From the Ludwig Institute for Cancer Research,
Lausanne Branch, University of Lausanne and ¶ Institute for
Biochemistry, University of Lausanne, 1066 Epalinges, Switzerland
Received for publication, August 29, 2002, and in revised form, October 28, 2002
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ABSTRACT |
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Fluorescence-labeled soluble major
histocompatibility complex class I-peptide "tetramers" constitute a
powerful tool to detect and isolate antigen-specific
CD8+ T cells by flow cytometry. Conventional
"tetramers" are prepared by refolding of heavy and light chains
with a specific peptide, enzymatic biotinylation at an added C-terminal
biotinylation sequence, and "tetramerization" by reaction with
phycoerythrin- or allophycocyanin-labeled avidin derivatives. We show
here that such preparations are heterogeneous and describe a new
procedure that allows the preparation of homogeneous tetra- or
octameric major histocompatibility complex-peptide complexes. These
compounds were tested on T1 cytotoxic T lymphocytes (CTLs), which
recognize the Plasmodium berghei circumsporzoite peptide 252-260 (SYIPSAEKI) containing photoreactive 4-azidobenzoic acid on
Lys259 in the context of H-2Kd. We report that
mutation of the CD8 binding site of Kd greatly impairs the
binding of tetrameric but not octameric or multimeric
Kd-PbCS(ABA) complexes to CTLs. This mutation
abolishes the ability of the octamer to elicit significant
phosphorylation of CD3, intracellular calcium mobilization, and CTL
degranulation. Remarkably, however, this octamer efficiently activates
CTLs for Fas (CD95)-dependent apoptosis.
CD8+ T cells and thymocytes recognize with their
T-cell antigen receptor
(TCR)1 cognate MHC-peptide
complexes on the surface of antigen-presenting cells (1). CD8, by
binding to the constant domain of MHC class I molecules, can increase
the avidity of TCR-ligand binding but can also act as an adhesion
molecule and strengthen CTL-target cell conjugate formation (1-3). The
coordinate binding of CD8 to TCR-associated MHC molecules brings
CD8-associated Lck to TCR/CD3, which promotes tyrosine phosphorylation
of its immunoreceptor tyrosine-based activation motifs, which is an
initial crucial event of TCR-mediated T-cell activation (1-4). The CD8
binding site on MHC class I molecules contains an acidic loop (residues 222-229), and charge inversion in position 227 (e.g.
KdD227K) impairs CD8 binding by about 85% (3, 4).
The recognition of sensitized target cells by CD8+ CTLs
involves rapid and avid conjugate formation, followed by CTL
degranulation in the contact zone, resulting in
perforin/granzyme-mediated target cell killing (5). With a slower
kinetic, CTLs also express surface Fas ligand, which, by interacting
with Fas on other cells, induces Fas-mediated cytotoxicity (5, 6).
Although perforin-mediated cytotoxicity and Fas-mediated cytotoxicity
are both induced by TCR triggering, the activation requirements are
very different. For example, certain altered peptide ligands that are
unable to elicit perforin-dependent cytotoxicity or
cytokine production can efficiently induce Fas-mediated killing (7, 8).
We observed previously that blocking of CD8 greatly impairs calcium
mobilization, degranulation of CTL, and IFN- The idea of eradicating antigen-specific CD8+ T cells or
thymocytes via Fas-dependent apoptosis is attractive
because it takes place in the absence of full, potentially harmful
T-cell activation. Because in vivo application of blocking
CD8 by antibodies or the use of MHC-peptide-coated microspheres
is risky, we investigated here whether soluble MHC-peptide complexes
with impaired CD8 binding can be used to the same end.
Soluble fluorescence-labeled MHC class I-peptide multimers, so-called
"tetramers," are widely used for the detection and isolation of
antigen-specific CD8+ T cells (10, 11). The conventional
way to prepare such molecules involves enzymatic biotinylation of
an added C-terminal biotinylation sequence (BSP) with the biotin ligase
BirA (12). The biotinylated MHC-peptide monomers are then reacted with
phycoerythrin (PE)- or allophyco-cyanin-labeled avidin derivatives.
Although PE and allophyco-cyanin have very high fluorescence
intensities, their conjugates with avidin are heterogeneous. Because of
their large size, their conjugation with streptavidin yields
ill-defined mixtures of conjugates with different stoichiometries and
configurations (the molecular mass of PE is about 240,000 Da, the
molecular mass of allophyco-cyanin is about 104,000 Da, and the
molecular mass of streptavidin is about 60,000 Da). In
consequence, saturation of such conjugates with biotinylated
MHC-peptide complexes results in heterogeneous MHC- peptide
complexes, referred to as multimers.
To prepare well-defined soluble MHC class I-peptide complexes of
different sizes, we introduced by point mutation a free cysteine at the
C terminus of the MHC heavy chain, which can be alkylated with biotin
containing iodoacetamide or maleimide derivatives. For oligomerization,
we used homogeneous streptavidin conjugates containing the low
molecular weight fluorochrome Cychrome Cy5. By using a branched peptide
containing one biotin and two maleimide moieties (DMGS), this strategy
allows the preparation of octameric MHC class I-peptide complexes. Here
we describe the preparation of well-defined soluble tetrameric and
octameric MHC-peptide complexes that co-engage or do not co-engage CD8
and their ability to activate perforin- and Fas-dependent
cytotoxicity on cloned T1 CTLs. T1 CTLs recognize the PbCS peptide
252-260 (SYIPSAEKI) containing photoreactive 4-azidobenzoic acid on
Lys259 (PbCS(ABA)) in the context of Kd (4,
13). We find that the binding of tetrameric but not octameric or
multimeric Kd-PbCS(ABA) complexes to T1 CTLs is greatly
reduced when CD8 co-engagement is ablated by the charge inversion
Kd-D227K. Although Kd-D227K-PbCS(ABA) complexes
fail to elicit significant tyrosine phosphorylation, calcium
mobilization, and degranulation, they efficiently induced
Fas-dependent cytotoxicity.
Chemicals--
The branched DMGS-biotin peptide was synthesized
using conventional solid phase Fmoc strategy. Biotin was introduced by
using Fmoc-K( Cells and Antibodies--
Cloned T1 CTLs were cultured and used
as described previously (13, 14). In brief, the CTLs were restimulated
weekly using Production of Refolding, Purification, and Alkylation of Kd-Peptide
Monomers--
Kd-BSP heavy chain and human
The enzymatic biotinylation of Kd-peptide-BSP was performed
overnight at 25 °C with ATP, biotin, and the biotin ligase BirA as
described previously (12). For alkylation, the Kd-peptide
complexes (1 mg/ml) were incubated in Tris buffer (20 mM
Tris, pH 8.0, 150 mM NaCl) for 1 h at 4 °C with 15 mM glutathione to reduce the C-terminal free cysteine.
