This suggest that low cytokine receptor expression and

increased IFNγ production are independent consequences of immune activation, where

the small increase in NK cell IFNγ responses does not compensate for low CD56lo

NK

cell proportions.

In conclusion; I identified three mechanisms that may affect NK cell function in HIV

patients with a stable virological response to ART. Firstly, there is increased perforin

expression by CD56hi

NK cells from HIV patients compared to healthy controls. The

second mechanism depresses IFNγ responses in direct proportion to loss of CD56lo

NK

cells. Finally, indirect evidence that immune activation depresses levels of mRNA for

IL-10 and IL-12 receptors were observed. Future studies to dissect mechanisms of

persistent defects in NK cell must include TLR and NK receptor signalling and

measurement of critical cytokines.


112

Table 7.1 Demographics of study cohort

CMV-lo CMV-hi Healthy

Controls

N 8 10 9

Age

(years) 54 (42-65) 50 (43-67) 53 (32-59)

Nadir CD4+ T-cell count

(cells/µµµµl) 8 (0-48)

b 31 (0-45) NA

Current CD4+ T-cell count

(cells/µµµµl) 594 (120-961) 652 (168-1152) 774 (525-1260)

Time on ART

(months) 102 (17-112) 102 (49-105) NA

IFNγγγγ response to CMV

(mediated by CD4+ T-cells)

a

47 (19-145)c 372 (203-850) 216 (37-1106)

IFNγγγγ response to K562 cells

(mediated by NK cells)a

451 (190-655)d 384 (107-861)

d 1089 (777-2499)

Values are presented as median (range), NA = Not applicable

a ELISpots per 200,000 PBMC

b Lower than CMV-hi (p = 0.05)

c Lower than CMV-hi and controls (p < 0.001)

d Lower than controls (p < 0.01)

Note: The selection of patients, compilation of routine laboratory data and CMV

ELISpot assays were performed by Dr Sonia Fernandez (UWA).


113

Figure 7.1 CD56lo

NK cells produced more IFNγ in response to K562 cells and

expressed more CD16 and perforin than CD56hi

NK cells.

Gating strategies were based on expression of (A) CD56 and CD16, and (B) CD56 and

Perforin. Expression of intracellular IFNγ following stimulation of PBMC with K562

cells.


114

Figure 7.2 Proportion of CD56lo

NK cell was low in CMV-lo and CMV-hi

patients

Proportions of each NK cell subset as a percentage of lymphocytes (A). Expression of

perforin (B) and CD16 (C) as a percentage of each NK cell subset (left panel) and MFI

(right panel).


115

Figure 7.3 Low NK cell IFNγ responses were associated with low frequency of

CD56lo

NK cells

Numbers of NK cells producing IFNγ after stimulation with K562 cells was low in

CMV-lo and CMV-hi patients (A). IFNγ production by NK cells was directly related to

the proportion of CD56lo

NK cells in HIV patients (B).


116

Figure 7.4 Expression of mRNA for IFNγ, IL-10 receptor and IL-12 receptors

by RT-PCR in purified CD56+ cells.

(A) Expression of IFNγ, IL-10 receptor and IL-12 receptors was similar in all groups.

(B) Expression of receptors was inversely related to co-expression of perforin and CD16

(except IL-12Rβ2) as well as NK cell IFNγ ELISpot responses to K562 cells in HIV

patients.

117

Chapter 8

Conclusions

&

future studies

Chapter 8 - Summary and future studies

118

Chapter 8 - Conclusions and future studies

Two major themes are highlighted in this thesis: 1) Immune Restoration Disease (IRD)

and 2) understanding immune recovery occurring in immunodeficient HIV patients with

a virological response to ART. HIV patients with severe immunodeficiency before

starting ART are at risk of IRD and poor immune recovery so patients involved in my

work all had <200 cells/µL pre-ART. My objective was to determine the immunological

characteristics of these unfavourable outcomes to give insights into the causes and

markers that may help us predict and diagnose them. A better understanding of the

underlying mechanism could lead to the development of immunotherapies and other

treatment strategies for HIV patients starting ART.

