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Cellular Immunology Section, Neuroimmunology Branch, National Institute of Neurological Disorders and Stroke and
Lymphocyte Biology Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain phosphorylation were observed, with the ratios of TCR
isoforms relating to ligand agonist potency. The appearance of
partially phosphorylated TCR isoforms was paralleled by relative
changes in certain response thresholds within the hierarchy. Thus, a
combination of density, potency, and quality of signaling all
contribute to the distinct effects of agonist ligands on T cell
immunity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain phosphorylation patterns and association of ZAP-70 with the
-chain without detectable kinase activation were seen in murine T
cell clones (TCC)3 upon
exposure to TCR antagonists (19, 20) or partial agonists (21, 22).
Because this specific pattern of TCR subunit phosphorylation and
associated kinase activation was not seen at any concentration of
wild-type ligand, the change in the proximal signaling pattern was
considered responsible for the qualitatively altered functional
responses. Other data suggest, however, that even for a conventional
agonist TCR ligand, the elicitation of different effector/cytokine
responses may not be proportional at low and high Ag densities. These
more recent observations concerning the effect of Ag concentration on
the quality of T cell responses, especially polarization to the Th1 vs
Th2 phenotype (23, 24, 25), may explain older reports on the humoral vs
cell-mediated response bias in animals administered widely different
amounts of Ag (26). Most studies examining these issues have been performed using mouse models and TCC, and only limited data are available concerning the influence of ligand structure and amount on the response phenotype of human T cells (27, 28, 29, 30, 31). Even less is known about the TCR signaling events that occur in human T cells when exposed to variant ligands (27). Determining how the quality and the quantity of a TCR ligand together regulate the multiple potential responses of T cells is critical to understanding what controls not only the extent but also the humoral vs inflammatory character of immune responses. Information on human lymphocytes is particularly important to provide a rational basis for understanding the pathogenesis of autoimmune diseases and for designing Ag-dependent interventions in these diseases or for vaccine purposes.
For these reasons, we have systematically examined the relationship between Ag concentration and the induction of a large number of different effector functions characteristic of human CD4+ TCC. These functional studies have been complemented by the measurement of TCR down-modulation as an indicator of TCR engagement and by an analysis of early TCR-associated biochemical signaling events. These parameters have been assessed using for each TCC a series of related receptor ligands. The stimuli include the prototypic agonist as well as ligands with higher (superagonists) or lower (weak agonists) potency or some that function as antagonists (18, 32, 33).
Based on a comparison of Ag dose responses for various effector
functions, we demonstrate a hierarchical arrangement of response
thresholds for each clone. Alterations in ligand structure result in a
general shift in the set of response thresholds (potency change) that
correlates with the degree of phosphorylated ZAP-70 kinase and the
extent of TCR down-modulation induced by each ligand at comparable
peptide concentrations. In addition, shifts in the relative positions
of some response thresholds and altered patterns of -chain
phosphorylation are seen with the less potent ligands. Thus, the
selective activation effects reported for the latter peptide-MHC
molecule combinations may reflect both the potency change that makes
some responses difficult to elicit at achievable Ag densities and the
relative changes in response thresholds within the hierarchy.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Myelin basic protein peptide 87–99
(MBP87–99)-specific TCC were generated,
characterized, and maintained as previously described (34). The
restriction elements used by the TCC are DRB5*0101 for MS18 and
DRB1*1302 for GDBP. Clonality was shown by RT-PCR amplification using
21 V
and 22 Vß family-specific primers (34). TCR usage is
V23/Vß21 for GDBP and V3.1/Vß5.2 for MS18. EBV-transformed B
cell lines GP-B (HLA-DR1*0404/1*1302) and AF-B (HLA-DR1*1501/5*0101)
were used as APCs in all assays. The peptide MBP87–99
(one-letter code, VHFFKNIVTPRTP) and its variants (indicated as the
original residue at position X, followed by the substituted residue,
e.g., T95-A) were synthesized using Merrifield’s solid phase
methodology as previously described (35).
