By
From the * Transplantation and Immunobiology Group, The John P. Robarts Research Institute; the Department of Microbiology and Immunology, The University of Western Ontario, London, Ontario,
Canada; the § Committee on Immunology, Ben May Institute for Cancer Research, University of
Chicago, Chicago, Illinois 60615; and the
Lymphocyte Biology Section, Laboratory of Immunology,
National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda,
Maryland 20892-1892
One hypothesis seeking to explain the signaling and biological properties of T cell receptor for
antigen (TCR) partial agonists and antagonists is the coreceptor density/kinetic model, which proposes that the pharmacologic behavior of a TCR ligand is largely determined by the relative
rates of (a) dissociation of ligand from an engaged TCR and (b) recruitment of lck-linked coreceptors to this ligand-engaged receptor. Using several approaches to prevent or reduce the association of CD4 with occupied TCR, we demonstrate that consistent with this hypothesis,
the biological and biochemical consequence of limiting this interaction is to convert typical agonists into partial agonist stimuli. Thus, adding anti-CD4 antibody to T cells recognizing a
wild-type peptide-MHC class II ligand leads to disproportionate inhibition of interleukin-2
(IL-2) relative to IL-3 production, the same pattern seen using a TCR partial agonist/antagonist. In addition, T cells exposed to wild-type ligand in the presence of anti-CD4 antibodies
show a pattern of TCR signaling resembling that seen using partial agonists, with predominant
accumulation of the p21 tyrosine-phosphorylated form of TCR-, reduced tyrosine phosphorylation of CD3
, and no detectable phosphorylation of ZAP-70. Similar results are obtained
when the wild-type ligand is presented by mutant class II MHC molecules unable to bind
CD4. Likewise, antibody coligation of CD3 and CD4 results in an agonist-like phosphorylation pattern, whereas bivalent engagement of CD3 alone gives a partial agonist-like pattern. Finally, in accord with data showing that partial agonists often induce T cell anergy, CD4 blockade during antigen exposure renders cloned T cells unable to produce IL-2 upon restimulation.
These results demonstrate that the biochemical and functional responses to variant TCR
ligands with partial agonist properties can be largely reproduced by inhibiting recruitment of
CD4 to a TCR binding a wild-type ligand, consistent with the idea that the relative rates of
TCR-ligand disengagement and of association of engaged TCR with CD4 may play a key role
in determining the pharmacologic properties of peptide-MHC molecule ligands. Beyond this
insight into signaling through the TCR, these results have implications for models of thymocyte selection and the use of anti-coreceptor antibodies in vivo for the establishment of immunological tolerance.
Recent evidence indicates that the TCR transduces different signals depending on the precise structure of
the peptide-MHC molecule ligand to which it binds (1).
This differential signaling has been associated with selective
cytokine induction (by TCR partial agonists) or inhibition
of secretion (by TCR antagonists) (4), selective upregulation of surface molecule expression (7), or development
of T cell anergy in the presence of adequate CD28-related
costimulation (1, 8, 10). The mechanistic basis for these
distinct pharmacologic properties of closely related peptide-
MHC molecule ligands recognized by the same TCR is
not well understood. A number of models have been proposed to explain how ligand structural variation can be
translated into altered TCR signaling and T cell activation
(3, 11). Among these, the most widely accepted hypothesis postulates that the affinity of the receptor and,
most probably, the dissociation rate of the ligand from the
receptor, determines what effect antigen recognition has on
the T cell (3, 12). In its simplest form, this model argues
that different biochemical events take place at distinct times
after the TCR has bound ligand, and premature dissociation results in the occurrence of only some but not others of
these. Increasing the amount of a ligand would then increase the amount of those signals occurring before dissociation, but would not lead to the generation of any of those
events that typically require a longer time of TCR-ligand
association to occur. This model provides a simple explanation for the reported data on the effects of TCR interaction
with partial agonists or antagonists on early TCR-related tyrosine phosphorylation events, which show an altered pattern that varies in quantity with ligand amount, but which
remain distinct from that seen using agonist at all tested
concentrations of ligand (1, 2, 16). It is also in general
agreement with very recent studies that have correlated the
functional properties of peptide-MHC molecule ligands with
their measured affinity for soluble versions of the TCR studied in the absence of coreceptor (17, 18).
The major alternative models focus on structural rather
than affinity issues. One argues for an efficacy component
to TCR-ligand engagement that reflects a conformational
change in the TCR upon ligand binding that is necessary
for all the typical downstream biochemical events seen using agonist ligands. Alternatively, emphasis is placed on a
narrow requirement for alignment of the various proteins
(TCR, CD4, MHC-peptide) involved in an effective signaling complex, such that misalignment, irrespective of the absolute ligand-TCR affinity, would result in aberrant signaling and altered functional response (19, 20). Only very
limited data offer support for these hypotheses, such as the
apparent realignment of TCR on the MHC surface when
the side chains of a peptide are altered (21), and the ability
of only some but not other monovalent anti-TCR antibody fragments to synergize with cross-linking antibodies
in T cell activation (22).
Independently of whether kinetic or architectural models or both are correct, one still has to explain in biochemical terms how either type of change in ligand-TCR interaction gives rise to distinct early TCR-mediated signaling
events. Several investigators have pointed out how variations in ligand structure leading to either diminished TCR
affinity or altered receptor architecture could affect proper
recruitment of the CD4 or CD8 coreceptors and associated
lck molecules. Coligation of CD4 or CD8 with the TCR is
well documented to augment markedly functional T cell
responses to ligand (23), as well as to change the overall
pattern of intracellular phosphorylation (24). For peptide-MHC class I ligands, cobinding of CD8 has been
demonstrated to decrease the rate of ligand dissociation
from the TCR (28). Recent functional studies have shown
that CD8 blockade can convert a poor antagonist peptide
into a good TCR antagonist (29) and that reductions in
available CD4 levels can change a partial or weak agonist
into an antagonist (30, 31). However, none of these studies
has examined the relationship between the extent of successful TCR-coreceptor coassociation and early TCRassociated intracellular signaling events. Such studies could
prove especially helpful in relating coreceptor function to
the properties of variant TCR ligands, in light of the recent
data on the consistent pattern of altered early TCR-associated tyrosine phosphorylation seen using partial agonists
and antagonists (12, 16, 32). Here, we have directly tested
the hypothesis that the particular pattern of TCR signaling seen using partial agonists and antagonists might be the result of inefficient TCR-coreceptor interactions. Results
obtained from three different approaches all show that limiting recruitment of CD4 to TCR engaged by either a
wild-type peptide-MHC molecule ligand that under normal circumstances shows agonist function, or by antibody,
leads to biochemical and functional responses closely resembling those elicited by partial agonist ligands, including deficient IL-2 production resulting in induction of clonal
anergy. These data are consistent with a key role of coreceptor recruitment to engaged TCR complexes in determining both the signal transduction and functional properties of TCR ligands. Together with previous studies, these
data may help explain some apparently contradictory results
involving thymocyte positive selection, as well as provide
insight into the phenomenon of T cell tolerance in vivo after coadministration of nondepleting anti-CD4 antibody and antigen.
Cells.
