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*
Medizinische Klinik II, Hämatologie-Onkologie, Krankenhaus Nordwest, Frankfurt, Germany;
Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213;
Istituto Tumori Milano, Milano, Italy;
Department of Medical Microbiology, University of Mainz, Hochhaus Augustusplatz, Mainz, Germany; and
¶ Immunomics Operations, Beckman Coulter, Marseille, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain down-regulation, pattern, and quantity of
cytokine secretion. As a paradigm, we analyzed the reactivity of a
Melan-A/MART-1-specific and HLA-A2-restricted CD8+ T
cell clone to either soluble or solid-phase presented peptides,
including the naturally processed and presented Melan-A/MART-1 peptide
AAGIGILTV or the peptide analog ELAGIGILTV presented either by the
HLA-A2 wild-type (wt) or mutant (alanine
valine aa 245) MHC class I
molecule, which reduces engagement of the CD8 molecule with the HLA-A2
heavy chain. Soluble MHC class I complexes were used as either
monomeric or tetrameric complexes. Soluble monomeric MHC class I
complexes, loaded with the Melan-A/MART-1 peptide, resulted in CD3/CD8
and TCR -chain down-regulation, but did not induce measurable
cytokine release. In general, differences pertaining to
CD3/CD8/-chain regulation and cytokine release, including IL-2,
IFN-
, and GM-CSF, were associated with 1) the format of Ag
presentation (monomeric vs tetrameric MHC class I complexes), 2) wt vs
mutant HLA-A2 molecules, and 3) the target Ag (wt vs analog peptide).
These differences are to be considered if T cells are exposed to
recombinant MHC class I Ags loaded with peptides implemented for
detection, activation, or sorting of Ag-specific T
cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One of the major advantages of soluble MHC class I molecules is that a
defined amount of Ag can be used to stimulate or to sort Ag-specific T
cells. Earlier studies showed that the amount of Ag and the exposure
time of T cells to their nominal target Ag critically influence the
quality and the quantity of cytokines released after the interaction of
the TCR with the MHC/peptide complex (7). In addition, the
encounter of T cells with the target epitope leads to TCR
down-regulation, which represents a pivotal event in T cell activation
(8). In general, CD3 
and the T cell coreceptor CD4 or
CD8 are down-regulated in a parallel fashion (9). The same
is true for the TCR -chain, which is promptly degraded after TCR
triggering (10). Some authors postulated that the
magnitude of TCR down-regulation is correlated with T cell effector
functions (11). Others suggested that TCR engagement and
down-regulation may not be associated directly with T cell responses
(12). Thus, events associated with TCR engagement and
ultimately TCR down-regulation are not only of interest in the context
of T cell biology, but also in the experimental and clinical
application of tetramer-guided sorting of Ag-specific T cells.
We have examined CD3/CD8 and TCR -chain down-regulation,
implementing the Ag-specific CD8+ T cell clone
SK-Mel-29.1.1, which is directed against the HLA-A2-restricted and
melanoma-associated differentiation Ag
Melan-A/MART-1 (AAGIGILTV)
(13). This T cell clone appears to be associated with
immunosurveillance and protection from tumor recurrence in the patient
SK29 (14). Soluble as well as solid-phase bound monomeric
and tetrameric HLA-A2 complexes were loaded with the naturally
processed and presented Melan-A/MART-1 Ag AAGIGILTV
(15), or the peptide analog ELAGIGILTV, which has been
reported to induce enhanced immune effector functions in
anti-Melan-A/MART-1-directed T cells (16). HLA-A2
molecules were used as the wild-type (wt)3 HLA-A2.1
molecule or, alternatively, as an HLA-A2 mutant protein, which reduces
nonspecific background binding of nonspecific T cells due to an altered
interaction of the CD8 molecule with the HLA-A2 
3 domain
(17). We tested whether these reagents affect
CD3/CD8/-chain modulation and T cell immune effector functions
defined by cytolysis and cytokine release.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HLA-A2 wt or HLA-A2 mutant tetramer complexes loaded either with
AAGIGILTV or ELAGIGILTV were prepared as described in detail recently
(17). Mutated (alaninevaline at position 245, termed
HLA-A2 m) HLA-A2 tetrameric complexes have been shown to reduce the
background staining of nonspecific CD8-binding T cells by affecting the
interaction of the CD8 molecule with the MHC class I heavy chain.
