Human virus-specific T cells in peripheral blood and lymph nodes: Phenotype, function and clonal relationships - Thesis (complete)

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Human virus-specific T cells in peripheral blood and lymph nodes: Phenotype,

function and clonal relationships

Remmerswaal, E.B.M.

Publication date

2014

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Final published version

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Remmerswaal, E. B. M. (2014). Human virus-specific T cells in peripheral blood and lymph

nodes: Phenotype, function and clonal relationships.

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HUMAN VIRUS-SPECIFIC T CELLS IN

PERIPHERAL BLOOD AND LYMPH NODES:

phenotype, function and clonal relationships

H

U

MAN VI

R

US-SPE

C

IFIC T CELLS IN PE

R

IPHER

A

L

B

LOOD

A

ND

LYMPH NODES:

phenotype, fun

ct

ion and c

lona

l r

elat

ionsh

ip

s

Ester B.M. Remmerswaal

Ester B.M. Remmerswaal

2014

HUMAN VIRUS-SPECIFIC T CELLS IN PERIPHERAL

BLOOD AND LYMPH NODES:

PHENOTYPE, FUNCTION AND CLONAL RELATIONSHIPS

This research was performed at the department of Experimental Immunology (EXIM) and the Renal Transplant Unit, Department of Internal Medicine, at the Academic Medical Centre (Amsterdam, the Netherlands).

Title: Human virus-specific T cells in peripheral blood and lymph nodes: phenotype, function and clonal relationships Cover: "verdedigingswerken", 2014

by Marjolein Spitteler (www.marjoleinspitteler.nl) Printing and layout: Off Page

ISBN: 978-94-6182-492-9

   

HUMAN VIRUS-SPECIFIC T CELLS IN PERIPHERAL

BLOOD AND LYMPH NODES:

PHENOTYPE, FUNCTION AND CLONAL RELATIONSHIPS

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam op gezag van de Rector Magnificus

prof. dr. D.C. van den Boom

ten overstaan van een door het college voor promoties ingestelde commissie, in het openbaar te verdedigen in de Agnietenkapel

op woensdag 19 november 2014, te 14:00 uur door

Elisabeth Bernardina Maria Remmerswaal geboren te Voorburg

Promotiecommissie

Promotores: Prof. Dr. R.A.W. van Lier

Prof. Dr. R.J.M. ten Berge

Overige leden: Prof. Dr. A. Akbar

Dr. V. Appay

Prof. Dr. T.B.H. Geijtenbeek Prof. Dr. T.W. Kuijpers Dr. G.M.G.M. Verjans Prof. Dr. H.L. Zaaijer

TABLE OF CONTENTS

Chapter 1 General introduction 9

Chapter 2 Blood and beyond: Properties of circulating 39

and tissue-resident human virus-specific

αβ

CD8+ _{T cells}

Chapter 3 Two dimensions in human CD8+ _{T-cell development: 63}

cell surface phenotype in conjunction with T-bet and Eomes expression levels predicts the functional potential

of antigen-experienced CD8+ _T-cells

Chapter 4 Deep sequencing of antiviral T-cell responses to hCmv 97

and EBv in humans reveals a stable repertoire that is maintained for many years

Chapter 5 Human virus-specific effector-type T cells accumulate 125

in blood but not in lymph nodes

Chapter 6 Clonal evolution of CD8+ _{T cell responses against latent 149}

viruses: relationship between phenotype, localization and function

Chapter 7 Emergence of a CD28

⁻

granzyme B+_CD4+ _{cytomegalovirus 183}

-specific T cell subset after recovery of primary cytomegalovirus infection

Chapter 8 Strong selection of virus-specific cytotoxic CD4+ _{T-cell 205}

clones during primary human cytomegalovirus infection

Chapter 9 General discussion 225

Appendix Summary 241

Samenvatting voor niet ingewijden 245

List of Publications 253

Abbreviations 257

PhD portfolio 261

Curriculum vitae 263

General introduction

1

THE IMMUNE SYSTEM

The complex system of cells and molecules that deals with invading foreign entities, like bacteria and viruses, is called the immune system. The immune response can be divided into the innate response and the adaptive response. Upon invasion of our body with a foreign entity the innate response occurs first and is generally non-specific. NK cells and phagocytes like macrophages are key players of the innate immune response. The innate immune response creates a window in which the adaptive immune system can be instructed. The adaptive immune response is specific and in general it will provide long lasting immunological memory. This response depends on the capture and presentation on antigen presenting cells (APC). Dendritic cells are highly specialized professional

APCs. B cells, providing humoral immunity by producing antibodies, and CD4+ _and

CD8+ _{T cells, providing cellular immunity, are cells of the adaptive immune response.}

This thesis describes the analysis of CD8+ _{and CD4}+ _{T cell responses against}

(persistent) viral infections in peripheral blood and lymph nodes in ‘steady state’ as well as during primary infection and reactivation. Flow cytometry, chromium release

assays, stimulation assays and high throughput sequencing of the TCR-V

β

were used

to detect and study the phenotype, function and relationships of virus-specific T cells.

