biology of hiv-nef in vivo - universiteit utrecht · frank miedema, kiki tesselaar department of...

BIOLOGY OF HIV-NEF IN VIVO

-STUDIES IN INDUCIBLE TRANSGENIC MICE-

Paulus Gerardus Franciscus Koenen

CIP-GEGEVENS KONINKLIJKE BIBLIOTHEEK, DEN HAAG

© 2007 Paul Koenen (Snok Flagga productions)

Biology of HIV-Nef in vivo; studies in inducible transgenic mice

Paul Koenen - Utrecht; Universiteit Utrecht, Faculteit Geneeskunde

Proefschrift Universiteit Utrecht - met een samenvatting in het Nederlands

Used with permission: Chapter 2, © The American Association of Immunologists,

Inc.

Cover picture: © Picture of oocyte injection by Frans Hofhuis

Print: Gildeprint, Enschede

ISBN: 978-90-393-4480

The research described in this thesis was performed a the the Department of

Immunology, University Medical Centre Utrecht and was fi nancially supported by

grant number 6011 from Aids Fonds Netherlands.

BIOLOGY OF HIV-NEF IN VIVO

-STUDIES IN INDUCIBLE TRANSGENIC MICE-

BIOLOGIE VAN HIV-NEF IN VIVO

-STUDIES IN INDUCEERBARE TRANSGENE MUIZEN-

(met een samenvatting in het Nederlands)

PROEFSCHRIFT

ter verkrijging van de graad van doctor aan de

Universiteit Utrecht op gezag van de rector magnifi cus,

prof. dr. W.H. Gispen, ingevolge het besluit van het

college voor promoties in het openbaar te verdedigen op

donderdag 22 maart 2007 des middags te 4.15 uur

door

Paulus Gerardus Franciscus Koenen

geboren op 30 maart 1978, te ’s-Hertogenbosch

Promotor: Prof. dr. F. Miedema

Co-promotoren: Dr. M.A. Oosterwegel

Dr. K. Tesselaar

Er zijn geen feiten, alleen interpretaties

Friedrich Nietzsche

Aan mijn moeder

Commissie: Prof. dr. P.J. Coffer

Prof. dr. W. van Eden

Dr. J.S. Verbeek

Prof. dr. J. Verhoef

Prof. dr. H. Schuitemaker

Paranimfen: Bastiaan Tops

Frans Hofhuis

De uitgave van dit proefschrift werd mede mogelijk gemaakt door fi nanciële

steun van; Corning BV, BD Biosciences, Edward Jenner Stichting, Eijkman Gradu-

ate School, de Universiteit Utrecht, Tebu-bio, Dr. Ir. Van de Laar Stichting en de

J.E. Juriaanse stichting.

CONTENTS

Chapter 1 Introduction: HIV pathogenesis: a brief history of Nef 9

Chapter 2 T cell activation and proliferation characteristic for HIV-1 21

Nef transgenic mice is lymphopenia induced

Chapter 3 An inducible HIV-Nef transgenic mouse: a reappraisal of 39

Nef-mediated pathology

Chapter 4 The two faces of Nef: opposing in vivo functional effects 61

of Nef expression in murine T cells compared to dendritic

cells

Chapter 5 Evaluation of doxycycline use in T cell specifi c inducible 85

transgenic mouse models

Chapter 6 GENERAL DISCUSSION 101

SUMMARY 107

NEDERLANDSE SAMENVATTING 111

NAWOORD 117

CURRICULUM VITAE 123

HIV Pathogenesis: a Brief History of Nef

1

Paul G.F. Koenen, Mariëtte A. Oosterwegel, Frank Miedema, Kiki Tesselaar

Department of Immunology, University Medical Center, Utrecht, The Netherlands.

Manuscript submitted

CHAPTER

CHAPTER 110

Introduction

That the mechanism of action of the pathogenicity factor Nef of HIV is still not clear is

immediately apparent from the studies published last year. Distinct and sometimes even

incompatible mechanisms of action are ascribed to this enigmatic protein (1-3). It is

intriguing that despite more than 20 years of research the question of how Nef contributes

to pathogenesis of HIV infection is still unsolved. New insights into the dominant role of HIV

mediated hyper-immune activation in disease progression, culminating in the proposal that

microbial translocation, due to immediate irreversible damage to the gastrointestinal tract

upon HIV infection, may drive this persistent T cell activation, call for the role of Nef in viral

replication to be revisited (4). Here, we briefl y review two decenniums of Nef history and will

focus on consistent functional properties of Nef related to viral and immune pathogenesis

that may be compatible with current ideas of HIV pathogenesis.

Establishment of Nef as a pathogenicity factor in HIV and SIV

Many functions have been ascribed to the 25-27kD sized regulatory protein of HIV, since its

discovery in 1985 (5-7). The fi rst described functional activity of this cytoplasmic protein

was negative regulation of HIV transcription, and based on this data its name, Negative

Factor (Nef), was coined (8, 9). Since at that time the high HIV viral dynamics were

not yet discovered (9, 10) this negative regulation of HIV transcription observed in vitro,

was believed to be critical for establishment of viral latency in vivo in early asymptomatic

infection. Yet, several other studies using identical Nef isolates were unable to detect any

negative effect on HIV-1 transcription or replication and it was generally concluded that

Nef was not a negative factor (11, 12). On the contrary, it was observed that Nef although

probably not essential for in vitro HIV replication, apparently was crucial for in vivo replication

and progression to AIDS in rhesus monkeys infected with the HIV related SIV (13). The

importance of Nef as a pathogenicity factor in HIV for development to AIDS was revealed

in a few seminal studies that clearly showed that HIV Nef deletion mutants could establish

infection but were associated with slow progression to HIV-1 disease (14, 15). From that

moment on, Nef was established as a crucial pathogenicity factor in HIV and the search for

a molecular mechanism explaining its contribution to AIDS pathogenesis was started.

Immune escape

In the early 90s, Nef research focused on observations that Nef could down modulate the

cell surface receptors CD4 (16) and later MHC class I (17). In the course of this work, much

was revealed about how and by which interactions Nef could target these receptors and

how this disturbed cell surface expression occurred (reviewed in Doms and Trono, 2000)

(18). Besides immune evasion by MHC-I down regulation, Nef has also been suggested to

HIV pathogenesis: a brief history of Nef 11

interfere with apoptosis, inhibiting apoptosis to prolong life of cells it has infected (19,

20) or enhancing apoptosis to kill T cells that might destroy its host cell (21-23). These

immune-escape mechanisms would be effective in escaping the adaptive immune response,

it remained however diffi cult to envision how immune-escape could account for the profound

effect of Nef deletion on viral load and replication, especially in the early phase of the

infection.

Increased viral infectivity and replication

In addition to immune-evasion and down regulation of CD4 molecules a completely distinct

primary effect of Nef affecting viral infectivity seemed to be involved in Nefs pathogenicity.

This indeed became apparent from two studies in 1994. One study showed that Nef expression

increases the viral particle production by human quiescent CD4 lymphocytes by at least 10

fold (24). This fi nding was extended in a study, where decrease of viral replication was seen

in CD4 lymphocytes as well as in primary macrophages after Nef deletion (25). Besides the

signifi cant effect of Nef on viral replication in vitro this study reported also a direct effect

on virion infectivity. Virions produced from cells infected with Nef-deleted HIV, had a fi ve to

six fold lower infectivity for human target cells (25). A later complementation study showed

that the phenotype of Nef-defective HIV-1 could be restored to near wild-type infectivity by

co-expressing Nef in the cell line producing the virus (26). In this regard it is of interest that

it was recently proposed that Nef increases synthesis of cholesterol, which when recruited

to the virions ensures higher infection rates (27-29).

Increased viral replication by Nef-mediated T cell activation.

From the studies described above it became clear that Nef has a predominant, selective

effect on HIV-1 replication of primary T cells undergoing transition from a resting to an

activated state. When virus infection occurs in pre-stimulated, highly proliferating cells, the

effect of Nef is greatly diminished (24, 25). The fi nding that Nef expression accounts for

80% of early double spliced transcripts, just after integration of the virus (30, 31), led to

the speculation that Nef infl uences the activation state of CD4 T cells in order to facilitate

and stimulate viral replication. Two different mechanisms to induce this activation state are

proposed: either by direct stimulation of infected T cells or indirect via infected APCs.

Direct effects of Nef on T cells

One of the fi rst studies showing Nef-mediated activation in the tumor cell line Jurkat,

reported that stable expression of a hybrid CD8-Nef molecule led to activation of the TCR

signaling pathway in Jurkat cells (32). The use of a chimeric CD8-Nef protein to mimic the

CHAPTER 112

function(s) of native Nef protein, and its toxicity, complicate interpretation of these data.

A similar spontaneous activation was observed in a study, where induction of Nef in Jurkat

cells sets off a transcriptional program that is almost identical to that of anti-CD3 meditated

stimuli (33). The infl uence of Nef on TCR signaling was also shown in two studies, where

Nef expression led to enhancement, but not to initiation of T cell activation (34, 35). In a

recent study it was suggested that the loss of Nefs capability to down regulate the TCR-CD3

complex during its evolution from SIV to HIV-1 was responsible for the observed immune

activation during HIV-1 infection (3). In total contrast to these studies the group of Schwartz

reported that Nef interfered with the formation of the immunological synapse which results

in improper signaling and a higher threshold for T cell activation in cells that express Nef (2).

Thus, totally different effects on T cell signaling were found. This might be explained by the

variety of interactions with well defi ned signaling proteins acting in the TCR environment

such as PAK2 (36), Lck (37), the TCR-ζ chain (38) and Vav (39) which have been described

for Nef. What the functional consequences of these interactions, if any, are in real life HIV

infection remains unclear. Nef transgenic mice have been used to study effects of Nef in

primary cells in vivo.

Nef transgenic mice

Strong functional evidence for Nef-mediated disturbance of signaling events in thymocytes

comes from studies in Nef transgenic mice models. Although in these models T cell lineage

expression of Nef is under control of different promoters, in all models Nef-mediated thymic

atrophy is observed (40-42), at least in the single positive CD3+ CD4+ compartment (43).

Restoration of thymic cellularity by introducing active Lck in Nef transgenic thymocytes,

points to an important role for Nef in disturbing (pre) TCR signaling (44). Peripheral T cells

in these Nef transgenic models show an activated phenotype (42, 44, 45), compatible with

a direct effect of Nef on peripheral T cells as has been reported in Jurkat cells.

However, by studying Nef transgenic cells in a full T cell compartment we showed

that, the observed activation in the constitutive Nef transgenic mice was a secondary effect

of lymphopenia, rather than a direct effect of Nef on T cell activation in the periphery

(Koenen et al., submitted). This was further substantiated in a recently developed inducible

Nef transgenic mouse model. In this model we showed upon Nef induction a rapid loss of

thymocytes over time and again no effects on the activation status of the peripheral T cell

compartment were observed (Koenen et al., manuscript in preperation).

This discrepancy between strong effects on thymocytes on the one hand, and no

effects on T cells in the periphery on the other, could be explained by the difference in

utilization of TCR signaling in thymocytes and T cells. During T cell development, thymocytes

repeatedly use the TCR signaling pathway to check for functionality. During thymopoiesis

disturbed TCR signaling is much more prevalent than in the peripheral T cell compartment


where no active TCR signaling occurs, except for the rare event of antigen (Ag) encounter.

To study Nef expressing T cells during Ag encounter, our inducible Nef transgenic mice were

challenged with infl uenza virus. Nef expression in T cells led to reduced antiviral CD8 T cell

responses (manuscript in preparation), which can be explained by disturbed T cell signaling

after triggering of the TCR by Nef.

Nef increased viral permissiveness of T cells via APCs

The importance of dendritic cells (DCs) in HIV infection has been known for more than

a decade and DCs are considered key players in the maintenance of HIV infection. DCs

function as transport vehicles between the site of infection and the lymphoid organs, as

hide out places for non-replicating quiescent virus and as crucial stimulating factors during

intense HIV replication (46). Several studies have shown that viral replication in DC-T cell

co-cultures is much higher than in T cells alone and that in these co-cultures replication

occurs in conjugates formed among DCs and T cells (47-51). A crucial factor in forming

these conjugates and generating effi cient viral replication for both SIV and HIV is Nef (52,

53). In the absence of Nef, replication is sharply decreased. This effect is most apparent

in cultures of T cells with immature DCs (52-54). Cultures of mature DCs with T cells or

superantigen activated T cell-immature DC cultures led to comparable replication of Nef

defi cient and wild-type virus (52, 53). Subsequently, it appears that the presence of Nef

only works as an activating stimulus on viral replication in immature DC-T cell cultures. An

early study, using a highly pathogenic SIV strain, described an extensive Nef-dependent T

cell activation upon in vitro infection of PBMCs (55). The need of cell-cell contact between

macrophages and T cells for this highly activated phenotype and for SIV replication in T cells

pointed to an important role of membrane receptors on APCs in mediating Nef-dependent

enhancement on viral replication. In line with this idea Nef was shown to upregulate DC-

SIGN, a C type lectin, which facilitates contact of DCs with T cells through binding with

ICAM -3 (56). Upregulation of DC-SIGN could explain the increased DC-T cell clustering

and viral replication in the presence of Nef. However, report of confl icting fi ndings might

result in re-evalation of this mechanism (57, 58). Other studies suggested that besides

direct cell-cell contact between APCs and T cells, Nef mediates cytokine and chemokine

production of infected macrophages or DCs and thereby T cell activation (57, 59, 60). To

study the relevant outcome of these in vitro described effects in vivo, we decided to study

Nef function in APCs in vivo. Therefore, we generated a mouse model in which we could

induce Nef expression in MHC class II positive cells. After a challenge with infl uenza virus

we observed that Nef-expressing APCs were able to alter the T cell response, resulting in

increased numbers and enhanced activation of antigen specifi c CD8 T cells early in the

response, pointing to enhanced kinetics (manuscript in preparation).

CHAPTER 114

So altogether, although the underlying mechanisms are not clear yet, a consistent

observation was that Nef plays an important role in enhancing T cell activation by infl uencing

APC function in vitro and in vivo.

Nef in HIV pathogenesis

Over the years several hypotheses explaining HIV mediated pathogenesis have been

postulated. One of these hypotheses proposes that HIV infection of the thymus reduces

thymic output, which eventually will lead to reduced T cell numbers in the periphery and life

threatening immuno-defi ciency (61, 62). A role for Nef in thymic decline has been shown

in several human and murine systems and Nef-mediated thymic decline is observed in the

context of infections with the complete virus as well as by singly Nef protein expression in

thymocytes. Perturbation by Nef of thymic function is thus consistently supported by a large

body of evidence arguing for Nef-mediated reduction of de novo naïve T cell production in

human HIV infection.

Next to direct loss of CD4 cells by HIV infection, persistent hyper-activation of the

immune system in combination with failure of thymic homeostasis has been proposed to

cause erosion of the CD4 T cell pool (63-65). Sustained HIV replication during the latent

phase of HIV infection is thought to provoke a constant T cell response most likely involving

innate and adaptive immune responses, leading to a gradual exhaustion of T cell resources

and ultimately to a collapse of the CD4 T cell pool. As major disparities in the effects of

Nef on T cell activation have been reported, we tried to delineate a role for Nef in activation

of primary T cells. It appeared that Nef expression in T cells in mice with a full T cell

compartment did not infl uence the activation state of these cells. This fi nding however, does

not formally exclude an effect of Nef on the level of hyper-activation in the context of HIV

infection.

Despite many discrepancies concerning potential pathogenic effects of Nef,

a consistent effect of Nef on the pathogenicity of HIV and SIV by enhancing viral replication

stands out. Recently is was recognized that high level viral replication during the fi rst days

of HIV infection causes major and probably irreversible damage both on the memory CD4

T cell compartment and the epithelial lining of the gastrointestinal tract (4, 66-68). These

fi ndings re-emphasize the importance of viral replication and the associated CD4 T cell death

during the primary HIV infection. It is conceivable that Nef-mediated enhancement of viral

replication in T cells and even more in T cells in conjunction with APCs may play an important

role in HIV pathogenesis.


Scope of this thesis

HIV-Nef is established as an important factor for pathogenicity during HIV infection. The

exact role and underlying mechanisms however are still unclear. Several studies have

shown that Nef expression in T cells results in increased T cell activation. This Nef-mediated

T cell activation has been suggested to result in higher viral replication and increased

pathogenicity. As most studies were performed in cell lines, we addressed the function of Nef

in T cell activation in constitutive Nef transgenic mice (chapter 2). During the examination

of characteristics of T cell activation in constitutive Nef transgenic mice, we came to the

conclusion that the observed activation in this model was caused by homeostatic mechanisms,

resulting from Nef-mediated thymic atrophy, rather than by direct Nef-mediated effects on

T cells. This suggested that Nef expression does not lead to spontaneous T cell activation

upon expression in primary T cells in vivo. This fi nding was substantiated in conditional

Nef transgenic mice (chapter 3), with tightly regulated Nef protein induction in the T cell

compartment, which were generated in order to circumvent Nef-mediated effects on thymic

development and secondary effect in the periphery. In these mice upon Nef expression, a

rapid thymic atrophy occurred, but no effect on peripheral T cell activation was observed.

Under normal conditions, the majority of T cells in mice are not activated. To study

effects of Nef on T cell activation after TCR stimulation, T cells of inducible Nef transgenic

mice were stimulated by infection of the mice with infl uenza virus. We showed that Nef

expression in T cells leads to a reduced anti-viral T cell response, suggesting a role for Nef

in the disturbance of T cell function and possibly TCR signalling (chapter 4).

The infection of antigen presenting cells (APCs) by HIV is important for viral spread

and replication. Since several studies reported a Nef-mediated stimulation of HIV replication

in APCs, we generated mice with inducible Nef expression in antigen presenting cells (APCs)

and studied Nef effects in APCs during infl uenza infections (chapter 4). We showed that Nef

expression in APCs results in increased T cell responses. This suggests a role for Nef in the

stimulation of T cell activation via APCs, in this way increasing the likelihood of HIV infection,

as activated T cells are more prone to HIV infection than naïve T cells.

In our studies we used doxycycline as a reagent to induce gene transcription, the

side-effects of this compound on T cell function are described in chapter 5.

CHAPTER 116

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T Cell Activation and Proliferation Characteristic for HIV-Nef Transgenic Mice is Lymphopenia Induced

2

Paul G.F. Koenen, Frans M. Hofhuis, Mariëtte A. Oosterwegel, Kiki Tesselaar


Manuscript accepted for publication in the Journal of Immunology

CHAPTER

CHAPTER 222

ABSTRACT

The HIV-Nef protein has been implicated in generating high viral loads and T cell activation.

Transgenic mice with constitutive T cell specifi c Nef expression show a dramatic reduction

in T cell number and highly increased T cell turnover. Previous studies in Nef transgenic

mice attributed this T cell activation to a direct effect of Nef at the cellular level. Given

the strongly reduced peripheral T cell numbers we examined whether this enhanced T cell

division might instead be lymphopenia induced. Adoptively transferred naïve wild-type T

cells into lymphopenic Nef transgenic mice showed high T cell turnover and obtained the

same effector/memory phenotype as the autologous Nef transgenic T cells, supporting the

idea that the micro-environment determines the phenotype of the T cells present. Moreover

in bone marrow chimeras from mixtures of wild-type and Nef transgenic bone marrow,

with a full T cell compartment containing a small proportion of Nef transgenic T cells, Nef

transgenic T cells kept a naïve phenotype. These results demonstrate that T cell activation in

the Nef transgenic mice is lymphopenia-induced rather than due to a direct T cell activating

effect of Nef.

HIV-Nef does not directly activate primary peripheral T cells 23

INTRODUCTION

HIV-Nef is one of the key determinants in the pathogenesis of AIDS. HIV strains with a

modifi ed or deleted Nef gene were found in HIV-infected long-term non-progressor patients

who remain asymptomatic for more than 10 years and maintain low viral loads and stable

CD4 T cell counts (1, 2). In analogy, rhesus monkeys infected with Nef deleted SIV strains

maintain low or undetectable levels of virus and show a strongly attenuated clinical course

of infection (3, 4).

Additional evidence for the importance of Nef in development of AIDS comes from

a transgenic mouse model in which constitutive expression of the whole HIV genome in CD4

T cells and in cells of the monocyte/macrophage lineage leads to severe AIDS-like pathology

(5). Further dissection of this mouse model leads to the conclusion that Nef alone was the

major disease determinant (5, 6). Yet, the mechanism of Nef causing this severe phenotype

in mice is still not understood.

Nef is a small protein of 27kD that is expressed abundantly in the early stages of

viral replication. It is posttranslationally modifi ed by myristilation of the N-terminus, which

targets Nef to the plasma membrane (reviewed in (7). Nef enhances viral replication (3). The

best documented functions of Nef in vitro are down regulation of CD4 and MHC class I cell

surface expression via the endocytosis machinery (8, 9) and interference with TCR signaling

(reviewed in (7)). Simmons and coworkers have demonstrated that Nef expression in Jurkat

T cells induces a very similar gene expression profi le as TCR signaling (10). Other groups

have shown that Nef leads to enhanced activation after stimulation of the TCR and CD28,

resulting in increased IL-2 production (11, 12). Two recent studies showed an effect of Nef

on the threshold of T cell activation either by infl uencing formation of the immunological

synapse or by down regulation of the TCR-CD3 complex (13, 14). Numerous proteins have

been reported to interact with Nef, many of which are part of the TCR signaling cascade,

which is compatible with effects of Nef expression on T cell activation in cell lines (reviewed

in (7)). Yet, functional evidence for such effects of Nef in primary T cells is very limited.

To study the consequence of Nef expression in primary cells in vivo, various Nef

transgenic murine models have been generated (6, 15-17). All, except one, which expresses

Nef in CD4 T cells as well as in cells of the monocyte/macrophage lineage (6), express Nef

specifi cally in the T cell compartment. In all these transgenic mouse strains depletion of

the T cell compartment is observed. In addition, T cells derived from secondary lymphoid

organs showed increased activation (15, 18, 19). Upon viral challenge with VSV and LCMV,

it appeared that Nef transgenic T cells were less capable of inducing virus specifi c CTL

responses and to clear the virus (15). In line with this fi nding Nef transgenic T cells had

decreased in vitro proliferative capacities compared to wild-type T cells (15, 18, 19).

Altogether, these models show that Nef expression in the T cell compartment leads to T

cell activation, decreased functional capacity of T cells and thymic atrophy, all features of

CHAPTER 224

the T cell compartment of HIV infected humans. These studies suggest that Nef expression

in peripheral T cells of transgenic mice leads directly to HIV-like hyper-activation of T cells

and thereby implies a dominant role for Nef on peripheral T cell functioning during HIV

infection.

During HIV infection, T cells are chronically activated and have high proliferation

rates (20-23). Different causes for this activation have been proposed, varying from the

presence of HIV viral load, which continuously drives immune-activation (20) to particular

activating effects of HIV proteins including Nef (20-23). We studied whether the T cell

activation seen in Nef transgenic mice is indeed due to direct effects of Nef expression on T

cells, compatible with HIV induced hyper-activation, or whether this activation is lymphopenia-

induced, secondary to Nef-mediated thymic atrophy and T cell depletion. Adoptive transfer

and chimeric mouse models demonstrated that in Nef transgenic mice peripheral T cell

turnover results from lymphopenia, which is caused by Nef-mediated thymic atrophy.


RESULTS

Strongly reduced T cell numbers and a highly activated T cell compartment in Nef

transgenic mice

In order to determine the cause of peripheral T cell activation in vivo, we studied a Nef transgenic

mouse model as described earlier by Dzierzak and co-workers (founder line F (17) (18)).

In these mice HIV-1BRU

Nef gene expression is under control of the CD2 promoter and locus

control region resulting in constitutive transgene expression in all T cells, starting during the

earliest stages of T cell development. The most striking phenotype in these mice is a dramatic

depletion of thymocytes (17). Total thymocyte cellularity of Nef transgenic mice was ten-fold

reduced compared to wild-type littermates (Fig.1A) as shown before (17). Nef expression

disturbs thymocyte development, as evidenced by changes in thymic subset distribution,

resulting in low numbers of single positive (SP) thymocytes (17, 18) (data not shown).

FIGURE 1. Nef transgenic mice have a strong reduced and highly activated T cell compartment. Thymocytes and T cells derived from Nef transgenic mice and wild-type littermates of 6-8 weeks old were counted and analyzed by fl ow cytometry. Absolute cell numbers of the thymus were determined (A). CD4 expression levels on thymocytes and T cells derived from lymph nodes were determined (B). Lymph nodes and spleens were recovered from transgenic mice and wild-type littermates of 6-8 weeks old, absolute CD4 and CD8 T cell numbers were determined (C). Spleen derived CD4 and CD8 T cells were analyzed for differential expression of CD44 and CD62L by fl ow cytometry, percentages of naïve (N) and effector/memory (E/M) T cells are shown (D). Indicated are mean numbers ± SD of 3 mice (*, p<0.005; **, p<0.0005). Shown is a representative of three independent experiments.