Functional and protein expression assays
For functional assays, TCC were rested for 8 days after Ag
stimulation. All functional and biochemical experiments were conducted
under the same conditions of T cell expansion and rest. For
proliferation and immunofluorescence staining assays, irradiated GP-B
and AF-B as APC (5000 rad) were incubated for 2 h with different
concentrations of peptide ligands and washed twice. B cells (2 x
104) and T cells (5 x 104) were
added to each well of 96-well round-bottom plates to a final volume of
200 µl (Iscove’s modified Dulbecco’s medium, 5% human serum, 1%
glutamine, 1% penicillin/streptomycin, and 0.2% gentamicin). For
proliferation assays cells were cultured for 48 h at 37°C. Fifty
microliters of the supernatant of each well were removed for cytokine
measurements and stored at -70°C. One microcurie
[3H]thymidine was then added to each culture well. After
another 8 h cells were harvested, and incorporated radioactivity
was measured by scintillation counting. For the detection of cytokines,
supernatants from three wells for each concentration of the ligand were
pooled, and the amounts of IL-4, IFN-
, and TNF- were determined
by ELISA (BioSource, Camarillo, CA). Cells for immunofluorescence
staining were harvested after 12 h, washed twice in PBS, and
stained with FITC-labeled anti-TCRß Ab, phycoerythrin-labeled
anti-CD69 or anti-CD25 Ab (all from Becton Dickinson, San Jose,
CA), or FITC- or phycoerythrin-labeled control IgG. Staining was
analyzed on a FACScan (Becton Dickinson, Mountain View, CA) for 2000
gated events. For cytotoxicity (CTL) assays, target cells (5 x
105) were labeled overnight at 37°C in 1000 µl of CTL
medium (RPMI, 5% FCS, and 1% glutamine) with 50 µCi of
51Cr (DuPont-New England Nuclear, Boston, MA) and then
incubated with different concentrations of peptide for 4 h, washed
twice (200 x g, 20°C, 10 min), and adjusted to
2 x 104 cells/ml. Target cells (2 x
103) were plated into 96-well U-bottom microtiter plates
containing 2 x 104 T cells. After 4-h incubation
(37°C), supernatants were counted in a gamma counter (ME Plus, ICN
Micromedic, Huntsville, AL). Specific lysis was calculated according to
the following formula: (test release (cpm) - spontaneous release
(cpm))/(total incorporation (cpm) - spontaneous release (cpm)) x 100.
The data in Figure 1
are expressed as
percentages of the maximum response. The 100% value in multiple
experiments ranged from 43 to 62% (GDBP) and from 40 to 65% (MS18)
for cytolysis, from 10,505 to 30,054 cpm (GDBP) and from 19,975 to
27,091 cpm (MS18) for proliferation, from 925 to 5,547 pg/ml (GDBP) and
from 410 to 4,165 pg/ml (MS18) for IFN-, from 34 to 179 pg/ml (GDBP)
and from 808 to 2,450 pg/ml (MS18) for IL-4, and from 158 to 234 (GDBP)
and from 128 to 510 median fluorescence (MS18) for CD69
up-regulation.
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GP-B or AF-B APC (1 x 106/ml) were incubated for 2 h at 37°C at the indicated concentration of peptide, then washed twice. T cells (1 x 106/ml) were added and, after an 8-s spin in an Eppendorf centrifuge, were incubated for 5 min at 37°C and washed with PBS. The pellet was immediately solubilized for 30 min in ice-cold lysis buffer containing 1% Nonidet P-40, 10 mM Tris-HCl (pH 7.2), 140 mM NaCl, 2 mM EDTA, 5 mM iodoacetamide, complete protease inhibitor (Boehringer Mannheim, Mannheim, Germany), and 1 mM Na3VO4 (Sigma, St. Louis, MO). After removal of nuclear debris by centrifugation, the supernatants were analyzed by immunoprecipitation and immunoblotting. Rabbit antiserum to ZAP-70 (36) was used for immunoprecipitation. Immunoprecipitation and immunoblotting analysis were performed as previously described (37). An anti-phosphotyrosine mAb 4G10 (Upstate Biotechnology, Lake Placid, NY) was used for detection. Immunoblots were developed by SuperUltraSignal chemiluminescence (Pierce, Rockford, IL). Quantitative data were obtained from film exposures with a Molecular Dynamics laser densitometer (Molecular Dynamics, Sunnyvale, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first explored the activation of different functional responses using two human Th0 CD4+ TCC specific for the native self-peptide MBP87–99. To facilitate quantitative comparison among the several measured response parameters, they were all expressed as fractional responses in relationship to the maximum absolute response observed.