8.1 Immune restoration disease: Elevation of innate and


Up to 40% of HIV patients in resource limited countries develop IRD after starting ART

(section 1.5). IRD is increasing as a clinical problem due to the increased access to ART

in these countries. Many clinical descriptions of IRD have been published but the

pathogenesis and causes of IRD remain unclear. Numerous reviews conclude that IRD

reflect the restoration of pathogen-specific immune responses that are

immunopathological rather than protective, but further details are only now beginning to

emerge from prospective studies such as those in this thesis.

I chose to begin with the role of IFNγ responses to relevant antigens. IFNγ was selected

as it is an important mediator of Th1 or cell-mediated immune responses (Chapter 3).

IRD presenting as TB lymphadenitis and Cryptococcal meningitis were associated with

increased IFNγ responses to PPD and mannoprotein from Cryptococcus neoformans,

respectively. This is the first demonstration in HIV patients who experienced

Cryptococcal IRD and supports data from patients who experienced TB IRD in South

Africa, France and Cambodia (Bourgarit et al. 2006; Meintjes et al. 2008c; Elliott et al.

2009). It was noted that some patients whose IFNγ responses to PPD increased on ART

did not develop TB IRD, whilst cryptococcal-specific IFNγ responses were low in all

non-cryptoccocal IRD patients. This may reflect less common exposure of the

population to Cryptococcus neoformans than Mycobacterium tuberculosis.


119

I also correlated proportions of CD4+ T-cells with either an activation (HLA-DR

hi) or

regulatory (CTLA-4+ or CD25

+CD127

lo) phenotype with the development of IRD

(Chapter 3). IRD patients had higher proportions of activated CD4+ T-cells, mostly

peaking during their IRD. Interestingly, regulatory CD4+ T-cells were not lacking in

IRD patients, rather they were increased in parallel with immune activation. Data from

South Africa showed slightly higher frequency of CD4+ T-cells expressing Foxp3 (a

marker for T-reg) in patients who experienced paradoxical TB IRIS (at presentation)

than in patients who did not (Week 2 of ART), but this was not significant (p=0.13)

(Meintjes et al. 2008c). IRD may associate with impaired T-reg function (Seddiki et al.

2009), though this awaits confirmation.

Next, the role of NK cells was evaluated as they can mediate Th1 responses through

production of IFNγ (Chapter 4). A role in IRD was suggested by our finding that

Australian HIV patients who experienced herpes virus IRD or CMV as an AIDS

defining illness had NK cell genotypes consisting of more activating KIR genes (Price

et al. 2007). Such patients have persistently low systemic T-cell IFNγ responses to

relevant antigens suggesting a pathogenesis distinct from mycobacterial or cryptococcal

IRD. No studies have addressed the role of NK cells in these IRD.

I showed that NK cell IFNγ responses were low in all HIV patients after ART

regardless of IRD, so they may not contribute to the increased IFNγ production

observed mycobacterial or cryptococcal IRD. However, CD56lo

NK cells from IRD

patients have lower expression of CD16 prior to ART. This may relate to less efficient

clearing of pathogens by NK cells, thereby increasing the risk of developing IRD. New

assays are being developed to assess the other arms of NK cell-mediated immune

protection such as cytotoxicity (CD107a degranulation) and secretion of pro-

inflammatory cytokines. Target cells will include human fibroblasts infected with CMV

(assayed with and without antibody to elicit ADCC) and the donors will be grouped

according to HIV status and KIR genotype. In the longer term, these assays will be

applied to longitudinal sample sets including patients with IRD.

Data from studies in South Africa, Cambodia, France and Malaysia (Chapter 3)

associate TB IRD with increased MTB-specific Th1 responses (Bourgarit et al. 2006;

Meintjes et al. 2008c; Elliott et al. 2009). However, increased responses were also

evident in patients who did not develop TB IRD and amelioration of TB IRD symptoms


120

by steroids did not depress IFNγ responses (Meintjes et al. 2008c; Skolimowska et al.

2009). Hence, IFNγ responses may not explain all cases of TB IRD (e.g. paradoxical vs.

ART-associated TB) but likely to associate with responses to pathogen-specific antigens

in the system.