3C6 and A.E7 are mouse CD4+ Th1 clones specific
for cytochrome c fragment 81-104 (PCC[81-104]) bound to I-Ek
(33); TK.G4 is a CD4+ Th1 clone specific for sperm whale myoglobin fragment 102-118 (SWmyo[102-118]) bound to I-Ad,
provided by Dr. J.A. Berzofsky (National Cancer Institute, National Institutes of Health, Bethesda, MD) (34). T cell clones
were maintained by cycles of antigen stimulation with irradiated
spleen cells, cytokine expansion, and rest, as previously described
(2). They were used for functional or biochemical experiments
10-14 d after they had last seen antigen. P13.9 L cells transfected with cDNA constructs encoding E Monoclonal Antibodies.
The following mAbs were used in
these experiments: purified RM4.5, a rat IgG against mouse CD4
(Pharmingen, San Diego, CA); GK 1.5 (37), a rat IgG against
mouse CD4; 14-4-4S (38), a mouse antibody specific for class II
molecules containing E Peptides.
The following peptides were used for these experiments: PCC(81-104) (IFAGIKKKAERADLIAYLKQATAK);
PCC(88-104); and Swmyo (102-118) (KYLEFISEAIIHVLHSR).
Peptides were synthesized in the Peptide Synthesis Facility of the
National Institute of Allergy and Infectious Diseases (Dr. J. Coligan, NIH, Bethesda, MD), or commercially provided by Procyon
Biopharma, Inc. (London, Ontario, Canada). They were purified
to greater than 85% by HPLC before use.
Tyrosine Phosphorylation Analysis.
Detection of tyrosine phosphorylation of proteins in CD3 Functional Assays.
Measurement of proliferation and cytokine
(IL-2, IL-3) production were performed following standard procedures (2). In brief, for proliferation assays, 5 × 104 T cells were
incubated with either 5 × 104 mitomycin c-treated L cells and
the indicated concentration of peptide or with the indicated concentration of anti-CD3-fos, anti-CD4-jun, or anti-CD3-fos × antiCD4-jun bivalent antibody for 28 h, after which the cultures were
pulsed with 1 µCi [3H]thymidine for another 20 h before harvesting and counting of incorporated label. Cytokine production
was measured by ELISA of 24-h supernatants from 5 × 104 T
cells stimulated with peptide and 5 × 104 APC or antibody.
Induction of T Cell Anergy.
P13.9 L cells were first loaded with
magnetic beads (Dynal, Inc., Lake Success, NY) by incubating
these cells (30 × 106 in 30 ml of complete medium) with 200 × 106
beads at 37°C for 48 h. P13.9 having ingested beads cells were then harvested by magnetic sorting. A.E7 T cells (1 × 106 per
group in duplicate) were incubated with mitomycin c-treated bead-containing P13.9 cells as APC (1 × 106 per group in duplicate) and 10 or 100 nM PCC(81-104), in the presence of increasing concentrations of anti-CD4 mAb (GK1.5) in 24-well plates in
a final volume of 2 ml at 37°C for 24 h. The T cells were harvested by two rounds of magnetic depletion of APC, followed by
Ficoll centrifugation, and one wash in complete medium. Recovered antigen-free viable T cells (0.5-1.5 × 106) were then rested
in 2 ml of complete medium in 24-well plates for at least 5 d, before
being rechallenged with PCC(81-104) and mitomycin c-treated
P13.9 cells (3 × 104 T cells plus 3 × 104 P13.9 and increasing
concentrations of peptide). IL-2 production was measured by
ELISA using 24-h supernatants from these restimulation cultures.
We have previously reported that 3C6
proliferates and secretes both IL-2 and IL-3 in response to a
mutant I-Ek molecule agonist expressed on transfected L
cell APC (7). Addition of PCC(81-104) to the culture results in the formation of an altered TCR ligand consisting
of the PCC peptide and the mutant I-E molecule (7). The
pattern of protein tyrosine phosphorylation in anti-CD3
The response of 3C6 cells to antigen is CD4 dependent,
as indicated by dose-dependent inhibition of proliferation
or cytokine production using increasing concentrations of
anti-CD4 mAb in culture (Madrenas, J., and R.N. Germain, unpublished observations). This blocking effect, or
the decreased antigen sensitivity seen following loss of surface CD4 expression in T hybridomas, has generally been
considered to result from lower TCR occupancy and, hence, less receptor signaling for activation. However, some
published reports document changes in the pattern of overall intracellular protein phosphorylation in cell lysates when
CD4 or CD8 are coligated with the TCR, and shifts between agonist and antagonist behavior of the same peptide-
MHC molecule combination have been observed when
peptide-MHC molecule ligands are tested under conditions of variable coreceptor expression or availability. Therefore, we examined whether anti-CD4 antibody inhibition of the
3C6 response led to a simple quantitative loss of signal at
any given ligand concentration, or whether it actually altered the pattern of cytokine response to and/or proximal
signaling events associated with TCR recognition of ligand.
3C6 cells were exposed to PCC(88-104)-I-Ek complexes
(wild-type ligand) on APC in the presence of increasing
concentrations of a blocking mAb specific for CD4 (RM4.5).
This was compared with stimulation of the cells in the
presence of a blocking anti-class II mAb specific for I-E
(14-4-4S), to limit ligand availability by a means other than
peptide titration as had been done previously (2) and
shown here in Fig. 2. Addition of anti-CD4 inhibits both
IL-2 and IL-3 secretion, but IL-2 release is disproportionately sensitive to the antibody. Thus, any given fractional
decrease in IL-2 production requires less anti-CD4 than a
similar decrease in IL-3 production, and IL-2 secretion is
abolished using much less anti-CD4 than is necessary to
achieve the same effect on IL-3. In contrast, anti-class II
MHC antibody inhibits IL-2 and IL-3 production to a similar extent. The results obtained using anti-CD4 resemble
those seen upon simultaneous exposure of 3C6 to both agonist and antagonist, under which conditions IL-2 production is preferentially inhibited (7). If PCC(81-104) peptide
is used to stimulate 3C6 instead of PCC(88-104), production of IL-2 requires less TCR occupancy than production
of IL-3, as indicated by the fact that 50% maximal response
for IL-2 production is reached at a lower concentration of
peptide than is required for 50% maximal IL-3 production
(7). Strikingly, under these conditions, exposure to antiCD4 mAb leads to an inversion in the IL-2 and IL-3 dose- response relationship, with relatively greater fractional IL-3 production than IL-2 production at each point in the dose-
response (data not shown). Thus, blockade of CD4 leads to
a functional response similar to that seen upon engagement
of the 3C6 TCR with a variant ligand, and given the distinct results obtained with anti-class II antibody (Fig. 2),
this effect cannot be explained by a simple decrease in occupancy of the TCR.