After GFC over a Superdex S75 column (1 × 30 cm;
Pharmacia), the reduced monomers were reacted in Tris buffer
containing 5 mM EDTA with a 5-fold molar excess of
alkylation reagent under argon overnight at 4 °C. The alkylated Kd-peptide complexes were purified by gel filtration on a
Superdex S75 column. Dimeric Kd-DMGS-biotin complexes were
obtained by reacting monomeric Kd-DMGS-biotin with a 2-fold
excess of Kd-cysteine monomers. Dimeric
Kd-peptide complexes were separated from monomers by anion
exchange chromatography on a Resource Q fast flow anion exchange column (Amersham Biosciences). The biotinylation efficiency was determined by
SDS-PAGE (15%, nondenaturing) of 5-µg aliquots of
Kd-peptide complexes that were reacted or not reacted with
a 2-fold molar excess of avidin (Molecular Probes). The Coomassie
Blue-stained gels were evaluated by densitometry, and the alkylation
efficiency, calculated as a percentage, was as follows: (amount
of avidin-bound Kd-peptide-avidin)/(amount of
Kd-peptide) × 100.
Preparation of Fluorescence-labeled Kd-Peptide
Oligomers--
Kd-peptide tetramers and octamers were
obtained by reacting the biotinylated Kd-PbCS(ABA)
complexes with Cy5-labeled streptavidin (Amersham Biosciences), and the
multimers were obtained by reacting biotinylated
Kd-PbCS(ABA) monomers with Extravidin-PE (Sigma) at a molar
ratio of 4:1. Labeled oligomers were purified by GFC on a Superdex 200 column (1 × 30 cm; Pharmacia), which was eluted in Tris buffer (20 mM, pH 8.0, 150 mM NaCl) at a flow rate of
0.7 ml/min.
Kd-Peptide Oligomer Binding Assays--
For binding
studies, T1 CTLs were washed with Optimem (Invitrogen) containing 1%
bovine serum albumin, 0.02% sodium azide, 15 mM HEPES, and
2 µg/ml human Bystander Cytolytic Assay--
T1 CTLs (5 × 105 cells/ml) were incubated in Dulbecco's modified
Eagle's medium (Invitrogen) supplemented with 2 µg/ml human Esterase Release--
T1 CTLs were adhered in 96-well plates
previously coated with super fibronectin (Sigma) and incubated in
Optimem medium (Invitrogen) supplemented with 2 µg/ml human Immunoprecipitation and Western Blotting--
T1 CTLs (1 × 107 cells/ml) were incubated or not incubated with
Kd-PbCS(ABA) complexes (25 nM) for 3 min at
37 °C. After washing with chilled phosphate-buffered saline, the
cells were lysed on ice for 1 h in phosphate-buffered saline
containing Brij78 (1%) and protease inhibitor mixture (Roche Molecular
Biochemicals), and from the detergent-soluble fraction, TCR/CD3
was immunoprecipitated with anti-TCR mAb H57. The immunoprecipitates
were resolved on SDS-PAGE (15%, reducing) and Western blotted using
anti-phosphotyrosine mAb 4G10 or anti-CD3 Confocal Microscopy--
T1 CTLs were incubated with Cy5-labeled
Kd-PbCS(ABA), KdD227K-PbCS(ABA), or
Kd-Cw3 170-179 octamer for 30 min at 37 °C or 18 °C.
After washing, cells were fixed with 3% paraformaldehyde for 10 min at
room temperature and laid onto poly-L-lysine-coated slides
for 10 min and mounted. Internalization of Cy5-conjugated octamer was
analyzed by confocal microscopy on an Axiovert 100 microscope (LSM510;
Carl Zeiss, Jena, Germany) with a ×63 oil objective. Cy5 fluorescence
was measured upon excitation with neon/helium laser at 633 nm. Each image was the average of four scans. Digital images were prepared using
Adobe Photoshop.
Apoptosis Assay--
T1 CTLs (0.5 × 106
cells/ml) were resuspended in Dulbecco's modified Eagle's medium
supplemented with 5% fetal calf serum and 20 mM HEPES and
incubated in 50-µl aliquots at 37 °C for 30 min with 25 nM Kd-PbCS(ABA), KdD227K-PbCS(ABA),
or Kd-Cw3 170-179 octamers or left untreated. After one
wash, the cells were incubated for 4.5 h in the same medium,
stained with Cy5-labeled annexin V, and analyzed by FACS.
Intracellular Calcium Mobilization--
T1 CTLs (1 × 106 cells/ml) were incubated with 5 µM Indo-1
(Sigma) at 37 °C for 45 min, washed, and incubated at 37 °C with 25 nM Kd-peptide octamers or medium, and
calcium-dependent Indo-1 fluorescence was measured by FACS
on a FACStarTM as described previously (14).
Preparation of Fluorescence-labeled Soluble Kd-Peptide
Complexes--
We prepared and examined three different types of
soluble Kd-peptide complexes: multimers, tetramers, and
octamers. All complexes were produced with either wild type
Kd or KdD227K, which has greatly impaired CD8
binding (4). Monomeric Kd-PbCS(ABA) complexes were obtained
by refolding of different Kd heavy chains and human
Alternatively, biotinylation was accomplished by site-specific
alkylation of a free cysteine introduced by point mutation at the
C-terminal portion of the Kd heavy chain. To find out what
position is most suitable, a free cysteine was introduced in position
273, 275, or 277 (Fig. 1B). The former two flank the
conserved Trp274, which is the last residue of the folded
The alkylation efficiency of the different Kd-PbCS(ABA)
cysteine mutants was assessed after incubation at 4 °C overnight
with a 5-fold molar excess of iodoacetyl-PEO-biotin (Fig.
1C). The alkylation efficiency was 16% for
KdR273C, 24% for KdK275C, and 12% for
KdA277C (Fig. 1S, B). Based on these results,
KdK275C was selected. Its alkylation efficiency was
increased to about 80% upon reduction of KdK275C-PbCS(ABA)
complexes with 15 mM glutathione before the alkylation (Fig
1S, C). The alkylation was selective for the free cysteine because Kd-PbCS(ABA) wild type complexes were not
significantly alkylated (Fig. 1S, B and C). A
slightly higher alkylation efficiency (about 85%) was obtained for the
mono-alkylation of KdK275C-PbCS(ABA) complexes with
bi-malemide-biotin reagent DMGS-biotin (Fig. 1D). The
purified mono-alkylated Kd-DMGS-biotin complexes
were then reacted with a 2-fold molar excess of reduced
KdK275C-PbCS(ABA). The efficiency for this reaction was
60-70%. Together, these results show that KdK275C can be
refolded and biotinylated by alkylation with the same efficiencies as
by the BSP/BirA strategy. The same findings were obtained for HLA-A2
(data not shown).
