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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
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
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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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
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