CD5-MEDIATED INHIBITION OF T LYMPHOCYTE SIGNALING · CD5-MEDIATED INHIBITION OF T LYMPHOCYTE SIGNALING ... A central role for Fyn in the CD5-mediated inhibition of T lymphocyte signaling

CD5-MEDIATED INHIBITION OF

T LYMPHOCYTE SIGNALING

Ph.D. Thesis

Dissertation to obtain the Degree of Doctor in Biomedical Sciences, speciality in

Immunology, submitted to the Abel Salazar Institute of Biomedical Sciences,

University of Porto

MARTINA BAMBERGER

Porto, 2008

Supervisor: Dr. Alexandre M. Carmo

Co-supervisor: Dr. Mónica A. Castro

Institute for Molecular and Cell Biology

Rua do Campo Alegre, 823, 4150-180 Porto, Portugal

Department of Molecular Immunology and Pathology

Abel Salazar Institute for Biomedical Sciences

Largo Prof. Abel Salazar, 2, 4099-003 Porto, Portugal

The research described in the present thesis was performed at the Institute for Molecular

and Cell Biology, Porto, Portugal, at the Dana-Farber Cancer Institute, Harvard Medical

School, Boston, USA and at the Institute of Immunology, Medical University of Vienna,

Austria. Martina Bamberger was the recipient of a studentship from the Fundação para a

Ciência e a Tecnologia – Programa Operacional Ciência, Tecnologia, Inovação (POCTI).

Additional support was from the Federation of European Biochemical Societies (FEBS),

the Boehringer Ingelheim Fonds (BIF), and the American Portuguese Biomedical

Research Fund (APBRF).

TABLE OF CONTENTS

Summary, Sumário, Sommaire

Publications

I General Introduction

1

II Results

CHAPTER 1

The accessory receptor CD5 down-regulates T lymphocyte

proliferation

CHAPTER 2

A central role for Fyn in the CD5-mediated inhibition of T

lymphocyte signaling

CHAPTER 3

The membrane distribution of CD5 and CD5-associated signaling

effectors

Concluding Remarks

39

40

49

67

81

III Material and Methods

88

IV Appendix

Abbreviations

References

Acknowledgments

96

97

100

122

Summary

The coordinated action of antigen receptors and accessory surface molecules

enables immune cells to provide an adjusted response to foreign elements. The CD5

glycoprotein is considered to be an accessory receptor that modulates signals arising from

the T cell receptor (TCR). Analysis of CD5-deficient mice has indicated that CD5

attenuates TCR responses, an effect that is greatly amplified in the CD2/CD5 double-

knockout mouse. Although these data gave important insights into the inhibitory role of

CD5, they have not been adequately complemented by biochemical analysis of the

mechanisms involved and by studies of the human cell system. In this study, we focused

on the human system, and established that CD5 plays an important role in the initiation of

TCR signaling by specifically modulating tyrosine phosphorylation of the Src family kinase

Fyn, a crucial kinase in the early steps of T cell activation. CD5 induced the

phosphorylation of Fyn at the negative regulatory tyrosine residue (Y531) which resulted

in a reduction of the kinase’s activity. The ensuing downstream signaling was significantly

restrained, namely the activity of another central kinase of early T cell signaling events,

ZAP-70. Moreover, the phosphorylation of the adapter PAG at Y317, a tyrosine residue

that is uniquely targeted by Fyn, was down-regulated upon CD5 engagement. Given that

many of the downstream enzymes and adapters of these signaling pathways localize

within lipid rafts, we investigated whether CD5 would move from the fluid phase of the

membrane once stimulated. Confirming our assumptions, CD5 molecules are in fact

massively translocated to lipid rafts upon antibody-mediated stimulation. We provide

evidence that CD5 forms dimers on the surface of resting T cells, a feature that can help

explaining the extent of CD5 translocation between the different membrane phases.

Based on the results of this study, we suggest a model of CD5-controlled T cell inhibition:

CD5 triggering induces its relocalization to lipid rafts, where it interacts with important

signaling effectors, including Fyn, Lck, PAG and LAT. A CD5-associated kinase is then

responsible for Fyn tyrosine phosphorylation and decreased Fyn activity, following which

PAG phosphorylation and association of PAG with the C-terminal Src kinase Csk is

reduced. This CD5-controlled modulation of the PAG/Csk/Fyn loop, together with the

CD5-mediated recruitment of other accessory surface receptors (like CD2) ultimately

down-regulates long-term T cell responses, such as T cell proliferation. Overall, this study

indicates that CD5 plays an inhibitory role in TCR signaling by controlling one of the

earliest steps of T cell signaling, namely the activation of Fyn. The model we propose here

may help to understand the molecular basis of beneficial therapeutic effects that have

been demonstrated for anti-CD5 antibodies in certain autoimmune disorders and in bone

marrow transplantation.

Sumário

A acção coordenada do receptor da célula T (TCR) para antigénios e das

moléculas acessórias presentes na superfície de linfócitos T permitem a estas células do

sistema imune coordenar uma resposta adequada para combater os elementos estranhos

ao sistema. A glicoproteína CD5 é considerada um receptor acessório que tem um efeito

modulatório sobre o sinal proveniente do TCR. A análise de ratinhos CD5-deficientes

indica que o CD5 atenua as respostas via TCR, um efeito que é aumentado

significativamente na dupla-deficiência CD2/CD5. Apesar de os dados obtidos a partir de

animais knock-out terem sido fundamentais para o estabelecimento do CD5 como um

mediador inibitório da sinalização, não foi no entanto ainda estabelecida uma correlação

entre as observações verificadas nos modelos animais e os dados bioquímicos obtidos

nos estudos in vitro usando células humanas. Neste estudo, focámo-nos num modelo

humano de activação celular, e estabelemos que o CD5 desempenha um papel

importante no controlo da iniciação das vias de sinalização via TCR através da

modulação específica da actividade da cinase Fyn. Esta cinase de tirosinas, pertencente

à família Src, é uma cinase crucial nos primeiros passos da activação das células T, e

verificámos que a estimulação de CD5 induz a fosforilação de Fyn num resíduo de

tirosina regulatório negativo (Y531), o que resultou numa redução da actividade da

cinase. A sinalização subsequente foi significativamente afectada, nomeadamente a

actividade de outros enzimas envolvidos nas cascatas de sinalização, tais como a cinase

ZAP-70 cuja actividade foi deveras reprimida. Igualmente, a fosforilação da Y317 do

adaptador PAG, um resíduo de tirosina unicamente fosforilado por Fyn, diminuiu após a

estimulação do CD5. Muitos dos enzimas e adaptadores destas vias de sinalização

localizam-se nos lipid rafts, microdomínios existentes na membrana celular, pelo que

investigámos se de facto o CD5 é transferido da fase fluída da membrana para os lipid

rafts após estimulação. A nossa hipótese confirmou-se, uma vez que as moléculas de

CD5 são massivamente translocadas para os rafts após estimulação mediada por

anticorpos contra CD5. Providenciamos igualmente evidências de que o CD5 forma

dímeros na superfície de células não activadas, uma característica que ajuda a explicar a

extensão da translocação entre as fases da mebrana. Baseados nos resultados deste

estudo, propomos um modelo da inibição das células T controlada pelo CD5: a activação

do CD5 induz a sua relocalização para os lipid rafts onde interage com importantes

efectores de activação, como Fyn, Lck, PAG e LAT. Uma cinase associada ao CD5 é

depois responsável pela fosforilação da tirosina inibitória de Fyn e pela diminuição da sua

actividade, com a consequente diminuição da fosforilação de PAG e decréscimo da

associação PAG-Csk. Esta regulação do loop PAG/Csk/Fyn através do CD5, em conjunto

com o recrutamento de outros receptores de superfície acessórios (como o CD2) provoca

por fim, uma diminuição da respostas subsequentes das células T, tais como a

proliferação. De uma forma global, este estudo indica que o CD5 desempenha uma papel

inibidor na sinalização via TCR ao controlar um dos primeiros passos descritos na

sinalização: a activação da cinase Fyn. O modelo aqui proposto pode ajudar a

compreender a base molecular dos efeitos terapêuticos benéficos que foram

anteriormente demonstrados para anticorpos anti-CD5 em algumas doenças autoimunes

e no transplante de medula óssea.

Sommaire

L’action cordonnées des récepteurs a antigène et des molécules accessoires de

surfaces permettent au système immunitaire d’induire une réponse adéquat aux éléments

étrangers. La glycoprotéine CD5 est considérée comme étant un récepteur accessoire qui

module le signal provenant du récepteur des cellules T (TCR). L’analyse de souris

mutante pour CD5 indique que CD5 atténue les réponses du TCR, cet effet étant amplifie

chez le souris double mutante CD2/CD5. Bien que ces résultats indiquent le rôle inhibiteur

de CD5, aucune études biochimiques ainsi que sur un modèle cellulaire humains n’a à ce

jour été présenté. Dans cette étude, nous avons concentré nos efforts sur un modèle

humain, et nous avons établi que CD5 joue un rôle dans l’initiation de la réponse TCR par

le régulation de la phosphorylation des résidus tyrosine de la kinase Fyn appartenant à la

famille Src, une kinase essentiel dans l’activation des cellules T. CD5 induit la

phosphorylation de Fyn sur un résidu tyrosine (Y531) ayant pour effet la réduction

l’activité kinase. La réponse en aval est ainsi réduite, notamment l’activité de ZAP-70, une

kinase essentiel au évènement initiaux de l’activation des cellules T. De plus, la

phosphorylation de la protéine adaptatrice PAG au résidu Y317, un résidu tyrosine cible

de l’activité kinase de Fyn, est réduite après CD5 activation. Par le fait que la plupart des

enzymes et protéines adaptatrices des ces voies de signalisation localisent au niveau du

lipid rafts, nous avons étudié la motilité de CD5 depuis la partie fluide de la membrane

après activation. Confirmant nos prédictions, les molécules de CD5 re-localisent après

anticorps stimulation au niveau du lipid rafts. Nos résultats indiquent que CD5 dimérisent

à la surface des cellules T inactivent, ce qui peut aider à expliquer l’ampleur de la re-

localisation entre les différentes phases de la membrane. Avec ces résultats, nous

proposons un modèle de l’inhibition des cellules T par CD5: l’activation de CD5 induit ça

re-localisation au niveau du lipid rafts, où ils inter-agit avec des effecteurs tels que Fyn,

Lck, PAG et LAT. Un complexe CD5-kinase est responsable de la phosphorylation du

résidu tyrosine et de la diminution de l’activité de Fyn, qui est suivi par la

dephosphorylation de PAG et une diminution de l’association de PAG avec la kinase Csk.

Le contrôle par CD5 de la loupe PAG/Csk/Fyn, en association avec le recrutement par

CD5 d’autres molécules de surfaces (tel que CD2) réduit la réponse à long terme des

cellules T, tel que la prolifération des cellules T. Nos travaux indiquent que CD5 est un

inhibiteur de la voie TCR en contrôlant les évènements précoces de la réponse des

cellules T, notamment l’activation de Fyn. Le modèle que nous proposons peut aider à

comprendre au niveau moléculaire les effets thérapeutiques bénéfiques des traitements

par anticorps CD5 dans certaines maladies auto-immunes et durant les greffes de moelle

osseuse.

Publications

The following articles were published upon contributions during this Ph.D.:

Mayr, C., Bund, D., Schlee, M., Bamberger, M. , Kofler, D.M., Hallek, M., and Wendtner,

C.M. 2006. MDM2 is recognized as a tumor-associated antigen in chronic lymphocytic

leukemia by CD8+ autologous T lymphocytes. Exp Hematol 34(1): 44-53.

Nunes, R.J., Castro, M.A., Goncalves, C.M., Bamberger, M. , Pereira, C.F., Bismuth, G.,

and Carmo, A.M. 2008. Protein interactions between CD2 and Lck are required for the

lipid raft distribution of CD2. J Immunol 180(2): 988-997.

Submitted:

Bamberger, M., Castro, M.A., Oliveira, M.I., James, J.R., Davis, S.J., Lozano, F., and

Carmo, A.M. A central role for Fyn in the CD5-mediated inhibition of T lymphocyte

signaling. Submitted to J Immunol

In preparation:

Castro, M.A., Bamberger, M. , Nunes, R.J., Maia, A.R., and Carmo, A.M. Lck-dependent

translocation of the T cell glycoproteins CD2 and CD5 to lipid rafts. Manuscript.

Batista, A., Bamberger, M. , Rodriguez-Rodriguez, S., Carlesso, N., Carmo, A.M., and

Cardoso, A. The Notch pathway is regulated by leukemia microenvironmental cues and

positively modulates IL-7 signaling. Manuscript.

Chance favours only the prepared mind

Louis Pasteur

1

I – General Introduction

General Introduction

2

1. Introduction: T cell activation 3

2. Initiation of T cell activation 5

2.1. The TCR/CD3 complex 5

2.2. The major histocompatibility complex 6

2.3. Molecular scanning and binding of pMHC by TCRs 7

2.4. TCR triggering 8

3. The TCR signal transduction machinery 10

3.1. Signaling motifs and domains: ITAM, ITIM, ITSM, SH2, SH3, PH 11

3.2. Protein tyrosine kinases and phosphatases in TCR signaling 12

3.2.1. Src family kinases 13

3.2.2. Syk family kinases 15

3.2.3. Tec family kinases 16

3.2.4. The C-terminal Src kinase Csk 16

3.2.5. Protein tyrosine phosphatases 17

3.3. Adaptor proteins 18

3.3.1. The signalosome organized by LAT 19

3.3.2. The PAG/Csk/Fyn loop 21

3.3.3. The SLAM/SAP/Fyn interaction 22

3.4. Accessory signaling receptors 23

3.4.1. The Coreceptors CD4 and CD8 23

3.4.2. Costimulation 25

3.4.3. The accessory receptor CD5 26

3.4.4. The accessory receptor CD2 28

4. TCR signaling platforms and networks 29

4.1. The cytoskeleton 29

4.2. Lipid rafts 30

4.3. The immunological synapse 32

5. TCR signaling pathways 34

5.1. The Ca2+/PLC pathway 34

5.2. The Ras/MAPK pathway 37

6. Scope of the thesis 38


3

1. Introduction: T cell activation

The immune system is complex, intricate and interesting. Its main function is the

discrimination between self and non-self. This ability is necessary to protect the organism

from different types of invading pathogens. Our body’s first line of defense against

infectious agents, the innate immune system, provides an immediate non-specific

response that includes anatomical barriers (e.g. skin), humoral barriers (e.g. complement

system, coagulation system), and cellular barriers (e.g. phagocytes, natural killer cells).

The adaptive (also known as acquired) immune response acts as a second line of

defense and is more complex and specific. The pathogen must first be processed and

recognized, then immune cells are specifically designed to attack that antigen. Adaptive

immunity also develops a memory that makes future responses against a pathogen more

efficient.

In order to prevent immunological disorders and to provide an effective immune

response against a wide range of pathogens, a well balanced collaboration of the complex

components of the immune system is required. B lymphocytes and T lymphocytes play a

central role in this immunological network as the major types of cells of the adaptive

immune system. They derive from the same pluripotent hematopoietic stem cell in the

bone marrow, where B cells develop. T cell progenitors travel to and mature in the

thymus. In adults, the peripheral lymphoid organs contain a mixture of B and T cells in

three stages of differentiation: 1) Naïve cells that have matured, left the bone marrow or

thymus, have entered the lymphatic system, but that have not yet encountered their

cognate antigen 2) Effector cells that have been activated by their cognate antigen, and

are actively involved in eliminating a pathogen and 3) Memory cells – the long-lived

survivors of past infections.

Whereas B lymphocytes recognize native, unprocessed antigens by means of their

B cell receptor (BCR) and respond by secretion of the soluble receptor (antibody,

immunoglobulin) and marking of the antigen for subsequent agglutination and

neutralization, T lymphocytes recognize only processed antigen, that is presented on the

surface of antigen presenting cells (APCs) as a peptide fragment of the original antigen, in

association with MHC (major histocompatibility complex) molecules. During development

in the thymus, T cells are screened for their ability to recognize self-MHCs (positive

selection) that present antigenic non-self peptides (negative selection), and only those

that bind with the appropriate affinity leave the thymus as matured naïve T cells and begin

to circulate throughout the body, including the lymph nodes. Like all T cells, they express

the T cell receptor (TCR) which determines what antigen the T cell can respond to. During

immune response, APCs endocytose foreign material (typically bacteria or viruses),


4

process it and then travel from the site of infection to the lymph nodes, where they

encounter the T cells and activate them in two steps (Two-signal model of T cell

activation:

Signal 1 (recognition) is the activation of the TCR by its specific ligand, which consists of

the antigenic peptide in association with self-MHC molecules (pMHC) on the surface of

APCs.

Signal 2 (verification) is a costimulatory signal, that is independent of antigen specific

recognition and delivered through accessory receptors on the T cell surface. If this second

signal is not present during initial antigen exposure, the T cell presumes that it is

autoreactive (binding to self-pMHC) and becomes, as a protective measure,

unresponsive, a state called anergy (taken from Macian et al. 2004). Anergic cells will not

respond to any antigen in the future, even if both signals are present later on (Schwartz

2003), except when the TCR signal is extremely strong.

The aftermath of the activation depends on the subset of T cell that encountered

the antigen:

• CD4+ T cells (Helper T cells, Th cells) divide rapidly and secrete cytokines that

regulate the immune response.

• CD8+ T cells (Cytotoxic T cells, Tc cells) destroy infected cells and tumor cells by

release of cytolytic molecules.

• Memory T cells may be either CD4+ or CD8+. They are antigen-specific T cells that

persist long-term after an infection and can quickly expand upon re-exposure to

their cognate antigen.

• Regulatory T cells (Treg cells) shut down T cell mediated immunity towards the end

of an immune reaction and suppress autoreactive T cells that escaped the process

of negative selection in the thymus.

• Natural Killer T cells (NKT cells) recognize glycolipid antigen presented by CD1d.

Once activated, they can perform functions ascribed to both Th and Tc cells.

Upon successful T cell activation several signal transduction pathways that involve

second messengers, protein kinases, protein phosphatases and other enzymes and key

intermediates are triggered. This signaling cascade culminates with the induction of gene

transcription according to defined genetic programmes that are characteristic of the

different T cell subsets, leading to the differentiation and proliferation of these cells. In

addition, the proper development and selection of immature T cells in the thymus also

depends on similar signaling events that determine the selection of T cells with the

appropriate antigen specificity. In the following, the current state of our understanding of

this T cell activation process will be reviewed.


5

2. Initiation of T cell activation

2.1. The TCR/CD3 complex

The TCR is a cell surface heterodimer consisting of either disulfide-linked α- and

β- chains (in most of the T cells), or γ- and δ-chains (in a small subset of T cells). Each

TCR chain is a member of the immunoglobulin (Ig) superfamily, possessing one N-

terminal variable (V) and one constant (C) Ig-like domain, followed by a connecting

peptide, a transmembrane domain and a short cytoplasmic tail at the C-terminus. The V

domain has three hypervariable or complementarity determining regions (CDRs),

generated by somatic recombination of the TCR gene segments V(D)J during thymic

development, a process that results in a high variation in the TCR repertoire (Lewis 1994).

CDR3, which exhibits the greatest degree of genetic variability, is the main CDR

responsible for recognizing processed antigen and thereby the principal determinant of

specificity (Davis et al. 1998), whereas MHC contacts are mainly mediated through CDRs

1 and 2 (Garcia et al. 1999).

Four distinct polypeptides are noncovalently associated with the TCR heterodimer:

the CD3 subunits δ, ε, γ, and ζ that associate to form the CD3δε and CD3γε heterodimers

and the disulfide-linked ζζ homodimers (Koning et al. 1990; Manolios et al. 1991). Stable

cell surface expression and normal development of αβTCRs rely on the presence of the

CD3 components (Berkhout et al. 1988; Wang et al. 1998). CD3δε and ζζ are directly

associated with TCRα whereas CD3γε associates with TCRβ. This TCR/CD3 complex

possesses three basic residues in the transmembrane (TM) regions of the TCR αβ

heterodimer, and two acidic residues in each of the three different CD3 signaling dimers.

These potentially charged TM residues were proposed to drive the associations between

the TCR and CD3 components by forming pairwise ionic interactions (Manolios et al.

1990; Cosson et al. 1991). In addition, the extracellular domains of CD3δε and CD3γε also

appear to contribute to these associations (Kuhns et al. 2006; Kuhns and Davis 2007).

Concerning the stoichiometry (the number of copies of each subunit per complex)

and the valency (the number of ligand-binding TCR αβ heterodimers per complex) of the

TCR/CD3 complex, two major groups of models were proposed. The monovalent model

holds that there is a single TCR heterodimer and one copy of each of the three signaling

dimers per complex (Manolios et al. 1991; Hou et al. 1994; Punt et al. 1994; Kearse et al.

1995; Call et al. 2002; Call et al. 2004), whereas a second set of (multivalent) models

proposes that two or more TCR heterodimers are present per complex (Exley et al. 1995;

Jacobs 1997; San Jose et al. 1998; Fernandez-Miguel et al. 1999). Furthermore, a


6

unifying model of TCR/CD3 valency was proposed, in which multivalent TCR/CD3s are

co-expressed with monovalent receptors (Schamel et al. 2005; Alarcon et al. 2006). A

recent report revealed convincing evidence that the dominant form of the TCR complex is

composed of a single heterodimer (James et al. 2007).

2.2. The major histocompatibility complex

The term “MHC” refers to the region of the genome that encodes for the HLA

(human leukocyte antigen) proteins in humans. Nevertheless, in biomedical literature

MHC is frequently and canonically used (instead of HLA) when referring to the proteins as

well, as will be in the following. In humans, the MHC genomic region at chromosomal

position 6p21 is highly polymorphic and polygenic, leading to a vast amount of HLA

phenotypes within the human population.

During thymic development, T cell progenitors are strictly screened for recognition

of non-self peptide/self-MHC complexes. However, up to 10% of mature T cells recognize

and respond to non-self-MHC (Sherman and Chattopadhyay 1993), a phenomenon

termed alloreactivity, which is the molecular reason for graft rejection and, in

immunocompromised individuals, graft-versus-host disease. Monoclonal antibodies

(mAbs) specific for human CD3ε have been used or tested as immunomodulating agents

in preventing transplant rejection (Kjer-Nielsen et al. 2004; Chatenoud 2005).

The most intensely studied MHC genes are the nine classical (antigen-presenting)

MHC genes HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1,

HLA-DRA and HLA-DRB1. The A, B and C genes belong to MHC class I, whereas the D

genes belong to MHC class II. MHC class I molecules are expressed by nucleated cells

and present peptides primarily derived from degradation of intracellular pathogens. Class

II MHCs are expressed by immune cells that are able to endocytose extracellular

antigens, the presented peptides originate from proteolytic antigen degradation.

Both classes of MHCs are noncovalently linked heterodimers composed of three

domains, one α-helix/β-sheet (αβ) superdomain that forms the peptide-binding site and

two Ig-like domains. Peptides presented by MHC class I molecules are typically 8 – 10

amino acids long (Cresswell et al. 1994), whereas MHC class II molecules display

peptides with an average length of 12 – 25 residues (Wolf and Ploegh 1995). Polymorphic

residues were shown to cluster within and around the peptide binding groove of the MHC

molecules in order to provide the required variation in shape and chemical properties that

accounts for the specific peptide-binding motifs identified for each MHC allele (Rudensky

et al. 1990; Falk et al. 1991; van Bleek and Nathenson 1991).


7

2.3. Molecular scanning and binding of pMHC by TCRs

T cell activation is initiated by binding of pMHC complexes by TCRs. Whereas

γδTCRs bind directly to pathogen-derived glycoproteins or so-called nonclassical (not

antigen-presenting) MHC molecules (Morita et al. 1995; Chien et al. 1996; Belmant et al.

1999; Moody et al. 1999; Adams et al. 2005), αβTCRs recognize peptides presented by

either class I MHCs (CD8+ T cells) or class II MHCs (CD4+ T cells), depending on the

class of MHC to which they are restricted. Scanning a huge amount of pMHCs requires a

fast mode of TCR binding and disengagement. The association rate of most TCRs is

rather slow, a fact that cannot be explained by random collision and electrostatic

interactions only. This suggests that conformational changes are occurring after or during

binding. A crystal structure database of 27 class I and class II TCR/pMHC complexes has

been accumulated so far that reveals a high degree of structural variability in TCR/pMHC

recognition, making it difficult to deduce a common binding mode (Rudolph et al. 2006). In

the two-step mechanism model proposed by Wu and colleagues the TCR first contacts

the MHC molecule, then samples the MHC peptide-binding groove with the antigenic

peptide mainly by means of its CDR3 loops (Wu et al. 2002c), thereby subdividing TCR

binding into peptide-independent and peptide-dependent steps. This two-step binding

mechanism would explain how the same TCR could interact with different peptides bound

to the same MHC. Also, it could be the basis for the positive selection of TCRs in the

thymus based on binding to self-pMHC (Goldrath and Bevan 1999). Nevertheless, the

model has been controversial and challenged by a recent kinetic study (Davis-Harrison et

al. 2007).

A frequent trend appears to be that the TCR docks on the pMHC in a diagonal

orientation over the center of the binding groove. Electrostatic effects seem to be

important for orienting the TCR relative to the pMHC (McCoy et al. 1997). In addition, the

orientation of the CD3 modules relative to the T cell membrane was assumed to be an

essential prerequisite for TCR binding (Arnett et al. 2004). The main structural changes

that accompany TCR/antigen engagement were found in the TCR CDR3 loop regions

(Reiser et al. 2002; Rudolph and Wilson 2002), whereas the pMHC rarely changes its

conformation. A recent study comparing free and bound TCR structures for both wild-type

and a CDR3 mutant revealed an induced fit mechanism in which restructuring of CDR3

loops leads to improved TCR/pMHC surface complementarity, better peptide binding and

higher affinity (Sami et al. 2007).

Recently, seminal studies revealed that T cells can detect even one single agonist

pMHC, three peptides are sufficient for killing by Tc cells and ten peptide ligands already

lead to TCR-induced calcium influx (Irvine et al. 2002; Purbhoo et al. 2004). This was


8

surprising, as the affinity of the TCR-pMHC interaction is low (1 - 50 µM) (Davis et al.

1998; van der Merwe and Davis 2003) and monomeric ligands were shown to be not

stimulatory in solution (Boniface et al. 1998). Furthermore, T cells respond to a wide range

of pMHC concentrations (Bachmann et al. 1998; Cochran et al. 2000; Irvine et al. 2002).

How does one explain the ability of small numbers of agonist pMHCs to trigger a much

larger number of TCRs? How can we explain the paradox of high specificity and

sensitivity, but low affinity in the TCR-pMHC interaction? Despite long interest, the exact

relationship between the biochemistry of the TCR/pMHC interaction and T cell responses

remains unsolved, but good progress has been made in the last years. Some of the

currently debated hypotheses will be discussed in the following section.

2.4. TCR triggering

The TCR/CD3 complex is the primary trigger for the clonal expansion of antigen-

specific cells from the T cell repertoire (Davis et al. 1998). The structure of the TCR/CD3

complex requires that the ligand-binding components communicate to the signaling chains

on the cytoplasmic side of the membrane upon appropriate interaction with pMHC. The

intracellular domains of each of the CD3 chains contain immunoreceptor tyrosine-based

activation motifs (ITAMs) that serve as the central point for the intracellular signal

transduction machinery upon TCR engagement (Kane et al. 2000). The CD3δ, ε and γ

chains each contain one ITAM, whereas CD3ζ contains three ITAMs. One of the earliest

biochemical events that has been detected following TCR engagement by pMHC is

phosphorylation of these TCR/CD3 ITAMs. This step is believed to be essential for TCR

signal transduction. Consequently, models of TCR triggering typically focus on explaining

how TCR binding to pMHC stimulates this ITAM phosphorylation. Many models have

been proposed for how TCR signaling is initiated, comprising aggregation (coreceptor

heterodimerization and pseudodimer), conformational change and segregation (kinetic

segregation and lipid raft) models (Choudhuri and van der Merwe 2007).

Aivazian and colleagues suggested that interactions between the cytoplasmic

domains of the signaling components and the plasma membrane may play an important

role in TCR activation. Their results indicate that the cytoplasmic tail of the CD3ζ chain is

reversibly bound to the inner leaflet of the plasma membrane sequestering the ITAM

phosphorylation sites from important tyrosine kinases (Aivazian and Stern 2000).

Dislodging of the cytoplasmic domains from the membrane by ligand-induced receptor

clustering could thus permit receptor phosphorylation and trigger the subsequent cascade

of signaling events.