When studying virus-specific CD8+ _{and CD4}+ _{T cells it is also important to be aware}

of the expression profile of the protein / epitope studied as well as properties of the virus that might influence such an immune response. This introduction will therefore contain background information on each virus containing mode of infection, tropism, composition of the virion, and immune escape mechanisms. The function and expression profile of the proteins against which the T cell responses were studied will be explained in more detail. For the techniques used, it will provide an introduction on the T cell receptor and

its rearrangement and on the detection-methods of virus-specific CD8+ _{and CD4}+ _{T cells.}

The T cell receptor

The T cell has a T cell receptor (TCR) on its surface for recognition of antigens presented in HLA (human leukocyte antigen) on the surface of an APC. In the thymus somatic

rearrangement of the germ line TCR genes takes place. First the

β

-chain of the TCR is

rearranged so that it contains only one variable (V), diversity (D), joining (J) and constant

(C) segment (figure 1). The

α

-chain is only rearranged once the

β

-chain has been proven

productive. The

β

-chain of the TCR is rearranged from 52 V regions, 2 D regions and 13

J regions and during the rearrangements of the V and D region and the D and J region nucleotides will be randomly added in between by terminal deoxynucleotidyl transferase

(TdT) or subtracted by exonuclease. The

α

-chain rearranges from approximately 70 V

regions and 61 J regions. Random addition and removal of nucleotide occurs at only one location: the joining region between the V and J region.

All these different VDJ rearrangements together with the nucleotide addition and

General introduction

1

FIGURE 1: TCR rearrangement in the thymus.

of a TCR when recognizing a HLA-peptide complex also allows for recognition of different HLA-peptide complexes by one TCR. The highly variable region around

the D segment in the

β

-chain and the junction between the V and J segment in the

α

-chain encodes the CDR3 region.

Through the process of negative and positive selection in the thymus, both auto-reactive T cells as well as non-functional or low binding T cells are largely eliminated. It is estimated that only approximately 2% of the developing thymocytes will finally leave the thymus to enter the periphery. Upon recognition of their cognate antigen-HLA complex presented on DCs, T cells will start to proliferate. Since the TCR is fixed after its rearrangement in the thymus, the offspring of the proliferating T cell will all carry the same TCR. These T cells with identical TCRs are referred to as a clone and the CDR3 region of a TCR can be used as a fingerprint to identify all T cells from the same parent.

CD8

+

_{T cells}

CD8+ _{T cells recognize peptides presented in the context of HLA-I. HLA-I is expressed}

on every cell in our body, with the exception of erythrocytes. Peptides expressed in

HLA-I reflect the intracellular components of a cell, allowing CD8+ _{T cells to detect}

cells that are infected or malignant. Because of their function CD8+ _{T cells are often}

referred to as cytotoxic T cells or CTL. When a naïve T cell recognizes its cognate HLA-peptide complex on a professional APC it starts to proliferate, leaves the lymph

node and migrates to the site of infection. The CD8+ _{T cells will start producing large}

amounts of granzyme and perforin, allowing them to effectively kill infected cells. Once

the infected cells are eliminated the majority of the CD8+ _{T cells will go into apoptosis.}

A few cells however remain, providing immunological memory. These memory CD8+ _T

cells swiftly regain effector functions upon a reinfection with the same virus.

Virus-specific CD8+ _{T cells can be visualized on a fluorescence activated cell sorter}

(FACS) by incubating them with HLA-I-peptide complex tetramers labeled with a fluorochrome (Figure 2).

General introduction

1

FIGURE 2: Visualization of virus-specific CD8+ _{T cells with HLA-I tetramers.}

CD4

+

_{T cells}

CD4+ _{T cells recognize peptides in the context of HLA-II. HLA-II is expressed on}

(professional) APCs, like DCs, B cells and monocytes, but also on T cells, endothelial and epithelial cells when they become activated, infected or stressed. Exogenous peptides

are presented in HLA-II. CD4+ _{T cells are often named T helper cells, because they}

support other immune cells, like CD8+ _{T and B cells in their function. However, the CD4}+

T cell population also comprises a subset that expresses granzyme B and perforin (1).

Virus-specific CD4+ _{T cells can be visualized on a FACS after re-stimulation ex vivo with}

their cognate peptide or antigen in the presence of antigen presenting cells (figure 3).

FIGURE 3: Visualization of virus-specific CD4+ _{T cells by stimulation with virus, viral proteins or}

General introduction

1

VIRAL INFECTIONS

Most of the viral infections, humans encounter during their life are effectively cleared

by the immune system. Examples of such viruses are influenza and respiratory syncytial virus (RSV). Not all viruses however are dealt with so effectively. All herpes viruses, like hCMV and EBV, but also other viruses like HIV persist after the primary infection, hiding or constantly escaping from the immune system.

Persistent viral infections

Herpesviridae

V i r a l s t r u c t u r e

A herpes virion consist of double stranded DNA contained by the icosahedral nucleocapsid, surrounded by a series of tegument proteins which in turn are enveloped by a lipid bilayer, containing the envelope proteins, which are usually involved in binding and entry of the target cell (figure 4).

FIGURE 4: Schematic representation of a herpesvirus virion.

Herpesviruses have co-evolved with their vertebrate hosts for more than 100 million years (2). Over 130 herpesviruses have been identified so far and 8 of them are herpesvirus types that have been shown to infect humans: herpes simplex viruses 1 and 2, varicella-zoster virus, EBV (Epstein-Barr virus), human cytomegalovirus, human herpesvirus 6, human herpesvirus 7, and Kaposi’s sarcoma-associated herpesvirus (3).