To determine whether this functional switch in response
to one resembling variant ligand stimulation also reflects a
corresponding change in TCR signaling, we examined TCR
subunit tyrosine phosphorylation in T cells responding to
wild-type ligand in the presence of CD4 blockade. At concentrations of anti-CD4 affecting cytokine production, the
early TCR-dependent signaling response clearly changes from one typical of agonist to one close to that characteristic of partial agonists (preferential accumulation of the p21 tyrosine phosphorylated form of TCR-
To examine whether the lack of CD4 recruitment to the
TCR leads to a partial agonist pattern of response under
conditions not involving antibody ligation of CD4, we
examined the functional and biochemical responses of a
CD4+ T cell clone to its specific peptide presented by either wild-type class II molecules or by class II molecules in
which the main CD4 binding site has been mutated (36,
42, 43). TK.G4 is a Th1 clone specific for sperm whale
myoglobin fragment 102-118 (SWmyo[102-118]) bound
to I-Ad molecules. The proliferative response of TK.G4 to
this peptide-MHC class II molecule combination is CD4
dependent as shown by a significant reduction in the magnitude of the response when CD4 cannot bind to the I-Ad
molecule present on the APC (Fig. 4 a). Under these conditions involving presentation of antigen by a mutant class
II molecule, a pattern of signaling similar to that typical of
partial agonist stimulation is observed (Fig. 4 b). This cannot be simply attributed to lower overall signaling from the
TCR because pp21 TCR-
The differences in TCR-mediated signaling and response seen upon
TCR engagement with peptide-MHC ligands with or
without adequate CD4 coengagement are also observed
when activation of T cells with heterobivalent antibodies
with specificity for both CD3 and CD4 (anti-CD3-fos × anti-CD4-jun) or with bivalent antibody specific for CD3
alone (anti-CD3-fos) are compared. Immunoblotting of
proteins in lysates of T cells exposed to anti-CD3-fos × anti-CD4-jun antibody shows the presence of ligand-induced
tyrosine-phosphorylated species of approximately 36, 38, 42-44, 50, 60, 70, 80, 90, 120, 140, and 150 kDa. In contrast, immunoblotting of lysates of T cells exposed to antiCD3-fos antibody only shows induction of tyrosine-phosphorylated 44, 60, 90, and 120 kDa species, the latter being
of variable magnitude (Fig. 5 a). When analyzed by blotting of anti-ZAP-70 immunoprecipitates, coengagement of
CD3 and CD4 results in a clear induction of tyrosine-phosphorylated p21 and p23 TCR-
Recently, it has been shown
that some partial agonists preferentially induce T cell clonal
anergy rather than cytokine secretory activity even when
presented by live APC able to provide costimulatory function due to expression of CD80/CD86 (1, 10, 44). The ability of these variant TCR ligands to induce anergy appeared to correlate with their ability to induce an altered
pattern of TCR signaling (1). However, our own studies
have indicated that the controlling factor in anergy induction is not the pattern of early phosphorylation itself, but
the combination of a certain level of TCR signaling in the
face of inadequate production of IL-2 due to either agonist
exposure without costimulation, or partial agonist exposure
even with costimulation (10). The ability of anti-CD4 to
selectively inhibit IL-2 production while permitting substantial TCR signaling of the variant pattern suggested that anergy induction might accompany TCR engagement by
agonist ligand on costimulatory APC in the face of antiCD4 blockade. This hypothesis would explain published
findings showing that coadministration of nondepleting
anti-CD4 mAb and antigen can induce a state of antigenspecific tolerance (45, 46). To investigate this possibility, A.E7 cells exposed to PCC(88-104) on I-Ek expressing,
ICAM-1+, CD80+ L cells in the presence of anti-CD4
mAb were recovered after 24 h, rested for 7 d, and restimulated by agonist in the absence of anti-CD4. As shown in
Fig. 6 A, the presence of anti-CD4 mAbs in the primary
challenge of A.E7 T cells with antigenic peptide renders
these cells less able to produce IL-2 in a subsequent rechallenge with the same antigenic peptide in the absence of the
blocking antibody. The effectiveness of anergy induction
by CD4 blockade correlates with the amount of anti-CD4
mAb present in the primary culture (Fig. 6 b). These results
further confirm that CD4 blockade mimics the functional
effects of TCR partial agonists and also provides a possible
explanation for the induction of tolerance using anti-CD4
antibody in vivo.
The mechanistic basis of TCR partial agonism and antagonism is still unknown. Our data show that both the signaling events and functional effects characteristic of TCR
engagement by variant ligands can be largely reproduced
by preventing effective CD4 recruitment during TCR recognition of wild-type ligand. These observations are consistent with the hypothesis that the properties of these altered ligands may be secondary to an inability to support effective TCR-CD4 interaction during ligand recognition.
This conclusion is in agreement with previous data showing differential TCR- CD4 and CD8 bind to monomorphic regions of MHC
class II and class I molecules, respectively (49, 50). A variety of experiments indicate that during antigen presentation these proteins interact with the same MHC molecule-
peptide complex bound to a clonally distributed TCR (51).
This ligand-induced coassociation of the CD4 or CD8
molecule with the TCR complex is analogous to the cytokine or growth factor-induced association of distinct receptor chains (52), and both of the consequences of such
heterotypic protein association as described for cytokine receptors has been observed for CD4 or CD8 and the TCR
(53). Thus, the simultaneous interaction of the TCR
and the CD4 or CD8 molecules with a single MHC molecule decreases the dissociation rate of the MHC ligand
from the TCR (increases the affinity of binding) (28). In
addition, the recruitment of the src kinase lck that is noncovalently associated with the cytoplasmic tail of CD4 or
CD8 contributes directly to biochemical events involved in
signal transduction, just as apposition of kinases associated
with the cytoplasmic regions of cytokine receptors initiates
such events (24, 25, 59). Because of these corecognition and cosignaling roles in antigen-specific T cell activation,
CD4 and CD8 have been termed coreceptors (60, 61).
We have previously proposed that the reason these coreceptors are not an integral part of the TCR complex arises
from the need of the T cell recognition system to discriminate among very structurally similar ligands (related peptides bound to identical MHC molecules) (62). The total
pool of such related ligands is very large compared with the
number of specific ligands whose recognition is intended to
initiate cellular activation. If the TCR had a substantial
MHC-related affinity for the peptide-MHC complex, as
would be the case if the monomorphic recognition activity of the coreceptor were part of the TCR complex itself,
then the mass action of the high total MHC molecule density on APC would constantly initiate T cell responses unless the threshold for activation was set very high. But then
it would be difficult to have high sensitivity to specific ligands
present at low density. By allowing the TCR to interact
first on its own with potential ligands, and then permitting
the coreceptor to associate with a preformed TCR-ligand
complex, one could set a threshold for discrimination that
is dependent on the time it takes for a coreceptor to reach
such an occupied TCR on the membrane, in comparison to the dissociation rate of the TCR-ligand complex. Ligands
that dissociate more rapidly than the coreceptor can bind
the complex would be incapable of being stabilized or of having the coreceptor-associated lck contribute to downstream
signaling events. Those that survive until the coreceptor
can enter into association, stabilize the binding, and contribute lck to the signaling complex would produce an enhanced or even a different biochemical signal suitable for full T cell activation.
This model is a specific form of the kinetic proofreading
model that proposes that, in an analogous way to kinetic
proofreading of DNA replication and protein synthesis,
there is a time lag between TCR occupancy and the onset
of full signaling from the receptor (14). It is consistent with
the finding that the density of coreceptor expression may
determine susceptibility to TCR antagonism (19, 29,
48), recent evidence that the agonist versus antagonist properties of ligands are correlated with their affinities for the TCR (17, 18), and the new evidence we present here
from three independent approaches that limiting recruitment of CD4 to the engaged TCR results in a qualitative
change in the pattern of early tyrosine phosphorylation
events from that typically seen with complete agonists to
one resembling that seen with partial agonists/antagonists.