9

The pseudodimer model of TCR triggering postulates that one TCR binds a single

agonist pMHC complex, while a second TCR interacts with self-pMHC and its associated

coreceptor with the agonist pMHC complex (bound by the first TCR), hence forming a

TCR pseudodimer (Figure 1A). Therefore, self-pMHCs could be used by TCRs in close

proximity to an agonist to fully activate the T cell (Irvine et al. 2002). Recently, a study

suggested that both receptor aggregation and conformational changes are required for full

TCR activation (Minguet et al. 2007).

Figure 1. Two models for the initiation of T cell a ctivation. (A) The pseudodimer model postulates that one TCR binds an agonist pMHC complex and a second TCR, bound to self-pMHC and associated with its coreceptor, is dimerized with the agonist-engaged TCR. A pseudodimer is hence formed by dual interaction of the second TCR with self-pMHC and its associated coreceptor with the agonist pMHC complex. (B) The kinetic segregation model proposes that TCR binding to pMHC traps the TCR/CD3 complex in close contact zones, thereby segregating it form inhibitory tyrosine phosphatases with large extracellular domains, leading to phosphorylation of TCR/CD3 ITAMs by tyrosine kinases (adapted from Choudhuri and van der Merwe 2007).


10

Another type of mechanism that has been proposed for TCR triggering is binding-

induced segregation or redistribution of the TCR/CD3 complex with respect to other cell

membrane associated molecules. The kinetic segregation model, first proposed in 1996

(Davis and van der Merwe 1996), postulates that multiple zones of close contact (∼ 15 nm

apart) form at the cell-cell interface from which molecules with large extracellular domains,

such as certain inhibitory tyrosine phosphatases, are excluded (Figure 1B). TCR binding

to pMHC in close contact zones leads to triggering by trapping the TCR/CD3 complex in

regions where their phosphorylation is unopposed by the excluded tyrosine phosphatases.

A large body of evidence supports this model. Representative, in an elegant study

Choudhuri and colleagues showed that elongation of the pMHC complex leads to

increased intermembrane distance and inhibition of TCR triggering (Choudhuri et al.

2005).

However, several of the proposed models are consistent with the available

evidence and the mechanism(s) of TCR triggering remain(s) controversial. It is important

to note that the suggested triggering mechanisms are not mutually exclusive. Instead it is

likely that TCR triggering involves a combination of these mechanisms.

3. The TCR signal transduction machinery

An important question asked by immunologists concerns the propagation of the

initial TCR-mediated signal. Once T cell activation is initiated, the long-established

“canonical” TCR signaling pathway begins with phosphorylation of TCR/CD3 ITAMs by

the Src familily kinases (SFKs) Lck and Fyn followed by recruitment, phosphorylation and

activation of the protein tyrosine kinase ZAP-70 (zeta chain-associated protein of 70 kDa),

phosphorylation of adaptor proteins and activation of respective downstream signaling

pathways, leading to gene regulation, proliferation and actin-reorganization responses

(Weiss and Littman 1994). In the following, several steps, important players and signaling

domains of the TCR signaling pathway will be elaborated.

Of note, at least in some circumstances, TCRs may use alternative “non-

canonical” pathways of signaling, different from the SFK-mediated tyrosine

phosphorylation pathway (Gil et al. 2002; Call and Wucherpfennig 2005; Campi et al.

2005; Gil et al. 2005; Yokosuka et al. 2005; Bueno et al. 2006; Tewari et al. 2006;

Zamoyska 2006).


11

3.1. Signaling motifs and domains: ITAM, ITIM, ITSM , SH2, SH3, PH

Biological specificity requires that receptors and their cytoplasmic targets are

delivered to the appropriate site in the cell so that they are activated at the right time and

in the right place. The transmission of signals from the cell surface to the nucleus involves

coordinated protein-protein and protein-phospholipid interactions. Proteins are frequently

constructed in a cassette-like fashion from interaction domains (35 – 150 amino acids in

length) that can target them to a specific subcellular location, provide a means of

recognition of protein posttranslational modifications or second messengers and mediate

the formation of multiprotein complexes. Enzymes can generate modified amino acids or

lipids on their substrates that are then recognized and bound by these interaction

modules. Thereby catalytic and interaction domains collude to control the dynamic state of

the cell. Out of the vast amount of protein interaction domains identified so far, only the

most prominent domains in TCR signaling, the domains SH2, PTB, SH3 and PH, and their

counter-motifs will be discussed here.

TCR-mediated signaling processes are controlled by the ten ITAMs present in the

CD3 subunits of the TCR/CD3 complex. The “classical” ITAM consensus sequence is

defined as YxxI/Lx6-12YxxI/L (x = any amino acid) (Reth 1989), including two potentially

phosphorylated tyrosine residues. Phosphotyrosine (pTyr or pY) sites, as those present in

the ITAMs, are formed by the action of tyrosine kinases and were first described by Tony

Hunter and colleagues almost three decades ago (Eckhart et al. 1979). Seven years later,

Tony Pawson and co-workers discovered that pTyr sites are bound by SH2 (Src homology

2) domains (Sadowski et al. 1986), the first modular signaling domain that was recognized

to bind to its ligand in a phosphorylation dependent manner. The SH2 domain now serves

as a prototype for a collection of interaction domains that recognize not only proteins, but

also phospholipids, nucleic acids and small molecules (Pawson, Nash 2003).

SH2 domains are usually about 100 amino acids long, with the N- and C-termini

closely juxtaposed, leaving the ligand binding surface exposed. The pTyr site fits into the

conserved pTyr binding pocket and is captured by an invariant arginine residue at the

base of the pocket (Waksman et al. 1992). Substrate binding specificity is achieved by

recognition of residues that lie 3 – 5 amino acids C-terminal to the pTyr in a fashion that

differs from one SH2 domain to another (Songyang et al. 1993). Binding of a pTyr site can

either target a SH2-containing protein to the membrane or induce its phosphorylation or

activity. Several proteins contain two SH2 domains in tandem (e.g. ZAP-70), which can

confer enhanced binding to ligands that contain several pTyr motifs (Ottinger et al. 1998).

Furthermore, phosphorylated tyrosine residues are also recognized by phosphotyrosine

binding (PTB) domains (Blaikie et al. 1994). In contrast to SH2 domains, it is residues that


12

are N-terminal to the phosphorylated tyrosine residue that confer PTB domain binding

specificity.

SH3 domains, consisting of 50 – 60 amino acids, recognize proline-rich motifs with

the minimal consensus PxxP (Ren et al. 1993). Contrary to SH2 domains, they bind with

low affinity (Kd of 10-8 for SH2, Kd of 10-6 – 10-4 M for SH3) and independent of

posttranslational modifications. Typically, the proline-rich peptide adopts a conformation,

in which one residue each three amino acids is oriented toward the SH3 domain. The

presence of a basic residue at a N-terminal (R/KxxPxxP) or C-terminal (PxxPxR/K)

position in the peptide gives rise to two modes of recognition, depending on whether the

ligand binds with its N- or C-terminus near the acidic cluster of the SH3 domain (Lim et al.

1994; Feng et al. 1995).

PH (pleckstrin homology) domains are protein modules of approximately 120

amino acids, that bind to inositol phospholipids, such as inositol-1,4,5,-trisphosphate (IP3),

thereby allowing PH proteins to respond to lipid messengers by relocation to membrane

regions where the relevant phosphoinositides are generated (Haslam et al. 1993).

Individual PH domains possess specificites for inositol phospholipids phosphorylated at

different sites within the inositol ring. Consequently, the recruitment of PH proteins is

sensitive to the activities of enzymes that either phosphorylate (e.g. phosphatidylinositol 3-

kinase (PI3K)) or dephosphorylate (e.g. phosphatase and tensin homolog deleted on

chromosome 10 (PTEN)) these sites.

As opposed to ITAM motifs, the immune system can engage negative regulators in

the form of receptors bearing a immunoreceptor tyrosine-based inhibition motif (ITIM),

consensus S/I/V/LxYxxI/V/L (Ravetch and Lanier 2000). ITIM phosphorylation may recruit

cytoplasmic phosphatases having a SH2 domain, resulting in decreased tyrosine

phosphorylation of activation pathway effectors. Furthermore, the immunoreceptor

tyrosine-based switch motif (ITSM) TxYxxV/I was described to modulate downstream

signaling based on the differential binding of SH2 domain-containing molecules

(Sidorenko and Clark 2003). Unlike ITAM and ITIM motifs, tyrosine phosphorylation of the

ITSM motif does not seem to be a prerequisite for binding of SH2 proteins (Sayos et al.

1998).

3.2. Protein tyrosine kinases and phosphatases in T CR signaling

Many receptors of the Ig superfamily employ a similar mechanism of signal

transduction, which involves protein tyrosine kinases (PTKs), that transfer phosphate onto

tyrosine residues of substrate proteins, adaptor proteins and effector enzymes in a highly

organized tyrosine phorphorylation cascade. This cascade is equally dependent on the


13

class of enzymes that remove phosphate from PTK substrates and from PTKs

themselves, the protein tyrosine phosphatases (PTPases). 90 PTK genes (Manning et al.

2002) and 107 PTPase genes (Alonso et al. 2004) have been identified in the human

genome so far, indicating the abundance and importance of these molecule families.

Tyrosine phosphorylation is rapidly reversible and generally of a very low stoichiometry.

Hence, a minor change in the PTK/PTPase balance can have a major impact on net

tyrosine phosphorylation and thereby on the process of T cell activation.

3.2.1. Src family kinases

The Src family of kinases (comprising the nine members Src, Lck, Fyn, Hck, Lyn,

Yes, Fgr, Blk and Yrk) play a central role in many cellular processes (Bjorge et al. 2000)

and the necessity for their regulation is evident by the fact that many members of this

family were identified as oncogenes. In addition to colon cancer (Aligayer et al. 2002),

breast cancer (Reissig et al. 2001), leukemias, lymphomas, and the metastatic potential of

tumors (Boyer et al. 2002), Src kinases have been implicated in a number of T-cell

mediated disease models (Kamens et al. 2001).

All members of the family of SFKs have the following domain organization in

common: N-terminal unique region, SH3 domain, SH2 domain, kinase domain (SH1) and

C-terminal regulatory region (Figure 2). The presence of an SH3 and SH2 domain

indicates that they not only have tyrosine kinase enzymatic function, but can also function

as adaptor proteins. The kinase activity is regulated at two levels. First, through

interactions of the SH3 and SH2 domains with proline-rich sequences and pTyr sites,

respectively, resulting in conformational changes that make the kinase domain accessible

(Gonfloni et al. 1997; Superti-Furga and Gonfloni 1997). Second, by the phosphorylation

status of the two principal regulatory tyrosine phosphorylation sites, that are present in the

kinase domain and in the C-terminal tail. When phosphorylated, the inhibitory tyrosine

residue (Y505 in Lck and Y531 in Fyn) residing in the C-terminal tail binds to the SH2

domain of the same kinase molecule thereby maintaining a closed conformation

(stabilized by the interaction of the SH3 domain with a region in the linker between the

SH2 and the kinase domain) and keeping the kinase in an inactive state (Liu et al. 1993).

Dephosphorylation of this tyrosine residue, potentiates kinase activity. Additionally, SFKs

are activated by phosphorylation (autophosphorylation, intramolecular

transphosphorylation or phosphorylation by other tyrosine kinases) of the positive

regulatory tyrosine (Y394 in Lck and Y420 in Fyn) within the kinase domain activation loop

(Veillette and Fournel 1990).


14

T cells primarily express Lck and Fyn (sometimes also termed FynT (T for thymus)

to distinguish it from the brain isoform FynB). Both proteins are targeted to the membrane

due to (cotranslational) myristoylation of glycine-2, a common feature of all SFKs

(Marchildon et al. 1984). Most SFKs, including Lck and Fyn, also undergo reversible

palmitoylation at cysteine-3 and at either cysteine-5 (Lck) or cysteine-6 (Fyn) (Paige et al.

1993; Koegl et al. 1994). Lck interacts with the coreceptors CD4 and CD8 through a

dicysteine motif present in its N-terminal unique domain and with two cysteines in the

cytoplasmic domains of CD4 and CD8 (Turner et al. 1990).

Figure 2. The domain structure of Src family kinase s. The architecture of Src family kinases consists of four domains: the unique region, which varies among family members, followed by the SH3, SH2, and tyrosine kinase (SH1) domains. The SH2-kinase linker, the activation loop (A-loop) of the kinase domain, and the positive and negative regulatory tyrosines are indicated. In the autoinhibited form of Src family kinases, the SH2 domain binds the phosphorylated C-terminal tyrosine, and the SH3 domain binds the linker segment between the SH2 and kinase domain.

Analyses of cells from Lck- and Fyn-deficient mice and cell lines expressing only

one or the other kinase have indicated that both of these kinases function as positive

regulators of TCR signaling, increasing sensitivity to stimulation and potentially influencing

the differentiation outcome after TCR triggering (Appleby et al. 1992; Stein et al. 1992;

Straus and Weiss 1992; Denny et al. 2000; Palacios and Weiss 2004). Lck was suggested

to be the main SFK responsible for TCR/CD3 ITAM phosphorylation (van Oers et al.

1996), and Fyn was shown to be important in activating Lck tyrosine kinase activity (Lee

et al. 1994). Nevertheless, recent studies revealed that Fyn may also play a role as a

negative regulator, as it interacts with molecules which may downregulate the activation of

SFKs themselves (Yasuda et al. 2002) or which influence T cell proliferation and

differentiation (Latour et al. 2003; Davidson et al. 2004).

The activation of Lck and Fyn is the earliest event upon TCR engagement. Once

activated, Lck and Fyn phosphorylate the two critical tyrosine residues within the ITAMs of

the TCR/CD3 complex, generating binding sites for proteins bearing SH2 domains. At

present, it remains unclear whether the activation of Lck and Fyn is accomplished by


15

dephosphorylation of the inhibitory tyrosine residue, by reduced phosphorylation of that

residue or by enhanced phosphorylation of the activatory tyrosine residue (possibly by

transphosphorylation due to higher local SFK concentration upon membrane redistribution

or mediated by the recruitment of the coreceptors CD4 or CD8). These mechanisms are

not mutually exclusive, and probably all operate in concert.

3.2.2. Syk family kinases

The Syk family tyrosine kinase member ZAP-70 is the predominant effector protein

that links TCR ligation to downstream cellular events. ZAP-70 contains two tandemly

arranged SH2 domains that recognize and bind the tyrosine phosphorylated ITAM motifs

of the TCR/CD3 complex. Due to the presence of 10 ITAMs in the TCR complex, up to 10

ZAP-70 molecules may cluster on the fully phosphorylated receptor. Little is known at

present about the mechanism by which recruitment of ZAP-70 to ITAMs triggers its

activation. The phosphorylation of two pairs of tyrosine residues in ZAP-70 is crucial for

the activation process. One pair (Y492 and Y493) is located in the activation loop of the

kinase domain, the phosphorylation of these tyrosines is most likely initiated by Lck (Watts

et al. 1994; Chan et al. 1995) or by transautophosphorylation (Brdicka et al. 2005). The

other two critical tyrosine residues (Y315 and Y319) are located in the SH2-kinase linker.

These are phosphorylated upon recruitment of ZAP-70 to the TCR, also most likely by Lck

(Williams et al. 1999; Brdicka et al. 2005) or by ZAP-70 itself (Di Bartolo et al. 1999). The

recently determined crystal structure of autoinhibited ZAP-70 revealed that Y315 and

Y319 are involved in interactions that connect the linker to the kinase domain (in contrast

to SFKs, where the linker interacts with the SH3 domain) and thereby stabilize the inactive

form. ITAM engagement by ZAP-70 might disrupt this conformation and thereby activate

the kinase (Deindl et al. 2007).

The importance of Syk family kinases in lymphocyte signaling and development

became apparent with the discovery of patients lacking a functional ZAP-70 gene. They

show severe immunodeficiency characterized by the absence of CD8+ T cells and TCR-

unresponsive CD4+ T cells (Arpaia et al. 1994; Elder et al. 1994). Mice lacking ZAP-70

have neither CD8+ nor CD4+ T cells (Negishi et al. 1995). Mice lacking Syk, the

eponymous member of the Syk family, die shortly after birth and show blocked B cell

development, whereas T cell development appears normal (Turner et al. 1995).

Nevertheless, double knock-out mice show a much more severe defect in T cell

development, underlining the importance of both Syk family kinases in pre-TCR signaling

(Cheng et al. 1997).


16

3.2.3. Tec family kinases

All five Tec family PTKs described so far (Itk, Rlk, Tec, Btk and Bmx) share an

overall similar domain organization that resembles the organization of SFKs. Starting at

the N-terminus they comprise a SH3 domain, follwed by a SH2 domain and the kinase

domain. Tec family kinases are the only PTKs that additionally possess a N-terminal PH

domain that preferentially binds IP3. As a result these kinases are cytosolic in resting

lymphocytes, where levels of IP3 are low (discussed in section 5.1.). Following T cell

activation, IP3 levels in the plasma membrane increase due to PI3K activity, leading to

recruitment of the Tec kinases to the membrane. This localization is essential for the

subsequent phosphorylation and activation of Tec kinases (August et al. 1997).

Besides by subcellular localization, Tec kinase activity can be regulated by

phosphorylation. Similar to SFKs, phosphorylation of a conserved activation loop tyrosine

in the kinase domain enhances enzymatic activity. However, contrary to SFKs, Tec

kinases do not autophosphorylate. Instead, the phosphorylation is mediated by SFKs. In

the case of Itk, the critical residue Y511 is phosphorylated by Lck (Heyeck et al. 1997). Itk

activation also depends on ZAP-70 and the adaptor protein LAT (linker for activation of T

cells) (Shan and Wange 1999). Because ZAP-70 does not directly phosphorylate Itk, this

requirement is most likely indirect, via the need to recruit Itk to the LAT signalosome (see

section 3.3.1.). Furthermore, Itk was shown to bind to a variety of other important

signaling effectors, such as Fyn and CD28 (August et al. 1994).

Concerning substrates of Tec kinases, from the three members of the Tec family

PTKs that are expressed in T cells (Itk, Rlk and Tec) Itk is most strongly implicated in the

regulation of phosholipase Cγ1 (PLCγ1), as discussed in section 3.3.1. Itk-deficient mice

show reduced numbers of peripheral T cells, diminished TCR induced responses,

including Calcium influx, and reduced tyrosine phosphorylation and activation of PLCγ1

(Liao and Littman 1995; Liu et al. 1998b).

3.2.4. The C-terminal Src kinase Csk

The PTK responsible for the suppressive phosphorylation of Lck at Y505 and Fyn

at Y531 is the C-terminal Src kinase Csk, a widely expressed cytoplasmic 50 kDa enzyme

(Okada et al. 1991; Bergman et al. 1992; Chow et al. 1993). Overexpression of Csk in T

cells showed to cause a marked reduction in TCR-induced tyrosine phosphorylation and

IL-2 production (Chow et al. 1993). Accordingly, the disruption of the CSK gene leads to

constitutive activation of SFKs in early embryos (Nada et al. 1993) and embryonic lethality

in mice (Imamoto and Soriano 1993).


17

Two different mechanisms have been reported to regulate Csk activity. The cAMP-

dependent protein kinase A (PKA) induces Csk activity through phosphorylation of S364

and thereby inhibits TCR signaling (Vang et al. 2001). In addition, Csk activity is increased

upon its recruitment to lipid rafts, membrane microdomains that play an important role in T

cell signaling (see section 4.2.), by a mechanism that will be discussed in section 3.3.2.

3.2.5. Protein tyrosine phosphatases

In the control and coordination of the TCR signaling cascade PTPases are as

important as PTKs. They play a crucial role in keeping T cells in a resting state, as well as

in activating them by removal of inhibitory tyrosine residues and in the reversion of

activated T cells back to the resting phenotype. About 30 PTPases are expressed in T

cells, including the transmembrane PTPase CD45 and the intracellular PTPases PEP,

SHP-1 and PTEN.

Counteracting the activity of Csk, CD45 is the main PTPase that dephosphorylates

the negative regulatory tyrosine residue of SFKs and thereby activates them (Mustelin and

Altman 1990). Underlining its importance, CD45-deficient humans and mice develop a

severe-combined immunodeficiency (SCID) phenotype (Kung et al. 2000; Tchilian et al.

2001). CD45 knock-out mice show impaired T cell maturation with dysfunctional signaling

through the pre-TCR and TCR and hyperphosphorylation of Lck and Fyn (Kishihara et al.

1993; Byth et al. 1996; Mee et al. 1999). Expression of an active mutant form (Y505F) of

Lck was shown to rescue T cell development in these mice (Seavitt et al. 1999). These

reports confirmed earlier studies with T cells lacking CD45, that failed to respond to

stimulation by antigen or mitogenic antibodies (Pingel and Thomas 1989). Several reports

confirmed the positive regulatory role of CD45 in TCR signaling, disclosing, that CD45

was required for TCR-triggered tyrosine phosphorylation of cellular proteins (Koretzky et

al. 1990) and for calcium mobilization (Volarevic et al. 1992). However, there are reports

showing that CD45 may as well dephosphorylate the positive regulatory tyrosine within

the kinase domain (D'Oro and Ashwell 1999). Additionally, according to the kinase

segregation model of TCR triggering, PTPases with large extracellular domains, such as

CD45, are mainly excluded from the TCR-APC interface. The puzzling question is: How

can CD45 activate SFKs by dephosphorylation of their inhibitory tyrosine residue, if CD45

is excluded from the TCR triggering platform? Studies using CD45-deficient cell lines and

thymocytes indicate that in an unstimulated T cell, CD45 dephosphorylates both

regulatory tyrosines of Lck with an overall effect of limiting kinase activity (D'Oro et al.

1996; D'Oro and Ashwell 1999). It is proposed that upon T cell activation, CD45 loses its

proximity to Lck, resulting in rephosphorylation of Y394 and activation of Lck (Thomas and


18

Brown 1999). On the other hand, Hermiston and colleagues proposed a model in which

Lck is maintained in a primed state in resting cells, due to the positive regulation by CD45.

During antigen recognition, CD45 is segregated from the contact area and Lck activity

sustained in the initial phase of T cell activation (Hermiston et al. 2002).

PEP (PEST domain-enriched PTPase) belongs to the family of PEST-rich tyrosine

phosphatases and is a negative regulator of TCR signaling, that dephosphorylates the

positive regulatory tyrosine residues of Lck (Y394) and Fyn (Y420) (Cloutier and Veillette

1999; Gjorloff-Wingren et al. 1999). PEP associates with the SH3 domain of Csk (Cloutier

and Veillette 1996), a domain that seems to be required for the inhibitory function of Csk

(Cloutier et al. 1995). It was suggested that the PEP-Csk association provides a tight

synergistic negative regulation of SFK activity (Cloutier and Veillette 1999). Another

PEST-rich tyrosine phosphatase family member that modulates T cell activation, PTP-

PEST, counteracts Fyn activity by dephosphorylating the Wiskott-Aldrich syndrome

protein (WASP) (for details see section 4.1.).

The SH2 domain-containing protein tyrosine phosphatase-1 (SHP-1) contains two

SH2 domains, that can bind to phosphorylated ITIM motifs. This binding activates SHP-1

and juxtaposes it to its substrates. Thymocytes and peripheral T cells of SHP-1 knock-out

mice were shown to be hyperresponsive to TCR stimulation (Pani et al. 1996), suggesting

that SHP-1 plays a role as a negative regulator of TCR signaling. Consistently, SHP-1

was shown to dephosphorylate the active site of Lck (Y394) (Chiang and Sefton 2001)

and to dephosphorylate ZAP-70 (Brockdorff et al. 1999)

PTEN is a dual specificity phosphatase acting on both protein phosphotyrosine

and phosphothreonine/serine residues, as well as on 3-phosphorylated inositol

phospholipids (hydrolysis of IP3 to PIP2).

3.3. Adaptor proteins

Adaptor proteins are proteins that do not possess an enzymatic activity or receptor

function, but function as scaffolds recruiting other proteins. They are comprised

exclusively of protein-protein or protein-lipid interaction domains and motifs that promote

formation of multiprotein signaling complexes (Figure 3). Adaptor proteins can be divided

into two main groups. The cytoplasmic adaptor proteins (CAPs) comprise, amongst many

others, the proteins Grb2, Gads, SLP-76, ADAP, SKAP55 and SKAP-HOM. The

transmembrane adaptor protein (TRAP) family includes LAT, PAG, LIME, SIT and TRIM.

TRAPs display a short extracellular domain, a single transmembrane segment, and a long

cytoplasmic region bearing multiple potential sites of tyrosine phosphorylation and proline-

rich sequences.


19

Figure 3. The domain structure and potential intera ction partners of selected adaptor proteins involve d in TCR signaling. Adaptor proteins are composed of signaling motifs and domains that allow for interaction with a multitude of effector proteins. For details see main text (adapted from Leo and Schraven 2001).

3.3.1. The signalosome organized by LAT

The 36 kDa phosphoprotein LAT was the first TRAP to be described in 1998

(Zhang et al. 1998a) and seems to be the most important one. Its essential role in T cell

signaling became clear when LAT-deficient T cell lines (J.CaM2 and ANJ3) were found to

have defects in key elements of the TCR-mediated signaling cascade that lead to IL-2


20

gene expression (Finco et al. 1998; Zhang et al. 1999a). LAT-/- mice show a total absence

of T lymphocytes in the periphery and block of thymic development at an early stage

(Zhang et al. 1999b). LAT localization in lipid rafts, through palmitoylation of its membrane

proximal CxxC palmitoylation motif (Zhang et al. 1998b), is required for T cell activation

(Lin et al. 1999; Zeyda et al. 2002).

Upon TCR triggering, LAT becomes tyrosine phosphorylated primarily by ZAP-70

(and to a lesser extent by Lck and Itk), creating binding sites for SH2-containing

molecules such as Grb2 (growth factor receptor-bound protein 2), Gads (Grb2-related

adaptor downstream of Shc), PLC-γ1 and PI3K, thereby connecting TCR signals with

downstream intracellular pathways. One path involves the connection LAT-Grb2-Sos,

which leads to the activation of the Ras/MAPK pathway. Another connection involves

LAT-Gads-SLP76, which induces the activation of PLCγ1, triggering calcium flux and

subsequent gene activation. Both pathways are discussed in section 5.

The four distal tyrosine residues of LAT (Y132, Y171, Y191 and Y226 in human)

seem to be crucial for the function of LAT. The phenotype of LAT knock-in mice,

expressing a LAT molecule in which these four tyrosine residues were mutated to

phenylalanine, is similar to that seen in LAT-/- mice (Sommers et al. 2001). Y132 is

responsible for binding PLCγ1, Y171 for binding PI3K, whereas Gads binds Y171 and

Y191 and Grb2 binds to all three distal tyrosines (Zhang et al. 2000). SLP-76 is recruited

to LAT (and thereby to the plasma membrane) indirectly, together with Gads, as it binds

constitutively to the SH3 domain of Gads (Liu et al. 1999). SLP-76 also associates with

the SH3 domain of PLCγ1 in a constitutive manner. Once associated with the LAT

signalosome, SLP-76 gets phosphorylated and recruits Itk, through the interaction with

both the SH2 and SH3 domain of Itk (Bunnell et al. 2000). Itk (activated by Lck) then

phosphorylates PLCγ1 at Y775 and Y783 (Bogin et al. 2007), the two phosphorylation

sites that are critical for PLCγ1 activation, and LAT, the latter promoting the recruitment of

the Rac-specific guanine nucleotide exchange factor Vav to Y171 and Y226 (Perez-Villar

et al. 2002) and thereby connecting T cell activation to the Ras pathway and to

cytoskeletal rearrangement (Fischer et al. 1998; Villalba et al. 2000). Vav may also bind to

LAT indirectly via either Grb2 or SLP-76 (Ye and Baltimore 1994; Tuosto et al. 1996).

Furthermore, Grb2 also recruits Itk, Sos (which can activate the Ras pathway as well),

Cbl, SHP-2, WASP and Shc. And SLP-76 interacts with several other signaling molecules,

such as Lck (Sanzenbacher et al. 1999), non-catalytic region of tyrosine kinase (Nck)

(Wunderlich et al. 1999) and adhesion- and degranulation-promoting adaptor protein

(ADAP) (da Silva et al. 1997). ADAP, formerly known as Fyn SH2-binding protein (Fyb) or

SLP-76-associated phosphoprotein of 130 kDa (SLAP-130), binds to Fyn and to the


21

adaptors Src kinase-associated protein of 55 kDa (SKAP55) and SKAP-HOM (da Silva et

al. 1993; Liu et al. 1998a; Marie-Cardine et al. 1998). SKAP55 is recruited to lipid rafts

upon TCR stimulation and was shown to mediate Fyn dephosphorylation by CD45 (Wu et

al. 2002a; Wu et al. 2002b). Taken together, LAT is a central adaptor protein that

functions as a molecular scaffold to assemble important signaling effectors in form of the

LAT signalosome and thereby links early TCR signaling events with the downstream

signaling pathways.