Defined Kd-peptide tetramers and octamers were obtained by
reacting Kd-peptide-PEO-biotin and dimeric
Kd-peptide-DMGS-biotin complexes with homogeneous
Cy5-labeled streptavidin. For multimeric Kd-peptide
complexes, Kd-BSP-biotin-peptide monomers were reacted with
heterogeneous PE-extravidin. The different compounds were analyzed by
gel filtration on a Superdex S200 column and anion exchange
chromatography on a Source 15Q column, respectively. As shown in Fig.
2, the Kd-PbCS(ABA) monomers,
dimers, tetramers, and octamers were homogenous, except for some minor
contaminants, and eluted in both types of chromatography as expected.
By contrast, the PE-labeled Kd-PbCS(ABA) multimers eluted
in a heterogeneous manner from the anion exchange column. The majority
of the PE-labeled Kd-PbCS(ABA) multimers eluted from the
Superdex S200 column at around 10 min, i.e. in the void
volume. Because the size exclusion of this column is about
Mr 600,000, and PE-extravidin
Kd-peptide tetramers have a Mr of
about 470,000, this preparation contained mainly conjugates that were
larger than tetramers containing one PE. In agreement with this is the
late elution of these complexes from the anion exchange column. The
same results were obtained for the Kd-peptide complexes
containing the D227K mutation (data not shown).
Binding of Soluble Kd-Peptide Complexes to T1
CTLs--
To study the binding of Cy5-labeled Kd-peptide
complexes, we first measured the binding kinetics of the
Kd-PbCS(ABA) octamers and multimers. As shown in Fig.
3, A-D, the binding of both
complexes was rapid at 37 °C, 18 °C, and 4 °C, with
We next assessed the binding isotherms at 37 °C and 18 °C for the
compounds under study (Fig. 3, E-H). At 37 °C, the
binding of Cy5-labeled Kd-PbCS(ABA) octamers increased
steeply in the concentration range of up to 30 nM and then
gradually up to 100 nM, the highest concentration tested
(Fig. 3E). By contrast, for the tetramer, much lower levels of binding were observed, and the increase in binding required higher
concentrations. For the corresponding Kd-D227K-PbCS(ABA)
complexes, the binding of the octamer was reduced by about 10%, but
binding of the tetramer was close to the background binding observed
for the noncognate Kd-Cw3 171-179 peptide complexes. A
similar binding pattern was observed at 18 °C, except that nearly
maximal binding was reached already at 12.5 nM for the
octamer and at 30 nM for the tetramer (Fig. 3G).
This difference seems mainly accounted for by endocytosis, which is
taking place at 37 °C, but not at 18 °C (see below).
The binding of PE-labeled multimers increased continuously over the
range of concentrations tested at 37 °C (Fig. 3F) and at
18 °C (Fig. 3H). Strikingly, at both temperatures,
Kd wild type and Kd-D227K complexes exhibited
about the same binding patterns. Because these multimer complexes
contain higher order complexes (see above), these results argue that
the CD8 dependence of the binding of MHC-peptide complexes decreases as
their valence increases. Essentially the same findings were obtained on
the PbCS(ABA)-specific S14 CTL clone.
Kd-PbCS(ABA) and KdD227K-PbCS(ABA) Octamers
Induce TCR and CD8 Down-modulation and Internalization--
To
assess whether octamers induce down-modulation of TCR and CD8, T1 CTLs
were incubated with saturating concentrations of Kd-PbCS(ABA) and KdD227K-PbCS(ABA) octamers for
different periods of time, and TCR and CD8 expression was measured by
FACS. At 4 °C, the surface expression of TCR and CD8 remained
essentially unchanged (Fig. 4,
A and B). At 18 °C, a scant reduction of TCR
and CD8 expression was observed after 30 and 60 min of incubation (Fig.
4, C and D). At 37 °C, there was a
time-dependent down-modulation of TCR and CD8 of about 40%
after 1 h of incubation with Kd-PbCS(ABA) octamer
(Fig. 4E). The KdD227K octamer induced the same
TCR down-modulation, but the down-modulation of CD8 was less than half
(18% after 1 h; Fig. 4F).
To visualize endocytosis of Cy5-labeled Kd-PbCS(ABA) and
KdD227K-PbCS(ABA) octamers, they were incubated with T1
CTLs for 30 min at 18 °C or 37 °C and analyzed by confocal
microscopy. As shown in Fig. 4G, at 18 °C,
Kd-PbCS(ABA) complexes were distributed on the cell surface
in a wide cap. This was also true for the
Kd-D227K-PbCS(ABA) octamer, but the cap formation was less
pronounced. By contrast, at 37 °C, the majority of
Kd-PbCS(ABA) complexes were internalized in the form of a
bright patch. For KdD227K-PbCS(ABA), about half of the
complexes were localized in a wide cap on the surface, and half were
internalized in patch, distal to the cap. In the presence of Fab'
fragments of the anti-Kd Kd-PbCS(ABA) Octamers Elicit Fas-mediated
Cytotoxicity--
To assess the ability of Kd-PbCS(ABA)
octamers to elicit Fas-dependent cytotoxicity, T1 CTLs were
pulsed with different Kd-peptide complexes, washed, and
then incubated for 4 h with 51Cr-labeled P815 cells
transfected with Fas. As shown in Fig.
5A, tetrameric, octameric, and
multimeric Kd-PbCS(ABA) and Kd-D227K-PbCS(ABA)
complexes induced bystander cell killing. The most efficient killing
was observed for Kd-D227K-PbCS(ABA) octamer (65%). The
Kd-PbCS(ABA) octamer induced slightly less efficient
killing, but for both tetramers and multimers, target cell killing was
about 20% lower. T1 CTLs pulsed or not pulsed with the corresponding Kd-Cw3 170-179 complexes exhibited only faint background
lysis. By contrast, very strong lysis was observed in the presence of anti-Fas antibody, which induces apoptosis by cross-linking of Fas on
the target cells.