Whether the TCR is stimulated using a wild-type peptide-
MHC ligand and CD4 availability is reduced by anti-CD4 antibodies or mutation of the CD4 binding site of the class
II molecule, or whether the corecruitment of TCR and
CD4 is controlled using antibodies of various specificities,
we consistently find that TCR engagement alone promotes
accumulation primarily of the p21 form of phosphorylated
A striking feature of the data derived from antibodyblocking experiments is the very rapid switch from the agonist to variant ligand signaling patterns with low amounts
of blocking antibody. We have not yet specifically measured the fraction of CD4 bound by antibody under nonsaturating conditions, but clearly only a modest proportion
of the CD4 molecules need to be blocked for a switch in
the signaling pattern. These data agree with functional results showing that only a small change in coreceptor expression (less than a two to threefold shift) can alter the
pharmacologic properties of peptide-MHC molecule ligands
in vitro (29), and also can switch the pattern of thymocyte
selection from positive to negative, or vice versa, in vivo
(63). In the context of the kinetic model proposed above for
coreceptor function, these data suggest that many T cells
may operate at the limit of the system, such that even a
modest decrease in coreceptor availability (a small delay in
recruitment time) is sufficient to change stimulatory agonist
recognition to the partial agonist pattern and an altered response. This high sensitivity to antibody blocking is also
consistent with the hypothesis that CD4 does not function
as a monomer, but as an oligomer (36, 64, 65), which
would amplify the consequences of coreceptor blockade.
In terms of thymocyte selection, this concept of a critical
role for coreceptor density in the signaling properties of
self-ligands can help explain the results obtained in organ
culture analysis of the peptide requirements for positive selection. Data obtained from fetal thymic organ cultures using Several investigators have shown that antigen administration in the presence of nondepleting anti-coreceptor antibodies induces the development of an antigen-specific state
of tolerance that does not appear to result from deletion
of the antigen-specific T cells (49, 71). Here, we provide a possible explanation for these results by showing that
the blocking of coreceptors is distinct in its effects from decreasing the density of ligand, with the former producing
a change in signaling that can have a selective effect on
IL-2 secretion despite continued intracellular biochemical
changes due to receptor ligation. This combination of partial TCR signaling without adequate IL-2 production has been shown to induce anergy in established The 1 clones
when using altered ligands (10); here, we demonstrate that
wild-type ligands for the TCR offered in the presence of
blocking anti-CD4 antibody also induce an anergic state in
these clones. If such a phenomenon occurs with primary T
lymphocytes in vivo, they would be likely candidates for
the suppressors cells seen in antibody plus antigen-treated animals by Waldmann and associates (74, 76), as they
would fail to generate effector function but could either
compete for growth factors with potential effectors in their
environment, or secrete a subset of cytokines that deviate
the response away from that being measured.
In summary, we have shown that interference with CD4
recruitment to an engaged TCR complex leads to a change
in early TCR-mediated signaling, with conversion from an
agonist to a partial agonist-like pattern. As a consequence,
the response to what was initially an agonist ligand when
CD4 is fully available becomes a partial agonist response,
with a change in the pattern of cytokine production and/or
the induction of T cell anergy. These findings support the
concept that the relative affinity of a ligand for the TCR and the density of coreceptor together define the functional
properties of TCR ligands in both the thymus and periphery, and provide additional insight into the possible molecular basis of variant ligand function.
and E
, as well as the costimulatory molecules ICAM-1 and CD80 (B7-1), were used as
APCs (2, 35, 36).
; 4G10, a mouse IgG2b mAb to phosphotyrosine (Upstate Biotechnology, Inc., Lake Placid, NY), and
500A2 (39), a hamster IgG against mouse CD3
(PharMingen,
San Diego, CA). Blocking experiments were performed using
deaggregated Abs prepared by ultracentrifugation (100,000 g) for
1 h at 4°C. The F(ab
)2 fragment, anti-CD3-fos, has been previously described (40). Anti-CD4-jun F(ab
)2 was derived from
GK 1.5 (Yun Tso, J., and J. Bluestone, unpublished observations), and the bispecific F(ab
)2 anti-CD3-fos × anti-CD4-jun was prepared according to Kostelny et al. (40). Anti-ZAP-70 rabbit antiserum was the gift of Dr. L. Samelson (Cell Biology and
Metabolism Branch, National Institute of Child Health and Development, NIH, Bethesda, MD).
or anti-ZAP-70 immunoprecipitates was performed as described (2). 4G10 mAb was used for
immunoblotting, and 500A2 or rabbit antiserum to ZAP-70 was
used to immunoprecipitate TCR subunits or ZAP-70 and associated proteins, respectively. Immunoprecipitations were performed from 1 × 107 cells per group at 10 min after ligand exposure in the form of antigen-bearing L cells or soluble antibodies,
using culture conditions previously described (2). Signal intensity
was quantitated using an imaging densitometer (Bio Rad, model
GS 700, Hercules, CA) and the Molecular Analyst® Software
(version 1.0, 1994, Bio Rad Laboratories).
Antibody Blockade of CD4 Changes Agonist-induced Cytokine
Secretion and Early Phosphorylation Events to a Partial Agonist/
Antagonist Pattern.
immunoprecipitates is different for 3C6 cells exposed to an
agonist consisting of wild-type I-Ek and PCC(81-104) or
to this peptide-mutant MHC class II combination (2). The
former is characterized by the presence of similar levels of
p21 and p23 tyrosine-phosphorylated forms of TCR-
, tyrosine-phosphorylated CD3
, and kinase-active tyrosinephosphorylated ZAP-70. In contrast, the latter leads to the
predominant presence of the p21 tyrosine phosphorylated
form of TCR-
, little or no tyrosine phosphorylated CD3
,
and the presence of ZAP-70 that is neither detectably phosphorylated nor kinase active, as reported previously (2) and
shown here (Fig. 1 a). This PCC peptide-mutant I-E molecule ligand selectively antagonizes IL-2 production, while also acting as a partial agonist capable of modestly stimulating IL-3 production (7) (Fig. 1 b).
Fig. 1.
The IL-2 response of 3C6 T cells to mutant I-Ek is selectively antagonized by the addition of PCC(81-104). (a) Tyrosine phosphorylation analysis of proteins in CD3 immunoprecipitates from 3C6
cells stimulated with increasing concentrations of agonist (PCC[81-104]- I-Ek) or antagonist (PCC[81-104]-mutant I-E) ligands. T cells were stimulated for 10 min with APC plus PCC(81-104) and CD3
immunoprecipitates (9 × 106 cell equivalents/lane) were electrophoresed and
immunoblotted with an anti-phosphotyrosine antibody. (b) 3C6 T cells (5 × 104) were stimulated with mitomycin C-treated, wild-type I-Ek, or mutant I-E-transfected L cells and PCC(81-104) (100 µM). Production of
IL-2 and IL-3 was measured in 24-h supernatants by ELISA. Crosshatched bars show IL-2 and closed bars show IL-3.
[View Larger Version of this Image (37K GIF file)]
Fig. 2.