IRD with different clinical manifestations may involve different immuno pathogeneses

including defects in innate immune responses. This is highlighted in the study from

Cambodia which show more prominent Th1 responses (IFNγ) in ART-associated TB

(Elliott et al. 2009), with higher plasma levels of IL-18 but lower CXCL10 compared to

paradoxical TB IRIS (Oliver et al. 2010). This suggest that adaptive T-cell responses

may involve in IRD presenting as ‘unmasking’ of an infection after starting ART

(possibly to viable pathogens), while innate responses may be more important in

paradoxical type cases (possibly in the absence of viable pathogens due to treatment).

I decided to investigate the role of the innate immune system focusing on antigen-

presenting cells (APC) and TLR-mediated responses (Chapter 5). APC such as DC and

monocytes encounter pathogens first and present antigenic peptides to T-cells and

regulate their responses through cytokines. For example, mycobacterial cell wall

products such as lipomannan (LM) can be recognized by TLR2 on mDC and monocytes

which induce the production of pro-inflammatory cytokines (e.g. TNFα), Th1-related

cytokines (e.g. IL-12) and regulatory cytokines (e.g. IL-10). Persistently high TLR2

expression and increased in LM-induced TNFα and IL-12p40 responses were observed

in some TB IRD patients but were not seen in non-IRD patients. IL-10 responses were

low in these IRD patients. This suggests the role of TLR2-induced pro-inflammatory

cytokines without parallel production of regulatory cytokines in driving the

development of TB IRD.

This work warrants further investigations in larger cohort with other known

mycobacterium-derived TLR2 ligands such as LAM which may induce different

profiles of immune responses. LAM derived from MTB inhibited rather than induced

IL-12 production by monocytes or DC (Nigou et al. 2001). Another PhD student in our

unit showed increased levels of IL-18 in plasma in patients who developed TB IRD

compared to non-IRD controls (Oliver et al. 2010). Production of monocyte–derived

cytokines like IL-18 and IL-1 should be investigated after stimulation with TLR2


121

agonists. If the role of TLR2 can be confirmed and defined, it may present as a novel

therapeutic target for preventing IRD associated with mycobacterial infection.

TLR7/8- and TLR9 induced-cytokine responses were not associated with the

development of TB IRD in my cohort (Chapter 5). These TLR may be less important

since they do not specifically recognize mycobacterial products but rather are involved

in the recognition of viral RNA. A collaborative study has been established with the

University of Indonesia involving HIV/HCV co-infected patients of whom some

patients develop exacerbation of hepatitis (Appendix 1). This is defined as HCV-related

IRD. Longitudinal samples of PBMC have been collected from these patients and I have

been helping our new PhD student (Heni Saraswati) to set up flow cytometry protocols

to enumerate DC able to produce cytokines in response to TLR3, 7, 8 and 9 agonists.

We are using ELISpot assays to detect the small proportion of cells that produce IFNγ

in response to these agonists and HCV antigens. We hypothesize that these immune

parameters will increase on ART in patients who experience HCV IRD and not in non-

IRD patients.

8.2 Recovery of CD4+ T-cells: Associated with good DC

function with a potential for functional compensation by

NK cells

Functional recovery of CD4+ T-cells, NK cells or DC may be inadequate in HIV

patients with a virological response to ART. These patients have increased risk of HIV

disease progression and may develop other opportunistic infections or malignancies.

Here proportions and functions of NK cells and DC were associated with CD4+ T-cell

counts.

In Chapter 6, I showed that higher CD4+ T-cell counts after ART paralleled robust IFNα

and IL-12 responses in DC. This is first demonstrated in HIV patients from South-East

Asia and in patients early in ART (3-6 months). It supports a study which collected

samples after >2 years of ART (Sachdeva et al. 2008). The effect on either the DC or

the CD4+ T-cells may be causative or both may respond to a common factor such as

lymphoid tissue damage before ART. Similar assays are being applied to our samples

from patients treated in Jakarta, so we expect to refine this result in a separate South-

East Asian population.