V i r a l l i f e c y c l e

After binding of the virus to the cell, the viral envelope is fused with the cell membrane and the contents are released into the cytoplasm. The double-stranded viral genome

General introduction

1

within the icosahedral capsid is subsequently transported from the cytoplasm through

the nuclear pore via an active process. Once in the nucleus, the capsid is removed and the viral DNA is transcribed. First the immediate early (IE) genes are expressed (4). The IE proteins act as trans-activators for the early and late gene expression and change the host cell environment. Once transcription of the viral genome has commenced, orchestrated by the early genes, the late genes are transcribed. These late viral genes encode the structural proteins required for the assembly of the new virion. Once the viral particles are assembled, they bud off and start a new wave of infection (5). In contrast to the far majority of the human genes, almost all of the early and late genes from herpes viruses do not have introns (6). Transport from the nucleus and accumulation in the cytoplasm of these intronless mRNA’s is solely depending on viral proteins, like UL69 in HCMV (7) and BMLF1 in EBV (8). Without these proteins, production of new viral particles is ceased.

In some cells, the IE genes are suppressed, halting the lytic cycle. When this happens, hardly any proteins are transcribed and no infectious viral particles are produced: the virus enters a state of latency. Upon environmental clues, latency can be broken causing reactivation of the lytic cycle and resulting in the production of infectious viral particles once again. Suppression of the immediate-early genes by microRNA’s to enter and maintain latency has been shown for EBV (9) and hCMV (10, 11).

Human cytomegalovirus (hCMV)

HCMV (also called human herpesvirus 5) is a

β

-herpesvirus that causes a chronic

systemic infection. More than half of the adult population is latently infected with hCMV. Transmission occurs via bodily fluids like saliva, urine and breast milk. Transplantation of both solid organ as well as hematopoietic stem cells of an hCMV-seropositive donor into an hCMV-seronegative recipient can also cause primary hCMV infection. This latter mode of infection allows for the unique opportunity to study primary infections in humans: the exact date of infection (the day of transplantation) is known and close monitoring by hCMV-PCR and hCMV IgG and IgM elisa can be used to follow the course of infection.

HCMV infection is usually symptomless, but in some cases it can cause infectious mononucleosis. In immunocompromised patients such as transplant recipients and HIV infected individuals hCMV can lead to hepatitis, pneumonia, gastrointestinal disease and/or retinitis. Transmission of hCMV from the mother, undergoing a (symptomless) primary infection, to the unborn child can lead to mental retardation and deafness. HCMV-latency has also been associated with vascular disease (12).

The genome of hCMV is 235kb long and comprises at least 166 open reading frames (ORFs) (13). Some parts of the genome are highly diverse between strains. By deep sequencing it has been shown that primary infection usually occurs with only one strain. However, individuals can get infected with more strains during life as they have been shown to contain up to 6 different strains at a time. Usually one or two strains were shown

General introduction

1

to represent over 80% of all strains found, but with new reactivations subdominant types could become dominant (14), suggesting that not all different strains reactivate at the

same time. The genetic content of each hCMV strain was shown to be very stable. HCMV has been shown to infect a large variety of cell types varying from diverse types of endothelial and epithelial cells as well as leukocytes. Viral entry steps vary per cell type (15, 16). So far, four complexes have been shown to mediate viral entry: gCI (gB) (17), gCII (gM/gN) (18), gCIII (gH/gL/gO) (19), and the pentameric gH complex (gH, gL, pUL128, pUL130 and pUL131) (20, 21). Latent hCMV infection has been shown in, but might not be restricted to cells of the myeloid lineage, like hematopoietic stem cells and monocytes, which makes these cell types a source for reactivation (22, 23).

The capability of hCMV to infect many cell types is crucial in its viral spread (24, 25). Since hCMV is usually transmitted via bodily fluids the first cells to become infected are usually the mucosal epithelial cells (26). Once these locally infected epithelial cells produce virions they infect leukocytes (and circulating endothelial cells), which in their turn disseminate the virus through the blood stream infecting organ- and tissue-specific cells like vascular endothelial cells and epithelial cells of the kidney, liver, lung and salivary glands (27, 28). A recent paper showed that mCMV

can in fact recruit CX₃CR1 expressing patrolling monocytes (PM) to the site of primary

infection by production of the viral chemokine MCK2. As a result, PM became infected and disseminated mCMV. They even showed that PM became latently infected by transferring the mCMV infection by transferring the PM monocytes long after primary infection into a naïve recipient (29). In a similar fashion hCMV has been shown to encode vCXCL1 (UL146) and vCXCL2 (UL147) which allows CXCR1 and CXCR2 expressing neutrophil recruitment, infection and dissemination (30). Another viral gene US28 has

been reported to encode a vCX₃CR1 homologue, steering hCMV infected leukocytes

towards CX3CL1 (fraktalkine) expressing immune activated endothelial and epithelial

cells, resulting in viral spread (31). Infection of a cell can occur via cell free virus, but also via virion free cell to cell transmission (32). US28 has been shown to facilitate cell to cell spread (33). Other G protein-coupled receptors (GPCRs) that likely are chemokine receptors, are encoded by hCMV US27, UL33 and UL78.

Besides the infectious virions, non-infectious enveloped particles, lacking only the double-stranded viral genome, and dense bodies (tegument proteins, mostly pp65, in viral envelope) have been shown to be produced by hCMV infected cells (5, 34). However, the significance of these particles is not known (34).