Effect of anti-CD4 and anti-MHC class II mAbs on IL-2 and
IL-3 production by 3C6 T cells responding to the wild-type ligand PCC(88-104)-I-Ek. T cells (5 × 104 per well) were stimulated with I-Ekexpressing L cells and increasing concentrations of PCC(88-104) for 24 h
in the absence or the presence of the indicated concentrations of antiCD4 or anti-class II mAb. Supernatants were then collected and IL-2
(open circles) and IL-3 (closed triangles) measured by ELISA. Results are
expressed as the percent of cytokine produced considering the maximal
cytokine measured in each experiment in the absence of blocking antibody as 100%.
[View Larger Version of this Image (30K GIF file)]
, with less phosphorylated CD3
, and no detectable phosphorylated ZAP-70)
(Fig. 3 a). This shift is unlikely to reflect active signaling
following antibody interaction with the CD4 molecule
because we used soluble deaggregated antibodies (41), the
L cell APC do not express Fc receptors, and no significant tyrosine phosphorylation of TCR subunits is observed when
antigen is omitted from cultures containing anti-CD4, T cells,
and APC (data not shown). The effect of anti-class II mAb
is different from that of the anti-CD4 and similar to what
has been reported previously for changes in antigenic peptide concentration (1, 2, 16), namely, a uniform decrease in
phosphorylation of all TCR subunits or the associated
ZAP-70. These results seen using anti-CD4 versus anticlass II antibodies were confirmed by quantitative densitometric analysis of the pp21, pp23, and pZAP-70 bands from
three different experiments (Fig. 3 b). The ratio between pp23 and pp21 is markedly decreased with higher concentrations of mAb against CD4, whereas this ratio remains
stable for samples treated with anti-class II MHC. In addition, the ratio between pZAP-70 and pp21 falls to zero in
those samples with higher concentrations of anti-CD4
mAb. Nevertheless, as reported previously (2, 3), ZAP-70
is recruited to the TCR complex upon TCR engagement
of ligand even if anti-CD4 antibody is present and no
phosphorylated ZAP-70 is observed (Fig. 3 c).
Fig. 3.
Effect of anti-CD4 antibody on antigen-induced tyrosine
phosphorylation of TCR subunits. (a) 3C6 T cells (1 × 107) were stimulated with I-Ek-transfected L cells and PCC(88-104) (100 µM) for 10 min in the presence of increasing concentrations of anti-CD4 mAb (RM
4.5) or anti-class II mAb (14-4-4S). Cell were lysed with lysis buffer containing 1% Triton X-100, and TCR subunits were immunoprecipitated using a mAb against mouse CD3 (500A2). Immunoprecipitates were immunoblotted using a mAb against phosphotyrosine (4G10). (b) Optical
density of the pp21, pp23, and the pZAP-70 signals from three independent experiments was measured using an imaging densitometer, and the
pp23/pp21 and pZAP-70/pp21 ratios displayed. (c) 3C6 T cells (1 × 107)
were stimulated with I-Ek-transfected L cells and PCC(88-104) (100 µM)
for 10 min in the presence of anti-CD4 mAb (RM 4.5) or anti-class II
mAb (14-4-4S). Cell were lysed with lysis buffer containing 1% Triton
X-100, and TCR subunits were immunoprecipitated using a mAb against
mouse ZAP-70. Immunoprecipitates were immunoblotted using a mAb
against phosphotyrosine (4G10).
[View Larger Versions of these Images (40 + 39K GIF file)]
reaches a higher level under
these conditions than when the same clone is stimulated
with 10 µM SWmyo(102-118) and wild type I-Ad, yet the
latter produces a higher proliferative response and shows
substantial pp23 TCR-
accumulation not seen with the
mutant class II ligand (data not shown).
Fig. 4.
TK.G4 T cell responses to cognate peptide bound to wildtype MHC class II molecules or to mutant MHC class II molecules unable to bind CD4. (a) Proliferative response of TK.G4 cells stimulated with Swmyo(102-118) + wild-type I-Ad or Swmyo(102-118) + I-Ad
mutated at the primary CD4 binding site. (b) Tyrosine phosphorylation analysis of cell lysates (top) and anti-CD3 (bottom) immunoprecipitates from TK.G4 cells stimulated under the same conditions.
[View Larger Version of this Image (28K GIF file)]
, CD3
, and ZAP-70, similar to the pattern seen with agonist peptide-MHC class II
ligands (1, 2, 16). Activation of T cells with CD3 engagement alone instead induces a pattern of TCR-associated phosphoproteins resembling that seen using partial agonists/antagonists, with pp21 TCR-
predominating, a very
low amount of pp23 TCR-
, and no detectable phosphorylated ZAP-70 (Fig. 5 b). In some, but not other experiments, a limited amount of pp21 TCR-
was formed in response to anti-CD4-jun alone (data not shown), as reported previously (24). Functionally, substantial cell proliferation and IL-2 production is seen upon stimulation with antiCD3-fos × anti-CD4-jun, but not using anti-CD3-fos only
or anti-CD4-jun only (Fig. 5 c), in agreement with previous observations that IL-2 production most closely tracks pp23
TCR-
accumulation and/or phosphorylation of ZAP-70
(1, 2).
Fig. 5.
Biochemical and functional consequences of anti-CD3-fos,
anti-CD4-jun, or anti-CD3-fos × anti-CD4-jun bivalent cross-linking. Tyrosine phosphorylation in cloned T cells after CD3 cross-linking, CD3/CD4 cocross-linking, or CD4 cross-linking. T cells (1 × 107 per
sample) were stimulated with the 10 µg/ml of antibody in 100 µl of medium for 10 min. Cells were then lysed and a portion of the lysate used
for immunoprecipitation with an antiserum against ZAP-70. Both cell lysates (a) and ZAP-70 immunoprecipitates (b) were electrophoresed and
immunoblotted using anti-phosphotyrosine antibody. (c) Cell proliferation and IL-2 production by T cells stimulated with soluble anti-CD3-
fos, anti-CD4-jun, or anti-CD3-fos × anti-CD4-jun antibodies.
[View Larger Version of this Image (25K GIF file)]
Fig. 6.
Anergy induction by stimulation with wild-type ligand in
the presence of anti-CD4 antibody. (a) A.E7 T cells were incubated with
I-Ek-expressing L cells without or with PCC(81-104) (100 nM) and antiCD4 mAb (1:200 dilution of supernatant) for 24 h. T cells were then isolated and rested for 7 d. At this point, T cells were restimulated with
I-Ek-transfected L cells and PCC(81-104) (1 µM) for 24 h and IL-2 production was measured by ELISA. (b) Effect of variation in the concentration of anti-CD4 mAb in the primary culture on the extent of anergy induction. A. E7 cells were treated as in (a), but using various concentrations
of anti-CD4 antibody (1:200, open squares; 1:2,000, closed diamonds;
1:20,000, half-filled squares; 1:200,000, half-filled diamonds; no antiCD4, closed triangles). After rest, each of these cell populations was restimulated as in (a) and IL-2 in the medium measured after 24 h by
ELISA.
[View Larger Version of this Image (13K GIF file)]
phosphorylation in CD4+ versus
CD4
T cells exposed to altered peptide-MHC class II
complexes (47), and also with reports indicating that blockade of coreceptor function enhances TCR antagonism
(29), and converts partial agonist ligands into antagonist
ligands (30, 31, 48).
