Aus dem Institut für Virologie und Immunbiologie · Aus dem Institut für Virologie und...

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Aus dem Institut für Virologie und Immunbiologie der Universität Würzburg Lehrstuhl für Immunologie Vorstand: Professor Dr. rer. nat. Th. Hünig Effects of desialyation on TCR-cross-linking and antigen sensitivity of CD8 positive T lymphocytes Inaugural-Dissertation zur Erlangung der Doktorwürde der Medizinischen Fakultät der Bayerischen Julius-Maximilians-Universität Würzburg vorgelegt von Lars Eichler aus Vechta Würzburg, November 2005

Transcript of Aus dem Institut für Virologie und Immunbiologie · Aus dem Institut für Virologie und...

Aus dem Institut für Virologie und Immunbiologie der Universität Würzburg

Lehrstuhl für Immunologie

Vorstand: Professor Dr. rer. nat. Th. Hünig

Effects of desialyation on TCR-cross-linking and antigen sensitivity of CD8 positive T lymphocytes

Inaugural-Dissertation

zur Erlangung der Doktorwürde der

Medizinischen Fakultät

der

Bayerischen Julius-Maximilians-Universität Würzburg

vorgelegt von

Lars Eichler

aus Vechta

Würzburg, November 2005

Referent: Professor Dr. rer. nat. Th. Hünig

Korreferent: Professor Dr. med. H. Klinker

Dekan: Professor Dr. med. G. Ertl

Tag der mündlichen Prüfung: 11. August 2006

Der Promovend ist Arzt

Table of contents

Table of contents……………………………………………………………….I List of Figures…….…………….……………………………………………..III Abbreviations…………………………………………………………………..IV 1 Introduction.............................................................................................. 1

1.1 T lymphocytes ........................................................................................ 1

1.1.1 Structural basis of T cell antigen recognition................................... 1 1.1.2 T cell-APC contact .......................................................................... 5 1.1.3 Lymphocyte activation..................................................................... 6

1.2 Synthesis and function of N- and O-linked polyglycans on glycoproteins .................................................................................................................9

2 Materials and Methods .......................................................................... 12

1.3 Monoclonal antibodies (mAb)............................................................... 12

1.4 Reagents.............................................................................................. 13

1.5 Laboratory instruments......................................................................... 14

1.6 Materials............................................................................................... 15

1.7 Buffers and media ................................................................................ 16

1.7.1 Buffers for cell preparation and FACS analysis............................. 16 1.7.2 Buffers and media for tissue culture.............................................. 16 1.7.3 ELISA Buffer ................................................................................. 17 1.7.4 Buffer for purification of MHC/Ig dimer .......................................... 17 1.7.5 Buffers for peptide loading of MHC/Ig dimer ................................. 18

1.8 General procedures.............................................................................. 18

1.8.1 Flow cytometry .............................................................................. 18 1.8.2 Spectrophotometry........................................................................ 19 1.8.3 FPLC/HPLC .................................................................................. 20

1.9 Animal model ....................................................................................... 20

1.9.1 Testing mice for 2C TCR expression ............................................ 21

1.10 Production of FITC labeled dimeric MHC/Ig protein ............................. 22

1.10.1 Dimeric MHC/Ig protein................................................................. 22 1.10.2 Tissue culture of J 558 hybridoma cells ........................................ 23 1.10.3 Cell count/ viability test.................................................................. 23 1.10.4 Cryopreservation of cells............................................................... 24

I

Table of contents

1.10.5 Purification of MHC/Ig dimer ......................................................... 25 1.10.6 Quantitation of MHC/Ig dimer by ELISA........................................ 25 1.10.7 FITC-labeling of MHC-Ig dimer ..................................................... 27 1.10.8 Peptide loading of fluorescently labeled MHC/Ig........................... 27

1.11 Cell preparation.................................................................................... 28

1.11.1 Preparation of splenocytes............................................................ 28 1.11.2 Isolation of CD 8+/ DN- T cells...................................................... 29 1.11.3 Neuraminidase treatment of cells.................................................. 31 1.11.4 CFSE labeling of 2C splenocytes.................................................. 32 1.11.5 Generation of activated T cells by Mixed Lymphocyte Reaction…32

1.12 Equilibrium binding assay..................................................................... 33

1.13 Functional assays................................................................................. 36

1.13.1 Tyrosine phosphorylation assay.................................................... 36 1.13.2 In vitro proliferation assay ............................................................. 37

3 Results.................................................................................................... 38

1.14 Effects of desialyation on MHC/Ig binding on T cell surface................. 38

1.14.1 Desialyation enhances binding of specific peptideMHC ligand on T cell surface. ................................................................................... 38

1.14.2 Enhanced binding of peptideMHC ligand is a result of increased TCR cross-linking. ................................................................................. 39

1.14.3 Desialyation of DN cells results in decreased cross-linking. ......... 42 1.14.4 Neuraminidase treatment does not affect non-specific binding of

peptideMHC/Ig to T cells ................................................................... 43

1.15 Effects of desialyation on cellular reactions upon in vitro stimulation ... 44

1.15.1 Increased TCR cross-linking results in enhanced signaling .......... 45 1.15.2 Desialyation accelerates and enhances T cell proliferation in vitro47

4 Discussion.............................................................................................. 49

5 Objectives and summary ...................................................................... 54

6 References ............................................................................................. 55

II

List of Figures

List of Figures Fig.1. 2 C TCR bound to peptideMHC class I molecule H-2Kb …………...3 Fig.2. Molecular model of soluble CD8αα-homodimer…………………..4 Fig.3. Mixed lymphocyte reaction of 2C TCR transgenic splenocytes

and sublethally irradiated Balb C splenocytes…………………….7 Fig.4. Schematic and ribbon model of MHC/Ig molecule………………22 Fig.5. Cell purification ………………………………………………….….30 Fig.6. Control of desialyation by PNA staining………………………….31 Fig.7. Binding of peptideMHC/Ig dimer is a sum of specific and

non-specific binding……………………………………….…….….35 Fig.8. Desialyation of 2C TCR transgenic cells results in increased

avidity of TCR-peptideMHC interaction……………………..……….39 Fig.9. Deconvolution of binding isotherms shows increases in double-

bound ligand on desialyated T cells……………………..………..42 Fig.10. Desialyation of DN cells results in decreased cross-linking

…..…………………………………………………………………….43 Fig.11. Non-specific binding does not increase upon desialyation

…..…………………………………………………………………….44 Fig.12. Early tyrosine kinase signaling is enhanced in desialyated cells. …………………………………………………….………………….46 Fig.13. Desialyated cells show enhanced proliferative response.

……………..………………………………………………………….48

III

Abbreviations

Abbreviations Å Angstrøm; measure of length; 1 Å = 0,1 nm

Asn asparagine

APC antigen presenting cell

B7.1 /.2 T cell co-stimulatory molecules

BCR B cell receptor

CD cluster of differentiation

CDR complementarity determining regions

CFSE carboxyfluoresceine diacetate

CTL cytotoxic lymphocyte

DMSO dimethyl sulfoxide

DN cell “double-negative” T lymphocyte; lacks CD4- and

CD8-expression

DNA desoxyribonucleic acid

ELISA enzyme-linked immunosorbent assay

ER endoplasmic reticulum

FACS fluorescence activated cell sorting

FCS fetal calf serum

FITC fluoresceine isothiocyanate

FPLC fast protein liquid chromatography

GAM goat-anti-mouse

HPLC high pressure liquid chromatography

ICAMs intercellular adhesion molecules

Ig Immunoglobulin

IL Interleukin

H-2 Kb murine MHC-class I gene product; syngenic ligand

of 2C TCR

Kd dissociation constant; measure for affinity of

molecular interactions, expressed in mole

KD kilo dalton

Lck tyrosine kinase associated with CD4 and CD8

H-2 Ld murine MHC-class I gene product; allogenic ligand

of 2C TCR

IV

Abbreviations

LFAs leukocyte functional antigens

M mole

mAb monoclonal antibody

MCF mean channel fluorescent

MCMV non-cognate peptide used with H-2 Ld;

Mgat5 gene encoding murine

β1,6 N-acetylglucosaminyltransferase

MHC major histocampatibility complex

MLR mixed lymphocyte reaction

N-glycan carbohydrate molecule glycosidically linked to

asparagine side chains of proteins

NP nitrophenol

O-glycan carbohydrate molecule glycosidically linked to

threonine, serine or hydroxylysine side chains of

proteins

PBS phosphate buffered saline

PE phycoerythrine

PI phosphatidylinositole

PKC Protein kinase C

PNA peanut agglutinin

QL9 cognate peptide presented by H-2 Ld; QLSPFPFDL

RPMI Roswell Park Memorial Institute

SIY cognate peptide presented by H-2 Kb; SIYRYYGL

SIIN non-cognate peptide used with H-2 Kb; SIINFEKL

SMAC supramolecular activation cluster

TCR T cell receptor

ZAP-70 ζ chain associated protein; tyrosine kinase involved

in TCR signalling

V

Introduction

1 Introduction

The following studies on the effect of surface sialic acid on T cell antigen

recognition focus on a process, which plays a central role not only in the

maintenance of health, but also the pathogenesis of several diseases.

Ligand recognition by T cells is a key event in the body`s specific defence

against pathogens and malignantly transformed cells. It is dependend on

the presentation of degraded antigen on the surface of target cells or

professionally antigen presenting cells (APC) and results in activation of the

T cell, which subsequently impacts on infested target cells and other

components of the immune system.

Besides introducing structure and function of T cells and their antigen

receptor, this section will also give a brief overview on the synthesis and

biological role of sialic acid carrying glycoconjugates on proteins.

1.1 T lymphocytes

1.1.1 Structural basis of T cell antigen recognition

The T cell receptor (TCR) belongs to the group of tyrosine kinase coupled

receptors. These cell surface glycoproteins associate with Src family

tyrosine kinases translating an extracellular stimulus into a cascade of

intracellular tyrosine phosphorylation events that result in production of

second messengers, Ca2+ influx and finally modifications of cellular

functions on the DNA level (reviewed in Schenk and Snaar-Jagalska 1999).

T cells can express two different types of TCRs. Both are protein

heterodimers linked by a disulfide bond. The first type, the αβ TCR, is

expressed on the majority of peripheral T cells. Only a small fraction of T

cells expresses the second form of receptor, the γδ TCR.

αβ T cells can be devided into two functionally distinct subclasses

depending on the type of coreceptor they express in conjunction with their

TCR. These glycoproteins, referred to as CD4 and CD8, enhance T cell

signaling by making contact with the MHC molecules of APCs and by

interacting with cytosolic components of the TCR signaling machinery.

1

Introduction

CD4 contributes to antigen recognition in the context of MHC class II

molecules on professionally antigen presenting cells, such as dendritic

cells. These antigenic peptides are derived from lysosomal degradation of

ingested protein (e.g. phagocytosed bacteria). Activated CD4+ lymphocytes

subsequently promote T- and B cell differentiation and pathogen elimination

by macrophages. CD8 in contrast, supports recognition of antigen

presented by cells expressing MHC class I molecules on their surface. At

various levels this applies to basically every cell of the body; an important

exception is represented by red blood cells. Antigenic peptide presented in

the context of MHC class I molecules is derived from cytosolic degradation

of the cell`s own protein components. Therefore cells exhibit the whole

range of molecules produced by their protein production apparatus on their

surface. This allows for recognition of aberrant proteins, as seen in

malignantly transformed or virally infected cells. Subsequent activation of

CD8+ lymphocytes results in killing of the infested cell (Janeway et al.,

2001).