3.3.2. The PAG/Csk/Fyn loop

Phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG),

also referred to as Csk-binding protein (Cbp), is an ubiquitously expressed

transmembrane adaptor protein with an apparent molecular weight of 68 – 85 kDa. Its

long cytoplasmic domain includes ten tyrosine residues, two proline-rich sequences, and a

C-terminus capable of interacting with ezrin/radixin/moesin-binding phosphoprotein 50

(EBP-50), thereby linking PAG to the actin cytoskeleton (Brdickova et al. 2001; Itoh et al.

2002). Due to the palmitoylation of two cysteines in the proximal portion of the cytoplasmic

domain, PAG is largely localized in lipid rafts and, like LAT, considered a raft marker.

PAG is involved in the negative regulatory mechanism of SFKs by recruiting Csk to

lipid rafts through binding of the Csk SH2 domain via a specific phosphorylated tyrosine

residue of PAG (Y317 in humans, Y314 in mice). The interaction was shown to increase

the kinase activity of Csk and Csk-mediated inhibition of SFKs (Brdicka et al. 2000;

Kawabuchi et al. 2000; Takeuchi et al. 2000; Torgersen et al. 2001; Veillette et al. 2002;

Davidson et al. 2003).

Whereas in resting T cells, PAG was shown to be constitutively tyrosine

phosphorylated and associated with Csk, these modifications are rapidly lost upon

engagement of the TCR/CD3 complex (Brdicka et al. 2000; Torgersen et al. 2001;

Davidson et al. 2003). It was proposed that the PAG-Csk complex may prevent activation

of resting T cells and that TCR-mediated release of Csk from PAG (and thus from the

plasma membrane) allows activation of SFKs due to reduced Csk-mediated

phosphorylation of their negative regulatory tyrosine. Termination of this activating

signaling event occurs by rephosphorylation of PAG at Y317 and recruitment of Csk back

to lipid rafts.

PAG tyrosine phosphorylation and binding of PAG to Csk were noted to be

dramatically reduced in Fyn-deficient T cells (Yasuda et al. 2002), corroborating the

importance of Fyn for the PAG-Csk interaction. Solheim and colleagues demonstrated

recently that Fyn binds PAG via both its SH2 domain (to PAG phosphotyrosines) and its


22

SH3 domain (to the membrane-proximal proline-rich region of PAG). They postulated that

this dual domain docking activates the kinase, which subsequently phosphorylates PAG

tyrosine residues, including Y317. Subsequent Csk recruitment to pY317 leads to C-

terminal phosphorylation of Fyn, but inhibition of Fyn activity is not effective until

phosphorylated Fyn dissociates from PAG (Solheim et al. 2008). According to this model,

the crucial step in rendering Fyn inactive would be the PAG-Fyn dissociation. However,

the only reported trigger for PAG-Fyn dissociation so far is TCR engagement, which was

shown to activate Fyn. Elaborating on this dissociation step, two conflicting studies

propose that the PAG-Fyn association is either not modulated by TCR stimulation

(Brdicka et al. 2000) or is rapidly lost in response to TCR stimulation (Davidson et al.

2007). In the latter study, dissociation of the PAG-Fyn complex preceded PAG

dephosphorylation and PAG-Csk dissociation after TCR engagement. In contrast to the

study of Solheim and colleagues, where the PAG-Csk complex was lost prior to the PAG-

Fyn dissociation.

Clearly, further studies are in need to understand the precise mechanism involved

in the regulation of the PAG/Csk/Fyn loop. Moreover, the phosphatase responsible for

PAG dephosphorylation has not been clearly identified yet, even though several PTPases

(CD45, SHP-1, PEP, PEP-H1 and CD148) were suggested to be implicated (Davidson et

al. 2003; Lindquist et al. 2003). The most prominent candidate, CD45, was shown to be

required for efficient TCR stimulated PAG dephosphorylation (Davidson et al. 2003).

Although several studies evidenced that PAG is implicated in the negative

regulation of cellular processes mediated by Src kinases (Brdicka et al. 2000; Kawabuchi

et al. 2000; Ohtake et al. 2002; Davidson et al. 2003), two groups reported that mice

lacking PAG exhibited little or no phenotype (Dobenecker et al. 2005; Xu et al. 2005).

Given that a severe phenotype was observed in Csk-deficient mice (Imamoto and Soriano

1993; Nada et al. 1993), it was postulated that other, PAG-independent mechanisms of

Csk recruitment may exist.

3.3.3. The SLAM/SAP/Fyn interaction

SAP (SLAM-associated protein) is a small SH2 domain adaptor protein that was

shown to have important roles in intracellular signaling pathways elicited through the TCR,

such as regulating the activation of PKC and its recruitment to the TCR-APC contact

zone, activation of the transcription factor nuclear factor-κB (NF-κB) and modulating

interferon-γ (IFN-γ) production (Latour et al. 2001; Cannons et al. 2004). Since its

identification as the protein defective in the human immunodeficiency X-linked

lymphoproliferative disease (Coffey et al. 1998; Nichols et al. 1998; Sayos et al. 1998),


23

several studies have been performed in an attempt to identify SAP-regulated pathways.

SAP is constitutively associated with the cell surface receptor SLAM (signaling

lymphocyte activation molecule). A conserved arginine at position 32 (R32) within its SH2

domain allows SAP to bind to the ITSM present in the cytoplasmic domain of SLAM, even

in the absence of tyrosine phosphorylation. Furthermore, R78 of SAP (interestingly,

located within a motif that does not contain any proline) binds the SH3 domain of Fyn

(Latour et al. 2003) and recruits Fyn to the SLAM/SAP complex (Chan et al. 2003). When

SLAM is crosslinked on T cells, it becomes tyrosine phosphorylated through a SAP- and

Fyn-dependent mechanism (Latour et al. 2001; Chan et al. 2003; Latour et al. 2003; Li et

al. 2003). In a comprehensive study, Latour and colleagues demonstrated that these

tyrosine-phosphorylated residues act as docking sites for the PTPase SHIP, which

becomes phosphorylated and binds to the adaptor proteins Dok1 and Dok2. Tyrosine-

phosphorylated Dok2 protein was shown to bind the SH2 domain of rasGAP (Latour et al.

2001). These adaptors may allow the recruitment of additional effectors of this signaling

cascade. Nonetheless, the exact mechanism(s) by which SAP-mediated pathways affect

T cell responses and whether this contributes to the phenotypes of XLP remain to be

determined.

3.4. Accessory signaling receptors

The coordinated action of antigen receptors, coreceptors, costimulatory molecules,

cytokine and chemokine receptors and inhibitory receptors enables immune cells to

provide a customized and adjusted response to foreign elements and to prevent disease

states. Although the specificity of the T cell–APC interaction is determined by the

recognition of antigen-peptide bound to the MHC molecules, signals delivered by

accessory adhesion molecules are necessary for a full immune response. A multiplicity of

coordinately engaged accessory molecules ensures the efficiency of immune recognition

as well as modulates the T cell activation process. Accessory signal receptors and their

counter-receptors involved in T cell–APC interactions include, amongst others: CD4 with

class II MHC, CD8 with class I MHC, CD28 with B7-1/B7-2, LFA-1 with ICAM-1 and

ICAM-2, CD2 with CD58 and CD6 with ALCAM.

3.4.1. The Coreceptors CD4 and CD8

During thymic development, the earliest thymocytes express neither CD4 nor CD8

(CD4-CD8- or double-negative cells). As they progress through their development they

become double-positive (CD4+CD8+) thymocytes that eventually mature to single-positive


24

helper T cells (CD4+) or cytotoxic T cell (CD8+). CD4 and CD8 play major roles in both the

differentiation and selection of T cells during thymic development as well as in the

activation of mature T lymphocytes.

The coreceptor CD4 has been viewed as a monomeric molecule, although

Moldovan and colleagues claimed that CD4 dimers are required for T cell activation

(Moldovan et al. 2002). CD8 is expressed as either a classical αβ heterodimer or as a αα

homodimer, the latter being the principal ligand of a non-classical MHC molecule

(Leishman et al. 2001). The extracellular parts of CD4 and CD8 coreceptors contain Ig-

like domains and bind to MHC II and MHC I molecules, respectively (Doyle and

Strominger 1987; Norment et al. 1988). Whereas the determination of a crystal structure

of a CD8αβ heterodimer in complex with pMHC has been elusive, the CD4 coreceptor

was shown to bind MHC via the N-terminal domain (Wang et al. 2001).

The cytosolic domains of CD4 and CD8 bind non-covalently to Lck (Marth et al.

1986). TCR triggering requires, according to the coreceptor heterodimerization model

(Trautmann and Randriamampita 2003), simultaneous engagement of the same pMHC by

TCR with either CD4 or CD8 coreceptors (Emmrich et al. 1986), causing the intracellular

juxtaposition of coreceptor-bound Lck with TCR signaling motifs and thus initiating TCR

signaling (Veillette et al. 1989; Abraham et al. 1991; Kersh et al. 1998; Li et al. 2004).

However, coreceptor-independent TCR signaling has been reported (Locksley et al. 1993;

Schilham et al. 1993) and is presumably mediated by coreceptor-unbound Lck that is

passively captured within ligand-induced TCR aggregates. Notably, Julius and colleagues

reported 16 years ago that binding of Lck by CD4 specifically impaired coreceptor-

independent TCR signaling by sequestering Lck and making it unavailable to the TCR

unless CD4 was coengaged (Haughn et al. 1992). These results were confirmed later

(Wiest et al. 1996) and a recent study proposed that the sequestration of Lck by

coreceptors prevents the selection of non-MHC reactive TCRs (TCRs that recognize

ligands other than MHC) during thymic development, thus imposing MHC specificity

during positive selection (Van Laethem et al. 2007).

Besides Lck, the coreceptors CD4 and CD8 were shown to associate with CD45

(Mittler et al. 1991; Bonnard et al. 1997), thereby bringing CD45 to close proximity to its

substrate Lck. The predominant effect of CD45 appears to be dephosphorylation of Lck

pY505, since T cell development in CD45-/- mice can be largely restored by back-crossing

to mice expressing the active mutant LckY505F (Pingel et al. 1999; Seavitt et al. 1999).

In a recent study using the mild non-ionic detergent Brij97 to prevent weak protein-

protein interactions, a total of 26 proteins were found to be associated with CD4, including

components of the cytoskeleton and heat shock proteins (Krotov et al. 2007).


25

3.4.2. Costimulation

Almost all physiological responses of naïve T cells require costimulation, that is

simultaneous engagement of the TCR by the appropriate pMHC (signal 1) and ligation of

a costimulatory receptor (signal 2). Only under exceptional circumstances, triggering of

the TCR alone is sufficient to induce proliferation, e.g. stimulation with CD3-specific Abs at

high surface density (Viola et al. 1999).

The most prominent costimulatory receptor is CD28 (Hara et al. 1985; Christensen

et al. 2002), a 44 kDa transmembrane glycoprotein that is expressed as a homodimer on

the surface of the majority of CD4+ T cells and of a third of CD8+ T cells (June et al. 1990;

Azuma et al. 1993). CD28 consists of an extracellular part with two Ig-like domains and a

41 amino acids long cytoplasmic part with four tyrosine residues (Aruffo and Seed 1987).

By means of a MYPPPY-motif in the extracellular domain (Peach et al. 1994; Metzler et

al. 1997) CD28 binds to its ligands B7-1 (CD80) and B7-2 (CD86), expressed on the

surface of APCs. Studies using T cells derived from mice deficient in CD28 or in both B7.1

and B7.2 demonstrate their crucial roles in T cell activation and proliferation (Shahinian et

al. 1993). CD28 brings the TCR activation threshold down (Viola and Lanzavecchia 1996),

enhances early TCR signals such as phosphorylation of the ζ-chains and ZAP-70 as well

as Lck activity (Tuosto and Acuto 1998; Salojin et al. 1999; Viola et al. 1999), prolongs the

T cell response and reduces the apoptosis rate (Boise et al. 1995), mainly by enhanced

production of the cytokines IL-2 and IL-4 (Fraser et al. 1991; Thompson 1995; Chen et al.

1998), enhanced expression of anti-apoptotic genes (Boise et al. 1995), expression of cell

cycle kinases (Nagasawa et al. 1997) and degradation of the cell cycle inhibitor p27Kip

(Firpo et al. 1994). A recent study indicates that T cells require CD28 costimulation due to

negative regulation of TCR signals by the phosphatase PTEN (Buckler et al. 2006).

In an approach to identify molecules engaged upon CD28 ligation, CD28-specific

antibodies known to induce proliferation in the absence of signal 1 (TCR engagement),

referred to as superagonists (Tacke et al. 1997; Luhder et al. 2003), and the CD28 ligands

B7-1 and B7-2 were used. The cytoplasmic tail of CD28 definitely possesses the potential

for signal transduction. It contains four tyrosine residues, one of which organized in the

motif YMNM, as well as two proline-rich motifs. The p85 subunit of PI3K can associate

with phosphorylated Y170 within the YMNM motif (Pages et al. 1994). Y170 mediates

several functions of CD28, such as proliferation and cell survival, but is dispensable for IL-

2 production (Okkenhaug et al. 2001). The YMNM motif and the proximal proline rich

region of the CD28 tail are mandatory for association of Grb2, that connects CD28 via Sos

to the Ras/MAPK pathway (Kim et al. 1998). Itk and Tec can associate via their SH3

domain with the YMNM-proximal proline motif of CD28 and regulate a variety of


26

downstream events through activation of PLCγ1 and Erk (Miller and Berg 2002). Apart

from CD28-induced recruitment of signaling effectors that are mainly shared with the TCR,

CD28 ligation was shown to trigger another posttranslational modification, protein arginine

methylation (Blanchet et al. 2005), maybe by a novel TCR-independent pathway.

The importance of CD28-B7.1/B7.2 as a costimulatory receptor-ligand pair in T cell

activation is unquestioned. Nevertheless, in the last decade, other members of the

CD28/B7 superfamily have been discovered, e.g. ICOS-ICOSL, the B7 homologues B7-

H3 and B7-H4, both binding to an unknown receptor on T cells, and the CD28 homologue

BLTA with an unknown ligand. Next to members of the B7 costimulatory family, other

surface molecules have also been found to provide costimulatory signals, such as tumor

necrosis factor (TNF)-related family members (CD27, 4-1BB, CD40L), adhesion

molecules (LFA-1, CD2) and CD4 (Watts and DeBenedette 1999; Rogers and Croft 2000;

Watts 2005).

3.4.3. The accessory receptor CD5

CD5, erstwhile referred to as T1, Leu-1 or Tp67 in humans and Ly-1 in mice, is a

67 kDa transmembrane glycoprotein belonging to the scavenger receptor cysteine-rich

(SRCR) superfamily (Jones et al. 1986; Resnick et al. 1994). Expression of CD5 has been

reported on thymocytes, all mature T cells and a subset of mature B cells (B-1) (Thomas

et al. 1984; Antin et al. 1986). CD5 appears early in thymocyte development, being

considered as one of the first surface markers of committed T cells. Low CD5 expression

by CD4-CD8- thymocytes is up-regulated soon after thymocytes receive the pre-TCR

signal. The level of CD5 surface expression on developing thymocytes, as well as on

mature T cells parallels the avidity of the TCR/MHC-ligand interaction (Azzam et al. 1998).

In return, CD5 modulates thymocyte development and selection. Thymocytes expressing

distinct transgenic TCRs were shown to undergo abnormal selection in CD5-deficient

mice, in a manner suggestive of enhanced TCR signaling (Tarakhovsky et al. 1995; Pena-

Rossi et al. 1999). Surprisingly, the inhibitory effect of CD5 on TCR signals during thymic

development is independent of the extracellular domain of CD5, and thereby does not

involve CD5 binding of an extracellular ligand in the thymus (Bhandoola et al. 2002).

There is evidence for interaction of CD5 with different B cell surface receptors: CD72 (Van

de Velde et al. 1991), gp40-80 (Biancone et al. 1996; Bikah et al. 1998) and IgV(H)

framework region sequences (Pospisil et al. 1996; Pospisil et al. 2000) have been

described as potential CD5 ligands. Another surface molecule, gp150, was shown to

interact with CD5 and has a broader expression range (Calvo et al. 1999).


27

It is broadly accepted that CD5 is implicated in the modulation of T and B

lymphocyte activation and differentiation, but the exact contribution of CD5 depends

strongly on the type of cells analyzed, as well as on the maturation state. When

crosslinked with mAbs, CD5 was shown to deliver a signal to T cells that augments their

proliferation in response to TCR stimulation (Imboden et al. 1990; Spertini et al. 1991;

Alberola-Ila et al. 1992). Initially, this type of effect suggested that CD5 could perform a

costimulatory or amplification role during certain types of T cell activation processes.

However, generation of mice deficient in CD5 clearly revealed an inhibitory role,

conferring a hyperresponsive phenotype to thymocytes and peripheral T cells when they

are stimulated through their TCRs (Tarakhovsky et al. 1995; Pena-Rossi et al. 1999).

Upon TCR/CD3 ligation, thymocytes of CD5-deficient mice showed enhanced cell

proliferation, increased intracellular Calcium and tyrosine hyperphosphorylation of PLCγ1,

TCRζ, LAT and Vav (Tarakhovsky et al. 1995). Furthermore, new technologies allowed

the observation that CD5 delivers negative signals during the formation of the TCR-APC

contact zone (Brossard et al. 2003).

Structurally, the extracellular part of CD5 is 348 amino acids long and comprises

three SRCR domains. It is followed by a hydrophobic transmembrane region of 29 amino

acids and a 94 amino acid cytoplasmic domain (Figure 4) that contains several putative

sites for tyrosine and serine/threonine phosphorylation and signaling motifs, suggesting a

role in signal transduction.

Figure 4. Schematic structure of the CD5 molecule. CD5 consists of an extracellular region (EC), a single transmembrane domain (TM) and a short cytoplasmic tail (CY). The cytoplasmic domain includes four tyrosine residues (Y378, Y429, Y441 and Y463), three of which are potentially embedded into known tyrosine-based motifs. The amino acid sequence neighboring Y429 and Y441 resembles an ITAM, with only residue P432 (indicated in gray) varying from the ITAM consensus sequence YxxI/Lx6-12YxxI/L. The membrane proximal residue Y378 as well as Y441 are contained within an ITIM (consensus S/I/V/LxYxxI/V/L). aa, amino acids; x, any amino acid.

Furthermore, CD5 associations have been reported with ZAP-70, TCRζ (Gary-

Gouy et al. 1997), Vav (Dennehy et al. 1997), casein kinase II (CK2) (Calvo et al. 1998),

rasGAP and Cbl (Dennehy et al. 1998). Cbl is an adapter protein that can negatively


28

regulate TCR signaling. It has been shown to associate with and inhibit both Syk and

ZAP-70 PTKs (Ota and Samelson 1997; Thien et al. 1999). Interestingly, Cbl functions as

a ubiquitin-protein ligase that can promote ubiquitination of tyrosine phosphorylated

proteins as a recognition tag for subsequent degradation by the proteasome (Joazeiro et

al. 1999). Therefore, recruitment of Cbl could contribute to CD5-mediated suppression of

TCR signaling. Cbl is also a binding partner of Grb2, raising the possibility that, in addition

to promoting receptor internalization, it can influence Ras signaling (Buday et al. 1996).

The CD5 cytoplasmic residue Y378 is contained within an ITIM-like sequence, which

suggests that CD5 may interact with other intermediate proteins involved in down-

regulatory function. In fact, SHP-1 was shown to bind to Y378 (Perez-Villar et al. 1999).

However, at least in B cells, receptor constitution studies have shown that the inhibitory

activity of CD5 is retained in CD5 constructs lacking the ITIM sequence (Gary-Gouy et al.

2000).

3.4.4. The accessory receptor CD2

The CD2 family of receptors is part of the Ig superfamily, and currently includes 11

cell surface molecules, amongst them: CD2, CD58, CD48, SLAM and CD244. CD2 is a 45

- 58 kDa type I transmembrane protein expressed on virtually all T lineage and NK cells

(Moingeon et al. 1989). The extracellular domain of CD2 is composed of two Ig

superfamily domains (Jones et al. 1992), of which the N-terminal V-like domain is involved

in the binding to the ligand (Davis and van der Merwe 1996). Engagement of CD2 to its

ligand (CD58 in humans and CD48 in rodents) facilitates adhesion between T cells and

APCs and is proposed to promote the formation of an optimal intercellular membrane

spacing suitable for TCR recognition of pMHC (Wang et al. 1999).

Concomitantly with its function as an adhesion molecule, the accessory receptor

CD2 promotes T cell activation. CD2 has long been known to transduce mitogenic

stimulation in T cells, either alone, or in synergistic combination with the TCR (Tiefenthaler

et al. 1987; Bierer et al. 1988). CD2 physically associates with the TCR, which may recruit

the TCR into the T cell–APC contact area and thereby modulate T cell responses (Brown

et al. 1989; Beyers et al. 1992). Nevertheless, crosslinking of CD2-transfected, TCR/CD3-

deficient cell lines resulted in IL-2 secretion, suggesting that CD2 can transmit mitogenic

signals independent of TCR expression (Ohno et al. 1991). Surprisingly, CD2-deficient

mice did not reveal any obvious phenotype and could mount effective immune responses,

challenging the role that CD2 plays in T cell activation (Killeen et al. 1992; Evans et al.

1993).


29

As CD2 has no intrinsic enzymatic activity, its role in signal transduction is

attributable to the association of its cytoplasmic domain with intracellular signaling

mediators. Via proline-rich sequences CD2 interacts with the SH3 domains of Lck and Fyn

(Carmo et al. 1993; Bell et al. 1996). Furthermore, CD2 associates with CD5 (Carmo et al.

1999; Castro et al. 2002), and thereby is able to amplify modulatory signals at the T cell

surface (Teh et al. 1997; Carmo et al. 1999). In addition, the cytoplasmic tail of CD2 is

linked to the cytoskeleton via a variety of adaptors that influence cytoskeletal polarization,

adhesion, and activation (Dustin et al. 1998; Li et al. 1998; Nishizawa et al. 1998; Tibaldi

and Reinherz 2003). In particular, CD2 interacts with CD2AP, CD2BP1 and subsequently,

WASP, thereby coupling WASP translocation to the TCR-APC contact area and T cell

activation (Badour et al. 2003).

4. TCR signaling platforms and networks

The past years have seen the beginning of a shift in the way that TCR signal

transduction is studied. Although many new molecules have been identified, particularly

adaptor proteins, attempts have been made to look at signaling events in a larger cellular

context. Thus, the identification of distinct formations of signaling molecules at junctions

between T cells and APCs, the role of the cytoskeleton and the partitioning of molecules

into specialized lipid subdomains have been the subjects of many studies. Such concepts

are helping to assemble a scheme of how the multitudinous receptors, adaptors, kinases

and phosphatases fit together to effect T cell activation. Of note, in an ambitious project,

Schraven and co-workers set up an in silico tool, a computer model of the T cell signaling

network, that holds the potential in foreseeing new signaling pathways, the effects of

drugs and network modifications (Saez-Rodriguez et al. 2007).

4.1. The cytoskeleton

T cell-APC interaction leads to rapid cytoskeletal polarization, which involves

filamentous (F)-actin polymerization, reorientation of the microtubule-organizing center

(MTOC) towards the region of interaction and formation of an actin-rich structure known

as the distal-pole complex on the opposing side of the T cell. Disruption of F-actin itself or

the depletion of cytoskeletal regulators impairs T cell activation, emphasizing the

importance of the actin and microtubule sytems (Valitutti et al. 1995; Campi et al. 2005).

As regards actin dynamics, the LAT signalosome (discussed in section 3.3.1.),

formed following TCR ligation, recruits actin-regulatory proteins like PLCγ1, Nck and Vav.

PLCγ1 leads to calcium mobilization, which is essential for F-actin remodeling (Bunnell et


30

al. 2001). Vav activates the small Rho GTPases Cdc42 and Rac1 (Turner and Billadeau

2002). Binding of active Cdc42 to WASP, which is recruited by SLP-76 and Nck to the

LAT signalosome (Zeng et al. 2003), allows WASP to polymerize F-actin through its

association with the actin-related protein 2/3 (ARP2/3) complex. Active Rac1 interacts with

the WASP-family verprolin-homologous protein-2 (WAVE2) complex and is thought to

either localize or activate WAVE2-mediated activation of the ARP2/3 complex (Zipfel et al.

2006). Moreover, through an interaction with Vav, dynamin-2 (DNM2) is localized to the

TCR-APC contact area where it participates in regulating F-actin reorganization (Gomez

et al. 2005). DNM2 itself recruits Nck, thereby linking DNM2 to WASP- and WAVE2-

mediated actin dynamics. Furthermore, TCR stimulation induces WASP tyrosine (Y291)

phosphorylation through Fyn kinase, while CD2BP1-associated PTP-PEST causes

tyrosine dephosphorylation, thereby acting in an opposing manner to WASP-mediated

Arp2/3 activation to regulate T cell activation (Badour et al. 2003; Badour et al. 2004).

The reorientation of the MTOC to a region beneath the T cell-APC contact site is a

hallmark of T cell activation. Once the MTOC is polarized, lytic granules and cytokines

travel along microtubules using minus-end-directed movement to the MTOC, which directs

their secretion towards the APC surface. The mechanism of MTOC movement after TCR

engagement is not clearly defined yet. Recently it has been shown that the microtubule

motor protein dynein participates through an interaction with ADAP (that is recruited to the

LAT signalosome by SLP-76) (Combs et al. 2006). This indicates a potential mechanism

of connecting actin dynamics to MTOC reorientation.

Another outcome of T cell activation that involves cytoskeletal reorganization is the

activation of integrins, leading to integrin clustering and enhanced lymphocyte adhesion to

integrin ligands (Kinashi 2005). The process by which intracellular signals result in the

activation of a cell surface receptor is generally referred to as “inside-out signaling”. LFA-1

is believed to be, through its interaction with ICAM-1 on APCs, the most important integrin

involved in T cell-APC conjugate formation (Dustin and Springer 1989; Grakoui et al.

1999). Many of the proteins that are recruited to the LAT signalosome are also required

for TCR-mediated integrin activation, including PLCγ1, Vav, SLP-76 and Itk, although the

exact mechanisms remain elusive. A potential LAT-independent mechanism of integrin

activation might involve ADAP and its constitutive binding partner SKAP-55 (Kliche et al.

2006).

4.2. Lipid rafts

The fluid mosaic model of the plasma membrane, proposed by Singer and

Nicolson in 1972 (Singer and Nicolson 1972), suggested that proteins and lipids were free


31

to move laterally within the plasma membrane. In the last decade, however, it has become

clear that the plasma membrane is no longer considered a uniform structure but rather a

patchwork of microdomains that compartmentalize signaling. The plasma membrane

microdomains that have received by far the most attention are the lipid rafts, also referred

to as glycolipid-enriched domains (GEMs) or detergent-insoluble glycolipid domains

(DIGs) (Simons and Ikonen 1997). Lipid rafts are rich in sphingolipids (sphingomyelin and

glycosphingolipids) and cholesterol, which mediate a compact and thickened bilayer

structure (Yeagle 1985; Ramstedt and Slotte 2002), forming a liquid-ordered state within

the disordered glycerophospholipids of a bulk membrane. The most common biochemical

method to analyze lipid rafts is based on the partial insolubility of membranes in non-ionic

detergents such as Triton X-100 at 4°C. As a consequence, when such membranes or cell

lysates are subjected to density gradient ultracentrifugation, the detergent-insoluble

membranes float to low-density fractions and can be separated from soluble and non-

membrane fractions. Together with lipid components, membrane proteins are also

separated into detergent soluble and insoluble fractions.

Signals targeting proteins to rafts can be divided into lipid modifications and

protein-based signals. Glycosylinositolphosphatidyl (GPI)-anchored proteins are generally

targeted to lipid rafts, by insertion into the outer leaflet of the membrane, and have

frequently been used as lipid raft markers (Cinek and Horejsi 1992). The second type of

lipid modification that targets proteins to lipid rafts is acylation such as myristoylation and

palmitoylation, which allows the modified proteins to attach to the inner leaflet of the cell

membrane (Shenoy-Scaria et al. 1993; Zacharias et al. 2002). Moreover, protein-based

interactions with membrane lipids or other raft proteins may target proteins to rafts as well

(Scheiffele et al. 1997).