A concern of the present experiments was that during the assay,
Kd-PbCS(ABA) complexes decay, and liberated PbCS(ABA)
peptide binds to cell-associated Kd and induces target cell
killing. To consolidate this, we assessed the stability of
Kd-PbCS(ABA) monomers at 37 °C. As shown in Fig. 2S,
~50% of the Kd-PbCS(ABA) complexes were decayed after
2.5 h of incubation, and after 3.5 h, nearly 80% were
decayed. However, in the presence of
The same experiment performed on normal P815 cells showed no
significant lysis, except in the incubation where a high concentration of free PbCS(ABA) peptide was used (Fig. 5B). This is
consistent with the finding that normal P815 cells, which express low
amounts of Fas, in 4-h cytolytic assays are sensitive to
perforin/granzyme-mediated but not Fas-dependent killing
(8). Taken together, these results indicate that CTLs pulsed with
soluble KdD227K-PbCS(ABA) octamer induce strong
Fas-dependent but no
perforin/granzyme-dependent bystander cell killing.
Kd-PbCS(ABA) but not KdD227K-PbCS(ABA)
Complexes Induce Degranulation of Adherent CTLs--
We next examined
the ability of the different soluble Kd-peptide complexes
to elicit degranulation of T1 CTLs, which reflects perforin/granzyme-dependent lysis. Because degranulation
requires adhesion and polarization of CTLs (18-20), we adhered T1 CTLs
to immobilized fibronectin and incubated them with the soluble
Kd- peptide complexes. As shown in Fig.
6, all Kd-PbCS(ABA) complexes
elicited CTL degranulation. The strongest degranulation was observed
for PE-labeled Kd-PbCS(ABA) multimers (75%) and
Kd-PbCS(ABA) octamer (68%); Kd-PbCS(ABA)
tetramers induced about 48% esterase release. In the presence of
concanamycin A, which blocks CTL degranulation (21), the strong
degranulation induced by Kd-PbCS(ABA) tetramers was reduced
to background levels, as observed for the Kd-Cw3 170-179
complexes.
Remarkably, the corresponding KdD227K-PbCS(ABA) complexes
elicited no or scant esterase release, which was ablated upon blocking of the residual CD8 co-engagement by anti-CD8 mAb H35 (Fig. 6; Ref. 4).
Together, these results indicate that soluble Kd-PbCS(ABA)
complexes, namely, octamer and multimers, efficiently elicit CTL
degranulation by soluble MHC-peptide complexes under the condition that
they co-engage CD8. The same observations were made on S14 CTL clones
(data not shown).
Kd-PbCS(ABA) but not KdD227K-PbCS(ABA)
Octamers Induce Calcium Mobilization and Strong Tyrosine
Phosphorylation--
A hallmark of TCR/CD8-mediated T-cell activation
is a rapid increase in intracellular calcium and tyrosine
phosphorylation of CD3. To assess the ability of
Kd-PbCS(ABA) octamers to elicit intracellular calcium
mobilization, Indo-1-labeled T1 CTLs were incubated with
Kd-PbCS(ABA) and KdD227K-PbCS(ABA) complexes,
and calcium flux was measured by FACS. As shown in Fig.
7A, wild type octamer induced
strong calcium mobilization that was maximal about 2 min after the
addition of octamer. By contrast, KdD227K-PbCS(ABA) octamer
elicited a calcium flux close to background levels, i.e.
unpulsed CTLs. The same findings were obtained on S14 CTLs (data not
shown). This is consistent with our previous finding that soluble
MHC-peptide complexes elicit intracellular calcium mobilization only
when they co-engage CD8 (14, 22).
Moreover, upon brief incubation with Kd-PbCS(ABA) octamers,
T1 CTLs exhibited strong tyrosine phosphorylation of the CD3 Kd-PbCS(ABA) Octamers Induce Apoptosis of T1
CTLs--
Based on the observation that Kd-PbCS(ABA)
octamers induce Fas-dependent cytotoxicity of bystander
cells (Fig. 5), we investigated whether they also induce apoptosis of
the CTLs. As shown in Fig. 8, T1 CTLs
exhibited a marked increase in annexin V, which is a marker for
apoptotic cells (9), upon incubation with Kd-PbCS(ABA) and
a slightly smaller increase in annexin V upon incubation with
KdD227K-PbCS(ABA) octamers. For CTLs incubated with
Kd-Cw3 170-179 octamer, the annexin V expression was at
the background level.
The present study shows that conventional "tetramers" are
ill-defined mixtures of MHC-peptide conjugates (Fig. 2) and that this
precludes precise binding studies (Fig. 3). The cause for this
heterogeneity is the high molecular weight of PE (or
allophyco-cyanine), which renders defined conjugation with the smaller
avidin or avidin derivatives difficult, if not impossible. We find that
defined MHC-peptide complexes can be obtained by using Cy5-labeled
streptavidin. Although the fluorescence intensity of Cy5-labeled
streptavidin is 4-5-fold lower as compared with PE-streptavidin, it
is, unlike PE, remarkably resistant to photobleaching, which allows
analysis other than FACS. Other low molecular weight fluorochromes can be used instead of Cy5, such as Cy3 or various Alexa dyes.
Moreover, the conventional strategy to derivatize monomeric MHC-peptide
complexes by enzymatic biotinylation of an added BSP sequence permits
only the preparation of avidin-based MHC-peptide "tetramers." To
produce different soluble MHC-peptide complexes, we investigated the
derivatization of MHC-peptide monomers by site-specific alkylation. It
has been reported that the heavy (25) or light chain (26) of MHC class
I molecules can be biotinylated by alkylation of a free cysteine with
maleimide containing biotin derivatives. Because on living cells under
physiological conditions The biotinylation of MHC peptide complexes has several important
advantages compared with the conventional enzymatic biotinylation. 1)
The biotinylation can be performed in the cold, which is advantageous in particular in case of thermo-labile MHC-peptide complexes. 2) It is
significantly lower in terms of cost because alkylation reagents are
much cheaper than BirA. 3) The thioether bond formed by alkylation of a
free cysteine is very stable and resists proteolytic and chemical
degradation. 4) The alkylation method is remarkably versatile. In
addition to biotinylation of MHC class I-peptide complexes (Figs. 1 and
2) (25, 26), site-specific alkylation allows the preparation of
MHC-peptide complexes of diverse valence and configuration by using
branched, maleimide containing linkers for alkylation. Also,
fluorescence-labeled MHC-peptide complexes can be prepared by
alkylation with fluorescence-labeled maleimides or maleimide containing linkers.