*

CD62L

CD

44

CD4 T-cells

CD8 T-cells

Wt Nef tg

E/M=22%

E/M=42% E/M=88%

E/M=76%

N=77% N=22%

N=12%N=57%

WtNef tg

Thymus Lymph Nodes

Ce

ll C

ou

nts

CD4

Wt

Nef tg

0

50

100

150

thym

ocyte

nu

mb

er

x10

6

** ** ** **

WtNef tg

CD4 CD8

Lymph Nodes

CD4 CD8

Spleen

0.0

2.5

5.0

7.5

10.0

Tcell

nu

mb

er

x10

6

A B

C D

CHAPTER 226

As described before, Nef mRNA could be detected in thymocytes and peripheral

T cells by RT PCR (data not shown). Nef expression resulted in a reduced level of CD4

expression on both thymocytes (wild-type mean fl uorescence intensity (MFI) of ~ 300 vs

Nef transgenic ~ 135) and peripheral CD4 T cells (wild-type MFI of ~ 900 vs Nef transgenic

~650) (Fig. 1B), indicative for the presence of functional Nef protein. Analysis of peripheral

lymphoid organs revealed a marked decrease in T cell numbers in both peripheral lymph

nodes (PLN) and spleen (17) (Fig. 1C).

Phenotypical analysis of transgenic peripheral T cells in previous studies using these

Nef transgenic mice showed enhancement of T cell activation as evidenced by increased

expression of the cell surface molecules CD25, CD69 and CD44 (17). Next to induction

of these activation markers we determined the composition of the peripheral T cell pool.

Antigen inexperienced naïve T cells can be distinguished from antigen experienced effector

and memory T cells on basis of their CD62L and CD44 expression levels. While T cells

expressing CD44medium CD62Lhigh are defi ned as naïve, CD44high CD62Llow and CD44high CD62Lhigh

are defi ned as effector/memory cells (25-27).

Phenotypical analysis of spleen derived T cells showed that in wild-type mice

approximately 77% of all CD4 T cells and ~57% of all CD8 T cells were naïve, compared to

~22% naïve CD4 and ~12% naïve CD8 T cells in Nef transgenic mice (Fig. 1D). Calculation

of absolute T cell number showed a reduction of all T cell subsets in the Nef transgenic mice,

which was most pronounced for naïve T cells (Table I). Expression of activation markers was

increased in Nef expressing mice as was shown before.

Despite their severely decreased T cell numbers, Nef expressing mice, housed in an

open facility or under SPF conditions, had a normal life span and showed no overt increase in

infection or other physiological abnormalities when studied macroscopically. Taken together,

in these transgenic mice Nef expression led to strong depletion of the thymus and to a

depleted, activated effector/memory like peripheral T cell compartment.

CD4 T cells (x106) CD8 T cells (x106)

N E/M N E/M

Wt 6.1 ±0.76 2.08±0.77 3.18 ±0.76 1.8±0.73

Nef tg 0.13 ±0.04 0.48±0.04 0.05 ±0.02 0.32±0.02

Table 1. Shifted ratio of naïve vs effector / memory T cells in Nef transgenic

mice. CD4 and CD8 T cells derived from spleen of mice of 6-8 weeks old were stained for differential expression of CD44 and CD62L by fl ow cytometry. Absolute cell numbers of respectively naïve (N) and effector/memory (E/M) subsets were calculated. Depicted are mean numbers x 106 ± SD of 4 mice.

Increased T cell turnover in Nef transgenic mice

Once we had observed low thymic cellularity and an activated peripheral T cell compartment,


we examined the dynamics of the T cell pool. The proliferation of transgenic versus wild-

type T cells was examined in vivo by measuring BrdU incorporation in DNA of dividing cells.

Analysis of CD4 and CD8 T cells revealed a six-fold increase in BrdU incorporation in both

CD4 and CD8 T cells derived from PLN of transgenic mice (as depicted in Fig. 2A), indicative

for a six-fold increased proliferation compared to wild-type littermates. As a measure for cell

death we determined the fraction of AnnexinV positive cells ex vivo by FACS analysis. A more

than seven-fold increase in the percentage of death CD4 T cells versus a three-fold increase

among CD8 T cells derived from the PLN was measured in the transgenic mice (Fig. 2B). The

fraction of dividing cells in the various T cell subsets did not differ between transgenic and

wild-type mice. Increased overall turnover rates in T cells from transgenic mice were thus

due to the increased proportion of effector/memory cells which have a larger fraction of

dividing cells compared to the naïve T cell subset (data not shown) (26, 28).

In conclusion, increased proliferation and apoptosis in Nef transgenic T cells

was observed and the majority of transgenic cells showed an activated effector/memory

phenotype. This resembled T cell characteristics observed in lymphopenic mice (27, 29,

30) and raised the question whether proliferation and activation is driven by Nef directly or

indirectly due to homeostatic effects.

Activation of normal T cells in Nef transgenic mice

We studied whether activation of the T cells was caused by an intrinsic Nef effect on

peripheral T cells or was brought about by the strongly reduced thymic output and the

concomitant T cell depleted environment in Nef transgenic mice. Wild-type T cells, which

could be distinguished from host cells by Ly5 allele expression, were adoptively transferred

into Nef transgenic or wild-type mice of similar age. Before transfer, PLN derived Ly5.1

FIGURE 2. The T cell compartment of Nef transgenic mice has an increased cell turn-over. The level of proliferation of peripheral T cells derived from PLN was determined by BrdU in-corporation. Mice of 6-8 weeks old were injected 3 times with 1 mg BrdU one day before analysis. The percentage of T cells that had incorporated BrdU is depicted (A). CD4 and CD8 T cells derived from PLN of transgenic mice and wild-type littermates of 6-8 weeks old were analyzed for the ex-pression of Annexin V by fl ow cytometry (B). Indicated are mean percentages of positive cells ± SD of 3 mice (*, p<0.01; **, p<0.001). Shown is a representative of three independent experiments.

* * ** **WtNef tg

0

5

10

15

CD4 CD8

%B

rdU

+T

-cell

s

0

10

20

30

CD4 CD8

%A

nV

+T

-cell

s

A B

CHAPTER 228

expressing lymphocytes were labeled with CFSE, to monitor their proliferative behavior. The

fate and phenotype of the transferred cells was determined in blood at day 3 and 6 after

transfer and in spleen at day 10 after transfer (for overview experiment see Fig. 3A). At day

3 after injection of the wild-type Ly5.1 T cells no CFSE dilution was observed in either of the

acceptor mice. Yet 6 days after injection proliferation was detectable in Nef transgenic mice

(Fig. 3B). In wild-type recipients proliferation was only detected at day 10 after transfer. At

this time point a fraction of ~85% of the transferred wild-type donor Ly5.1 CD4 T cells had

not divided when transferred to wild-type mice, compared to ~54% when transferred to

Nef transgenic mice (Fig. 3B). Of the transferred wild-type Ly5.1 CD8 T cells ~87% had not

divided in wild-type recipients versus ~63% in Nef transgenic recipients by day 10 (Fig. 3B).

Based on the CFSE profi les, it seemed that the cell cycle progression of transferred wild-type

Ly5.1 T cells is similar in wild-type and Nef transgenic mice, however the number of cells

entering the cell cycle seemed to be increased in Nef transgenic mice. Upon proliferation,

CD44 was up regulated and CD62L down modulated, resulting in a higher proportion of

day 3

day 10

day 6

blood

blood

spleen

100%

63%87%54%85%

70%95%78%97%

95%98%97%

CFSE

Even

ts

Wt + Wt Ly5.1 Wt + Wt Ly5.1Tg + Wt Ly5.1 Tg + Wt Ly5.1

CD4-T cells CD8-T cells

CD

44

CD

62L

CD

44

CD

62L

CFSE

Wt +Wt Ly5.1 Tg + Wt Ly5.1

CD4-T cells

Wt + Wt Ly5.1 Tg + Wt Ly5.1

CD8 T-cells

63%

63%

87%

87%

54%85%

84% 54%

iv Ly5.1 lymphocytes sacrifice

Tg Ly5.2

day 0 day 3/6 day 10

blood analysis

Wt Ly5.2

** *

0

5

10

15Wt + Wt Ly5.1

Tg + Wt Ly5.1

CD4 CD8

%B

rdU

+T

-cell

s

FIGURE 3. Micro-environment in Nef transgenic mice leads to activation of T cells. PLN were isolated from 8 weeks old C57Bl/6 Ly5.1 mice, lymph node suspensions were CFSE labeled and 5 x 106 cells were transferred into 6-8 weeks old Nef transgenic and wild-type mice. Three, six and ten days after transfer blood and respectively spleens were recovered and analyzed, an overview of the experiment is shown (A). CFSE profi les of donor Ly5.1 CD4 and CD8 T cells derived from either Nef transgenic (Tg + Wt Ly5.1) or wild-type (Wt + Wt Ly5.1) mice were determined by fl ow cytometry in the blood at day 3 and 6 and in the spleen at day 10 post transfer. Percentages of undivided cells are indicated (B). Splenocytes derived at day ten post transfer, were incubated with mAbs directed against CD44 and CD62L. CFSE expression together with CD44 or CD62L on donor Ly5.1 T cells was determined, percentages of undivided cells are indicated (C). The level of proliferation of donor Ly5.1 T cells derived from either wild-type or Nef transgenic spleens were determined by BrdU incorpora-tion (for method see Figure 2). The proportion of Ly5.1 donor T cells that had incorporated BrdU is depicted (D). Data are representative of 2 mice per group and the experiment is performed 3 times.

A B

C D


CD44high and CD62Llow (Fig. 3C) wild-type donor T cells in the transgenic mice compared to

wild-type littermates. In vivo BrdU incorporation was measured to quantify cell division.

Higher division rates were observed for donor wild-type Ly5.1 T cells transferred into Nef

transgenic mice then for cells transferred into wild-type mice (Fig. 3D). This was in good

agreement with the CFSE data.

Thus, wild-type T cells transferred into T cell depleted Nef transgenic animals become

activated effector/memory cells suggesting that the micro-environment in Nef transgenic

mice is able to cause T cell activation irrespective of Nef expression in the peripheral T

cells.

Normal phenotype of Nef transgenic T cells developing in a full T cell

compartment

To dissect the effect of the T cell depleted environment from the effect of expression of Nef

on division and activation of Nef transgenic T cells, we examined the T cell activation status

of transgenic cells in a non-lymphopenic environment. Therefore we generated mixed bone

marrow chimeras, where co-transfer of Nef transgenic and wild-type bone marrow resulted

in Nef transgenic T cells in a normally developed T cell compartment of a mostly wild-type

origin. Host and donor cells could be distinguished on basis of Ly5 allele expression. Wild-

type bone marrow recipient mice of 10-12 weeks old, which expressed both Ly5.1 and Ly5.2

on their haematopoietic cells were sublethally irradiated and reconstituted with wild-type

Ly5.1 bone marrow in combination with either Ly5.2 wild-type (wt/wt group) or Ly5.2 Nef

transgenic (wt/tg group) bone marrow, in a ratio of 1:10 (Ly5.1:Ly5.2).

Blood analysis revealed that after three weeks naïve T cells numbers had increased, indicative

of de novo thymic output. Six weeks after bone marrow transplantation, lymphocyte numbers

in blood had stabilized and mice were sacrifi ced for ex vivo analysis of lymphoid organs (for

overview experiment, see Fig. 4A).

Absolute cell numbers of the thymus derived from mice of the wt/wt group contained

~80x106 thymocytes, whereas thymi derived from mice of the wt/tg group contained

~50x106 thymocytes (Fig. 4B). Thymi derived from mice of the wt/wt group were composed

of approximately 90% Ly5.2 thymocytes, due to the administered ratio of Ly5.1 and Ly5.2

bone marrow (1:10). The thymus of mice from the wt/tg group however, contained around

15% thymocytes of the Ly5.2 lineage; the remaining 85% was predominantly from the

Ly5.1 wild-type donor lineage (Fig. 4C). In order to substantiate functionality of the thymus,

subset distribution within the thymus was determined by analyzing CD4 and CD8 expression

profi les for the different Ly5 subsets in the thymus of mice from both wt/wt and wt/tg

reconstituted groups. CD4 and CD8 SP thymocytes of all Ly5 subtypes were present (Fig.

4D), which indicated normal thymic development and an effl ux of mature CD4 and CD8 T

cells into the peripheral T cell compartment. Examination of total cellularity of the secondary

lymphoid organs demonstrated rather normal T cell numbers among PLN and spleens of

CHAPTER 230

sacrifice

0 h 6h 6 weeks

Wt Ly5.1/2

irradiation- Ly5.1 wt- Ly5.2 wt

iv BM:

- Ly5.1 wt- Ly5.2 tg

Cell C

ou

nts

CD4

Ly5.1 wt

Ly5.2 tg

Wt/Tg

Wt/Wt

CD62L

CD

44

84%

76%

85%

88%

CD4 T-cells

Ly5.1 wt Ly5.2 wt

Ly5.1 wt Ly5.2 tg Ly5.

1w

t

Ly5.

1w

t

Ly5.

2tg

Ly5.

2w

t

CD62L

68% 74%

56%64%

Ly5.1 wt Ly5.2 wt

CD8 T-cells

Ly5.1 wt Ly5.2 tgCD

44

Ly5.

1w

t

Ly5.

1w

t

Ly5.

2tg

Ly5.

2w

t

** **

0

2 5

50

75

10 0

Wt/Wt Wt/Tgthym

ocyte

s (

x10

)6

0 %

2 0 %

4 0 %

6 0 %

8 0 %

10 0 %

Wt/Wt Wt/Tg

% t

hym

ocyte

s o

f to

tal

Ly5.1

Ly5.1/2

Ly5.2

Wt/Wt

Wt/Tg

Ly5.1 Ly5.1/2 Ly5.2

CD8

CD

4

8

22

88 12

33

82 14

52

80

26

71

66 8

121

80 15

421

60

Ly5.2 wt

Ly5.2 tg

% o

f to

tal T

cell n

um

ber

FIGURE 4. Nef transgenic T cells which developed in a full T cell compartment show a normal naïve phenotype. Bone marrow was isolated from 6-10 weeks old C57Bl/6 Ly5.1, Nef transgenic Ly5.2 and wild-type Ly5.2 littermates. After T cell depletion, 5x106 bone marrow cells were injected in 10-12 weeks old irradiated Ly5.1 / Ly5.2 mice. One group of irradiated mice was injected with a mixture of Ly5.1 wild-type together with Ly5.2 wild-type bone marrow (wt/wt group) and the other group of irradiated mice was injected with a mixture of wild-type Ly5.1 together with Nef transgenic Ly5.2 bone marrow (wt/tg group), the ratio of injected Ly5.1:Ly5.2 bone marrow was 1:10 for both groups. Six weeks after transfer, lymphoid organs were dissected and analyzed by fl ow cytometry, an overview of the experiment is shown (A). Single cell suspensions derived from thymus and PLN were incubated with antibodies directed against Ly5.1, Ly5.2, CD4 and CD8 and analyzed by fl ow cytometry. Absolute (B) and relative proportions (C) of the different Ly5 subsets in the thymus were determined for both the wt/wt and wt/tg group. The different Ly5 subsets present in the thymus were analyzed for differential expression of CD4 and CD8 (D). Relative proportions of the Ly5.2 subset among CD4 and CD8 T cell derived from PLN were determined for both the wt/wt and wt/tg group (E). Ly5.1 wild-type and Ly5.2 transgenic lymphocytes derived from PLN of wt/tg group were analyzed for CD4 expression levels by fl ow cytometry (F). T cells derived from PLN of mice from wt/wt and wt/tg group were analyzed for differential expression of CD62L and CD44. Dot plots of one representative mouse from each group is shown, percentages of naïve (CD44mediumCD62Lhigh) CD4 and CD8 T cells are depicted in the dot plots, mean percentages of 3 mice are shown in bar charts (G). Data are representative of two experiments (one experiment with n=3 per group and one experiment with n=5 per group). No signifi cant differences were found, using the student’s t test. Indicated are mean percentages ± SD of 3 mice (*, p<0.005; **, p<0.0005).

A B C

D E F

G


both the wt/wt and the wt/tg group (data not shown). The PLN derived T cells from wt/wt-

reconstituted mice consisted for about 50% of wild-type Ly5.2 T cells, whereas, the wt/tg-

reconstituted group contained only 1-2% of Nef transgenic Ly5.2 T cells in their PLN derived

T cells (Fig. 4E). This poor outgrowth of transgenic Ly5.2 T cells is compatible with the Nef-

mediated block in T cell development.

To confi rm Nef expression in peripheral T cells, the CD4 level on wild-type Ly5.1 and

Nef transgenic Ly5.2 cells was measured in the wt/tg group. Wild-type cells expressed CD4

at MFI of ~ 124, whereas Nef transgenic cells expressed CD4 at MFI of ~ 73. This CD4 down

regulation is typical for a functional Nef protein being present in transgenic peripheral T cells

(Fig. 4F). In order to study the activation state of the Nef transgenic T cells which developed

in a full T cell compartment, the subset distribution of the different Ly5 populations in the

wt/wt and wt/tg group were examined by measuring CD44 and CD62L expression by fl ow

cytometry. As expected, no signifi cant difference was found between the percentage of

naïve wild-type Ly5.1 and wild-type Ly5.2 T cells within the wt/wt group. This was observed

in both CD4 and CD8 T cells (Fig. 4G). Strikingly, the Ly5.2 Nef transgenic T cells had the

same naïve phenotype as the wild-type Ly5.1 T cells, which had developed together in the

wt/tg group. This was observed for CD4 as well as for CD8 T cells (Fig. 4G).

These data show that when Nef transgenic T cells develop in a T cell repleted normal

environment, their behavior is similar to wild-type cells and they have a predominantly naïve

phenotype. We conclude that the T cell activation seen in Nef transgenic mice is mediated

by lymphopenia-induced mechanisms rather than by an intrinsic effect of Nef expression on

T cell activation and division.

CHAPTER 232

DISCUSSION

In this study we show that the cellularity of the thymus and, as a consequence, thymic output

and T cell numbers in secondary lymphoid organs are dramatically reduced in Nef transgenic

mice. Under these circumstances, the transgenic T cells are highly activated and show an

increased turnover. By generating bone marrow chimeras we were able to study transgenic

T cells in a full T cell compartment. In contrast to the highly activated phenotype that was

observed in Nef transgenic mice, Nef transgenic T cells in a full compartment showed an

overall naïve phenotype. This strongly suggests that the T cell activation observed in Nef

transgenic mice is a secondary effect of Nef-mediated abrogation of thymus output and

that in this model Nef does not lead to T cell activation directly. Our data closely resemble

classical experiments in which T cells were adoptively transferred into lymphopenic mice

leading to homeostatic proliferation and an effector/memory like phenotype (25, 28, 30).

Our results thus are reminiscent of lymphopenia-induced mechanisms.

Based on the in vitro interference of Nef with TCR signaling, many of the in vivo

effects of Nef in transgenic mice models have been ascribed to effects on T cell activation (7,

10). Studies by Jolicoeur and coworkers in CD4C/HIV-Nef transgenic mice have shown similar

thymic depletion and increased peripheral T cell activation and proliferation as we report

here (19). To test whether this increased activation was TCR dependent, these investigators

crossed their Nef transgenic mice with TCR transgenic mice (AD10). Also in these double

transgenic mice CD4 T cells had an activated effector/memory phenotype, suggesting an

intrinsic antigen-independent effect of Nef on T cell activation (19). However, it was shown

that under lymphopenic conditions these TCR transgenic CD4 T cells can proliferate in the

absence of their cognate antigen, resulting in the acquisition of an effector/memory-like

phenotype (27), thereby questioning the former interpretation of their results.

Correlation between the expression levels of activation markers and the amount

of Nef protein expressed in CD4 T cells in a Nef transgenic mice further suggests an in vivo

role for Nef in T cell activation (19). In these Nef transgenic mice different CD4 populations,

designated as CD4high and CD4low, with respectively low and high Nef expression have been

described (19). In agreement with a direct effect of Nef on T cell activation these two

subpopulations also differed in their activation status, i.e. the more Nef protein expressed,

the lower the CD4 expression, the more the cells appeared to be activated. However, when

we evaluated the activation status of low and high CD4 expressing T cells derived from

wild-type mice, expression of activation markers was also found to be unevenly distributed.

Even in wild-type mice the CD4low expressing T cells contained a higher fraction of cells

expressing activation markers (unpublished data). Apparently this inverse correlation of

CD4 and activation marker expression levels is a general phenomenon independent of Nef

effects on T cell activation.


Next to TCR dependent effects of Nef, in vitro studies have shown effects of Nef on

CD28 mediated co-stimulation (11, 12). Crossing of our Nef transgenic mice on a B7 defi cient

background did not lead to any changes in the observed phenotype or in vitro characteristics

of Nef transgenic T cells (unpublished data), which questions the effect of Nef on CD28

mediated co-stimulation. Recently it was proposed that two different mechanisms drive

proliferation in a lymphopenic environment; homeostatic proliferation which is slow and IL-7

dependent and endogenous proliferation which is fast and depends on competition of TCRs

for stimulatory self-peptide/MHC interactions. It has been reported that CD28-mediated

signals are not required for homeostatic proliferation, but are necessary for endogenous

proliferation. Since in our mice absence of CD28 mediated co-stimulation did not result in

any change in the observed phenotype this would point to homeostatic proliferation as the

driving mechanism in our model (31).

Taken together, our data show that Nef expression in the T cell lineage of transgenic

mice does not directly activate primary peripheral T cells. Instead, Nef expression severely

affects T cell development in the thymus, resulting in lymphopenia and causing lymphopenia-

induced division and differentiation of the remaining Nef transgenic T cells.

CHAPTER 234

MATERIALS AND METHODS

Mice

The CD2 Nef transgenic mice, have been described previously (17) and were a kind gift of Elaine Dzierzak.

Experiments were performed with mice 6 to 12 weeks of age. Transgenic mice were compared with non-

transgenic littermates. C57BL/6.SJL (Ly5.1) and C57BL/6 (Ly5.2) mice were obtained from The Jackson

Laboratories and were maintained as breeding colony in our animal facility. Ly5.1 / Ly5.2 mice were

bred by crossing (C57BL/6.SJL Ly5.1 × C57BL/6-Ly5.2.). All mice were kept under specifi c pathogen-

free conditions and housed in accordance with institutional guidelines of American Association of

Accreditation of Laboratory Animal Care, in the mouse facility of the Central Laboratory Animal Institute

Utrecht University (Utrecht, the Netherlands). The Institutional Animal Ethics Committee approved all

experiments.

Antibodies and reagents

(BrdU)-APC fl ow kit, Annexin V, mAbs (CD4 (RM4-5), CD8 (53-6.7), CD16/32 (2.4G2), CD44 (IM7),

CD45.1 (A20), CD45.2 (104) and CD62L (MEL-14)), and streptavidin, unlabeled or labeled with appropriate

fl uorochromes, were all purchased from BD Biosciences (San Gosé).

CFSE labeling and adoptive transfer

Single cell suspensions were prepared from PLN. Cells were labeled with CFSE (Molecular Probes Inc.,

Eugene, OR.) as previously described (24). Briefl y, cells were washed twice in phosphate-buffered saline

(PBS) and resuspended at 107 cells/ml in PBS. CFSE was added to a fi nal concentration of 0.5µM, and

cells were incubated for 10 min at 37ºC. Unbound CFSE was quenched by washing labeled cells twice with

RPMI 1640 medium supplemented with 10% fetal bovine serum (Integro b.v., Dieren, the Netherlands).

5x106 Cells were resuspended in 200 μl PBS and injected i.v. into the tail vein of the mice.

Bone marrow transplantations

Bone marrow was obtained by the passage of iced RPMI through the tibias and femurs. T cells were

depleted by use of CD90.2 (Thy1-2) labeled magnetic beads and LD columns (Miltenyi Biotec GmbH,

Bergisch Gladbach), following the manufacturer’s instructions. Recipient mice were irradiated with a

single sublethal dose of 7.0 Gy from an X-ray source. Within six hours the recipient mice were injected

i.v. with a total number of 5x106 bone marrow cells.

Measurement of BrdU incorporation in vivo

BrdU (1mg/mouse, Sigma, St. Louis, Mo) was injected three times intraperitoneally with a time interval

of 4h, mice were sacrifi ced 24h after the fi rst injection. Single cell suspensions of lymphoid organs were

prepared and cells were labeled with the appropriate antibodies. Next, cells were fi xed, permeabilized and

stained for BrdU with a BrdU-APC fl ow kit according to the manufacturer’s instructions.