The observation that many of the proteins that partition into lipid raft fractions are

involved in signaling has led to the hypothesis that lipid rafts are hot spots for signal

transduction (Simons and Ikonen 1997; Simons and Toomre 2000). Much of the evidence

for lipid rafts serving as signaling platforms comes from the study of T lymphocytes (Janes

et al. 2000; Langlet et al. 2000). TCR and associated signaling molecules were shown to

be enriched in lipid rafts following TCR activation, and TCR signaling could be inhibited by

disrupting these microdomains by cholesterol depletion (Xavier et al. 1998). Essential T

cell signaling molecules, such as Lck (Kabouridis et al. 1997), Fyn (Koegl et al. 1994),

LAT (Zhang et al. 1998b) and PAG (Brdicka et al. 2000) are acylated at their N-termini.

Using a transmembrane Lck chimera, it was shown that the localization of Lck at the

membrane is not sufficient for its function, but targeting of Lck to rafts reconstituted Lck-

mediated signaling in Lck-deficient T cells (Janes et al. 1999). In addition, LAT function


32

revealed to be associated with its recruitment to lipid rafts (Zhang et al. 1998b; Zeyda et

al. 2002).

A further function suggested for lipid rafts is to link the signaling machinery with the

cytoskeleton. Rafts and F-actin were found to be colocalizing (Harder and Simons 1999)

and the actin cytoskeleton was shown to drive the aggregation of the detergent-insoluble

microdomains (Rodgers and Zavzavadjian 2001; Villalba et al. 2001; Valensin et al. 2002).

In return, lipid rafts were shown to promote signal-induced association of TCRζ with the

actin cytoskeleton (Moran and Miceli 1998). Furthermore, CD2, which segregates to lipid

rafts upon ligation (Yang and Reinherz 2001), is linked to the actin cytoskeleton and the

raft protein PAG was shown to bind to EBP-50.

Despite the vast amount of evidence underlining the role of lipid rafts, the lipid raft

model has been challenged, mostly due to the nature of the standard biochemical method

for lipid raft isolation and its limitations (Munro 2003). Therefore, novel techniques for

analyzing lipid rafts have rapidly been applied. Conventional microscopy is not suitable to

prove the existence of rafts in resting cells. However, crosslinking raft-resident proteins

induces coalescence of rafts into patches that can be viewed by flurescence microscopy

(Harder et al. 1998). The use of fluorescence resonance energy transfer (FRET)

microscopy provided evidence of raft existence under physiological conditions (Silvius

2003). Fluorescence recovery after photobleaching (FRAP) analysis and novel single

particle tracking techniques combined with single fluorophore video imaging surprisingly

revealed that raft molecules are highly mobile and indicate raft heterogeneity that includes

the existence of different subsets of rafts as well as zones within one raft (McCabe and

Berthiaume 2001; Schade and Levine 2002; Douglass and Vale 2005; Kiyokawa et al.

2005).

4.3. The immunological synapse

Upon T cell activation, recognition of agonist pMHC leads to visible TCR

aggregation or clustering in two stages. Initially, so-called microclusters (or patches)

containing an estimated 10 – 20 TCRs form by a process dependent on actin

polymerization (Campi et al. 2005; Yokosuka et al. 2005; Varma et al. 2006). These

microclusters are then transported towards the center, forming a large central

supramolecular activation cluster (cSMAC) that is 2 – 3 µm in diameter (Monks et al.

1998; Grakoui et al. 1999) and pMHC complexes are clustered opposite them (both

agonist pMHCs and self-pMHCs). The tight, but transient contact site between a T cell

and a cell it is recognizing (typically an APC) is termed immunological synapse (IS) and


33

was first defined by Norcross (Norcross 1984) due to its reminiscence of neurological

synapses (Davis et al. 2007).

The so-called mature IS forms the cSMAC, enriched with TCRs and other

signaling receptors, surrounded by the peripheral SMAC (pSMAC), which is enriched

mainly with LFA-1 (Bromley et al. 2001). When they first form, TCR microclusters are sites

of signaling that include important signaling molecules, such as ZAP-70 and LAT.

Surprisingly, as microclusters are transported towards the cSMAC, signaling declines and

is very weak or absent. Phosphorylated forms of Lck and ZAP-70 were observed in the

peripheral region of the T cell-APC interface rather than in the central region at early time

points of the conjugation. The recruitment of signaling molecules, such as ZAP-70, LAT,

Grb2, Gads and SLP-76, to TCR microclusters was shown to begin within 30 seconds of

receptor engagement (Bunnell et al. 2002), but the majority of kinases and adaptors do

not move to the center, but rather dissociate from the microclusters (Yokosuka et al.

2005). Also, imaging analyses revealed that the early phosphorylation of Lck and ZAP-70

occurred before the formation of the mature IS (Lee et al. 2002). Therefore, the rapid

kinetics of T cell activation is not consistent with the kinetics for formation of the mature

IS. Biochemical analyses demonstrated that most of the phosphorylation events of

signaling molecules are induced within a few seconds to minutes after TCR engagement.

However, studies applying a planar bilayer system developed by Dustin and co-workers

(Grakoui et al. 1999) revealed that T cell activation leads to formation of the mature IS

within 10 min, and the structure is stable for several hours. In addition, recent studies

demonstrated that T cells can be activated in the absence of cSMAC formation (O'Keefe

and Gajewski 2005; Saito and Yokosuka 2006).

Recapitulatory, whereas the importance of the IS as the TCR-APC contact zone in

T cell activation is unquestioned, the role of the centripetal transport of TCR microclusters

to form the cSMAC, and thereby the mature IS, is highly controversial (van Der Merwe

and Davis 2002). It was suggested that it is part of the process of TCR/CD3 internalisation

and degradation which follows triggering (Lee et al. 2003; Varma et al. 2006). Besides,

several other possible functions have been proposed, including determination of T cell

polarity and orientation for the secretion of cytokines and chemokines or promotion of

endocytosis. A potential role of the mature IS could be the mediation of additional signals,

e.g. signals that stabilize adhesion, required for sustained activation. It was shown that

whereas initial T cell activation signals are induced within a few minues, continuous

stimulation of T cells for at least several hours (between 4 and 10 hours) is required for

the final induction of T cell activation, such as cytokine production and proliferation

(Huppa et al. 2003). A recent report shows that the formation the mature IS influences the

stimulatory potency of T cell antigens (Cemerski et al. 2007).


34

5. TCR signaling pathways

There are numerous intracellular biochemical events initiated through the TCR that

trigger downstream pathways and finally result in nuclear transcription, changes in T cell

morphology and proliferation (Figure 5). The most important “second messenger”

signaling pathways in T cell activation are the Ca2+/PLC pathway and the Ras/MAPK

pathway.

5.1. The Ca2+/PLC pathway

Calcium (Ca2+) ions act as second messengers in signal transduction. The resting

concentration of Ca2+ in the cytoplasm is normally in the range of 50 – 100 nM, that is four

orders of magnitude lower than the extracellular and organellar Ca2+ concentration. To

maintain this low concentration, Ca2+ is actively pumped from the cytosol and

accumulated in the endoplasmic reticulum (ER), which is the major Ca2+ storage organelle

in most cells. One of the crucial early steps in T lymphocyte activation is the release of

Ca2+ from the ER (Ca2+ influx) and the increase of the cytoplasmic free Ca2+ concentration

([Ca2+]i) (Tsien et al. 1982).

T cell stimulation leads to activated PLCγ1 (discussed in section 3.3.1.) that

hydrolyzes the membrane lipid phophatidylinositol-4,5-bisphosphate (PIP2), generating

1,2-diacylglycerol (DAG) and inositol-1,4,5,-trisphosphate (IP3) (Berridge 1993). IP3

diffuses in the cytoplasm and binds to its receptor IP3R (Ferris et al. 1989), which is an

intracellular Ca2+ release channel localized primarily in the ER membrane (Ross et al.

1989), and controlled by both the cytoplasmic [Ca2+] and the [IP3]. The subsequent [Ca2+]i

signals are organized at three broad levels. At low [IP3] during weak agonist stimulation, a

single or few IP3R(s) bind IP3 and highly localized small Ca2+ signals, called blips (Parker

et al. 1996), are generated, raising cytoplasmic Ca2+ concentration. Ca2+ release from one

channel stimulates, together with higher [IP3], opening of nearby channels through a

process of Ca2+-induced Ca2+ release (CICR), thus stronger signals (called puffs) arise

from the concerted opening of multiple channels organized within a cluster (Swillens et al.

1999). At the third level, with higher [IP3] associated with stronger extracellular agonist

stimulation, Ca2+ released at one cluster site can trigger Ca2+ release at adjacent sites by

CICR, leading to the generation of Ca2+ waves (Foskett et al. 2007). The resulting

reduction of the level of free Ca2+ in the ER activates a more sustained influx through Ca2+

release-activated Ca2+ (CRAC) channels that are located in the plasma membrane, a

mechanism called store-operated Ca2+ entry (SOCE). Prolonged Ca2+ entry through these

channels, also termed Ca2+ release-activated Ca2+ current (ICRAC), is crucial for activating


35

transcription factors that initiate many of the changes in gene expression that drive T cell

proliferation and cytokine production (Feske et al. 2001). Accordingly, abrogated signaling

through CRAC channels results in a lethal severe combined immunodeficiency (SCID)

syndrome in human patients that is characterized by defective T cell activation and

proliferation (Le Deist et al. 1995).

Although the SOCE hypothesis was first decribed 22 years ago (Putney 1986), not

until recently the sensor for Ca2+ depletion, the stromal interaction molecule STIM1 (Liou

et al. 2005; Roos et al. 2005) and the CRAC-channel pore subunit Orai1 (Feske et al.

2006; Vig et al. 2006; Zhang et al. 2006) were identified. STIM1 is primarily located in the

ER. It senses the [Ca2+] decrease in the ER through its Ca2+-binding domain and

communicates this event to the CRAC/Orai1 channels in the plasma membrane. Store

depletion causes STIM1 and Orai1 to accumulate at the ER-plasma membrane junctions,

triggering CRAC/Orai1 channel opening and local Ca2+ influx.

Increase in [Ca2+]i results in binding of Ca2+-calmodulin to the calcineurin complex

and activation of its phosphatase activity. Subsequent desphosphorylation of the

cytoplasmatic subunits (NFATc) of the nuclear factor of activated T cells (NFAT)

transcription complexes by calcineurin unmasks the nuclear localization sequence and

results in their import into the nucleus (Clipstone and Crabtree 1992; Loh et al. 1996). In

the nucleus, NFATc and its nuclear partner, NFATn, cooperatively bind to DNA and

regulate the expression of several T cell proliferation-associated genes. One prominent

target is the promoter of the gene encoding IL-2, which harbous several NFAT-binding

sites (Serfling et al. 1995; Hogan et al. 2003). Consequently, a frequent experimental

read-out of the T cell activation process is measurement of IL-2 production or IL-2

promotor activity.

Besides its effect on NFAT activation, released endogenous Ca2+ binds to

cytosolic PKC (more precisely, to classical PKC isoforms) (Spitaler and Cantrell 2004).

Binding of Ca2+ exposes the phospholipid-binding site of PKC and thereby translocates

PKC to the membrane, where it interacts with membrane-associated DAG and is

transformed into a fully active enzyme. One important PKC target is the ubiquitous

transcription factor NF-κB, that regulates, amongst many others, IL-2 gene expression.

Of note, one other route for metabilization of inositol phospholipids is mediated by

PI3K, which phosphorylates PIP2 to generate IP3. PLCγ1 and PI3K-regulated signaling

pathways can be interdependent, because IP3 binds to the PH domain of certain Tec

family kinases, such as Itk and Tec, localizing them to the plasma membrane, where they

regulate PLCγ1 (Ward and Cantrell 2001).

The two major systems that actively pump Ca2+ against its large concentration

gradient out of the cell or back into the ER are the plasma membrane Ca2+ ATPases


36

(PMCAs) and the sarco/ER Ca2+-ATPases (SERCAs), respectively. Interestingly, Ca2+

ions themselves were shown to be involved in ICRAC inactivation (Zweifach and Lewis

1995b; Zweifach and Lewis 1995a). Hence, any organelle and transport system that

regulates [Ca2+]i could also modulate CRAC/Orai1 channel activity. In this context, it was

shown that mitochondria were able to reduce Ca2+-mediated inhibition of CRAC/Orai1

channels by sequestering Ca2+ ions entering through those channels (Hoth et al. 1997).

Interestingly, mitochondria were shown to translocate to the vicinity of the IS upon T cell

activation and this mitochondrial redistribution was necessary to maintain Ca2+ influx

across the plasma membrane and for Ca2+-dependent T cell activation (Quintana et al.

2007).

Figure 5. Downstream responses induced by TCR ligat ion. Following TCR engagement, Lck and Fyn are activated, resulting in phosphorylation of CD3 modules of the TCR/CD3 complex and activation of ZAP-70. Activated ZAP-70 phosphorylates LAT and SLP-76. Tyrosine-phosphorylated LAT then recruits several SH2 domain-

containing proteins, including Grb2, Gads and PLCγ1. Activation of PLCγ1 results in the hydrolysis of PIP2, generating IP3 and DAG. IP3 production leads to increases of intracellular free Ca2+, whereas DAG can activate PKC isoforms. Phosphorylated LAT also recruits Grb2, and thereby Grb2-associated Sos, providing a mechanism of Ras activation through LAT. Eventually, TCR downstream signaling events result in nuclear transcription, proliferation and T cell effector functions. See main text for detailed explanation. (Taken from Abraham and Weiss 2004)


37

5.2. The Ras/MAPK pathway

Ras proteins belong to the superfamily of monomeric GTPases, that cycle

between an inactive, GDP-bound, and an active, GTP-bound, form. Conversion to the

active state is mediated by guanine nucleotide-exchange factors (GEFs) that stimulate the

exchange of GDP for GTP. GTP binding induces Ras activation by causing a

conformational change of regions that contribute to the effector-binding domain, a domain

that engages (and thereby regulates the function of) downstream signaling elements only

when Ras is in the GTP-bound stage (Chung et al. 1993). This process is reversed by

GTPase activating proteins (GAPs), that activate the intrinsic GTPase activity of Ras and

thereby the hydrolysis of GTP to GDP.

Ras is a central player in signal transduction and is mutated in about one third of

all human cancers, highlightening the great impact of Ras on human health (Vojtek and

Der 1998). The commonly occurring mutations that render Ras oncogenic are those that

make the GTPase insensitive to the action of GAPs and thereby lock it in the GTP-bound,

active state (Barbacid 1987; Lowy and Willumsen 1993).

The best characterized of the signaling pathways regulated by Ras is the MAPK

(mitogen-activated protein kinase) pathway. In this pathway, MAPK signaling modules are

organized as signaling cascades in which MAPKs are activated by dual-specificity

(serine/threonine and tyrosine) MAPK kinases (MAPKKs), which in turn are activated by

multiple MAPKK kinases (MAPKKKs).

Ras is localized on the cytosolic leaflet of the plasma membrane due to a C-

terminal hydrophobic domain that is created posttranslationally by virtue of a series of

modifications that include prenylation, proteolysis, carboxyl methylation and palmitoylation

(Clarke 1992). The main GEF for Ras is Sos. Upon signal induction, Sos is recruited to

the LAT signalosome and thereby to the plasma membrane via Grb2 (see section 3.3.1.).

Once recruited, Sos catalyzes the exchange of GDP attached to inactive Ras for GTP,

which leads to Ras activation. The function of Sos was shown to be counteracted by the

calcium-sensitive Ras GAP CAPRI (Lockyer et al. 2001). RasGTP has several

downstream effectors, including Raf-1, a serine/threonine MAPKKK that is recruited to the

membrane and subsequently phosphorylated and activated (Morrison and Cutler 1997).

Once active, Raf-1 phosphorylates and activates MEK, a MAPKK, that in turn

phosphorylates and activates the MAPKs Erk1 and Erk2. Phospho-Erk forms dimers that

are transported into the nucleus, where they activate transcription factors that are involved

in cytokine gene induction.

More recently, Ras/MAPK signaling has been observed in cell compartments other

than the plasma membrane. Surprisingly, an important organelle for Ras signaling in


38

lymphocytes seems to be the Golgi apparatus (Mor and Philips 2006). There, the calcium-

and DAG-sensitive Ras exchange factor RasGRP1, proved to be responsible for Ras

activation (Bivona et al. 2003). Interestingly, because both RasGRP1 and CAPRI are

activated by Ca2+, this second messenger controls Ras signaling in opposite directions

simultaneously on different subcellular compartments. The physiological relevance of Ras

signaling on the Golgi remains to be elucidated.

6. Scope of the thesis

The relevance of the accessory receptor CD5 in lymphocyte function is supported

by clinical and pathological studies that revealed the involvement of CD5 in diseases as

diverse as B cell chronic lymphatic leukemia (Gary-Gouy et al. 2007; Perez-Chacon et al.

2007), systemic lupus erythematosus (Youinou and Renaudineau 2007) or experimental

autoimmune encephalomyelitis (Axtell et al. 2004). Also, beneficial therapeutic effects of

anti-CD5 mAbs have been demonstrated in certain autoimmune disorders (Sun et al.

1992; Plater-Zyberk et al. 1994) and in bone marrow transplantation (Martin et al. 1996).

CD5 is viewed as an important attenuator of signals arising from the TCR. So far,

most data on the inhibitory role of CD5 were accumulated in CD5-deficient mice, where

absence of the receptor rendered T cells more responsive. These data gave important

insight into the modulatory role of CD5. However, they have not been adequately

complemented by biochemical analysis of the mechanisms involved and by studies of the

human cell system. Instead, many aspects of the molecular function of CD5 remain

elusive. Therefore, the aim of this thesis was to biochemically characterize the inhibitory

role of CD5 in human T lymphocyte activation.

The specific aims were:

• The delineation of CD5-controlled proliferative responses of T lymphocytes

• The biochemical characterization of the role of CD5 in T lymphocyte signaling and

the mechanisms involved in CD5-mediated inhibition

• To study the membrane distribution of CD5 and its interaction with important TCR

signaling effectors

39

II – Results

Results – Chapter 1

40

CHAPTER 1

THE ACCESSORY RECEPTOR CD5 DOWN-REGULATES

T LYMPHOCYTE PROLIFERATION

1.1. Introduction

41

1.2. CD72 does not modulate proliferation of human T cells 42

1.3. CD5-Ig does not bind to CD72-transduced Bw cells

44

1.4. CD5 engagement down-regulates T cell prolifera tion

45

1.5. Discussion

47


41

1.1. Introduction

The outcome of a T cell activation process is tightly regulated by additional stimuli

provided by both positive and negative accessory molecules. In early studies, the

accessory molecule CD5 was described as a positive regulator of late T cell responses.

Anti-CD5 mAbs were shown to augment TCR/CD3-mediated production of IL-2 and

proliferation of primary human T cells under conditions where perturbation of CD3 alone

elicited suboptimal responses and anti-CD5 mAbs as a sole stimulus did not alter T cell

responses (Ledbetter et al. 1985; Ceuppens and Baroja 1986; Imboden et al. 1990).

Besides this reported costimulatory role of CD5 in TCR activation, several studies suggest

that CD5 can act as a TCR/CD3-independent signal transducing molecule. Some anti-

CD5 mAbs were shown to be by themselves mitogenic to peripheral human T

lymphocytes, although they are restricted to the presence of monocytes, phorbol esters or

anti-CD28 mAbs (Spertini et al. 1991; Vandenberghe and Ceuppens 1991; Alberola-Ila et

al. 1992; Verwilghen et al. 1993).

More recent studies with CD5 knockout mice suggest that CD5 negatively

regulates antigen receptor-mediated signaling in peritoneal B-1 cells, thymocytes, and

peripheral T cells. The absence of CD5 in these animals rendered thymocytes

hyperresponsive to stimulation through the TCR/CD3 complex, leading to enhanced cell

proliferation (Tarakhovsky et al. 1995). CD5-deficient B-1 cells of these mice show, in

comparison to normal B-1 cells, enhanced proliferation upon IgM crosslinking (Bikah et al.

1996). Furthermore, T cell hybridomas and TCR-restricted peripheral T cells are more

responsive to TCR stimulation in the absence of CD5 (Pena-Rossi et al. 1999). Taken

together, these reports support the idea that under certain circumstances, CD5 acts as a

negative regulator of cellular activation.

The comparison of results attained from human T cells with data from knockout

animal systems is defensible, but it might lead to discrepancies. In this study, we focused

on human cell systems. We addressed the modulatory role of CD5 in late T cell activation

responses in both, human peripheral T cells and human T cell lines. Therefore, we applied

a novel system based on engineered T cell stimulator cells that enables us to study the

contribution of the potential CD5 ligand CD72 to T cell activation in a cellular system but

detached from the context of numerous molecules regulating T cell activation that are

present on cell membranes of APCs. Furthermore, upon mAb crosslinking of CD5 on the

surface of human T cell lines, we evaluated the signal transducing capacity of CD5 in a

system independent of TCR/CD3 activation.


42

1.2. CD72 does not modulate proliferation of human T cells

Reports in the early nineties showed that the surface molecules CD5 and CD72

form a receptor-ligand pair. However, so far no data have been published indicating that

the potential CD5-CD72 interaction delivers a signal towards T lymphocytes. In order to

assess the role of human CD72 in T cell activation in a cellular system that allows

stimulating primary human T cells, we used previously described engineered T cell

stimulator cells (Pfistershammer et al. 2006). These cells were generated by retroviral

transduction of the murine thymoma cell line Bw5147 (referred to as Bw cells) to express

membrane-bound single-chain Ab (scFv) to human CD3 (referred to as aCD3). The

presence of aCD3 on the surface of Bw cells (BwaCD3 cells or T cell stimulator cells)

allows activation of human T cells by transferring a signal to the TCR/CD3 complex that

induces moderate T cell proliferation. In this cellular context, the coexpression of a

stimulatory or inhibitory molecule evokes enhanced or decreased proliferative responses,

respectively (Figure 1.1A).

To generate CD72-expressing T cell stimulator cells, the cDNA of human CD72

was PCR amplified from an expression library of human B cells (kindly provided by P.

Steinberger, Institute of Immunology, Medical University of Vienna, Austria). The PCR

product was cloned into a retroviral expression vector and the integrity of the resulting

retroviral expression construct was confirmed by DNA sequencing. Subsequently, 293T

cells were transfected with the construct and the generated infectious retroviral material

(supernatant) was used for spin-infection of T cell stimulator cells. Precisely, BwaCD3

cells were retrovirally transduced to additionally express CD72 or, as a stimulatory control,

the strong costimulator CD80. Single cell clones were selected and moderate expression

of aCD3, as well as high expression of CD80 and CD72, was confirmed by flow cytometry

(Figure 1.1B). To evaluate the contribution of CD72 to the activation of T cells, we

cocultured human T cells from different donors with irradiated BwaCD3 stimulator cells

expressing CD72 (BwaCD3/CD72). Cell proliferation was determined by 3H-thymidine

incorporation during the last 16 hours of culture. For control purposes, mock-transduced

stimulator cells expressing aCD3 only (BwaCD3/Control) or stimulator cells coexpressing

aCD3 and CD80 (BwaCD3/CD80) were included.

In line with its well-established stimulatory role, the presence of CD80 on these

cells stongly enhanced T cell responses, inducing a 2.5-fold increase in DNA-synthesis,

as measured by 3H-thymidine incorporation (Figure 1.2). In contrast, stimulator cells

expressing CD72 did not modulate T cell proliferation as compared to mock-transduced

control stimulator cells expressing aCD3 only (Figure 1.2). In order to exclude the

influence of different densities of surface expression on the modulatory role of CD72,


43

proliferation assays were performed with several single cell clones, representing various

expression levels, or with polyclonal cell populations (data not shown). T cell proliferation

was in no condition affected by expression of CD72. We therefore conclude, that CD72

does not modulate proliferation of human T cells.

Figure 1.1. Characterization of the T cell stimulat or cell lines used in this study. (A) To generate T cell stimulator cells the Bw5147 (Bw) cell line was retrovirally transduced to express membrane bound anti-CD3 antibody fragments at medium density (BwaCD3). The BwaCD3 cell line moderately induces T cell proliferation, which can be down-regulated by inhibitory molecules (INHIB.) or enhanced when expressing a costimulatory molecule (STIM.). (B) The expression profiles of the T cell stimulator cell lines used in this study. T cell stimulators were transduced to coexpress CD80 (BwaCD3/CD80), CD72 (BwaCD3/CD72) or mock-transduced (BwaCD3/Control). Stable single cell clones were selected and cell surface expression was examined by flow cytometry. For detection of aCD3, cells were stained with PE-conjugated anti-mouse IgG that reacts with the V-regions of the murine OKT3 antibody (left panels). For detection of CD80 and CD72, cells were stained with anti-CD80 mAb (middle panels) or anti-CD72 mAb

(right panels), respectively, followed by PE-labelled goat anti-mouse antibody. All antibodies were used at 10 µg/ml. Cpm, counts per minute; PE, phycoerythrin.


44

Figure 1.2. CD72 does not modulate proliferation of human T cells. Human peripheral T cells of two independent donors (A and B) were cocultured with irradiated stimulator cell lines expressing membrane-bound anti-CD3 (BwaCD3/Control) and the indicated molecules (CD80 or CD72) for 72 h. For determination of T cell proliferation,

tritiated thymidine (0.5 µCi/well) was added during the last 16 hours of culture. Cells were harvested on fiber filters

und incorporated thymidine determined by scintillation counting. Results show thymidine update (cpm, mean ± SD of triplicates). Cpm, counts per minute; SD, standard deviation.

1.3. CD5-Ig does not bind to CD72-transduced Bw cel ls

Our proliferation studies with CD72-transduced BwaCD3 cells showed that CD72

does not alter T cell proliferation. It has been proposed that CD72 is the ligand for CD5

(Van de Velde et al. 1991). Nevertheless, this study has been challanged (Bikah et al.

1998). We therefore wanted to verify if CD72 and CD5 do effectively interact. For this

purpose, we generated a chimeric protein consisting of the extracellular domain of human

CD5 attached to the Fc portion of human IgG1 (CD5-Ig) as a soluble form of CD5. After

PCR amplification of the extracellular domain of CD5, using a cDNA library from human T

cells as template, we cloned the PCR product into a vector containing the hIgG1-Fc

sequence (kindly provided by P. Steinberger). The resulting construct was transiently

transfected into 293T cells, and the secreted chimeric protein was purified on a Protein A

column. The purified fusion protein was analyzed by immunoblotting and binding to anti-

hIgG1 and to anti-CD5 was confirmed by ELISA.

Direct binding of CD5-Ig to CD72 was examined by staining CD72-transfected

BwaCD3 cells with soluble CD5-Ig. Control-Ig, representing the Fc-part of hIgG1 only, was

used as control fusion protein. Results in Figure 1.3 demonstrate that anti-CD72 mAb

specifically stained the transduced cells (Figure 1.3B), whereas an excess of CD5-Ig

fusion protein (20 µg/ml) failed to directly stain these same cells (Figure 1.3A). We

therefore conclude that CD5 does not bind to CD72.


45

Figure 1.3. The fusion protein CD5-Ig does not bind to CD72. (A) BwaCD3 cells were transduced to express

CD72 (BwaCD3/CD72) or mock-transduced (BwaCD3/Control). Cells were incubated with CD5-Ig (20 µg/ml) or

Control-Ig (20 µg/ml), followed by staining with PE-conjugated anti-hFc antibody and flow cytometry. (B) The expression of CD72 was confirmed by flow cytometry. BwaCD3/Control and BwaCD3/CD72 cells were stained with

anti-CD72 antibody (10 µg/ml), followed by PE-labelled goat anti-mouse antibody. PE, phycoerythrin.

Additionally, to verify that the purified CD5-Ig fusion protein can bind to any

potential ligand, we sorted Bw cells that were transfected with an expression library

generated from human T cells and dentritic cells (provided by P. Steinberger) due to their

binding property to CD5-Ig. Subsequent flow cytometry, performed 6 days after sorting,

showed that incubation of these transfected and sorted Bw cells with CD5-Ig leads to a

1.7-fold increase in binding (as measured by mean fluorescence intensity) when

compared to Control-Ig (data not shown). Although these data are preliminary, they

suggest that the purified CD5-Ig fusion protein has a potential binding partner on the

surface of human T cells and/or dendritic cells.