, reduced CD3
accumulation, and receptor association of
ZAP-70 without detectable phosphorylation. In contrast, effective coassociation of the TCR with CD4 leads to formation of both p21- and p23-phosphorylated
, strong
CD3
phosphorylation, and phosphorylation of recruited
ZAP-70. These different patterns of signaling are accompanied by distinct functional responses that match those associated with partial agonist recognition and with agonist recognition, respectively. Although these data do not directly
demonstrate that a limitation in coreceptor recruitment is
the proximal cause of altered signaling in the case of variant peptide-MHC molecule ligands, when the available data
on TCR affinity are taken together with the present results,
they make a strong case that this is one likely origin of the
distinct pattern of signaling that accompanies exposure of T
cells to these modified ligands. Moreover, the present results emphasize the concept that the pharmacologic properties of a TCR ligand are defined not by its absolute affinity
of interaction with the TCR, but by its relative affinity in
the context of a variable time to recruitment of functional
coreceptors whose surface density can vary from cell to
cell.
2m
/
donors expressing an OVA-specific transgenic
TCR clearly show that no concentration of a potent agonist can induce net positive selection, and that if an antagonist or a partial agonist for the mature T cell bearing this
same TCR is offered to the developing thymocytes, the
cells that emerge as CD8+ mature cells cannot respond to
this same ligand (66, 67). However, in another transgenic
model an agonist for the mature T cell source of the TCR
was shown to be capable of promoting differentiation to
the CD8+ state (68, 69). Nonetheless, these cells also do
not show responses to the same peptide used to promote
their differentiation, even though this is an agonist ligand
for the donor T cell (70). In the OVA case, the lack of response correlates with a small but clearcut reduction in
mean CD8 expression by the effectively selected cells, and
this has been shown to be associated with a loss of partial
agonist function for the selecting ligand (29). One might
predict that with the other system, a similar effect is occurring such that among the thymocyte precursors with a
wider range of coreceptor expression than on the TCR donor clone, those cells with the correct CD8 density to signal primarily in the antagonist/partial agonist mode rather
than agonist mode are the ones that avoid negative selection and mature, emerging as unresponsive to the same
ligand that has agonist properties for the donor clone. Thus, rather than operating in a strict avidity mode, with
just the intensity of signaling controlling thymocyte selection, these other data could be reconciled with the OVA
results to argue that successful selection demands a match of
ligand-TCR affinity and thymocyte coreceptor expression
level that results in the altered signaling pattern characteristic
of variant ligands. Interactions outside this range that allow
very effective coreceptor recruitment and support agonist
pattern signaling would instead lead to negative selection.
Address correspondence to Ronald N. Germain, Laboratory of Immunology, Building 10, Room 11N311, 10 Center Drive, MSC-1892, National Institutes of Health, Bethesda, Maryland 20892-1892.
Received for publication 11 September 1996
This work was partially supported by the Medical Research Council of Canada, the Kidney Foundation of Canada, and PO1 AI29531. J. Smith was supported by HL 07605.1. |
Sloan-Lancaster, J.,
A.S. Shaw,
J.B. Rothbard, and
P.M. Allen.
1994.
Partial T cell signaling: altered phospho-![]() |
2. |
Madrenas, J.,
R.L. Wange,
J.L. Wang,
N.A. Isakov,
L.E. Samelson, and
R.N. Germain.
1995.
![]() |
3. | Kersh, G.J., and P.M. Allen. 1996. Essential flexibility in the T-cell recognition of antigen. Nature (Lond.). 380: 495-498 [Medline] . |
4. | Evavold, B.D., and P.M. Allen. 1991. Separation of IL-4 production from Th cell proliferation by an altered T cell receptor ligand. Science (Wash. DC). 252: 1308-1310 [Medline] . |
5. | De Magistris, M., J. Alexander, M. Coggeshall, A. Altman, F.C. Gaeta, H.M. Grey, and A. Sette. 1992. Antigen analog- major histocompatibility complexes act as antagonists of the T cell receptor. Cell. 68: 625-634 [Medline] . |
6. | Windhagen, A., C. Scholz, P. Hollsberg, H. Fukaura, A. Sette, and D.A. Hafler. 1995. Modulation of cytokine patterns of human autoreactive T cell clones by a single amino acid substitution of their peptide ligand. Immunity. 2: 373-380 [Medline] . |
7. | Racioppi, L., F. Ronchese, L.A. Matis, and R.N. Germain. 1993. Peptide-major histocompatibility complex class II complexes with mixed agonist/antagonist properties provide evidence for ligand-related differences in T cell receptor-dependent intracellular signaling. J. Exp. Med. 177: 1047-1060 [Abstract] . |
8. | Sloan-Lancaster, J., B.D. Evavold, and P.M. Allen. 1993. Induction of T-cell anergy by altered T-cell-receptor ligand on live antigen-presenting cells. Nature (Lond.). 363: 156-159 [Medline] . |
9. | Ruppert, J., J. Alexander, K. Snoke, M. Coggeshall, E. Herbert, D. McKenzie, H.M. Grey, and A. Sette. 1993. Effect of T-cell receptor antagonism on interaction between T cells and antigen-presenting cells and on T-cell signaling events. Proc. Natl. Acad. Sci. USA. 90: 2671-2675 [Abstract] . |
10. | Madrenas, J., R.H. Schwartz, and R.N. Germain. 1996. IL-2 and not TCR signaling patterns control anergy induction by agonists or partial agonists. Proc. Natl. Acad. Sci. USA. In press. |
11. | Evavold, B.D., J. Sloan-Lancaster, and P.M. Allen. 1993. Tickling the TCR: selective T-cell functions stimulated by altered peptide ligands. Immunol. Today. 14: 602-609 [Medline] . |
12. | Madrenas, J., and R.N. Germain. 1996. Variant TCR ligands: new insights into the molecular basis of antigen-dependent signal transduction and T cell activation. Semin. Immunol. 8: 83-101 [Medline] . |
13. | Jameson, S.C., and M.J. Bevan. 1995. T cell receptor antagonists and partial agonists. Immunity. 2: 1-11 [Medline] . |
14. | McKeithan, T.W.. 1995. Kinetic proofreading in T-cell receptor signal transduction. Proc. Natl. Acad. Sci. USA. 92: 5042-5046 [Abstract] . |
15. | McConnell, H.M., H.G. Wada, S. Arimilli, K.S. Fok, and B. Nag. 1995. Stimulation of T cells by antigen-presenting cells is kinetically controlled by antigenic peptide binding to major histocompatibility complex class II molecules. Proc. Natl. Acad. Sci. USA. 92: 2750-2754 [Abstract] . |
16. | Reis e Sousa, C., E.H. Levine, and R.N. Germain. 1996. Partial signaling by CD8+ T cells in response to antagonist ligands. J. Exp. Med. 184: 149-157 [Abstract] . |
17. | Alam, S.M., P.J. Travers, J.L. Wung, W. Nasholds, S. Redpath, S.C. Jameson, and N.R.J. Gascoigne. 1996. T-cell-receptor affinity and thymocyte positive selection. Nature (Lond.). 381: 616-620 [Medline] . |
18. | Lyons, D.S., S.A. Lieberman, J. Hampl, J.J. Boniface, Y. Chien, L.J. Berg, and M.M. Davis. 1996. A TCR binds to antagonist ligands with lower affinities and faster dissociation rates than to agonists. Immunity. 5: 51-59 . |
19. | Yoon, S.T., U. Dianzani, K. Bottomly, and C.A. Janeway Jr.. 1994. Both high and low avidity antibodies to the T cell receptor can have agonist or antagonist activity. Immunity. 1: 563-569 [Medline] . |
20. | Janeway, C.A. Jr.. 1995. Ligands for the T-cell receptor: hard times for avidity models. Immunol. Today. 16: 223-225 [Medline] . |
21. | Ehrich, E.W., B. Devaux, E.P. Rock, J.L Jorgensen, M.M. Davis, and Y.-h. Chien. 1993. T cell receptor interaction with peptide/major histocompatibility complex (MHC) and superantigen/MHC ligands is dominated by antigen. J. Exp. Med. 178: 713-722 [Abstract] . |
22. | Janeway, C.A. Jr.. 1992. T cell receptor signaling: high fives or hand clasps? Curr. Biol. 2: 591-593 [Medline] . |
23. | Janeway, C.A. Jr., and K. Bottomly. 1994. Signals and signs for lymphocyte responses. Cell. 76: 275-285 [Medline] . |
24. | Veillette, A., M.A. Bookman, E.M. Horak, L.E. Samelson, and J.B. Bolen. 1989. Signal transduction through the CD4 receptor involves the activation of the internal membrane tyrosine-protein kinase p56lck. Nature (Lond.). 338: 257-259 [Medline] . |
25. | Abraham, N., M.C. Miceli, J.R. Parnes, and A. Veillette. 1991. Enhancement of T-cell responsiveness by the lymphocyte-specific tyrosine protein kinase p56lck. Nature (Lond.). 350: 62-66 [Medline] . |
26. |
Dianzani, U.,
A. Shaw,
B.K. al-Ramadi,
R.T. Kubo, and
C.J. Janeway Jr..