Flow cytometry

Thymus, spleen and PLN were forced through cell strainers (Falcon, BD Biosciences) in the presence of

RPMI 1640 with 10% FCS to obtain single cell suspensions. Erythrocytes were lysed by incubation of the

cells in 0.15 M NH4Cl, 0.01 M KHCO3, 0.1 mM EDTA, pH 7.4, for 2 min on ice. Cells were preincubated

with Fc-block (mAb to CD16/32, 2.4G2; BD Biosciences) and washed in staining buffer (PBS, 0.5%BSA,

0.01% sodium azide). Afterwards cells were incubated with antibodies and/or Annexin V. Stained cells


were analyzed using a FACSCalibur or BD LSR II and analyzed by CELLQuest or BD FACSDiva software

(BD Biosciences).

Statistical analysis

Results were analyzed using a Student’s t test (two-tailed). Differences between groups were considered

statistically signifi cant at the p < 0.05.

ACKNOWLEDGEMENTS

We acknowledge F. Miedema, L. Meyaard and J. Borghans for critically reading the

manuscript. We thank A. Martens for helpful discussions and practical assistance concerning

the bone marrow transfer experiments. We thank E. Dzierzak for kindly providing the CD2

Nef transgenic mice and R. Arens for supplying C57Bl/6 Ly5.1 derived lymphocytes.

CHAPTER 236

REFERENCES


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HIV-Nef severely impairs thymocyte development and peripheral T cell function by a CD4-independent



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Hamann. 2000. T cell division in human immunodefi ciency virus (HIV)-1 infection is mainly due

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An Inducible HIV-Nef Transgenic Mouse: A Reappraisal of Nef-mediated Pathology

3

Manuscript in preperation

CHAPTER

Paul G.F. Koenena, Frans M. Hofhuisa, Elise H.R. Schrijvera, Dietlinde Wolfb, Derk Amsenc, Patrick E. Fieldsd, Richard A. Flavelle, Sjef Verbeekf, Kiki Tesselaara, Mariëtte A. Oosterwegela

aDepartment of Immunology, University Medical Center, Utrecht, The Netherlands; bDepartment of Microbiology and Immunology, University of Miami, School of Medicine, Miami, USA; cDepartment of cell Biology and Histology, The Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; dDepartment of Pathology, University of Kansas Medical Center, Kansas City, USA; eHoward Hughes Medical Institute, Section of Immunology, Yale University School of Medicine, New haven, USA; fDepartment of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands.

CHAPTER 340

ABSTRACT

HIV infection leads to hyper-activation of the T cell compartment and thymic atrophy. Both

mechanisms may play an important role in the development towards AIDS. To investigate the

role of the viral protein Nef in these two distinct mechanisms of HIV pathogenicity, inducible

Nef transgenic mice were generated. Induction of Nef expression in the peripheral T cells

did not have any infl uence on the activation status of the peripheral T cell compartment. In

contrast, induction of Nef expression in thymocytes led to a fast and rapid reduction of all

thymic subsets, subsequently resulting in reduced peripheral T cell numbers. These fi ndings

show that Nef expression by itself does not lead to hyper-activation of the peripheral T cell

compartment and underscore an important role for Nef in thymic atrophy as seen during

HIV.

An inducible HIV-Nef transgenic mouse: a reappraisal of Nef-mediated pathology 41

INTRODUCTION

Acute HIV infection has been shown to cause a major loss of memory CD4 T cells from

gastrointestinal mucosa (1, 2). During the subsequent chronic phase of HIV-1 infection

a further gradual loss of CD4 T cells is observed. Given the low number of HIV-infected

CD4 T cells, systemic effects of HIV infection have been proposed which could, next to the

direct killing due to HIV infection, contribute to loss of CD4 T cells. Persistent hyper-immune

activation associated with HIV infection has been proposed to be the main determinant for

CD4 T cell loss and HIV disease progression (3). This immune activation hypothesis is based

on the fi nding that in the chronic phase HIV infection is highly dynamic, with high daily viral

production rates and rapid turnover of HIV-infected cells (4). Over time both CD4 and CD8

T cell activation and turnover is increasing, which has been shown to be induced directly or

indirectly by HIV infection (4-9). These fi ndings indicate that a highly active infection is going

on in the asymptomatic phase, which has been shown to lead to a constantly increased

recruitment and differentiation of peripheral naïve CD4 and CD8 T cells, together with direct

infection and death by HIV, ultimately leading to CD4 T cell depletion (3, 10-12).

It was assumed that increased T cell renewal, for instance by homeostatically

increased thymic output, could initially compensate for the increased loss of naïve and

memory T cells (4). However several lines of evidence have suggested that HIV infection

actively interfered with thymus function and output, thereby not only preventing possible

compensatory T cell renewal but even implying reduced thymic output as a major

mechanism contributing to CD4 T cell depletion in chronic HIV infection (13, 14). Compatible

with HIV-induced thymic impairment, peripheral T cells from HIV infected patients have a

decreased content of TCR excision circles (TRECs) that are formed during intrathymic TCR

rearrangement (13, 15-17). Interestingly, it was recently shown that intrathymic T- cell

proliferation is reduced in HIV infected patients and was restored by antiviral therapy (18).

This restoration of thymopoiesis after treatment with effective antiretroviral therapy in

a proportion of infected individuals provides further evidence for this mechanism of T cell

depletion (13, 18-20). More direct evidence for severely decreased thymic output resulting

from HIV infection came from the severe combined immunodefi ciency mouse grafted with

human tissue (SCID-hu) model. In this model HIV infection unambiguously causes thymic

involution and thymocyte depletion (21, 22).

The viral regulatory protein Nef has been implicated in HIV pathogenesis and CD4

T cell depletion. HIV strains with a non-functional or deleted Nef gene were found in HIV-

infected long-term non-progressor patients who remained asymptomatic for more than

10 years and maintained low viral loads and stable CD4 T cell counts (23, 24). Evidence

for a direct role of Nef on T cell activation comes from a study, which demonstrates that

induction of Nef expression in Jurkat T cells induces a very similar gene expression profi le

as TCR signaling (25). Other groups have shown that Nef leads to enhanced activation after

CHAPTER 342

stimulation of the TCR and CD28, resulting in increased IL-2 production (26, 27). As direct

binding of Nef was reported towards Src-family kinase p56lck (28-31) and the TCR-ζ chain

(32, 33), reviewed in (34)) these fi ndings can be explained by direct interference of Nef with

components of the TCR signaling cascade. Collectively these studies indicate a role for Nef

in disturbance of T cell function in order to increase HIV production levels. In a recent study,

the different activating capacities of Nef derived from either SIV or HIV-1 were suggested to

refl ect the different pathogenicity of these two viruses. The authors proposed that Nef gene

evolution from SIV to HIV-1 has been associated with a functional evolution from a non-

pathogenic to a pathogenic variant, which is refl ected in lack of pathogenicity of SIV in Sooty

Mangabyes, the natural host for SIV. By down regulating TCR-CD3 on T cells of infected

monkeys, an excess of immune activation by SIV infection is prevented. HIV-1 Nef on the

other hand was shown to allow for high levels of immune activation because it has lost the

capacity to down regulate TCR-CD3 in human T cells (35). An opposite effect of HIV-1 Nef

on T cell activation was recently shown by Thoulouze et al., who showed that Nef disturbs

Lck distribution towards the immunological synapse in HIV-1 infected CD4 T cells, leading to

reduced T cell activation (36).

Besides these effects on T cell activation, Nef also exerts a dominant role in HIV

mediated pathology of the thymus as shown in a study by Duus et al. In this study effects

of HIV isolate Lai/IIIB with and without a functional Nef gene were compared. No signifi cant

thymic depletion was found in the SCID-hu model when the Nef-defi cient strain was used.

Restoration of the Nef gene resulted in an almost complete loss of CD4 thymocytes 6 weeks

post infection (37). Similar observations were reported using an in vitro human-murine fetal

thymic organ culture (FTOC) model, demonstrating that human T cell precursors, expressing

the Nef protein, are impaired in generating T cells (38, 39).

In order to study the effect of Nef protein expression in vivo, several constitutive

Nef transgenic mouse models with T cell specifi c Nef expression have been generated

(40-43). It was consistently reported that the T cells of the Nef transgenic mice have

an activated phenotype, compatible with the fi nding on Nef effects in human T cell lines

discussed above (40, 44, 45). We and others showed that in these animals the T cells also

had an increased turnover rate (Koenen et al., submitted) (44), altogether implying a role

for Nef in T cell activation in HIV infection. Besides this peripheral T cell activation, all Nef

transgenic mice showed thymocyte depletion, resulting in decreased thymic output and

peripheral lymphopenia (40-43).

Recently, we showed that the peripheral T cell activation observed in a constitutive

Nef transgenic model was lymphopenia induced, rather than mediated by a direct effect of

Nef on T cell activation (Koenen et al., submitted). These data suggested that constitutive

expression of Nef throughout the T cell compartment leads to thymic involution and

lymphopenia from birth on and subsequently to lymphopenia-induced T cell proliferation in

the periphery.


Thus due to its immediate effect on thymic output, constitutive Nef expression

dramatically alters the T cell compartment during early ontogeny and impedes studies of the

effects of Nef on the peripheral T cell compartment. To address this problem we generated

inducible Nef transgenic mice. These mice do develop normally and Nef expression can be

induced when all T cell compartments have fully developed. Here we show that induction of

Nef protein in the T cell compartment of these transgenic mice did not lead to autonomous

activation of peripheral T cells. In contrast to this lack of effect on peripheral T cells,

thymic cellularity decreased rapidly after the Nef protein became expressed, resulting in

subsequently reduced T cell numbers in the periphery. This study demonstrates that Nef

expression in the T cell lineage does not directly induces peripheral T cell aberration but has

a dominant effect in causing thymus atrophy.

CHAPTER 344

RESULTS

Tetracycline-responsive Nef expression in the T cell lineage of transgenic mice

To mimic the effects of Nef expression in vivo during HIV infection, we aimed to generate

transgenic mice in which HIV-1 Nef expression can exclusively be induced in the T cell

lineage. We made use of the tetracycline-responsive gene induction system that has been

described previously as the “tet-on” system (53-55). In this double transgenic system

expression of a target gene is dependent on binding of a reverse tet transactivator (rtTA)

to tet-response elements (tetO) upstream of the target gene. The transactivator normally

does not activate the target gene, but in the presence of doxycycline (dox), a derivate

of tetracycline) binds and activates transcription (Figure 1A) (53-55). Cell specifi c rtTA

expression is attained using a cell type specifi c promoter to drive the expression of the

transactivator. Possible background transcription can be repressed by a third transgene that

encodes a β-actin driven repressor of transcription (KTR), which binds in the absence of

dox (Figure 1A, ii). To overcome the detection limitations of intracellular Nef expression, a

reporter gene, either tailless human CD2 or EGFP was simultaneously induced with Nef to

visualize protein expression (Figure 1A). A more detailed description of the model system is

given in the materials and methods section.

To verify protein induction in T cells of double or triple transgenic mice, splenocytes

were cultured in the presence of αCD3 mAbs, with and without dox induction. After 48h the

T cells were analyzed for reporter protein expression by fl ow cytometry (Figure 1B) and

for Nef protein expression by western blot analysis (Figure 1C). Without dox, no reporter

expression was observed, but in the presence of dox almost 100% of both CD4 and CD8

T cells expressed the reporter protein, the CD2-rtTA-M2/β-actin-KTR/TetO7-Nef-huCD2TL

transgenic line, with highest in vitro induction is shown. The presence of reporter expression

was accompanied by the expression of Nef as shown in Figure 1C.

tetO NEFmCMVEGFP/huCD2TL mCMV

rtetRCD2 VP16

+ doxycycline (dox)

VP16

rtetR

rtTA:

NEF:

tetO NEFmCMV mCMV

rtetR-M2CD2 VP16

- dox VP16

rtetR

rtTA:

NEF:

tetR ß-actinKRAB : KTR

KTR

tetR

tetO NEFmCMVEGFP/huCD2TL mCMV

rtetR-M2CD2 VP16

+ dox

VP16

rtetR

rtTA:

NEF:

tetR ß-actinKRAB : KTR

KTR

tetR

i) ii)

EGFP/huCD2TL

A


Since functional Nef expression is characterized by down regulation of CD4

molecules from the cell surface (56), stimulated T cells were analyzed after 48h for CD4

expression by fl ow cytometry. A specifi c down regulation of CD4 expression was observed on

dox treated cells, while CD8 expression levels were unchanged, the CD2-rtTA-M2/β-actin-

KTR/TetO7-Nef-huCD2TL transgenic line, with highest in vitro induction is shown (Figure

1D). This downregulation of CD4 indicates that upon induction a functional Nef protein is

expressed.

In short, we successfully generated an inducible Nef transgenic mouse line, with

expression in the T cell lineage, without any background expression in the absence of

induction in vitro. The induced Nef protein is functional, as it is able to down regulate CD4

molecules from the cell surface. Furthermore, Nef induction is linked with reporter protein

expression, which makes reporter expression a good representation for Nef expression.

Kinetics of induction of tetracycline-responsive Nef transgenic mice in vivo

In order to study induction of Nef expression in vivo, kinetics of protein expression upon dox

treatment in transgenic mice were studied. Inducible Nef transgenic mice were exposed to dox

and at various time points cells were analyzed for reporter expression by fl ow cytometry. No

expression of reporter protein could be detected on T cells or thymocytes before administration

of dox, emphasizing the tight regulation of transgene transcription in vivo. Also no differences

with regard to background expression in the absence of dox were observed among CD2-rtTA

and CD2-rtTA-M2/β-actin-KTR inducer lines, neither line showed any background expression

(data not shown). Administration of dox led to detectable reporter protein expression in

dox

Nef

- +

negpos

reporter

even

tsCD4 T-cells CD8 T-cells

- dox+ dox

even

ts

CD4 CD8

FIGURE 1. Doxycycline (dox)-respon-sive Nef transgenic mice. Schematic view of the dox-dependent double (i) or triple (ii) transgenic system (A). A re-verse transactivator (rtTA) consisting of the reverse- tet-repressor (rTetR) fused to the VP16-activating domain is expressed via the human CD2 promoter. Binding of the transactivator tot tet operator (TetO) regulatory sequences fl anked by two mini-mal CMV promoters is necessary to drive expression of both Nef and a reporter

for gene induction, either EGFP or tailles human CD2 (huCD2TL), this only occurs in the presence of dox, therefore called ‘tet’-on system. Possible background transcription can be repressed by a tTA driven repressor of transcription (KRAB), binding in the absence of dox (A, ii). Splenocytes derived from CD2-rtTA-M2/ β-actin-KTR /TetO7-Nef-huCD2TL transgenic mice were cultured in the presence of 0.5 µg/ml αCD3 mAb, with or without 1µg/ml dox. After two days the cells were either incubated with mAbs direct-ed to mouse CD4, CD8 and human CD2 and analyzed by fl ow cytometry (B) and (D) or they were lysed in NP40 lysis buffer and after immunoprecipitation of Nef analyzed by western blot for Nef protein (neg= negative control, pos= positive control for Nef protein, for details see materials and methods section) (C).

B

D

C

CHAPTER 346

blood-derived T cells, from 4h onwards and reached a maximum after ~12h hereafter the

percentage of reporter protein expressing T cells stayed relatively constant, the CD2-rtTA/

Nef- TetO7-Nef-EGFP transgenic line, with highest peripheral expression is shown (Figure

2A). Also in the thymus, reporter protein expressing thymocytes could be detected upon

dox administration, the CD2-rtTA-M2/β-actin-KTR/ TetO7-Nef-huCD2TL transgenic line, with

highest thymus induction is shown (Figure 2B).

Tissue specifi city for both inducer lines was determined by FACs analysis of different

cell populations after induction. While CD2-rtTA controlled induction drove expression

specifi c to the T cell compartment, the CD2-rtTA-M2/β-actin-KTR induced expression in T

cell compartment as well as in B cells (data not shown). No expression was observed on

other immune cells. After analyzing the specifi city and levels of induction in offspring from

various double and triple transgenic lines (data not shown), two lines for our studies were

selected; one line with two transgenes, CD2-rtTA/TetO7-Nef-EGFP driving Nef expression

predominantly in the periphery and another line with three transgenes, CD2-rtTA-M2/β-

actin-KTR/TetO7-Nef-huCD2TL driving Nef expression predominantly in the thymus.

Together, these data show that there is regulation and fast induction of protein

specifi cally in the T cell compartment upon dox administration in vivo. Our inducible Nef

transgenic mice model thus provides us with an elegant system to study transient Nef

expression in primary T cells in vivo.

Transient Nef expression does not induce T cell activation or turnover

In contrast to constitutive Nef transgenic mice, where thymic atrophy leads to a severely

lymphopenic periphery and high fractions of activated T cells, a normal T cell compartment

developed in the inducible Nef transgenic mice in the absence of dox (data not shown).

CD4+ T-cellsCD8+ T-cells

even

ts

reporter

- dox+ dox

0 10 20 300

20

40

60

induction time [hours]

%in

du

ced

T-c

ell

s

FIGURE 2. Doxycycline-controlled Nef expression in the T cell compartment. CD2-rtTA/ TetO7-Nef-EGFP transgenic mice were fed with 2mg/ml dox in their drinking water. Blood was drawn two to three times per mouse at different time points. After lysis of erythrocytes blood cells were incubated with mAbs directed towards mice CD4 and CD8, subsequently cells were analyzed for EGFP expression by fl ow cytometry. Data derived from nine different mice is depicted (A). CD2-rtTA-M2/ β-actin-KTR /TetO7-Nef-huCD2TL transgenic mice were fed with 2mg/ml dox for 12h , thymi were removed and single cell suspensions were incubated with antibodies directed towards human CD2, to determine reporter expression by fl ow cytometry. Shown is a representative example of ten mice (B).

A B


Therefore, the tet system offered the possibility of studying direct effects of Nef expression

on the peripheral T cell compartment and T cell activation in particular.

Nef effects in the periphery were studied in the CD2-rtTA/TetO7-Nef-EGFP transgenic

mice with predominant transgene expression in the periphery. After dox administration to

double transgenic mice for three days, lymphoid organs were collected. To control for any

dox or rtTA related effects, dox treated transgenic mice expressing rtTA only were used as

controls. Among double transgenic mice, individual mice showed variable induction levels,

ranging from 0.2 - 48% among CD4 T cells and from 0.1 - 60% among CD8 T cells, with

average induction levels of 19% among CD4 and 26% among CD8 T cells. The average

induction levels among CD4 and CD8 T cells were respectively ~20% and ~26% (Figure

3A). In transgenic mice expressing Nef, the total number of T cells showed a modest but

signifi cant reduction, compared to control mice fed with dox. This reduction was observed

among CD4 as well as CD8 T cells, although only the reduction among CD8 T cells was

signifi cantly different (Figure 3B). To investigate whether this decline in T cells was due to

Nef-mediated T cell death, Annexin V expression was measured by fl ow cytometry. However,

no difference in percentage of apoptotic cells could be detected between double transgenic

and control mice (Figure 3C). To interpret data derived from double transgenic mice with

different induction levels, the percentage of induced T cells in relation to the percentage

of Annexin V+ T cells was studied (Figure 3C). No correlation between the percentage of

induction and the proportion of AnV expressing T cells was found further stressing that T cell

loss in double transgenic mice is not caused by increased apoptosis in the periphery.

Since we were interested in the activating capacities of Nef in T cells, the expression

of CD25 on both CD4 and CD8 T cells was examined. No differences in CD25 expression

level between double transgenic and control T cells was observed (Figure 3D). When

double transgenic mice with low percentage of induction were compared with mice with

higher percentage of induction, no correlation between the percentage of induction and the

proportion of CD25 expressing T cells was found either (Figure 3D). These same results were

obtained when the T cells were analyzed for the expression of CD69, an early marker of T

cell activation, (Figure 3E). So this analysis showed that after 3 days of Nef expression in T

cells no effects on the activation status of these cells could be demonstrated.

Next to the analysis of CD25 and CD69, the composition of the peripheral T cell pool,

ie. the fractions of T cells in respectively the naïve and, memory and effector compartments,

was determined. TCR-ligation dependent differentiation leads to naïve T cell activation and

progression from the naïve stage into either effector or memory stage. The different stages

of T cell differentiation can be distinguished on basis of their CD62L and CD44 expression. T

cells expressing CD44medium CD62Lhigh are defi ned as naïve, CD44high CD62Lhigh as memory and

CD44highCD62Llow as effector cells (57, 58). Enhanced activation signals in T cells can skew

the T cell population from naïve towards effector and memory cells, thus if Nef expression

infl uences T cell activation, this might lead to a different composition of the T cell pool.

CHAPTER 348

Therefore, lymph node suspensions were analyzed for CD44 and CD62L expression. First,

percentages of naïve T cells were compared between the control and double transgenic

mice after dox administration. No differences could be found in the CD4 or the CD8 T cell

subsets (Figure 3F). Further analysis of double transgenic mice, comparing mice with a

low and high percentage of induced cells, did not reveal any differences in T cell subset

distribution related to Nef expression either (Figure 3F). Similar analyses of effector and

memory subsets revealed no difference between mice with and without Nef expression in

peripheral T cells (Figure 3G).

Three days of Nef expression did not affect the activation state and phenotype of

the T cell compartment in the transgenic mice. To investigate whether this also was the case

after prolonged Nef expression, Nef expression was induced for six days. However, also after

0

25

50

75

CD4 CD8

% in

du

ced

T-c

ells

0

5

10

15 *rtTA

rtTA/Nef

CD4 CD8

T-c

ells x

10

6

0 10 20 30 40 50 60 70

0.0

2.5

5.0

7.5

% induced T-cells

% C

D69+

T-c

ells

0 10 20 30 40 50 60 70

0

10

20

% induced T-cells

% A

nV

+ T

-cells

0 10 20 30 40 50 60 70

0.0

2.5

5.0

7.5

10.0

CD4-rtTA

CD4-rtTA/Nef

CD8-rtTA

CD8-rtTA/Nef

% induced T-cells

% C

D25+

T-c

ells

0 10 20 30 40 50 60 70

0

10

20

30

40

50

% induced T-cells

% E

/M T

-cells

0 10 20 30 40 50 60 70

0

25

50

50

60

70

80

90

100

% induced T-cells

% n

aïv

e T

-cells

FIGURE 3. Nef expression in T cell compartment does not lead to T cell activation or cell death. CD2-rtTA/TetO7-Nef-EGFP (rtTA/Nef) and CD2-rtTA (rtTA) transgenic mice were supplied with 2mg/ml dox in their drinking water for three days. Mice were sacrifi ced and peripheral lymph nodes (PLN) were isolated, single cell suspensions were incubated with anti-CD4 and CD8 mAbs and analyzed for EGFP expressing cells by fl ow cytometry to determine induction levels (A). Total CD4 and CD8 T cell numbers in PLN were calculated (B) and CD4 and CD8 T cells were analyzed for Annexin V (AnV) expression by fl ow cytometry to determine cell death (C). PLN derived CD4 and CD8 T cells were analyzed for membrane expression of CD25 (D), CD69 (E) and differential expression of CD44 and CD62L, percentages of naïve (CD44mediumCD62Lhigh) (F) and effector/memory (CD44highCD62Lhigh/CD44highCD62Llow) (G) T cells are indicated. CD4 and CD8 T cells derived from rtTA control mice were indicated with CD4-rtTA (r) and CD8-rtTA (p) and CD4 and CD8 T cells derived from rtTA/Nef transgenic mice were indicated with CD4-rtTA/Nef (m) and CD8-rtTA/Nef (l). Each symbol represents an individual mouse and dashes represent mean values. (*, p<0.05), using a two-tailed Students t test with equal variance. Shown is data of ten mice per group.

A B

E F

C D

G


six days of Nef expression effects on the activation state of T cells were not observed.

All together these data show a variable induction rate among double transgenic

mice after several days of dox administration, resulting in a slightly decreased CD8 T cell

number, without any effect on the activation status of T cells. So, apparently transient Nef

expression by itself did not lead to peripheral T cell activation.

Nef expression in thymocytes depletes all thymic subsets and results in reduced T

cell number in periphery

As infection of the thymus in HIV infected individuals has a great impact on thymic output (13,

18), we wanted to study the role of transient HIV-Nef expression on thymocyte development

in our inducible Nef transgenic mouse model. For this study we used our triple transgenic

CD2-rtTA-M2/β-actin-KTR/TetO7-Nef-huCD2TL mouse line, as this line had highest induction

levels in the thymus. After exposing the triple transgenic and control mice (CD2-rtTA-M2/β-

actin-KTR) with dox for 6 days, thymi were collected, and cell numbers were determined.

In contrast with the unaffected peripheral T cell compartment upon Nef induction,

in the thymus a 2-3 fold reduction of thymocyte numbers occurred after six days of Nef

induction (Figure 4A). To examine whether this reduction was specifi c to one thymic subset,

subset distribution in the thymus was studied. On the basis of CD4 and CD8 expression,

thymocytes can be divided in four subsets, as shown in fi gure 4B. The earliest stages are

CD4-CD8- thymocytes, (double negative (DN) thymocytes). After the DN stages thymocytes

proceed towards the CD4+CD8+, double positive (DP) stage where positive and negative

selection of the T cell receptor occurs. After proper selection, thymocytes either become CD4

or CD8 single positive (CD4SP, CD8SP) thymocytes, and subsequently leave the thymus and

enter the periphery.