1.4. CD5 engagement down-regulates T cell prolifera tion

The optimal method of testing the role of a surface molecule in modulating cell

responses would utilize the molecule’s ligand. The true identity of the physiologically

relevant ligand of CD5 remains to be established. Therefore, in absence of the ligand, the

conventional method of using mAbs allows us to specifically activate individual

receptors/pathways in order to selectively analyze the response. By utilizing anti-CD5

mAbs, we addressed the modulatory role of CD5 as an independent signal transducing

molecule. Hence, we activated T cells by engagement of CD5, in absence of any

additional stimulus. In our first approach, we cultured primary human T cells and T cell

lines in the presence of the anti-CD5 antibody UCHT2 and quantified T cell proliferation

72 hours and 96 hours after cultivation start by 3H-thymidine incorporation. The results

revealed a very stong inhibitory effect of UCHT2 when compared to the medium-only


46

control, leading to a 5-fold reduction of DNA-synthesis. Nevertheless, upon replacing the

medium-only control with the correct controls (isotype-matched antibody in the same

storage buffer as UCHT2 and azide-containing medium-only control) we established that

this strong effect on T cell proliferation was accredited to the toxic effect of azide, present

as preservative in the storage buffer. It is noteworthy that, in several reports, published in

renowned journals, azide-containing antibody solutions were used to activate cells for

long-term responses without comparison to the correct azide-containing control.

For our further studies, we utilized the azide-free cell culture supernatant of

hybridoma cells generated to secrete the anti-CD5 mAb Y-2/178. This antibody has been

described to bind to the extracellular portion of CD5 (Carmo et al. 1993). We cultured

Jurkat E6.1 cells in presence of medium, Y-2/178 (1 µl/well, corresponding to a 1:200

dilution of the hybridoma supernatant) or in presence of an excess amount of IgG without

azide (100 µg/ml) as control. The antibodies were added in solution, in the absence of

secondary antibody for crosslinking purposes. As the results in Figure 1.4 show, the

engagement of CD5 by Y-2/178 induces a strong inhibitory effect on T cell proliferation.

DNA-synthesis, as indicated by 3H-thymidine incorporation, was down-regulated 1.7-fold,

when compared to the IgG control. We here demonstrate that in the absence of any other

activating stimulus and without mAb crosslinking, ligation of CD5 down-regulates T cell

proliferation.

Figure 1.4. CD5 engagement down-regulates proliferation of Jurkat T cells. Jurkat E6.1 T cells were cultured in medium or in presence of the anti-CD5 mAb Y-2/178 (1:200 dilution of hybridoma supernatant) or IgG

(100 µg/ml) at 37°C for 72 h. Cell proliferation was determined by 3H-thymidine incorporation during the last 16 h of culture. The Proliferation Index (PI) was calculated as: PI = [cpm experimental condition/cpm control condition (medium)]. Results are given as mean

± SD of three independent experiments, cells were cultured in triplicates. Cpm, counts per minute, SD, standard deviation.


47

1.5. Discussion

CD72 is a 42 kDa type II glycoprotein, which is expressed as a homodimer at all

stages of B cell differentiation, but not on plasma cells (Von Hoegen et al. 1990). Reports

in the early nineties showed that the surface molecules CD5 and CD72 form a receptor-

ligand pair. Native human CD5 was found to bind specifically to CD72 expressed on B

lymphocytes and to non-B cells transfected with the cDNA encoding for human CD72

(Van de Velde et al. 1991). The CD5-CD72 interaction was confirmed in the murine

system using the same strategy (Luo et al. 1992). Furthermore, native soluble CD5 was

shown to bind in ELISAs to immobilized recombinant CD72 antigen, an interaction that

could be blocked by anti-CD72 mAb. Giving a functional evidence for this interaction, CD5

was shown to provide a costimulatory signal to human IgM-activated B cells, which was

shown to be inhibited by soluble CD72 (Van de Velde and Thielemans 1996). On the

other hand, M. Brown and co-workers demonstrated that recombinant CD5 fusion protein

does not bind to CD72 on B cells (Brown and Barclay 1994). The discrepancies between

native and recombinant molecules may have been caused by differences in glycosylation,

structure, and/or affinity. Nevertheless, in a later study, recombinant CD5-Ig fusion protein

was shown to induce proliferation of resting B cells, but the same chimeric protein did not

bind to CD72-transfected cells (Bikah et al. 1998).

In this study, we addressed the role of CD72 in delivering a signal towards T

lymphocytes, rather than B lymphocytes, in the presence of endogenous CD5. Upon

coculturing of CD72-expressing T cell stimulator cells with primary human T cell, we did

not observe a modulatory role for CD72 in T cell proliferation. These results can be

interpreted in two ways. First, CD72 interacts with CD5 in this cellular system, but the

CD72-CD5 contact does not transfer a signal towards T lymphocytes that influences the

proliferative response. Second, CD72 and CD5 do in fact not interact. To address the

latter possibility, we generated a CD5-Ig fusion protein for CD5-CD72 binding studies. In

accordance with previously published data that challenged CD72 as the ligand of CD5,

CD5-Ig did not bind to CD72-transduced cells, in fact indicating that CD72 is not a ligand

of CD5. This does not exclude the possibility, that CD72 binds to any other T cell surface

receptor. However, in the system of engineered T cell stimulator cells applied in this study,

such a hypothetical interaction did not influence the proliferative response of primary

human T cells.

CD72 was the first molecule described as a potential CD5 ligand. However, later

studies could not reproduce the data, but revealed the interaction of CD5 with a protein

(gp40 – 80) that is expressed on T cells and activated B cells (Biancone et al. 1996; Bikah

et al. 1998). CD5 was also shown to bind to BCR Ig framework sequences (the product of


48

a single V(H)1 gene) of rabbits (where all B cells express CD5) (Pospisil et al. 1996) and

humans (Pospisil et al. 2000). These potential ligands do not explain all the aspects of

CD5 function. Instead, a recombinant human CD5, as well as a natural soluble CD5 form,

were shown to bind to a receptor (gp150) more ubiquitously expressed on monocytes,

lymphocytes and various cell lines of lymphoid, myelomonocytic and epithelial origin

(Calvo et al. 1999). Gp150 shows a similar expression profile to the ligand of CD6,

ALCAM (Bowen et al. 1995). It is also expressed on thymic epithelial cells and both CD5

and CD6 can mediate important interactions between thymocytes and epithelial cells for

the maturation and differentiation of T cells. However, the description of gp150 has been

limited to binding and competition-binding assays so far, whereas functional data are still

missing. In other words, the ultimate nature of the CD5 ligand is still a controversial

matter. Therefore, in the absence of the CD5 ligand, we used mAbs directed at the CD5

molecule for our further studies of the role of CD5 in T cell activation, taken into account

that receptor stimulation via antibodies does only mimic the physiological CD5-CD5 ligand

interaction.

In this work we clearly establish a strong inhibitory effect of CD5 on T cell

proliferation. We present here for the first time, that CD5 acts as an inhibitory molecule of

late T cell responses in the human cell system. Additionally, these data prove that CD5

can act as an independent signal transducing molecule. It is striking that ligation of an

accessory receptor as a sole stimulus can strongly modulate T cell responses, suggesting

that the function of CD5 is not limited to fine-tuning of TCR-induced signals only.

Furthermore, neither crosslinking with a secondary antibody in solution, nor immobilization

on a carrier via crosslinking of CD5 molecules with multimeric binding of the Fc part of the

anti-CD5 antibody to a solid support or to sepharose beads, were required. This suggests,

that CD5 exists at the T cell surface in a form that can readily be activated and that does

not require extensive protein crosslinking to initiate a response.

We conclude that even in the absence of TCR/CD3 stimulation, engagement of

CD5 is sufficient to efficiently down-regulate T cell proliferation. The data presented in this

report extend the existing evidence for the functional role of CD5 as an inhibitory

molecule.


49

CHAPTER 2

A CENTRAL ROLE FOR FYN IN THE CD5-MEDIATED

INHIBITION OF T LYMPHOCYTE SIGNALING

2.1. Introduction

50

2.2. CD5 stimulation induces Fyn phosphorylation in the C-terminal

inhibitory tyrosine residue, and down-modulates the kinase

activity of Fyn

52

2.3. The tyrosine kinases responsible for CD5-induced Fy n

phosphorylation, and for CD5 phosphorylation, inter act with

distinct sequences of the cytoplasmic tail of CD5

57

2.4. CD5 stimulation inhibits downstream signaling

60

2.5. Discussion

63


50

2.1. Introduction

The fact that CD5 interacts with the antigen receptor of T cells (Beyers et al. 1992;

Osman et al. 1992; Osman et al. 1993), as well as with other accessory receptors at the T

cell surface (Beyers et al. 1992; Carmo et al. 1999) emphasizes the capacity of CD5 to

directly modulate TCR signals. Moreover, the cytoplasmic tail of CD5, although lacking

intrinsic catalytic activity, contains multiple tyrosine and serine/threonine residues that are

susceptible to posttranslational modifications and to associations with intracellular

signaling mediators. The analysis of the role of CD5 in T cell activation has mainly been

based on experiments in which CD5 was clustered with the TCR. This type of approach

has allowed dissection of T cell responses such as cell proliferation or IL-2 production.

Although the initial studies of antibody-mediated crosslinking had characterized CD5 as a

costimulatory molecule in peripheral T cells, capable of enhancing TCR-mediated cell

proliferation, the current view is that CD5 is one of the major attenuators of signals arising

from the engagement of the TCR with cognate pMHC complexes expressed on APCs.

However, the signaling pathway engaged by CD5 has scarcely been described and the

idea of CD5 behaving as a signaling molecule per se has largely been ignored.

The earliest event upon TCR engagement is the activation of Lck and Fyn, the two

predominant Src-family kinases (SFKs) in T-lineage cells. Once activated, Lck and Fyn

phosphorylate the two critical tyrosine residues within the ITAMs of the TCR/CD3

complex, generating binding sites for proteins bearing SH2 domains, and thereby initiating

downstream signaling events. Despite the central role of Lck and Fyn in this first step in

TCR signaling, it remains unclear how their activation is accomplished exactly, hindered

by the complex regulation of their kinase activities. The phosphorylation and activities of

Lck and Fyn are modulated by an interactive network of kinases and phosphatases that is

still not well understood. The two principal regulatory tyrosine phosphorylation sites play a

critical role in modulating their kinase acitivities. When phosphorylated, the inhibitory

tyrosine residue (Y505 for Lck and Y531 for Fyn), residing in the C-terminal tail, binds to

the SH2 domain of the same kinase molecule, thereby maintaining a closed conformation

and keeping the kinase in an inactive state (Liu et al. 1993). Dephosphorylation of this

tyrosine residue potentiates kinase activity. Additionally, SFKs are activated by

phosphorylation of the positive regulatory tyrosine (Y394 for Lck and Y420 for Fyn) within

the kinase domain activation loop (Veillette and Fournel 1990). Evidently, PTKs and

PTPases that target these two critical residues are principal modulators of TCR signaling.

In T cells, the transmembrane protein CD45 is the most prominent phosphatase that

dephosphorylates the negative regulatory tyrosine residue of SFKs and thereby activates

them (Mustelin and Altman 1990). CD45-deficient T cell lines contain Lck and Fyn


51

molecules that are hyperphosphorylated at their C-terminal negative regulatory tyrosines

and exhibit a drastic reduction in TCR-stimulated phosphotyrosine induction (Ostergaard

et al. 1989; Koretzky et al. 1991; Stone et al. 1997). Furthermore, PTPα was shown to

activate SFKs by dephosphorylation of the C-terminal regulatory tyrosine residue as well

(Zheng et al. 1992; Ponniah et al. 1999). Counteracting the activity of these

phosphatases, the C-terminal Src kinase Csk is the only kinase described so far (in T

cells) that phosphorylates this inhibitory residue (Okada et al. 1991). Moreover, the activity

of SFKs is regulated through interactions of their SH3 and SH2 domains with proline-rich

sequences and pTyr sites, respectively (Gonfloni et al. 1997; Superti-Furga and Gonfloni

1997).

The cytoplasmic tail of CD5 is constitutively phosphorylated (Calvo et al. 1998)

and undergoes rapid hyperphosphorylation upon TCR activation (Burgess et al. 1992;

Davies et al. 1992), with the cytoplasmic residues Y429 and Y463 being the prime targets

for phosphorylation (Dennehy et al. 2001; Vila et al. 2001). Lck appears to be accountable

for most of the tyrosine kinase activity, although Fyn and Itk may complement or regulate

Lck function (Dennehy et al. 2001; Vila et al. 2001; Castro et al. 2003). The induction of

tyrosine phosphorylation of CD5 allows Lck itself to dock onto the cytoplasmic tail of CD5,

with the Lck SH2 domain interacting primarily with pY429, resulting in a reported increase

in kinase activity (Raab et al. 1994; Dennehy et al. 2001). However, these coupled effects

were obtained using coinfection of Lck and CD5 in an insect cell line system, or using

short peptides or fusion proteins in cells stimulated with pervanadate. In fact, docking of

Lck to CD5 upon cell activation through the physiological receptor has not been

demonstrated. Furthermore, direct stimulation of CD5 with mAbs has little effect on Lck

phosphorylation in Jurkat cells (Perez-Villar et al. 1999) and no reported effect on CD5

phosphorylation itself.

It has been shown that the inhibitory function of CD5 on TCR signaling during

thymocyte development is dependent on functional integrity of the CD5 cytoplasmic

domain, as a transgene encoding a mutant form of the protein lacking the imperfect ITAM

and distal sequences failed to rescue the CD5-/- phenotype (Azzam et al. 1998). Likewise,

in peripheral T cells, negative regulation of T cell responses does not occur in the

absence of the CD5 cytoplasmic domain, suggesting that the inhibitory effect depends on

intracellular interactions mediated with signaling effector molecules (Pena-Rossi et al.

1999). Moreover, this down-modulatory effect of TCR signaling in developing thymocytes

does not involve the extracellular domain of CD5, hence, does not require engagement of

an extracellular ligand (Bhandoola et al. 2002). One hypothesis raised to explain the

inhibitory biochemical properties of CD5 was the involvement of SHP-1. CD5 has been

reported to associate with SHP-1 in Jurkat T cells, murine thymocytes and B-1 cells (Pani


52

et al. 1996; Carmo et al. 1999; Perez-Villar et al. 1999; Sen et al. 1999) and the

recruitment of SH2-containing phosphatases to phosphorylated tyrosine residues

contained within ITIM sequences is a common feature among negative regulatory

receptors (Vivier and Daeron 1997). A tyrosine residue contained within an ITIM-like motif

(LAY378KKL) that straddles the CD5 transmembrane/cytoplasmic junction was indicated

as the binding site of SHP-1 in Jurkat T cells (Perez-Villar et al. 1999). However, other

reports claim that this residue is not targeted for phosphorylation and is dispensable for

CD5 negative regulation of TCR responses in Jurkats (Pena-Rossi et al. 1999; Dennehy

et al. 2001). Moreover, in B cells the CD5 inhibitory effect seems to irradiate from Y429,

outside a second putative ITIM motif (Gary-Gouy et al. 2002). In addition to SHP-1,

several other signaling molecules may associate with the cytoplasmic tail of CD5,

potentially conferring a negative regulatory function to CD5. These include CKII, RasGAP,

Cbl and PI3K (Dennehy et al. 1997; Calvo et al. 1998; Dennehy et al. 1998).

Although the inhibitory role of CD5 has been mostly defined as a result of

association of CD5 with signaling attenuators, the targets of these inhibitors have not

been fully described, neither has the possibility been exploited that signal modulation

mediated by CD5 may also result from the active down-regulation of activatory enzymes.

In this chapter we focused on CD5-mediated molecular intracellular signaling events that

could provide the basis for its immunomodulatory properties. We have addressed the

activity and function of SFKs during CD5-induced signaling in T cells, and whereas Lck

remains mostly unresponsive following CD5 triggering, Fyn is very rapidly phosphorylated

and its activity modulated following stimulation of CD5.

2.2. CD5 stimulation induces Fyn phosphorylation in the C-terminal inhibitory

tyrosine residue, and down-modulates the kinase act ivity of Fyn

The rapid stimulation of PTK activity and the subsequent tyrosine phosphorylation

of the TCR/CD3 ITAMs and of numerous other receptors and cytoplasmic proteins is a

hallmark of T cell activation. Therefore, we addressed the early response of T cells to

mAb-induced CD5 stimulation by analyzing the global protein tyrosine phosphorylation of

T cell lysates. For this purpose, Jurkat T cells were stimulated for different periods of time

with anti-CD5 at 37°C, and rapidely collected and l ysed. DNA was cleared from the

supernatants by centrifugation and lysates were subjected to SDS-PAGE and

immunoblotted with the anti-phosphotyrosine mAb 4G10. Consistently with previous

observations (Perez-Villar et al. 1999), CD5 triggering induced a very slight increase in

phosphorylation of a number of cellular proteins within 30 sec of activation, peaking at 2

min (Figure 2.1A). The substrates phosphorylated after CD5 triggering corresponded to


53

those of the same apparent molecular weight phosphorylated upon CD3 engagment.

Nevertheless, global tyrosine phosphorylation was markedly delayed and impaired

following CD5 stimulation, compared to equivalent stimulations with anti-CD3 mAb (Figure

2.1A). Despite been much reduced in comparison to the activation induced via CD3, this

fast response to CD5 stimulation suggested that the role of CD5 is not solely to down-

modulate signals transduced through the physiological receptor for antigens.

Figure 2.1. CD5 triggering induces changes in the p rotein tyrosine phosphorylation of Src-family kinas es. (A) Jurkat E6.1 cells (5 x 106 cells per sample) were incubated with anti-CD3 mAb OKT3 (2 µg/ml) or with anti-CD5

mAb OKT1 (10 µg/ml) and rabbit anti-mouse (20 µg/ml) at 37°C. At the indicated times, cells were p elleted and lysed in 1% NP-40 lysis buffer. The global phosphotyrosine content in lysates derived from these samples was revealed by Western blotting (WB) with the phophotyrosine-specific mAb 4G10. Numbers on the left represent molecular masses (MM) of proteins in kilodaltons (kDa). (B) 1 x 106 JKHM cells were activated with the anti-CD5 mAb Y-2/178 for 2 min or incubated with isotype-control antibody (NS) for the same time at 37°C. Subsequently, cells were c ollected, fixed and permeabilized for 30 min at 4°C. For flow cytom etric analysis, cell were stained with anti-pSrc(+), anti-pSrc(-) or anti-pLck(Y505) antibody, followed by FITC-labelled anti-rabbit antibody. Details of the histograms are shown. FITC, fluorescein-5-isothiocyanat.

CD5 triggering revealed pronounced phospho-protein bands at the size range of

120 kDa and 55 kDa, the latter correlating with the size of SFKs and thereby suggesting

CD5-induced changes in the tyrosine phosphorylation status of Src-family kinases. Using

a cytometry-based simple and rapid detection method for intracellular tyrosine

phosphorylation, we examined whether stimulation through CD5 in Jurkat cells would


54

induce phosphorylation of SFKs, and which tyrosine residues would be targeted. We used

an antiserum specific to the activatory tyrosine residues of Src-like kinases, which in T-

lineage cells should recognize the phosphorylated form of Y394 of Lck as well as the

homologous residue in Fyn, Y420. In parallel, we used a polyclonal antibody detecting

phosphorylation of the C-terminal inhibitory tyrosine residues, Y505 in Lck and Y531 in

Fyn. JKHM Jurkat cells were stimulated for two minutes with the anti-CD5 antibody Y-

2/178, or left undisturbed (incubation with the isotype-control antibody IgG1), following

which cells were fixed, permeabilized, incubated with the phospho-specific antibodies and

analyzed by flow cytometry.

As shown in Figure 2.1B, CD5 stimulation resulted in a small but consistent

increase in the phosphorylation of C-terminal inhibitory tyrosine residues of the Src-type

kinases, whereas no such increase could be detected in the phosphorylation of the

activatory tyrosine residues. However, when we used a polyclonal serum specifically

reacting with Y505 of Lck (Figure 2.1B, right panel), again no increased signal was

perceived, suggesting that the detected C-terminal tyrosine phosphorylation was not

targeting Lck. By immunoblotting of Lck immunoprecipitates, we confirmed that no

significant increase in the overall tyrosine phosphorylation of Lck was induced upon CD5

stimulation of Jurkat E6.1 or JKHM cells (Figure 2.2A).

Fyn thus became a central focus of our study, and we analyzed the effect of

cellular stimulation via CD5 on Fyn phosphorylation and activity. JKHM and E6.1 cells

were stimulated for different times with Y-2/178 mAb, cells were lysed and Fyn was

immunoprecipitated. Following SDS-PAGE and Western blotting, we could detect a

significant increase in Fyn phosphorylation, which peaked at 2 min and progressively

decreased in subsequent times in both Jurkat lines (Figure 2.2B). We then confirmed that

the inhibitory Fyn Y531 residue was indeed phosphorylated upon CD5 stimulation (Figure

2.2C): after different times of CD5 stimulation, lysates from JKHM cells were subjected to

immunoprecipitation using a polyclonal Ab reactive with phosphorylated C-terminal

tyrosine residues of Src-family kinases. Immunoprecipitates were transferred to

nitrocellulose membranes and probed with Lck- and Fyn-specific Abs. Whereas the

inhibitory Lck tyrosine residue was only marginally phosphorylated, and its

phosphorylation level did not change significantly upon CD5 ligation, the level of

phosphorylation of the corresponding residue in Fyn had an increase of over 10-fold at the

2 min time point.


55

Figure 2.2. CD5 ligation induces Fyn tyrosine phosp horylation at the C-terminal inhibitory residue. 5 x 107 Jurkat T cells (E6.1 or JKHM) were incubated at 37°C with the anti-CD5 mAb Y-2/178 (1:5 dilution of hybridoma

supernatant) for the indicated time points or with the isotype control antibody IgG1 (10 µg/ml) for 2 min (corresponding to time point 0 of activation). Subsequently, cells were rapidely collected and resuspended in 1% NP-40 lysis buffer. Lysates were immunoprecipitated with polyclonal (A) anti-Lck, (B) anti-Fyn or (C) anti-pSrc(-) antibodies, resolved by 7.5% SDS-PAGE and blotted onto PVDF filters. Membranes were incubated with the HRP-conjugated phosphotyrosine-specific mAb 4G10 (A and B, top panels) or with polyclonal anti-Lck (C, top panel) or anti-Fyn (C, lower panel), followed by HRP-labelled goat anti-rabbit antibody. Proteins were visualized by enhanced chemoluminescence. In A and B, lower panels correspond to loading controls to verify equal amounts of protein (membranes were stripped and reprobed with the indicated antibodies). In C, values below the panels correspond to densitometric analysis of exposed films from non-saturated signals. The black spot at the lower panel (10 min time point) was excluded from the analysis. IP, immunoprecipitation; WB, Western blot.

To investigate the mAb presentation requirements for Fyn tyrosine

phosphorylation, we activated Jurkat T cells with the anti-CD5 mAb BL1a, that identifies a

different epitope of the CD5 molecule than Y-2/178. Under the conditions tested, BL1a

provided the signal necessary to induce Fyn tyrosine phosphorylation (Figure 2.3). The

kinetics of BL1a-induced Fyn phosphorylation was delayed in comparison to Y-2/178-


56

mediated responses (Figure 2.2.B), the former peaking at 10 min. Nevertheless, the

response was comparably strong. Therefore, CD5-mediated Fyn tyrosine phosphorylation

seems to be epitope-independent. Furthermore, the response is not related to the

antibody isotype because Y-2/178 and BL1a belong to IgG1 and IgG2a isotypes,

respectively.

Figure 2.3. CD5-induced Fyn tyrosine phosphorylatio n is epitope-independent. 5 x 107 JKHM cells were

activated at 37°C with the anti-CD5 mAb BL1a (10 µg/ml) for the indicated time points. For control purpose (time

point 0, unstimulated cells), cells were incubated with IgG1 (10 µg/ml) for 2 min. Subsequently, cells were lysed in 1% NP-40 lysis buffer. Anti-Fyn immunoprecipitates of the lysates were subjected to 7.5% SDS-PAGE under non-reducing conditions and immunoblotted with the HRP-conjugated anti-phosphotyrosine mAb 4G10 (upper panel). For loading control, membranes were stripped and reprobed with anti-Fyn Ab (lower panel). IP, immunoprecipitation; WB, Western blot.

As the results from Figure 2.1B and Figure 2.2C indicated that the C-terminal

inhibitory tyrosine residue of Fyn was being phosphorylated upon CD5 triggering, we

questioned what the functional effect of the observed phosphorylation in the ensuing

activity of Fyn would be. For this purpose we assessed the kinase activity of total cellular

Fyn, performing in vitro kinase assays. JKHM cells were stimulated for different times with

Y-2/178 mAb following which cells were lysed and Fyn immunoprecipitated with a

polyclonal antibody. Fyn immunoprecipitates were washed and subjected to a kinase

reaction in the presence of [γ-32P]-ATP, and radioactive Fyn could be detected on X-ray

films following exposure to the dried gel. As can be seen in Figure 2.4 (upper panel), the

activity of Fyn, as measured by its autophosphorylation, decreased sharply from non-

stimulated cells (time point 0) until 2 minutes, with a decline of over 2.5-fold as evaluated

by densitometry of the autoradiography. Interestingly, CD5 immunoprecipitation and in

vitro kinase reactions of resting versus CD5-activated cells resulted in a marked

enhancement, 2.8-fold, of [γ-32P]-ATP incorporation into CD5 at the 2 minute reaction

(Figure 2.4, lower panel).


57

Figure 2.4. CD5 stimulation down-modulates Fyn activity and enhances CD5-associated kinase acitivity. 5 x 107 JKHM cells were activated with Y-2/178 (1:5 dilution of hybridoma supernatant) for the indicated time points at 37°C and lysed in 1% NP-40 lysis buffer. In vitro kinase assays of anti-Fyn (upper panel) and anti-CD5 (lower panel) immunoprecipitates were performed. Phosphorylated products were separated by 11% SDS-PAGE and visualized by exposure of the dried gels to Kodak BioMax MR films. pFyn, pCD5 and x-fold modulation of phosphorylation in comparison to non-stimulated control (time point 0), as evaluated by densitometric analysis, are indicated. IP, immunoprecipitation

2.3. The tyrosine kinases responsible for CD5-induc ed Fyn phosphorylation, and for

CD5 phosphorylation, interact with distinct sequenc es of the cytoplasmic tail of

CD5

As CD5 does not have intrinsic enzymatic activity, its phosphorylation is most likely

catalyzed by tyrosine kinases that coprecipitate and are thus present in CD5 immune

complexes at the time of reaction. Using a set of Jurkat cell line variants expressing

different CD5 truncation mutants, we investigated which kinases could be present in CD5

immunoprecipitates that induced CD5 phosphorylation, and whether these or other

kinases could be responsible for the CD5-induced phosphorylation of Fyn. For this study

we used CD5-expressing JKHM cells, the CD5-negative cell line 2G5, reconstituted 2G5

cells expressing the 471 amino acids long wild-type CD5 protein (2G5/CD5.WT), and also

2G5 transfectants expressing the truncation mutants CD5.K384stop, CD5.E418stop and

CD5.H449stop (Figure 2.5A).

As assessed by FACS, all cells expressed CD5 at comparable levels, except for

2G5 that were completely CD5-deficient (Figure 2.5B). SDS-PAGE analysis of

immunoprecipitated CD5 from cell surface biotinylated cells confirmed additionally that all

CD5 mutants and wild-type molecules were at the expected size (Figure 2.5C, top). Cell

lines were grown in media, and aliquots of 5 x 107 non-stimulated cells were lysed in

Triton X-100 lysis buffer. Using Y-2/178 mAb, CD5 was immunoprecipitated from lysates

and subjected to in vitro kinase assays. As expected, the most prominent phospho-protein

in each lane corresponded to the CD5 polypeptides at their respective mass (Figure 2.5C,

middle). While slight variations in the intensity of the gel bands result from minor changes


58

in CD5 expression as well as from the number of tyrosine residues present in each mutant

(one tyrosine residue in the E418stop mutant, three in the H449stop mutant and four in the

full-length molecule), the lack of detection of the CD5.K384stop mutant (that also contains

one tyrosine residue, compare to E418stop), suggests that the stretch of amino acids

between K384 and E418 suffices for associating with the protein tyrosine kinase(s)

responsible for, at least, partial phosphorylation of CD5.