As shown in Figure 1
, different concentrations of the native peptide
were required to activate distinct effector functions as well as to
stimulate the expression of surface markers. With the clone GDBP,
perforin-mediated cytolysis and CD69 expression were induced at lower
peptide concentrations than proliferation, IFN-, or IL-4 production
(Fig. 1A). In contrast, clone MS18 required low
levels of Ag for CD69 up-regulation, cytolysis, and IL-4 production,
but up to 1000-fold more ligand for proliferation and IFN- secretion
(Fig. 1B). Thus, effector functions were activated
according to a clone-specific hierarchy and, at certain concentrations,
even the native Ag could lead to selective activation of only some T
cell functions. Interestingly, in these and other TCC, the order in the
hierarchy was similar for certain functions, with perforin-mediated
cytolysis and CD69 elicited first, followed by proliferation. In
contrast, the relative hierarchical positions for certain cytokines
differed among the clones in a stable manner.
Altered peptide ligands differ in their overall potency from the native ligand and alter the agonist-defined response hierarchy
For autoreactive CD4+ TCC, modifications of
native antigenic peptides that do not affect binding to the HLA-DR
molecule can lead to the production of more potent (superagonist), less
potent (weak agonist), or inhibitory (antagonist) ligands, as assessed
by examining proliferative responses of the TCC (18, 32, 33). The
demonstration of clone-specific response hierarchies, therefore, raised
the question of whether the rank order of the entire set of response
thresholds would be the same using such altered TCR ligands. Two TCC,
GDBP and MS18, were studied in detail because of the availability of a
well-characterized set of modified ligands for each. Superagonist
(T95-K for GDBP; T95-A for MS18), weak agonist (I93-A and T95-A for
GDBP; P99-A and truncated peptide 87–98 for MS18), antagonist/weak
agonist peptide (P96-A for MS18) and antagonist peptide (N92-A for
GDBP), as well as the native peptide MBP87–99 were tested
for their abilities to induce different T cell functions (Fig. 2). In general, the potency change seen
for proliferative responses was found for all responses to a given
ligand. The antagonist peptide N92-A did not elicit any T cell function
in GDBP. The antagonist-weak agonist peptide P96-A induced marginal
up-regulation of CD69 and some cytolysis in MS18, the two functions
requiring the least amount of peptide for their induction by agonist
ligands (Fig. 2B). The perforin-mediated cytolysis
observed with these clones does not require gene transcription, and
activation of this effector mechanism might indeed be expected to be
less stringent than cytokine production in terms of signal quality,
level, and duration.
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). Such changes in the
relative ligand requirements for two events evoked by engagement of a
single receptor are characteristic of a class of ligands traditionally
defined as partial agonists. The degree of TCR down-modulation reflects ligand potency
TCR/CD3 complexes are down-modulated after engagement with MHC
molecule/peptide ligands, providing a single cell measure of receptor
engagement and, presumably, effective signaling (27). We therefore
evaluated TCR expression after stimulation of the clones by different
ligands. In both TCC, TCR down-modulation occurred homogeneously on all
T cells rather than on a fraction of the cell population (Fig. 3A). The experiments
demonstrated a clear relationship between TCR down-modulation and the
concentration and nature of the ligand. As expected, for each ligand,
down-modulation increased with increasing ligand concentration. More
importantly, the hierarchy of ligand potency in functional assays
(superagonist > native peptide > partial/weak agonist
> antagonist) was reflected in the extent of TCR loss at equivalent Ag
concentrations (Fig. 3, B and D). For each
clone, the induction of a particular functional response appeared to
require a specific level of TCR down-modulation regardless of the
potency of the ligand and thus of the actual Ag concentration needed to
achieve this level of TCR internalization (Fig. 3, C and
E). The extent of TCR down-modulation needed to
elicit the various responses showed the same hierarchical relationship
observed by titration of offered peptide. This provided direct evidence
that the hierarchy reflects the amount of TCR signaling required for
each response to be induced. Interestingly, TCR down-modulation
occurred even at concentrations that induced no (GDBP; Fig. 2A) or little (MS18; Fig. 2B)
functional activity, and even the antagonist/weak agonist P96-A (MS18)
induced some receptor loss at very high concentrations, indicating that
TCR down-modulation may in some cases be a more sensitive measure of
engagement by ligand than are traditionally examined functional
responses.
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How do these findings concerning response hierarchies,
ligand potency, and TCR down-modulation relate to previous data on
qualitatively altered proximal TCR signaling in response to
weak/partial agonists and antagonists (7, 20, 21, 38, 39, 40)? We have
recently begun analyzing proximal TCR signaling events in human T cells
in response to peptide/MHC molecule ligands and have demonstrated that
several distinguishable phosphorylated isoforms of ZAP-70-associated
TCR are produced in response to distinct ligands (I. Stefanova et
al., manuscript in preparation). To determine the relationship between
these patterns of proximal TCR signaling and the functional studies
detailed above, we immunoprecipitated ZAP-70 from the lysates of
stimulated TCC GDBP and MS18 and analyzed the level of tyrosine
phosphorylation of ZAP-70 together with phosphorylation of the
associated TCR -chain.