Thymocytes were analyzed for CD4, CD8 and reporter expression by fl ow cytometry.

The different thymic subsets expressed reporter protein and no difference in distribution

among the different subsets in Nef expressing thymi was observed, compared to controls,

so there was no evidence for a specifi c decline of one thymic subset (Figure 4B). In addition,

absolute cell numbers of different thymic subsets demonstrated that a reduction in cell

number in Nef expressing transgenic mice was apparent in all thymocyte subsets (Figure

4C). When total number of Nef expressing thymocytes was depicted as a percentage of

control values (Figure 4D) the strongest reduction appeared to be among the DN and DP

subsets. The increased reduction in cell number correlated with the height of induction

levels, as DN and DP subsets showed the highest induction levels (Figure 4B). Annexin V

stainings were performed to see whether Nef expression led to increased cell death among

thymocytes, but no difference in the proportion of Annexin V+ thymocytes with and without

Nef was demonstrated (data not shown).

Since a decrease in the number of peripheral T cells upon Nef induction was

observed (Figure 3B), we investigated whether the observed reduction of peripheral T cells

CHAPTER 350

was the consequence of decreased thymic output. Therefore, for the different experiments,

thymocyte numbers were correlated with the peripheral T cell numbers. Firstly we correlated

the data obtained with the CD2-rtTA-M2/β-actin-KTR/TetO7-Nef-huCD2TL mouse line after

6 days of induction. As depicted in fi gure 4E, thymocyte numbers correlated tightly with

numbers of peripheral T cells. To further substantiate this fi nding we also correlated data

derived from CD2-rtTA/TetO7-Nef-EGFP transgenic mice after 3 days of induction (Figure

3), the same correlation was found with this data set. This suggested that Nef-mediated

reduction of thymic output resulted in a rapid decrease in T cell numbers in the periphery.

3 day-induction

6 day-induction

rtTA rtTA/Nef

0.08 0.11

0.26 0.08

13.1 30.4

17.1 5.1

% induction

CD4

CD

8

4.3

7.93.7

84.1

CD4

CD

8

3.9

12.83.3

80

% induction% induction% induction

**

rtTArtTA/Nef

0

25

50

75

100

thym

ocyte

s(x

10

6)

DN D

PCD4S

P

CD8S

P

** ** *

0

5

25

50

75

100

thym

ocyte

s(x

10

6)

DN D

PCD4S

P

CD8S

P0

25

50

75

100

%rt

TA

/Nef

thym

ocyte

so

fco

ntr

ol

0 50 100 150 200 2500

10

20

thymocytes (x106)

T-c

ell

s(x

10

6)

FIGURE 4. Nef expression in thymocytes depletes all thymic subsets and results in reduced T cell number in the periphery. CD2-rtTA-M2/β-actin-KTR /TetO7-Nef-huCD2TL (rtTA/Nef) and CD2-rtTA-M2/ β-actin-KTR (rtTA) transgenic mice were treated with dox (2mg/ml) for 6 days, where after thymi were removed and absolute cell numbers were determined (A). Single cell suspensions of thymocytes were incubated with antibodies directed against mouse CD4 and CD8 and human CD2 and were subsequently analyzed by fl ow cytometry. Dot plots indicating CD4 and CD8 expression are shown and percentage of human CD2 induction per thymic subsets is indicated in histograms for rtTA and rtTA/Nef transgenic mice. One representative example is shown (B). Absolute cell numbers per thymic subset were calculated (C) and values of rtTA/Nef transgenic mice were indicated as percentage of rtTA control values per thymic subset (D). rtTA and rtTA/Nef transgenic mice were fed with dox (2mg/ml) for three (m) and respectively six days (n), total thymus number was correlated to total T cell numbers in the PLN (E). Indicated are mean numbers ± SEM of a minimum of 6 mice per group, derived from 2 different experiments (*, p<0.01, **, p<0.001), p values were obtained using a two-tailed Students t test with equal variance.

A B

EC D


Thus short-term transient Nef expression in a fully developed thymus led to a rapid

reduction of thymocyte number in all different thymic subsets, implying a general effect of

Nef on thymocytes of all different subsets, which rapidly led to a decreased amount of T cells

in the periphery.

CHAPTER 352

DISCUSSION

HIV-mediated thymic impairment and hyper-activation of peripheral T cells are distinct

mechanisms believed to be involved in CD4 T cell depletion and progression to AIDS. Nef

has been implicated to play a major role in HIV pathogenesis. The effects of Nef reported

to date are not easily reconciled in a consistent model of AIDS pathogenesis. We addressed

the role of HIV-Nef with regard to HIV pathogenic mechanisms in a unique mouse model.

To be able to study the effects of Nef expression in a fully developed T cell compartment, an

inducible T cell lineage specifi c Nef transgenic mouse model was generated. We show that

after induction of Nef protein in peripheral T cells these cells retained a normal, overall naïve

phenotype, without enhanced expression of activation markers. Nef expression in the thymus

of these mice however, led to a rapid loss of thymocytes in all different subsets, resulting in

a decrease of peripheral T cell numbers. These data clearly demonstrate that Nef expression

in peripheral T cells does not directly lead to abnormalities in T cell activation. The observed

Nef-induced thymic depletion however, is compatible with an important role for Nef in the

HIV-mediated thymus depletion, leading to decreased thymic output in infected patients.

The fi nding that Nef expression in primary T cells does not lead to T cell activation

is in contrast with several studies showing that Nef expression in T cells enhances T cell

activation (25-27, 59). The use of different biological model systems might account for

these discrepancies. First, most studies that showed Nef-mediated activation of T cells were

performed in Jurkat cells, a tumour-T cell line which lacks the PTEN tumour suppressor

phosphatase, resulting in an overactive phosphatidylinositol 3-kinase (PI3K) in these cells

(60-62). PI3K is a positive component of a multitude of signalling pathways that promote

cell growth, proliferation, differentiations, motility and cytoskeletal organization, which likely

explains the continuous proliferation and activation state of Jurkat cells (62). As a key player

in TCR signalling, it was shown that Jurkat cells are hyperresponsive to TCR stimulation (60),

so activation of Jurkat T cells by Nef may be related to these intrinsic properties of Jurkat

cells.

Evidence for a role of Nef in activating primary T cells comes from studies with

transgenic mice with constitutive Nef expression showing that peripheral T cells that express

Nef had an activated phenotype (40, 44, 45). However, Nef expression also had great impact

on thymic output, consequently leading to a lymphopenic peripheral T cell compartment from

birth on (40-43). In this context we have shown that the T cell activation in a constitutive

Nef transgenic mouse model was lymphopenia-induced rather than a direct effect of Nef

expression in peripheral T cells (Koenen et al., submitted).

To our knowledge this is the fi rst study where Nef expression is induced in a

normally developed, overall naïve peripheral T cell compartment. Under these circumstances

Nef expression apparently did not have any effect on the activation status and TCR reactivity

of peripheral T cells.


Besides Nef involvement in T cell activation, we studied the effect of Nef expression

in a normally developed thymus. Already after 6 days of Nef protein expression, the thymus

size of transgenic mice declined two- to three-fold. This immediate effect of Nef expression

on thymus size is suggestive for Nef-mediated cell death of thymocytes. However, increased

fractions of cell in apoptosis could not be detected (data not shown). Nef-mediated

thymic atrophy was also observed in several transgenic mouse models with constitutive

Nef expression (40-43). In most models reduction in DP and SP thymocyte subsets was

reported. Whether Nef-mediated T cell reduction was specifi c for these stages or whether

Nef had a general effect on all thymocytes was however not addressed in these studies. In

one study, constitutive Nef expression on early thymocyte development was examined (45).

It was shown that Nef expression led to a partial block in early thymic development of a

specifi c (DN3) double negative stage. Thymocytes going through this early developmental

stage need proper rearranged TCR β-chains and signalling through a functional pre-TCR

complex (reviewed in (63, 64). It was proposed that the partial developmental block of Nef

transgenic thymocytes at this transition point was caused by Nef-mediated perturbation of

pre-TCR signalling. This idea was supported by the the observation that the introduction of a

constitutive active form of Lck overruled the Nef-mediated thymic block (45). To investigate

whether we could fi nd a similar block in development in our inducible Nef transgenic mice,

we studied the different DN stages. Although we found induction of the Nef transgene in

these early thymic subsets, no specifi c block was observed, but rather a general reduction

in cell number among the different DN subsets was seen (data not shown). This general

Nef-mediated reduction among different thymic subsets was not only seen in the different

DN stages but also observed in later stages of thymocytes development in our inducible Nef

transgenic mice (as shown in fi gure 4C). Using OP9-DL1 cells to support T cell development

in vitro, as described by Zuniga-Pfl ucker (65, 66), we have tried to study this thymocyte

decline in more detail. Although we were able to obtain high levels of transgene induction

in this in vitro system no differences in thymocyte development in the presence of dox was

observed. This observation suggested that, Nef-mediated effects in vivo are either overruled

in vitro or they do not play a role in in vitro thymocyte development.

Our results presented here, will add to our insight in the role of Nef during HIV

infection. We clearly show that Nef expression does not contribute to an activated T cell

compartment. The fact that not all T cells were induced to express Nef upon dox administration

corresponds well with the situation during HIV infection, where only a small proportion of

T cells is infected with HIV and expresses Nef. So, it seems that Nef expression does not

contribute to the observed hyper-activation characteristic for HIV infection as has been

suggested before (35). Although Nef expression did not infl uence the activation state of T

cells, a strong effect of Nef on T cell development was observed. This rapid Nef-mediated

decrease in thymocyte number is compatible with an important role of Nef in HIV-mediated

thymic decline in HIV infected patients.

CHAPTER 354


Construction of transgenic mice

HIV-1BRU

Nef (46, 47) cDNA was cloned as a NotI – NotI fragment into one cloning site of the pBI vector

(BD Biosciences, San Jose), in which a multimer of TetO elements is fl anked by the minimal CMV promoter

in two directions. The other cloning site of pBI was used to clone a reporter for gene expression, either

EGFP as a MluI – EcoRV fragment, extracted from pd2EGFP plasmid (BD Biosciences) or tailless human

CD2 as a MluI – EcoRV fragment, generated by PCR from human CD2 cDNA as described before (48). An

AseI – AatII fragment derived from pBI was injected into either C57Bl/6xCBA F1 or C57Bl/6 oocytes and

6 transgene positive founders were generated.

A CD2-rtTA-M2/β-actin-KTR inducer line was generated by co-injection of the improved rtTA

M2 (49), cloned into the EcoR1 and BamH1 sites of VA-CD2 (50) and the KRAB domain containing

tetracycline responsive transcriptional repressor controlled by the beta actin promoter (51) into C57Bl/

6xC3H F1 oocytes. CD2-rtTA-M2/β-actin-KTR mice were backcrossed for more than 10 generations onto

C57Bl/6.

In order to acquire inducible T cell lineage specifi c Nef expression, the pBI founder lines were either

crossed with CD2-rtTA (founder line C, a kind gift of Dr. R. Zamoyska) transgenic mice as earlier

described, containing the original rtTA (52) or with the CD2-rtTA-M2/β-actin-KTR transgenic line which

were generated by Flavell and coworkers as just described. The obtained double and triple transgenic

lines were screened for tissue specifi c and level of reporter expression upon treatment with 2mg/ml

doxycycline (dox, BD Biosciences) in drinking water containing 0.4% sucrose, which was changed freshly

every 2-3 days.

Two lines were selected for our experiments: the double transgenic CD2-rtTA/TetO7-Nef- EGFP

line with predominant induction in peripheral T cells and the triple transgenic CD2-rtTA-M2/β-actin-KTR

/TetO7-Nef-huTLCD2 with predominant expression in the thymus. Double and triple transgenic mice were

always compared with the rtTA controls, either with CD2-rtTA or with CD2-rtTA-M2/β-actin-KTR, which

were both treated with dox in drinking water to control for any rtTA and/or dox related effects. All mice

were kept under specifi c pathogen-free conditions and housed in accordance with institutional guidelines

of American Association of Accreditation of Laboratory Animal Care, in the mouse facility of the Central

Laboratory Animal Institute Utrecht University (Utrecht, the Netherlands). The Institutional Animal Ethics

Committee approved all experiments.

Flow cytometry

Thymus, spleen and lymph nodes were forced through cell strainers (Falcon, Becton Dickinson) in the

presence of RPMI 1640 with 10% FCS to obtain single cell suspensions. For blood analysis, 50μl blood

was drawn from the vena Saphaena. Erythrocytes were lysed by incubation of the cells in 0.15 M NH4Cl,

0.01 M KHCO3, 0.1 mM EDTA, pH 7.4, for 2 min on ice. Cells were preincubated with Fc-block (mAb to

CD16/32, 2.4G2; BD, Biosciences) and washed in staining buffer (PBS, 0.5% BSA, 0.01% sodium azide).

Afterwards cells were incubated with Annexin V, anti-mouse CD3 (145-2C11), CD4 (RM4-5), CD8 (53-

6.7), CD25 (PC 61), CD16/32 (2.4G2), CD44 (IM7), CD62L (MEL-14), anti-human CD2 (RPA-2.10) mAbs

or streptavidin, unlabeled or labelled with appropriate fl uorochromes (BD Biosciences). Stained cells were

analyzed using a FACSCalibur or BD LSR II and analyzed by CELLQuest or BD FACSDiva software (Becton

Dickinson).


Immunoprecipitation and western blot

Splenocytes derived from double transgenic mice stimulated with mAb aCD3 (PV1) in the presence or

absence of 1µg/ml dox for 2 days, were collected by centrifugation and lysed in 0.5% RIPA lysis buffer

(0.5% RIPA, 5 mM MgCl2 10% Glycerol) for 30 min at 4ºC. The lysate was clarifi ed by centrifugation

at 15,000 rpm for 10 min at 4ºC and the supernatant was incubated with antibody-absorbed beads.

This mixture was incubated for 2 ½ hours at 4ºC by end-over-end rotation. Immunoprecipitates were

washed 3 times with wash buffer. Precipitates were resuspended in reducing Laemmli sample buffer and

analyzed by SDS-PAGE and western blotting. After SDS-PAGE, proteins were transferred to Nitrocellulose

Transfer Membranes (Whatman GmbH, Dassel, Germany), whereafter membranes were blocked with 5%

low fat milk powder in PBS for 1 hour at RT. For Nef detection the membranes were probed with sheep

polyclonal anti-Nef for 1 hour at RT followed by incubation for 1hour with Donkey-anti-Sheep IgG (H+L)-

HRP (Pierce, Rockford, IL). After extensive washing (3 x 5 min) in PBST (PBS, 0.05% tween-20), bound

antibodies were detected by chemiluminescence (Amersham Biosciences, Freiburg, Germany).

In case of the positive and negative control, 10 cm dishes of 293-T cells were transfected

(using CaPO4) with a HIV-1

SF2 Nef or mock expressing plasmids respectively. After 48h, cells were lysed

(see above) and 100 μg of total cellular protein was subjected to immunoprecipitation with the polyclonal

sheep anti-Nef antibody. Precipitates were treated as described above. The size of NefSF2

is 25kD whereas

the NefBRU

used in our transgenic system is 27kD.




ACKNOWLEDGEMENTS

We acknowledge F. Miedema for critically reading the manuscript. We thank Dr. R. Zamoyska

for kindly providing CD2-rtTA mice and human CD2 cDNA.

CHAPTER 356

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The Two Faces of Nef:Opposing in vivo Functional Effects of Nef Expression in Murine T Cells Compared to Dendritic Cells

4

Paul G.F. Koenen, Frans M. Hofhuis, Elise H.R. Schrijver, Mariëtte A. Oosterwegel, Kiki Tesselaar


Manuscript in preperation

CHAPTER

CHAPTER 462

ABSTRACT

HIV infected DCs have been shown to play an important role in stimulating viral replication

in CD4 T cells. Since Nef has been implicated as a crucial factor for high viral replication

and pathogenicity in HIV infection, we have investigated the functional outcome of Nef

expression in APCs and T cells in vivo. By the use of APC and T cell specifi c inducible Nef

transgenic mice models we show that Nef expressing T cells are less capable in forming anti-

viral T cell responses then wild-type T cells. This Nef disturbed CD4 and CD8 T cell function

is only observed after cognate antigen stimulation of the TCR in vivo, pointing towards Nef-

mediated disturbed TCR signalling in vivo. In contrast to Nef-mediated effects in T cells,

Nef expression in APCs during infl uenza infections resulted in heightened anti-viral CD8 T

cell responses, suggesting a stimulating role on T cell activation via APCs. These opposing

effects might explain how HIV-1 Nef may optimize viral spread in the infected host.

The two faces of Nef 63

INTRODUCTION

The regulatory viral protein Nef, expressed in HIV and SIV is an important factor in determining

viral replication and the pathogenicity of disease. This capacity is displayed by several long-

term- non-progressors, who remained asymptomatic for more than 10 years, maintained

low viral loads and stable CD4 T cell counts and appeared to be infected with Nef- deleted

HIV-1 variants (1, 2). Further in vivo evidence comes from experimental infection of rhesus

monkeys with Nef-deleted SIV variants. This infection is characterized by low levels of

viral replication and a strongly attenuated clinical course of infection (3, 4). Although the

mechanisms underlying Nefs pathogenic role during HIV and SIV infections remains unclear,

its importance for viral replication in T cells and DC-T cell co-cultures (5-12) might play an

important role.

In DC-T cell co-cultures viral replication occurs in conjugates between DCs and T

cells and is much higher than in isolated T cell cultures (6-12). Nef has been suggested to

promote conjugate formation and by this mean generate effi cient viral replication for both

SIV and HIV (12, 13). This effect is most apparent in cultures of immature DCs with T cells

(12-14). HIV replication in co-cultures of T cells with mature DCs or, superantigen activated

immature DC-T cell cultures leads to comparable replication of Nef defi cient and wild-type

virus (12, 13). Nef-mediated maturation of immature DCs and a consequential increased

T cell priming and viral replication has been proposed as mechanisms to explain these

fi ndings.

Concomitant with this mechanism, Nef-mediated upregulation of DC-SIGN on DCs

in vitro has been shown (15). DC-SIGN is a C type lectin, which is specifi cally expressed on

DCs and facilitates contact of DCs with T cells through binding with ICAM-3. It was shown

that the Nef-mediated upregulation of DC-SIGN promotes T cell-DC clustering and viral

spread (15). Nef-mediated DC-SIGN upregulation could however not be confi rmed by some

investigators (16, 17). Other studies suggested that besides direct cell-cell contact between

APCs and T cells, Nef mediates cytokine and chemokine production of infected macrophages

or DCs and thereby T cell activation (16, 18, 19).

Next to these DC and macrophage dependent, indirect ways of Nef to

promote T cell activation and viral production, Nef expression in T cells is also reported

to enhance T cell activation directly (20-23). Multiple interactions of Nef with molecules

involved in TCR signaling events have been described (for review see (20)). Two recent

studies showed effects of Nef on the threshold of T cell activation, either by infl uencing

formation of the immunological synapse or by down regulation of the TCR-CD3 complex (24,

25).

The relevance of in vitro obtained effects of Nef has been tested in different in

vivo mouse models. Hanna et al. generated a mouse model in which Nef is constitutively

expressed in CD4 expressing cells, i.e. thymocytes, CD4 T cells and cells of the monocyte/

CHAPTER 464

macrophage lineage (26). These mice suffer from a severe AIDS-like pathology which,

unexpectedly, does not depend on CD4 T cells (27). This lack of CD4 T cell involvement,

points to a crucial role for Nef in monocytes and macrophages in the induction of this AIDS-

like pathology. However, due to the constitutive expression of Nef, disturbed myelopoiesis

and lymphopoiesis should be considered in the interpretation of this model.

Former studies in our laboratory showed that constitutive Nef expression in the T

cell lineage leads to thymic atrophy and lymphopenia induced activation and proliferation

of T cells, rather than to Nef-mediated activation of peripheral T cells (Koenen et al.,

manuscript in preperation), as was suggested before (20, 27-29). We confi rmed these

fi ndings using inducible Nef transgenic mice. In these mice induction of Nef expression in a

full T cell compartment leads to rapid thymic atrophy but not to any T cell activation of the

normal naïve T cell compartment (Koenen et al., submitted), highlighting the drawbacks of

a constitutive transgenic model.

To study functional consequences of Nef expression in vivo we have extended our

analyses to transgenic mice with inducible Nef expression in APCs and/or T cells and have

analysed T cell responses after challenges with infl uenza virus. We show that mice with

Nef expression in both the T cell lineage and in APCs were less capable of generating an

infl uenza specifi c CD8 T cell response. Further analysis revealed that in this model Nef

negatively affected both CD4 and CD8 T cell function but not APC function. On the contrary,

we here show that restricted Nef expression in APCs resulted in higher T cell responses.

Thus, inducible Nef expression in APCs and T cells revealed functional evidence for disturbed

TCR signalling in T cells and for increased T cell activation due to Nef expression in APCs in

vivo.


RESULTS

Reduced virus specifi c T cell responses in mice expressing Nef in APCs and T cells

To study the infl uence of Nef expression on APC and T cell function in vivo, we generated

double transgenic mice in which Nef expression could be transiently induced in T cells

and APCs. We used the tetracycline (tet)-dependent transgene expression system that

has been described previously as the “tet-on” system (37-39). In this double transgenic

system expression of a target gene is dependent on binding of a tet-sensitive transactivator

to regulatory sequences upstream of the target gene. The transactivator will only bind

in the presence of doxycycline (dox), a derivate of tet, and thereby activate target gene

transcription (37-39). To obtain APC and T cell restricted Nef expression we used the earlier

described Ii-rtTA transgenic line (30), in which protein expression is under the control of the

Eα enhancer and Ii promoter and thereby drives gene expression in MHC II positive cells.

This transgenic line was crossed with our previously described Nef-huCD2TL responder line

(Koenen et al., manuscript in preperation), which drives concomitant expression of Nef, and

tailless human CD2 as a reporter of induction.

To test APCs and T cell specifi c induction Ii-rtTA and Ii-rtTA/TetO7-Nef-huCD2TL

(Ii-rtTA/Nef) double transgenic mice were fed with dox for 6 days, draining lymph nodes

(DLNs) were collected and macrophages (defi ned on basis of CD11b expression), dendritic

Ii-rtTA

Ii-rtTA/Nef

**

*

*

Ii-rtTA

Ii-rtTA/Nef

CD11bCD11c

CD19CD4

CD8

dox

Day 0

intranasal fluinfection

blood analysis

Ii-rtTA

Ii-rtTA/Nef

dox

Day 1 Day 9,11,13

dox

0

5

10

15

203550

%in

du

ced

cell

s

6 8 10 120.0

2.5

5.0

7.5

days post infection

%te

tr+

CD

8T

-cell

s

FIGURE 1. Nef expression in APCs and T cells leads to reduced virus specifi c CD8 T cell responses in mice. Ii-rtTA and Ii-rtTA/TetO7-Nef-huCD2TL (Ii-rtTA/Nef) transgenic mice were treated with 2 mg/ml doxycycline (dox)-containing drinking water for six days. Splenocytes were analyzed by fl ow cytometry for tailless human CD2 reporter expression on different cell subsets distinguished on basis of CD11b, CD11c, CD19, CD4 or CD8 expression (A). Ii-rtTA and Ii-rtTA/Nef transgenic mice were fed with 2 mg/ml dox-containing drinking water during the experiment, one day after start of dox treatment mice were infected intranasally with infl uenza virus. On the indicated days, blood was drawn and analyzed (B). Blood was stained with anti-CD8 mAb and NP

366-374/ H-2Db tetramers and

analyzed by fl ow cytometry (C). Indicated are mean numbers ± SD of 6 mice (*, p<0.05, **, p<0.01), using a two-tailed Students t test with equal variance. Shown is a representative of two independent experiments.

A B

C

CHAPTER 466

cells (DCs, CD11c), B-cells (CD19) and CD4 and CD8 T cells were analyzed for human CD2

reporter expression. Induction of CD2 reporter expression was clearly shown in a proportion

of all cell types (Figure 1A).

To determine functional effects of Nef expression on DC mediated T cell priming,

we analyzed the immune response after intranasal infection with infl uenza virus (strain A/

HK/2/68). In this model system, infl uenza virus causes a localized infection in the respiratory

tract which is characterized by an infl ux of both CD4 and CD8 effector T cells into the lung

(40). The CD8 T cell response can be monitored with H-2Db MHC class I tetramers, loaded

with NP366-374

(ASNENMDAM) peptide.