Our MHC-peptide binding studies allow three conclusions. First, the
increase of Kd-PbCS(ABA) binding to T1 CTLs is dependent on
the valence of the complexes. CD8 increased the binding of monomeric
complexes about 10-fold at 37 °C (4, 14), about 5-fold for
tetrameric complexes, and <2-fold and hardly at all for multimeric
complexes (Fig. 4). With regard to the CD8 dependence of multimer
binding to CD8+ T cells, there exists a controversy in the
literature. Whereas according to some studies multimer binding is
markedly CD8-dependent (28, 29), it is not so according to
others (23, 30). This discrepancy may be explained in part by
differences in the MHC-peptide multimer composition used in the
different studies. However, we observed that under the same conditions
as described here, the multimer binding to HLA-Cw3-specific CTLs, which
express low affinity TCR, is substantially strengthened by
CD8.2 Similarly, Daniels and Jameson (28) found that the
CD8 dependence of multimer binding depends on the affinity of the TCR
of the cells under study. It thus appears that the binding of
MHC-peptide complexes to CD8+ T cells is essentially
determined by the overall binding avidity. Thus, the higher the
affinity of the TCR and the higher the valence of the complexes, the
less important the contribution of CD8 is to the binding. In our hands,
the binding of MHC-peptide octamers, but not of smaller complexes, is
not CD8-dependent, except on CTLs that express
exceptionally low affinity TCR.
Second, the kinetics of Kd-PbCS(ABA) octamer binding to T1
CTLs is remarkably rapid, taking place within few minutes at all temperatures tested (Fig. 3, A, C, and D). This
was also true for tetrameric complexes and on S14 CTLs.2 By
contrast, the binding of monomeric Kd-PbCS(ABA) complexes
was considerably slower, especially in the cold (4). This argues that
the kinetics of MHC-peptide complex binding increases with their valence.
Third, the heterogeneity of MHC-peptide multimers (Fig. 2) precludes
precise binding studies. For example, the binding of Kd-PbCS(ABA) multimers increased continuously with
concentration and also at 18 °C, where internalization is scant,
whereas octamer binding at 18 °C reached saturation at low
concentrations (Figs. 3 and 4). It thus appears that low valence
complexes in multimer preparations bind significantly only at higher
concentrations, whereas high valence ones bind already at low
concentrations. However, it is also conceivable that MHC-peptide
complexes that have an appropriate configuration can elicit TCR (and
CD8) aggregation. Such aggregation effects may explain why tetrameric
Kd-PbCS(ABA) complexes fail to reach the high levels of
binding observed for octameric complexes (Fig. 4G) (tested
up to 500 nM).
A key finding of the present study is that soluble MHC class I-peptide
complexes that are unable to co-engage CD8 induce strong Fas-mediated
cytotoxicity but no perforin-mediated cytotoxicity (Figs. 5, 6, and 8).
This is in accordance with the previous observations that blocking of
CD8 by antibody blocks perforin- but not Fas-dependent killing of target cells (8) and that target cells or microspheres expressing MHC class I-peptide with ablated CD8 binding induce Fas-dependent apoptosis of human CTLs (9). Common to these strategies is that CD8 co-receptor function is blocked. In the present
study, where soluble MHC-peptide complexes are used, CD8-mediated adhesion and MHC-peptide binding or involvement of other auxiliary molecules are excluded (Fig. 3). It thus appears that engagement and
cross-linking of TCR in the absence of CD8 co-engagement induce Fas-dependent cytotoxicity, including Fas-mediated
apoptosis of the CTLs, in the absence of other cell activation (Figs.
5-8).
What implications has blocking of CD8 co-receptor function on CTL
activation? On one hand, the lack of CD8 co-engagement by MHC-peptide
complexes impairs the avidity of TCR-ligand binding (4, 14, 28, 29). We
show here that for soluble MHC-peptide complexes, this can be
compensated for by increasing their valence (Fig. 3). On the other
hand, the lack of CD8 co-engagement impairs Lck-mediated
phosphorylation of CD3. This is so because normally the coordinate
binding of MHC-peptide to CD8 and TCR brings CD8-associated Lck to CD3,
which upon cross-linking-mediated activation of Lck results in their
phosphorylation (14, 22-24, 31). Once phosphorylated by Lck, CD3 and
The lack of CD8 co-engagement hence results in impaired Lck activation
and by consequence reduced tyrosine phosphorylation of CD3 and
recruitment and activation of Zap-70 (Fig. 7B) (8, 9, 23,
32). As a result of this, two downstream signaling pathways are
compromised. The first is the recruitment and activation of
phospholipase C The second signaling pathway that is compromised involves the
recruitment of phosphatidylinositol 3-kinase to phosphorylated CD3 and
The physiological significance of this is not clear. Because CTLs are
prone to apoptosis once they express Fas ligand (Fig. 8) (9), it is
conceivable that this way, CD8+ T cells with defective CD8
co-receptor function are eliminated. Indeed, it has been shown that
misselected CD8+ T cells, which express TCRs that are not
MHC class I-restricted, are eliminated this way (41). The observation
that large soluble MHC-peptide complexes with ablated CD8 binding
permit eradication of antigen-specific CTLs (Fig. 8) gives them a
therapeutic potential. They are more attractive to this end than the
previously described use of anti-CD8 antibodies (8) or
MHC-peptide-coated microspheres (9) because such molecules can be
produced in a well-defined form in adequate quantities and purity.
Also, because soluble MHC-peptide complexes with ablated CD8 binding
are unable to elicit any cell activation other than
Fas-dependent cytotoxicity, they harbor a minimal risk to
induce unwanted, potentially harmful immunological reactions (Figs. 5,
6, and 8) (23).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
release but has no
effect on Fas-dependent cytotoxicity (8). More recently, it
has been shown that antigen-presenting cells or microspheres expressing
MHC-peptide complexes with ablated CD8 binding selectively induce F
as-mediated apoptosis of CTLs (9).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Ahx-biotin) (Bachem AG, Bubendorf, Switzerland). The
deprotected GS-biotin peptide was purified by GFC on a Superdex
peptide column (1 × 30 cm; Amersham Biosciences) and reacted with
N-
-maleimidobutyryloxysuccinimide ester (GMBS; Pierce) in
DMSO containing 1% di-isopropyl-ethylamine. The resulting di-maleimide
conjugate was purified by high pressure liquid chromatography on a C18
reverse phase column (2 × 30 m; Machery & Nagel, Oensingen,
Switzerland). The column was eluted with 0.1 trifluoroacetic
acid and a linear gradient of acetonitril rising in 1 h
from 0 to 75%. The DMGS-biotin peptide eluted at 26.4 min, and the
mono-maleimide derivative eluted at 25.3 min. All products had the
correct molecular weight as assessed by mass spectrometry.
Iodoacetyl-PEO-biotin was from Pierce, and SYIPSAEK(ABA)I (PbCS(ABA))
was prepared by the conventional solid phase Fmoc strategy using
Fmoc-Lys(ABA) (Bachem) following previously published procedures (13,
14).