Each individual peptide elicited a distinct pattern of phosphoproteins
that was characteristic for a given ligand (Fig. 4), with the intensity, but not the
pattern, of phosphorylation changing with the concentration of a
particular peptide (Fig. 4A, lower panel, shown
for superagonist T95-K for GDBP). The signaling patterns of the most
potent ligands (superagonists; T95-K for GDBP and T95-A for MS18) were
characterized by the appearance of substantial amounts of
tyrosine-phosphorylated ZAP-70 and three forms of TCR -chain that
migrate under nonreduced conditions as dimers with apparent molecular
masses of 32, 35, and 38 kDa. Superagonist ligands induced the
accumulation of the pp32 and/or pp35 forms and the pp38 form of TCR
in almost an equal ratio. Native ligand induced pp38 in lesser
proportion to pp35 or pp32, along with lower levels of ZAP-70
phosphorylation. The weak agonists stimulated only low levels of
phosphorylated ZAP-70 and pp38, and for TCC GDBP, some pp35 but high
levels of TCR pp32 were obtained. Finally, antagonist ligand N92-A
for TCC GDBP induced no detectable phosphorylated ZAP-70 and little or
no TCR pp38 or pp35, but still yielded significant levels of TCR
pp32 (Fig. 4A). The rank order of the amounts of
phosphorylated ZAP-70 and pp38 detected using all the ligands at the
same offered peptide concentrations was the same as the potency order
seen in the functional assays and when examining TCR down-modulation
(Fig. 5). However, in contrast to TCR
pp38, the absolute amount of ZAP-70 induced by the different ligands at
the highest Ag doses did not directly correlate with the functional
activity seen. The relatively high level of ZAP-70 phosphorylation by
superagonist ligands that induced little TCR pp32 and the relatively
low level of ZAP-70 phosphorylation by weak agonists that induced high
levels of TCR pp32 may indicate that the abundance of pp32
correlates with a biochemical process that interferes with ZAP-70
phosphorylation, providing a possible explanation for the relative
shifts in response thresholds by weak agonists.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The difference in offered peptide concentration required to evoke the
most readily elicited vs the least readily elicited responses in the
hierarchy can exceed 2 to 3 orders of magnitude. It is thus easy to
expose T cells to Ag amounts that allow relatively selective induction
of some and not other effector activities. For the Th0 cells tested,
IL-4 and IFN- show distinct activation thresholds. These differences
result in a changing ratio of these counter-regulatory cytokines as the
Ag concentration increases, which, in turn, could result in a different
degree of polarization of the immune response toward the humoral or
inflammatory mode depending on the Ag dose. The early observations of
Parish (26) and Bretscher et al. (23) demonstrating that low Ag doses
favor cell-mediated immunity, whereas higher doses favor humoral
immunity may be explained in part by this hierarchical relationship of
cytokine response thresholds. In vivo, when T cell responses to an Ag
are heterogeneous, the activation hierarchy of the majority of cells
within the bulk population will determine which function(s) will be
elicited at a given Ag load. If, for example, clones such as MS18
predominate in a bulk population, stimulation by a weak ligand or a low
concentration of native peptide will lead to full IL-4 but little or no
IFN- secretion. Similarly, in an autoimmune disease or experimental
models such as experimental allergic encephalomyelitis, the activation
hierarchy and the functional repertoire of disease-mediating cells will
determine whether a certain dose of Ag will promote proinflammatory
responses and disease or, instead, induce an anti-inflammatory
cytokine milieu and be therapeutic (42, 43, 44, 45).
The modified peptide ligands tested tend to elicit effector functions in a hierarchical order similar, but not identical, to that seen with the prototypic agonist. They differ, however, in the amount of ligand required to induce any of the responses. Some ligands are more potent than the native ligand and induce each activity at a lower concentration of peptide (superagonists). Other peptides are less potent, and the weakest among these are unable to induce a measurable level of those functions that require the greatest amount of Ag to be stimulated. By itself, the loss of only these most difficult to induce responses upon alteration of a peptide structure classifies such ligands as weak agonists. Some ligands induce little or no functional activity, but efficiently inhibit responses to simultaneously offered native ligand, falling into the category of functional receptor antagonists.