Figure 2.5. Lck binds to the CD5 cytoplasmic tail b etween K384 and E418 and phosphorylates CD5. (A) Schematic representation of wild-type and cytoplasmic tail-mutated CD5 molecules used in this work. CD5 truncation mutants (CD5.K384stop, CD5.E418stop and CD5.H449stop) are named according to the amino acid, in which a premature stop codon was introduced, and its position in the wild-type sequence. The four tyrosine residues of the CD5 cytoplasmic domain are indicated by arrows. (B) Expression of CD5 in the indicated cell lines was measured by flow cytometry of cells stained with anti-CD5 mAb, followed by FITC-labelled rabbit anti-mouse antibody. (C) Indicated cell lines were surface biotinylated, washed, lysed in 1% Triton X-100 lysis buffer, and either subjected to immunoblotting with HRP-conjugated ExtrAvidin (top panel) or to immunoprecipitation with the anti-CD5 mAb Y-2/178. In vitro kinase assays were performed with 50% of the CD5 immunoprecipitates. Samples were separated by 11% SDS-PAGE and visualized by exposure of Kodak BioMax MR films to the dried gel (middle panel). The rest of the CD5 immunoprecipitates were denatured in 2% SDS and reprecipitated with polyclonal anti-Lck antibody. Lck reprecipitates were separated by SDS-PAGE, and signals in dried gels were detected on Kodak BioMax MR films (lower panel). CY, cytoplasmic; EC, extracellular; FITC, fluorescein-5-isothiocyanat; IP, immunoprecipitation; RP, reprecipitation; TM, transmembrane.


59

Lck has been described as the tyrosine kinase that most efficiently phosphorylates

CD5 (Dennehy et al. 2001; Vila et al. 2001; Castro et al. 2003), therefore we used the

CD5 immune complexes obtained from the kinase reactions of the different cell lines, and

attempted to reprecipitate Lck. As can be seen in Figure 2.5C (bottom) active Lck could

be well recovered from JKHM cells, and also from 2G5 cells reconstituted with full-length

CD5 and with the truncation mutants E418stop and H449stop, but was not detected in 2G5

cells and 2G5/CD5.K384stop cells. This suggests that Lck binds to the cytoplasmic tail of

CD5 after lysine residue K384 and, confirming most previous reports, may indeed be the

major kinase phosphorylating CD5.

We next assessed whether the CD5 truncation mutants could induce Fyn

phosphorylation when challenged with CD5 mAb. Activation of 2G5/CD5.WT with Y-2/178

mAb more than recapitulated the CD5-induced Fyn phosphorylation seen in JKHM cells,

with a sharp increase in Fyn phosphorylation immediately after activation, and a peak at 2

minute post-activation (Figure 2.6A).

Figure 2.6. CD5-induced Fyn tyrosine phosphorylatio n depends on CD5 C-terminal residues. (A) 2G5/CD5.WT cells and (B) 2G5/CD5.H449stop and 2G5/CD5.K384stop cells (5 x 107 cells per condition) were activated with the anti-CD5 mAb Y-2/178 (1:5 dilution of hybridoma supernatant) for the indicated time points at 37°C or incubated for 2 min with isotype-control antibody (non-stimulated, time point 0). Subsequently, cells were rapidely collected and resuspended in 1% NP-40 lysis buffer. Lysates were subjected to anti-Fyn immunoprecipitation, followed by 7.5% SDS-PAGE and immunoblotting with the HRP-conjugated anti-phosphotyrosine mAb 410G. In A, the lower panel represents the amount of Fyn protein present in the reaction (membrane was stripped and reprobed with anti-Fyn antibody). In B, 2G5/CD5.WT cells treated in the same conditions were activated in parallel for 2 min (positive control) and showed a clear increase in Fyn tyrosine phosphorylation, indicating that the cells were properly stimulated.


60

However, even the CD5 mutant with the longest sequence, 2G5/CD5.H449stop,

failed to deliver any significant signal to Fyn (Figure 2.6B), a result confirmed with the

other mutant cell lines, 2G5/CD5.K384stop (Figure 2.6B), and 2G5/CD5.E418stop and 2G5

(data not shown). Based on these results, we conclude that the tyrosine kinase(s)

involved in CD5-induced Fyn phosphorylation interact(s) with the C-terminal section of

CD5, between amino acids H449 and L471.

2.4. CD5 stimulation inhibits downstream signaling

As the result from Figure 2.4 (upper panel) showed that CD5 stimulation down-

regulates Fyn activity, we consequently addressed downstream targets of Fyn, in order to

evaluate how signals to important Fyn proximate molecules were modulated by CD5

stimulation. For that purpose we first assessed tyrosine phosphorylation and activity of

ZAP-70, the key kinase that links SFKs to downstream signaling events. Upon

conventional TCR/CD3 stimulation using OKT3, ZAP-70 is rapidely phosphorylated, as

can be seen in Figure 2.7A. Total cellular ZAP-70 was immunoprecipitated at different

time points of TCR/CD3 activation, and global ZAP-70 phosphorylation determined using

phosphotyrosine antibodies for immunoblot detection (Figure 2.7A, top panel).

Additionally, we subjected total cellular lysates of the activated cells to SDS-PAGE,

followed by immunoblotting with a phosphospecific antibody specifically recognizing

phosphorylated Y493 of ZAP-70. As shown in Figure 2.7A (middle panel), we detected a

clear increase of the phosphorylation of this activatory residue. Both global ZAP-70

tyrosine phosphorylation and phosphorylation of ZAP-70 Y493 peaked upon 2 min of

activation. By contrast, JKHM cells stimulated through CD5 displayed a sharp decrease of

total ZAP-70 tyrosine phosphorylation with phosphorylation levels coming under the

detection limit right after activation (Figure 2.7B, top panel). Moreover, phosphorylation of

Y493 of ZAP-70, which correlates with functional positive signaling via ZAP-70, presented

a slight decrease at the 2 min time point of CD5 ligation, and a more pronounced

reduction at earlier and later time points (e.g. 30 sec or 10 min). In all CD5-activated cells,

the detected phosphorylation of Y493 was below the level detected in non-stimulated cells

(Figure 2.7B, middle panel). These low levels of ZAP-70 tyrosine phosphorylation were

paralleled by a decrease in the activity of ZAP-70 following CD5 stimulation, with a low

peak of ZAP-70 autophosphorylation at 2 min of activation (Figure 2.7C).


61

Figure 2.7. CD5 ligation down-modulates ZAP-70 phos phorylation and activity. 5 x 107 JKHM cells were

activated with (A) anti-CD3 mAb OKT3 (1 µg/ml) or (B) anti-CD5 mAb Y-2/178 (1:5 dilution of hybridoma supernatant) for the indicated time points. Subsequently, cells were rapidely collected and resuspended in 1% NP-40 lysis buffer. Aliquots (90%) of the lysates was subjected to anti-ZAP-70 immunoprecipitation, resolved by 10% SDS-PAGE and immunoblotted with HRP-labelled anti-phosphotyrosine mAb 410G (A and B, top panels). The rest (10%) of the lysates was directly immunoblotted with anti-phospho-ZAP-70(Y493) antibody, followed by HRP-conjugated goat anti-rabbit antibody (A and B, middle panels). For loading control, membranes were stripped and reprobed with anti-ZAP-70 antibody (A and B, lower panels). (C) JKHM cells were CD5-activated (Y-2/178, 1:5 dilution of hybridoma supernatant) for the indicated times, lysed, and ZAP-70 immunoprecipitates were denatured and

reprecipitated with anti-ZAP-70 antibody, followed by an in vitro kinase reaction in the presence of [γ-32P]-ATP. Autophosphorylated ZAP-70 was separated on 11% SDS-PAGE and visualized by exposure of the dried gel to Kodak BioMax MR films. IP, immunoprecipitation; RP, reprecipitation; WB, Western blot.

Besides ZAP-70, we also addressed the raft-resident adaptor PAG, an important

direct downstream target of Fyn. Similarly to ZAP-70, the phosphorylation of PAG at its

tyrosine residue 317, reported to be mediated by Fyn, steadily and significantly decreased


62

at 2 and 10 min following CD5 engagement, as seen by cytometric analysis, using anti-

phospho-PAG antibody specific for Y317 (Figure 2.8A). Total phosphorylation of PAG, as

measured in kinase assays of immunoprecipitated PAG, also decreased at the same time

points, despite an initial rise at 30 sec (Figure 2.8B). Tyrosine 317 of PAG has been

identified as the docking site for the SH2 domain of C-terminal Src kinase Csk, the protein

tyrosine kinase that targets the C-terminal inhibitory tyrosine residue of SFKs, and thus

modulates their activity. Csk was shown to be specifically recruited to phospho-PAG.

Figure 2.8. CD5 stimulation induces PAG dephosphory lation and PAG-Csk dissociation. (A) 1 x 106 JKHM cells were incubated with the anti-CD5 mAb Y-2/178 (1:5 dilution of hybridoma supernatant) for 2 min or 10 min, or with isotype-control antibody for 2 min (NS). Subsequently, cells were collected, fixed and permeabilized for 30 min at 4°C. For flow cytometric analysis, cells were st ained with anti-pPAG(Y317) antibody, followed by FITC-labelled anti-rabbit antibody. Details of the histograms are shown. (B) JKHM cells were activated for the indicated times at 37°C, lysed, immunoprecipitated with PAG-C1, denatu red and reprecipitated with polyclonal anti-PAG antibody. In

vitro kinase assays were performed in the presence of [γ-32P]-ATP, and labelled PAG was detected on X-ray films following exposure to the dried gel. (C) 5 x 107 JKHM cells were stimulated with anti-CD5 (Y-2/178) or anti-CD3 (OKT3) for the indicated time periods at 37°C and l ysed in 1% NP-40 lysis buffer. PAG immunoprecipitates were resolved by 7.5% SDS-PAGE and immunoblotted with anti-Csk polyclonal antibody. The x-fold modulation of PAG-associated Csk was evaluated by densitometric analysis and is indicated as values under the panels. IP, immunoprecipitation; FITC, fluorescein-5-isothiocyanat; RP, reprecipitation; WB, Western Blot.


63

We therefore tested whether CD5-induced PAG dephosphorylation would result in

the release of Csk from the PAG adaptor. JKHM cells were stimulated with Y-2/178 mAb,

or OKT3 as a positive control for activation, and reactions stopped at different time points.

Cell lysates were subjected to immunoprecipitations using PAG-C1, and immune

complexes analyzed by immunoblotting with a Csk polyclonal. As shown in Figure 2.8C,

CD5 stimulation resulted in a gradual dissociation of Csk from PAG, confirming our

prediction. On the other hand, stimulation through CD3 resulted in a sharp release of Csk

from PAG, as reported (Brdicka et al. 2000; Davidson et al. 2003), but we observed an

almost immediate reassembly of the complex at 2 min, and more evidently at 5 min upon

activation.

2.5. Discussion

Protein tyrosine kinases (PTKs) are key mediators of signaling through cell surface

receptors as many of the critical early phosphorylation events that occur within a cell

following stimulation are performed by these kinases. Therefore, specificity and regulation

of their activities is critical for creating the correct responses to external stimuli. In T cells,

a number of PTKs are known to play significant roles in the generation of a response to

the engagement of the TCR, including members of the Src family and Syk family of PTKs.

Ligand engagement of the TCR leads to activation of the SFKs Lck and Fyn. While

the generality of their activation has been delineated, many questions concerning the

mechanism(s) involved remain. SFKs adopt specific conformations that largely dictate

their level of activity. When the crystal structure of inactive Hck and Src and active Lck

were solved, a common mechanism of activation was proposed (Sicheri et al. 1997; Xu et

al. 1999), where SFKs adopt a closed conformation, effectively inactivating the kinase

domain, when the C-terminal inhibitory tyrosine residue is phosphorylated. In contrast,

phosphorylation of the activatory tyrosine residue promotes an active conformation and

increases their catalytic activity. Therefore, Lck and Fyn may exist in an equilibrium state,

where subpopulations of molecules simultaneously exist as (1) open and activated

(phosphorylated on the activatory residue), (2) open and not activated (“primed”), and (3)

closed and not activated. Supporting the crystallographic studies, biochemical studies

revealed that loss of the phosphorylation-based inhibitory switch, by deletion or mutation

of the C-terminal residue, results in constitutively active Lck (Marth et al. 1988) or

oncogenic activation of Src (Schwartzberg 1998). Dephosphorylation of that residue by a

specific PTPase may be a mechanism favoring the adoption of the “open” kinase-active

state. Such a mechanism appears to account for the PTPase CD45 in TCR signaling

pathways involving Lck and Fyn. In contrast, phosphorylation of the C-terminal tyrosine


64

residue of SFKs is mediated by Csk (Nada et al. 1991). Thus, CD45 and Csk function to

positively and negatively regulate SFK function in T cells, respectively, with direct

antagonistic actions.

For Lck and Fyn to be involved in TCR signal transduction, they must each be

recruited to the receptor complex. It was suggested, that the interaction of Lck with the

coreceptors CD4 and CD8 plays a role here. Coreceptor ligation might shift the kinase

activity equilibrium of Lck towards open and activated states by clustering and activating

Lck molecules. In contrast to Lck, Fyn can instead interact directly with the CD3 subunits

(Samelson et al. 1990; Timson Gauen et al. 1992). Furthermore, the adaptor Unc119 was

suggested to link both Lck and Fyn to TCRζ and CD4 (Gorska et al. 2004). It is unclear

how the TCR initially activates each kinase. Filipp and colleagues demonstrated in the

murine system, that after crosslinking primary T cells with anti-TCR and anti-CD4

antibodies, Lck activity peaked early, upon 1 min of activation, before Fyn activity, which

showed a peak at 2 min, when Lck activity had already decreased to control levels (Filipp

et al. 2003). This suggests that Fyn activation follows different kinetics and potentially

involves a different mechanism of regulation than Lck.

In this study we delineate an important role of the accessory receptor CD5 in Fyn

regulation. CD5-induced signals revealed to specifically down-modulate the activity of Fyn

via enhanced phosphorylation of its C-terminal inhibitory tyrosine residue. As CD5 does

not possess intrinsic enzyme activity, the change in Fyn tyrosine phosphorylation must be

mediated either by a CD5-controlled tyrosine kinase or through the CD5-mediated down-

regulation of a tyrosine phosphatase. Using various CD5 truncation mutants, we

demonstrate, that Fyn tyrosine phosphorylation is dependent on the CD5 cytoplasmic

distal part, more precisely on the region between amino acids H449 and L471 (Figure

2.6B). The only cytoplasmic CD5 tyrosine residue located within this 23 amino acid

stretch, Y463, was shown to be phosphorylated by Lck and the phospho-peptide

pY463DLQ was shown to be bound by the SH2 domain of Lck, although with low affinity

(Dennehy et al. 2001). This suggested that Lck may be, through association with CD5,

involved in the CD5-induced modulation of Fyn tyrosine phosphorylation. Nevertheless, in

our experimental set-up, CD5 association with phospho-Lck revealed to be lost in CD5

mutants shorter than CD5.E418stop, suggesting that Lck binds to the stretch between K384

and E418 within the CD5 cytoplasmic tail (Figure 2.5C). This stretch does not coincide

with the minimal sequence of CD5 necessary for Fyn phosphorylation. Therefore,

although Lck may be involved in CD5-mediated Fyn regulation (probably independent of a

direct CD5-Lck interaction or independent of the tyrosine phosphorylation state of both

molecules), an additional tyrosine kinase could be responsible for Fyn phosphorylation at

the C-terminal tyrosine residue. Our preliminary results show that the activity of the


65

potential candidate Csk does not change upon CD5 activation (data not shown).

Nevertheless, the role of CD5 might be to position Csk in proximity of Fyn, rather than

changing the activity of Csk. As we demonstrate here, CD5 ligation modulates the

interaction of Csk with PAG. Upon CD5 activation, the relative amount of PAG-associated

Csk is 0.7- to 0.6-fold reduced compared to resting cells. Csk recruitment to PAG was

shown to be important for Csk-mediated inhibition of SFKs. Less Csk-PAG association

would therefore result in decreased phosphorylation of the C-terminal inhibitory residue of

SFKs. On the other hand, decreased phosphorylation of the C-terminal negative

regulatory residue of Fyn would increase Fyn activity and subsequently the

phosphorylation of PAG at Y317, a specific target of Fyn. Phosphorylation of PAG in turn

leads to recruitment of Csk, which binds via its SH2 domain to PAG pY317. Csk

translocation to the proximity of SFKs would down-modulate their activities, therefore

closing the PAG/Csk/Fyn feedback loop.

We demonstrate here, that CD5 interferes with the PAG/Csk/Fyn complex by

down-modulating Fyn kinase activity and subsequent Fyn-mediated phosphorylation of

PAG Y317. CD5 ligation induces a smooth reduction in the PAG-Csk association,

whereas T cell activation through the TCR/CD3 complex leads to an abrupt disassembly

with a fast PAG-Csk reassociation. The fact that PAG-Csk association, upon CD5

engagement, is reduced at the time point of maximum Fyn tyrosine phosphorylation

suggests that either (1) a tyrosine kinase different from Csk is responsible for CD5-

induced phosphorylation of Fyn, whereas Csk is released from the proximity of the plasma

membrane/of Fyn or (2) extensive Csk association with PAG is not necessary for Csk-

mediated Fyn phoshorylation. Supporting the latter idea, Csk was shown to be recruited to

the proximity of SFKs in PAG knock-out mice, suggesting that there are other

mechanisms of Csk recruitment. Moreover, additional interactions seem to be involved in

the inhibitory function of Csk, as the SH3 domain of Csk was shown to be required,

probably through its interaction with PEP (Cloutier et al. 1995; Cloutier and Veillette 1996;

Cloutier and Veillette 1999). Therefore, an adaptor molecule, binding to the distal part of

the CD5 cytoplasmic domain, may link CD5/Csk/Fyn and therefore be involved in Fyn

tyrosine phosphorylation and in the smooth release of the PAG-Csk complex.

Besides its role in modulating the PAG/Csk/Fyn loop, CD5-induced down-

regulation of Fyn activity may also influence the SLAM/SAP/Fyn interaction (see section

3.3.3). Furthermore, Fyn kinase was shown to phosphorylate WASP Y291, the major site

of TCR-induced WASP tyrosine phosphorylation. Mutation of Y291 was shown to

abrogate induction of WASP tyrosine phoshorylation and its effector activities, including

NFAT induction, actin polymerization and IS formation (Badour et al. 2004). Therefore,


66

CD5-induced down-regulation of Fyn activity may result in decreased WASP tyrosine

phosphorylation and thereby inhibit TCR signaling.

In this study, we reveal additional biochemical evidence for the inhibitory role of

CD5 in TCR signaling. We demonstrate that CD5 inhibits early TCR signaling events by

modulating the most prominent proximal downstream target of SFKs, ZAP-70. Upon CD5

ligation, both phosphorylation of total ZAP-70 and phosphorylation of the activatory ZAP-

70 residue Y493 were significantly down-regulated. In comparison, as reported before,

activation via the TCR/CD3 complex clearly enhanced ZAP-70 phosphorylation. We

therefore provide further evidence that CD5 ligation inhibits signaling downstream of the

SFKs, both by modulating PAG and ZAP-70. In addition, this indicates that Fyn may also

contribute to ZAP-70 regulation, at the absence of modulated Lck activity.

In conclusion, our results show that CD5 inhibits T cell signaling through its

selective down-regulaton of Fyn and decreased phosphorylation of ZAP-70 and PAG. We

demonstrate biochemical basis for a CD5-controlled signaling pathway and the

involvement of CD5 in regulation of the PAG/Csk/Fyn loop.


67

CHAPTER 3

THE MEMBRANE DISTRIBUTION OF CD5 AND

CD5-ASSOCIATED SIGNALING EFFECTORS

3.1. Introduction

68

3.2. CD5 associates with signaling effectors within lipid rafts, but

translocation to rafts is independent of cytoplasmi c tail

interactions

69

3.3. CD5 activation targets the accessory receptor CD2 t o raft

microdomains

73

3.4. Evidence for CD5 dimerization in resting cells

75

3.5. Discussion

77


68

3.1. Introduction

The T cell plasma membrane contains regions of distinct lipid composition,

enriched mainly in sphingolipids and cholesterol, immersed in a phospholipid-rich

environment, termed lipid rafts (discussed in section 4.2). A large body of evidence

supports a crucial role for lipids rafts in the initial stages of T cell activation. In resting

cells, key signaling proteins such as the adaptors LAT (Zhang et al. 1998b) or PAG

(Brdicka et al. 2000), are resident in rafts, whereas most integral proteins are found

outside these platforms. However, the composition of rafts-associated proteins may

change upon cell stimulation. Membrane compartmentalization and partitioning of

essential T cell-activating components in lipid rafts were shown to be involved in the initial

stages of T cell activation (Xavier et al. 1998; Alonso and Millan 2001). The TCR itself

seems to accumulate in lipid rafts after selective stimulation of T cells (Montixi et al. 1998;

Giurisato et al. 2003) and several accessory receptors on the T cell surface, such as CD2

(Mestas and Hughes 2001), and CD28 (Viola et al. 1999) can up-regulate TCR signals by

enhancing the association of the TCR with lipid rafts. This suggests that the

reorganization of membrane microdomains and the translocation of signaling effectors to

lipid rafts could contribute to the mechanisms of signal modulation.

CD5 was shown to accumulate at the TCR-APC contact zone in Jurkat cells

(Gimferrer et al. 2003) and in various murine cells systems (Brossard et al. 2003). This

recruitment of CD5 was correlated with its inhibitory role in TCR signaling (Brossard et al.

2003). Elaborating on the membrane sublocalization of CD5, a large portion of CD5

molecules was found in detergent-soluble non-raft fractions in unstimulated cells (Cerny et

al. 1996; Yashiro-Ohtani et al. 2000). However, unlike CD2, a stimulation-induced

membrane redistribution of CD5 has not yet been reported. CD5 associates with the

accessory receptor CD2 (Castro et al. 2002) and it has been suggested that CD2 can

potentiate the CD5 inhibitory effect in thymocyte selection, as demonstrated in the

CD2/CD5 double knockout mouse (Teh et al. 1997). Human CD2 is entirely non-raft

resident in resting cells and translocates to lipid rafts upon CD2 Ab crosslinking or

following binding to CD58 during conjugate formation (Yashiro-Ohtani et al. 2000). It was

proposed that the shift of CD2 between phases may result in the replacement of CD2BP2

by raft-located Fyn, which competes for the same proline-rich sequence of the

cytoplasmic tail of CD2 (Freund et al. 2002).

In this study, we focused on the mechanisms that regulate the equilibrium of CD5

to shift between different membrane microenvironments. We show that CD5 has the

capacity of controlling its own localization, and that this influences its interaction with


69

important intracellular signaling effectors. Furthermore, we show that CD5 controls the

membrane distribution of CD2 through a Lck-dependent mechanism.

3.2. CD5 associates with signaling effectors within lipid rafts, but translocation to

rafts is independent of cytoplasmic tail interactio ns

As a significant number of CD5 effectors and binding partners, such as Fyn, Lck

and PAG, among others, have been reported to associate with plasma membrane lipid

rafts, we investigated whether CD5 would target to these membrane microdomains

following CD5 stimulation. In parallel to JKHM cells, we analyzed CD5 membrane sub-

localization in PBMC as well, to validate our findings, as inconsistencies regarding the

lipid raft localization of some of these effectors in different cell lines have been

occasionally reported. Cells were activated with CD5 antibodies for 10 min, or left

undisturbed, following which they were lysed in MBS buffer containing 1% Triton X-100,

and lysates were subjected to sucrose gradient centrifugation. Fractions of approximately

1 ml were collected, and assayed by immunoblotting for the presence of CD5. As Figure

3.1 shows, in non-activated JKHM cells and PBMC, the large majority of CD5 molecules is

not associated with lipid rafts, as most CD5 is recovered from fractions containing

membrane freely-diffusing proteins, typically fractions 7 to 9. However, upon CD5

triggering, an extensive translocation of CD5 to the raft fractions (1 to 3) is observed both

in JKHM cells as well as in PBMC. In the meantime, Fyn and Lck do not change between

membrane phases upon CD5 stimulation, being detected mostly in the lipid rafts fractions

(Figure 3.1A and B).

In order to evaluate whether translocation of CD5 to lipid rafts would result in the

association with raft-resident molecules, we analyzed the CD5 fractions prior to, and after

activation via CD5. Using PBMC, we immunoprecipitated CD5 from the three different

sets of sucrose gradient fractions, the first corresponding to CD5 in soluble fractions (7 to

9) of non-activated cells (Figure 3.1B, Set I). Set II was obtained from sucrose gradient

fractions 7 to 9 of CD5-stimulated PBMC, corresponding to the portion of CD5 that did not

translocate to lipid rafts, whereas Set III included CD5 molecules that shifted to lipid rafts

upon CD5 stimulation. All sets of immunoprecipitates were subjected to kinase assays to

label the signaling molecules, and then the immune complexes were disrupted by heat

and individual molecules reprecipitated with polyclonal antibodies. The first clear

indication obtained from the autoradiographs is that CD5 does not associate with any of

the tested molecules outside lipid rafts, as the only reprecipitated detected

phosphoprotein in soluble fractions corresponds to CD5 itself.


70

Figure 3.1. CD5 translocates to lipid rafts and ass ociates with signaling effectors upon CD5 ligation. (A) JKHM cells or (B) PBMC were stimulated with anti-CD5 mAb Y-2/178 (1:5 dilution of hybridoma supernatant) or incubated with isotype-control antibody (NS) for 10 min at 37°C. Total Triton X-100 cell lysates were subjected to sucrose density centrifugation as described in Material and Methods. Fractions recovered were numbered 1 to 9 from the top of the gradient. An equal volume of each fraction was separated by SDS-PAGE and immunoblotted with either anti-CD5 mAb, or anti-Lck or anti-Fyn polyclonal antibodies. The preferential distribution of LAT into the rafts allowed for the identification of raft fractions (not shown). (B) Indicated (framed) fractions were pooled,

immunoprecipitated with anti-CD5 and subjected to in vitro kinase assays in the presence of [γ-32P]-ATP. CD5 immune complexes were reprecipitated with polyclonal CD5, ZAP-70, Lck, Fyn, Csk, PAG and LAT antibodies. Following SDS-PAGE, radioactive products were visualized by exposure of dried gels to Kodak BioMax MR films. Numbers on the right represent molecular masses (MM) of proteins in kilodaltons (kDa). IP, immunoprecipitation; RP, reprecipitation.


71

This is true for both CD5 obtained from unstimulated cells (Set I), as well as for CD5

molecules that did not migrate to lipid rafts in the activated set (Set II). By contrast, CD5

molecules that were recovered from lipid rafts did associate neatly with Lck, Fyn, PAG

and LAT, all detected at their expected molecular sizes, but not with ZAP-70 neither with

Csk (Figure 3.1B, Set III).

The use of different cells and cell lines provided additional important information

regarding CD5 associations with Lck and Fyn. Using JKHM, where most of Lck and Fyn

are located in raft microdomains (Figure 3.1A), we were not able to detect any interactions

between CD5 and Lck or Fyn in the non-raft fractions (data not shown). In PBMC, in which

a significant part of Lck still remains outside rafts, we could not detect any association of

“soluble” CD5 with both kinases as well. Therefore, the interaction between CD5 and both

SFKs seems to be restricted to the raft membrane domains. We next used E6.1 Jurkat

cells where, conversely, virtually all Lck lies within rafts, but an important amount of Fyn is

in the membrane fluid phase. Cells were stimulated with Y-2/178 mAb, which induced an

extensive translocation of CD5 to lipid rafts (Figure 3.2A), whereas once again, no net

movement of Lck or Fyn was observed upon CD5 triggering (data not shown). Analyzing

CD5 associations with Lck and Fyn in these cells, we observed that also in E6.1 Jurkats,

CD5 association with Lck and Fyn was exclusively detected within lipid rafts (Figure 3.2B).