The αβ TCR-CD3 complex is composed of the heterodimeric TCR molecule

and a non-covalently associated set of invariant proteins know as CD3

subunits (γδε2ζ2) whose main function is to couple the antigen recognition

process to the intracellular signaling machinery. The TCR α- and β-chain

consist of an N-terminal variable (V) region, which exposes three highly

variable loops, referred to as “complimentarity determinig regions” 1,2 and 3

(CDR 1-3) (Hedrick, Engel et al. 1988) This highly variable antigen

recognition site is formed during T cell development in the thymus, by

rearrangement of germline encoded gene segments. As implicated by their

name, the CDR determine the generally moderate binding affinity of the

TCR to the peptideMHC complex (Manning, Parke et al. 1999). The V region

is connected to a constant (C) region followed by a short hinge region that

connects the two polypeptide chains by forming a cystin bridge. The C-

terminal part of each chain is formed by a hydrophobic transmembrane

domain linking the receptor to the cell surface. The heterodimer is stabilized

by several non-covalent interactions of amino acid side chains and a

2

Introduction

carbohydrate residue at the Cα-region forming hydrogen bounds with the

Cβ-domain (Garcia, Degano et al. 1996).

The polymorphic MHC class I molecule consists of two proteins, a heavy

chain (43 KD) and a non-covalently bound light chain (11 KD), β2-

microglobulin. The heavy chain is composed of three globular domains.

While the α3-domain inserts into the cell membrane, the α1- and α2-domain

form a binding groove able to harbour 8-11 amino acids long peptides

(Fremont, Stura et al. 1995).

Fig.1. 2 C TCR bound to peptideMHC class I molecule H-2Kb (a), TCR binding site

projected onto the surface of the MHC molecule, showing the orientation of α- and

β-chain CDRs towards the bound peptide (b) (from Garcia et al., Science,1996)

3

Introduction

As previously mentioned, TCR antigen recognition is dependent on the cell

surface expression of coreceptors. In the case of CD8+ T cells this

coreceptor is represented by a 32-34 KD heterodimeric protein, referred to

as CD8. CD8 α- and β-chain are linked by a cystin bridge and each consist

of an N-terminal immunoglobulin-like domain, an extended stalk region, a

short hydrophobic transmembrane domain and a C-terminal intracellular

domain, which is associated with the Src family tyrosine kinase lck (Barber,

Dasgupta et al. 1989; Leahy, Axel et al. 1992). The protein is extensively

glycosylated with short, terminally sialyated O-glycans primarily located at

the extended stalk-like region and 4 putative N-glycans primarily located at

the membrane distal globular head region of the molecule (Classon, Brown

et al. 1992; Rudd, Elliott et al. 2001).

Fig.2. Molecular model of soluble CD8αα-homodimer, showing glycosylation sites

at the immunoglobulin-like domains and the membrane distal stalk region (Merry et

al., The Journal Of Biological Chemistry, 2003)

4

Introduction

1.1.2 T cell-APC contact

Crystallographic analysis revealed details of interaction between TCR and

its peptideMHC class I ligand. The TCR Vα- and Vβ-domain are diagonally

straddled by the peptideMHC binding site, with α-chain CDR1 and CDR2

contacting the peptideMHC class I α2-domain at the N-terminus of the peptide

and β-chain CDR1 and CDR2 making contact with the α1-domain at the C-

terminal region. The centrally juxtaposed CDR3 of both TCR V-chains bind

to the extended central part of the presented peptide and are therefore

primarily responsible for peptide specificity of the interaction (Garcia,

Degano et al. 1996). Though specific, the interaction of TCR and peptideMHC

is of low affinity with Kd`s in the range of 1-90 µM (Davis, Boniface et al.

1998).

The N-terminal Ig-like domains of the coreceptor molecule CD8 make

contact primarily with the membrane proximal MHC class I α3-domain, while

additional contacts of the molecule with α1- and α2-domain were reported

(Salter, Benjamin et al. 1990; Sun, Leahy et al. 1995; Gao, Tormo et al.

1997; Kern, Teng et al. 1998). This interaction was shown to have

extremely fast kinetics (Koff≈18s-1) and a very low affinity (∼0.2 mM) (Wyer,

Willcox et al. 1999). Nevertheless, its importance in T cell signaling is well

established, as CD8 increases the intrinsic affinity of the TCR-peptideMHC

interaction (Sykulev, Vugmeyster et al. 1998; Wyer, Willcox et al. 1999;

Daniels and Jameson 2000) and prevention of CD8 binding by knockout of

the molecule or competition with sCD8 or peptide antagonist prevents T cell

maturation and activation (Fung-Leung, Schilham et al. 1991; Krensky, Lyu

et al. 1993; Sewell, Gerth et al. 1999). In addition to the described antigen recognition, T cell activation also

involves a host of antigen independent cell-cell interactions including LFA-2

(CD2) binding LFA-3 (CD58) on APCs, CD 40L binding CD40, leukocyte

function-associated antigen (LFA-1) binding intercellular adhesion

molecule-1 (ICAM-1) and interactions of CD28 on T cells with B7.1 (CD80)

and B7.2 (CD86) on APCs. Beyond simply strengthening cell-cell interaction

the latter of these molecules were found important in T cell

5

Introduction

activation by acting as costimulating molecules (Jenkins, Taylor et al. 1991;

Bennett, Carbone et al. 1998). This second signal is especially important in

naïve T cells that have not previously been in contact with antigen, whereas

memory cells and activated T cells show relative independence of

costimulation. Upon encounter of an APC by a CD4+ T cell a process of

redistribution of surface molecules is initiated, leading to the formation of

complexly organized cell-cell interfaces, referred to as “immunological

synapses” (Grakoui, Bromley et al. 1999). These structures, also termed

supramolecular activation cluster (SMAC), were found to form a peripheral

accumulation of larger membrane molecules (pSMAC) like LFA-1, CD43,

the phosphatase CD45 and ICAM-1 and a central area (cSMAC) in which

TCR-CD3, its coreceptors CD4, CD28, peptideMHC , CD80 and CD86 are

spaced for optimal interaction. Similar structures were subsequently

observed during the interaction of CTL and target cells and shown to allow

for directed release of cytotoxic granule content towards the infested cell

(Stinchcombe, Bossi et al. 2001; Trambas and Griffiths 2003).

1.1.3 Lymphocyte activation

The described interactions of cell surface molecules and the following

changes in intracellular phosphorylation trigger the lymphocyte`s

transformation from a resting status into a metabolically highly active

effector T cell capable of specifically removing its targets. This transition is

reflected by phenotypical changes of the cell, which can be observed by

light microscopy, as shown in Figure 3.

6

Introduction

Fig.3. Mixed lymphocyte reaction of 2C TCR transgenic splenocytes and sublethally irradiated BalbC splenocytes

On day 1 (a) small circular cells at low density are visible. After three days of MLR on

day 4 (b) activated cells with blastoid shape show, surrounded by dead/ fragmented

cells.

Though current research is revealing more and more details on the

molecular interactions and TCR downstream events leading to T cell

activation a definite solution to the question how TCR ligation results in

productive signaling by the receptor complex and subsequent activation of

the cell is yet to be found. However, evidence strongly points towards a

combination of several suggested models including clustering of receptors

(Bachmann and Ohashi 1999), serial TCR triggering due to association-

dissociation processes of moderate affinity peptideMHC-ligands (Valitutti,

Muller et al. 1995) and subtle conformational changes of the receptor

complex (Aivazian and Stern 2000; Gil, Schamel et al. 2002) promoting a

signal from the cell surface to the cytosole.

7

Introduction

Closely linked to the question of how antigen contact generates a signal is

the question of how the cell controls its sensitivity to this stimulus.

Similar to the situation in B cells, during T cell development in the thymus a

huge repertoire of TCRs with different antigen specificities is formed by

rearrangement of germ-line encoded gene segments. However, as opposed

to BCRs, which undergo affinity maturation by somatic hypermutation,

resulting in an even broader repertoire of high affinity BCRs/ antibodies, a

given TCR will not change its affinity once the T cell has left the thymus

(Rudikoff, Pawlita et al. 1984). Therefore T cells have to imply different

mechanisms to increase their sensitivity.

Dynamic segregation and clustering of receptors as implemented by

formation of SMACs in T cell-APC contact is such an effective mechanism

of increasing a cell`s sensitivity to low densities of ligands, e.g. nutrients for

bacteria (Bray, Levin et al. 1998) and can amplify a weak binding event by

several orders of magnitude (DeLisi 1981). Responsible for the enhancing

effects of this mechanism are an increased reaction probability between the

clustered receptor and ligand and a prolonged interaction even between low

affinity receptor-ligand pairs. Therefore, the larger a cluster of receptors

gets, the longer a low affinity ligand will stick and the higher the cell`s

sensitivity will be.

Moreover, clustering of TCR complexes facilitates efficient signal

transduction by concentrating cytoplasmic signaling motifs, present in the

CD3 domains (Bu, Shaw et al. 1995).

Since signaling by intracellular tyrosine phosphorylation is the result of the

balanced activity of kinases, which phosphorylate proteins, and

phosphatases, which dephosphorylate proteins, distribution of these

enzymes is important in generating a signal. This reveals another functional

aspect of SMACs, as kinases like ZAP-70, lck and PKC are recruited to the

cSMAC and the phosphatase CD45 is transported to the pSMAC. This

enzyme redistribution could shift the phosphorylation-dephosphorylation

balance towards activation (Shaw and Dustin 1997).

A well described molecular mechanism of controlling receptor clustering

during T cell activation is the action of Cbl proteins. Binding

phosphotyrosine residues through their SH domains these molecules

8

Introduction

promote intracellular degradation of the Src family kinase fyn and the PI3’-

kinase and directly inhibit the kinase ZAP-70. This leaves the PI3’-kinase

substrate Vav1 in a hypophosphorylated state, unable to initiate activation

of WASP-family proteins, which drive cytoskeletal rearrangement by actin

polymerisation. The importance of this mechanism shows in animals

deficient for either Cbl or Vav1. While the former suffer from autoimmunity

due to uncontrolled T cell activation, the latter are unable to mount a strong

T cell immune response, due to inability of forming TCR clusters.

(Krawczyk, Bachmaier et al. 2000; Krawczyk and Penninger 2001).

Consequently, lack of Wiskott-Aldrich-Syndrom-Protein (WASP) leads to an

immune-deficient phenotype with poor T cell antigen response (Dupre, Aiuti

et al. 2002).

Another proposed mechanism of negative regulation of TCR clustering

highlights the importance of glycosylation of the cell membrane and its

protein components. Mice lacking the enzyme β1,6 N-

acetylglucosaminyltransferase (Mgat5) were found to suffer from

autoimmune disease and showed enhanced delayed-type hypersensitivity.

Since Mgat5 catalyzes the synthesis of branched N-glycans carrying N-

acetyllactosamine, the ligand for the carbohydrate binding galectins, the

observed susceptibility to autoimmunity was attributed to increased lateral

mobility of TCRs lacking Mgat5 modified glycans (Demetriou, Granovsky et

al. 2001). Though it is not known whether regulation of Mgat5 plays a

physiological role in T cell activation, this example shows the importance of

glycan adducts for the function of membrane proteins.

1.2 Synthesis and function of N- and O-linked polyglycans on glycoproteins

The majority of the body`s protein components is modified by glycosylation.