Lanzavecchia and colleagues have introduced flow cytometric analysis of
TCR down-modulation as a convenient measure of effective receptor
engagement by ligand (27, 31, 40, 46) and have shown that for IFN-
production, similar levels of cytokine production occur with a strong
and weak agonist when the extent of TCR loss they induce is similar
(27). We have substantially extended these observations here, showing
that for the entire set of measured responses and for a number of
different ligands, the extent of down-modulation for various ligands at
the same concentration reflects the potency rank order of the ligands
in functional assays. With increasing concentrations of each ligand,
the down-modulation increases proportionally and in accord with the
increase in functional activation of the TCC. The several peptides
tested for each clone form the similar number of complexes with HLA-DR
at a given concentration (18), and it is likely that the rates of
initial physical interaction of TCR on a TCC with these different
ligands are comparable. Although the precise biochemical basis for
ligand-induced TCR down-modulation is not well defined (47, 48), these
data suggest that the fraction of such TCR-ligand contacts resulting in
an effective signal differs according to the structure of the ligand,
and it is specifically these effective engagement events that result in
T cell responses, at least of the type measured here. This hypothesis
is supported by the biochemical data showing a similar relationship
among the extent of ZAP-70 phosphorylation, TCR down-modulation, and
functional response. Although it is tempting to also suggest from these
relationships that ZAP-70 itself is the key proximal signaling molecule
whose activity is limiting for all the measured responses, we as yet do
not have direct evidence for this contention.
The biochemistry of TCR signaling in the human TCC does not, however,
fit completely with a simple model in which recognition of the less
potent agonist ligands by the TCR generates the same set of signals,
but only less frequently. If this were the case, then the pattern of
receptor-associated phosphorylation events should be identical when
using either a given amount of the less potent ligand or a lower amount
of strong agonist chosen to yield an equivalent functional response,
extent of ZAP-70 phosphorylation, or TCR down-modulation. In contrast
to this expectation, reproducible and significant differences in the
ratios of phosphorylated TCR isoforms (pp32, pp35, and pp38) were
seen upon receptor engagement with distinct peptide-MHC class II
complexes. The combination of high levels of pp32 with little or no
pp35 or pp38, as seen with weak agonists and antagonists, could not be
reproduced by titration of agonist.
This argues for a more complex view of these early signaling events.
Given past data with mouse T cell clones (19, 20, 21) and the present
results, it appears likely that any individual TCR on a cell can signal
in either of at least two modes: one giving rise to the full
phosphorylation pattern including pp38 and phosphorylation of
ZAP-70 and TCR down-modulation, and the other producing only pp32
and associated, but not phosphorylated, ZAP-70 and no TCR
down-modulation. The appearance of both types of signals in varying
ratios when using different ligands with the same clone presumably
reflects the relative proportions of engaged TCR signaling in each of
the two modes, dictated by such parameters as dissociation rate of
TCR-ligand pairs (39, 49) and rate of coreceptor (CD4 or CD8)
recruitment to the engaged receptors (50, 51). The overall potency of a
ligand would reflect the number of effective signaling events obtained
per available ligand on the APC. In addition to the potency change,
alterations in the relative positioning of specific responses within
the hierarchy may be due to variations in the pattern of intracellular
signaling that accompany the changing ratios of fully and incompletely
signaling TCR elicited by distinct ligands. Such changes presumably
affect the balance of activated transcription factors within the cell,
which, in turn, may impact the regulation of individual target genes in
distinct ways.
Given 1) the simultaneous generation of two distinct types of proximal
TCR signaling complexes in a single T cell and 2) the correlation of
pp32 phospho- generation with the inhibitory activity of
antagonists, it is tempting to propose that those TCR showing the
pp32/pp35 -associated signaling pattern may in some manner
contribute to an active inhibition of the effective signaling that
would typically occur upon engagement of other TCR. Recent data
obtained using human CD4+ as well as CD8+ TCC
indicate that the phosphatase SHP-1 can be recruited by TCR-associated
pp32/pp35 phospho- (I. Stefanova et al., manuscript in preparation).
The presence of this phosphatase in clusters of engaged TCR may modify
the level and the nature of downstream signaling, contributing to both
the potency shifts and the changes in hierarchical relationships seen
using distinct ligands of the TCR. Clearly, additional investigation is
needed to examine the roles of such putative active inhibitory
processes in TCR-dependent events evoked by ligands of distinct
structures.