H C M V T c e l l e p i t o p e s a n d t h e f u n c t i o n a n d

e x p r e s s i o n p r o f i l e o f t h e p r o t e i n s t h e y d e r i v e f r o m

The majority of the hCMV-specific CD8+ _{T cells recognizes the tegument protein pp65}

and the immediate early protein 1, but T cell responses against other proteins like gB and pp50 have been shown to exist too (35-40).

General introduction

1

Te g u m e n t p r o t e i n p p 6 5 ( U L 8 3 )

The HCMV tegument contains at least 32 proteins, most of which are phosphorylated. Ten tegument proteins are conserved across herpesviruses (41-43). The most abundant tegument protein of hCMV is pp65 (44). Upon infection pp65 does independently migrate to the nucleus, but it returns to the cytoplasm approximately 48h after infection (45). Pp65 is not required for the production of new infectious viral particles in fibroblasts. However, in macrophages viral replication has been shown to be impaired when pp65 is deleted (46). This protein is directly involved in immune modulation. Pp65 can selectively block the immediately early 1 protein from being presented in HLA-I by threonine-phosphorylation via the threonine kinase at the carboxy terminus of pp65 (47). Other functions of pp65 involve inhibition of the NKp30 activating receptor by acting as an antagonistic ligand (48), and degradation of the HLA-DR alpha chain via the accumulation of HLA-II in lysosomes (49). Pp65 has also been reported to stimulate the activity of the viral immediate-early promoter (50). Although pp65 is a late protein, pp65 from the virion can act before immediate early products are even transcribed, making this protein a potential target for drugs or vaccine development (51).

I m m e d i a t e e a r l y p r o t e i n 1 ( U L 1 2 3 )

As mentioned before, the immediate early proteins are the first proteins to be produced after hCMV infection. IE1 (or IE72) and IE2 (IE86) are the most prominent immediate early proteins of hCMV. They are splicing variants of the same primary transcript. IE1 consists of exons 2, 3 and 4. IE2 is identical, except instead of exon 4 it contains the transcript of exon 5, as a result IE1 and IE2 share the first 85 amino acids (AA) (52).

The majority of the CD8+ _{T cell responses that are directed towards the immediate}

early products, is directed against IE1. However, p65 selectively phosphorylates IE1 to prevent expression of IE1 peptides in the context of HLA (47). Interestingly, another

Table 1: hCMV tetramers used in this thesis

Name Protein Peptide Position AA HLA

hCMV pp65 YSE pp65 YSEHPTFTSQY 363-373 HLA-A*0101

hCMV pp65 NLV pp66 NLVPMVATV 495-504 HLA-A*0201

hCMV pp65 TPR pp65 TPRVTGGGAM 417-426 HLA-B*0702

hCMV pp65 IPS pp65 IPSINVHHY 123-131 HLA-B*3501

hCMV pp65 IIK pp65 IIKPGKISHIMLDVA 281-295 HLA-DR4

hCMV IE VLE IE-1 VLEETSVML 316-324 HLA-A*0201

hCMV IE QIK IE-1 QIKVRVDMV 88-96 HLA-B*0801

hCMV IE ELR IE-1 ELRRKMMYM 199-207 HLA-B*0801

General introduction

1

tegument protein, pp71, has recently been shown to enhance the expression of IE1 and IE2 by derepression of the major IE gene locus, resulting in a direct correlation

between the amount of pp71 in the viral particle and the amount of IE1 expressed in MHCI, independent of the effect of pp65 (53).

I m m u n e e v a s i o n

A staggering amount of the hCMV-genome has been shown to be dedicated to immune evasion and many immune evasion strategies of viral proteins have probably not been discovered yet. This is a short overview of some of these strategies. First of all, several gene products are dedicated to limit the expression of viral peptides on the surface of an infected cell. US2 and US11 have been shown to bind and transport HLA-I heavy chains from the endoplasmic reticulum (ER) to the cytosol, leading to proteasomal degradation (54, 55). It has also been shown that US3 blocks the transport of HLA-I from the ER to the golgi network (54, 56), US6 inhibits TAP (transporter associated with antigen processing) thereby preventing peptide translocation (57), US10 blocks HLA-G expression (58) and UL83 (pp65) actively blocks IE1 from presentation by phosphorylation (47). UL18 and UL142 can prevent NK cell mediated cell death as a result of the loss of expression of HLA-I on the cell surface, by encoding an HLA-I homologue (59-62). A large range of proteins (UL16 (63, 64), UL142 (65), US18, US20 (66) and miRUL112 (67)) have been implicated in the downregulation of the activating receptor NKG2D ligands (MICA, MICB and ULBP1-6) on infected cells, thereby

preventing activation of NK and CD8+ _{T cells (66, 68). UL141 has been shown to prevent}

TRAIL (TNF-related apoptosis-inducing ligand) induced cell death by intracellular

retention of the TRAIL death receptors (69). CD8+ _{T cell activation is also inhibited by}

UL144, a viral ortholog of HVEM, that binds the coinhibitory receptor BTLA (70, 71).