In a process that partially takes place in the cytosol and in the lumen of the

ER, nucleosid-diphosphate-activated sugars are connected to branched

polysaccharide chains which become N-glycosidically linked to asparagine

residues of the protein. Passing ER and Golgi, the resulting N-glycosylated

protein undergoes further modification. In a process referred to as

“trimming” glucose and mannose residues are cleaved off and replaced by

9

Introduction

N-acetylglucosamin, galactose, fucose and sialic acid derivatives. On their

way through the Golgi complex most proteins receive additional O-linked

sugar chains. Specific glycosyltransferases catalyze attachment of activated

monosaccharides to serines and threonines of the protein and elongation of

the polysaccharide by addition of further sugar molecules (Varki 1999).

Most glycan adducts on proteins are terminally modified by sialic acids , a

group of monosaccharides derived from neuraminic acid or keto-

deoxynonulosonic acid. These sugar molecules carry a carboxylate group

at the 1-carbon position, which is typically ionized at physiological pH. Since

first found as a major product of mild acidic hydrolysis of salivary mucins,

the family of molecules was given the name “sialic acids” (Blix, Gottschalk

et al. 1957). In many cases interactions of glycoproteins with their

environment are influenced by the presence or absence of this negatively

charged and intensively hydrated molecule. Sialic acids were first described

as recognition structures for invading pathogens such as myxoviruses, the

bacterium Escherichia coli and protozoa like Plasmodium and

Trypanosoma. Subsequently physiological roles in mammalians were

discovered, where negative charges of sialic acids, or lack thereof,

influence on cell proliferation, convey viscosity of mucins, shielding

epithelial surfaces of the body, protect glycproteins from the actions of

proteases and endoglycosidases and shield potentially antigenic glycan

structures from recognition by the immune system (Huenig 1983; Corfield

1992; Rutledge and Enns 1996; Fujita, Ohara et al. 2000). Further

instances in which the physico-chemical properties of sialic acids govern

cellular function were described with the identification of polysialic acid

(PSA). This linear homopolymer of α-2,8-sialic acid was found important in

cell migration during embryogenesis and growth of neurites in the central

and peripheral nervous system (Tang, Rutishauser et al. 1994; Yang, Major

et al. 1994; Hu, Tomasiewicz et al. 1996). A role for sialic acid as a

determinant for specific recognition processes in mammalian cell-cell

interactions was found with the discovery of the sialic acid specific lectin

sialoadhesin (Crocker, Kelm et al. 1991). This adhesion molecule is a

member of the immunoglobulin superfamily and the first one out of the

growing family of siglecs, which have distinct functions including inhibitory

10

Introduction

signaling in B cells (CD22), control of neuronal growth (MAG) and inhibition

of innate immune responses (CD-33 related siglecs)(reviewed by Crocker

2002).

11

Materials and Methods

2 Materials and Methods

1.3 Monoclonal antibodies (mAb)

Monoclonal antibodies have mainly been used in a fluorescently labeled

form, to specifically stain cellular proteins for FACS analysis. Antibody

polymers and antibody coated beads functioned as a stimulus for T cell

activation in vitro.

Depending on individual protocols antibodies were diluted in appropriate

buffer and a defined volume was applied to the cells. Samples were

subsequently incubated at 4°C in the dark to avoid photo bleaching of

fluorescently labeled mAbs. After incubation cells were washed and

analyzed by flow cytometry.

Please refer to individual protocols for details.

GAM IgG1 ,1mg/ml, Southern Biotechnologies (ELISA coating mAb)

Purified mouse IgG1 κ , 0.5 mg/ ml; BD (ELISA IgG1 standard)

GAM K-HRP, Southern Biotechnologies (ELISA secondary mAb)

FITC conjugated rat anti CD8 mAb, clone 53-6.7 (BD)

biotinylated 1B2 mAb (1mg/ ml); cells for production of 1B2 mAb (J 558) are

derived from Jonathan Schneck‘s liquid nitrogen stock and grown in spinner

flasks. Purification of the antibody was performed on a 5 ml HiTrap Protein

G column (SIGMA). 1B2 mAb was biotinylated using a Biotin Protein

Labeling Kit DSB-X™ (Molecular Probes).

PE conjugated rat anti CD25 mAb, clone PC 61 (BD)

PE conjugated Ar Ham anti CD69 mAb, clone H1.2F3 (BD)

12

Materials and Methods

P-Tyr-100 mAb (Cell Signaling, Beverly, MA)

Ar Ham anti CD3ε mAb, clone 145-2C11(BD), biotin-streptavidin complexes

(1 mg/ml)

1.4 Reagents

αCD3 beads

2C11 mAb, BD ; Dynabeads® M-450 Tosylactivated (140.04)

(Dynal Biotec.)

Biotin Protein Labeling Kit DSB-X™, Molecular Probes

CD 8+-/ CD 4+-lymphocyte isolation kit (R&D SYSTEMS, PN:950068)

Cytofix/Cytoperm™ (BD)

DAKO TMB One-Step Substrate System

Dimethyl sulfoxide (DMSO), (SIGMA)

FITC-1-isomer (Molecular Probes)

Gel Filtration Standard (Bio-Rad Laboratories)

N-Acetylheparin sodium salt (SIGMA)

IL-2 (PROLEUKIN®, CHIRON)

Neuraminidase Type III from Vibrio cholerae (SIGMA,N-7885)

Perm-Wash™ (BD)

peptideMHC/Ig-dimer biotin-streptavidin complexes ( 1 mg/ml)

13

Materials and Methods

Rocal™

Streptavidin PE (BD)

Streptavidin Cychrome (BD)

Trypan blue 0.4% (Gibco BRL)

Vybrant™ CFDA SE Cell Tracer Kit (Molecular Probes,V-12883

1.5 Laboratory instruments

Biological safety cabinet:

(sterilGARD Hood; the BAKER COMPANY; Sanford, Maine)

Centrifuges:

RC 5C plus, SORVALL®

LEGEND™RT, SORVALL®

Microcentrifuge 5415D, Eppendorf®

ELISA plate reader:

Fluoroskan™, MTX Labs.

Flow cytometry workstation:

FACScalibur, BD; Mac OS 9, Macintosh;CELLQuest software, BD

FPLC work station:

Amersham Pharmacia Biotech-Superdex® 180HR 10/30

Freezers:

-80°C freezer: -86 ULT FREEZER, ThermoForma

-170°C liquid nitrogen freezer: MVE 1411,CHART Industries

14

Materials and Methods

HPLC work station:

SHIMADZU VP SERIES™,SHIMADZU Inc.,Kyoto

Humidified incubator (37°C, 0.05 pCO2):

3326 Dual Chamber CO2 Incubator, FORMA Scientific

Magnetic stir plate:

Biostir®, Wheaton Science Products

Microscope:

binocular microscope (Zeiss), hemacytometer (Reichert)

Spectrophotometer:

SHIMADZU UV-2000 spectrophotometer (SHIMADZU Inc., Kyoto)

Stiring plate:

Thermolyne, cellgroTM

Tube rotator:

Labquake®, Barnstead

1.6 Materials

Conical tubes 15ml (BD,Falcon™)

Conical tubes 50 ml (BD,Falcon™)

96 well ELISA plate (BD,Falcon™)

Sterile cryovials (NUNC™)

Eppendorf micro centrifuge tubes (BD)

FACS tubes (BD)

Freezing chamber (NALGENE™)

Tissue culture flasks, 12.5-150 cm2 (BD,Falcon™)

Spinner flasks,1000 ml (CORNING),

5 ml HiTrap Protein G column (BD)

15

Materials and Methods

50 KD size exclusion centrifuge tubes (BD,Falcon™)

Cell strainer 70 µm Nylon (BD,Falcon™)

1 ml syringe(BD,Falcon™)

Petri dishes 100x15 mm (BD,Falcon™)

Polysterene serological pipets,1ml-50ml,(BD,Falcon™)

Portable Express™ Pipet-Aid®,(BD,Falcon™)

6 inch plastic Transfer pipet (BD,Falcon™)

Flat bottom 24 well tissue culture plate (BD,Falcon™)

U-bottom 96 well plate (BD,Falcon™)

V-bottom 96 well plate (BD,Falcon™)

1.7 Buffers and media

1.7.1 Buffers for cell preparation and FACS analysis

ACK Lysis Buffer: (filtered sterile through 0.2 µm membrane):

8.29 g NH4Cl (0.15 M), 1g KHCO3 (1 M),37.2 mg Na2EDTA (0.01 M)

Add 800 ml ddi H2O and adjust pH value to 7.4 with 1 N HCL. Add ddi H2O

to 1 liter.

Dulbecco`s Phosphate Buffered Saline (PBS), GIBCO™

FACS Buffer:

PBS, 10% FCS (heat inactivated),0.05% NaAzide

Freezing medium:

FCS (heat inactivated) + 10% DMSO (SIGMA)

1.7.2 Buffers and media for tissue culture

RPMI Medium1640, GIBCO™

Hybridoma SFM, GIBCO™

16

Materials and Methods

Primary Medium:

RPMI, 10% SFCS, 300 µg/ ml G 418 (SIGMA)

Secondary Medium:

Hybridoma SFM

Fetal Bovine Serum (FCS), HyClone™

Super Fetal Calf Serum (SFCS) (filtered sterile through 0.2 µm membrane):

330 ml FCS HyClone™, 80 ml L-glutamine (GIBCO BRL®), 40 ml MEM-

NON ESSENTIAL (GIBCO BRL®) , 40 ml HEPES (GIBCO BRL®), 1.1 mg

Gentamicin (GIBCO BRL®), 11 µl β-mercapto ethanol 1:500 in ddi water

(GIBCO BRL®)

1.7.3 ELISA Buffer

Carbonate Buffer:

5.26 g Na2CO3 anhydrous brought to 1L with ddi H2O;

pH value adjusted to 10.4 with 1 M HCL.

Wash Buffer:

100 ml 10x PBS; 10 ml heat inactivated FCS; 0.5 ml TWEEN 20

brought to 1L with ddi H2O

Diluent Buffer:

100 ml 10x PBS; 10 ml FCS (heat inactivated) brought to 1L with ddi H2O

Final Wash Buffer:

100 ml 10x PBS; 0.5 ml TWEEN 20 brought to 1L with ddi H2O

1 N H2SO4

1.7.4 Buffer for purification of MHC/Ig dimer

Elution Buffer (made freshly prior to use):

17

Materials and Methods

24 mg 3-nitro-4-hydroxyl-phenylacetyl-aminocaproic acid (NP-CAP-OH;

Biosearch Technologies) are dissolved in 275 µl DMSO. 225 µl are diluted

in 14.8 ml of PBS.

1.7.5 Buffers for peptide loading of MHC/Ig dimer

Denaturing Buffer for Kb-Ig:

15 mM Na2CO3, 150 mM NaCl

pH: 11.5

Neutralizing Buffer for Kb-Ig:

250 mM Tris/ HCL

pH: 6.8

Denaturing Buffer for Ld-Ig:

131 mM Citric Acid, 150 mM NaCl, 124 mM NaH2PO4

pH: 6.5

Neutralizing Buffer for Ld-Ig:

250 mM Tris/ HCL

pH : 8.8

1.8 General procedures

1.8.1 Flow cytometry

Flow cytometry is a method for quantitative and qualitative analysis of

particles in suspension. In this study it was used to analyse murine

leukocytes.

Fluorescently labeled cells in suspension are drawn into a capillary and

pass, one at a time, a set of laser beams. Light emission and scattering

caused by each single cell are detected with a photometer and processed

by a computer, allowing differentiation of cells by size, granulation and the

intensity of fluorescence.