122

There was no association observed between CD4+ T-cell recovery and the proportions

and IFNγ responses of NK cells in our Malaysian cohort (Chapter 4). The proportions of

CD56lo

and CD56hi

NK cells were relatively stable during the first year of ART. CD16


NK cells was impaired at baseline but recovered to normal levels

after 6 months of ART. Numbers of IFNγ-producing NK cells were lower in patients

than healthy controls prior to ART and increased significantly after 6 months. However,

this level remained lower compared to healthy controls. The studies of NK cells

described in Section 8.1 will allow us to correlate NK responses to CMV-infected target

cells with CD4+ T-cell recovery.

A small study carried out using samples collected and purified by a post doctoral fellow

of our lab (Dr. Sonia Fernandez) correlated NK cell function with CD4+ T-cell IFNγ

responses to CMV in Caucasian HIV patients who were on long term ART (2-5 years). I

found increased proportions and perforin expression in CD56hi

NK cells in patients who

had lower CD4+ T-cell responses to CMV. This suggests a mechanism by which NK

cells may partially compensate for impaired CD4+ T-cell mediated responses. Assays

described in Section 8.1, will be applied to a cohort consisting of older patients (stable

on long-term ART) and will include other NK cell receptors such as the NCR, KIR and

C-lectin type receptors (section 1.3.4).

8.3 Conclusions and study limitations

Results presented in this thesis suggest that IRD (TB and cryptococcal-associated) are

related to increased IFNγ responses to antigens from provoking pathogens and increased

TLR-induced pro-inflammatory cytokines from DC or monocytes may drive the

development of some TB IRD. This mechanism fits with emerging evidence implicating

persistent pathogen load in susceptibility to IRD. Furthermore, the ability of DC to

produce cytokines is related to CD4+ T-cell recovery. Future studies should address the

role of DC and their innate responses in IRD and whether impaired DC function is a

cause or effect of poor CD4+ T-cell recovery on ART. Impaired NK cell function before

ART may be a risk factor for IRD, while the CD56hi

NK cells may compensate for poor

function of CD4+ T-cells after ART.


123

Based on the data presented in this thesis, immune modulatory therapies could be

developed to regulate immune responses and prevent any IRD or better recovery in HIV

patients commencing ART. However, interpretations were derived from a small number

of cases (e.g. patients who experienced IRD) which were heterogeneous in nature (e.g.

paradoxical vs. unmasking IRD). Hence, the findings should be confirmed in larger

cohorts distinguished by their clinical history and presentation, to elucidate the

immunopathological mechanisms of IRD and the determinants of optimal immune

recovery.

Chapter 9 - References

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Appendix

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Appendix 1 - HCV IRD Study (Indonesia)

Fifty HIV/HCV co-infected patients from the Pokdisus AIDS Clinic, Cipto

Mangunkusumo Hospital, Jakarta, Indonesia were recruited into a prospective cohort

study managed by Dr Evy Yunihastuti. Blood samples were collected at weeks 0, 4, 8,

12 and 24 after ART from all patients. Additional samples were taken during an IRD

event. PBMC, plasma and serum were isolated and cryopreserved. Routine CD4+ T-cell

counts and serum alanine transaminase (ALT) levels were available at all timepoints.

Plasma HIV RNA levels were only available at baseline, 6 months after ART and

during an IRD event while HCV RNA levels were only measured at baseline.

Inclusion criteria:

1) Age: 17-50 years old

2) Starting ART with <200 CD4+ T-cells/µL

3) HCV sero-positive

4) Risk factors for HIV/HCV: Intravenous drug use

5) Treatment status: ART naïve and have not received anti-HCV therapy

Exclusion criteria:

1) Co-infection with Hepatitis B virus (i.e. positive for HBsAg)

2) Defaulted ART

3) Pregnancy

4) Kidney, heart or end-stage liver failure

HCV IRD is defined as:

1) Elevated serum ALT

- 5 times higher than upper normal limit or 3 times higher than baseline

2) Achieved 1 log decreased of HIV RNA from baseline

IRD observed in the cohort include HCV (n=9), TB (n=5), HZV (n=5), Mycobacterium

avium complex (n=1) and CMV (n=1).

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