The modified ratios of phosphorylated TCR isoforms elicited in human
TCC by TCR antagonists resemble those previously reported for mouse T
cells (19, 20, 21). The major difference from the mouse data is that with
weak agonist ligands for the human TCC, phosphorylated ZAP-70
associated with the TCR complex is detected at the same time as an
altered ratio of isoforms is seen. Application to mouse Th1 clones
of the same methods able to detect low level ZAP-70 phosphorylation
signals in human TCC in this study have not revealed ZAP-70
phosphorylation upon stimulation with the altered ligands (I. Stefanova
and R. N. Germain, unpublished observations), and in contrast to
the ligands employed in this report, the stimulatory activities of the
native ligand and the altered peptides in the previous mouse studies
differed by several orders of magnitude (7, 19, 21). It is therefore
difficult to assess whether the patterns observed with our TCC using
weak agonist ligands are unique to human T cells or a result of the
widely different stimulatory potencies of the ligands examined in the
two species.
Taken as a whole, the data presented here indicate that the amounts of the cytokines produced by a CD4+ TCC in response to a given dose of Ag depend on ligand potency, a factor that correlates with the efficacy of inducing adequate signaling upon ligand interaction with the TCR. At the same time, the ratio of the cytokines produced depends on the hierarchical arrangement shown by that T cell in the signaling thresholds for the individual responses. Finally, altered TCR ligands differ from the parental ligand mainly in potency and also in the relative positionings of individual thresholds within the hierarchy. These latter two effects result in changes in the cytokine response to Ag offered at various concentrations 1) as a consequence of a failure of attainable Ag levels to engender enough signaling for the highest response thresholds to be reached, and 2) as a result of the altered positioning of individual thresholds within the overall hierarchy. Although the activation of cloned cells may not necessarily reflect the activation of T cells in vivo, the data could explain reports of a polarization of immunity toward a humoral or cell-mediated response at different levels of Ag exposure, of selective Th1 vs Th2 cytokine production at distinct Ag levels (23, 24, 25, 26), and of the ability of TCR partial agonists to modulate immune response quality or the balance between thymocyte-positive and -negative selections (52, 53, 54).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Roland Martin, Neuroimmunology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Building 10, Room 5B-16, 10 Center DR MSC 1400, Bethesda, MD 20892-1400.
3 Abbreviations used in this paper: TCC, T cell clone; MBP, myelin basic protein peptide.
Received for publication December 4, 1997. Accepted for publication February 13, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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phosphorylation without ZAP-70 activation induced by TCR antagonists or partial agonists. Science 167:515.
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ß-transgenic model. J. Exp. Med. 182:1579. and tumor necrosis factor production. J. Exp. Med. 180:2227.This article has been cited by other articles:
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A. K. Stanic, R. Shashidharamurthy, J. S. Bezbradica, N. Matsuki, Y. Yoshimura, S. Miyake, E. Y. Choi, T. D. Schell, L. Van Kaer, S. S. Tevethia, et al. Another View of T Cell Antigen Recognition: Cooperative Engagement of Glycolipid Antigens by Va14Ja18 Natural TCR J. Immunol., November 1, 2003; 171(9): 4539 - 4551. [Abstract] [Full Text] [PDF]
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C. M. Bonefeld, A. B. Rasmussen, J. P. H. Lauritsen, M. von Essen, N. Odum, P. S. Andersen, and C. Geisler TCR Comodulation of Nonengaged TCR Takes Place by a Protein Kinase C and CD3{gamma} Di-Leucine-Based Motif-Dependent Mechanism J. Immunol., September 15, 2003; 171(6): 3003 - 3009. [Abstract] [Full Text] [PDF]
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D.-G. Lim, J. M. Slavik, K. Bourcier, K. J. Smith, and D. A. Hafler Allelic Variation of MHC Structure Alters Peptide Ligands to Induce Atypical Partial Agonistic CD8+ T Cell Function J. Exp. Med., July 7, 2003; 198(1): 99 - 109. [Abstract] [Full Text] [PDF]