The dynamics of the virus-specifi c T cell response were studied in Ii-rtTA and Ii-

rtTA/Nef double transgenic mice which were fed with dox during the course of the experiment

and infected with infl uenza virus one day after start of dox treatment (for the outline of the

experiment see Figure 1B). Blood analysis revealed decreased percentages of virus specifi c

CD8 T cells at day 8,10 and 12 after infection in mice expressing Nef, amounting upto a four-

fold reduction at day 8 (Figure 1C). Mice sacrifi ced at day 10 after virus infection contained

decreased numbers of virus specifi c T cells in the lung, but not in the DLN or spleen (data not

shown). So, concomitant Nef expression in APCs and T cells alters specifi c CD8 T response

to infl uenza virus leading to reduced fl u specifi c T cell in the periphery.

It has been previously observed, that inducible gene expression in vivo can lead

to CTL responses towards the newly produced protein and subsequent cytolysis of the cells

expressing the induced protein. In order to rule out that the observed effects were mediated

by Nef specifi c CTLs, lung suspensions were stimulated with Nef peptide pools to test for

IFN-γ producing Nef- specifi c CTLs. Analysis of lung suspensions of infl uenza challenged Ii-

rtTA/Nef double transgenic mice at day 10 after infection indeed showed Nef specifi c CTLs.

A possible infl uence of the Nef specifi c CTLs on the anti-viral response was examined by

analyzing the relation between the percentage of Nef specifi c CTLs and the percentage of

fl u-specifi c CD8 T cells per mouse. High Nef specifi c CTL responses correlated with high fl u-

specifi c viral responses (data not shown). This rendered the possibility that the reduction

in virus specifi c T cells in Nef expressing mice could be explained by the presence of Nef

specifi c CTLs unlikely. So these data indicated an intrinsic effect of Nef on APCs and/or T cells

during an anti-viral immune response.

Nef expression in B and T cells leads to reduced virus specifi c T cell responses in

mice

In order to dissect whether Nef-mediated reduction of anti-viral CD8 T cells upon infl uenza

infection was mediated by Nef expression in APCs, in T cells or both, we used the previously

described CD2-rtTA-M2/β-actin-KTR/TetO7-Nef-huCD2TL triple transgenic mouse line

(Koenen et al., manuscript in preperation). These transgenic mice, express Nef in thymocytes,

T cells and B cells upon induction but not in DCs. The approach was similar to the fi rst


experiment except for the length of dox administration (Figure 2A).

In this model system at day 10 after viral infection, as much as 24% of CD8 T

cells present in the lung were directed against the ASNENMDAM epitope in control mice. In

the DLN and the spleen this was respectively 1.4% and 8.7%. Double transgenic animals

however, showed a two-fold decrease in the percentage of virus specifi c CD8 T cells in

lung and DLN and a three-fold decrease in the spleen when compared to control animals

(Figure 2B). Reduced numbers of virus specifi c CD8 T cells in Nef expressing mice was

confi rmed by a two-fold reduction in peptide specifi c IFN-γ producing CD8 T cells in the

lung suspensions of Nef expressing mice compared to controls (Figure 2C). Thus, anti-

viral responses towards the dominant fl u epitope ASNENMDAM were diminished upon Nef

expression in T and B cells. In order to extend this fi nding to the total anti-viral response,

total infl uenza specifi c CD4 and CD8 T cell responses were investigated. Firstly, total CD4

and CD8 T cell numbers were determined in the lung to examine possible difference of T cell

infl ux upon infection. No differences were found in T cell number in the lung. Secondly, to

study T cell phenotype, lung suspensions were stained with mAb directed against CD44 and

CD62L to distinguish between naïve (CD44medium CD62Lhigh), memory (CD44high CD62Lhigh) and

effector (CD44highCD62Llow) T cells (41, 42). As anti-viral T cells present in the lung at day

CD2-rtTA

CD2-rtTA/Nef

Lung DLN Spleen

24.1% 1.4%

12.7%

8.7%

0.7% 2.9%tetr

am

er

CD8

dox

Day 0


sacrifice

CD2-rtTA

CD2-rtTA/Nef

dox

Day 1 Day 6 Day 11

dox

stop doxtreatment

*

CD2-rtTA

CD2-rtTA/Nef

0

10

20

%IF

N-?

+C

D8

T-c

ell

s

* *

0

25

50

75

100

CD4 CD8

%eff

ecto

rT

-cell

s FIGURE 2. Nef expression in B and T cells leads to reduced virus specifi c T cell responses in mice. Schematic view of experimental setup for infl uenza infections in CD2-rtTA-M2/β-actin-KTR and CD2-rtTA-M2/ β-actin-KTR/TetO7-Nef-huCD2TL transgenic mice. The same protocol was followed as described in Figure 1, only in this experiment, dox was supplied for six days and organs were collected at day 10 after infection (A). Cells at day 10 after infection were isolated from the lung,

DLN, and spleen. Cell suspensions were stained with anti-CD8 mAb and NP366-374

/ H-2Db tetramers and analyzed by fl ow cytometry. Indicated are percentages of tetramer positive cells within the CD8 T cells present in the indicated organs, shown is a representative example of 6 mice per group (B). Cells derived from the lung were stimulated for 4h with 1µg/ml NP

366-374 peptide and analyzed for expression of CD8

and IFN-γ by fl ow cytometry (C). Lung derived cells were stained with anti-CD4, CD8, CD44 and CD62L mAbs and proportion of effector T cells (CD44highCD62Llow) was determined by fl ow cytometry (D). Each symbol represents an individual mouse (at least fi ve per group) and dashes represent mean values. (*, p<0.05), using a two-tailed Students t test with equal variance. Shown is a representative of two independent experiments.

A B

C D

CHAPTER 468

10 of the anti-infl uenza response will be of the effector type, the percentage of effector CD4

and CD8 T cells may provide a measure for effi ciency of the total T cell response. We found

that the proportion of effector CD4 and CD8 T cells was signifi cantly reduced in lungs of the

inducible Nef transgenic mice compared to controls (Figure 2D). This suggest that not only

the ASNENMDAM specifi c but also the total anti-infl uenza T cell response is declined upon

Nef expression in T and B cells.

In short, Nef expression in T and B cells leads to reduced anti-viral responses of

CD4 as well as CD8 T cells. Since DCs and not B cells are implicated as APCs in primary CTL

responses to a virus, this suggests a role for Nef in T cells during anti-infl uenza responses.

Nef expression in APCs increases ant-viral CTL responses

We next addressed the question whether the reduced virus specifi c T cell response

was caused by a Nef effect in T cells or was brought about by Nef expression in APCs,

either DCs or B cells. The possible infl uence of Nef expressing APCs on a T cell response

was tested in two independent mice models. We adoptively transferred either CFSE labeled

OT-I (31) or non-labeled F5 TCR transgenic T cells (43) into Ii-rtTA and Ii-rtTA/TetO7-Nef-

huCD2TL mice. Two days after transfer of the cells, mice were either infected with the

WSN-I or the A/HK/2/68 infl uenza strain. The WSN-I virus is modifi ed in such a way that it

expresses the OVA epitope which is recognized by the TCR of CD8 OT-I transgenic T cells

(36). The F5 transgenic TCR recognizes the ASNENMDAM epitope, which is presented upon

A/HK/2/68 infection. WSN-I infected mice were analyzed 4 days post infection to examine

early responses whereas the A/HK/2/68 infected mice were analyzed 8 days post infection

to examine the established response (Figure 1A).

Lungs, DLNs and spleens from WSN-I infected mice were collected and single cell

suspensions were stained with anti-CD8, Vβ5 and CD25 mAbs. On basis of Vβ5 expression,

CFSE signal and CD25 expression, OT-I transgenic CD8 T cells could be distinguished from

endogenous cells by fl ow cytometry. To examine the response of CD8 OT-I T cells in the

different organs of Nef expressing versus control mice, CFSE dilution profi les of CD8 OT-I T

cells were examined. As expected at this time after infection, control mice showed the most

vigorous proliferation in the DLNs, only 3% of cells had not divided (Figure 3B). Of the OT-I

T cells derived from lung and spleen of control mice 42-43% had not divided. CD8 OT-I T

cells derived from Nef expressing mice showed a similar CFSE profi le in DLN and spleen.

However, CD8 OT-I T cells drived from the lungs of Nef expressing mice, showed increased

proliferation, compared to controls. Only 14% of these cells had not divided, showing a three

fold decrease in undivided cells compared to control animals (Figure 3B). To examine if this

increased level of proliferated CD8 OT-I cells in Nef expressing mice had also resulted in

increased T cell numbers in the lung, total CD8 OT-I T cell numbers were determined in all

organs of both groups of mice. Indeed a signifi cantly higher number of CD8 OT-I T cells was

observed in the lungs of Nef expressing mice (Figure 3C).


A/HK/2/68 infected mice were sacrifi ced at day 8 post infection, organs were

removed and single cell suspensions were stained with anti-CD8, Vβ11 and earlier described

tetramers. On basis of Vβ11 expression and tetramer binding, F5 transgenic CD8 T cells

could be distinguished from endogenous cells by fl ow cytometry. Total CD8 F5 T cell numbers

were determined in all organs and are shown in Figure 3D. No difference of cell numbers

were found in the lungs, but in the DLN and spleen an increased cell number was found in

Nef expressing mice (not signifi cant for DLN , p<0.05 for spleen). This fi nding substantiated

the Nef-mediated increased viral response when expressed in APCs.

Altogether this shows that Nef-mediated decreased anti-viral T cell response was

not due to Nef expression in APCs. To the contrary, expression of Nef in APCs resulted in

increased T cell responses, pointing towards a potentiation of T cell activation.

Lymphopenia induced proliferation of T cells is not infl uenced by Nef expression

Our experiments suggested that the reduced anti-viral T cell response was mediated by Nef

expression in T cells. Since Nef has been described to act on TCR signaling we next analyzed

spontaneous T cell proliferation in a lymphopenic environment. Lymphopenia driven

proliferation is proposed to dependent on two mechanisms. Homeostatic proliferation, which

is slow and depends on IL-7 and spontaneous proliferation which is fast and depends on

*

F5 T-cells

Day 10

3%

2%14%

43% 42%

43%

DLNLung Spleen

Ii-rtTA

Ii-rtTA/Nef

even

ts

CFSE

dox

Day 0


sacrifice

Ii-rtTA

Ii-rtTA/Nef

dox

Day 2 Day 6,10

iv lymphocytes:- F5 or OT-I tg

dox

*Ii-rtTA

Ii-rtTA/Nef

Day 6

OT-I T-cells

0

1

2

3

4

Lung

CD

8+

OT

-IT

-cell

s(x

10

4)

0.0

0.5

1.0

1.5

DLN Spleen

CD

8+

OT

-IT

-cell

s(x

10

6)

0

11

2

3

4

Lung DLN

CD

8+

F5

T-c

ell

s(x

10

6)

0

1

2

3

4

Spleen

CD

8+

F5

T-c

ell

s(x

10

6)

FIGURE 3. Increased virus specifi c T cell responses upon Nef expression in APCs. Ii-rtTA and Ii-rtTA/TetO7-Nef-huCD2TL (Ii-rtTA/Nef) transgenic mice were fed with 2 mg/ml dox-containing drinking water for six days. Mice were either i.v. injected with 107 CFSE labelled OT-I or with unlabeled F5 TCR transgenic lymphocytes. Two days later OT-I injected mice were infected with WSN-I virus, while F5 injected mice were infected with A/HK/2/68 virus. Organs were harvested four and eight days post infection and CD8 OT-I or F5 transgenic T cells were analyzed respectively (A). Four days post infection OT-I injected mice were analyzed. CFSE profi les of CD8 OT-I T cells either derived from Ii-rtTA or Ii-rtTA/Nef transgenic mice were analyzed by fl ow cytometry. Indicated are proportions of undivided cells. A representative example of fi ve mice per group is shown (B). Total CD8 OT-I T cell numbers were calculated for lung, DLN and spleen (C). Eight days post infection F5 injected mice were analyzed. Total CD8 F5 T cell numbers were calculated for lung, DLN and spleen (D). Each symbol represents an individual mouse (at least fi ve per group) and dashes represent mean values. (*, p<0.05), using a two-tailed Students t test with equal variance. One experiment is shown.

A B

DC

CHAPTER 470

competition of TCRs for stimulatory self-peptide/MHC interactions (44, 45).

To measure the infl uence of Nef in this system we intravenously co-injected 107

CFSE labeled Nef inducible (Ly5.2, CD2-rtTA-M2/β-actin-KTR/TetO7-Nef-huCD2TL) and wild-

type (Ly5.1, C57B/6) T cells in a 1:1 ratio into T cell deplete, Rag 2 defi cient mice. The use

of the allelic marker Ly5 enabled us to distinguish between the different T cells. One group

of Rag 2 -/- mice was fed with dox throughout the experiment, whereas another group of

Rag 2 -/- mice received normal drinking water (Figure 4A). Six days after injection, DLN and

spleens of Rag 2-/- mice were harvested and analyzed. Firstly, reporter protein induction

levels on Ly5.2 CD4 and CD8 T cells were analyzed and all dox treated Ly5.2 T cells showed

increased reporter expression (Figure 4B), indicating the presence of Nef as was shown

before (Koenen et al., manuscript in preperation). Secondly, CFSE dilution profi les were

analyzed and showed proliferation of both CD4 and CD8 T cells. No difference in profi les

could be observed among wild-type and Nef inducible T cells within one mouse, either in the

presence or absence of induction (data not shown). To determine if these similar CFSE profi les

were also refl ected in total T cell number, transgenic (Ly5.2) CD4 and CD8 T cell numbers

were calculated per 105 Ly5.1 CD4 and CD8 T cells respectively. When Nef interferes with

- dox

+ dox

CD4 T-cells CD8 T-cells

huCD2TL

even

ts

- dox+ dox

* **- dox

+ dox

CD25

even

ts

- dox+ dox

- dox

+ dox

CD69

even

ts

- dox+ dox

- dox

+ dox

dox

Day 0

sacrifice

Rag 2 -/-

dox

Day 2 Day 8

Rag 2 -/-

- Ly5.1 Wt- Ly5.2 CD2-rtTA/Nef

iv lymphocytes:

0.00

0.05

0.10

0.15

0.20

CD4 CD8

#L

y5.2

Tcell

s/

10

5

Ly5.1

T-c

ell

s(x

10

6)

0

100

200

300

400

CD4 CD8

MF

Ih

uC

D2T

L

0

10

20

30

CD4 CD8

%C

D25+

T-c

ell

s

0

10

20

30

40

CD4 CD8

%C

D69+

T-c

ell

s

FIGURE 4. Lymphopenia induced proliferation of T cells is not infl uenced by Nef expression. Rag 2 -/- mice were either fed with regular or 2 mg/ml dox-containing drinking water. Two days later a mixture of CFSE labelled C57Bl/6 Ly5.1 and CD2-rtTA-M2/β-actin-KTR/TetO7-Nef-huCD2TL transgenic (Ly5.2) derived lymphocytes in a ratio of 1:1 was injected iv. Five days after injection DLN were harvested and cells were analyzed (A). Cells were stained with anti-mouse CD4, CD8, Ly5.1, Ly5.2 and anti-human CD2 mAbs, tailless human CD2 levels on transferred Ly5.2 T cells was determined as a measure for induction (B). Subsequently, transgenic (Ly5.2) CD4 and CD8 T cell numbers were calculated per 105 Ly5.1 CD4 and CD8 T cells in animals treated with and without dox (C). Cells derived from DLN were incubated with anti-CD4, CD8, Ly5.1, Ly5.2, CD25 and CD69 mAbs. CD25 and CD69 expression on CD4 and CD8 Ly5.2 derived T cells either treated or untreated with dox were measured by fl ow cytometry (D and E). Each symbol represents an individual mouse (fi ve per group) and dashes represent mean values. (*, p<0.05, **, p<0.001), using a two-tailed Students t test with equal variance. Shown is a representative of two independent experiments.

A B

DC

E


spontaneous proliferation one would expect to fi nd a difference between the dox treated, Nef

expressing, and untreated, control group. However no differences were found between the

treated and untreated group (Figure 4C). We then analyzed the expression of the activation

markers CD25 (Figure 4D) and CD69 (Figure 4E), which are upregulated in lymphopenic

situations. No differences between Nef expressing T cells and control T cells were found.

To conclude, Nef expressing CD4 and CD8 T cells in lymphopenic situations function

as normal T cells, and no effect of Nef on lymphopenia driven proliferation was observed.

Nef increases IFN-γ production during early anti-viral response of CD8 T cells

Nef expression did not seem to affect proliferation of T cells under lymphopenic conditions.

As signals stimulating proliferation of T cells under lymphopenic conditions are qualitatively

different from signals stimulating T cells to undergo cognate antigen-driven proliferation

(46), we further investigated the effect of Nef expression on antigen driven proliferation of

*

Ly5.2

Ly5.1

**

rtTA/OT-I

rtTA/Nef/OT-I

dox

Day 0

sacrifice

dox

Day 2 Day 5

dox

intranasal WSN-Iinfection

- Ly5.1 Wt- Ly5.2 rtTA/OT-I

iv lymphocytes:

- Ly5.1 Wt- Ly5.2 rtTA/Nef/OT-I

Bl/ 5.1/5.26 Ly

Bl/ 5.1/5.26 Ly

rtTA/OT-I rtTA/Nef/OT-I

76% 71%

CFSE

ev

en

ts

0

10

20

30

40

%in

du

ced

Ly5.2

-CD

8+

T-c

ell

s

0

25

50

75

100

%C

D25+

Ly5.2

-CD

8+

T-c

ell

s

0

25

50

75

100

%C

D69+

Ly5.2

-CD

8+

T-c

ell

s

0

10

20

%IF

N-?

+L

y5.2

-CD

8+

T-c

ell

s

FIGURE 5. IFN-γ production increased during early anti-viral response of CD8 T cells upon Nef expression. C57Bl/6 Ly5.1/5.2 host mice were injected with CFSE labeled C57Bl/6 Ly5.1 lymphocytes in a 1:1 ratio with either CFSE labeled CD2-rtTA-M2/β-actin-KTR/OT-I (rtTA/OT-I) or CD2-rtTA-M2/β-actin-KTR/TetO7-Nef-huCD2TL/OT-I (rtTA/Nef/OT-I) transgenic lymphocytes. Subsequently mice were fed with 2 mg/ml dox-containing drinking water. Two days after lymphocyte transfer mice were intranasally infected with WSN-I infl uenza virus. Cells were isolated from the DLNs at day 3 after infection (A). Obtained cell suspensions were stained with anti mouse-CD8, Ly5.1, Ly5.2 CD25, CD69 and anti-human

CD2 mAbs and were analyzed by fl ow cytometry. An example of Ly5.1 and Ly5.2 stained DLN derived cell suspension is shown (B). Ly5.2 CD8 T cells were examined for expression of human CD2 (C), CD25 and CD69 by fl ow cytometry (D). CFSE profi les from rtTA/OT-I or rtTA/Nef/OT-I transgenic Ly5.2 CD8 T cells were analyzed by fl ow cytometry, indicated are percentages of undivided cells (E). Cells derived from DLN were stimulated for 4h with 1µg/ml OVA

257-264 (SIINFEKL) peptide and analyzed for expression of

Ly5.2, CD8 and IFN-γ by fl ow cytometry (F). Each symbol represents an individual mouse (six per group) and dashes represent mean values. (*, p<0.05, **, p<0.001)), using a two-tailed Students t test with equal variance. One experiment is shown.

A B

D

C

E

F

CHAPTER 472

CD8 T cells. To this extend we generated CD2-rtTA-M2/β-actin-KTR/TetO7-Nef-huCD2TL, OT-

I TCR transgenic mice, of which we used the CD8 T cells in adoptive transfer experiments. In

these experiments CSFE labeled CD2-rtTA-M2/β-actin-KTR/OT-I/Ly5.2 (rtTA/OT-I ) or CD2-

rtTA-M2/β-actin-KTR/TetO7-Nef-huCD2TL /OT-I/Ly 5.2 (rtTA/Nef/OT-I) derived lymphocytes

were co-injected intravenously in a 1:1 ratio with wild-type Ly5.2 cells into Ly5.1/5.2

recipient mice which were fed with dox as indicated (Figure 5A). On the basis of differential

Ly5 expression, host cells, expressing both Ly5.1 and Ly5.2 could be distinguished from

wild-type C57B/6 Ly5.1 and either rtTA/OT-I or rtTA/Nef/OT-I transgenic Ly5.2 cells (Figure

5B).

Two days after adoptive transfer of cell suspensions, mice were infected with the

earlier described WSN-I infl uenza virus to trigger OT-I TCR transgenic CD8 T cells. In order

to study the early priming of the CD8 T cell response, DLNs of infected mice were harvested

and Ly5.2 CD8 T cells were analyzed three days after infection by fl ow cytometry. The

reporter induction level (huCD2TL) of Ly5.2 CD8 T cells expressing Nef was approximately

25% compared to no induction in Ly5.2 CD8 control T cells (Figure 5C). Next T cell activation,

proliferation and function were analyzed, by determination of CD25 and CD69 expression

levels, CFSE dilution profi les and levels of IFN-γ production respectively. As expected at

this time in the response CD69 and CD25 were highly upregulated on the OT-1 cells and

cells started to proliferate. Expression levels of these activation markers were the same

among Nef+ and Nef- CD8 T cells (Figure 5D). Approximately 30 % of the OT-I cells had

undergone multiple rounds of divisions. However, no difference between Nef expressing cells

and control cells was found regarding cell division (Figure 5E). No response to the virus of

wild-type CD8 Ly5.1 control T cells was observed which underscores the antigen specifi c

response of OT-I transgenic cells. To examine the ex vivo IFN-γ response of Ly5.2 OT-I CD8

T cells, DLN suspensions were stimulated with the SIINFEKL peptide, recognized by the

OT-I TCR, and IFN-γ production by CD8 T cells was measured intracellulary. Surprisingly,

an increased IFN-γ response was found in the Nef expressing CD8 OT-I cells compared to

control CD8 OT-I cells (Figure 5F).

Thus, we showed in this experiment that Nef expression in CD8 T cells during the

initiation of an infl uenza response did not result in different activation or proliferation of CD8

T cells. However, IFN-γ responses were increased in Nef expressing CD8 T cells.

Reduced Antigen mediated clonal expansion of CD4 T cells expressing Nef

To address the question whether Nef also affects CD4 T cells upon triggering with

their cognate antigen, we used a similar approach as for the CD8 T cells. Only in this case

CD2-rtTA-M2/β-actin-KTR/TetO7-Nef-huCD2TL transgenic mice were bred with OT-II TCR

transgenic mice (32) and recipient mice were challenged with ovalbumin together with alum

as an adjuvant to trigger CD4 T cell activation. Furthermore, both CD2-rtTA-M2/β-actin-KTR

(rtTA/OT-II) and CD2-rtTA-M2/β-actin-KTR /TetO7-Nef-huCD2TL /OT-II (rtTA/Nef/OT-II)


transgenic CD4 T cells were studied in the presence and absence of dox (Figure 6A).

Four days after antigen challenge mice were sacrifi ced and cells were harvested

from peripheral lymphoid organs. On basis of differential Ly5 expression, wild-type Ly5.1

cells could be distinguished from either rtTA/OT-II/Ly5.2 or rtTA/Nef/OT-II/Ly5.2 transgenic

cells. Analysis of CFSE dilution profi les by fl ow cytometry showed that Ly5.2 CD4 T cells

had divided upon ovalbumin-alum challenge, whereas the Ly5.1 derived CD4 T cells had

not (Figure 6B). The groups of mice injected with rtTA/OT-II derived T cells were studied as

controls, as no effect of dox was observed only data of CD4 rtTA/Nef/OT-II/Ly5.2 transgenic

T cells in the presence or absence of dox will be presented.