Figure 3.2. CD5 associates with Lck and Fyn exclusi vely in lipid rafts. (A) 1.5 x 108 Jurkat E6.1 cells were stimulated with anti-CD5 mAb Y-2/178 (1:5 dilution of hybridoma supernatant) or incubated with isotype-control

antibody (10 µg/ml) for 10 min at 37°C. Subsequently, cells were collected, washed and lysed in 1% Triton X-100 lysis buffer. Cell lysates were prepared and subjected to sucrose density centrifugation as described in Material and Methods. Equal volumes of the collected fractions 1 – 9 were separated by SDS-PAGE and immunoblotted with anti-CD5 mAb, followed by HRP-labelled rabbit anti-mouse antibody (lower panels) or with anti-Lck or anti-Fyn polyclonal antibodies, followed by HRP-conjugated goat anti-rabbit antibody (top panels). Proteins were visualized by enhanced chemoluminescence. (B) Rafts fractions 1 – 3 (R) and soluble fractions 7 – 9 (S) of non-stimulated (NS) cells or of CD5-stimulated cells were combined, immunoprecipitated with anti-CD5 and subjected to in vitro kinase assays. Labelled CD5 immune complexes were denatured in 2% SDS and reprecipitated with polyclonal anti-CD5, anti-Lck or anti-Fyn antibodies. Products were subjected to SDS-PAGE and visualized by exposure of Kodak BioMax MR films to the dried gels. IP, immunoprecipitation; RP, reprecipitation; WB, Western blot.


72

CD5 stimulation with specific mAb induced its insertion within lipid rafts, where it

was able to interact with Lck and Fyn. However, the fact that under no circumstances

could we detect any association of CD5 with the kinases in “soluble” fractions would

strongly argue against a role of either Lck or Fyn in partitioning of CD5 within rafts. We

tested whether Lck was required for lipid raft targeting of CD5. Using the Lck-deficient

J.CaM1 cell line, we confirmed that upon CD5 triggering, most of “soluble” CD5 would

translocate to the raft fractions. (Figure 3.3A). Furthermore, we tested CD5 distribution in

the ZAP-70-deficient Jurkat cell line P116. As shown in Figure 3.3B, CD5 translocation

was unaffected by the absence of ZAP-70. Moreover, the tailless mutant CD5.K384stop

could also be found at significant amounts in lipid raft fractions following stimulation of

cells with the CD5 mAb (Figure 3.3D). This translocation of CD5 was not as complete as

in the control reconstituted cells 2G5/CD5.WT (Figure 3.3C). Nevertheless, it provided

evidence that the cytoplasmic domain of CD5, and hence its cytoplasmic associations, are

not fully required for the shifting of CD5 between the different phases of the membrane.

Figure 3.3. CD5 translocation to lipid rafts is ind ependent of intracellular associations of CD5. (A) J.CaM1, (B) P116, (C) 2G5/CD5.WT and (D) 2G5/CD5.K384stop cells (1.5 x 108 cells per condition) were stimulated with anti-CD5 mAb for 10 min at 37°C or left undisturbed. Cel ls were collected, washed and lysed. Total Triton X-100 cell lysates were subjected to sucrose density centrifugation as described in Material and Methods. Equal volumes of each fraction were resolved by SDS-PAGE and blotted onto PVDF filters. Membranes were incubated with anti-CD5 mAb, followed by HRP-conjugated rabbit anti-mouse antibody, and proteins were visualized by enhanced chemoluminescence. WB, Western blot.


73

3.3. CD5 activation targets the accessory receptor CD2 to raft microdomains

In contrast to CD5, the accessory receptor CD2 seems to depend on cytoplasmic

interactions for its translocation to lipid rafts. We recently reported that rat CD2 depends

on Lck for lipid rafts distribution (Nunes et al. 2008). CD5 and CD2 do physically interact

(Castro et al. 2002) and show functional synergism in the CD2/CD5 double knockout

mouse (Teh et al. 1997). We therefore addressed the CD5-dependence of the CD2

receptor for lipid raft localization, in the human system.

We examined the membrane distribution of CD2 in resting versus CD5-stimulated

cells. E6.1 Jurkat cells were either left untreated, or stimulated with anti-CD5 mAb.

Following cell lysis and sucrose gradient centrifugation, the localization of CD2 was

evaluated by immunoblotting. As shown in Figure 3.4A, CD2 molecules localize mostly

outside lipid rafts in unstimulated cells. Upon stimulation of CD5 with the CD5 mAb Y-

2/178, we could observe that CD2 was efficiently recovered from the raft fractions.

Translocation into lipid rafts of an accessory T cell molecule induced by another

accessory molecule is a very unusual event. To further investigate the signaling

mechanisms involved for the activation-dependent translocation of CD2 to lipid rafts, we

utilized the Src-family kinase inhibitor PP2. Interestingly, CD2 translocation was

completely abolished in the presence of the inhibitor, suggesting that the mechanism is

strictly dependent on active Src-family kinases.

To assure that the translocation of CD2 induced by ligation of CD5 was not a

particularity of the cell line utilized, we confirmed the data in primary T cells. Following

CD5 stimulation, we could clearly detect CD2 translocation to the detergent-insoluble

ordered membrane domains when compared to non-stimulated cells in PBMC (Figure

3.4A, lower panels). As in Jurkat E6.1, CD2 shifiting was efficiently inhibited by PP2,

confirming that the mechanism is strictly dependent on kinases of the Src-family. We

therefore analyzed the CD2 membrane distribution in the Lck-deficient cell line J.CaM1. In

these cells, CD5 activation did not cause a change in CD2 membrane sublocalization,

suggesting that the mechanism is dependent on Lck. Confirming this assumption,

recruitment of CD2 to the rafts was readily reestablished In J.CaM1 cells reconstituted

with Lck (J.CaM1/Lck), as shown in Figure 3.4B.


74

Figure 3.4. CD5-induced recruitment of CD2 to lipid rafts is Lck-dependent. (A) Jurkat E6.1 cells and PBMC (1.5 x 108 cells per condition) were stimulated with the anti-CD5 mAb Y-2/178 (1:5 dilution of hybridoma supernatant) or incubated with isotype-control antibody (NS) for 10 min at 37°C. For inhibition of Src-family kinas e activity, cells

were pre-treated with the Src kinase inhibitor PP2 at 10 µg/ml (lower panel of each cell line). After stimulation, cells were collected, washed, lysed in 1% Triton X-100 lysis buffer and subjected to sucrose density centrifugation. Equal volumes of each fraction were separated by 10% SDS-PAGE and immunoblotted with anti-CD2 polyclonal antibody, followed by HRP-linked goat anti-rabbit antibody. (B) J.CaM1 and J.CaM1/Lck cells were treated as in described in A. WB, Western blot.


75

3.4. Evidence for CD5 dimerization in resting cells

Although CD5 stimulation using mAb did not induce exuberant responses in terms

of general phosphorylation of intracellular substrates, tyrosine phosphorylation of the

inhibitory residue of Fyn was very consistent, and the ensuing modulation of cellular

signaling was indeed robust (see Chapter 2). Furthermore, CD5 activation efficiently

down-regulated T cell proliferation, as described in Chapter 1. One striking feature,

however, is that antibody-mediated stimulations were performed without using a

secondary antibody to induce crosslinking of CD5. As shown in Figure 3.5A, Y-2/178

readily effects the Fyn phosphorylation level at very low primary antibody concentrations,

even in the absence of secondary antibody. At a concentration as low as 0.5 µg/ml Y-

2/178 (corresponding to a 1:100 dilution of hybridoma supernatant), the anti-CD5 mAb

revealed to increase Fyn tyrosine phosphorylation. Only Y-2/178 at a concentration of 0.1

µg/ml shows a response comparable to the non-stimulated (time point 0) control. This

suggests that even a small amount of the primary antibody Y-2/178 is able to initiate

signals to Fyn, and this effect is independent of crosslinking with a secondary antibody in

solution. For our studies, Y-2/178 was typically used at a concentration of 10 µg/ml or 2.5

µg/ml to ensure maximum responses.

To elaborate on this effect, we performed comparative studies. Figure 3.5B and

3.5C show examples of CD5-mediated responses in the presence or absence of

secondary antibody. Anti-CD5 in solution, in the absence of both secondary antibody or a

immobilization platform, revealed at least as effective as anti-CD5 crosslinked by the

addition of anti-mouse Ig (Figure 3.5B). Even the massive translocation of CD5 to lipid

rafts did not require secondary crosslinking of the Y-2/178 mAb used as soluble reagent,

as the comparative example in Figure 3.5C shows. This suggests, that CD5 exists at the

surface of resting T cells in a form that can readily be activated and that does not require

extensive protein crosslinking to initiate a response. We therefore tested whether the CD5

glycoprotein could be present at the cell surface in a dimeric form, which could in part

explain why CD5 stimulation could be so effective and independent of antibody

crosslinking. For that purpose, full-length CD5 as well as mutants were genetically fused

to either the bioluminescent protein luciferase (Luc) or to the green fluorescent protein

(GFP) and expressed as “BRET pairs”, as described previously (James et al. 2006). In

brief, if the two partners do not interact, only luciferase signals are emitted. However, if

they come into close proximity due to protein-protein interactions, energy transfer can

occur between Luc and GFP and an additional signal emitted by the GFP can be

detected. BRET efficiency (BRETeff) is the ratio of GFP emission to that of Luc emission.


76

Figure 3.5. CD5-induced Fyn tyrosine phosphorylatio n and CD5 membrane redistribution do not require antibody crosslinking. 5 x 107 JKHM cells were activated at 37°C with (A) the indicated concentrations of Y-2/178

hybridoma supernatant for 2 min in the absence (w/o) of secondary antibody or with (B) 10 µg/ml Y-2/178 (1:5 dilution of hybridoma supernatant) for the indicated times in presence or absence (w/o) of secondary antibody. For

control purpose (time point 0, unstimulated cells), cells were incubated with 10 µg/ml IgG1 for 2 min. Subsequently, cells were lysed in 1% NP-40 lysis buffer. Anti-Fyn immunoprecipitates of the lysates were subjected to 7.5% SDS-PAGE under non-reducing conditions and immunoblotted with the HRP-conjugated anti-phosphotyrosine mAb 4G10 (upper panels). For loading control, membranes were stripped and reprobed with anti-Fyn Ab (lower panels). C) 1.5 x 108 Jurkat E6.1 cells were stimulated with anti-CD5 mAb Y-2/178 (1:5 dilution of hybridoma supernatant) or

incubated with control antibody IgG1 at 10 µg/ml (NS). Y-2/178 was either crosslinked with rabbit anti-mouse

antibody (20 µg/ml) or left undisturbed (anti-CD5 w/o, middle panel). After activation for 10 min at 37°C, cells wer e collected, washed and lysed in 1% Triton X-100 lysis buffer. Cell lysates were prepared and subjected to sucrose density centrifugation as described in Material and Methods. Equal volumes of the collected fractions 1 – 9 were separated by 7.5% SDS-PAGE and immunoblotted with anti-CD5 mAb, followed by HRP-labelled rabbit anti-mouse antibody. IP, immunoprecipitation; WB, Western blot.

Expression of full-length CD5 as a BRET pair revealed that it is able to form weak

dimers at the cell membrane, with the BRETeff values exhibiting a dependence on the

acceptor:donor ratio (Figure 3.6). To assess the involvement of the extracellular domain of

CD5 in dimerization, we generated a chimeric protein, CD5Ext/CD2Int, composed of the


77

extracellular and transmembrane regions of CD5 fused to the cytoplasmic tail of the CD2

molecule. CD2 was already confirmed as a monomer in BRET assays. The BRETeff levels

obtained for the CD5Ext/CD2Int chimera were indistinguishable from those obtained for

the wild-type molecule, implying a role for the extracellular domain of CD5 in its

dimerization (Figure 3.6). These results complete our previous findings and suggest that

CD5 may exist as a dimer on the surface of resting T cells.

Figure 3.6. CD5 is able to self-associate at the ce ll surface through its extracellular domain. BRET analysis of CD5, CD2 and a CD5/CD2 chimera. The CD5/CD2 chimera (CD5Ext/CD2IntBP) shows BRETeff values that follow the same profile as seen for the CD5 wild-type BRET pair (CD5BP), which can readily be fitted by a dimerization model. CD2 (CD2BP) shows low BRETeff values and independence of the acceptor:donor ratio, characteristics of monomers. BRET analysis was performed by Marta I. Oliveira and John R. James in the laboratory of Simon J. Davis (Nuffield Departement of Clinical Medicine, University of Oxford, UK). BRETeff, BRET efficiency; BP, BRET pair; Ex, extracellular; Int, intracellular; GFP, green fluorescent protein; Luc, luciferase; WB, Western Blot.

3.5. Discussion

It is increasingly clear that lipid rafts play an important role in T cell signaling. The

aggregation of lipid rafts to form signaling platforms where relevant molecules and

adaptors are clustered seems critical to the modulation of TCR signaling. We show in this

study that CD5 is not constitutively present in these membrane microdomains, but is

massively recruited to the rafts upon crosslinking with anti-CD5 mAb. Inducible raft

association of surface molecules upon crosslinking with antibodies has been previously

demonstrated. The first studies reported the aggregation-dependent association of FcεRI

with lipid microdomains (Field et al. 1997). Later, the IL-2 receptor was shown to partially

translocate to lipid rafts after selective IL-2 stimulation (Goebel et al. 2002). Likewise, the


78

TCR/CD3 complex translocates, at least partially, to lipid rafts upon TCR engagement

(Montixi et al. 1998) and the costimulatory receptor CD28 was shown to be recruited to

rafts upon engagement with either anti-CD28 mAbs or the natural ligand B7-2 (Sadra et

al. 2004). What is striking in the partition of CD5 to the detergent-insoluble ordered

membrane domains is that, firstly it presents an extensive redistribution of CD5 molecules

at the cell membrane, and secondly, CD5 has been linked to down-modulation. Hence,

the capacity of CD5 recruitment to lipid rafts must correlate with its inhibitory role in TCR

signaling.

It is noteworthy that the association of CD5 with lipid rafts was readily detected

under highly stringent detergent conditions (1% Triton X-100). Previous studies also

reported that human CD2 translocated to lipid rafts when 1% Triton X-100 was used for

membrane solubilization (Yang and Reinherz 2001). In contrast, the interaction between

aggregated FcεRI and lipid rafts demonstrated to be very sensitive to solubilization

conditions, as it was disrupted when concentrations of Triton X-100 rose above 0.05%

(Field et al. 1997). Similarly, the association of CD3 with rafts, induced by engagement of

the TCR, is only preserved when a mild detergent (Brij) is used (Montixi et al. 1998). One

property that has been attributed to Triton X-100 is that it can partially solubilize the liquid-

ordered phase of the plasma membrane (see General Introduction, section 4.2), which

may lead to partial loss of raft components, especially if they are weakly associated.

Demonstration of raft association under these stringent conditions might therefore indicate

a strong raft “affinity” of the protein examined. In the last years, the existence of rafts has

been challenged. The main argument of discussion has been the questioned reliability of

the standard technique of raft isolation due to their detergent-insolubility. Nevertheless, it

has been clearly demonstrated that detergent does not induce artifacts such as ordered

domains or association of solubilized components with liquid-ordered microdomains

(Schroeder et al. 1998). Taken together, the results of this study suggest that the

mechanism underlying weak interactions of multichain immune recognition receptors (e.g.

TCR or FcεRI) with the rafts may be distinct from that mediating stronger associations of

receptors, like CD5, with these microdomains.

In this report, we further demonstrate that CD5 associates with major signaling

effectors exclusively in the raft domains. We reveal inducible associations of CD5 with

both the Src family kinases Lck and Fyn and with the membrane adaptors PAG and LAT.

16 years ago, the group of C. E. Rudd demonstrated the presence of Lck, Fyn, CD3

subunits, and TCRζ in CD5 immunoprecipitates of Brij 96-based detergent lysates derived

from peripheral blood T cells (Burgess et al. 1992). 2 years later, the same group showed

that CD5 and Lck (but not Fyn) could be coprecipitated under more stringent detergent

conditions, including NP-40 and Triton X-100, in a variety of Jurkat cell lines and in a


79

recombinant insect cell system (Raab et al. 1994). Up to date, no direct interaction

between CD5 and Fyn has been reported. Here we clearly demonstrate, that CD5

interacts with both SFKs Lck and Fyn in Triton X-100 lysates and that this interaction is

raft-restricted. The direct interaction of CD5 with Fyn, exclusively in membrane

subdomains, could provide a mechanism for regulating CD5 “activity”. Although CD5 does

not possess intrinsic enzymatic activity, CD5-induced translocation to rafts and raft-

restricted CD5 binding to Fyn might sequester Fyn from possible interaction partners (e.g.

phosphatases that dephosphorylate the negative regulatory tyrosine of Fyn and thereby

activate the kinase). Another hypothetical scenario includes the inducible recruitment of a

CD5-associated kinase to the proximity of Fyn. This kinase could then phosphorylate the

C-terminal regulatory tyrosine residue of Fyn and thereby increase the kinase activity. As

discussed in Chapter 2, this kinase might bind to the C-terminal stretch of CD5, between

amino acids H449 and L471.

The observed association of CD5 with the two most important T cell adaptors LAT

and PAG potentiates the complexity of CD5-controlled events, but also interlines the

importance of CD5 in early signaling events, including the involvement of CD5 in the

PAG/Csk/Fyn loop. Although we could not detect a direct interaction of CD5 with Csk, this

does not exclude a possible weaker association between those molecules, which could

have been lost under the stringent detergent conditions tested. This might be the case for

ZAP-70. CD5 association with ZAP-70 was not detected under the conditions tested in

this study. However, ZAP-70 was shown to associate with CD5 in human thymocytes and

in C58 cells under mild detergent conditions (Brij 96) before (Gary-Gouy et al. 1997;

Castro et al. 2003). Recapitulatory, the raft exclusivity of the CD5 associations with crucial

signaling effectors suggests that CD5 translocation to lipid rafts may provide a mechanism

for regulating CD5-induced inhibition of TCR signaling.

In this report, we also demonstrate, that CD5 translocation to lipid rafts is

independent of CD5 cytoplasmic interactions, as the tailless mutant CD5.K384top was still

recruited to the membrane microdomains, although to a lesser extend, compared to the

wild-type CD5 molecule. Underlining these data, we could also show that CD5 membrane

redistribution was independent of Lck and ZAP-70. It is puzzling how, in a system where

CD5 has lost its interactions with the signaling apparatus, and where CD5 undeniably no

longer exerts a signaling effect (as described in Chapter 2), it still is capable to produce

major changes in the membrane reorganization. This example is not unique, however.

Rafts reorganization induced by FcγR ligation has been shown to occur independently of

the FcγR cytoplasmic receptor sequences (Kono et al. 2002). Therefore, it is possible that

inducible raft coalescence following receptor crosslinking may depend in the first instance

on extracellular interactions.


80

Unlike CD5, the induced translocation of CD2 is dependent on cytoplasmic

interactions, as it is abolished by the Src-family kinase inhibitor PP2 or in Lck-deficient T

cells. Structural and functional studies on CD2 have clearly assigned signaling properties

to the CD2 cytoplasmic domain. Proline-rich sequences of the cytoplasmic tail of CD2

bind to the SH3 domains of Lck and Fyn (Bell et al. 1996; Lin et al. 1998), and deletion of

the cytoplasmic tail completely abolishes signaling via CD2 (He et al. 1988; Bierer et al.

1990). We can thus expect some of the results we observe in the CD5-induced

translocation of CD2 to be determined by the signaling machinery associated with CD2.

In conclusion, we demonstrate here that CD5 tends to form dimers, driven through

the extracellular parts of CD5, on the surface of resting T cells. In this form, CD5 can

readily be activated by a sole anti-CD5 mAb stimulus. This engagement induces massive

membrane redistribution of CD5 molecules, resulting in the raft-restricted association with

molecules that control early T cell signaling events.

Results – Concluding Remarks

81

CONCLUDING REMARKS

A MODEL FOR CD5-MEDIATED INHIBITION OF

T LYMPHOCYTE SIGNALING


82

Immune processes need to be strictly controlled to counteract any immunological

disorders or pathological events and maintain a healthy balance in the body. The

importance of T lymphocytes in the immunological network is underlined by the fact that

they have been a therapeutic target for many years. The first mAb drug approved by the

Food and Drug Administration (FDA, in 1986) for treatment of transplant rejection, was

directed against the TCR/CD3 complex (Muronomab-CD3, also known as Orthoclone

OKT3). Since then, our knowledge of the nature of T cells has improved and we realized,

that T lymphocyte signaling is a delicately balanced process. It requires the coordinated

action of both kinases and phosphatases and, depending upon the combination of

receptors activated, can lead to a variety of cellular responses. Protein tyrosine

phosphorylation, which plays a key regulatory role in many aspects of eukaryotic cell

biology, is a reversible and dynamic process, and the elucidation of protein

phosphorylation signaling pathways is an important undertaking. Besides establishing the

identity of protein substrates and the specific residues that are phosphorylated, the

determination of which kinases and phosphatases catalyze these reactions represents a

major challenge. The many challenges in investigating signal transduction pathways

include the determination of the combinatorial action of kinases and phosphatases, the

rates of phosphorylation and dephosphorylation, and the actual activity of the substrate

protein.

In this present study, we focussed on the contribution of the accessory surface

receptor CD5 to the balanced T lymphocyte signaling network. CD5 inhibits T lymphocyte

activation without decreasing APC-T cell adhesion or interfering with IS formation

(Brossard et al. 2003). Instead, CD5 associates with signaling inhibitors such as SHP-1,

rasGAP and Cbl, all possibly contributing to the role of CD5 as a signaling attenuator

(Pani et al. 1996; Dennehy et al. 1998; Carmo et al. 1999; Perez-Villar et al. 1999). In the

present report we show that CD5 also inhibits cell activation through a previously

unsuspected mechanism that leads to the phosphorylation of Fyn at the C-terminal

inhibitory tyrosine residue, thus reducing the activity of the kinase and the subsequent

downstream signaling.

In the functional modulatory interaction with Fyn, different domains of CD5 may be

involved in differential tasks. The cytoplasmic domain should account for the molecular

associations that CD5 holds with signaling effectors, as using several CD5 truncation

mutants, we show that the cytoplasmic distal part of CD5, more precisely the region

between amino acids H449 and L471, is required to induce Fyn inhibitory tyrosine

phosphorylation (Figure 2.6B). This stretch does not coincide, however, with the minimal

sequence of CD5 necessary for the association with Lck, and for CD5 phosphorylation,

because CD5 mutants as short as CD5.E418stop can still be phosphorylated, and


83

associate with Lck (Figure 2.5C). Although Lck has been reported as the major kinase

phosphorylating CD5, it is possible that it is not the only one. Following

immunoprecipitation and in vitro kinase assays, several CD5 mutants displayed exuberant

tyrosine phosphorylation while exhibiting modest levels of association with Lck. Moreover,

these kinase assays were performed using non-stimulated cells, a situation in which CD5

and CD5 mutants are adrift in the soluble phase of the membrane, having little contact

with the signaling machinery assembled at lipid rafts.

Although not completely excluding the role of Lck in CD5-mediated Fyn regulation,

an additional tyrosine kinase is involved in the phosphorylation of Fyn at the C-terminal

tyrosine residue. Csk appears to be the most relevant kinase that phosphorylates Src-

kinases at the inhibitory C-terminal tyrosine residue (Nada et al. 1991; Okada et al. 1991)

However, we were not able to detect a direct association between CD5 and Csk (Figure

3.1B) or any change in the activity of Csk upon CD5 activation (preliminary data, not

shown). This raises the possibility that another, still elusive, protein tyrosine kinase may

be involved in the direct phosphorylation of the C-terminal tyrosine residue of Fyn.

However, it is just likely that an indirect interaction between CD5 and Csk may occur,

mediated by an adaptor that binds to the distal part of the CD5 tail, and it is in fact Csk

that conventionally phosphorylates Fyn at Y531. Furthermore, we cannot completely

exclude a potential direct interaction between Csk and CD5, possibly of weak nature, as

our co-precipitation studies were performed under stringent detergent conditions (Triton X-

100). Clearly, further experiments need to be performed, in order to define whether CD5

and Csk do associate directly.

A potential (direct or indirect) interaction between Csk and CD5 might position the

kinase in the proximity of PAG, and therefore interfere with the assembly/disassembly of

the PAG/Csk/Fyn ternary complex. CD5 triggering modulates the interaction of Csk with

PAG, reducing it 0.6-fold after 5 min of activation, compared to the association detected in

resting cells (Figure 2.8C). In contrast, TCR-mediated activation produces a much more

pronounced and fast release of Csk from PAG, with a fast reassembly of the PAG-Csk

complex as well, and termination of the response. The Csk interaction with PAG was

shown to be important for maintaining high levels of Csk activity and keeping Src-family

kinases down-regulated (Kawabuchi et al. 2000; Takeuchi et al. 2000; Davidson et al.

2003). Interruption of the Csk-PAG association would therefore result in a decreased

phosphorylation of the C-terminal inhibitory residue of Src-kinases, which can allow them

to become active. As CD5, once targeted to lipid rafts, can associate with Fyn and PAG

(Figure 3.1B), its role may be to promote a stable interaction between these two proteins,

together with Csk, to prevent their fast dissociation. CD5-associated Fyn would be able to

associate with PAG and continuously phosphorylate PAG at the Csk-docking site.


84

Consistent with this interpretation, our results show that no abrupt dislodging of Csk from

PAG is induced following CD5 stimulation (Figure 2.8C). It can be argued that while Csk is

complexed with PAG, Fyn would be subjected to the inhibitory phosphorylation promoted

by nearby Csk. However, as Solheim and colleagues have suggested, if Fyn is bound via

its SH2 domain to phospho-PAG, it remains in an open conformation, still resistant to a

complete Csk-dependent inhibition (Solheim et al. 2008). Hence, Fyn’s own kinase

capacity, although possibly slightly decreased, can be maintained for longer periods and

keep the Csk docking site for PAG phosphorylated.

Interestingly, once engaged by CD5 antibodies, CD5 is massively transported to

lipid rafts independently of its cytoplasmic domain. While this establishes that shifting

between membrane phases is not phosphorylation driven and does not require

associations with Src kinases, it also means that molecular associations between the

extracellular domain of CD5 and raft-associated molecules must be facilitated to allow

CD5 to dock onto the membrane microdomains once its extensive reorganization at the

membrane is induced via antibody binding. One obvious candidate that may link CD5 to

lipid rafts is the surface glycoprotein CD2, with whom CD5 associates through the

extracellular and intracellular domains (Carmo et al. 1999; Castro et al. 2002) and that

targets to rafts via a protein-protein-based interaction with Lck (Nunes et al. 2008). In

absence of a CD2-negative human T cell line, the involvement of CD2 in linking CD5 to

lipid rafts could be tested by blocking the interaction between CD2 and CD5 with peptide

blockers, e.g. peptide sequences that comprise the stretch of the CD5 extracellular

sequences potentially involved in the interaction with CD2. In such an experimental set

up, blockade of the interaction with other surface molecules must not be excluded.

Nevertheless, the portions of the extracellular domain of CD5 involved in its raft

recruitment could be identified. Furthermore, the involvement of the extracellular domain

of CD5 in membrane relocalization events could be tested with a chimeric molecule,

comprising the intracellular region of CD5 and the extracellular domain of a signaling

receptor, e.g. the epidermal growth factor receptor (EGFR) or the erythropoietin receptor

(EpoR). Both receptors form dimers in the plasma membrane in a ligand-independent

manner and signaling through these receptors can be readily induced by their ligands

EGF and Epo, respectively (Constantinescu et al. 2001; Yu et al. 2002). Ligation of such a

chimeric CD5 molecule and subsequent analysis of its lipid raft localisation would give

important insight in the involvement of the CD5 extracellular domain in the recruitment

process.

A model of CD5-mediated inhibition of T cell signaling can thus start to emerge

(Figure 4): Upon T cell-APC interaction, CD5 translocates to the IS, either driven by

binding to a yet undetermined counter receptor expressed on the APC, or co-transported


85

by an associated surface receptor. Upon reorganization of the membrane and lipid raft

coalescence into the TCR-APC zone (Xavier and Seed 1999; Nunes et al. 2006), CD5

can then closely interact with kinases such as Lck and Fyn, which may be present in

different populations of lipid rafts.

Figure 4. A model for CD5-mediated inhibition of T lymphocyte signaling. Upon CD5 ligation, CD5 molecules (expressed as dimers) are readily recruited to lipid rafts, where they can bring together signaling effectors such as Lck, Fyn and PAG. Within the complex of pooled rafts, CD5 interactions with PAG and Fyn result in a continuous phosphorylation (although reduced in comparison to resting cells) of the tyrosine residue 317 of PAG catalyzed by Fyn, blocking or slowing down the release of Csk that binds to phosphorylated Y317 of PAG (in comparison to activation through the TCR/CD3 complex, that results in an abrupt disassembly of the PAG/Csk complex). Captured or unreleased Csk targets Fyn at the C-terminal inhibitory tyrosine, but as Fyn is docked through the SH2 domain and thus kept in an open conformation, is it resistant to total inhibition. The activity of Fyn is nevertheless decreased and consequently impairs downstream signaling, including ZAP-70 activity. This CD5-controlled modulation of the PAG/Csk/Fyn loop ultimately down-regulates long-term T cell responses, such as T cell proliferation.