Substitution at position 245, located on the fifth 
-pleated sheet,
may either directly affect the interaction of the TCR/CD8 with MHC
class I, or it may alter the MHC class I conformation at a distant
site, i.e., the major CD8 contact site that includes position 227 in
the HLA-A2 molecule (18). T cells were either exposed to
monomeric or tetrameric HLA-A2 complexes in solution or, alternatively,
attached to a solid surface. The
anti-2-microglobulin mAb clone B1G3
(IgG2a) obtained from Beckman Coulter (Krefeld, Germany) was dissolved
at 2 µg/ml in PBS, and 50 µl of this solution was used per well of
a 96-well microtiter plate (Maxisorb; Nunc, Wiesbaden, Germany) and
incubated for 2 h at 37°C. Plates were washed twice with PBS
before adding either monomeric or tetrameric class I complexes, as
indicated in individual experiments.
To ensure that an equal number of MHC/peptide complexes, composed of
either monomers or tetramers, are presented to CTL, the
anti-2 mAb was attached to 96-well plates,
as described above, followed by incubation of monomer or tetramer
MHC/peptide complexes either at 1, 0.1, or 0.01 µg MHC/peptide
equivalent per well for 1 h at room temperature and washed twice
with PBS, followed by incubation with the biotin-conjugated mAb w6/32
(mIgG2A; Leinco Technologies, Ballwin, MO), 50 µl/well, a 1/400
dilution of a 0.1 mg/ml concentration for 30 min, followed by two
washing steps with PBS/0.005% Tween. This mAb defines a monomorphic
determinant on MHC molecules that is dependent on the correctly folded
MHC heavy chain/peptide/2-microglobulin
complex (19). A total of 50 µl avidin-peroxidase was
added for 1 h at room temperature, washed four times with
PBS-Tween, and developed with tetramethylbenzidine. The reaction was
terminated using 50 µl 1.8 N
H2SO4, and the OD was
determined at 450 nm (Fig. 1
). The
titration of monomer or tetramer complexes attached to plastic surfaces
yielded a similar number of MHC/peptide complexes, as defined by the
mAb w6/32.
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-chain expression determined by flow cytometry.
Alternatively, T cells were exposed to MHC class I complexes, to the
HLA-A2+ and Melan-A/MART-1-positive target cell
line Mel624, or to plate-bound anti-CD3 mAb (Okt3, 2 µg/ml, 50
µl/well), and supernatants were harvested for detection of IFN-,
GM-CSF, or IL-2 by ELISA, according to the manufacturer’s
instructions.
The staining pattern of HLA-A2 molecules loaded either with the
naturally processed and presented Melan-A/MART-1 peptide AAGIGILTV or,
alternatively, with the T cell superagonist ELAGIGILTV loaded either
onto HLA-A2m or HLA-A2wt molecules, was defined in a time course
experiment using the SK-Mel-29.1.1 T cell clone as effector cells. No
differences between A2wt or A2m molecules presenting the superagonist
peptide, but reduced staining of the T cell clone implementing the
AAGIGILTV peptide (Fig. 2A), could be observed. This
staining pattern could also be reproduced using tetramer complexes to
stain Ag-specific T cells in PBL (Fig. 2B). A2wt or A2m
molecules loaded with ELAGIGILTV stain 4% of the
CD3+ CD8+ T cell population
from a patient with melanoma. In contrast, the A2m tetramer loaded with
AAGIGILTV stained only 1% of T cells. We have been able to show
earlier that the TCR repertoire binding to either HLA-A2wt or
HLA-A2m MHC/peptide complexes loaded with ELAGIGILTV is almost
identical in patients with melanoma (20).