Expression of US2, 3, 6 and 11 has been shown to be critical for the evasion of CD8+

T cell immunity and super-infection by cytomegalovirus in rhesus monkeys (72). Also humoral immunity is intervened with by hCMV. Viral gp34 (RL11) and gp68 (UL119-118)

type I transmembrane glycoproteins bind Fc

γ

I, II and III on the cell surface, interfering

with host Fc

γ

R activation and inhibiting antibody-dependent NK cell degranulation

(73). Membrane bound and soluble UL7, a viral SLAM- (signalling lymphocyte-activation molecule) like protein, can also impair monocyte derived dendritic (DC) cell and monocyte function by attenuating the production of proinflammatory cytokines

Table 2: Expression and localization of the above hCMV proteins

Name virion lytic cycle latency cellular localization

pp65 + + - Cytoplasm

General introduction

1

TNF

α

, IL-8 and IL-6 (74). Another immune evasion mechanism involves the blocking

of chemokine and cytokine production. For example, UL122 (IE2) has been shown to

prevent

β

-interferon, RANTES, MIG, MCP-2 and MIP1

α

production by virus-infected

cells (75). Many more escape mechanisms will exist and probably only few have been deciphered so far. The last one that should be mentioned is the fact that only few proteins are expressed during latency (23, 76-78), which allows these latently infected cells to become undetectable by many hCMV-specific T cells. One of these latent proteins is the viral homolog of cellular IL10 UL111a, which has been shown to polarize monocytes to the anti-inflammatory M2c subset (79).

Epstein-Barr virus (EBV)

EBV (or human herpesvirus 4) is a

γ

-herpesvirus. Over 95% of the adult population

is latently infected with EBV. Infection with EBV naturally occurs by the oral transfer of saliva, blood-blood contact and in clinical situations after transplantation of solid organs or stem cells.

EBV infects B cells and epithelial cells (80). In rare cases, other cell types like T cells and NK cells can also become infected (81). Once the primary infection is controlled, EBV latently persists in memory B cells. Four different latent programs do exist, all of which do occur in B cells. The last three are each associated with a different type of malignancy (82).

EBV causes infectious mononucleosis and is associated with various forms of B cell malignancies such as Hodgkin’s lymphoma and Burkitt’s lymphoma as well as epithelial cell cancer: nasopharyngeal carcinomas (83). It is also associated with posttransplant lymphoproliferative disorder (PTLD) (84).

Table 3: Latency programs, viral proteins expressed and associated malignancies latency

viral proteins expressed

associated malignancies

EBNA LMP

0 none none none

I 1 2A Burkitt’s Lymphoma

II 1 1, 2A, 2B Hodgkin’s disease, peripheral T cell lymphoma, nasal T/NK cell

lymphoma, nasopharyngeal carcinoma, lymphoepithelioma

III 1, 2, 3, 4, 5, 6 1, 2A, 2B Infectious Mononucleosis, post-trans lymphoproliferative

disorder (PTLD), AIDS-related immunoblastic B-cell lymphoma

The genome of EBV is 172 kb long and encodes more than 86 proteins. There are two subtypes of EBV, which differ in their composition of EBNA3 loci (85). Type 1 is dominant in the western half of the world and south-east Asia and both types are equally found in Africa (85, 86).

General introduction

1

of the BMRF-2 protein with Upon oral infection, EBV infects the oral mucosal epithelial cells by interaction

β

1 integrins (87-89). Fusion of the virion with the cell

membrane is subsequently triggered by EBV gH/gL envelope protein interaction with

α

v

β

6/8 integrins on the cell surface (90). BMRF-2 also facilitates cell to cell spread of

EBV between the epithelial cells (88). The epithelial cell produced virions in their turn infect B cells. Viral gp350 binds to CD21 on the B cells (91), followed by fusion as a result of the viral gp42 and HLA-II binding (92). Infection of epithelial cells by infected B cells has also been shown to occur via cell cell contact (93).

At the onset of the lytic cycle BZLF-1 (ZEBRA, Z, EB1, ZTa) is the first gene to be transcribed, directly followed by BRLF-1 (Rta, R, EB2). Protein peak levels are reached 4 hours after infection (94). Both IE genes encode transcriptional activators inducing the expression of the E genes (95). Over 30 E genes are expressed and their functions include replication of the viral DNA, activation of transcription of the L genes and blocking of antigen-processing. The over 30 L genes that are subsequently subscribed, encode the structural proteins like the viral capsid antigens (VCA).

During latency, the genome replication depends on the host replication machinery and is initiated by host factors like ORC2 and Cdt1. EBNA1 binds the latent origin of replication (oriP) of the EBV genome and via its chromosome-binding-domains it also binds mitotic chromosomes and interphase chromatin (96). In this way, the closed circular plasmid of EBV genomic DNA is replicated together with the host chromosomal DNA.

During the lytic cycle, however, the host replication cycle is suppressed, the EBV genome is replicated from the lytic origin of replication (oriLYT) in a linear fashion more than 100-fold by the viral replication machinery and new virions are produced (97).

EBNA2 is essential for cellular transformation. It is a transactivator that regulates several viral genes (like other key players of cellular transformation: latent membrane protein 1 (LMP1) and LMP2A) and many cellular genes (98). LMP1 acts like a constitutively active CD40 receptor supplying constant survival signals (99). LMP2A mimics the B Cell Receptor also promoting cell survival (100), while it also prevents activation of the B cell and the subsequent onset of the lytic replication cycle, which would cause the infected cell to dye (101).