18

Materials and Methods

Cells analyzed by flow cytometry were derived from mouse spleens and

peripheral blood. Depending on the individual assay, cells were stained with

fluorescently labeled mAb, MHC/Ig-dimer, CFSE, PNA-FITC or a

combination of the listed. In each experiment a sample of unlabeled cells

was used to determine autofluorescence. Isotype control using fluorescently

labeled mouse IgG with irrelevant epitope was performed. Since emission

spectra of the used fluorescent dyes partially overlap, single colour and

pairwise stainings were prepared to allow for compensation.

1.8.2 Spectrophotometry

This method is used to determine concentrations of protein solutions.

Aromatic amino acids (Tryptophane,Tyrosine and Phenylalanine) absorb

UV-light having a maximum at 280 nm wave length. Knowing the content of

those amino acids in a given protein, an extinction coefficient can be

calculated to determine the concentration of protein in solution.

An extinction coefficient was derived from the following equation, relating

the extinction coefficient of a protein to the numbers of tryptophans

(W),tyrosines (Y) and cystines (C) (Gill and von Hippel 1989):

εc = 5690 (# of W) + 1290 (# of Y) + 120 (# of C)

For a typical MHC class I molecule, the number of W, Y and C are 11, 22, 5

respectively. Accounting for two MHCs and one IgG molecule this yields an

extinction coefficient of approximately 390000 M-1cm-1.

Concentration of MHC-Ig in solution was calculated using the following

equation:

C = ∆E / d * εc

In which E stands for extinction (dimensionless) and d for the distance the light has to

travel through the sample (cm).

19

Materials and Methods

1.8.3 FPLC/HPLC

Liquid chromatography was performed to isolate proteins from solutions

(FPLC) and to control quality of protein reagents (HPLC).

Protein solution is forced to run over a column, resulting in separation of

different protein fractions by size. Leaving the column, proteins are detected

by a spectrophotometer. Since proteins of higher molecular weight are

bigger, the number of interactions per time with the porous column bed is

smaller than in proteins of lower molecular weight. Large proteins therefore

travel the column faster followed by proteins of gradually decreasing size.

Using a protein standard of known molecular weights sample weight can be

determined as a function of time.

In both procedures columns are equilibrated with column buffer until a

stable photometric baseline is achieved. HPLC is performed at a pressure

of 600-800 psi. The column is loaded with a 20 µl sample and resulting

photometric profile is compared to profile of a standard protein solution.

FPLC is performed using Protein G columns and size exclusion columns.

1.9 Animal model

All experiments in this study were performed on 2C TCR transgenic mice,

bred heterozygously on a C57BL/6 background in the Johns Hopkins

Hospital animal facility. BalbC mice were purchased from Jackson

Laboratories (Bar Harbor, MA). Preliminary experiments including non-

specific T cell stimulation were performed on non transgenic C57BL/6

mice. Animal care and handling followed the rules set by the Office of

Laboratory Animal Welfare (OLAW), National Institutes of Health.

The transgenic animal is created by injecting cloned DNA for the rearranged

2C TCR α- and β-chain into a fertilized F2 egg, derived from maiting mice

strains lacking the Vβ8 gene. The offspring is checked for transcription and

expression of the transgene by Northern blot and flow cytometry. Animals

expressing the 2C TCR are inbred repeatedly to create a stable transgenic

strain (Sha, Nelson et al. 1988; Sha, Nelson et al. 1988).

20

Materials and Methods

This animal model was chosen, because transgenic CD 8+ T cells

expressing the 2C TCR do not form a repertoire of different antigen

specificities and can therefore be studied using well defined peptideMHC

class I ligands. The syngenic ligand recognized by the 2C TCR is the

mouse MHC class I molecule H-2 Kb presenting the peptide SIY

(SIYRYYGL), whereas its allogenic ligand is H-2 Ld presenting the peptide

QL9 (QLSPFPFDL). Moreover TCR transgenic mice, as opposed to

hybridoma cells which have to be kept under in vitro stimulating conditions,

allow comparative studies on naïve and activated cells.

1.9.1 Testing mice for 2C TCR expression

2C TCR transgenic mice have to be bred heterocygously, because of

increased incidence of lymphoproliferative disease in homocygous animals.

This protocol allows for the identification of animals expressing the

transgene. 2C TCR expression on murine CD 8+ lymphocytes is detected

using a biotinylated 2C TCR specific mAb (1B2) which is subsequently

stained with cychrome labeled streptavidine. The CD 8+ lymphocyte

population is identified by staining with FITC labeled αCD8 mAb.

Mice are weaned at 4 weeks of age and separated by sex. A 50-100 µl

blood sample is taken from each mouse`s tail vene. Each animal is labeled

by colouring its tail. Collected samples are centrifuged at 400g for 5 min.,

supernatants are decanted and RBC are depleted by incubation in 1 ml of

ACK Lysis Buffer at roomtemperature for 10 min.. Cells are resuspended in

FACS Buffer and transferred to FACS tubes. 1 ml of FACS Buffer is added

to each tube, cells are centrifuged at 400 g for 5 min. and supernatant is

decanted. 50 µl of biotinylated 1B2 mAb (1:5000 in FACS Buffer) are

added and cells are incubated at 4°C in the dark for 1 h.. Cells are washed

twice with 2 ml of FACS Buffer and 50 µl of streptavidine PE (1:1000 in

FACS Buffer) + αCD8 mAb FITC (1:100 in FACS Buffer) are added to each

tube. Cells are incubated at 4°C in the dark for 30 min.. Cells are washed

twice and samples are analyzed by flow cytometry.

21

Materials and Methods

1.10 Production of FITC labeled dimeric MHC/Ig protein

1.10.1 Dimeric MHC/Ig protein

Soluble MHC/Ig chimera were generated by fusing the extracellular domain

of a class I MHC molecule to the N-termini of the immunoglobulin heavy

chain (IgG1) (Dal Porto, Johansen et al. 1993; Fahmy, Bieler et al. 2001). In

this construct the immunoglobulin molecule forms the flexible molecular

backbone of a dimeric MHC ligand. The fusion proteins were constructed

using a pXIg plasmid containing the cDNA encoding for the extracellular

domain of the MHC and the variable heavy chain of IgG1. These plasmids

were subsequently transfected into a murine plasmocytoma cell line that

only expresses the λ light chain (J558L). To increase the yield of secreted

fusion protein cells were co-transfected with genomic human β2-

microglobulin DNA.

Fig.4. Schematic and ribbon model of MHC/Ig molecule

22

Materials and Methods

1.10.2 Tissue culture of J 558 hybridoma cells

This procedure aims for in vitro expansion of MHC/Ig transgenic hybridoma

cells (J 558L) and subsequent isolation of secreted protein from tissue

culture supernatant.

Under aseptic conditions general tissue culture techniques adapted from

those defined by the American Type Culture Collection (ATCC) were

performed.

These include: supply of cells with nutrients, monitoring and adjusting of

temperature, pCO2 and pH value, media exchange and control of cell

density.

All media used for tissue culture were kept at 4°C in the dark for a

maximum time of 6 weeks. Before use, bottles were warmed to 37°C in a

water bath and cleaned with Rocal™ to prevent contamination of the

working area. Cells from the –170°C liquid nitrogen stock were brought up

as described in the protocol for cryopreservation and adjusted in Primary

Medium to a density of 5x105 - 5x106 viable cells/ml. Over a period of 3-6

days cells were expanded until a final number of 5 x 107 –1 x 108 was

reached. During expansion every second day cell density was adjusted to 1

x 106/ ml by adding Primary Medium. One liter of prewarmed Hybridoma

SFM was transferred to an autoclaved spinner flask and 5 x 107 – 1 x 108

cells were added. Constantly agitated on a stiring plate the culture was

allowed to grow for 6 – 10 days. After this time cell viability had usually

dropped below 40 % and the flask was harvested by centrifuging the cell

suspension at 500g for 10 minutes and sterile filtering of the supernatant.

To allow storage of the supernatant 0.05 % sodium azide was added and

pH value was adjusted to 7.5 with Tris buffer (pH 9). Until purification, the

protein solution was stored at 4° C in the dark.

1.10.3 Cell count/ viability test

This procedure allows determinig of the concentration of cells in a given

suspension and the ratio of live to dead cells.

For light microscopy cells are stained with trypan blue. Viable cells exclude

the dye and their cytoplasm appears clear, whereas dead cells stain blue.

23

Materials and Methods

A 10 µl aliquot of cell suspension is diluted 1:10 in 90 µl of trypan blue 0,4%

(0,4% trypan blue in isotonic NaCl solution). 10 µl of this suspension are

applied to a hemacytometer and analyzed by light microscopy. Cells within

all 16 squares of one of the 1x1 mm fields are counted and cell density is

defined using the following equation:

Cells / ml = number of cells counted x 10000

Calculation of cell viability:

viable cells (%) = number of viable cells x 100 / total number of cells

1.10.4 Cryopreservation of cells

Storage of cell lines in liquid nitrogen at –170°C is a standard method of

maintaining a source of healthy cells to start tissue culture from.

Frozen cell lines are not endangered of contamination nor genetical

alteration as observed after prolonged culture. Furthermore frozen cells can

be stored space sparingly and shipped to distant places by express mail.

By slowly freezing cells in a medium containing the organic solvent

dimethyl sulfoxide (DMSO) cells are dehydrated. The danger of ice crystals

damaging cell membranes and organells is therefore minimized.

All following steps are performed in the aseptic environment of a biosafety

cabinet.

Cells to be frozen must be in the logarithmic phase of their expansion.

Cryovials are labeled with name of cell line, number of cells, date and

initials of person who prepared them for freezing. After cell count and

assesment of viability (> 98%) cells are transferred to 50 ml conical tubes

and centrifuged at 400g for 5 min. Supernatant is decanted and cells are

resuspended in freezing medium at a concentration of 1 x 107 / ml. 1 ml of

cell suspension is transferred to each vial. Vials are put in an ethanol filled

freezing chamber which allows a constant and slow decrease of

temperature (∼1°C / min.), resulting in a higher viability of cells after

24

Materials and Methods

thawing. Freezing chamber is put in a –80°C freezer for 24 h and vials are

afterwards transferred to –170°C liquid nitrogen.

Starting tissue culture from liquid nitrogen stocks requires rapid thawing of

frozen cells in a 37°C water bath until just a small piece of frozen medium is

visible. To eliminate toxic DMSO, cells are washed with 10 ml of 37°C warm

RPMI Medium, centrifuged at 200 g for 7 min. and supernatant is decanted.

Cells are subsequently seeded in Primary Medium at a concentration of 5 x

105 – 5 x 106 cells / ml.

1.10.5 Purification of MHC/Ig dimer

The antigen binding site of the IgG1 molecule which builds the backbone of

the MHC/Ig dimer is specific for 4-hydroxy-3-nitrophenol (NP). This allows

for affinity purification of the protein construct from tissue culture

supernatant using a NP-sepharose column (Schneck et al, 2000).

Protein purification is performed at 4°C in the dark. After washing the

column with 15 ml of PBS, tissue culture supernatant is allowed to circulate

over the column at a speed of 1 ml / min. for 4 days. Bound protein is eluted

from the column by washing with excess NP, without the need of harsh

changes of pH value. For this purpose the column is washed with 25 ml of

PBS and allowed to drain. The column is washed with 15 ml Elution Buffer,

while eluat is collected. Another 15 ml of PBS is run over the column and

eluat is collected. The column is washed with 20 ml of PBS and stored in

PBS + 0.02% sodium azide at 4°C in the dark. Excess NP is separated from

yielded protein using size exclusion centrifuge filters. Based on a protein

standard of known molecular weights protein integrity is checked by HPLC.

1.10.6 Quantitation of MHC/Ig dimer by ELISA

This assay is used to determine the amount of soluble dimeric MHC-Ig

protein after final purification and for purposes of quality control, at different

times during protein production.