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T cells were stained with an anti-CD3 (clone UCHT1) mAb
labeled with energy-coupled dye, with anti-CD8 -chain mAb (clone
B9.11, murine IgG1) labeled with PE-Cyanin (PC5) for 15 min at 4°C,
and flow cytometric analysis was performed using a Beckman Coulter
Epics XL flow cytometer. Intracellular TCR -chain detection was
carried out after staining cells with cell surface markers, followed by
permeabilization with Intraprep (Beckman Coulter) using the
anti--chain mAb directly labeled with FITC (clone 6B10.2, mouse
IgG1) obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The
human T cell clone SK-Mel-29.1.1 expresses the TCR V region -chain
2S1 and V region -chain 13S3, and has already been described
in detail (13).
Functional assays
Cytotoxicity was analyzed in a standard 4-h
51Cr release assay, and supernatants were tested
for IL-2, IFN-, or GM-CSF by ELISA. Target cells were
HLA-A/B- B cells (C1R) transfected with either
the A2wt or A2m molecule (18). The E:T ratio was 10:1,
unless otherwise indicated.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The anti-Melan-A/MART-1-specific T cell clone SK-Mel-29.1.1
was incubated with monomeric or tetrameric HLA-A2 wt or HLA-A2 m MHC
class I complexes loaded with the Melan-A/MART-1 peptide analog
ELAGIGILTV. In each experiment, tetramer complexes as well as monomers
were at 1 µg in a reaction volume of 0.5 ml with 5 x
105 T cells. Aliquots of T cells were harvested
at different time points ranging from 5 to 360 min, and CD3/CD8
expression as well as intracellular TCR -chain expression was
evaluated and compared with CD3/CD8/-chain expression before Ag
exposure (i.e., 100%). Differences were observed in T cells exposed to
tetramer complexes compared with monomeric MHC class I molecules (Fig. 3A). CD3/CD8 expression was
down-regulated on T cells exposed to monomeric HLA-A2 wt molecules 10
min after Ag exposure (80% of the original expression, or 20%
reduction, respectively). After 360 min, T cells regained similar
expression levels as compared with TCR expression before Ag exposure.
HLA-A2wt tetramer complexes resulted already after 5 min in 80% of the
original CD3/CD8 and -chain expression and 
90% of the original
CD3, CD8, or TCR expression was observed 360 min after Ag
exposure.
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down-regulation was observed after 10 min,
similarly to the HLA-A2 wt molecule. In contrast, CD3/CD8 and TCR
-chain expression was reduced to 60% of the original expression
levels after 360 min of Ag exposure to monomeric HLA-A2 m complexes.
Similarly, HLA-A2 m tetramer complexes resulted in 70% of the
original TCR expression level after 360 min of Ag contact. Thus,
exposure of the anti-Melan-A/MART-1 T cell clone to HLA-A2wt class
I complexes resulted in restoration of the TCR expression level after
360 min of Ag exposure, and the mutant HLA-A2 molecule yielded a more
pronounced down-regulation of the TCR complex, as defined by CD3/CD8
and -chain expression. Notably, the stimulating Ag (i.e., monomer or
tetramer) was present throughout the entire experiment (Fig. 3
A). However, exposure of T cells for 10 min to tetramer
complexes, followed by two washing steps, showed similar results
regarding CD3/CD8 and -chain down-regulation, respectively (data not
shown). Pronounced TCR down-regulation in T cells exposed to solid-phase monomeric or tetrameric class I complexes
In a control experiment, monomeric or tetrameric HLA-A2 complexes
were attached to a solid phase using an
anti-2-microglobulin mAb, as described in
Materials and Methods. Thus, Ag presentation occurred as
multimeric HLA-A2 complexes composed of either monomeric complexes or
tetrameric complexes (Fig. 3B). We tested whether
Melan-A/MART-1-specific T cells reacted 1) similarly or differently as
defined by CD3/CD8 and -chain expression by comparing HLA-A2 wt or
HLA-A2 m molecules, and 2) differently to monomer or tetramer complexes
in fluid phase as compared with MHC class I Ag-presenting molecules
attached to a solid phase. Two patterns of reactivity emerged: CD3/CD8
and -chain expression was greatly reduced at 360 min after Ag
contact, yielding 50% of TCR expression in T cells exposed to
solid-phase HLA-A2wt monomeric complexes or HLA-A2m tetramer complexes.