Induction levels of CD4 T cells were determined. Approximately 30% of CD4 T

cells were induced in the presence of dox compared to almost no induction of CD4 T cells

in the absence of dox (Figure 6C). To analyze proliferation rates upon antigen challenge,

CFSE profi les were compared between dox treated and untreated CD4 T cells. To allow for

quantitative comparison of CFSE profi les the analysis was correlated to Ly5.1 non responder

CD4 T cells. Accordingly, CFSE profi les of cells treated with and without dox could be compared

(Figure 6D). Interestingly, this approach highlighted that both populations act similarly

regarding cell cycle entry. However, in all division cycles, fewer cells had accumulated in the

presence of Nef. Examination of total numbers of OT-II transgenic CD4 T cells confi rmed this

*

- dox+ dox

Ly5.2

CF

SE

CFSE

even

ts

- dox

+ dox

dox

Day 0

ip OVA-ALUM sacrifice

Bl/6 Ly5.1/5.2

Day 2 Day 6

iv lymphocytes:

- Ly5.1 wt- Ly5.2 rtTA/OT-II

- Ly5.1 wt- Ly5.2 rtTA/Nef/OT-II

Bl/6 Ly5.1/5.2

dox

dox

0

25

50

75

%in

du

ced

Ly5.2

-CD

4+

T-c

ell

s

0.00

0.05

0.10

0.15

Ly5.2

CD

4+

T-c

ell

s

(x10

6)

- dox+ dox

FIGURE 6. Reduced Antigen mediated clonal expansion of CD4 T cells expressing Nef. C57Bl/6 Ly5.1/5.2 host mice were injected with a mixture of CFSE labeled C57Bl/6 Ly5.1 lymphocytes in a 1:1 ratio with either CFSE labeled CD2-rtTA-M2/β-actin-KTR/OT-II (rtTA/OT-II) or CD2-rtTA-M2/β-actin-KTR/TetO7-Nef-huCD2TL/OT-II (rtTA/Nef/OT-II) transgenic lymphocytes. Subsequently mice were fed with regular or 2 mg/ml dox-containing drinking water. Two days after lymphocyte transfer mice were i.p. injected with a mixture of 5µg OVA and 5mg ALUM. Cells were isolated from spleens at day 4 after OVA injection (A). Obtained cell suspensions were stained with anti mouse-CD4, Ly5.1, Ly5.2 and anti-human CD2 and were analyzed by fl ow cytometry. An example of Ly5.1 and Ly5.2 CD4 T cells analyzed for CFSE dilution is shown (B). Human CD2 levels on rtTA/Nef/OT-II transgenic CD4 T cells in the presence and absence of dox were analyzed (C). To standardize recovery of OT-II transgenic CD4 T cells, 500 Ly5.1 CD4 T cells were collected and rtTA/Nef/OT-II CD4 T cells treated with and without dox were quantatively compared by overlay of CFSE histograms (D). Total OT-II transgenic CD4 T cell numbers were determined in mice injected with rtTA/Nef/OT-II transgenic cells treated with and without dox (E). Indicated are mean numbers ± SD of 3 mice (*, p<0.01), using a two-tailed Students t test with equal variance. One experiment is shown.

A B

D

C

E

CHAPTER 474

fi nding, revealing a fi ve-fold reduction in the total CD4 T cell number in the presence of Nef

(Figure 6E).


DISCUSSION

Nefs capacity to manipulate DCs and T cell function has been implicated in Nef-mediated

pathogenicity during HIV and SIV infections. Different mechanisms to explain this capacity,

like modulation of several membrane receptors, perturbation of signaling events and

modulation of cytokine and chemokine production, have been described. In this study we

investigated the in vivo functional correlates of these in vitro studied pathogenic properties

of Nef. For that purpose we generated transgenic mice in which Nef expression could be

transiently induced in T cells and APCs. Functional effects of Nef expression on T cell function

and DC-mediated T cell priming were determined during an infl uenza virus initiated immune

response. We observed that Nef expression in T cells interfered with the anti-viral CD8 and

CD4 T cell response. Further analysis of CD4 OT-II and CD8 OT-I TCR transgenic T cells

revealed that cognate antigen stimulation in the presence of Nef led to increased IFN-y

production in CD8 T cells but to reduced clonal expansion of CD4 T cells. In contrast and

quite unexpectedly, Nef expression in APCs resulted in enhanced T cell response, which

provides important in vivo evidence for a role of Nef in stimulating T cell activation and

thereby increasing viral replication and HIV pathogenicity.

After intranasal infection, priming, activation and expansion of naïve infl uenza-

specifi c CD8 T cells occur in the draining mediastinal lymph node 3-4 days after infection

(47-49). The magnitude of this CTL response is infl uenced by antigen presentation by the

CD8α DCs on one hand and the virus specifi c CD8 T cells on the other (50). It has been

shown that deletion of CD4 T cells does not affect the magnitude of primary CTL responses

in infl uenza infection (51, 52). It is thus the interaction between the DCs and the CD8 T

cell which determines the CTL response. In the experiment where we introduced either OT-

I or F5 transgenic CD8 T cells in Ii-rtTA/Nef transgenic mice which were infected with the

appropriate infl uenza virus, we excluded Nef expression in APCs as a factor determining

the lower anti-viral CD8 and CD4 T cell response in Nef expressing mice. Accordingly, direct

effects of Nef on T cells seem the most logical explanation for the reduced anti-viral T cell

responses. So both anti-viral CD4 and CD8 T cell responses seem to be diminished due to

Nef expression.

This diminution of the response could be explained by decreased priming of T cells

as a result of down regulated CD4 or CD8 expression and consequent diminished MHC-TCR

interactions. We observed however, no down regulation of CD4 or CD8 expression. The proto-

typic co-stimulatory molecule CD28 has also been described to be down regulated by Nef

(53). This co-stimulatory molecule is very important in priming T cells for clonal expansion

(54) and reduced CD28 expression might be a possible explanation for the reduced T cell

responses we observed.

Disturbance of signaling events in T cells is believed to be important for Nef-

mediated pathogenicity. A crucial region within the Nef molecule for its involvement in T

CHAPTER 476

cell signaling is the proline rich region, PxxP, located near the N-terminus of the molecule

(reviewed in (55)). The importance of this region for pathogenicity of Nef in SIV infection

is shown in a study by Khan et al., where mutation of the PxxP motif of Nef, led to a fast

functional restoration during infection (56). However, confl icting fi ndings have also been

reported (57). The PxxP region in Nef can bind the tyrosine kinase Lck, which acts proximal

in TCR signaling and is crucial for T cell activation. A recent study showed that Nef disturbs

Lck distribution towards the immunological synapse in HIV-1 infected CD4 T cells (25).

Binding of Nef to Lck has previously been shown to impair the kinase activity of Lck leading

to reduced IL-2 production (58). As this cytokine is critical for clonal expansion of T cells

during anti-infl uenza responses (59), a Nef-mediated reduction in production of Il-2 by CD4

and/or CD8 T cells might be an alternative mechanism to explain the reduced anti-viral T

cell response .

Next to Lck the PxxP motif of Nef is reported to bind the ζ-chain of the CD3 signaling

complex, which is associated with the TCR and is involved in TCR-mediated signaling (60).

Disturbance of TCR signaling events by Nef binding to the ζ-chain could directly disturb T

cell responses. Nef interaction with the ζ-chain has also been shown to to upregulate FasL

on the cell surface of T cells. Xu et al., showed that in SIV infections of macaques, Nef-

mediated upregulation of FasL leads to increased apoptotic cell death of CD4 and CD8 T cells

thereby obstructing CTL responses directed to SIV infected cells (61). Studies on infl uenza

infections in mice report that CTL expansion is dependent on Fas-FasL interactions and mice

defi cient in FasL show a marked increase in generation of activated effector CD8 T cells (62).

Combining these observations would predict a Nef-mediated upregulation of FasL on T cells

causing a decreased clonal expansion of T cells during infl uenza infections in Nef transgenic

mice.

Previous studies in the CD2-rtTA/β-actin-KTR/TetO7-Nef-huCD2TL model showed

that expression of Nef in a naïve peripheral T cell compartment does not lead to spontaneous

activation (Koenen et al., manusript in preperation). In the current study we did not observe

any effects of Nef on T cell proliferation or activation under lymphopenic conditions in the

empty T cell compartment of RAG 2 -/- mice (Figure 4). However, we could show that Nef

affects TCR dependent T cell activation and division by antigenic stimulation. This fi nding

point to a specifi c effect of Nef after cognate antigen stimulation, displaying the qualitatively

different signals upon TCR interactions with either self-peptide/MHC complexes during

lymphopenia induced proliferation or cognate antigen/MHC complexes (46).

Our data reveal that Nef expression in APCs infl uences the functional properties of

these cells regarding T cell activation during an anti-viral response in mice. By activating

T cells via APCs, Nef can facilitate enhanced viral infection and replication, increasing HIVs

pathogenicity. The importance of cell-cell contact between APCs and T cells for vigorous

viral replication has been shown by experiments with the highly pathogenic YE variant of

SIVmac239. These experiments described extensive Nef-dependent T cell activation and SIV


replication in T cells upon in vitro infection of PBMC (63). The activation and viral replication

were strictly dependent on cell-cell contact between macrophages and T cells. The contact

dependency points to an important role of membrane receptors on APCs in mediating Nef-

dependent enhancement on viral replication. In line with this notion, Nef has been described

to upregulate DC-SIGN (15), the upregulation of DC-SIGN could explain the increased DC-T

cell clustering and viral replication in the presence of Nef. However, contrasting fi ndings have

been reported (16, 17). Other studies suggested that besides direct cell-cell contact between

APCs and T cells, Nef mediates chemotaxis and T cell activation by infected macrophages or

DCs (16, 18, 19). Confl icting data concerning the activating capacities of Nef expression in

DCs on T cells can probably be attributed to in vitro associated variations among different

studies, such as the use of different viral vectors, donor variation and different methodologies

used to generate DCs. Therefore, we will use our inducible Nef transgenic mouse model to

unravel the mechanism underlying the activating capacities of Nef expressing APCs.

To conclude, this is the fi rst study showing that Nef expression in a normally

developed T cell compartment affects T cell function in vivo, pointing towards a disturbed TCR

signaling in primary T cells in vivo. Furthermore, we show the fi rst in vivo evidence that Nef

expression in APCs stimulates T cell activation. Further analysis of underlying mechanisms

will help to better understand pathogenicity of HIV and can lead to new insights regarding

therapy of HIV infected individuals.

CHAPTER 478


Mice

The Ii-rtTA transgenic mice, have been described previously (30) and were a kind gift from Dr. H.-M.

van Santen. CD2-rtTA-M2/β-actin-KTR and TetO7-Nef/huCD2TL transgenic mice were described before

in Koenen et al., OT-I transgenic mice (31), OT-II transgenic mice (32), Rag 2 knock out mice (33),

C57BL/6 (Ly5.1) and C57BL/6 (Ly5.2) mice were obtained from The Jackson Laboratories and were

maintained as breeding colonies in our animal facility. Ly5.1/Ly5.2 mice were bred by crossing (C57BL/6J-

Ly5.1 × C57BL/6J-Ly5.2.). Mice were treated with doxycycline (dox, 2mg/ml, BD Biosciences) and 0.4%

sucrose containing drinking water which was refreshed every 2-3 days. Experiments were performed

with mice 6 to 12 weeks of age. The inducible Nef transgenic line (rtTA/TetO7-Nef-huCD2TL) was always

compared to rtTA control animals, to exclude any rtTA and/or dox related effect. All mice were kept under

specifi c pathogen-free conditions and housed in accordance with institutional guidelines of American

Association of Accreditation of Laboratory Animal Care, in the mouse facility of the Central Laboratory

Animal Institute Utrecht University (Utrecht, the Netherlands). The Institutional Animal Ethics Committee

approved all experiments.

Flow cytometry

Single cell suspensions of lung, spleen and lymph nodes were obtained by mechanical disruption in the

presence of RPMI 1640 supplemented with 10% fetal bovine serum (FCS) (Integro b.v., Dieren, the

Netherlands). Erythrocytes were lysed by incubation of the cells in 0.15 M NH4Cl, 0.01 M KHCO

3, 0.1

mM EDTA, pH 7.4, for 2 min on ice. Cells were preincubated with Fc-block (mAb to CD16/32, 2.4G2;

BD, Pharmingen) and washed in staining buffer (PBS, 0.5% BSA, 0.01% sodium azide). Next, cells

were incubated with specifi c antibodies directly conjugated to FITC, PE, PerCP, APC, PE.cy7, APC.cy7,

Pacifi c Blue or Biotin. APC or PE labeled tetramers of the murine MHC class I H2-Db heavy chain, β2

microglobulin, and the infl uenza virus nucleoprotein (NP)366-374

peptide ASNENMDAM were prepared as

described (34) and used for immunofl uorescence in combination with the indicated mAbs. After incubation

with respective antibodies and tetramers, cells were analyzed using a FACSCalibur or BD LSR II and

analyzed by CELLQuest or BD FACSDiva software (Becton Dickinson). mAbs used for immunofl uorescence

were anti-mouse CD4 (RM4-5), CD8 (53-6.7), CD11b (M1/70), CD11c (HL3), CD19 (1D3), CD25 (PC 61),

CD16/32 (2.4G2), CD44 (IM7), CD45.1 (A20), CD45.2 (104) and CD62L (MEL-14), CD69 (H1.2F3), Vβ5

(MR9-4), anti-human CD2 (RPA-2.10) and streptavidin were all purchased from BD PharMingen (San

Diego, Calif.).

Peptide stimulation and intracellular cytokine staining

Cell suspensions from lungs or lymph nodes containing 106 cells/ml in RPMI 1640 supplemented with 10%

FCS were stimulated either with Nef peptide pools (15mers with 11 overlap, HXB2, individual peptides

concentration 2µg/ml, (NIH AIDS Research and Reagent program, Bethesda, Maryland, United States)) or

with OVA257-264

SIINFEKL or NP366-374

ASNENMDAM peptides (Mimotopes Pty Ltd, Clayton Victoria, Australia)

at a concentration of 1µg/ml. Stimulations were performed for 4h at 37°C, in the presence of Brefeldin

A (5µg/ml, Beckton Dickinson, San José, California, United States), cells were subsequently stained with

anti-CD8, fi xed and permeabilised (permeabilisation kit, Beckton Dickinson) and stained with anti-IFNγ-

PE mAb (BD) for 20 min at 4 °C. Finally, cells were analyzed by fl ow cytometry.



Single cell suspensions were prepared from lymph nodes and spleens. Cells were labeled with CFSE

(Molecular Probes Inc., Eugene, OR.) as previously described (35). Briefl y, cells were washed twice in

phosphate-buffered saline (PBS) and resuspended at 107 cells/ml in PBS. CFSE was added to a fi nal

concentration of 0.5μM, and cells were incubated for 10 min at 37ºC. Unbound CFSE was quenched by

washing labeled cells twice with RPMI 1640 medium supplemented with 10% FCS. Up to 107 cells were

resuspended in 200 μl PBS and injected i.v. into the tail vein of the mice.

Virus infections

The H3N2 infl uenza virus strain A/HK/2/68 was a kind gift from Prof. J. Borst and Prof T.N.M. Schumacher.

The recombinant WSN-I virus was earlier described (36) and was a kind gift from Dr. D. Topham. Mice

were anesthetized by ether and infected intranasally with 50µl PBS containing either one hemagglutinin

unit of A/HK/2/68 virus or 104 PFU of WSN-I virus.

Ovalbumin (OVA)/alum sensitization

Mice received one intraperitoneal injection of 5μg of OVA (Sigma, St. Louis, MO) mixed with 5 mg of alum

(kindly provided by Dr. R. Pieters), in 200μl PBS.




ACKNOWLEDGEMENTS

We acknowledge Prof. dr. F. Miedema for critically reading the manuscript. We thank Dr.

H.-M. van Santen for providing Ii-rtTA transgenic mice, Dr. D. Topham for providing WSN-I

virus, Prof. dr. J. Borst and Prof. dr. T.N.M Schumacher for providing infl uenza virus strain

A/HK/2/68, Dr. R. Arens and Prof. dr. D. Kioussis for providing spleens of F5 transgenic

mice and Dr. R.Pieters for providing ALUM. The Nef-derived overlapping peptide pool (15-

mers with 11 overlap, HXB2) was obtained through the NIH AIDS Research and Reference

Reagent program, Division of AIDS, NIAID, NIH.

CHAPTER 480

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Evaluation of Doxycycline Use in T Cell Specifi c Inducible Transgenic Mouse Models

5

Paul G.F. Koenen, Frans M. Hofhuis, Kees J. Koeman, Elise H. R. Schrijver, Kiki Tesselaar


Manuscript submitted

CHAPTER

CHAPTER 586

ABSTRACT

Tetracyclines are clinically widely used for their antibiotic and non-antibiotic properties. In

addition, tetracyclines, in particular doxycycline, are commonly used in transgenic mice

as regulators of gene transcription. In our studies we focused on the T cell compartment

of mice, using the ‘tet-on’ system in combination with the transcriptional repressor tTS

in vivo. We observed several side-effects from doxycycline treatment alone on the T cell

compartment of mice. Our experiments show evidence for doxycycline-mediated reversible

redistribution of T cells from the blood, inhibition of in vitro and in vivo T cell proliferation

and a toxic effect on embryogenesis of doxycycline in combination with co-expression of

CD2 controlled rtTA-M2 and β-actin controlled tTS expression. This study provides evidence

for several side-effects of doxycycline on the function of T cells, underscoring the importance

of well-controlled experiments with inducible systems.

Evaluation of doxycycline use in T cell specifi c inducible transgenic mouse models 87

INTRODUCTION

Tetracyclines are one of the most commonly prescribed antibiotics in the treatment of

bacterial infections. Besides these well known anti-microbial properties, tetracyclines

also directly interact with the immune system (1, 2). This is evidenced by studies where

the non-antibiotic properties of tetracycline derivatives were exploited in the successful

treatment of diseases such as rheumatoid arthritis, periodontal disease and abdominal

aortic aneurysms in animal models (3-7). These diseases share similar pathological features

including chronic infl ammatory infi ltration and local extracellular matrix destruction where

extensive infl ammatory cell infi ltration of predominantly T cells and macrophages are critical

for progression of these diseases.

The exact mechanism underlying tetracycline-mediated reduction of infl ammation is

not known, however several immune-modulating effects of tetracyclins have been described.

The inhibiting effect of tetracyclines on metalloproteinases (MP) (1, 8), which are secreted

during infl ammation and which leads to extracellular matrix damage, has been suggested to

reduce infl ammation (3, 7, 9). Furthermore, tetracyclines have been shown to specifi cally

decrease levels of inducible NO synthase in activated macrophages by regulating the stability

of its mRNA (10, 11). In addition to these effects, tetracyclines also inhibit B-cell function.

In vitro studies have shown that class-switching and proliferation of B cells is decreased in

the presence of doxycycline (12). Besides the inhibition of B-cell function and macrophage

activation, several studies indicated that doxycycline, a derivate of tetracycline, induces

apoptosis in tumour T cell lines (13, 14). Tetracyclines have also been reported to suppress

lymphocyte proliferation in vitro, although this effect was variable and not observed in all

systems (1, 2, 9, 13, 15-19).

Tetracyclines, and in particular doxycycline, are commonly used in cell lines and

transgenic mice as a regulator of gene transcription in inducible systems. The most widely

used inducible transgenic system is based on the tetracycline-controlled transcriptional

regulator developed by Gossen and co-workers (20-23). There are two basic variants used,

either inducing transcription upon addition of doxycycline (tet-on) or inducing transcription

upon withdrawal of doxycycline (tet-off) (23). In order to prevent possible leakage of gene

transcription, the tet-on system can be used in combination with the tetracycline-controlled

transcriptional silencer system (tTS). This consists of a fusion between the TetR of E.coli and

the KRAB-AB domain of Kid-1 protein, a transcriptional repressor (23, 24).

In our studies we have been using the tet-on system in combination with the

tTS in vivo. However, taking in account the specifi c activities of tetracyclines in eukaryotic

organisms, results obtained from doxycycline treated versus untreated groups should be

interpreted carefully. As we studied the infl uence of our gene of interest in inducible T cell

specifi c transgenic mice, we observed several doxycycline-mediated side-effects on the T

cell compartment of these mice.

CHAPTER 588

RESULTS

Temporal reduction of T cell numbers in blood and secondary lymphoid organs

upon doxycycline administration

We generated inducible transgenic mice in order to study the infl uence of our target gene on

T cell function, without interfering with T cell development caused by constitutive expression

during embryogenesis. We used the tetracycline-dependent transgene expression system

that has been described previously as the “tet-on” system (22, 23, 27, 28). In this double

transgenic system expression of a target gene only occurs in the presence of doxycycline

(22, 23, 27, 28).

During the analysis of our CD2-rtTA-M2/β-actin-KTR /TetO7-Nef-huCD2TL (Koenen

et al., manuscript in preperation) transgenic mice we studied T cell number and characteristics

in blood. We induced the transgenic mice with one injection containing 2mg doxycycline

followed by doxycycline administration into the drinking water (2mg/ml). Blood was drawn

before and at two time points during doxycycline administration and T cell numbers were

determined by quantative fl ow cytometry. At 24h after doxycycline administration a sharp

FIGURE 1. Temporal reduction of T cell numbers in the blood upon doxycycline administration. Transcription of CD2-rtTA-M2/β-actin-KTR /TetO7-Nef-huCD2TL transgenic mice was induced with one injection containing 2mg doxycycline followed by doxycycline administration into the drinking water (2mg/ml). Blood was drawn before and at 24h and 72h after the start of doxycycline treatment. T cells were analyzed after incubation of the cells with anti-CD4 and anti-CD8 directed antibodies by fl ow cytometry. Total blood T cell numbers were quantitated by administration of a fi xed amount of latex beads to a defi ned volume of blood. On basis of collected beads, T cell numbers were calculated, as depicted. Data is shown of three mice, shown is a representative of three independent experiments (A). C57Bl/6 mice were injected with doxycycline as described above, blood was drawn before and 16h after doxycycline injection and CD4 and CD8 T cell numbers were determined as described above. Data is shown from six mice (B). Each symbol represents a single mouse.

CD4 T cells CD8 T cells

Tg

Wt

0 25 50 750.0×10 -00

2.5×10 05

5.0×10 05

7.5×10 05

time (hours)

CD

4T

-cell

s/m

l

0 25 50 750.0×10 -00

1.0×10 05

2.0×10 05

3.0×10 05

4.0×10 05

5.0×10 05

time (hours)

CD

8T

-cell

s/m

l

0 5 10 15 200.0×10 -00

2.5×10 05

5.0×10 05

7.5×10 05

1.0×10 06

time (hours)

CD

4T

-cell

s/m

l

Wt

Tg

0 5 10 15 200.0×10 -00

5.0×10 05

1.0×10 06

1.5×10 06

time (hours)

CD

8T

-cell

s/m

l

A

B


reduction in the total amount of T cells in the blood, for CD4 as well as for CD8 T cells (Figure

1A), was observed. After this rapid reduction, T cell levels started to recover towards normal

values (Figure 1A). The extent of the observed reduction made it very unlikely that the

reduction was caused by the induction of our target gene, as gene induction only occurred

in a small proportion (2-8%) of T cells. Therefore in a subsequent experiment we injected

wild-type C57Bl/6 mice with 2mg doxycycline and examined T cell numbers in the blood.

Before and 16h after doxycycline injection blood was drawn and analyzed by fl ow cytometry

and again, a large reduction of CD4 and CD8 T cell numbers in blood was observed. (Figure

1B). Thus administration of doxycycline and not the expression of our target gene resulted

in a transient reduction of T cells.

To investigate whether this T cell loss was only apparent in the blood or whether

it was a general phenomenon, we determined T cell counts of the secondary lymphoid

organs after doxycline administration. C57Bl/6 mice were injected with 2mg doxycycline

and were either sacrifi ced 24h later, or were fed with 2mg/ml doxycyline-containing drinking

water for 4 days and than sacrifi ced. Total CD4 and CD8 T cell numbers in the spleen were

determined. A transient reduction of T cell numbers as observed in the blood was observed

in the spleen (Figure 2). After 24h a drop in T cell number occurred, whereas after four days

of doxycyline treatment, normal T cell numbers were observed. Altogether, we showed in

these experiments that upon doxycycline administration T cell counts in blood and secondary

organs dropped rapid, but transiently. This effect was however more prominent in the blood

than in the secondary lymphoid organs.

Doxycycline inhibits T cell proliferation in vitro and in vivo, but does not induce

cell death

Measurement of proliferation after in vitro stimulation of T cells is a widely used assay to

*

24h 4 days

- dox

+ dox

0

10

20

CD4 CD8

T-c

ell

s(x

10

6)

0

10

20

CD4 CD8

T-c

ell

s(x

10

6)

FIGURE 2. Doxycycline treatment decreases total T cell numbers in secondary lymphoid organs temporarily. C57Bl/6 mice were injected with 2mg doxycycline and were either sacrifi ced 24h later, or were fed with 2mg/ml doxycyline-containing drinking water and sacrifi ced 4 days later. Single cell suspensions derived from spleens were incubated with anti-CD4 and CD8 antibodies and analyzed by fl ow cytometry. Total T cell numbers from spleens were calculated and depicted Each symbol represents an individual mouse and dashes represent mean values. P values were determined using a two-tailed Students t test with equal variance (*, p<0.05). Shown is one experiment.

CHAPTER 590

test T cell function in vitro. To test the described effect of doxycycline on in vitro T cell

proliferation in our system, we performed T cell stimulations in the presence and absence

of doxycycline. T cells were extracted from peripheral lymph nodes (PLN), stimulated with

different concentrations of αCD3 mAb alone, or in combination with αCD28 mAb. Proliferation

was measured by [3H] Thymidine incorporation at different time points after stimulation.

A doxycycline-dependent decrease on T cell proliferation at 48h and 72h after

stimulation was observed under sub-optimal stimulation conditions (Figure 3). However,

no effect of doxycycline on T cell proliferation was observed when T cells were stimulated

with a higher concentration of αCD3 mAb in combination with αCD28 mAb (Figure 3). So

these data point out that doxycycline does interfere with T cell proliferation, but that the

doxycycline-mediated effects can vary under different stimulation conditions.

To investigate whether the reduction in proliferation also occured in vivo, we next

analyzed spontaneous T cell proliferation in a lymphopenic environment. To measure the

infl uence of doxycycline in this in vivo system of T cell proliferation, we intravenously injected

5x106 CFSE labeled wild-type C57B/6 lymphocytes into T cell deplete, Rag 2 defi cient mice.