1992.
Physical association of CD4 with the
T cell receptor.
J. Immunol.
148:
678-688
|
27. | Caron, L., N. Abraham, T. Pawson, and A. Veillette. 1992. Structural requirements for enhancement of T-cell responsiveness by the lymphocyte-specific tyrosine protein kinase p56lck. Mol. Cell. Biol. 12: 2720-2729 [Abstract] . |
28. | Luescher, I.F., E. Vivier, A. Layer, J. Mahiou, F. Godeau, B. Malissen, and P. Romero. 1995. CD8 modulation of T-cell antigen receptor-ligand interactions on living cytotoxic T lymphocytes. Nature (Lond.). 373: 353-356 [Medline] . |
29. | Jameson, S.C., K.A. Hogquist, and M.J. Bevan. 1994. Specificity and flexibility in thymic selection. Nature (Lond.). 369: 750-752 [Medline] . |
30. |
Mannie, M.D.,
J.M. Rosser, and
G.A. White.
1995.
Autologous rat myelin basic protein is a partial agonist that is converted into a full antagonist upon blockade of CD4. Evidence
for the integration of efficacious and nonefficacious signals
during T cell antigen recognition.
J. Immunol.
154:
2642-2654
|
31. | Vidal, K., B.L. Hsu, C.B. Williams, and P.M. Allen. 1996. Endogenous altered peptide ligands can affect peripheral T cell responses. J. Exp. Med. 183: 1311-1321 [Abstract] . |
32. | Sloan-Lancaster, J., and P.M. Allen. 1996. Altered peptide ligand-induced partial T cell activation: molecular mechanisms and role in T cell biology. Annu. Rev. Immunol. 14: 1-27 [Medline] . |
33. |
Matis, L.A.,
D.L. Longo,
S.M. Hedrick,
C. Hannum,
E. Margoliash, and
R.H. Schwartz.
1983.
Clonal analysis of the
major histocompatibility complex restriction and the fine
specificity of antigen recognition in the T cell proliferative
response to cytochrome C.
J. Immunol.
130:
1527-1535
|
34. |
Berkower, I.,
H. Kawamura,
L.A. Matis, and
J.A. Berzofsky.
1985.
T cell clones to two major T cell epitopes of myoglobin: effect of I-A/I-E restriction on epitope dominance.
J.
Immunol.
135:
2628-2634
|
35. | Ronchese, F., R.H. Schwartz, and R.N. Germain. 1987. Functionally distinct subsites on a class II major histocompatibility complex molecule. Nature (Lond.). 329: 254-256 [Medline] . |
36. |
König, R.,
X. Shen, and
R.N. Germain.
1995.
Involvement
of both major histocompatibility complex class II ![]() ![]() |
37. |
Dialynas, D.P.,
Z.S. Quan,
K.A. Wall,
A. Pierres,
J. Quintans,
M.R. Loken,
M. Pierres, and
F.W. Fitch.
1983.
Characterization of the murine T cell surface molecule, designated
L3T4, identified by monoclonal antibody GK1.5: similarity
of L3T4 to the human Leu-3/T4 molecule.
J. Immunol.
131:
2445-2451
|
38. |
Ozato, K.,
N. Mayer, and
D.H. Sachs.
1980.
Hybridoma cell
lines secreting monoclonal antibodies to mouse H-2 and Ia
antigens.
J. Immunol.
124:
533-540
|
39. | Havran, W.L., M. Poenie, J. Kimura, R. Tsien, A. Weiss, and J.P. Allison. 1987. Expression and function of the CD3antigen receptor on murine CD4+8+ thymocytes. Nature (Lond.). 330: 170-173 [Medline] . |
40. |
Kostelny, S.A.,
M.S. Cole, and
J.Y. Tso.
1992.
Formation of
a bispecific antibody by the use of leucine zippers.
J. Immunol.
148:
1547-1553
|
41. | Janeway, C.A. Jr.. 1992. The T cell receptor as a multicomponent signaling machine: CD4/CD8 coreceptors and CD45 in T cell activation. Annu. Rev. Immunol. 10: 645-674 [Medline] . |
42. | König, R., L.Y. Huang, and R.N. Germain. 1992. MHC class II interaction with CD4 mediated by a region analogous to the MHC class I binding site for CD8. Nature (Lond.). 356: 796-798 [Medline] . |
43. |
Cammarota, G.,
A. Scheirle,
B. Takacs,
D.M. Doran,
R. Knorr,
W. Bannwarth,
J. Guardiola, and
F. Sinigaglia.
1992.
Identification of a CD4 binding site on the ![]() |
44. | Tsitoura, D.C., W. Holter, A. Cerwenka, C.M. Gelder, and J.R. Lamb. 1996. Induction of anergy in human Th0 cells by stimulation with altered T cell antigen receptor ligands. J. Immunol. 156: 2801-2808 [Abstract] . |
45. | Alters, S.E., H.K. Song, and C.G. Fathman. 1993. Evidence that clonal anergy is induced in thymic migrant cells after anti-CD4-mediated transplantation tolerance. Transplantation. 56: 633-638 [Medline] . |
46. | Waldmann, H., and S. Cobbold. 1993. The use of monoclonal antibodies to achieve immunological tolerance. Immunol. Today. 14: 247-251 [Medline] . |
47. | Vignali, D.A., and J.L. Strominger. 1994. Amino acid residues that flank core peptide epitopes and the extracellular domains of CD4 modulate differential signaling through the T cell receptor. J. Exp. Med. 179: 1945-1956 [Abstract] . |
48. | Rabinowitz, J.D., C. Beeson, C. Wülfing, K. Tate, P.M. Allen, M.M. Davis, and H.M. McConnell. 1996. Altered T cell receptor ligands trigger a subset of early T cell signals. Immunity. 5: 125-135 [Medline] . |
49. | Doyle, C., and J.L. Strominger. 1987. Interaction between CD4 and class II MHC molecules mediates cell adhesion. Nature (Lond.). 330: 256-259 [Medline] . |
50. | Norment, A.M., R.D. Salter, P. Parham, V.H. Engelhard, and D.R. Littman. 1988. Cell-cell adhesion mediated by CD8 and MHC class I molecules. Nature (Lond.). 336: 79-81 [Medline] . |
51. |
Saizawa, K.,
J. Rojo, and
C.A. Janeway Jr..