HLA-A2m monomeric complexes ablated CD3/CD8 and TCR expression after
exposure of 360 min. A different pattern was observed in HLA-A2wt
tetramer complexes: CD3/CD8/-chain expression was reduced to 80%
after 5 min and completely regained TCR expression after 60
min.
Cytokine expression in T cells reacting to fluid- or solid-phase monomer or tetramer complexes
Aliquots from supernatants of each experiment (Fig. 3, A and B) were harvested at 3, 6, and 24 h
after Ag exposure and tested for IL-2, IFN-, or GM-CSF by ELISA
(Fig. 4, A and B).
No significant cytokine release could be detected in T cells exposed to
monomeric HLA-A2 complexes in fluid phase independent of the HLA-A2wt
or HLA-A2m Ag-presenting molecule (Fig. 4A). In contrast,
tetrameric complexes in fluid phase resulted in IL-2, IFN-, and
GM-CSF secretion independent of the HLA-Awt- or HLA-A2m-presenting
molecules (Fig. 4A). HLA-A2m monomeric complexes induced
higher IL-2 levels as compared with the HLA-A2m tetrameric molecules
(up to 420 pg/ml in 24 h using monomeric complexes) as compared
with 200 pg IL-2 in 24 h elicited by HLA-A2 tetramer complexes
(Fig. 4B). Differences regarding cytokine secretion were
observed for IL-2, but not for IFN- or GM-CSF. Note that alterations
in CD3/CD8 or -chain down-regulation (e.g., T cells exposed to
solid-phase tetrameric HLA-A2m molecules) were not associated with
elevated levels of cytokine secretion.
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The previous set of experiments was performed using a
Melan-A/MART-1 peptide analog ELAGIGILTV, which has been described to
represent a superagonistic peptide leading to enhanced cytolysis and
more efficient cytokine release as compared with the wt epitope
(E)AAGIGILTV (Fig. 5A). As a
control, we used the naturally processed and presented Melan-A/MART-1
peptide AAGIGILTV, the plate-bound anti-CD3 mAb OKT3, as well
as the Melan-A/MART-1+ and
HLA-A2+ melanoma cell line Mel624 (Fig. 5B) to activate T cells. Solid-phase monomeric HLA-A2 m
molecules loaded with AAGIGILTV yielded only 10% reduction of
CD3/CD8/-chain expression after 360 min of Ag exposure. In contrast,
tetrameric HLA-A2 m molecules, plate-bound anti-CD3 Ab, or melanoma
cells Mel624 showed a similar pattern of TCR reactivity: the TCR
expression level remained stable up to 60 min after Ag exposure and was
followed by a significant TCR down-regulation: 20% of the original
expression level as a reaction to HLA-A2m molecules loaded with
AAGIGILTV, 40% induced by anti-CD3, or 60% as a response to
melanoma cells. Evaluation of cytokine secretion (Fig. 6) in supernatants harvested at 3, 6, and
24 h of Ag exposure elicited by TCR engagement resulted in
significant IL-2 secretion: 300 pg/ml/24 h upon OKT3 stimulation, 370
pg/ml/24 h upon exposure to Mel624 cells, and 500 pg/ml/24 h upon
exposure to solid-phase monomeric HLA-A2m molecules loaded with the
AAGIGILTV peptide. Similar to solid-phase HLA-A2m tetramer complexes
loaded with ELAGIGILTV (Fig. 3B), tetramer HLA-A2m molecules
loaded with AAGIGILTV yielded less IL-2 secretion (120 pg/ml/24 h) as
compared with the multimeric class I complexes composed of monomeric
HLA-A2 molecules. As a control, fluid-phase HLA-A2m monomeric molecules
loaded with AAGIGILTV did not yield cytokine secretion. Similar to the
(fluid-phase) HLA-A2m tetramer complexes loaded with ELAGIGILTV (Fig. 4A, top panel), the HLA-A2m tetramer molecule
loaded with AAGIGILTV (Fig. 6) resulted in IL-2, IFN-, and GM-CSF
secretion (e.g., 80 pg IL-2/ml/24 h), albeit to a lesser extent as
compared with the ELAGIGILTV peptide analog (i.e., 420 pg
IL-2/ml/24 h).