A round bottom 96 well ELISA plate is coated with an α mouse IgG1 mAb.

25

Materials and Methods

Along with a standard of known concentration (Purified mouse IgG1),

samples for protein quantification are applied to wells of the plate. After

incubation in a humidified box and repetitive washing, bound IgG1

molecules and MHC-Ig dimers, respectively, are detected by an enzyme

linked α IgG1 mAb. After adding the enzyme`s substrate, the catalyzed

reaction leads to a colored product which can be quantified by

photometrical analysis. By correlating extinction differences of sample and

standard the concentration of analyte can be determined.

50 µl of coating mAb are diluted in 5 ml of Carbonate Buffer and 50 µl are

applied to each well of the plate. The plate is incubated in a humidified box

at room temperature for 1 h. To block nonspecific binding sites on the

coating mAb, 50 µl of FCS are diluted in 5 ml of Carbonate Buffer and 50 µl

are applied to each well (on top of 50 µl coating solution). The plate is

incubated in a humidified box at room temperature for at least 1 h.

IgG1 standard is diluted to 100 ng / ml in Diluent Buffer. Samples are diluted

in Diluent Buffer to fit standard range.

Before use ELISA plate is washed 3 times with 100 µl of Wash Buffer / well.

50 µl of Diluent Buffer are added to each well. Well A1 and A2 are left as

blanks. 50 µl of prepared standard are added to well B1 and B2. Samples

are applied to wells A3 to A12 .The content of each well is serially diluted,

by transferring 50 µl from row A to row B, from row B to row C, and so on.

The plate is incubated in a humidified box at room temperature for 1 h.

1 µl of enzyme linked secondary mAb is diluted in 5 ml of Dilution Buffer.

The plate is washed three times with 100 µl of Wash Buffer / well. The last

wash is discarded and 50 µl secondary mAb solution are applied to each

well. The plate is incubated in a humidified box at room temperature for 30

to 45 min.

Plate is washed three times.50 µl of substrate solution are added to each

well. Plate is incubated at room temperature in the dark for 2-15 min..

After slight blue staining of highest standard dilution is visible, reaction is

stopped by adding 25 µl of 1 N H2SO4 to each well.

Bottom of plate is cleaned and bubbles, if present, are eliminated from

wells. Plate is read at 450 nm wave length.

26

Materials and Methods

1.10.7 FITC-labeling of MHC-Ig dimer

Fluorescently labeled MHC-Ig dimers allow antigen specific staining of

CD8+ T cells and exploration of TCR-MHC binding kinetics based on the

experimental protocols developed by T. Fahmy.

A fluoresceine molecule with an amine reactive isothiocyanate (ITC) group

is covalently bound to the N-terminus of the protein.

MHC/Ig dimers are fluorescently labeled with fluoresceine isothiocyanate

(FITC) at pH 7.4. FITC is dissolved in N,N-Dimethylforamide at 10 mg / ml.

At room temperature, the protein is adjusted to a concentration of 10 mg /

ml in PBS and incubated with 100 fold molar excess of FITC for 1 h in the

dark on a stir plate. Excess FITC, aggregats and fragmented protein are

removed by FPLC.By spectrophotometry the fluoresceine / protein ratio is

determined using the following equation:

F / P = A496 / A280 – (0.35 A496) * (εp / εf)

εf = 0.69 x 105 M-1cm-1 is the extinction coefficient of fluoresceine and εp is the extinction

coefficient of the protein.

1.10.8 Peptide loading of fluorescently labeled MHC/Ig

To create a complete ligand, that allows determining of specific/non-specific

binding to 2C TCR transgenic lymphocytes, MHC/Ig dimers are loaded with

cognate/non-cognate peptide.

By changing pH value, MHC/Ig dimer is mildly denatured and allowed to

refold in the presence of excess peptide.

1.10.8.1 Kb-Ig loading

Kb-Ig at a concentration of 1mg/ ml is diluted 10 fold with Denaturing Buffer.

Solution is tumbled at room temperature for 15 min..A 40 fold molar excess

of peptide is added. The amount of peptide needed is calculated using the

following equation:

27

Materials and Methods

Vp= 0.352 CKb x VKb / Cp

Cp = concentration of peptide in mg / ml

CKb = concentration of Kb/Ig in µg / ml

VKb = ml of Kb / Ig at the concentration CKb

Vp = µl of peptide to add

An equivalent amount of Neutralizing Buffer is added and the solution is

incubated at 4°C for 48h in the dark.After 48 h protein is washed twice with

PBS and concentrated using a size exclusion centrifuge tube.

1.10.8.2 Ld-Ig loading

Ld-Ig at a concentration of 0.5-1 mg / ml is diluted 10 fold with Denaturing

Buffer. A 40 fold molar excess of peptide is calculated, using the equation

above, and added. The solution is allowed to incubate at 37°C for 2 h.

After 2 h pH value is adjusted to 7 with Neutralizing Buffer.

A 2 fold molar excess of β2-microglobulin is added and protein solution

washed and concentrated twice with PBS, using a size exclusion centrifuge

tube.

Peptide loaded MHC/Ig at a concentration of 1-3 mg/ml is stored at 4°C in

the dark.

1.11 Cell preparation

1.11.1 Preparation of splenocytes

This procedure allows for isolation of leucocytes from mouse spleens.

The spleen is a large secondary lymphatic organ and therefore an easily

accessable source of lymphocytes in mice. Splenocytes are extracted by

homogenizing the organ and depleting red blood cells by ammonium

chloride lysis.

28

Materials and Methods

The sacrified mouse is rinsed with ethanol and splenectomy is performed

employing aseptical surgical technique.The organ is transferred to a petri

dish and gently homogenized in 5 ml of PBS. To increase cell yields the

strainer is rinsed with another 5-10 ml of PBS. Cell suspension is

transferred to a 15 ml conical tube and centrifuged at 400g for 5 min..

Supernatant is decanted and cells are incubated in 5 ml ACK Lysis

Buffer/spleen at room temperature for 10 min. Cells are subsequently

centrifuged again (400g for 5 min.), supernatant is decanted and the pellet

is resuspended in desired buffer or medium.

1.11.2 Isolation of CD 8+/ DN- T cells

This procedure allows enrichment of splenocytes for CD 8+ / DN-

lymphocytes by negative separation. The resulting cell population is of high

purity (>95%), which facilitates analysis by flow cytometry.

Splenocytes are incubated with a cocktail of mAb specific for epitopes on

irrelevant cells, i.e. leucocytes other then CD 8+- or DN lymphocytes,

respectively. Cells are separated by trapping mAb labeled cells on a

column, filled with α FC mAb coated beads.

For detailed instructions see protocol of CD 8+/ CD 4+-lymphocyte isolation

kit (R&D SYSTEMS, PN:950068). In brief, after depletion of red blood cells

splenocytes are incubated with mAb cocktail, subsequently washed twice

and loaded on separation columns. After 15 min. of incubation at

roomtemperature cells are eluted with 10 -15 ml of Column Buffer,

centrifuged at 400 g for 5 min. and resuspended in desired buffer or

medium.

In order to isolate DN lymphocytes, splenocytes are incubated with both

commercially available mAb cocktails (CD 8+- and CD 4+- lymphocyte

isolation kit).

29

Materials and Methods

Fig.5. Cell purification Flow cytometrical analysis of 2C splenocytes before

purification (top panel), after CD8-enrichment (middle panel) and after additional

CD4-enrichment, resulting in a primarily DN cell population. FL2: αCD8, FL3: 1B2

(α2C TCR mAb)

30

Materials and Methods

1.11.3 Neuraminidase treatment of cells

Sialic acid carries a strong negative charge and influences interactions of

glycoproteins. The following procedure results in removal of terminal sialic

acid residues from cell surface glycan stalks.

The enzyme neuraminidase derived from Vibrio cholerae is able to catalyze

the cleavage of sialic acid molecules from glycan chains.

In a 15 ml conical tube cells were adjusted to a concentration of 2 x 106 / ml

in RPMI Medium. 0,022 U of neuraminidase / 1x106 cells were added. Cells

were incubated for 20 min. at room temperature. Cells were washed with

4°C cold RPMI Medium, centrifuged at 400 g for 5 min. and supernatant

was decanted. This washing step was repeated twice.

After staining with PNA-FITC, desialyation is verified by flow cytometry

comparing native and neuraminidase treated cells.

Fig.6. Control of desialyation by PNA staining

31

Materials and Methods

1.11.4 CFSE labeling of 2C splenocytes

This procedure was performed to monitor in vitro expansion of 2C

lymphocytes.

2C splenocytes were incubated with the succinimidyl ester of

carboxyfluoresceine diacetate (CFSE). Intracellular esterases cleave

acetate groups of the CFSE molecule, turning it into a fluorescent amine-

reactive form, that gets covalently bound to amine groups of intracellular

proteins. Expansion of labeled cells can be monitored, since the amount of

CFSE labeling drops by half with each subsequent cell devision.

A 10 mM stock solution of CFSE in DMSO was prepared and stored at

–20°C. In a 50 ml conical tube cells to be labeled with CFSE were adjusted

to a concentration of 4 x 106 / ml in 37°C RPMI. 1 µl of CFSE / 1 x 107 cells

was diluted in a volume of 37°C RPMI equal to the given volume of cell

suspension and mixed with the cells. Cells were incubated for 75 sec. and

reaction was stopped by filling the tube with 4°C RPMI+10%FCS. Cells

were centrifuged at 400 g for 5 min., supernatant was decanted and cells

were washed a second time with RPMI. After second wash was decanted,

cells are resuspended in desired medium.

1.11.5 Generation of activated T cells by Mixed Lymphocyte Reaction (MLR)

This protocol was developed to activate and expand 2C T cells in vitro by

allogenic stimulation.

2C splenocytes (H2 Haplotype b) are cultured in the presence of sublethally

irradiated Balb/C splenocytes (H2 Haplotype d).

The difference in expressed MHC molecules results in allorecognition,

subsequent activation / expansion of 2C T cells and killing of Balb/C cells

(Figure 3).

Due to previous irradiation, Balb/C cells are unable to proliferate upon

allogenic stimulus.

All following steps were performed in the aseptic environment of a biosafety

cabinet.

32

Materials and Methods

From each spleen leukocytes were isolated as previously described and

resuspended in 2 ml of 37°C Primary Medium. A cell count was performed.

Aiming for a final Balb/C- : 2C - cell ratio of 1.75 x 106 : 1.25 x 106/ ml , the

highest possible volume of MLR suspension was calculated.

Balb/C splenocytes were sublethally irradiated with 3000 RAD (Gammacell

40 Exactor™, MDS Nordion).

The previously calculated volume of MLR suspension was set up in Primary

Medium. 100 U IL-2 / ml were added. 2 ml of cell suspension was applied to

each well of the tissue culture plate.

The plate was incubated at 37°C; 5% CO2 and development of the culture

was checked daily by light microscopy.

Cells were used after 4-5 days of culture.

1.12 Equilibrium binding assay

In this assay FITC-labeled MHC/Ig dimer was used to quantitatively

measure

TCR-peptideMHC binding on lymphocytes.

2C T cells were incubated with varying concentrations of FITC-labeled

MHC/Ig dimer loaded with cognate and non-cognate peptide until

equilibrium was reached. To maintain an accurate measure of equilibrium

binding cells were analyzed by flow cytometry without previous washing.

Specific TCR-peptideMHC binding was assessed by substracting MCF values

for cells incubated with non-cognate ligand from MCF values for cells

incubated with cognate ligand.