-irradiated PbCS(ABA) pulsed P815 cells and Balb/c
feeder cells and Dulbecco's modified Eagle's medium supplemented with
5% fetal calf serum, 5 nM 2-mercaptoethanol, and 30 units/ml recombinant interleukin 2. Normal and Fas-transfected
mastocytoma P815 cells were cultured and used as described previously
(8). The following antibodies were used: Cy5-labeled annexin V and
anti-Fas antibody were from BD Biosciences, anti-CD8
mAb H35 and
anti-TCRC
mAb H57 were from American Type Culture Collection
(Manassas, VA), anti-phospho-tyrosine mAb 4G10 was from Upstate
Biologicals (Lake Placid, NY), and anti-CD3
antibody M-20 was from
Santa Cruz Biotechnology (Santa Cruz, CA). All stainings for FACS were
performed at 4 °C for 30-60 min.
2m and Kd Heavy Chains Containing a
Free Cysteine--
The cDNA encoding the Kd heavy
chain was cloned into the pET3a vector by PCR amplification,
using primers 5'-GCCATATGGGCCCACATTCGCTGAG-3' (forward
primer) and 5'-GCGGATCCTCAAGCCAGCTTCCATCTCA-3' (reverse primer). The restriction sites NdeI and BamHI
used for cloning are underlined. A free cysteine was
introduced by point mutation in positions 273, 275, and 277 (R273C, K275C, and A277C) of the Kd heavy chain, using the
QuikChange mutagenesis kit (Stratagene). For PCR amplification,
the following primers were used:
5'-GAGCCTCTCACCCTGTGCTGGAAGCTGGCTTGA-3' (forward primer) and
5'-TCAAGCCAGCTTCCAGCACAGGGTGAGAGGCTC-3' (reverse primer),
5'-CCTCTCACCCTGAGATGGTGCCTGGCTTGAGGATCCGGC-3' (forward primer) and 5'-GCCGGATCCTCAAGCCAGGCACCATCTCAGGGTGAGAGG-3'
(reverse primer), 5'-CCCTGAGATGGAAGCTGTGTTGAGGATCCGGCTGC-3'
(forward primer) and
5'-GCAGCCGGATCCTCAACACAGCTTCCATCTCAGGG-3' (reverse
primer). The codons in italics indicate the introduced cysteine. After digestion of the parental DNA template with DpnI, the
mutated strands were transformed in BL21 DE3 pLysS bacteria. For
expression of recombinant Kd heavy chain and human
2m,
BL21 DE3 pLysS bacteria were grown at 37 °C in Luria-Bertani medium
supplemented with 100 µg/ml ampicillin. The expression of the
recombinant proteins was induced by addition of
isopropyl-
-D-thiogalactopyranoside (1 mM,
final concentration). The recombinant proteins were produced as
insoluble inclusion bodies and extracted as described previously (15).
As judged by SDS-PAGE and Coomassie Blue staining, the purity of both
proteins was 80-90%. Aliquots of these solutions were stored at
80 °C.
2m were
refolded in the presence of the peptide using the dilution method
essentially as described previously (15). In brief, the Kd
heavy chain- and
2m-containing urea solutions were added at 4 °C
under agitation within 2 h in a 100-fold larger volume of refolding buffer (100 mM Tris, pH 8.1, 400 mM
L-arginine, 2 mM EDTA, 5 mM
glutathione, 0.5 mM oxidized glutathione, and 0.5 mM phenylmethylsulfonyl fluoride) containing 5 µM of the specific peptide. After stirring for 72 h
at 4 °C, insoluble components were removed by filtration on 0.2 µm
membrane filters (Nalgene), and the mixture was concentrated about
40-fold on an Amicon ultrafiltration concentrator equipped with a
polyethersulfone membrane (Mr 10,000 cutoff;
Amicon). The concentrate was passed over a 26/10 desalting column
(Amersham Biosciences) in Tris buffer (20 mM Tris, pH 8.0), and the Kd-PbCS(ABA) monomers were purified by anion
exchange chromatography using a Source Q15 fast flow column (Amersham
Biosciences). The column was eluted in the same buffer with a NaCl
gradient rising in 60 min from 0 to 500 mM. The average
refolding efficiency for the PbCS(ABA) peptide was 20%, with 100%
being the amount of Kd heavy chain invested. The purified
Kd-peptide complexes were supplemented with 2 mM glutathione, 5 mM EDTA, and 2 µg/ml
2m.
2m. The cells (0.5 × 106 cells/ml)
were incubated for 30 min with fluorescence-labeled Kd-peptide complexes in 50-µl aliquots at 18 °C or
37 °C, washed once in cold medium, and analyzed by flow cytometry on
a FACSCalibur (BD Biosciences).
2m
and 10 µM PbCS 252-260 peptide at 37 °C with 25 nM Kd-PbCS(ABA) octamers. After three washes,
the CTLs were incubated in 96-well plates (15,000 cells/well) with
51Cr-labeled P815 cells (5,000 cells/well) at 37 °C for
4 h. The specific chromium release, calculated as a percentage,
was as follows: (experimental release
spontaneous
release)/(total release
spontaneous release) × 100. The
total release was the esterase content after lysis of the cells with
1% Triton X-100.
2m and
10 µM PbCS 252-260 peptide for 90 min with 25 nM Kd-PbCS(ABA) octamer in the absence or
presence of 100 nM concanamycin A (Sigma),
anti-CD8
mAb H35 (10 µg/ml), or PbCS(ABA) peptide (1 µM). Released esterases were measured in the supernatants
as described previously (16). All incubations were performed in triplicates.
antibody. For detection,
the enhanced chemiluminescence Western blotting detection kit (ECL;
Amersham Biosciences) was used as recommended by the supplier.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2m
in the presence of PbCS(ABA) peptide. The refolding efficiency of
Kd-PbCS(ABA) complexes containing the heavy chain
comprising residues 1-277 or the heavy chain containing an added BSP
sequence was, on average, 20% (Fig.
1A). The BSP-containing
Kd-PbCS(ABA) complexes were biotinylated by using the
biotin ligase BirA (12). The efficiency of the biotinylation was
70-80%, and the efficiency of the refolding of
Kd-PbCS(ABA) or Kd-PbCS(ABA)-BSP complexes, on
average, was 20% (Fig. 1S).
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Fig. 1.
Linkers under study. A, the
sequences of the Kd 3 residues 270-271 with the BSP
sequence (underlined) added via a Gly-Ser spacer. The lysine
in the BSP sequence is biotinylated by BirA. B, the
C-terminal sequence (270-277) of the Kd heavy chain, in
which residue 273, 275, or 277 (triangles) was mutated to
cysteine. Structures of iodoacetyl-PEO-biotin (C) and
DMGS-biotin (D).