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HLA-A2wt and HLA-A2m tetrameric molecules showed substantial
differences as compared with CD3/CD8/-chain expression in T cells
after Ag exposure (Fig. 3, A and B; Fig. 5, A and B) as well as differences in cytokine
release (Fig. 4, A and B; Fig. 6). However, these
experiments do not address potential differences in the cytolytic T
cell effector function. To test for cytolytic capacity,
HLA-A,-B-negative C1R cells transgenic for HLA-A2wt or the HLA-A2m
molecules were loaded with the ELAGIGILTV or the AAGIGILTV peptide and
tested for cytolysis and cytokine release (Fig. 7). No significant differences in IL-2,
IFN-, or GM-CSF secretion as well as for cytolysis could be observed
in HLA-A2wt or HLA-A2m molecules presenting the peptide AAGIGILTV.
Similar data were obtained for the Melan-A/MART-1 peptideanalog
ELAGIGILTV (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One of the salient findings of this study is that monomeric HLA-A2
complexes loaded with the nominal target epitope are able to induce TCR
down-regulation, but not T cell effector functions, e.g., cytokine
release (Fig. 3, A and B; Fig. 4, A
and B). A recent study in a murine system showed that T cell
clones are indeed able to respond to monomeric MHC class I complexes,
as defined by calcium influx or proliferation if the coreceptor CD8
chain is available at the cell surface (21).
An early step in T cell activation is the phosphorylation of
immunoreceptor tyrosine-based activation motifs (ITAMs), either in the
TCR - or -chain mediated by Lck. Once phosphorylated, the ITAMs
recruit the tyrosine kinase ZAP-70, which facilitates further
phosphorylation and leads to more downstream signaling events and
ultimately to cytokine secretion (for review, see Ref.
22). The observation that anti-CD3 cross-linking
(using the anti-CD3 mAb OKT3) is able to induce T cell
activation supports this model (Figs. 5B and 6):
the signaling cascade can be triggered by tyrosine kinases that are
already associated with the -chains in the responsive T cell. This
model is supported by experiments in which dimers of peptide/MHC
complexes are able to elicit IL-2 secretion in CD8-negative T cells;
thus, in a minimalistic model, TCR homodimerization is able to trigger
T cells (23).
Nonmutually exclusive, the initial triggering event is mediated by the apposition of ITAM-bound ZAP-70 with the coreceptor Lck. This model is supported by the observation that bispecific Abs (directed against CD3 and the coreceptor) are able to induce T cell activation (24). Thus, homodimerization of the TCR chain and/or heterodimerization of the TCR with the coreceptor are also able to induce T cell activation.