All binding experiments were performed at 4°C.

Cells were adjusted to a concentration of 1x107 / ml in FACS Buffer. A row

of FACS tubes was equipped with 10 µl of cell suspension per tube. An

Eppendorf tube with 40 µl of FITC-labeled MHC/Ig was prepared. 20 µl of

FITC-labeled MHC/Ig were added to the first tube. Using a 20 µl pipet

concentration of dimer was serially diluted with FACS Buffer and 20 µl

aliquots were applied to following tubes. Described serial dilutions were

perfomed, using FITC-labeled MHC/Ig loaded with cognate and non-

cognate peptide. For determining of background fluorescence an additonal

33

Materials and Methods

FACS tube was equipped with a 10 µl aliquot of cell suspension without

labeled ligand. Cells were incubated in the dark for 2 h..After incubation

samples were analyzed by flow cytometry without previous washing.

Data analysis: Specific binding was calculated by subtracting MCF values for non-cognate

ligand from MCF values for cognate ligand. Specific binding in MCF values

was normalized to the plateau and plotted as a function of MHC/Ig dimer

concentration.

Binding data were fit to a model of equilibrium dimerization of homogenous

monovalent receptors by divalent ligand (Perelson,1984) :

The equilibrium solution to the dimerization reaction is:

[RL]=Rtβ (-1+(1 + 4δ)1/2) / 2δ

[R2L]= Rt (1+2δ-(1 + 4δ)1/2) / 4δ

where β = 2L/(Kd+2L), δ = β(1-β)KxRt

The total concentration of bound ligand is [Lb] = [RL]+[R2L] and the fraction

of ligand bound is [Lb] / Rt. Three parameters are unknown in these

equations: Kd, Kx and Rt. To determine these parameters, fits of the binding

data were performed using the nonlinear fitting algorithm of Microcal Origin

6.1 (Origin Lab Corporation, Northampton, MA). The resulting three

parameters, Kd, Kx, Rt were used to approximate the avidity constant at low

concentration, Kv~ Kd/KxRt and to calculate the concentration of singly

bound ligand [RL] and concentration of crosslinks [R2L].

34

Materials and Methods

0

5

10

15

20

25

30

35

40

0,00E+00 5,00E-08 1,00E-07 1,50E-07 2,00E-07 2,50E-07 3,00E-07 3,50E-07 4,00E-07

Total BindingNon-Specific BindingSpecific BindingR ih 4

Fig.7. Binding of peptideMHC/Ig dimer is a sum of specific and non-specific

binding. Specific binding (squares) was determined by subtracting binding of non-

cognate ligand (grey dots) from overall binding of cognate ligand (black dots).

35

Materials and Methods

1.13 Functional assays

1.13.1 Tyrosine phosphorylation assay

This flow cytometrical assay was developed to visualize early changes of

intracellular tyrosine phosphorylation after TCR engagement.

Cells are stimulated in vitro and sequentially fixated in a formaldehyde

containing solution. After permeabilization of the cell membrane intracellular

phosphotyrosine residues are stained with a biotinylated mAb +

streptavidine PE.

After preparation cells are adjusted to a concentration of 1x107/ml in RPMI

and allowed to rest at 37°C, 0.05 pCO2 for 1h..

A V-bottom 96 well plate was put on ice and 100 µl of Cytofix / Cytoperm™

are applied to each well. Using 37°C RPMI a volume of stimulus equal to

the given volume of cell suspension was prepared. In a 37°C waterbath the

timed reaction was started by adding stimulus to cell suspension. After

defined time intervals 25 µl aliquots of cell suspension were repetetively

transferred to rows of the 96 well plate. Cells were incubated on ice for 30

min. and subsequently washed three times with 4°C Perm-Wash™ . Third

wash was discarded and cells were allowed to rest at room temperature for

30 min.. pTyr100 mAb was diluted 1:200 in 4°C Perm-Wash™ and 50 µl of

mAb solution were added to each well. Samples were incubated on ice for

1h. Cells were washed three times with 4°C Perm-Wash™. Third wash was

discarded. Streptavidin PE was diluted 1:100 in 4°C Perm-Wash™ and 50

µl were added to each well. Cells were incubated on ice for 30 min..

Samples were washed three times with 4°C Perm-Wash™ and analyzed by

flow cytometry.

Data analysis: Data analysis was performed using Microcal Origin 6.1 (Origin Lab

Corporation, Northampton, MA).

Resulting MCF values for stimulated cells were normalized to MCF values

determined in unstimulated cells (baseline phosphorylation) and plotted as

a function of time.

36

Materials and Methods

The “area under the curve” (AUC) as a measure of overall phosphorylation

is determined after 180 and 300 sec.

1.13.2 In vitro proliferation assay

This in vitro experiment was performed to compare the proliferative

response of neuraminidase treated and untreated 2C splenocytes.

Using beads coated with α CD3 mAb as a stimulus, CFSE labeled cells

were expanded in culture for three days. Proliferation was monitored daily

by flow cytometry.

Cells were labeled with CFSE and half of the cells were subsequently

treated with Neuraminidase. At a concentration of 1x105 / ml in RPMI+10%

FCS cells were mixed with 1x105 beads / ml. 100 U of IL-2 / ml were added.

200 µl of cell suspension were applied to each well of the plate and culture

was incubated at 37°C, 5% CO2 for three days. Control experiments in the

absence of beads, IL-2 and both were performed simultaneously. Using

flow cytometry each day two wells of the neuraminidase treated and

untreated cells, respectively, were analyzed independently along with their

control wells.

Data analysis: FACS data is analysed on FACSexpress 2.0 software.

37

Results

3 Results

Influence of terminal desialyation on TCR membrane organization has been

verified by equilibrium binding of fluorescently labeled, genetically

engineerd dimeric MHC class I ligands. To check whether the observed

changes in receptor ligand interaction impact on the cell`s reaction upon in

vitro stimulation, an assay to visualize early intracellular tyrosine

phosphorylation events was developed. The in vitro proliferative response

of neuraminidase treated and native cells was compared in a three day

CFSE assay.

1.14 Effects of desialyation on MHC/Ig binding on T cell surface

1.14.1 Desialyation enhances binding of specific peptideMHC ligand on T cell surface.

After treating naïve CD8+ 2C T cells with neuraminidase, individual

equilibrium binding assays were performed using both SIYKb-Ig (syngenic

ligand) and QL9Ld-Ig (allogenic ligand). Values for non-specific binding were

determined in binding assays of the corresponding non-cognate ligands SIINKb-Ig and MCMVLd-Ig, respectively. Specific binding isotherms were gained

by subtracting values for non-specific binding from overall binding values of

cognate ligands. Accordingly, binding assays were also performed on naïve

CD8+ 2C T cells without previous neuraminidase treatment as well as on 2C

T cells after three days of allogenic in vitro stimulation.

Comparing the binding isotherms derived from these experiments, a strong

increase of SIYKb-Ig - (Fig.8a), as well as QL9Ld-Ig -ligand binding (Fig.8c) is

apparent in neuraminidase treated, naïve cells as compared to untreated

naïve cells. In both cases the amount of ligand bound approaches values as

determined for activated cells. This effect is most obvious at low ligand

concentrations and more pronounced in those cells incubated with the

allogenic ligand (Fig.8c).

To visualize increased ligand binding, especially at low ligand

concentrations, data were also analyzed in a Scatchard plot (Fig.8b and

Fig.8d). Scatchard analysis of dimeric ligand binding to monovalent

receptors highlights enhanced avidity resulting from increases in receptor

38

Results

cross-linking by pronounced curvilinearity (Fahmy, Bieler et al. 2001). In

both peptideMHC-TCR interactions desialyation of naïve cells led to

increased curvilinearity approaching values as seen in activated cells.

0

0.2

0.4

0.6

0.8

1

1 E-10 1 E-09 1 E-08 1 E-07

0.0E+00

5.0E+08

1.0E+09

1.5E+09

0

0,2

0,4

0,6

0,8

1

Naive

Desialyated

Activated

0.0E+00

5.0E+08

1.0E+09

1.5E+09

0 0.2 0.4 0.6 0.8 1

a SIYKb-Ig b

dc QL9Ld-Ig

Concentration [M]

Frac

tion

Boun

d

Fraction Bound

Boun

d/Fr

ee

Fig.8. Desialyation of 2C TCR transgenic cells results in increased avidity of

TCR-peptideMHC interaction.

1.14.2 Enhanced binding of peptideMHC ligand is a result of increased TCR cross-linking.

To further quantitate the indicated increase in TCR cross-linking, binding

data were fit to a model of equilibrium dimerization of homogeneous

monovalent receptors by divalent ligands (Perelson,1984). In this model the

first monovalent binding step (R+L→RL) creates a complex which increases

the local concentration of ligand for a potential second receptor. Depending

on the local concentration of TCRs a second binding event (R+RL→R2L)

39

Results

may occur, resulting in TCR dimerisation. Since it is assumed that all

receptors are identical, the second binding event increases the “apparent

binding affinity”, i.e. the avidity. The avidity is a measure for the stickyness

of receptor-ligand interactions and can be estimated from parameters

derived from this model: Kd, the single site dissociation constant,

characterizes the intrinsic affinity of a single peptideMHC-Ig binding site to one

TCR. The cross-linking potential of the second association step (Kx) and the

total number of receptors (Rt) are expressed in inverse units. Therefore, the

dimensionless cross-linking constant (KxRt) can be derived to characterize

the ability of the MHC-Ig-TCR complex to recruit another TCR. Knowing

these three parameters, one can calculate the estimated avidity (Kv) using

the following equation:

Kv = Kd / KxRt

As shown in table 1, activation of T cells leads to a clear increase in cross-

linking potential (Kx). A ∼6 fold increase of Kx values in the case of SIYKb-Ig

and a ∼70 fold increase in the case of QL9Ld-Ig are mainly responsible for

higher cross-linking constants (KxRt), since the total number of receptors

(Rt) remains fairly unaffected. These increases of KxRt again are the main

reason for the observed enhancement of avidities (Kv=Kd/KxRt), since

intrinsic affinities (Kd) in both cases just showed a ∼2 fold increase. In the

case of SIYKb-Ig Kv values show a ∼37 fold decrease upon activation,

reflecting a ∼37 fold increase in avidity. Using Ld-Ig a ∼270 fold increase in

avidity was seen. These observations are consistent with previous findings,

showing that increases in avidity of TCR-peptideMHC interaction due to

facilitation of TCR cross-linking are a likely mechanism for improving TCR

antigen sensitivity (Fahmy, Bieler et al. 2001). Analogous comparison of Kd,

Kx, Rt and the derived parameters KxRt and Kv in naïve, untreated and

naïve, desialyated T cells shows, that Neuraminidase treatment as well

results in increased avidities of TCR-peptideMHC interactions. Decreases of

determined Kv values (∼13 fold for Kb-Ig and ∼99 fold for Ld-Ig) are not as

strong as in the case of activated cells. Nevertheless, again primarily

40

Results

facilitation of cross-linking and not increases in intrinsic affinities are

responsible for this effect.