One group of mice received doxycycline throughout the experiment, whereas another

group of mice received normal drinking water (Figure 4A). Six days after injection, DLN of

Rag 2-/- mice were harvested and transferred donor T cells were analyzed. Firstly, CFSE

dilution profi les were analyzed by fl ow cytometry. The CFSE profi les showed similar cell

cycle progression of both doxycycline treated and untreated CD4 and CD8 T cells, however

the fraction of divided cells appeared to be higher in untreated CD8 T cells (Figure 4B).

Secondly, total CD4 and CD8 T cell numbers were calculated from DLN. Donor T cell numbers

obtained from Rag 2 -/- mice treated with doxycycline were decreased when compared to

donor T cell numbers obtained from untreated Rag 2 -/- animals (Figure 4C). The decrease

among CD8 T cells was more prominent than among CD4 T cells, probably refl ecting the

higher proliferation rate of CD8 T cells. To investigate whether the reduced T cell number was

Days of culture

[3H

] T

hym

idin

e (

cp

m)

- CD3 0.5 g/mlα μ - CD3 0.05 g/mlα μ

- CD28 2.5 g/mlα μ

- CD3 0.5 g/mlα μ

- CD28 2.5 g/mlα μ

- dox

+ dox

0 1 2 3 40

25000

50000

75000

0 1 2 3 40

5000

10000

15000

20000

25000

0 1 2 3 40

100000

200000

300000

FIGURE 3. In vitro response of doxycycline treated T cells. T cells from C57Bl/6 mice derived lymph nodes were purifi ed by αCD90-2 mAb labeled magnetic beads. Purifi ed T cells were stimulated with various concentrations plate-bound αCD3 (145-2C11) or αCD3- αCD28 (PV-1) as indicated. Cell proliferation was measured after 24, 48 and 72h by pulsing with [3H] Thymidine for 6h in the presence or absence of 1µg/ml doxycycline. Shown is a representative of three independent experiments.


caused by increased cell death, cell suspensions were stained with Annexin V. No differences

in proportion of death CD4 and CD8 T cells were observed between doxycycline treated and

untreated cells (Figure 4D), which indicated that the doxycycline-mediated decreased T cell

proliferation in Rag 2 -/- mice was not caused by cell death.

Tetracycline-controlled transcriptional silencer (tTS) in combination with

doxycycline is lethal

To study the effect of our transgene on thymopoeisis in the developing thymus, the transgene

should be expressed during embryogenesis. Therefore, we fed doxycycline in the drinking

water continuously to CD2-rtTA-M2/β-actin-tTS (rtTA/tTS) transgenic female mice paired

with the reciprocal male, transgenic for TetO7-Nef-huCD2TL (TetO7). rtTA and tTS elements

are co-integrated in the same locus and these fragments are passed to next generations

in conjunction (Koenen et al., manuscript in preperation). In these experiments both male

and female mice were heterozygous for their respective transgenes. To our surprise no pups

of interest, i.e. pups containing both transgenes, or rtTA/tTS single transgenic pups were

obtained. Only wild-type and TetO7 transgenic mice were born (Table I). After screening 22

pups, doxycycline containing drinking water was replaced by normal drinking water. During

the following 4 weeks on normal drinking water, 3 nests with in total 14 pups were born.

Genetic screening showed that under these conditions all 4 different possible genotypes

dox

Day 0

sacrifice

Rag 2 -/-

dox

Day 2 Day 7

Rag 2 -/-

iv lymphocytes

Even

ts

Annexin V

- dox+ dox

Even

ts

CFSE

CD4+ T-cells CD8+ T-cells

- dox+ dox

p=0.081 p=0.046

- dox

+ dox

0.0

2.5

5.0

7.5

10.0

CD4 CD8

T-c

ell

s(x

10

5)

CD4+ T-cells CD8+ T-cells

C D

A B

FIGURE 4. Doxycycline inhibits T cell proliferation in vivo, but does not induce cell death. Rag 2 -/- mice were either fed with regular or 2 mg/ml doxycycline-containing drinking water. Two days later CFSE labelled C57Bl/6 derived lymphocytes were injected iv. Six days after lymphocyte injection DLN were harvested and cells were analyzed (A). Cells were stained with anti-CD4 and CD8 mAbs and CFSE profi les were analyzed by fl ow cytometry (B). Total T cell numbers were calculated (C). DLN obtained T cells were incubated with Annexin V and analyzed by fl ow cytometry (D). Each symbol represents an individual mouse and dashes represent mean values. P values were determined using a two-tailed Students t test with equal variance. Shown is a representative of two independent experiments.

CHAPTER 592

were present among the pups (Table I). So, it seems that doxycycline in combination with

rtTA and tTS elements is lethal in early development.

nest dox tot.pups

Wt TetO7 rtTA/tTS TetO7rtTA/tTS

1 + 2 1 1 - -

2 + 2 1 1 - -

3 + 6 3 3 - -

4 + 6 3 3 - -

5 + 4 2 2 - -

6 + 2 2 - - -

tot. 22 12 10 0 0

7 - 8 3 2 1 2

8 - 3 1 - 1 1

9 - 3 1 - 1 1

tot. 14 5 2 3 4

TABLE I. tTS repressor in combination with doxycycline is embryonic lethal. 2mg/ml doxycycline containing drinking water was continuously administered to CD2-rtTA-M2/β-actin-KTR (rtTA/tTS) transgenic female mice paired with the reciprocal TetO7-Nef/huCD2TL (TetO7) male transgenic mice. rtTA and tTS elements are co-integrated in the same locus and these fragments are passed to next generations in conjunction. Six nests were born in the presence of doxycycline (+ dox). When doxycycline was withdrawn from the breeding mice, three more nests were born (- dox). Four different geno-types were present among the pups; wt, TetO7, rtTA/tTS, TetO7-rtTA/tTS.


DISCUSSION

Besides their diverse use in the treatment of several microbial and non-microbial caused

diseases, tetracyclines and specifi cally doxycycline are also used in inducible transgenic

systems as a regulator of gene transcription. Since doxycycline can have unexpected (side)

effects, the knowledge of its effects on cell functioning is essential for proper interpretation of

the results. We showed in this study that administration of doxycycline to mice led to a rapid

but transient reduction in total T cell numbers in the blood and the lymphoid organs. The fact

that it appeared to be a transient effect, suggest an effect of doxycycline on redistribution

of T cells.

L-(leukocyte) selectin (CD62L) and CD44 are both implicated in the migration of

T cells from blood towards infl amed tissues (29, 30). While CD62L is believed to play an

exclusive role in the initiation of homing of naïve lymphocytes to PLN (29-31), CD44 mediated

adhesion has been shown to initiate recruitment of activated cells at sites of infl ammation

(29, 30). CD44 and CD62L receptor expression is regulated by MP-mediated cleavage (32,

33). Since doxycycline is shown to inhibit MP activity (1, 8), it is conceivable that inhibited

MP activity can lead to disregulated CD44 and CD62L expression, consequently leading to a

change in lymphocyte distribution, as seen during our experiments. Since the decline in T

cell numbers was transient, with T cell numbers recovering to normal values after prolonged

doxycycline administration, this might refl ect normalization of receptor expression over

time. However, further research will be done, to clarify the mechanism underlying this effect

of transient T cell loss.

Furthermore, we showed that doxycycline decreased murine T cell proliferation

in vitro and in vivo. In vivo analysis of Annexin V expression did not reveal increased cell

death as a possible cause of decreased proliferation. This is in contrast with a previous study

describing specifi cally doxycycline-mediated inhibition of proliferation of Jurkat tumour cells

in vitro due to induction of Fas/Fas ligand-mediated apoptosis (13). The fact that we did

not observe any increased cell death upon doxycycline administration can refl ect a tumour

cell specifi c effect of doxycycline, which has been shown before (14, 34-36) and may not

be observed among primary T cells. Since the reduction in the number of T cells seems not

to be caused by increased cell death, it is probably due to doxycycline-mediated reduced

proliferation, which is suggested by the smaller proportion of CFSE negative cells in dox

treated mice (Figure 2B). To our knowledge this is the fi rst study showing doxycycline-

mediated inhibition of T cell proliferation in vivo.

Next to the effects of doxycycline on the distribution and proliferation of T cells, we

observed that doxycycline in combination with CD2 controlled rtTA and β-actin controlled tTS

protein expression was embryonic lethal. Previous studies have shown that CD2 controlled

rtTA expression in the presence of doxycycline during embryogenesis had no effect (37).

Therefore it is most likely that the broadly expressed tTS element under β-actin control is

CHAPTER 594

the cause of these toxic effects in combination with doxycycline. Whether this is a general

effect of the tTS element in combination with doxycycline, or whether this is specifi c to our

founder line only, we can not tell. Although the mechanism for this toxic effect remains

unclear, this is a caveat for future studies using this element of transcriptional repression.

Given the impact of the described side effects, novel doxycycline analogues

lacking doxycyclines’ unwanted side-effects could greatly improve the use of the tet-

controlled systems. In this context 4-epidoxycycline was proposed as a possible alternative

for doxycycline, 4-epidoxycycline lacks antibiotic properties, and thus does not disturb

the microbial status of mice during gene regulation (38). Still, even with newly developed

compounds, unexpected side-effects may occur, and have to be considered in the

interpretation of the results in te- regulated systems.



Mice

CD2-rtTA-M2/β-actin-KTR (rtTA/tTS) and TetO7-Nef/huCD2TL (TetO7) transgenic mice were described in

Koenen et al., manuscript in preperation, Rag 2 knock out mice (25) and C57BL/6 mice were obtained

from The Jackson Laboratories and were all maintained as breeding colonies in our animal facility. Mice

were treated either with doxycycline (2mg/ml, BD Biosciences, San Gose) and 0.4% sucrose containing

drinking water which was refreshed every 2-3 days or were i.p. injected with 2mg doxycycline solved

in 200μl PBS. Experiments were performed with mice 6 to 12 weeks of age. All mice were kept under

specifi c pathogen-free conditions and housed in accordance with institutional guidelines of American

Association of Accreditation of Laboratory Animal Care, in the mouse facility of the Central Laboratory

Animal Institute Utrecht University (Utrecht, the Netherlands). The Institutional Animal Ethics Committee

approved all experiments.

Flow cytometry

Single cell suspensions of spleen and lymph nodes were obtained by mechanical disruption in the

presence of RPMI 1640 supplemented with 10% fetal bovine serum (FCS) (Integro b.v., Dieren, the

Netherlands). For blood analysis, 50μl blood was drawn from the vena Saphaena. Erythrocytes were lysed

by incubation of the cells in 0.15 M NH4Cl, 0.01 M KHCO

3, 0.1 mM EDTA, pH 7.4, for 2 min on ice. Cells

were preincubated with Fc-block (mAb to CD16/32, 2.4G2; BD Biosciences) and washed in staining buffer

(PBS, 0.5% BSA, 0.01% sodium azide). Afterwards cells were incubated with Annexin V, anti-mouse CD3

(145-2C11), CD4 (RM4-5) or CD8 (53-6.7) labelled with appropriate fl uorochromes (BD Biosciences).

Stained cells were analyzed using a FACSCalibur or BD LSR II and analyzed by CELLQuest or BD FACSDiva

software (BD Biosciences).

Total blood T cells were quantitated by administration of a fi xed amount of latex beads

(Interfacial dynamics corp., Portland, USA) to a defi ned volume of blood.


Single cell suspensions were prepared from lymph nodes and spleens. Cells were labeled with CFSE

(Molecular Probes Inc., Eugene, OR.) as previously described (26). Briefl y, cells were washed twice in

phosphate-buffered saline (PBS) and resuspended at 107 cells/ml in PBS. CFSE was added to a fi nal

concentration of 0.5μM, and cells were incubated for 10 min at 37ºC. Unbound CFSE was quenched by

washing labeled cells twice with RPMI 1640 medium supplemented with 10% FCS. Up to 107 cells were

resuspended in 200 μl PBS and injected i.v. into the tail vein of the mice.

In vitro responses of T cells

T cells from wild-type derived lymph nodes were purifi ed by αCD90-2 mAb labeled magnetic beads

(Miltenyi Biotec). Purifi ed T cells were stimulated with various concentrations plate-bound αCD3 (BD

Biosciences, 145-2C11) or αCD3- αCD28 (BD Biosciences, PV-1) as indicated. Cell proliferation was

measured after 24, 48 and 72h by pulsing with [3H]Thymidine for 6h.




CHAPTER 596

ACKNOWLEDGEMENTS

We acknowledge F. Miedema and R. H. H. Pieters for critically reading the manuscript.


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GENERAL DISCUSSION6

Paul G.F. Koenen


CHAPTER

CHAPTER 6102

INTRODUCTION

In this thesis we presented our fi ndings regarding HIV-Nef functions in APCs and T cells

in different transgenic mouse models. In the last decade, many different and sometimes

confl icting fi ndings on Nefs function and interactions have been reported. In the research

described in this thesis, we have re-addressed the generally accepted idea of Nef-mediated

spontaneous T cell activation in transgenic mice (chapter 2 and 3). Here, I will discuss

possible explanations for the pleiotropy of functions ascribed to Nef and the heterogeneity

of data regarding Nef. Next, I will discuss our inducible Nef transgenic model and point out

future directions to further unravel the function of Nef in HIV pathogenesis.

The dynamic Nef protein

The most fascinating fact after two decades of Nef research is the number of different

and sometimes contrasting functions and interactions, which have been ascribed to this

molecule. It is very unlikely that all described, mostly in vitro effects are equally important

for HIV function in vivo, if relevant at all. Several reasons can explain the pleiotropy of

effects and contrasting results in Nef research.

First of all, Nef lacks any structural degree of homology to any other known molecule.

This lack of homology excludes functional insights on the basis of homologous proteins or

protein domains with known function. Secondly, structural analysis of different Nef alleles

revealed that HIV-1 Nef possesses a genetically diverse and structurally fl exible N-terminal

arm of ~70 residues, followed by a well-conserved and folded core domain of ~120 residues

(1, 2). The fl exibility of its structure makes Nef diffi cult to study, especially using mutational

analysis, where the introduction of mutations has been shown to lead to dramatic structural

changes in the secondary and tertiary structures of the protein (2-5). These changes in

protein conformation make it hard to dissect whether a loss of function is caused by a certain

amino acid alone or whether it is brought about by secondary conformational changes.

Thirdly, the non-structural regions of Nef have been suggested to provide an enormous

capacity to connect to other molecules and form multi-protein complexes (1, 2). This

adaptor-like function has been described to lead to Nef-associated signaling complexes (6).

Such a function of Nef could explain the multitude of binding molecules that have been

described for Nef, without implying functional interactions of any one of them. So, this would

mean that Nef functions as a scaffold to bring together molecules in order to change cell

function. Fourthly, an additional layer of complexity is introduced by the variable localization

of Nef in the cell. Nef has been localized in the membrane, cytosol and nucleus of the cell

(1, 3) and depending on Nefs localization different functions have been found (1, 7).The

question however remains, whether these are all relevant locations or were brought about

GENERAL DISCUSSION 103

by the abundant expression of Nef in cell lines.

Altogether, the fl exibility of the Nef protein, the great binding capacity and the

variable localization of the molecule, makes the more than thirty Nef binding partners,

which have already been reported more easy to understand. Still, in most cases a functional

relationship between Nef and its suggested interacting partners has not been demonstrated.

The high number of binding partners may refl ect a propensity of Nef to form non-specifi c

interactions in vitro. Thus, it is of extreme importance that putative Nef interacting proteins

are clearly demonstrated to functionally interact with Nef in relevant systems before they

are established as de facto binding partners.

Which model to study Nef function?

Nef studies with the tet-on system

Ideally, one would like to study the functional properties of Nef in primary cells, in vitro and

in vivo and with controlled levels of Nef expression. Based on these criteria we developed

the inducible Nef transgenic mouse model presented in this thesis.

Below we describe our case for the Nef transgenic tet-system we developed. First of all, we

chose the tet-system to induce transgenic expression in vivo, most importantly because this

was (and is) the only system successfully used in vivo. To minimize possible toxic effects of

doxycycline (dox), we chose the tet-on system, with gene induction upon dox administration

and short administration times, instead of the tet-off, system, with gene induction upon dox

withdrawal and extended administration times. As Nef is an intracellular protein a reporter

for intracellular Nef expression was used. The enhanced version of GFP (EGFP), with a

shorter half life than conventional GFP and consequently less toxicity for the cells was used.

Anticipating on the possibility of losing EGFP signal over mice generations, we chose to use

an extra, eukaryotic reporter, human tailless CD2 (used by Zamosyka (8)). This turned out

to be a good precaution, as with time the EGFP signal starts to diminish and in some mice

we have seen it disappear completely.

For our experiments, we used two different rtTA lines (chapter 3). One line was

obtained from Zamoyska and contained the conventional rtTA expressed under control of

an improved human CD2 cassette (9). The other rtTA line contained the improved rtTA-M2

under control of the conventional human CD2 cassette (10) in combination with a repressor

of transcription. Characterization of the mice showed that the improved human CD2 cassette

resulted in T cell specifi c expression, whereas the conventional human CD2 cassette resulted

in expression in T cells as well as B cells. Nevertheless, with regard to the conventional

rtTA and the improved rtTA-M2 we did not observe any difference regarding background

expression levels, as both inducer lines showed no leakage of gene transcription in the

absence of dox.

CHAPTER 6104

In contrast to the tight regulation in absence of dox, great variations in induction

levels among double transgenic littermates upon dox administration were observed (chapter

2). This variation was most prominent in vivo, as in vitro stimulations were comparable for

cells derived from double transgenic littermates. The reason for this variation is unknown

but it was made impossible to regulate protein levels in vivo by administering different

amounts of dox.

The transgenic models we generated supplied us with a source of genetically

uniform primary T cells with variable expression of the Nef protein, without the need to

stress the cells by transfection or viral transduction. Ultimately this approach also gave

us the opportunity to study in vivo Nef effects in the context of a normally developed

immune system. So, altogether, we presented here a powerful system to validate in vitro

described Nef interactions in vitro and in vivo. Future models to induce Nef expression in

vivo, should focus on the regulation of Nef protein levels, since we could not accomplish

uniform expression levels in our current models

HIV and SIV infections in vivo

Evidently the most physiologic way to study Nef function is by using Nef-deleted SIV and

HIV strains. By comparing Nef defi cient strains with either wild-type SIV or HIV strains, Nef

effects can be studied. The most convincing experimental evidence for Nef as a pathogenicity

factor comes from studies with Nef-defi cient SIV in rhesus monkeys (11) and this remains a

powerful model to study SIV function. However, an in vivo model to study HIV would be best.

This model now seems to become available with the recently developed humanized mouse

model. It was shown that Rag 2, common γ-chain defi cient mice reconstituted with human

CD34+ stem cells developed a “human adaptive immune system”, in which a functional

adaptive immune responses towards human viruses, i.e. EBV, (12) could develop. These

fi ndings were recently extended with a study which showed that these type of mice were

susceptible to HIV infection (13, 14). This break-trough will allow more detailed studies of

HIV infection in an experimental setting. Especially the fact that these in vivo models are

more accessible than monkey models, will give many more researchers the opportunity to

study the role of Nef during HIV infections in vivo.

Concluding remarks

With the development of the models we present in this thesis, we made a step forward in

Nef biology. These models provide great tools to verify in vitro described Nef interactions

and functions in vivo and will thereby aid to defi ne the crucial mechanisms which defi ne Nef

as a pathogenicity factor in HIV infection.

GENERAL DISCUSSION 105

REFERENCES

1. Arold, S. T., and A. S. Baur. 2001. Dynamic Nef and Nef dynamics: how structure could explain the complex

activities of this small HIV protein. Trends Biochem Sci 26:356-363.

2. Geyer, M., O. T. Fackler, and B. M. Peterlin. 2001. Structure--function relationships in HIV-1 Nef. EMBO Rep

2:580-585.

3. Arora, V. K., B. L. Fredericksen, and J. V. Garcia. 2002. Nef: agent of cell subversion. Microbes Infect 4:189-

199.

4. Craig, H. M., M. W. Pandori, N. L. Riggs, D. D. Richman, and J. C. Guatelli. 1999. Analysis of the SH3-binding

region of HIV-1 nef: partial functional defects introduced by mutations in the polyproline helix and the

hydrophobic pocket. Virology 262:55-63.

5. Garcia, J. V., and J. L. Foster. 1996. Structural and functional correlates between HIV-1 and SIV Nef isolates.

Virology 226:161-166.


497.

7. Baur, A. S., E. T. Sawai, P. Dazin, W. J. Fantl, C. Cheng-Mayer, and B. M. Peterlin. 1994. HIV-1 Nef leads to

inhibition or activation of T cells depending on its intracellular localization. Immunity 1:373-384.

8. Melton, E., N. Sarner, M. Torkar, P. A. van der Merwe, J. Q. Russell, R. C. Budd, C. Mamalaki, M. Tolaini,

D. Kioussis, and R. Zamoyska. 1996. Transgene-encoded human CD2 acts in a dominant negative fashion to

modify thymocyte selection signals in mice. Eur J Immunol 26:2952-2963.

9. Zhumabekov, T., P. Corbella, M. Tolaini, and D. Kioussis. 1995. Improved version of a human CD2 minigene

based vector for T cell-specifi c expression in transgenic mice. J Immunol Methods 185:133-140.

10. Lang, G., D. Wotton, M. J. Owen, W. A. Sewell, M. H. Brown, D. Y. Mason, M. J. Crumpton, and D. Kioussis.

1988. The structure of the human CD2 gene and its expression in transgenic mice. Embo J 7:1675-1682.



AIDS. Cell 65:651-662.

12. Traggiai, E., L. Chicha, L. Mazzucchelli, L. Bronz, J. C. Piffaretti, A. Lanzavecchia, and M. G. Manz. 2004.

Development of a human adaptive immune system in cord blood cell-transplanted mice. Science

304:104-107.

13. Zhang, L., G. I. Kovalev, and L. Su. 2006. HIV-1 infection and pathogenesis in a novel humanized mouse

model. Blood.

14. Baenziger, S., R. Tussiwand, E. Schlaepfer, L. Mazzucchelli, M. Heikenwalder, M. O. Kurrer, S. Behnke, J. Frey,

A. Oxenius, H. Joller, A. Aguzzi, M. G. Manz, and R. F. Speck. 2006. Disseminated and sustained HIV infection

in CD34+ cord blood cell-transplanted Rag2-/-gamma c-/- mice. Proc Natl Acad Sci U S A 103:15951-15956.

SUMMARY

108

In HIV infection CD4 T cells and antigen presenting cells get infected, which eventually

leads to the loss of CD4 T cells resulting in the development of AIDS. The implication of Nef,

one of the regulatory proteins of HIV, as an important factor for pathogenicity during HIV

infection was established in the early 90’s. It was observed that Nef was crucial for in vivo

replication and progression to AIDS in rhesus monkeys infected with the HIV related SIV. The

importance of Nef as a pathogenicity factor in HIV for development to AIDS was revealed

in a few seminal studies that clearly showed that HIV Nef deletion mutants could establish

infection but the infection was associated with slow progression to HIV-1 disease. To explain

its role in pathogenesis, many studies have been performed to study the function of this

protein. Two widely studied and general accepted effects of Nef are the down-regulation of

CD4 and MHC class-I, to circumvent super-infection and immune-escape respectively.

Besides these effects on receptor distribution at the cell membrane of T cells, several

studies have shown that Nef disturbs TCR signalling, resulting in increased T cell activation.

This Nef-mediated T cell activation has been suggested to result in higher viral replication

and increased pathogenicity. As most studies describing this Nef-mediated increase in T

cell activation were performed in cell lines, which have a much higher basal activation level

than primary T cells, we addressed the function of Nef in T cell activation in constitutive

Nef transgenic mice (chapter 2). The T cells in these mice had been earlier described to

be activated. During the examination of characteristics of T cell activation in constitutive

Nef transgenic mice, we came to the conclusion that the observed activation in this model

was caused by homeostatic mechanisms, resulting from Nef-mediated thymic atrophy,

rather than by direct Nef-mediated effects on T cells. This suggested that Nef expression

does not lead to spontaneous T cell activation upon expression in primary T cells in vivo.

This fi nding was substantiated in conditional Nef transgenic mice (chapter 3) with tightly

regulated Nef protein induction in the T cell compartment. We generated these mice in

order to circumvent Nef-mediated effects on thymic development and secondary effects on

peripheral T cell compartment. When Nef expression was induced, a rapid thymic atrophy

occurred, substantiating the Nef-mediated atrophy observed in constitutive Nef transgenic

mice.

Under normal conditions, the majority of T cells in mice are not activated. To study

effects of Nef on T cell activation after TCR stimulation, T cells of inducible Nef transgenic

mice were stimulated by infection of the mice with infl uenza virus. We showed that Nef

expression in T cells leads to a reduced anti-viral T cell response, suggesting a role for Nef

in the disturbance of T cell function and possibly TCR signalling (chapter 4).