86

Plasma membrane lipid rafts can be heterogeneous (Schade and Levine 2002; Kiyokawa

et al. 2005), of nanometer size (Sharma et al. 2004) and highly mobile (Douglass and

Vale 2005), therefore transmembrane receptors such as CD2 and CD5 may serve as

scaffolds that allow individual rafts of different subsets and their components to interact.

Of particular interest, the fact that CD5 may be present at the cell surface as a

homodimer, probably in equilibrium with the monomeric form, may help to explain not only

the massive translocation to lipid rafts with just primary antibody stimulation, but also the

multiple interactions it establishes with various effectors leading to functional interactions

between lipid raft protein components.

Taken together, in line with the phenotype of CD5-deficient mice (Tarakhovsky et

al. 1995; Bikah et al. 1996), CD5 may inhibit T cell responses through its role in different

pathways. For example, CD5 can associate with SHP-1, which has been shown to directly

dephosphorylate ZAP-70 and inhibit its activity (Plas et al. 1996; Brockdorff et al. 1999;

Denny et al. 2000). In parallel, CD5 may also inhibit the activity of Fyn, which has been

shown to be important for the normal activation of ZAP-70 (Denny et al. 2000). Probably

through the combination of different pathways converging to the same target, inhibitory

regulators such as CD5 may ensure a specific and effective control of critical check-points

of the signaling flow, and the controlled regulation of T cell activation.

Future perspectives to further investigate the contribution of CD5 to the inhibition

of T lymphocyte signaling would include the complementation of the present work with key

experiments performed in primary cells (peripheral blood lymphocytes or lymph node

lymphocytes). The validation of the functional relevance of the biochemical data of this

study in a more physiological setting would give us important mechanistic insights into the

contribution of the described CD5-induced pathway to T cell signaling. Furthermore, cell-

to-cell stimulation systems, e.g. with superantigen-stimulated Jurkat-Raji cell conjugates,

could be used to study the behaviour of the different CD5 mutants regarding Fyn and PAG

phosphorylation. In the absence of the physiological ligand of CD5, anti-CD5 antibodies

were used in this study to engage CD5. It can be argued that receptor activation via mAbs

only mimics the physiological CD5-CD5 ligand interaction and is prone to artifactual

protein aggregations. Therefore, for future studies it would be crucial to identify the ligand

of CD5. This could be done by phage display of potential CD5 ligands (present in a

lymphocyte cDNA library). The polyclonal phage library could be selected on either CD5

transfected cells or on the CD5-Ig fusion protein that was cloned and purified during this

study. The different tools available allow for screening of potential CD5 ligands either by

flow cytometry (binding to 2G5/CD5.WT cells versus 2G5 cells) or by ELISA (binding to

CD5-Ig versus Control-Ig).


87

The primary focus of this study was to investigate signaling pathways and effectors

engaged upon CD5 ligation as a sole stimulus. Nevertheless, co-activation studies of CD5

together with the TCR/CD3 complex would give us further insight into the inhibitory role

that CD5 holds in T cell activation. The difference between the very subtle CD5-induced

release of the Csk-PAG complex and the almost abortive disassembly of the complex

upon CD3 activation (at a very early time point) could contribute decisively to the signaling

outcome of the T cell activation process.

88

III – Material and Methods

Material and Methods

89

Cells and cell lines

Human PBMC from normal healthy adult volunteers were isolated by Ficoll-Hypaque

density gradient centrifugation. Jurkat cell lines used were E6.1 (Weiss et al. 1984),

obtained from A. Weiss (University of California, San Francisco, CA), JKHM (Carmo et al.

1999), donated by D. A. Cantrell (Imperial Cancer Research Fund, London, UK), Lck-

deficient J.CaM1 (Straus and Weiss 1992), kindly given by T. Mustelin (The Burnham

Institute for Medical Research, La Jolla), and the CD5-deficient 2G5 subclone (Simarro et

al. 1997), as well as 2G5 lines stably transfected with wild-type and cytoplasmic mutant

human CD5 molecules (Simarro et al. 1997; Calvo et al. 1998). Cell lines were maintained

in RPMI 1640, supplemented with 10% FCS, 1 mM sodium pyruvate, 2 mM L-glutamine,

penicillin G (50 U/ml) and streptomycin (50 µg/ml). Human embryonic kidney HEK293T

cells (DuBridge et al. 1987) were grown in at 37°C in a 5% CO2 humidified incubator, in

Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum, 5 mg/ml glucose,

penicillin (100 U/ml), streptomycin streptomycin (50 µg/ml) and 200 mM L-glutamine.

Antibodies and reagents

Monoclonal antibodies used were: The anti-phosphotyrosine mAb 4G10, HRP-

conjugated (Upstate Biotechnology); anti-CD5-Y-2/178 (Carmo et al. 1993), a kind gift

from J. Cordell and D. Y. Mason (John Radcliffe Hospital, University of Oxford, Oxford,

UK), anti-CD5-OKT1 (Kung et al. 1979) and anti-CD5-BL1a (Beckman Coulter); anti-

CD3-OKT3 (Kung et al. 1979); anti-PAG-C1 (Baumgartner et al. 2003), a kind gift from B.

Schraven and J. Lindquist (Institute of Immunology, Otto-von-Guericke University,

Magdeburg, Germany); anti-CD80 (Pfistershammer et al. 2006) and anti-CD72, given by

P. Steinberger (Institute of Immunology, Medical University of Vienna, Austria); and the

isotype control antibody IgG1 (BD Biosciences).

Polyclonal Abs were: anti-CD5, a gift from D. Y. Mason; anti-Lck (DA3), anti-Fyn

(BL90), and anti-ZAP-70, given by J. B. Bolen and M. G. Tomlinson (DNAX Research

Institute, Palo Alto, CA); anti-CD2 (CD2-300) (Brown et al. 1988), a gift from M. H. Brown

(Sir William Dunn School of Pathology, University of Oxford, Oxford, UK); anti-PAG and

anti-phospho-PAG (Y317), gifts from B. Schraven and J. Lindquist; anti-phospho-ZAP-70

(Y493), a gift from S. Valitutti (Institut Claude de Préval, Toulouse, France); anti-phospho-

Src Family Negative Regulatory Site (Biosource), anti-phospho-Src Family (Y416) and

anti-phospho-Lck (Y505) from Cell Signaling; anti-Csk (Santa Cruz Biotechnology); anti-

LAT (Upstate Biotechnology); goat anti-mouse peroxidase conjugate (Molecular Probes);

goat anti-rabbit peroxidase conjugate (Sigma-Aldrich); rabbit anti-mouse and rabbit anti-


90

mouse FITC-labeled from Dako and donkey anti-rabbit FITC-labeled from Jackson

ImmunoResearch. ExtrAvidin® peroxidase was purchased from Sigma-Aldrich and [γ-32P]ATP (>5000 Ci/mmol) was purchased from Amersham. Restriction enzymes BamHI,

NotI and NheI were purchased from Fermentas.

Cell surface biotinylation

For cell suface biotinylation, 2 x 107 cells were washed three times with ice-cold

PBS and resuspended with 1 ml PBS containing EZ-LinkTM Sulfo-NHS-LC-Biotin (Pierce)

at a final concentration of 0.5 mg/ml. After incubation for 10 min at RT cells were

thoroughly washed (four times with PBS) and lysed for 30 min at 4°C in 1% Triton X-100

lysis buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF and 1%

(v/v) Triton X-100).

Cellular activation

Cells were maintained in RPMI 1640 medium or serum-deprived 18 h prior to

stimulation. For activation, cells were washed and resuspended in RPMI 1640 (no FCS)

containing Y-2/178 at a 1:5 dilution of hybridoma supernatant (10 µg/ml), OKT3 at 2 µg/ml

or isotype-matched negative control antibody at 10 µg/ml. In a typical experiment,

stimulation was induced using only primary mAb, without the use of crosslinking

secondary Ab. In some indicated cases, secondary Ab (goat anti-mouse Ig) was added at

a concentration of 20 µg/ml. Cells were maintained at 4°C for 15 min and s ubsequently

incubated at 37°C for the indicated time points. Ce lls were then pelleted and lysed for 30

min in ice-cold 1% NP-40 lysis buffer (10 mM Tris-Cl (pH 7.4), 150 mM NaCl, 1 mM

EDTA, 1 mM PMSF, 1% (v/v) Igepal CA-630 and in some cases 1 mM sodium

orthovanadate). The nuclear pellet was removed by centrifugation at 11,000 x g for 10 min

at 4°C and the supernatants were subjected to immun oprecipitation or analyzed by

immunoblotting as described. In a typical experiment, 5 x 107 cells were activated per

condition.

For lipid rafts analysis of activated cells, approximately 1.7 × 108 cells were used

per sample. Cells were washed and resuspended in 1 ml RPMI medium containing Y-

2/178 or OKT1 at a 1:5 dilution of hybridoma supernatant. When indicated, cells were

crosslinked with RAM at 20 �g/ml. After 5 min incubation on ice, cells were activated at

37ºC for 15 min, collected and prepared for sucrose gradient centrifugation as described

below.


91

Immunoprecipitations and reprecipitations

Between 1 and 5 x 107 cells were lysed for 30 min on ice in lysis buffer (10 mM

Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF and 1% (v/v) NP-40 or TritonX-

100). The nuclear pellet was removed by centrifugation at 11,000 x g for 10 min at 4°C,

and the supernatants were mixed with 100 µl of a 10 % protein A Sepharose® CL-4B

(Amersham) slurry and with antibodies (1 – 10 µg) or antisera (1 – 3 µl). Samples were

incubated for 90 min or over night at 4°C. The bead s containing the immune complexes

were washed three times in 1 ml lysis buffer and either boiled for 5 min in SDS buffer for

immunoblotting or washed for two more rounds in kinase assay buffer and subjected to in

vitro kinase assays.

For reprecipitations, the beads containing the immune complexes were boiled for 5

min in 2% SDS and diluted 8-fold with lysis buffer. After centrifugation, the supernatants

were recovered and precleared for 30 min with 100 µl of protein A Sepharose® beads (10

% slurry). Proteins were incubated with antibodies (1 – 10 µg) or antisera (1 – 3 µl) and

100 µl of protein A Sepharose® beads (10% slurry) for 90 min. Immunoprecipitates were

washed three times with 1 ml lysis buffer. Samples were boiled for 5 min in SDS buffer

and subjected to SDS-PAGE.

Western blotting

Proteins were denatured in 2 x SDS buffer, separated by SDS-PAGE under

reducing (final concentration of β-mercaptoethanol: 5% (v/v)) or nonreducing conditions

and then transferred to HybondTM-C super membranes (Amersham) by electroblotting.

Membranes were blocked in 0.1% (v/v) TBST containing 5% (w/v) nonfat dried milk for 30

min, washed four times for 5 min with 0.1% TBST, probed with primary Ab (typically a

1:5,000 dilution) for 1 h at RT, washed four times for 5 min with 0.1% TBST, and

incubated with HRP-conjugated goat anti-mouse or goat anti-rabbit IgG (1:20,000 dilution)

for 1 h at RT. For phosphotyrosine detection, membranes were incubated with HRP-

conjugated 4G10. For detection of biotinylated cell surface antigens, membranes were

incubated with ExtrAvidin® peroxidase conjugate (Sigma). Membranes were washed again

four times for 5 min with 0.1% TBST and subjected to detection. Immunoblots were

developed using enhanced chemiluminescence – ECL, ECL Plus (Amersham) or Femto

(Pierce) – and exposed to BioMax MR films (Kodak).


92

In vitro kinase assays

NP-40 or Triton X-100 assay buffer (30 µl) containing 10 mM MnCl2, 1 mM

Na3VO4, 1 mM NaF, and 50 µCi (185 KBq) of [γ-32P]ATP was added to the beads

containing the immune complexes, and in vitro kinase reactions were allowed to occur for

15 min at 25°C. Reactions were stopped by the addit ion of 30 µl of 2 x SDS buffer,

following which the samples were boiled for 5 min. Products were separated on SDS-

PAGE gels, and autoradiography of the dried gels was done with BioMax MR films

(Kodak).

Flow cytometry

For the detection of surface receptors, cells were washed and resuspended in PBS

containing 0.2% BSA and 0.1% NaN3 (PBS/BSA/NaN3), at a concentration of 5 × 106

cells/ml. Staining was performed by incubation of 5 × 105 cells/well with mAbs (20 µg/ml)

for 15 min on ice, followed by rabbit anti-mouse FITC-labeled, in 96-well round-bottom

plates (Greiner, Nürtingen).

For measurement of intracellular phosphotyrosine proteins, activated cells (5 x 106

cells per sample) were collected and fixed for 20 min at 4°C with 4% PFA, washed twice

with ice-cold PBS and permeabilized for 20 min at 4°C with PBS/BSA/NaN 3 containing 0.4

% saponin. Intracellular staining was performed in 96-well round-bottom plates by

incubation of 1 × 106 cells/well with polyclonal phosphorylation site-specific antibodies for

15 min on ice, followed by FITC-labeled donkey anti-rabbit antibody. Cytometric analysis

was as previously described (Carmo et al. 1999), using a FACSCalibur (Becton

Dickinson).

Sucrose gradient centrifugation

Sucrose gradient centrifugation was performed as described previously (Nunes et

al. 2008). Briefly, activated cells were washed twice with ice-cold PBS and lysed for 30

min on ice in 1 ml MBS buffer (25 mM MES, 150 mM NaCl, pH 6.5), containing 1% Triton

X-100, 1 mM PMSF and cocktail protease inhibitors (1 mM AEBSF, 0.8 µM aprotinin, 50

µM bestatin, 15 µM E-64, 20 µM leupeptin and 10 µM pepstatin A; Calbiochem). The

lysates were homogenized by brief sonication for ten pulses on ice, using a Heat

Systems/Ultrasonics sonicator (model W-375) equipped with a microtip and set to 50%

duty cycle, output 3. To obtain the rafts fraction, cell lysates were mixed with an equal


93

volume of 85% sucrose in MBS buffer and transferred to the bottom of Sorvall

ultracentrifuge tubes. The samples were then overlaid with 6 ml of 35% sucrose followed

by 2 ml of 5% sucrose. After centrifugation at 200,000 × g for 17 h at 4°C in a TST41.14

swing-out rotor (Sorvall), 10 fractions of 1 ml were collected from the bottom of the tube

and analyzed by Western blotting. The fractions are labeled from the top of the gradient.

Proliferation assays

Proliferation assays were performed in flat-bottom 96-well cell culture plates. For T

cell stimulation, the aCD3 expressing Bw5147 transductants (T cell stimulators) co-

expressing CD80, CD72 or control T cell stimulators were irradiated (6000 rad) and added

to 96-well plates (2 x 104 cells/well). Human T cells were prepared as described

elsewhere (Pfistershammer et al. 2004), added (1 x 105 cells/well) and cocultured for 3

days. 16 h before cell harvest, [methyl-3H]-dThd (ICN Pharmaceuticals) was added at 1

µCi/well. Cells were harvested and measured on a microplate scintillation counter

(Packard, Topcount Instrument). All proliferation assays were done in triplicates and

means and SD are shown. For CD5 ligation, Jurkat E6.1 T cells were cultured at 4 x 105

cells/well, in medium or in the presence of Y-2/178 (1:200 dilution of hybidoma

supernatant) or IgG (100 µg/ml) for 3 days.

Generation of T cell stimulator cells

The scFv of the anti-human CD3ε Ab (clone OKT3, ATCC) that induces activation

of the human TCR/CD3 complex was used as a template for the amplification of the

variable heavy chain (VH) and variable light chain (VL) gene segments using primers VH-

for 5’-GGAATTCGCTAGCCCAGGTCCAGCTGCAGCAGTCT-3’, VH-rev 5’-

GGGGGATCCGGTGACCGTGGTGCCTTGGCCCCAGTA-3’ and VL-for 5’-

GGAATTCGAGCTCCCAAATTGTTCTCACCCAGTCTCCA-3’, VL-rev 5’-

GGGATCCCCACCGCCCCGGTTTATTTCCAACTTTGTCCC-3’. They were cloned

together with a (G3S)4 encoding linker and a CD5 leader (CD5L) sequence to the leader-

less human CD14 sequence into the retroviral vector pMMP (Pickl et al. 2001) to generate

a CD5L-OKT3scFv-CD14 expression construct, leading to a GPI-anchored expression of

the OKT3-scFv. The cDNA of human CD72 was PCR amplified from an expression library

generated from human B cells (provided by P. Steinberger, Institute of Immunology,

Medical University of Vienna, Austria) using primers CD72-for 5’-

GCGGGGGGATCCGAAGACGAGTGGGGGCAGAG-3’ and CD72-rev 5’-


94

GCGGGGGCGGCCGCGTCAACTCAGTGCAAAGGACTG-3’. PCR products were cloned

into the retroviral expression vector pBMN (Kinoshita et al. 1997) using the restriction sites

for BamHI and NotI. The integrity of the resulting construct was confirmed by DNA

sequencing.

Bw 5147 (referred to as Bw cells), a murine thymoma cell line, was retrovirally

transduced to express the constructs that encode membrane-bound aCD3. Single cell

clones that stably express these constructs (T cell stimulator cells) were established. Cells

derived from aCD3+Bw cell clones were then retrovirally transduced in parallel with pBMN

plasmids encoding CD80 ((provided by P. Steinberger, Institute of Immunology, Medical

University of Vienna, Austria), CD72 and for control purposes with vector containing the

lac Z gene (pBMNZ). For retroviral transduction, 293T cells were co-transfected with the

pEAK12-gag-pol vector (Steinberger et al. 2004). Cell culture supernatants were

supplemented with polybrene (5 µg/ml), sterile filtered and used to spin-infect

(centrifugation at 700 x g, 1 h, 30°C) the Bw5147 c ell lines described above.

Generation of the CD5-Ig fusion protein

The cDNA of the extracellular part of human CD5 was PCR amplified from an

expression library generated from human T cells (provided by P. Steinberger, Institute of

Immunology, Medical University of Vienna, Austria) using primers CD5-for 5’-

GCGGGGGCTAGCTGCCCAGGCTGAGGCAAGAG-3’ and CD5-rev 5’-

CGCGGGGGATCCCCACCGCCGCCTGCGGGGTTTGGATC-3’. PCR products were

cloned into pEAK-gag-pol, in frame with the sequence coding for the Fc part of the fusion

protein, using the BamHI and NheI restriction sites. The integrity of the resulting construct

was confirmed by DNA sequencing. For protein production, 293T cells were transfected

with the construct, supernatant was collected, sterile filtered and subjected to affinity

chromatography using a Hitrap Protein A column (Amersham). Collected fractions were

analysed for protein content, pooled, dialyzed and used for further applications. For

control purposes, Control-Ig protein was purified.

cDNA cloning and BRET assay

Human CD5 cDNA in pGFP-N1 (a kind gift from G. Bismuth, Institute Cochin,

Paris, France) was sub-cloned into pGFP-N3 (PerkinElmer) using the EcoRI restriction

site, and further to the prLuc-N3 (PerkinElmer) using BglII and BamHI. Human CD2 was

amplified from the Jurkat cell line E6.1 using as primers: 5’-

TAGTAGCTGCAGCCCCTAAGATGAGCTTTCC-3’ and


95

5´ATCATCGGATCCTTAGAGGAAGGGGACAATGAG-3’. The PCR product was then

cloned into both pGFP-N3 and prLuc-N3 using PstI and BamHI restriction sites. For the

CD5Ex/CD2Int chimera, two rounds of PCR amplification were performed. The

oligunucleotide pairs for the overlapping 5’ and 3’ products were: 5’-

TAGTAGGATATCGCCAGAAACCATGCCCATG-3’ and 5’-CGACTCCTCTGTTTTT

TCCTTTTGGCAAGGGGGCCGCACACGA-3’ using CD5 pGFP-N3 as template, and 5’-

TCGTGTGCGGCCCCCTTGCCAAAAGGAAAAAACAGAGGAGTCG-3’ and 5’-

CTACTACCGCGGCAGCCTCTGAGCCCCATGC-3’ using CD2 pGFP-N3 as template,

respectively. The amplified products were then used as templates in a chimeric PCR

reaction scheme. The resulting genes were digested using the appropriate restriction sites

and cloned into the BRET vectors. All constructs produced were sequenced to check

reading frame and integrity.

The BRET experiments were carried out as described previously, using the type-I

assay (James et al. 2006). No modifications were made to the assay. BRETeff values were

assayed by adding DeepBlueC substrate and measuring luminescence immediately.

Densitometry

Densitometric analysis was performed on a GS-800 densitometer (Bio-Rad) using

Quantity One quantitation software (Bio-Rad). Signals obtained from the volume analysis

of densitometric data are expressed in arbitrary units. All densitometric values were

calculated from non-saturated signals.

96

IV – Appendix

Abbreviations

97

Abbreviations

Ab antibody

ADAP adhesion- and degranulation-promoting adaptor protein

AP-1 activator protein 1

APC antigen presenting cell

ARP2/3 actin-related protein 2/3

BCR B cell receptor

BRET bioluminescence resonance energy transfer

C-terminal carboxy-terminal

CAP cytoplasmic adaptor protein

CAPRI calcium-promoted Ras inactivator

Cbp Csk-binding protein

CDR complementarity determining region

CICR Ca2+-induced Ca2+ release

CK2 casein kinase II

CRAC Ca2+ release-activated Ca2+

Csk C-terminal Src kinase

cSMAC central supramolecular activation cluster

DAG 1,2-diacylglycerol

DIG detergent-insoluble glycolipid domain

DNM2 dynamin-2

EBP-50 ezrin/radixin/moesin-binding phosphoprotein 50

e.g. for example (lat. exempli gratia)

ELISA enzyme-linked immunosorbent assay

ER endoplasmic reticulum

Erk extracellular-regulated kinase

et al. and others (lat. et alia)

F-actin filamentous-actin

FRAP fluorescence recovery after photobleaching

FRET fluorescence resonance energy transfer

Gads Grb2-related adaptor downstream of Shc

GAP GTPase activating protein

GDP guanosine diphosphate

GEF guanine nucleotide-exchange factor

GEM glycolipid-enriched domain

GFP green fluorescent protein

Grb2 growth factor receptor-bound protein 2

Abbreviations

98

GPI glycosylinositolphosphate

GTP guanosine triphosphate

HLA human leukocyte antigen

ICAM-1 intercellular adhesion molecule 1

ICOS inducible costimulator

IFN-γ interferon-γ

Ig immunoglobulin

IL interleukin

IP3 inositol-1,4,5,-trisphosphate

IP3R IP3 receptor

IS immunological synapse

ITAM immunoreceptor tyrosine-based activation motif

ITIM immunoreceptor tyrosine-based inhibitory motif

ITSM immunoreceptor tyrosine-based switch motif

Itk IL-2-inducible T cell kinase

LAT linker for activation of T cells

LFA-1 leukocyte function-associated antigen 1

LIME Lck-interacting membrane protein

Luc luciferase

mAb monoclonal antibody

MAPK mitogen-activated protein kinase

MHC major histocompatibility complex

MTOC microtubule-organizing center

NF nuclear factor

NFAT nuclear factor of activated T cells

NF-κB nuclear factor-κB

NK natural killer

N-terminal amino-terminal

PAG phosphoprotein associated with glycosphingolipid-enriched microdomains

PBMC peripheral blood mononuclear cell(s)

PEP PEST domain-enriched PTPase

PH pleckstrin homology

PHA phytohemagglutinin

PI phosphatidyl inositol

PIP2 phosphatidylinositol-4,5-bisphosphate

PI3K phosphatidylinositol 3-kinase

PKA protein kinase A

Abbreviations

99

PKC protein kinase C

PLC phospholipase C

pMHC peptide-MHC (non-self peptide presented on self-MHC)

pSMAC peripheral supramolecular activation cluster

PTB phosphotyrosine binding

PTEN phosphatase and tensin homolog deleted on chromosome 10

PTK protein tyrosine kinase

PTPase protein tyrosine phosphatase

pTyr phosphotyrosine

pY phosphotyrosine

RAG recombination-activating gene

RasGRP RAS guanyl-releasing protein

RIAM Rap1-interacting adaptor molecules

SAP SLAM-associated protein

SCID severe-combined immunodeficiency

SFK Src family kinase

SH1/2/3 Src homology 1/2/3

SHP-1 SH2 domain-containing protein tyrosine phosphatase 1

SIT SHP2-interacting TRAP

SKAP55 Src kinase-associated phosphoprotein of 55 kDa

SLAM signaling lymphocyte activation molecule

SLP-76 SH2 domain-containing leukocyte protein of 76 kDa

SMAC supramolecular activation cluster

SOCE store-operated Ca2+ entry

Sos son of sevenless

SRCR scavenger receptor cysteine-rich

STIM1 stromal interaction molecule 1

Syk spleen tyrosine kinase

TCR T cell receptor

TM transmembrane

TRAP transmembrane adaptor protein

TRIM TCR-interacting molecule

TNF tumor necrosis factor

WASP Wiskott-Aldrich syndrome protein

WAVE2 WASP-family verprolin-homologous protein 2

WT wild-type

ZAP-70 zeta chain-associated protein of 70 kDa

References

100

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Acknowledgments

This thesis would not have been possible without the contribution, support, and

friendship of many very important people (my VIPs):

To my supervisor and mentor Alexandre. Thank you for receiving me in your

laboratory, for your support, knowledge, patience and friendship. And for having given me

the freedom to follow my ideas.

To Prof. Maria de Sousa, for your support and inspiration, for appreciating O. K.

To Mónica, my co-supervisor, colleague and friend. For your enthusiasm and

passion for science. Thank you for helping me all along. I dedicate this thesis to you. To

your strength and positivity. We have no idea what you went through. It is very difficult for

me to accept this stroke of fate. I miss you.

To all other CAGE members: Alexandra, Carine, Mafalda, Raquel and Marta. To

Telmo, for the nice lunch hours, for our interesting conversations and for having great

taste in music! And to my dear Pedro, for your never-ending motivation and love for

science. What would I have done in the evenings and weekends without you? And for

appreciating classical music!!

To Angelo A. Cardoso. Thank you for receiving me at Dana-Farber Cancer

Institute. Especial thanks to Ana Batista and Pedro Veiga, I had a great time in Boston

and I won’t forget our fantastic trip to Maine!! To the Corbetts, for receiving me cordially in

Winchester. What an authentic experience to live with an American family (I miss our

Saturday morning pancake breakfasts ….)!

To Peter Steinberger and his team at the Institute of Immunology in Vienna. You

really received me with open arms! And thanks for finishing up the work I couldn’t do

anymore … To Judith, Christof, Anita and Katharina.

To João Barata and his team at the IMM in Lisbon. To João Taborda, the artist and

his music. To Ana Silva, for the great weekends in Lisbon, Santa Cruz, Porto … and Luís

Vinagre (have you ever heard of these things that measure temperature?).

To my dear friends Daniel and Stefano, you are my Portuguese family! Daniel, you

have been patient, listening, advising, helping … and how many fantastic dinners did you

cook for me?! I adore you! Joachim und die Kϋchenschaben! Stefano, for your cosmic

energy, for being so positive and spreading this out to the world (it’s contagious), for

appreciating the real things. To Alexandra (Sasha), I will go and visit you in Moscow after

the defense. Upon my life! I miss you so much!! For Charlotte, yeah Charly, remember

EURO2004?? To Eduardo, for listening. To Miguel T, thank you for helping me getting

started in Portugal, for your consistent friendship and for showing me around Nottingham

and London. To Miguel M, for being just as you are. To Miguel F and Susana C, thanks

Acknowledgments

123

for that crazy trip to Madeira and Porto Santo! AC/DJ, I hope to see you on the turntables

again!! Ralf, mein Lieblingswohnungskollege, es war echt eine tolle Zeit mit dir in der Rua

Barão Forrester! To Carla S, Carla G, Sergej, Henning, Simon, Heather, Reinhard, Mark,

Peter, Veronika and Deepak.

To my parents, who supported and believed in me all along. Ich danke euch für

eure Unterstützung. Ihr habt immer an mich geglaubt. Ohne euch hätte ich es niemals so

weit gebracht!! To my sister Anna.

And last, but not least: To Portugal!!! You received me with open arms, made me

feel at home, and made me stay … Obrigada!!

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CD5-MEDIATED INHIBITION OF T LYMPHOCYTE SIGNALING · CD5-MEDIATED INHIBITION OF T LYMPHOCYTE SIGNALING ... A central role for Fyn in the CD5-mediated inhibition of T lymphocyte signaling