62,59810,05096(± 0,43)36,4(± 0,005)0,0014(± 0,15)3,19DN naive desialiated

1,679691,2026(± 0,421)34,36(± 0,0086)0,035(± 0,11)2,02DN naiveQL9-Ld-Ig

1,44121,79017(± 0,68)48,646(± 0,01)0,0368(± 0,193)2,58Activated cells

3,934920,77562(± 0,42)43,09(± 0,0052)0,018(± 1,37)3,052Naive cells desialiated

390,2990,01773(± 0,781)39,4(± 0,0061)0,0005(± 0,47)6,92Naive cells

QL9-Ld-Ig

0,427985,304(± 0,6)20,40(± 0,07)0,26(± 0,37)2,27Activated cells

1,23453,6452(± 0,5)14,02(± 0,1)0,26(± 0,86)4,5Naive cells desialiated

15,8970,41706(± 0,36)9,93(± 0,06)0,042(± 1,02)6,63Naive cells

SIY-Kb-Ig

Kv KxRtRt (#/cell)Kx (cell/#)Kd (nM)

Table(1) Parameters derived from the model fit of binding data

Since binding of divalent ligands to monomorphic receptors includes single-

and double-bound ligand (Figure 9 a,b), increased cross-linking will favour

the latter of the two possible receptor ligand complexes. Deconvolution of

the binding isotherm results in separate graphs for RL and R2L,

respectively. Comparing the contribution of R2L to overall ligand binding

(Figure 9), increases in cross-linking upon desialyation become apparent. In

the case of SIYKb-Ig the maximum amount of cross-linking increased from

approximately 8% to 36% and in the case of QL9Ld-Ig it rose from virtually

undetectable to 14%. The classically bell shaped curve reflects the fact, that

there is a maximum in cross-linking at medium ligand concentrations.

Supraoptimal ligand concentrations result in decreased cross-linking due to

competition of ligand binding sites for available receptors. Accordingly,

41

Results

suboptimal ligand concentrations lead to less cross-linking due to

decreased probability of receptor-ligand encounter.

MHC-Ig [M]

Frac

tion

Boun

d

Kd

Kx

R L RL

RL R R2L

+

+

I

Y

a

0.0

0.1

0.2

0.3

0.4

0.5

1 E-11 1 E-10 1 E-09 1 E-08 1 E-07

d

0.0

0.1

0.2

0.3

0.4

0.5NaiveDesialyatedActivated

c

MHC-Ig [M]

Frac

tion

Cro

ss-L

inks

Y

IY

I I

Y

I I

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.E-10 1.E-09 1.E-08 1.E-07

bTotal bound

RL

R2L

SIYKb-Ig

QL9Ld-Ig

Fig.9. Deconvolution of binding isotherms shows increases in double-bound

ligand on desialyated T cells.

1.14.3 Desialyation of DN cells results in decreased cross-linking.

Since peptideMHC/Ig binding to T cells is dependent not only on the presence

of TCR molecules, but also their coreceptors CD8, possible targets of

desialyation should be discriminated, probing T cells lacking CD8 (DN

cells). In opposition to the enhancing effect of neuraminidase treatment on

peptideMHC/Ig binding to CD8+ T cells, a reverse effect was seen in DN cells.

Binding isotherms shifted to the right (Figure 10, left panel) and Scatchard

analysis did not show curvilinearity, indicating reduced cross-linking (Figure

10, right panel). Comparison of model fit parameters showed a ∼37 fold

decrease in avidity, again merely due to changes in cross-linking potential

42

Results

(Kx), since the overall affinity (Kd) and the total number of receptors (Rt)

virtually were not affected by neuraminidase treatment (Table 1).

0

0,2

0,4

0,6

0,8

1

1 E-10 1 E-09 1 E-08 1 E-07

DNDN desialiated

2E+07

4E+08

8E+08

1E+09

2E+09

2E+09

2E+09

0 0,5

Fraction Bound

Bou

nd/F

ree

1

Fig.10. Desialyation of DN cells results in decreased cross-linking

1.14.4 Neuraminidase treatment does not affect non-specific binding of peptideMHC/Ig to T cells

To rule out the possibility that the observed changes in ligand binding upon

desialyation were due to changes in non-specific binding of peptideMHC/Ig to

the surface of the T cell, binding of non-cognate peptideMHC/Ig (MCMVLd-Ig) to

2C T cells was compared in native and neuraminidase treated cells (Figure

11). Neither in CD8+ nor in DN cells did desialyation lead to remarkable

changes in non-specific binding of peptideMHC/Ig. This finding lends further

support to the hypothesis that by reduction of sialic acid residues on the cell

surface T cells can increase their antigen sensitivity without loosing

specificity for their peptideMHC ligand.

43

Results

0

5

10

15

20

25

1 E-11 1 E-08 2 E-08 3 E-08 4 E-08 5 E-08 6 E-08 7 E-08 8 E-08 9 E-08

Dimer Concentration [M]

MC

F

Naive

Naive Desialyated

DN Naive

DN Naive Desialyated

Fig.11. Non-specific binding does not increase upon desialyation

1.15 Effects of desialyation on cellular reactions upon in vitro stimulation

The former experimental data show an enhancing effect of neuraminidase

treatment on TCR-peptideMHC avidity. This effect shows to be dependent on

the presence of the T cell coreceptor CD8 and is mainly due to enhanced

TCR cross-linking. To evaluate possible physiological relevance of

enhanced TCR cross-linking after desialyation, a flow cytometrical assay to

measure early intracellular tyrosine phosphorylation upon in vitro stimulation

was developed. In a time dependent fashion native as well as

neuraminidase treated CD8+ T cells were stimulated with cognate/non-

cognate peptideMHC/Ig and antiCD3 mAb, respectively, and fixated in

formaldehyde containing solution. Cells were stained for intracellular

tyrosine phosphorylation and analysed by flow cytometry. In a second

experimental set up, effects of desialyation on the in vitro proliferative

response of T cells were investigated. Therefore cells were labeled with the

44

Results

intracellular, fluorescent dye CFSE, expanded in vitro over a time course of

three days and analysed daily by flow cytometry.

1.15.1 Increased TCR cross-linking results in enhanced signaling

Staining for intracellular phosphorylation was measured by flow cytometry

and resulting MCF values were normalized to baseline phosphorylation

values determined in unstimulated cells. Changes in intracellular

phosphotyrosine levels are plotted as fold increase of baseline

phosphorylation. Using antiCD3 mAb, a rapid increase of intracellular

tyrosine phosphorylation levels was regularly seen in the first 90 seconds of

stimulation. Accordingly, the accelerating and enhancing effect of

desialyation shows to be especially relevant in the first two minutes of

stimulation (Figure 12a). Stimulation with complexes of cognate

peptideMHC/Ig (SIYKb-Ig) led to a less rapid increase of phosphorylation levels,

assumingly due to the more complex kinetics of the interaction between

receptor and ligand. Again the tyrosine phosphorylation response was

enhanced while the overall shape of the profile was not affected by

desialyation (Figure 12b).

In both cases a pretreatment with neuraminidase resulted in similar

increases of phosphorylation, as asassed by calculating the ratios of “area

under the curve” (AUC) for neuraminidase treated an native cells after 180

and 300 seconds (Table 2). Neuraminidase by itself did not lead to

increases in intracellular phosphorylation.

45

Results

1

1,5

2

2,5

3

3,5

4

4,5

0 60 120 180 240 300 360 420 480 540 600Time [s]

Fold

Incr

ease

Naive Desialyated

1

1,5

2

2,5

3

3,5

4

4,5

0 60 120 180 240 300 360 420 480 540 600Time [s]

Fold

Incr

ease

Fig.12. Early tyrosine kinase signaling is enhanced in desialyated cells. The

profiles of intracellular tyrosine phosphorylation responses upon stimulation with

αCD3 mAb (top panel) and SIYKb-Ig (bottom panel) show increased signaling in

desialyated cells while the over-all shape of the phosphorylation curve remains

unchanged .

46

Results

180 sec.

300 sec.

AUC naive

AUC desialyated

fold increase

AUC naive

AUC desialyated

fold increase

α CD3 mAb

455.5

565.83

1.24

842,3

957.68

1.14

SIYKb-Ig

391.5

481.3

1.23

692.5

813

1.17

Table(2) Desialyated cells show increased over-all tyrosine phosphorylation upon

in vitro stimulation.

1.15.2 Desialyation accelerates and enhances T cell proliferation in vitro

During a three day in vitro proliferation assay with CFSE labeled 2C

splenocytes neuraminidase treated cells proliferated more rapidly and to a

higher extent than native cells. After two days in the presence of antiCD3

mAb coated beads and low dose IL-2, 70% of the desialyated cells had

undergone cell division, as compared to 56% of the untreated cells. On day

three, with 81% of neuraminidase treated cells being in proliferation as

compared to 75% of native cells, this effect was still detectable though less

pronounced, which is most likely attributable to resialyation by the cellular

sialytransferases (Fischer, Kelm et al. 1991). In the absence of antiCD3

mAb, neither native nor neuraminidase treated cells showed in vitro

proliferation.These findings indicate, that desialyation is in part sufficient to

mimic the phenotype of activated T cells, which show higher antigen

sensitivity and enhanced in vitro proliferation. Meanwhile, comparable in

vitro proliferation data has been published, which showed lowered threshold

of desialyated CD8 cells for antigenic stimulation, by titrating doses of

stimulatory peptide in a 48 h CFSE proliferation assay (Pappu and Shrikant

2004).

47

Results

Fig.13. Desialyated cells show enhanced in vitro proliferative response

48

Discussion

4 Discussion

Sialic acid is widely accepted as a key-molecule in cell-cell interactions. It

blocks or provides binding sites and influences on spatial orientation of cell

surface proteins (Kelm and Schauer 1997; Schauer 2000).

The featured experiments show that reduction of cell surface sialic acid on

CD8+ T lymphocytes leads to increased avidity of TCR-peptideMHC

interaction and enhances the cell`s response to TCR engagement in vitro.

Parameters derived from the model fit of binding data indicate that this

increase in avidity is due to enhanced cross-linking of TCR molecules by

dimeric peptideMHC, similar to the situation in activated CTL where

membrane reorganisation promotes antigen sensitivity (Fahmy, Bieler et al.

2001). The fact that activated T cells show lower levels of surface sialic acid

has long been known (Galvan, Murali-Krishna et al. 1998) and a gene

encoding a sialidase with limited substrate specificity has been located in

the MHC and is known to be upregulated upon T cell activation (Milner,

Smith et al. 1997). The observation that neuraminidase treatment is

sufficient to partially mimic an activated phenotype of CD8+ T cells and fails

to do so in DN- cells suggests a crucial role for the coreceptor molecule in

the observed “neuraminidase effect”.

Focusing on the importance of CD8, possible mechanisms of desialyation

induced increase of TCR cross-linking shall be discussed. In principle, three

different effects of neuraminidase treatment seem conceivable. Desialyation

might (I) change the conformation of T cell surface proteins resulting in

facilitation or enhancement of TCR-CD8-peptideMHC interaction, (II) unmask

binding sites resulting in a stronger interaction or even TCR clustering by

additional receptors, (III) allow for optimal TCR clustering by changing the

spatial arrangement of receptor molecules on the cell`s surface.

(I) In many instances striking effects of glycans on protein conformation and

therefore function where described, some of which even have major clinical

significance e.g. the transformation of low activity Antithrombin III into a

highly potent anticoagulant upon binding of the polysaccharide heparin

(Johnson and Huntington 2003). N-linked glycans are important in

promoting proper assembly and quality control of synthesised proteins in

the ER. They function as receptor structures for the ER based molecular

49

Discussion

chaperones calnexin and calreticulin which support folding of glycoprotein-

precursors (Parodi 2000). By means of their charged residues, e.g. by

prevention of intramolecular cystin-bridge formation, these fairly large

molecules (≈30 Å) influence on protein folding (Wormald and Dwek 1999).