The infection of antigen presenting cells (APCs) by HIV is important for viral spread

and replication. Since several studies reported a Nef-mediated stimulation of HIV replication

in APCs, we generated mice with inducible Nef expression in antigen presenting cells (APCs)

and studied Nef effects in APCs during infl uenza infections (chapter 4). We showed that Nef

expression in APCs results in increased T cell responses. This suggests a role for Nef in the

SUMMARY

109

stimulation of T cell activation via APCs, in this way increasing the likelihood of HIV infection,

as activated T cells are more prone to HIV infection than naïve T cells.

In our studies we used an inducible transgenic system, where doxycycline was used

as a reagent to induce gene transcription. To interpret our data correctly, the side-effects of

this compound on T cell function were studied (chapter 5).

With the development of the models we present in this thesis, we made a step forward in Nef

biology. The fi ndings that Nef expression in T cells does not lead to spontaneous activation

of T cells, that it does disturb T cel function, and that Nef expressing APCs stimulate T

cell activation are just the fi rst Nef-mediated in vivo functions to be discovered with these

models. The inducible Nef transgenic models provide great tools to verify in vitro described

Nef interactions and functions in vivo and will thereby aid to defi ne the crucial mechanisms

which defi ne Nef as a pathogenicity factor in HIV infection.

SUMMARY

NEDERLANDSE SAMENVATTING

112

Immuunsysteem

Het immuunsysteem bestaat uit een samenwerkingsverband tussen verschillende cellen

in het lichaam die samenwerken om schadelijke cellen en organismen (kankercellen,

bacteriën, schimmels en virussen) uit ons lichaam te weren. Om te voorkomen dat deze

organismen schade kunnen aanrichten, is het van groot belang dat ze snel herkend en

uitgeschakeld worden. Tijdens de evolutie hebben we ons ontwikkeld in de nabijheid van

dergelijke organismen en zijn er tal van afweermechanismen, tesamen het immuunsysteem,

in ons lichaam ontstaan die ons beschermen. Het immuunsysteem kan opgedeeld worden

in twee onderdelen. Een onderdeel is het aangeboren immuunsysteem, en zoals de naam

al doet vermoeden krijgen we de informatie benodigd voor dit systeem mee via het DNA

van onze ouders. De cellen behorend tot dit systeem herkennen goed geconserveerde

karakteristieke patronen van organismen, deze informatie is verzameld en opgeslagen in

ons DNA gedurende de evolutie.

Het andere onderdeel van het immuunsysteem heeft de capaciteit om zich aan te

passen aan specifi eke structuren en cellen. Dit systeem komt alleen voor in hoger ontwikkelde

dieren en is ontstaan om de snel veranderende virussen en bacteriën de baas te kunnen.

Dit systeem wordt gecoördineerd door cellen die door het hele lichaam reizen en op plekken

met problemen willekeurig celfragmenten en moleculen opnemen (antigenen genaamd)

die ze, eenmaal aangekomen in de lymfoide organen presenteren op hun celoppervlak.

T cellen, gevormd in de thymus reizen door de bloedbaan van het lichaam en controleren

de antigenen die antigeen presenterende cellen (APCn) in de lymfoide organen op hun

celoppervlak presenteren. Om antigenen te kunnen herkennen hebben ze een molecuul op

het oppervlak, de T cel receptor (TCR) genaamd. Als ze iets lichaamsvreemds herkennen

gaat er een signaal via de TCR de T cel in en wordt de T cel geactiveerd, waardoor een

afweerreactie van start gaat.

HIV-infectie en AIDS

Momenteel zijn er wereldwijd ongeveer 40 miljoen mensen geïnfecteerd met humaan

immunodefi ciëntie virus (HIV). HIV infecteert zowel de T cellen als de APCn van het

immuunsysteem. Direct na een HIV infectie ontstaat er een afweerreactie tegen het virus,

maar doordat het virus constant verandert ontsnapt het keer op keer aan het immuunsysteem.

Dit resulteert in een constante aanwezigheid van een hoeveelheid virus in het lichaam en

een constante afname van het aantal T cellen.

Gedurende de eerste jaren na HIV infectie is er niets aan de hand, HIV+ mensen zijn

relatief gezond en ondervinden geen last van de HIV infectie. Echter, na een aantal jaren komt

de hoeveelheid T cellen in het lichaam onder een kritieke grens en is het immuunsysteem

te verzwakt om goede afweerreacties te vormen. In dit stadium van de HIV infectie wordt er

gesproken van AIDS (Acquiered Immuno-defi ciency syndrome). Deze verzwakking van het

immuunsysteem maakt het voor organismen makkelijker om het lichaam binnen te dringen


113

en zichzelf te vermenigvuldigen. Dit resulteert vervolgens in allerlei infecties die uiteindelijk

tot de dood kunnen leiden van de met HIV geïnfecteerde persoon. Om de ziekte te kunnen

bestrijden is het van groot belang om te weten wat de exacte oorzaak is van de afname van

het aantal T cellen. De meest voor de hand liggende oorzaak, directe infectie van T cellen

met HIV, is zeer onwaarschijnlijk, aangezien maar 0.1% van de totale hoeveelheid T cellen

geïnfecteerd wordt.

Een andere mogelijke verklaring kan zijn dat door HIV infectie van de thymus er

een verlaging van de aanmaak van T cellen optreed, wat uiteindelijk resulteert in minder

T cellen. Weer een andere verklaring is gebaseerd op de observatie dat er een algehele

activatie van de T cellen plaatsvindt, wat zich uiteindelijk uit in en verhoogde dood van

de T cellen. Deze constante activatie van T cellen wordt momenteel gezien als de meest

waarschijnlijke oorzaak voor het verlies van T cellen en de ontwikkeling tot AIDS. Hoe HIV

deze constante activatie precies veroorzaakt is echter nog niet duidelijk.

HIV-Nef

HIV is opgebouwd uit meerdere moleculen, eiwitten genaamd. Ieder eiwit heeft een rol

in het optimaal laten functioneren van het virus. Een aantal eiwitten zijn nodig bij het

binnendringen van de cel, voor het inbouwen van viraal DNA en voor de replicatie van nieuw

virus. Naast deze eiwitten bevat HIV ook nog een aantal regulatoire eiwitten, waaronder Nef.

Het belang van Nef voor het voortbestaan van het virus werd voor het eerst aangetoond in

een studie waarbij apen werden geïnfecteerd met de apen variant van het HIV virus, SIV.

Door gebruik te maken van SIV met en zonder Nef, werd aangetoond dat Nef van groot belang

was voor een hoge virus replicatie en de ontwikkeling tot AIDS. Dezelfde conclusie werd

getrokken uit een aantal studies van HIV geïnfecteerde mensen die na zeer lange infectie-

tijd nog steeds geen AIDS ontwikkeld hadden. Het bleek dat deze mensen een HIV variant

zonder Nef hadden. De grote vraag daarna was, op wat voor manier het Nef eiwit betrokken

was bij het verhogen van de virus replicatie en de snellere ontwikkeling tot AIDS.

Na heel wat jaren van onderzoek kwamen er een aantal dingen aan het licht. In een

goed functionerende cel, wordt er constant informatie op het celoppervlak gepresenteerd, dit

maakt het mogelijk voor de cellen van het immuunsysteem om aan de buitenkant van een

cel te zien of deze cel nog goed funcioneert of dat hij geïnfecteerd is met een organisme.

Door dit proces te verstoren, zorgt Nef ervoor dat het immuunsysteem niet goed in staat is

om te zien dat een T cel geïnfecteerd is met HIV. Dit geeft het virus extra tijd om zichzelf

te vermenigvuldigen voordat de cel vernietigd wordt door het immuunsysteem. Verder werd

bekend dat Nef het signaal van de TCR manipuleert. Door manipuleren van dit signaal kan

Nef de activatie van T cellen stimuleren, waardoor het voor HIV makkelijker wordt om zich te

vermigvuldigen. Of het manipuleren van Nef van dit TCR signaal daadwerkelijk voor activatie

zorgt van T cellen was niet duidelijk en wij wilden dit fenomeen verder onderzoeken.


114

Dit Project

De studies die beschrijven dat Nef tot activatie van T cellen leidt, zijn over het algemeen

uitgevoerd in tumor-T cellijnen en niet in normale T cellen afkomstig uit mens of muis. Om

een delicaat proces als T cel activatie te bestuderen is het van groot belang om normale

T cellen te gebruiken. Aangezien de muis een immuunsysteem heeft wat behoorlijk goed

overeenkomt met het immuunsysteem van de mens, hebben we ervoor gekozen om muizen

te gebruiken om ons onderzoek in uit te voeren.

De Nef transgene muizen die we voor onze eerste studies gebruikten, waren zo

gemanipuleerd dat ze het Nef eiwit tot expressie brachten in al hun T cellen en thymocyten

vanaf vroeg in de embryonale ontwikkeling. Zoals al eerder was beschreven, leidt de expressie

van Nef in thymocyten uiteindelijk tot een sterke reductie van het aantal thymocyten en het

aantal T cellen. De nog wel aanwezige T cellen in deze muizen zijn enorm geactiveerd en

delen sneller dan normaal. Het leek er dus op dat Nef expressie in T cellen tot activatie

van T cellen leidde. Echter, een mogelijk andere verklaring was dat de lage hoeveelheid T

cellen in de Nef transgene muizen werden gestimuleerd om meer te delen om die leegte

op te vullen. Om uit te zoeken of de activatie veroorzaakt werd door een direct effect van

Nef op T cellen of door een indirect effect vanwege de lege omgeving, hebben we in eerste

instantie normale T cellen in Nef transgene muizen gespoten. We zagen dat deze T cellen

ook geactiveerd raakten, dus het leek erop dat de omgeving in deze muizen de activatie van

T cellen veroorzaakt. Om dit te bevestigen, lieten we Nef transgene T cellen ontwikkelen in

een normaal (vol) T cel compartiment, het bleek dat onder deze omstandigheden de Nef

transgene T cellen er normaal uitzagen en niet geactiveerd waren (hoofdstuk 2). Kortom,

Nef lijkt niet tot activatie in T cellen te leiden.

Aangezien dit Nef transgene muizenmodel niet ideaal leek om verdere studies van

Nef op de T cel functie te bestuderen, ontwikkelden we een nieuw muizenmodel waarbij we

het Nef eiwit ‘aan’ en ‘uit’ konden zetten wanneer we zelf wilden. Dit model noemden we

het induceerbare Nef transgene muizenmodel. Door de aanwezigheid van het Nef eiwit te

kunnen reguleren voorkwamen we effecten van Nef op de thymus tijdens de ontwikkeling

van T cellen met alle secundaire gevolgen van dien. Door toediening van doxycycline aan

de muizen konden we het Nef eiwit ‘aan’ en ‘uit’ zetten. Op verschillende dagen na de start

van Nef inductie werden de muizen geanalyseerd, het bleek dat de T cellen in de lymfoïde

organen van muizen wel geïnduceerd waren, maar geen enkel teken van activatie lieten

zien, ze zagen er normaal uit. De thymocyten daarentegen verdwenen snel na Nef inductie.

Dus we laten zien dat Nef expressie op zichzelf niet tot activatie leidt van T cellen maar wel

tot een snelle reductie van het aantal thymocyten (hoofdstuk 3).

In een normale situatie is het grootste gedeelte T cellen in een muis niet geactiveerd.

Om T cellen te activeren moet je ze o.a. stimuleren via hun TCR. Om te onderzoeken of

Nef een effect had op T cel functie nadat de TCR gestimuleerd was, werden de muizen

geïnfecteerd met infl uenza virus. Door deze infectie werden de muizen een beetje ziek en


115

ontwikkelden ze een immuunrespons tegen het virus met o.a. hun T cellen. Door deze T

cel respons te bestuderen in de aan- en afwezigheid van Nef eiwit, zagen we dat Nef eiwit

expressie in T cellen tot een minder goede T cel respons leidde. Dus het lijkt erop dat Nef

expressie de T cel functie verstoort (hoofdstuk 4).

De infectie van APCn door HIV is belangrijk voor de verspreiding van HIV naar

T cellen, verder wordt er meer HIV geproduceerd door T cellen die in contact staan met

APCn in vergelijking met T cellen die geen contact met APCn hebben. Om de rol van Nef

hierin te onderzoeken, maakten we een muizen model waarin Nef tot expressie kwam in

APCn. Ook deze muizen infecteerden we met infl uenza virus in de aan- en afwezigheid van

Nef eiwit. In tegenstelling tot de verminderede T cel respons wanneer Nef eiwit in T cellen

aanweizg was, leidde Nef eiwit expressie in APCn tot een toename van de anti-virale T cel

respons. Dus het lijkt erop dat Nef eiwit in APCn leidt tot verhoogde T cel activatie en een

verhoogde T cel respons. Een mogelijke verklaring is dat Nef T cel activatie stimuleert via

APCn om daarmee de infectie kans van HIV te vergroten, aangezien geactiveerde T cellen

gemakkelijker geïnfecteerd kunnen worden dan rustende T cellen. Hoe het Nef eiwit deze

activatie precies veroorzaakt is nog niet duidelijk en verder onderzoek zal dit uit moeten

wijzen (hoofdstuk 4).

Tijdens het gebruik van ons induceerbare transgene muizenmodel, observeerden

we een aantal directe effecten van doxycycline, de chemische stof om het Nef eiwit ‘aan’ te

zetten, in T cellen. Na toediening van doxycycline zagen we een tijdelijke reductie van het

aantal T cellen in het bloed en de lymfoïde organen. Verder remde doxycycline de deling van

T cellen in vivo en in vitro (hoofdstuk 5).

Conclusie

Met de ontwikkeling van de Nef transgene muizenmodellen beschreven in dit proefschrift

hebben we een stap voorwaarts gemaakt in het onderzoek naar de functie van Nef gedurende

HIV infectie. De bevindingen dat Nef eiwit expressie in T cellen niet leidt tot spontane T cel

activatie, dat het wel de T cel functie verstoord en dat Nef eiwit expressie in APCn tot T cel

activatie leidt, zijn nog maar de eerste Nef-gemedieerd in vivo effecten die ontdekt zijn

met behulp van onze modellen. De induceerbare Nef transgene muizenmodellen bieden

de mogelijkheid om in vitro gevonden bevindingen te verifi ëren in een in vivo model, en

zullen daardoor bijdragen aan het vinden van de cruciale mechanismen die Nef zo belangrijk

maken gedurende HIV infectie.


NAWOORD

118

na•woord (het ~), 1 slotwoord in een geschrift => epiloog, nabericht, narede

Nawoord, het woord erna, in dit geval dus het woord na mijn promotie onderzoek. Wat valt

er nog te zeggen nu ik al zoveel geschreven heb? Het nawoord handelt wat mij betreft over

de secundaire promotie-voorwaarden, je schrijf een heel boek vol over je wetenschappelijke

bevindingen en de mogelijke implicaties hiervan. Het nawoord gaat over hoe je het ervaren

hebt als persoon om je promotie onderzoek te volbrengen. Tja, een aantal termen, die

wat mij betreft van toepassing zijn op mijn promotie onderzoek: veelzijdig, uitdagend,

teleurstellend, gezellig, masochistisch, zelfstandig, lastig, leerzaam, eenzaam, sociale

ontwikkeling, doorzettingsvermogen, obsessief, analytisch denkvermogen, relativeren...

De grote vraag, hoewel die er nu niet meer toe doet, is natuurlijk: als ik geweten had wat

me te wachten stond, zou ik het dan nog steeds doen? JA, zeker wel, ik ben blij dat het af is,

maar ik vind het een zeer waardevolle periode waarin ik op heel veel vlakken ontzettend veel

geleerd heb, waar ik in de toekomst nog veel aan zal hebben. ‘Toekomst, here I come!’ Maar

goed, gedurende zo’n promotie periode zijn er allerlei mensen op verschillende manieren bij

betrokken geweest, die allemaal bijgedragen hebben aan het eindresultaat, dit brengt mij

tot het populairste onderdeel, het dankwoord.

dank•woord (~het), 1 dankbetuiging

Ik begin ermee om iedereen te bedanken die, op wat voor manier dan ook, betrokken is

geweest bij mijn onderzoek. Mensen helpen vind ik zelf zeer belangrijk, en ik waardeer

hulp van een ander dan ook enorm. Ik zal/kan niet iedereen die bijgedragen heeft bij naam

noemen maar ik weet wie me geholpen heeft en daarvoor dank.

Ik wil beginnen om mijn promotor Frank Miedema te bedanken, van een samenwerking

op afstand werd je ineens mijn promotor, dat pakte voor mij heel goed uit. Ik bewonder je

inzet en passie voor het vak enorm en ik ben zeer blij met je hulp en inzicht gedurende mijn

promotie.

Mariëtte, mijn co-promotor van het eerste uur. Jouw contacten en kennis van de transgenese

zijn van essentieel belang geweest voor het tot stand komen van ons induceerbare transgene

model. Wat heb ik een boel geleerd van jou. Je positieve en enthousiaste houding werkte

altijd zeer motiverend, zeker als ik het even niet zag zitten.

Kiki, mijn co-promotor van het tweede uur, ik heb het enorm gewaardeerd om met jou

samen te werken en ben zeer blij dat jij je ontfermt hebt over mijn project. Na jouw komst

zijn we andere paden ingeslagen en hebben we hele mooie experimenten gedaan. Het was

zeer leerzaam om met iemand te werken met zo’n scherpe kijk op ruwe data en zo’n goed

analytisch vermogen.

NAWOORD

119

Naast de categorie (co-) promotoren, is er nog een andere zeer belangrijke categorie van

mensen die me bijgestaan hebben met de meer praktische problemen tijdens mijn promotie

onderzoek. Om te beginnen in deze categorie, en met stip op één wil ik Frans (Fransie de

Pansie) enorm bedanken, zonder jouw hulp had ik al die enorme muizen proeven met een

record-aantal van 4 transgenen in een muis nooit uit kunnen voeren. Jouw expertise, inzet

en onophoudelijke optimisme zijn onmisbaar geweest voor dit resultaat, enorm bedankt, het

was me een eer om met jou te mogen werken, ouwe paranimf!

In dezelfde categorie met stip op twee, Elise, mijn buurvrouw op het lab, jouw nuchtere kijk

en jouw wil om altijd te helpen als je tijd had waren zeer welkom en ik ben je daar ook zeer

dankbaar voor.

Petra ook enorm bedankt voor de hulp tijdens het begin van mijn promotie onderzoek.

Dan zijn er natuurlijk ook nog mede-aio’s, in mijn geval één binnen de groep, Miranda, het

was fi jn dat jij al zoveel dingen had opgezet en dat je meedacht met de experimenten.

Verder wil ik Ronald en Kees-Jan als studenten werkzaam op o.a. mijn project ook bedanken

voor hun hulp en inzet.

Naast deze mensen die allemaal direct betrokken waren bij mijn project wil ik ook graag alle

andere collega’s bedanken voor de hulp, suggesties en gezelligheid. Ook de Coffers op de

1e verdieping natuurlijk, jullie korte aanwezigheid op het grote lab, zorgde voor een enorme

leegte toen jullie weg waren.

Een paar mensen die ik speciaal wil noemen zijn Jeffrey, Jantine, Annette en Mark, de oude

garde, ik heb zeer genoten van jullie aanwezigheid zowel binnen als buiten het lab en heb

met enorm veel plezier met jullie gewerkt. Tinus, als ik nog een 3e paranimf zou mogen

kiezen, zou jij dat zonder twijfel zijn, bedankt dat je er altijd was en dat ik alle ins en ‘autsj’

bij je kwijt kon. Succes met jouw eindsprint, je weet het: geen planning maken, gewoon

blijven ademhalen, niks aan de hand.

Guido, al een tijdje niet meer op de afdeling, maar onze gesprekken en jouw humor staan

mij nog steeds duidelijk voor de geest. Robert, ook al een tijdje weg, maar jouw hulp in

het begin met mijn FACs analyses en intstellingen en betrokkenheid bij het onderzoek heb

ik zeer gewaardeerd. Ludo, ook al een tijdje een oud-collega, het was me altijd een waar

genoegen om je te spreken.

Verder wil ik ook nog de mensen van AIO-kamer 3 bedanken voor de gezelligheid en wens

ik Rogier en Kees veel succes met het behouden van het evenwicht, ‘testosteron rules!’ Zorg

dat het kippen-hok-gehalte niet al te hoog wordt met al die vrouwen!

En dan natuurlijk het rustpunt van de afdeling, het secretariaat, altijd fi jn om even die oase

van rust binnen te stappen, Saskia en Yvonne bedankt.

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Ook wil ik alle mensen op het GDL bedanken die betrokken zijn geweest bij mijn onderzoek.

In het bijzonder, Toon, Anja, Sabine, Helma, Tamara, Joyce, Wendy en Kitty.

I’d like to thank Bruno Verhasselt, Veronique Stove, Andreas Baur, Dietlinde Wolf, Olivier

Schwartz and Nathalie Sol-Foulon who gave advice and provided technical assistance for

some Nef problems we encountered.

Tijdens deze periode van je leven kom je erachter hoe belangrijk en bepalend je opvoeding

is geweest, papa en mama bedankt voor de goede basis en de vrijheid om mijn eigen pad

te kiezen en het vertrouwen. Mijn enige echte grote zus, Esther en niet meer weg te denken

zwager Arne, bedankt voor jullie steun, het is altijd fi jn om wat afl eiding te vinden bij

mensen van wie je houdt. En dan natuurlijk mijn ‘nieuwe’ familie, Dorien, Yuri en Jantien,

bedankt voor jullie interesse en gezelligheid.

Vrienden, ik zou niet weten wat ik zonder jullie zou moeten beginnen! Er gaat toch niets

boven een verfrissende kijk op dingen naast de wereld die promoveren en biomedische

wetenschap heet. Lang leve het relativeren!

De oplossing voor alle problemen, een avond fl ink stappen en de volgende dag ziet het

leven er weer heel anders uit, toch? Femke, Frikke, Bas, Frank, Monique, Esther, Arne,

Maaike, Yvonne, Willem, Claire, Marije, Valentine, Esther B en Rens. Dat we dat nog maar

vaak mogen doen, 23 maart misschien? Ik weet nog wel een leuk feestje. Verder natuurlijk

Chantal en Ruben en Leila en Joost altijd gezellig om bij jullie te zijn!

En dan natuurlijk de twee-wekelijkse mannenavond; Frik, Bassie, Frankie en Willem, het is

altijd goed om met jullie te zijn, even alles eruit en de volgende dag weer door met het leven

(en koppijn), HEERLIJK!

Bas, het is altijd fi jn om met iemand te praten buiten de afdeling die wel precies weet hoe

het werkt, enorm bedankt voor je luisterend oor, of het nou bagger of emotie was (of iets

daar tussenin). Top(s) dat jij mijn paranimf bent.

Frikke, ouwe makker door dik en dun, al jaren en jaren gaan we terug en nog steeds beste

maatjes ik denk niet dat dat ooit zal veranderen, een heerlijk idee, dank dat je er altijd bent,

een dikke Snok erbovenop!

Fem, de vrouw van mijn leven, wat heb ik toch een geluk om met jou samen te mogen leven,

heerlijk dat je er altijd bent, door dik en dun, UCH!

Zo, dat was het dan... Nu door naar de volgende uitdaging!

P

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CURRICULUM VITAE

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De schrijver van dit proefschrift is geboren op 30 maart 1978 te ‘s-Hertogenbosch. In

1996 werd het VWO examen behaald, afgelegd aan het Prins Willem Alexander College

te Leerdam. In datzelfde jaar startte hij de studie Medische Biologie aan de Universiteit

Utrecht. Tijdens deze studie deed hij onderzoekservaring op tijdens twee stages. Een stage

werd afgelegd op de vakgroep Immunotoxicologie van de faculteit diergeneeskunde onder

begeleiding van Dr. R. Pieters en Dr. A. Gennari, onderzoek werd gedaan naar het effect van

organotins op thymocyten. De hoofdvak stage werd gevolgd bij de afdeling Immunologie van

het Universitair Medisch Centrum Utrecht/Crucell onder begeleiding van Dr. W. Germeeraad

en Prof. dr. T Logtenberg, onderzoek werd gedaan om nieuwe antilichamen te vinden tegen

gezuiverde eiwitten met behulp van de phage display techniek. Het doctoraal examen werd

afgelegd in 2001. In 2002, na een reis van zes maanden in Zuid-Oost Azië, startte hij zijn

promotie onderzoek aan de vakgroep Immunologie van het Universitair Medisch Centrum

Utrecht onder begeleiding van Dr. M.A. Oosterwegel en later ook van Dr. K. Tesselaar en Prof.

dr. F. Miedema. De resultaten van dit onderzoek zijn beschreven in dit proefschrift.

CURRICULUM VITAE

biology of hiv-nef in vivo - universiteit utrecht · frank miedema, kiki tesselaar department of...

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