E B V T c e l l e p i t o p e s a n d t h e f u n c t i o n a n d

e x p r e s s i o n p r o f i l e o f t h e p r o t e i n s t h e y d e r i v e f r o m

Most of the CD8+ _{T cell responses in healthy donors are directed against epitopes of}

the IE proteins (both BZLF1 and BRLF1), followed by the E proteins (BMLF1, BaRF1 and BMRF1). The responses against L proteins are hardly found (102, 103). For the latent proteins, responses against EBNA1, EBNA3A-C and LMP1 and 2 have been shown, with the latter two yielding the lowest responses (104-106). However, in cases of malignancies CD8 responses towards EBNA1 are reduced (107, 108).

General introduction

1

B Z L F - 1 ( B a m H I Z f r a g m e n t l e f t w a r d o p e n r e a d i n g f r a m e 1 ) ( Z t a , Z , Z E B R A , E B 1 )

Reactivation of the virus from latency depends on expression of the viral immediate-early gene, BZLF-1 (basic leucine zipper) (95, 109). At the onset of the lytic cycle, BZLF-1 is the first gene to be transcribed, directly followed by BRLF-1 (Rta, R, EB2). Protein peak levels are reached 4 hours after infection (94). Both IE genes encode transcriptional activators and together orchestrate the expression of E genes (95). One way of keeping latency is the direct targeting of BZLF-1 and BRLF-1 by miR-BART20-5p (9). Besides the transactivation

function, BZLF1 also has immune modulatory functions. BZLF1 reduces IFN-

γ

R

α

expression

at both mRNA and protein level (110). BZLF1 inhibits transcription of the MHC-II gene by binding to and repressing of the CIITA Promotor III (111). In addition, BZLF1 downregulates surface CD74 (a chaperone for MHC-II antigen presentation) post-transcriptionally (112).

Table 4: EBV tetramers used in this thesis

Name Protein Peptide Position AA HLA

EBV BZLF1 EPL BZLF-1 EPLPQGQLTAY 54-64 HLA-B*3501

EBV BZLF1 RAK BZLF-1 RAKFKQLL 190-197 HLA-B*0802

EBV BMLF1 GLC BMLF-1 GLCTLVAML 259-267 HLA-A*0201

EBV EBNA1 HPV EBNA-1 HPVGEADYFEY 407-417 HLA-B*3501

EBV EBNA3a RPP EBNA-3a RPPIFIRRL 247-255 HLA-B*0702

EBV EBNA3a FLR EBNA-3a FLRGRAYGL 193-201 HLA-B*0802

EBV EBNA3a RLR EBNA-3a RLRAEAQVK 603-611 HLA-A*0301

EBV EBNA3b IVT EBNA-3b IVTDFSVIK 416-424 HLA-A*1101

B M L F 1 ( B a m H I - M l e f t w a r d r e a d i n g f r a m e 1 ) ( E B 2 , M Ta a n d S M )

The early RNA-binding protein BMLF-1 influences stability, splicing, nuclear export and translation of RNA. It has been shown to be required for the efficient cytoplasmic accumulation of viral mRNAs derived from intronless genes (6, 8). BMLF1 negative EBV virus is replication-deficient.

E B N A 1 ( E p s t e i n B a r r v i r u s n u c l e a r a n t i g e n 1 )

EBNA1 is a bridging molecule that tethers EBV episomes, via binding to the latent origin of replication (oriP) of the EBV genome, to host mitotic chromosomes as well as to interphase chromatin (96). This interaction is required for latent replication of the virus DNA and its distribution to the daughter cells in proliferating cells (113). EBNA1 is expressed during the latent as well as the lytic cycle of EBV.

EBNA1 can strongly reduce its own expression in MHCI and MHC-II. A glycine–alanine repeat (GAr) domain of EBNA1 plays an important role in interfering with

antigen-General introduction

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processing by the proteasome and MHC-I-restricted presentation (114). The GAr domain also directly regulates the amount of EBNA1 protein by inhibition of messenger EBNA1

RNA translation (115) and in that way it reduces its accessibility to the macroautophagy pathway, limiting the amount of EBNA1 CD4 epitopes presented in MHC-II (116).

E B N A 3 A , B a n d C ( E B N A 3 , 4 a n d 6 )

The EBNA3 proteins share a 40% AA homology and all have several nuclear localization sequences (NLS) (117). The EBNA-3 gene family proteins act together to prevent cell cycle arrest by disrupting the G1/S, the S/G2 and the G2/M checkpoints (118, 119). They are also potent inhibitors of apoptosis. For example EBNA3A and EBNA3C together inhibit initiation of BIM transcripts (120), EBNA3C inhibits p53 transcription and promotes p53 degradation, and it also inhibits transcriptional activation of both p73 and Apaf-1(119).

Table 5: Expression and localization of the above EBV proteins

Protein virion lytic cycle latent cycle cellular localization

BZLF1 - + - Nucleus BMLF1 - + - Nucleus EBNA1 - + +/- Nucleus EBNA3a - - +/- Nucleus EBNA3b - - +/- Nucleus EBNA3c - - +/- Nucleus