The addition of terminal sialic acid residues to glycan stalks is part of the

late phase of glycoprotein “trimming” in the Golgi apparatus. Together with

synthesis of O-linked glycans it leads to the formation of mature

glycoproteins ready for export to e.g. the cell surface. Though functionally

relevant in various instances (Hanisch 2001), in contrast to N-glycosylation

these late glycosylation events are generally thought to have less impact on

the structure of the protein. Nevertheless, also O-linked glycosylation is

known to impact on protein conformation as it helps in stabilizing the

extended conformation of stalk like proteins such as mucin core proteins

(Carraway and Hull 1991). A comparable polypeptide structure is found in

the extended membrane proximal stalk region of the CD8 molecule which is

known to carry many sialyated O-glycans. Several studies investigated the

effect of developmentally regulated sialyation of the CD8 molecule on

antigen recognition by T cells (Daniels, Devine et al. 2001; Moody, Chui et

al. 2001; Daniels, Hogquist et al. 2002; Moody, North et al. 2003).

Assuming that desialyation would modify the conformation of the molecule

by changing the orientation of CD8α- and CD8β-chain towards each other,

improved TCR-peptideMHC interaction was attributed to a higher affinity of

non-cognate CD8-MHC interaction. This, however, does not seem to be the

case since a recent study showed that changes in terminal sialyation do not

alter CD8 conformation (Merry, Gilbert et al. 2003). Moreover, one would

expect to measure enhanced non-cognate binding if the highly conserved

CD8 component of the MHC-T cell interaction would be significantly

increased. In contrast to this assumption, the featured binding experiments

did not show increases in non-cognate interactions nor substantially

increased single site affinity (which is partially governed by the CD8

contribution) upon neuraminidase treatment and do not point to an influence

of desialyation on protein conformation.

(II)A second possible result of neuraminidase treatment would be the

generation of additional binding sites on the participating molecules or even

50

Discussion

recruitment of a previously uninvolved third party receptor. Sialic acid

residues indeed play a role in covering protein recognition motifs on glycan

stalks. Probably the most prominent examples are the macrophage

scavenger receptor for recognition of asialoglycans (ASG) on aged red

blood cells and the hepatic ASG receptor that recognizes galactose-

terminal glycans on aged plasmaproteins and mediates their uptake leading

to lysosomal degradation in hepatocytes (Jancik and Schauer 1974;

Schwartz 1984). Since in general protein-polyglycan recognition processes

have a relatively low affinity with KD`s in the range of 0.1-0.5 mM, the

observed increase of TCR-peptideMHC avidity would have to be due to a

“velcro-effect”, resulting from multiple desialyated binding sites interacting

with the dimeric ligand. This, however, does not seem to be a reasonable

explanation, taken the reductionist approach of equilibrium binding of

soluble dimeric peptideMHC to T cells into consideration. Moreover, for this

effect to be relevant, one would again expect a substantial increase in non-

specific binding and single site affinity to be detectable. Nevertheless,

looking at the CFSE assay, contribution of such non-specific binding events

to the observed enhancement of proliferation can not entirely be ruled out.

(III) Given the importance of membrane reorganisation in T cell signaling, a

third hypothesis to be made would be that elimination of terminal sialic acids

from surface glycoproteins changes their spatial orientation towards each

other thus favouring TCR clustering. Assuming the TCR/CD3 complex to

carry the relevant targets of desialyation, extinction of negatively charged

residues on these molecules might increase their lateral mobility in the

membrane and favour the formation of receptor clusters. The four

detectable N-glycosylation sites of 2C TCR have indeed been previously

suspected to sterically hinder receptor clustering (Rudd, Wormald et al.

1999). According to the parameters derived from the model fit, this

mechanism would allow for increases in avidity due to TCR-dimerisation

without grossly affecting the single site affinity.

Another explanation could focus on the membrane glycoproteins

surrounding the TCR. Considering the size of a typical N-glycan, which is

comparable to that of an immunoglobulin domain, a sterical influence on

TCR accesability for its peptideMHC ligand is conceivable. There are several

51

Discussion

proteins on a T cell`s surface that extend over the ∼6 nm small TCR (as

measured from crystal structure of 2C TCR (Garcia, Degano et al. 1996))

and carry apical sialyated N-glycans. On murine CD8, for instance, 4

potential N-glycosylation-sites, predominantly at the head of the molecule

(Asn70,Asn42 and Asn123 on the α-chain and Asn13 on the β-chain) can

be identified (Rudd, Elliott et al. 2001). Moreover, heavily sialyated proteins

like CD43 and CD45 neighbour the TCR complex and tower above it

(Pulido and Sanchez-Madrid 1990; Cyster, Shotton et al. 1991). These fairly

large molecules were found to be excluded from the center of the

immunological synapse, thus facilitating peptideMHC-scanning by TCRs

(Grakoui, Bromley et al. 1999; Leupin, Zaru et al. 2000). Therefore, one

could argue that depletion of sialic acid residues would decrease the

hydrodynamic radii of these molecules resulting in better accesability of

TCR by its peptideMHC-ligand. This is also compatible with the observation

that single site affinities remain fairly unaffected and non-specific binding

does not change, since the specificity of the TCR-petideMHC-interaction is

not altered. Considering the implied technique of soluble ligand equilibrium

binding, which makes the formation of complex synapse structures creating

niches for TCR-peptideMHC-interaction unlikely, this explanation is appealing.

However it does not give an answer to the question why the observed

“neuraminidase effect” is dependent on the presence of CD8. Though CD8

on 2C transgenic T cells is prooven to enhance ligand binding by the TCR

(10-100 fold in the case of Kb-SIY), its presence on the cell surface is not

crucial for TCR-peptideMHC binding. Using Ld-QL9-Ig, the allogenic ligand

was shown to bind almost equally well to CD8+- and DN-cells (Cho, Lian et

al. 2001). Therefore, for the above described mechanisms to be the most

relevant, one would expect to see improved ligand binding upon

desialyation on DN-cells as well. In fact, the opposite is the case,

suggesting a physiological role for desialyated CD8 that goes beyond

simply “making way”. Since desialyation of CD8 upon T cell activation is

known to primarily affect O-glycans of the β-chain (Casabo, Mamalaki et al.

1994; Moody, North et al. 2003), a role for these sugar residues as a

molecular spacer could be reasoned. Loss of sialic acid upon activation/

neuraminidase treatment would decrease the hydrodynamic radius of the

52

Discussion

molecule and eliminate negative charges, thus facilitating cross-linking by

favouring optimal TCR spacing. Hence, lack of this spacer in DN cells could

possibly explain failure of desialyation to enhance ligand binding. Indeed,

further experiments showed that suppression of surface CD8 by small-

interfering RNA reduces cross-linking of 2C TCRs by Ld-Ig-QL9 on activated

cells. As mentioned above, recognition of this allogenic ligand by the 2C

TCR is fairly independent of the CD8 binding component. Nevertheless,

finding CD8 important for the formation of TCR cross-links, lends further

support to the hypothesis that there is a role for the coreceptor molecule

that adds onto its interaction with the MHC molecule. Furthermore, the

proposed effect on lateral mobility of CD8 could also explain the slight

increases in single site affinity of the TCR-CD8-peptideMHC interaction as it

might allow for easier “swinging” of CD8 to its binding site at the MHC

molecule.

Considering the importance of tightly controlling T cell responsiveness, this

hypothetical mechanism could explain how CD8 on activated CTL can

enhance TCR-peptideMHC avidity without a need for increases in single site

affinity of the receptor-ligand interaction. Consistent with previous findings

(Fahmy, Bieler et al. 2001) the T cell could use this form of membrane

reorganisation to enhance its responsiveness to low doses of antigen

without compromising the specificity of the interaction with its peptideMHC

ligand.

53

Objectives and summary

5 Objectives and summary

The observation of increased TCR cross-linking in activated T cells agrees

with proposed models of receptor clustering as a central event in T cell

activation and provides a sensible mechanism of increasing the cell`s

sensitivity to low dose antigen (Fahmy, Bieler et al. 2001).

The featured experiments focus on changes in T cell membrane

glycosylation as a possible means of controlling TCR cross-linking. Taking

the long known fact that activated T cells show decreased levels of surface

sialic acid as a starting point, differences in ligand binding and cellular

reaction upon in vitro stimulation were investigated in naïve, activated and

enzymatically desialyated CD8+, 2C TCR transgenic mouse lymphocytes.

To detect differences in ligand binding lymphocytes were incubated with

various concentrations of fluorescently labeled, soluble MHC/Ig fusion

proteins until equilibrium was reached. Without previous washing, cells

were analyzed by flow cytometry, determined MCF values were normalized

to the plateau and fit to a mathematical model of equilibrium binding of

divalent ligands to monomorphic receptors (Perelson 1984). Parameters

derived from the model fit of binding data show, that neuraminidase

treatment of T cells was sufficient to mimic a partially activated phenotype,

showing enhanced TCR cross-linking. Enhanced TCR cross-linking was

found to be dependent on the presence of CD8, as neuraminidase

treatment of DN cells lead to decreased cross-linking. To elucidate the

physiological relevance of desialyation induced increases in TCR cross-

linking early tyrosine phosphorylation events and proliferative response

upon in vitro stimulation of T cells were investigated. Both were found

enhanced in neuraminidase treated cells, as compared to native cells.

In conclusion the featured experiments suggest a role of surface sialic acid

in controlling TCR cross-linking on naïve and activated T cells.

54

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62

Acknowledgements

First and foremost, I would like to thank my parents for their love and support.

Eventually, I owe it all to you.

Moreover, I say thank you to Jon Schneck and everybody at Schneck lab helping

me with my work.

Especially, Tarek and Georg, Dominic for creative input and worthwhile “off-lab”

conversation, Mathias, Puetz, Ben and Edda for being friends, Shiwen and Mily

for good neighborhood, Joany for being a caring “lab-mom” and Harry for

banking tips and insurance advice.

“Vielen Dank, mein Herr” to Cory for being an inspiring and memorable

roommate.

Last and certainly not least, I say thank you and hi to Tina and the good times for

us to come…

Curriculum vitae Lars Eichler geboren am 10.06.1978 in Münster/ Nordrheinwestfalen 1984 – 1988 Grundschule Dröper, Georgs-Marien-Hütte 1988 – 1990 Orientierungsstufe Schulzentrum Vechta Süd, Vechta 1990 – 1997 Gymnasium Antonianum, Vechta Juni 1997 Abitur

10.1997 – 10.1998 Zivildienst, Malteser Rettungswache, Vechta 10.1998 – 09.2000 Vorklinischer Abschnitt,

Christian-Albrechts-Universität, Kiel September 2000 Ärztliche Vorprüfung 10.2000 – 09.2001 1. klinischer Abschnitt,

Christian-Albrechts-Universität, Kiel August 2001 1. Staatsexamen September 2001 USMLE Step I CK 10.2001–09.2004 2. klinischer Abschnitt,

Julius-Maximilians-Universität, Würzburg 05.2002 – 04.2003 Research Traineeship, Department of Pathology,

Immunology Division Johns Hopkins University, Baltimore, USA

September 2004 2. Staatsexamen

10.2004 – 02.2005 Praktisches Jahr, ZOM, Universitätsklinik Würzburg Januar 2005 USMLE Step II CK 02.2005 – 05.2005 Unterassistent,

Neurologische Klinik Inselspital Bern, Schweiz 06.2005 – 09.2005 Praktisches Jahr,

Medizinische Universitätsklinik Würzburg September 2005 USMLE Step II CS

November 2005 3. Staatsexamen seit 12.2005 Wissenschaftlicher Mitarbeiter des

Zentrums für Anästhesiologie und Intensivmedizin Universitätsklinikum Hamburg Eppendorf

Hamburg, 11.08.2006