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V. Hurez, A. Saparov, A. Tousson, M. J. Fuller, T. Kubo, J. Oliver, B. T. Weaver, and C. T. Weaver Restricted Clonal Expression of IL-2 By Naive T Cells Reflects Differential Dynamic Interactions with Dendritic Cells J. Exp. Med., July 7, 2003; 198(1): 123 - 132. [Abstract] [Full Text] [PDF]
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A. E. Annenkov, G. M. Daly, T. Brocker, and Y. Chernajovsky Clustering of immunoreceptor tyrosine-based activation motif-containing signalling subunits in CD4+ T cells is an optimal signal for IFN-{gamma} production, but not for the production of IL-4 Int. Immunol., May 1, 2003; 15(5): 665 - 677. [Abstract] [Full Text] [PDF]
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 B. Chmielowski, R. Pacholczyk, P. Kraj, P. Kisielow, and L. Ignatowicz Presentation of antagonist peptides to naive CD4+ T cells abrogates spatial reorganization of class II MHC peptide complexes on the surface of dendritic cells PNAS, November 12, 2002; 99(23): 15012 - 15017. [Abstract] [Full Text] [PDF]
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C. Dumont, N. Blanchard, V. Di Bartolo, N. Lezot, E. Dufour, S. Jauliac, and C. Hivroz TCR/CD3 Down-Modulation and {zeta} Degradation Are Regulated by ZAP-70 J. Immunol., August 15, 2002; 169(4): 1705 - 1712. [Abstract] [Full Text] [PDF]
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A. D. Bitmansour, D. C. Douek, V. C. Maino, and L. J. Picker Direct Ex Vivo Analysis of Human CD4+ Memory T Cell Activation Requirements at the Single Clonotype Level J. Immunol., August 1, 2002; 169(3): 1207 - 1218. [Abstract] [Full Text] [PDF]
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 B. Bankamp, S. P. Kearney, X. Liu, W. J. Bellini, and P. A. Rota Activity of Polymerase Proteins of Vaccine and Wild-Type Measles Virus Strains in a Minigenome Replication Assay J. Virol., June 14, 2002; 76(14): 7073 - 7081. [Abstract] [Full Text] [PDF]
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 Z. Borovsky, G. Mishan-Eisenberg, E. Yaniv, and J. Rachmilewitz Serial Triggering of T Cell Receptors Results in Incremental Accumulation of Signaling Intermediates J. Biol. Chem., June 7, 2002; 277(24): 21529 - 21536. [Abstract] [Full Text] [PDF]
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W. Ise, M. Totsuka, Y. Sogawa, A. Ametani, S. Hachimura, T. Sato, Y. Kumagai, S. Habu, and S. Kaminogawa Naive CD4+ T Cells Exhibit Distinct Expression Patterns of Cytokines and Cell Surface Molecules on Their Primary Responses to Varying Doses of Antigen J. Immunol., April 1, 2002; 168(7): 3242 - 3250. [Abstract] [Full Text] [PDF]
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 K. Katagiri, M. Hattori, N. Minato, and T. Kinashi Rap1 Functions as a Key Regulator of T-Cell and Antigen-Presenting Cell Interactions and Modulates T-Cell Responses Mol. Cell. Biol., February 15, 2002; 22(4): 1001 - 1015. [Abstract] [Full Text] [PDF]
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 J. Rachmilewitz, G. J. Riely, J.-H. Huang, A. Chen, and M. L. Tykocinski A rheostatic mechanism for T-cell inhibition based on elevation of activation thresholds Blood, December 15, 2001; 98(13): 3727 - 3732. [Abstract] [Full Text] [PDF]
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T. N. J. Bullock, D. W. Mullins, T. A. Colella, and V. H. Engelhard Manipulation of Avidity to Improve Effectiveness of Adoptively Transferred CD8+ T Cells for Melanoma Immunotherapy in Human MHC Class I-Transgenic Mice J. Immunol., November 15, 2001; 167(10): 5824 - 5831. [Abstract] [Full Text] [PDF]
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K. E. de Visser, T. A. Cordaro, H. W. H. G. Kessels, F. H. Tirion, T. N. M. Schumacher, and A. M. Kruisbeek Low-Avidity Self-Specific T Cells Display a Pronounced Expansion Defect That Can Be Overcome by Altered Peptide Ligands J. Immunol., October 1, 2001; 167(7): 3818 - 3828. [Abstract] [Full Text] [PDF]
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H. M. Diepolder, N. H. Gruener, J. T. Gerlach, M.-C. Jung, E. A. Wierenga, and G. R. Pape Different Levels of T-Cell Receptor Triggering Induce Distinct Functions in Hepatitis B and Hepatitis C Virus-Specific Human CD4+ T-Cell Clones J. Virol., September 1, 2001; 75(17): 7803 - 7810. [Abstract] [Full Text] [PDF]