I m m u n e e v a s i o n

Like hCMV EBV has many immune evasion strategies. As mentioned before, the GAr domain of EBNA1 actively reduces MHC-I and MHC-II presentation of its antigens (114-116) and BZLF1 down regulates both MHC-II and it chaperone CD74 (111, 112). BGLF5 promotes mRNA degradation and induces host MHC-I gene shutoff (121), has

intrinsic RNA-activity (122) and thus impairs the CD8+ _{T cell recognition of endogenous}

antigens (123). TAP mediated transport of peptides into the ER is inhibited by BNLF2a (124). BILF1 down regulates the expression of MHC-I by enhancing their endocytosis and promoting lysosomal degradation (124, 125). Binding of the fusion protein gp42

to MHC-II can block TCR-binding and subsequent recognition by CD4+ _{T cells (112). In}

addition, just like hCMV, the EBV gene BCRF1 encodes vIL10 (126). The downregulation

of MHC-I on the surface on infected cells to prevent recognition by CD8+ _{T cells can}

trigger NK cells. Some herpesviridae have been shown to circumvent NK mediated lysis by expression of a viral MHC-I homolog (127). However, no viral homolog of MHC-I has been detected in the EBV genome. The only NK interference found until now is a microRNA (miR-BART2-5p) that inhibits MICB expression and possibly subsequent

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NK cell NKG2D mediated activation (128). EBV will without doubt utilize many more

immune escape mechanisms, many of which have not even been discovered yet.

Human Immunodeficiency Virus (HIV)

In 1983, Robert Gallo and Luc Montagnier simultaneously found HIV to be the cause of the acquired immunodeficiency syndrome (AIDS) (129, 130). HIV belongs to the genus lentivirus in the family of retroviridae. Two types of HIV exist: HIV1 and 2. HIV2 is predominantly restricted to Africa. HIV1 can be subdivided into nine subtypes, of which B is most prevalent in Western Europe. HIV1 has probably been introduced from chimpanzees into the human species some 100 years ago (131, 132).

V i r a l s t r u c t u r e a n d l i f e c y c l e

The HIV genome is composed of two identical copies of single stranded positive-sense RNA molecules, encoding nine open reading frames that produce 15 proteins. The HIV genome is contained within the conical nucleocapsid together with the viral enzymes reverse transcriptase, integrase and protease (figure 5). After binding of the gp120 trimer to the CD4 protein on the surface of the target cell, conformational changes and further binding of gp120 to the co-receptor takes place. Then the fusion protein gp41 gets exposed, leading to fusion and release of the viral contents. After the very error-prone reverse transcription and the generation of the complementary strand, the dsDNA is integrated into the host DNA. The viral long terminal repeats (LTR) act as a binding sites for host transcription factors hijacking the host machinery to transcribe the provirus and produce new virions.

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Infection with HIV via a mucosal site is generally established by one virus strain that CCR5 and CXCR4 have been identified as the principal co-receptors for HIV.

uses CCR5 as co-receptor (termed R5) (133). CXCR4-coreceptor using viruses (termed R4) are associated with the induction of syncytia.

As a result of the receptor and preferential co-receptors, HIV1 can infect CD4+ _T

cells, dendritic cells (DC), monocytes, macrophages and microglia (134). HIV1 also

preferentially infects HIV-specific CD4+ _{T cells (135). Uptake of HIV1 by DCs can occur}

via a wide variety of C-type lectins like langerin and DC-sign (136-139). However, where DC-sign expressing DCs have been shown to transmit HIV1 to T cells, Langerin (Langerhans cells) expressing DCs do not (140).

HIV infection and the tropism of this virus to infect CD4-expressing cells has a direct

effect on the CD4+ _{T cell numbers. The CD4}+ _{T cells are eliminated by a combination of}

mechanisms which include activation induced cell death (AICD), direct lysis as a result of the virion production upon infection, syncytia formation by fusion of infected and

non-infected CD4+ _{expressing cells and elimination of infected CD4}+ _{T cells by both the innate}

and the adaptive immune system. During primary infection only few of the circulating

CD4+ _{T cells are infected. However all memory CD4}+ _{T helper cells are eliminated from}

the gut-associated lymphoid tissue (GALT) during the first three weeks of HIV infection

(141). This loss of CD4+ _{T helper cells can affect the function of CD8}+ _{T cells and B cells.}

CD4+ _{T cell numbers below 200 cells/mm3 is one of the U.S. Centers for Disease}

Control and Prevention (CDC) criteria for the diagnosis of AIDS and is generally used as a surrogate marker. With highly active retroviral therapy (HAART), a combination of three or more drugs, each affecting a different part of the viral life cycle, progression to AIDS of patients infected with HIV can be halted.

During HAART treatment, latency has been shown to occur in vivo in primarily

resting (central) memory CD4+ _{T cells (142).}

H I V T c e l l e p i t o p e s a n d t h e f u n c t i o n a n d

e x p r e s s i o n p r o f i l e o f t h e p r o t e i n s t h e y d e r i v e f r o m

Table 6: HIV tetramers used in this thesis

Name Protein Peptide Position AA HLA

HIV gag EIY gag polyprotein EIYKRWII 260-267 HLA-B*0802

HIV nef FLK Nef FLKEKGGL 90–97 HLA-B*0802

G A G

During the maturation process, cleavage of HIV-1 polyprotein Gag (p55) by the viral protease yields the matrix protein (MA, p17), the capsid protein (CA, p7), nucleocapsid

General introduction

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protein (NC, p24), p6 protein and two spacer peptides, Sp1 and Sp2 (143). These

proteins are only produced during the lytic cycle, however since p17, p24 and p7 are structural parts of the virion, peptides of these proteins can be presented in HLA-I from the moment of infection.