3 domain of MHC class I molecules (17). Refolding under the same
conditions gave yields of 14% for KdK275C, 12% for
KdR273C, and 6% for KdA277C (Fig. 1S,
A). The refolding efficiency of KdK275C was
increased to 20% when 0.3 mM dithiothreitol was added to
the urea buffer.
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Fig. 2.
Characterization of the
Kd-peptide complexes under study.
Kd-PbCS(ABA) monomers-PEO-biotin, DMGS-biotin dimers,
Cy5-labeled tetramers, octamers, and PE-labeled multimers were analyzed
by gel filtration on a Superdex S200 column (A) or by anion
exchange chromatography on a Source Q15 column (B). The
Superdex column was eluted with phosphate-buffered saline at a flow
rate of 0.7 ml/min, and the Source Q15 column was eluted at a flow rate
of 1 ml/min with 20 mM Tris, pH 8.0, with a gradient of
NaCl rising in 70 min from 0 to 500 mM. The absorbance of
the effluent was measured at 280 nm.
90% of
maximal binding reached within the first few minutes of incubation. For
octamer at 37 °C (Fig. 3A), but not at 18 °C or
4 °C (Fig. 3, C and D), a transient binding
maximum was observed at about 10 min, followed by a modest decrease, to
reach a stable plateau after 1 h. A similar biphasic binding
kinetics was recorded at 37 °C for Kd-PbCS(ABA) monomer
and tetramer (data not shown) (4). In all cases, the binding of
noncognate Kd-Cw3 170-179 octamer or multimer was no more
than a few percent of the binding of the corresponding cognate
complexes, indicating that nonspecific binding, namely, binding to CD8
under these conditions, is insignificant.
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Fig. 3.
Binding of soluble Kd-PbCS(ABA)
complexes to T1 CTLs. T1 CTLs were incubated at 37 °C
(A and B), 18 °C (C), or 4 °C
(D) for the indicated periods of time (A D) with 25 nM Cy5-labeled Kd-PbCS(ABA) octamer (
),
Kd-Cw3 170-179 octamer (
), PE-labeled
Kd-PbCS(ABA) multimer (
), or Kd-Cw3 170-179
multimer (
). Cell-associated fluorescence was assessed by FACS as
mean fluorescence intensity (MFI). Alternatively, T1 CTLs
were incubated for 30 min in E and G with the
indicated concentrations of Cy5-labeled Kd-PbCS(ABA)
octamer (
), KdD227K-PbCS(ABA) octamer (
),
Kd-PbCS(ABA) tetramer (
), KdD227K-PbCS(ABA)
tetramer (
), Kd-Cw3 170-179 octamer (
), and
Kd-Cw3 170-179 tetramers (
) or in F and
H with Kd-PbCS(ABA) multimer (
),
KdD227K-PbCS(ABA) multimer (
), or Kd-Cw3
170-179 multimers (gray diamonds) at 37 °C (E
and F) or 18 °C (G and H), and
cell-associated fluorescence was assessed likewise. Representative
experiments are shown. Each experiment was repeated two to five
times.
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Fig. 4.
TCR down-modulation and internalization of
Kd-PbCS(ABA) and KdD227K-PbCS(ABA) octamers on
T1 CTLs. T1 CTLs were incubated at 4 °C (A and
B), 18 °C (C and D), or 37 °C
(E and F) for the indicated periods of time with
25 nM Cy5-labeled Kd-PbCS(ABA) (WT)
(A, C, and E) or
KdD227K-PbCS(ABA) (227) (B,
D, and F) octamers. The cells were washed and stained
with PE-labeled anti-TCR mAb H57 ( ) or FITC-labeled anti-CD8
mAb
53.6.72 (
), and cell-associated fluorescence was measured by FACS.
Mean values and S.D. of the mean fluorescence intensities were
calculated from three experiments. G, alternatively, T1 CTLs
were incubated in the absence or presence of Fab' of
antiKd
3 mAb SF1-1.1.1 (SF'; 20 µg/ml) with
Cy5-labeled Kd-PbCS(ABA) (WT) or
KdD227K-PbCS(ABA) (227) octamer for 30 min at
37 °C or 18 °C. After washing, cells were fixed, and the
distribution of Cy5 was analyzed by confocal microscopy. Representative
pictures from at least 100 cells analyzed are shown.
3 mAb SF1-1.1.1, which block
residual CD8 co-engagement (4), internalization and patch formation of
both complexes were strongly inhibited; in particular, the
KdD227K-PbCS(ABA) complexes were localized predominately at
the cell surface.
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Fig. 5.
Soluble Kd-PbCS(ABA) complexes
induce Fas-dependent killing of bystander cells. T1
CTLs in suspension were incubated at 37 °C for 30 min with 25 nM Kdwt, KdD227K PbCS(ABA), or
Kd-Cw3 170-179 peptide tetramers, octamers, or PE-labeled
multimers and washed and incubated at 37 °C for 4 h with
51Cr-labeled P815 cells overexpressing Fas (P815Fas)
(A) or normal P815 cells (B). In all incubations,
10 µM PbCS 252-260 peptide (competitor) and 2m (5 µg/ml) were present. As indicated, in some incubations, free
PbCS(ABA) peptide (10 nM or 10 µM) or
anti-Fas antibody (2 µg/ml) was added. The specific lysis was
calculated from the released chromium measured in supernatants. Mean
values and S.D. were calculated from triplicate values from three
different experiments.
2m, the dissociation was
greatly reduced; after 3.5 h, only 10% of dissociation took
place. The same results were obtained for KdD227K-PbCS(ABA)
complexes (data not shown). Thus, to reduce the liberation of PbCS(ABA)
peptide,
2m was added to all incubations. In addition, in all
assays, 10 µM PbCS 252-260 peptide was added to prevent
free PbCS(ABA) peptide from binding to cell-associated Kd.
Under these conditions, 10 nM PbCS(ABA) peptide caused no
significant lysis. However, at a very high concentration (10 µM), free PbCS(ABA) caused strong lysis (Fig.
5A).
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Fig. 6.
Kd-PbCS(ABA) but not
KdD227K-PbCS(ABA) complexes elicit degranulation of
adherent T1 CTLs. T1 CTLs adhered to fibronectin-coated plates
were incubated at 37 °C for 90 min with 25 nM
Kd-PbCS(ABA) (wt),
KdD227K-PbCS(ABA), or Kd-Cw3 170-179
(Cw3) tetramers, octamers, or PE-labeled multimers, and the
released esterases were measured in the supernatants. In some
incubations, anti-CD8 mAb H35 (10 µg/ml) was present (
). All
incubations contained 10 µM PbCS 252-260 peptide and 2 µg/ml
2m. As a positive control, 1 µM free PbCS(ABA)
or PbCS 252-260 peptide was used, and an incubation containing
concanamycin A (CMA; 100 nM), an inhibitor of
CTL degranulation, was used as a negative control. Mean values and S.D.
were calculated from three experiments, each performed in
triplicates.