Because TCR and CD8 coreceptor down-regulation occurs exclusively upon
Ag exposure, the T cell clone SK-Mel-29.1.1 was apparently activated by
monomeric MHC class I/peptide complexes (Fig. 3A). However,
this did not lead to significant downstream events, i.e., cytokine
secretion (Fig. 4B). It is interesting to note that the
CD3/CD8 or TCR -chain down-regulation was more pronounced in T cells
exposed to the monomeric class I complexes with the mutation in
position 245. This mutation reduces interaction of the CD8 molecule
with the MHC class I complex (18, 21): at first glance
surprising, since we observed a stronger TCR/CD3 down-regulation as a
response to a HLA-A2 (mutant) molecule with reduced capacity to engage
with the TCR as compared with the HLA-A2 wt molecule. The existence of
CD8-independent T cell clones (25) suggests that this
observation is not unique and that CD8 may, at least in some cases, not
be able to play its role as a coreceptor due to an orientation of the
TCR to its ligand that is not compatible with CD8 binding to HLA-A2
(discussed in Ref. 21). The mutation at position 245 in
HLA-A2 affecting the CD8 binding site (18) may thus allow
for a better engagement of the TCR/CD8 complex to HLA-A2 peptide
targets, resulting in TCR down-regulation.
However, coordinate TCR and CD8 down-regulation occurs also if the
coreceptor CD8 cannot engage with the MHC class I molecule: the
interaction of the coreceptor CD8 in initiating coordinate TCR/CD8
down-regulation may be dispensable if T cells are stimulated by optimal
ligands (26, 27). We prove that this is operational in the
Melan-A/MART-1-reactive T cell clone: the coordinate TCR/CD8
down-regulation is only observed as a response to the peptide analog
(superagonist) ELAGIGILTV presented by a monomeric MHC class I complex
(see Fig. 3A), but not to the monomeric class I peptide
complex loaded with the naturally processed and presented peptide
AAGIGILTV (see Fig. 5A). The proof that the monomeric MHC
class I/peptide (AAGIGILITV) complexes are properly folded is provided
by the control experiment, in which the association of monomeric
complexes loaded with the peptide AAGIGILTV to multimers by attachment
to plastic leads to pronounced TCR down-regulation and biologically
meaningful secretion of cytokines (Figs. 5 and 6).
In addition, the number of MHC/peptide complexes is identical
independent of monomeric or tetrameric MHC complexes (see Fig. 1).
However, even if the number of MHC/peptide complexes is identical, the
spatial arrangement of these Ag-presenting molecules impacts on
different spatial arrangements of TCR-associated molecules, e.g., CD8
or the adapter molecule linker for activation of T cells
(28), and ultimately on T cell effector functions
(29, 30). Thus, the spatial arrangement of MHC/peptide
molecules may represent an important aspect of the in vivo interactions
between T cells and APCs.
CD3/CD8 and TCR -chain down-regulation occurs only if the T cell
clone is exposed to the tetrameric complex, but not to monomers loaded
with the Melan-A/MART-1 peptide AAGIGILTV (Fig. 5). Thus, subtle
differences in response to either HLA-A2wt or HLA-A2m peptides are
observed if the dynamics of TCR down-regulation are measured. These
differences did not impact on downstream T cell effector functions: the
T cell clone secretes IL-2, IFN-, as well as GM-CSF in response to
multimeric MHC class I/peptide complexes and shows no differences
regarding cytolytic T cell responses if C1R-A2wt or C1R-A2m molecules
are expressed as a transgene in C1R APCs. Thus, at least in the
Melan-A/MART-1-specific T cell clone SK-Mel-29.1.1, TCR down-regulation
cannot be used as a measure to predict the biological outcome of T cell
activation. This notion is supported by additional experimental data
provided in this study: exposure of the T cell clone to
Melan-A/MART-1+ and HLA-A2+
melanoma cells did not show a pronounced CD3/CD8 reduction, but
resulted in a similar cytokine secretion pattern as compared with
multimeric HLA-A2 complexes loaded with the ELAGIGILTV peptide.
In addition, cell surface CD3/CD8 down-modulation in the
Melan-A/MART-1-specific effector cell clone exhibited a biphasic
pattern, i.e., CD3/CD8 down-modulation was observed after 10–30 min of
Ag exposure, followed by recovery of CD3/CD8 cell surface expression
and a subsequent CD3/CD8 down-modulation after 180 min (Fig. 3).