1987.
Evidence
for a physical association of CD4 and the CD3:![]() ![]() |
52. | Ullrich, A., and J. Schlessinger. 1990. Signal transduction by receptors with tyrosine kinase activity. Cell. 61: 203-212 [Medline] . |
53. | Tite, J.P., A. Sloan, and C.A. Janeway Jr.. 1986. The role of L3T4 in T cell activation: L3T4 may be both an Ia-binding protein and a receptor that transduces a negative signal. J. Mol. Cell. Immunol. 2: 179-190 [Medline] . |
54. | Saizawa, K., S. Haque, B. Jones, J. Rojo, J.P. Tite, J. Kaye, and C.A. Janeway Jr.. 1987. The L3T4 molecule is part of the helper T-cell antigen/Ia recognition complex. Ann. Inst. Pasteur Immunol. 138: 138-143 [Medline] . |
55. | McCluskey, J., A. Singer, R.N. Germain, and D.H. Margulies. 1987. The role of CD4/L3T4 in T-lymphocyte function. Ann. Inst. Pasteur Immunol. 138: 150-157 [Medline] . |
56. | Janeway, C.A. Jr., S. Haque, L.A. Smith, and K. Saizawa. 1987. The role of the murine L3T4 molecule in T cell activation: differential effects of anti-L3T4 on activation by monoclonal anti-receptor antibodies. J. Mol. Cell. Immunol. 3: 121-131 [Medline] . |
57. |
Anderson, P.,
M.L. Blue, and
S.F. Schlossman.
1988.
Comodulation of CD3 and CD4. Evidence for a specific association between CD4 and approximately 5% of the CD3:T cell
receptor complexes on helper T lymphocytes.
J. Immunol.
140:
1732-1737
|
58. | Eichmann, K., J.I. Jonsson, I. Falk, and F. Emmrich. 1987. Effective activation of resting mouse T lymphocytes by crosslinking submitogenic concentrations of the T cell antigen receptor with either Lyt-2 or L3T4. Eur. J. Immunol. 17: 643-650 [Medline] . |
59. | Veillette, A., M.A. Bookman, E.M. Horak, and J.B. Bolen. 1988. The CD4 and CD8 T cell surface antigens are associated with the internal membrane tyrosine-protein kinase p56lck. Cell. 55: 301-308 [Medline] . |
60. | Janeway, C.A. Jr.. 1988. T-cell development. Accessories or coreceptors? Nature (Lond.). 335: 208-210 [Medline] . |
61. | Janeway, C.A. Jr.. 1991. The co-receptor function of CD4. Semin. Immunol. 3: 153-160 [Medline] . |
62. | König, R., S. Fleury, and R.N. Germain. 1996. The structural basis of CD4-MHC class II interactions: coreceptor contributions to T cell receptor antigen recognition and oligomerization-dependent signal transduction. Curr. Topics Microbiol. Immunol. 205: 19-46 [Medline] . |
63. | Robey, E.A., F. Ramsdell, D. Kioussis, W. Sha, D. Loh, R. Axel, and B.J. Fowlkes. 1992. The level of CD8 expression can determine the outcome of thymic selection. Cell. 69: 1089-1096 [Medline] . |
64. | Sakihama, T., A. Smolyar, and E.L. Reinherz. 1995. Oligomerization of CD4 is required for stable binding to class II major histocompatibility complex proteins but not for interaction with human immunodeficiency virus gp120. Proc. Natl. Acad. Sci. USA. 92: 6444-6448 [Abstract] . |
65. | Sakihama, T., A. Smolyar, and E.L. Reinherz. 1995. Molecular recognition of antigen involves lattice formation between CD4, MHC class II and TCR molecules. Immunol. Today. 16: 581-587 [Medline] . |
66. | Hogquist, K.A., S.C. Jameson, and M.J. Bevan. 1994. The ligand for positive selection of T lymphocytes in the thymus. Curr. Opin. Immunol. 6: 273-278 [Medline] . |
67. | Hogquist, K.A., S.C. Jameson, W.R. Heath, J.L. Howard, M.J. Bevan, and F.R. Carbone. 1994. T cell receptor antagonist peptides induce positive selection. Cell. 76: 17-27 [Medline] . |
68. | Ashton-Rickardt, P.G., A. Bandeira, J.R. Delaney, L. Van Kaer, H.P. Pircher, R.M. Zinkernagel, and S. Tonegawa. 1994. Evidence for a differential avidity model of T cell selection in the thymus. Cell. 76: 651-663 [Medline] . |
69. | Sebzda, E., V.A. Wallace, J. Mayer, R.S. Yeung, T.W. Mak, and P.S. Ohashi. 1994. Positive and negative thymocyte selection induced by different concentrations of a single peptide. Science (Wash. DC). 263: 1615-1618 [Medline] . |
70. | Rothenberg, E.. 1995. Developmental biology of lymphocytes. The Immunologist. 3: 172-175 . |
71. | Alters, S.E., J.A. Shizuru, J. Ackerman, D. Grossman, K.B. Seydel, and C.G. Fathman. 1991. Anti-CD4 mediates clonal anergy during transplantation tolerance induction. J. Exp. Med. 173: 491-494 [Abstract] . |
72. | Pearson, T.C., J.C. Madsen, C.P. Larsen, P.J. Morris, and K.J. Wood. 1992. Induction of transplantation tolerance in adults using donor antigen and anti-CD4 monoclonal antibody. Transplantation. 54: 475-483 [Medline] . |
73. | Shizuru, J.A., S.E. Alters, and C.G. Fathman. 1992. AntiCD4 monoclonal antibodies in therapy: creation of nonclassical tolerance in the adult. Immunol. Rev. 129: 105-130 [Medline] . |
74. | Qin, S., S.P. Cobbold, H. Pope, J. Elliott, D. Kioussis, J. Davies, and H. Waldmann. 1993. "Infectious" transplantation tolerance. Science (Wash. DC). 259: 974-977 [Medline] . |
75. | Darby, C.R., A. Bushell, P.J. Morris, and K.J. Wood. 1994. Nondepleting anti-CD4 antibodies in transplantation. Evidence that modulation is far less effective than prolonged CD4 blockade. Transplantation. 57: 1419-1426 [Medline] . |
76. | Scully, R., S. Qin, S. Cobbold, and H. Waldmann. 1994. Mechanisms in CD4 antibody-mediated transplantation tolerance: kinetics of induction, antigen dependency and role of regulatory T cells. Eur. J. Immunol. 24: 2383-2392 [Medline] . |