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Fig. 7.
Kd-PbCS(ABA) but not
KdD227K-PbCS(ABA) octamers elicit calcium mobilization and
strong tyrosine phosphorylation in T1 CTLs. A,
Indo-1-labeled T1 CTLs were incubated at 37 °C with 25 nM Kd-PbCS(ABA) (black line) or
KdD227K-PbCS(ABA) (gray line) octamers or medium
(dotted line), and calcium-dependent Indo-1
fluorescence was measured by FACS. B, T1 CTLs, untreated
( ) or incubated for 3 min at 37 °C with 25 nM
Kd-PbCS(ABA) (wt) or
KdD227K-PbCS(ABA) (227), were washed and
lysed in 1% Brij78, and the detergent-soluble fraction was
immunoprecipitated with anti-TCR mAb H57. The immunoprecipitates were
resolved on SDS-PAGE (15%, reducing) and Western blotted with
anti-phospho-tyrosine (pY) mAb 4G10 and anti-CD3 antibody,
respectively. Data shown are from three representative experiments.
C, the purified Kd-PbCS(ABA) monomers
(lane 1) and DMGS-biotin dimers (lane 2) used in
the preparation of tetramers and octamers were resolved on SDS-PAGE
(15%, reducing), and the gel was stained with Coomassie Blue.
and
chain, including its pp23 phospho form, as is typically observed upon
TCR triggering with agonists (Fig. 7B) (14, 23, 24). By
contrast, KdD227K-PbCS(ABA) octamers induced only faint
phosphorylation of the pp21 phospho form of
chain, as typically
occurs upon T-cell triggering by weak agonists or antagonists (Fig.
7B) (14, 23, 24). Blotting with anti-CD3
antibody showed
that equal amounts were loaded in all lanes. The soluble
Kd-PbCS(ABA) complexes used were homogeneous according to
SDS-PAGE (Fig. 7C). The same results were obtained on S14
CTLs (data not shown). Taken together, these results show that soluble
Kd-PbCS(ABA) octamers efficiently induce intracellular
calcium mobilization and CD3 phosphorylation, given that they co-engage CD8.
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Fig. 8.
KdD227K-PbCS octamers induce
apoptosis of T1 CTLs. T1 CTLs were incubated or not incubated with
25 nM KdD227K-PbCS(ABA),
Kd-PbCS(ABA), or Kd-Cw3 170-179 octamers at
37 °C for 30 min, followed by an additional 4.5 h at 37 °C
in medium, and then analyzed by FACS after staining with Cy5-labeled
annexin V. Data from one out of two experiments are shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2m is rapidly exchanged (27), we examined
how site-specific alkylation of the heavy chain is best accomplished.
Our results show that position 275 of the heavy chain is most suitable.
For Kd the refolding and the alkylation efficiency of the
K275C heavy chain were higher as compared with the R273C and A277C
mutants (Fig. 1S). This is consistent with the fact that the conserved Trp274 marks the end of the folded
3 domain (17). Our
results further indicate that the efficiency of refolding and
alkylation critically depend on appropriate reduction of the introduced
free cysteine (Fig. 1S). Because the same results were obtained for
HLA-A2,2 this may be
generally applicable.
chain immunoreceptor tyrosine-based activation motifs recruit
ZAP-70 (and Syk), which upon phosphorylation by Lck phosphorylates LAT
and other substrates, thus initiating various downstream signaling
cascades (32-34).
, which is involved in the observation that in the
absence of CD8 co-engagement, there is no significant generation of
inositol 1,4,5-triphosphate, which in turn mediates the release of
intracellular calcium from stores (34, 35). In agreement with this is
our intracellular calcium mobilization (Fig. 7A) (8). It is
well established that CTL degranulation requires a rapid intracellular
calcium mobilization, followed by a sustained influx of extracellular
calcium, whereas for Fas-dependent killing, the latter is
sufficient (36, 37).
chain. This kinase phosphorylates phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-triphosphate and is critically involved in early TCR signaling, including calcium mobilization, activation of Rho family GTP-binding proteins, and cytoskeleton function (38). Inhibition of phosphatidylinositol 3-kinase by wortmannin blocks CTL degranulation but has no effect on
Fas-dependent cytotoxicity (39). It thus appears that
MHC-peptide complexes that do not co-engage CD8 are unable to
significantly activate Lck and hence most CTL effector function,
including degranulation, cytokine release, and proliferation. The
remarkable exception is Fas-dependent killing, which is
Lck-independent, which makes it possible to selectively induce this
cytotoxicity in the absence of any other cellular response (8, 40).
![]() |
ACKNOWLEDGEMENTS |
---|
We are gratefuls to Dr. M. Bézard for help with DMGS synthesis, Dr. G. Gachelin (Pasteur
Institute) for providing the cDNA encoding the Kd heavy
chain, Dr. S. Nathenson (Albert Einstein College) for the cDNA
encoding human 2m, and Dr. J.-P. Abastado (Pasteur Institute) for
the Kd-heavy chain-BSP fusion protein.
![]() |
FOOTNOTES |
---|
* This study was supported in part by grants from the Sandoz Foundation, the Stanley Thomas Johnson Foundation, and the Swiss National Science Foundation (Grant 3100-061946.00).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains supplementary Figs. 1S and 2S.
§ Supported by a grant from the Giorgi-Cavalieri Foundation Swiss.
To whom correspondence should be addressed. Tel.:
41-21-692-5988; Fax: 41-21-653-4474; E-mail:
iluesche@eliot.unil.ch.
Published, JBC Papers in Press, October 28, 2002, DOI 10.1074/jbc.M208863200
2 P. Guillaume, D. F. Legler, N. Boucheron, M.-A. Doucey, J.-C. Cerottini, and I. F. Luescher, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
TCR, T-cell antigen
receptor;
ABA, 4-azidobenzoic acid;
2m,
2-microglobulin;
BSP, biotinylation sequence;
DMGS, di-maleimide-di-glycine-serine;
FACS, fluorescence-activated cell sorting;
PEO, ethyleneglycol;
MHC, major
histocompatibility complex;
PbCS, Plasmodium berghei
circumsporzoite;
PE, phycoerythrin;
CTL, cytotoxic T lymphocyte;
Fmoc, N-(9-fluorenyl)methoxycarbonyl;
mAb, monoclonal antibody;
GFC, gel filtration chromatography.
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REFERENCES |
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