N e f ( n e g a t i v e f a c t o r )

Nef function has recently been reviewed by Basmaciogullari and Pizzato (144). Nef does not have enzymatic activity, it however does influence the expression of many proteins. It down-regulates CD4 by enhancing its uptake into the endosome–lysosome compartment (145). It is involved in both HLA-I (146) and HLA-II down regulation and up regulation of the invariant chain (147). Other molecules down regulated by nef are the CD3/TCR complex (148) and the co-stimulatory molecule CD28 (149). CD95L however is up regulated by nef, and this up regulation has been shown to trigger apoptosis of bystander cytotoxic cells (150).

Nef has also been to shown to influence signaling pathways and alters the threshold of activation. The result is a signaling cascade that resembles TCR activation which is thought to be favorable for viral replication (144).

Lastly, nef has been implicated in enhanced infectivity (151) and replication (152) of HIV.

Table 7: Expression and localization of the above HIV proteins

Name virion lytic cycle latency cellular localization

nef + + - cytoplasm

gag + + - cytoplasm

Transient viral infections

In this thesis, we also analysed the CD8+ _{T cell response against two transient viral}

infections, influenza A and RSV, in order to compare them with the CD8+ _{T cell response}

against the persistent viral infections (153, 154).

Influenza A

Influenza virus A belongs to the Orthomyxoviridae virus family. The genome of Influenza A consists of 8 pieces of negative-sense, single-stranded, segmented RNA. The name of each Infuenza A virus-strain is based on the H number (for the type of hemagglutinin, a protein that causes red blood cells to agglutinate) and an N number (for the type of neuraminidase, an enzyme that cleaves the glycosidic bonds of the monosaccharide, neuraminic acid). 18 different H antigens (H1 to H18) and 11 different N antigens (N1 to N11) have been found so far (155, 156).

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elderly, very young and otherwise immunocompromised individuals. As an example: Influenza A infection can cause severe respiratory problems and even death in the

during the outbreak of 1918 (Spanish Flu, H1N1) between 20 and 40% of the world’s population became ill and an estimated 50 million were killed.

The viral genome of influenza A is 13.5 kb long and encodes 11 proteins on 8 pieces of RNA: the before mentioned viral envelope proteins hemagglutinin and neuraminidase, the nucleoprotein (NP), the matrix proteins M1, M2 (M2 acts as an ion channel), the nonstructural proteins NS1 and NS2 (also called nuclear export protein (NEP) (157)) and the transcriptase complex proteins PA, PB1 (polymerase basic 1), PB1-F2 and PB2 [53] (figure 6). Because of the segmented genome, Influenza A virus-recombination of two strains can occur (antigenic shift). Also due to the error-prone RNA-based RNA polymerase, mutations in the genes encoding antibody binding sites do frequently occur (antigenic drift).

Table 8: Influenza A tetramers used in this thesis

Name Protein Peptide Position AA HLA

FLU M1 GIL Matrix Protein-1 GILGFVFTL 58-66 HLA-A*0201

FLU NP CTE Nucleoprotein CTELKLSDY 44-52 HLA-A*0101

FIGURE 6: Schematic representation of an Influenza A virion.

Respiratory syncytial virus (RSV)

RSV belongs to the subfamily Pneumovirinae within the family of Paramyxoviridae. RSV is the main cause of severe respiratory disease in the immune-compromised, young children and infants, and the elderly. Approximately 160.000 people die from RSV each year.

Natural RSV infection in young children does not elicit long-lasting immunity and individuals remain susceptible to repeated RSV infections throughout life. Because RSV

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infection is restricted to the respiratory tract, an RSV vaccine should elicit mucosal immunity

at upper and lower respiratory tracts in order to most effectively prevent RSV reinfection. The genome of RSV consists of one strain of negative-sense, single-stranded RNA with a length of 15 kb and it comprises 10 genes encoding 11 proteins: the RNA polymerase protein L. the cofactor for the L protein phosphoprotein P, the nucleocapsid protein N and the matrix proteins M1 and M2 (figure 7). M2 is also required for transcription and encodes M2-1 (an elongation factor) and M2-2 (involved in transcriptional regulation). The viral envelope is formed by three proteins: the SH protein, the heavily glycosylated attachment protein G and the fusion protein F, which is responsible for the formation of syncytia. The nonstructural proteins (NS1 and NS2) have been implicated in the inhibition of type I interferon induction and activity (158). Two subgroups of RSV exist, subgroup A and B. The F protein is highly conserved between the two subgroups, but the G protein differs considerably.

Under influence of immunological pressure the RSV genome can change within one individual (159).

Table 9: RSV tetramers used in this thesis

Name Protein Peptide Position AA HLA

RSV N NPK Nucleoprotein NPKASLLSL 306-314 HLA-B*0702

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SCOPE OF THIS THESIS

The research presented in this thesis focusses on the characterization of the T cell

response against (persistent) viral infections. We have studied the clonal evolution of

peripheral blood and lymph node derived virus-specific CD8+ _{and CD4}+ _{T cells during}

both primary infections and reactivations. We have analysed the impact of the tissue

(lymph node) on the phenotype and function of virus-specific CD8+ _{T cells. We also}

examined the relationship between transcription factor expression, phenotype and

the functional profile of virus-specific CD8+ _{T cells.}

Insight into the best cell providing long lasting immunological protection against such infections and the characteristics of the lymphocyte that maintains control throughout life, once a human being is infected, is invaluable in development and evaluation of the efficacy of vaccines. It also allows for diagnostic evaluation of patients suffering from chronic infections and might give indications for therapies that could influence the immunological system of the patient regaining control of the viral infection, either through compounds, antibodies or cell infusions.

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