Several mechanisms may account for this phenomenon: the first CD3/CD8
down-modulation may result from directly engaged receptors mediated by
endocytosis. In contrast, the CD3/CD8 down-modulation may represent
nondirectly engaged bystander TCRs that are internalized by
activation ofintracellular signaling mediators, i.e., protein
tyrosine kinase, phosphatidylinositol 3-kinase, or protein kinase C
(31, 32). Down-modulated CD3/CD8 complexes may impact on
the effector function(s) of freshly isolated Ag-specific T cells using
tetramer complexes. A strong antigenic stimulus may render these T
cells anergic or, alternatively, may induce apoptosis if
tetramer-reactive T cells are re-exposed to the nominal target
Ag.
Differences in TCR down-regulation in the Melan-A/MART-1-specific T cell clone in response to the naturally processed and presented peptide AAGIGILTV as compared with the peptideanalog ELAGIGILTV may also be associated with the nature of this individual T cell clone: this clone has been repeatedly isolated from the melanoma patient SK29, who is still in complete remission (13, 14). Recent evidence suggests that the time course of TCR down-regulation is different in naive and primed T cells (discussed in Ref. 33) and shows different requirements for costimulation.
In conclusion, we have been able to show that 1) monomeric class I
complexes are able to induce T cell activation, as defined by
coordinate TCR/CD8 down-regulation if an optimal T cell ligand is used
as the stimulating epitope, 2) multimeric class I complexes, either in
solution or attached to a solid phase, are able to induce cytokine
secretion in responding T cells, 3) CD3/CD8 and TCR -chain
down-regulation does not correlate with immune effector functions, and
4) differences pertaining to HLA-A2wt or HLA-A2m molecules exist if
CD3/CD8 down-regulation is analyzed, but both HLA-A2 molecules are able
to effectively induce cytokine secretion and cytolysis in
Melan-A/MART-1-reactive T cells. These observations impact on the
implementation of soluble class I molecules I in either detection or
sorting of MHC class I/peptide-specific T cells. In addition, monomeric
ligands may be used to gauge the affinity of the clonotypic TCR to the
respective peptide epitope: high affinity TCRs may respond with TCR
down-regulation even to monomeric MHC class I/peptide complexes.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Markus J. Maeurer, Department of Medical Microbiology, University of Mainz, Hochhaus Augustusplatz, 55101 Mainz, Germany. E-mail address: maeurer{at}mail.uni-mainz.de
3 Abbreviations used in this paper: wt, wild type; ITAM, immunoreceptor tyrosine-based activation motif; m, mutant.
Received for publication August 16, 2001. Accepted for publication January 15, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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complexes after antigenic stimulation. J. Exp. Med. 185:1859.3 domain of HLA-A2. Nature 345:41.[Medline]
molecules can inhibit T cell activation. J. Exp. Med. 190:1627.This article has been cited by other articles:
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  J. Yuan, J.-B. Latouche, J. Hodges, A. N. Houghton, G. Heller, M. Sadelain, I. Riviere, and J. W. Young Langerhans-Type Dendritic Cells Genetically Modified to Express Full-Length Antigen Optimally Stimulate CTLs in a CD4-Dependent Manner J. Immunol., February 15, 2006; 176(4): 2357 - 2365. [Abstract] [Full Text] [PDF]
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V. Dutoit, P. Guillaume, M. Ayyoub, C. S. Hesdorffer, I. F. Luescher, and D. Valmori Decreased Binding of Peptides-MHC Class I (pMHC) Multimeric Complexes to CD8 Affects Their Binding Avidity for the TCR But Does Not Significantly Impact on pMHC/TCR Dissociation Rate J. Immunol., May 15, 2003; 170(10): 5110 - 5117. [Abstract] [Full Text] [PDF]
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) together with the intracellular 