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Y. Zhao, B. Gran23, C. Pinilla, S. Markovic-Plese, B. Hemmer, A. Tzou, L. W. Whitney, W. E. Biddison, R. Martin, and R. Simon Combinatorial Peptide Libraries and Biometric Score Matrices Permit the Quantitative Analysis of Specific and Degenerate Interactions Between Clonotypic TCR and MHC Peptide Ligands J. Immunol., August 15, 2001; 167(4): 2130 - 2141. [Abstract] [Full Text] [PDF]
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 M Chen, B Gran, K Costello, K Johnson, R Martin, and S Dhib-Jalbut Glatiramer acetate induces a Th2-biased response and crossreactivity with myelin basic protein in patients with MS Multiple Sclerosis, August 1, 2001; 7(4): 209 - 219. [Abstract] [PDF]
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S. Krishnan, V. G. Warke, M. P. Nambiar, H. K. Wong, G. C. Tsokos, and D. L. Farber Generation and biochemical analysis of human effector CD4 T cells: alterations in tyrosine phosphorylation and loss of CD3{zeta} expression Blood, June 15, 2001; 97(12): 3851 - 3859. [Abstract] [Full Text] [PDF]
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S. G. Agrawal, J. Marquet, J. Plumas, H. Rouard, M.-H. Delfau-Larue, P. Gaulard, L. Boumsell, F. Reyes, A. Bensussan, and J.-P. Farcet Multiple co-stimulatory signals are required for triggering proliferation of T cells from human secondary lymphoid tissue Int. Immunol., April 1, 2001; 13(4): 441 - 450. [Abstract] [Full Text] [PDF]
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 B. Gran, L. R. Tranquill, M. Chen, B. Bielekova, W. Zhou, S. Dhib-Jalbut, and R. Martin Mechanisms of immunomodulation by glatiramer acetate Neurology, December 12, 2000; 55(11): 1704 - 1714. [Abstract] [Full Text] [PDF]
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F. Micheletti, A. Canella, S. Vertuani, M. Marastoni, L. Tosi, S. Volinia, S. Traniello, and R. Gavioli Supra-Agonist Peptides Enhance the Reactivation of Memory CTL Responses J. Immunol., October 15, 2000; 165(8): 4264 - 4271. [Abstract] [Full Text] [PDF]
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T. A. Colella, T. N.J. Bullock, L. B. Russell, D. W. Mullins, W. W. Overwijk, C. J. Luckey, R. A. Pierce, N. P. Restifo, and V. H. Engelhard Self-Tolerance to the Murine Homologue of a Tyrosinase-Derived Melanoma Antigen: Implications for Tumor Immunotherapy J. Exp. Med., April 3, 2000; 191(7): 1221 - 1232. [Abstract] [Full Text] [PDF]
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B. Hemmer, T. Kondo, B. Gran, C. Pinilla, I. Cortese, J. Pascal, A. Tzou, H. F. McFarland, R. Houghten, and R. Martin Minimal peptide length requirements for CD4+ T cell clones--implications for molecular mimicry and T cell survival Int. Immunol., March 1, 2000; 12(3): 375 - 383. [Abstract] [Full Text] [PDF]
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T. N. J. Bullock, T. A. Colella, and V. H. Engelhard The Density of Peptides Displayed by Dendritic Cells Affects Immune Responses to Human Tyrosinase and gp100 in HLA-A2 Transgenic Mice J. Immunol., March 1, 2000; 164(5): 2354 - 2361. [Abstract] [Full Text] [PDF]
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M. F. Denny, B. Patai, and D. B. Straus Differential T-Cell Antigen Receptor Signaling Mediated by the Src Family Kinases Lck and Fyn Mol. Cell. Biol., February 15, 2000; 20(4): 1426 - 1435. [Abstract] [Full Text]
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A. Chen, G. Zheng, and M. L. Tykocinski Hierarchical Costimulator Thresholds for Distinct Immune Responses: Application of a Novel Two-Step Fc Fusion Protein Transfer Method J. Immunol., January 15, 2000; 164(2): 705 - 711. [Abstract] [Full Text] [PDF]
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H. Liu and D. A. A. Vignali Differential CD3{zeta} Phosphorylation Is Not Required for the Induction of T Cell Antagonism by Altered Peptide Ligands J. Immunol., July 15, 1999; 163(2): 599 - 602. [Abstract] [Full Text] [PDF]
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Y. Itoh, B. Hemmer, R. Martin, and R. N. Germain Serial TCR Engagement and Down-Modulation by Peptide:MHC Molecule Ligands: Relationship to the Quality of Individual TCR Signaling Events J. Immunol., February 15, 1999; 162(4): 2073 - 2080. [Abstract] [Full Text] [PDF]
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S. H. Stotz, L. Bolliger, F. R. Carbone, and E. Palmer T Cell Receptor (TCR) Antagonism without a Negative Signal: Evidence from T Cell Hybridomas Expressing Two Independent TCRs J. Exp. Med., January 18, 1999; 189(2): 253 - 264. [Abstract] [Full Text] [PDF]
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