By
From the Center for Immunology and Department of Microbiology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235-8576
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The role of two central residues (K68, E69) of the fourth hypervariable loop of the V domain
(HV4
) in antigen recognition by an MHC class II-restricted T cell receptor (TCR) has been
analyzed. The TCR recognizes the NH2-terminal peptide of myelin basic protein (Ac1-11,
acetylated at NH2 terminus) associated with the class II MHC molecule I-Au. Lysine 68 (K68)
and glutamic acid 69 (E69) of HV4
have been mutated both individually and simultaneously
to alanine (K68A, E69A). The responsiveness of transfectants bearing wild-type and mutated
TCRs to Ac1-11-I-Au complexes has been analyzed in the presence and absence of expression of the coreceptor CD4. The data demonstrate that in the absence of CD4 expression, K68
plays a central role in antigen responsiveness. In contrast, the effect of mutating E69 to alanine
is less marked. CD4 coexpression can partially compensate for the loss of activity of the K68A
mutant transfectants, resulting in responses that, relative to those of the wild-type transfectants,
are highly sensitive to anti-CD4 antibody blockade. The observations support models of T cell
activation in which both the affinity of the TCR for cognate ligand and the involvement of
coreceptors determine the outcome of the T cell-antigen-presenting cell interaction.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
For the vast majority (90-95%) of T cells, the T cell receptor (TCR) is composed of a heterodimer of highly
diverse transmembrane and
chains, whereas a minor
population bear
TCRs. These
or
heterodimers
associate with the nonpolymorphic CD3 and TCR
chains
to form the functional TCR-CD3 complex. The
heterodimer interacts with cognate peptide-MHC (pMHC)
complexes, whereas the CD3-TCR
chain complex is
involved in signal transduction. These TCR-associated
polypeptides contain tyrosine motifs (called immune receptor tyrosine-based activation motifs, or ITAMs), which are the targets of phosphorylation following TCR triggering
(1). ITAM phosphorylation leads to recruitment of SH2
domain containing proteins, such as ZAP-70 and subsequent signaling cascades (1), and is one of the early events
of T cell activation (2, 3).
The extracellular domains of the TCR resemble the Fab
arms of an antibody at both the sequence and structural
levels, although there are also significant differences in both
complementarity determining regions (CDRs) and overall
fold for both and
chains (4). Structural modeling of
TCR-pMHC complexes led to the suggestion that the less
diverse CDRs 1 and 2 of the TCR
and
chain make contacts with the
helices of the MHC molecule, whereas
the highly variable CDR3 loops interact directly with the
antigenic peptide (7). More recent X-ray crystallographic structures of mouse and human TCR
-pMHC
class I complexes (10) are consistent with these proposed three-dimensional models insofar as CDR3 residues overlie, but do not always make intimate contact with, antigenic peptide, whereas CDR1 and CDR2 residues are
positioned to contact primarily MHC residues with more
limited peptide contacts. The X-ray structures (10)
demonstrate a diagonal orientation of the TCR with respect to pMHC in a configuration that is most likely to be
general, at least for pMHC class I complexes. Earlier structure-function studies of both MHC class I and class II restricted TCRs led to the proposal of a similar orientation
(14), but whether this configuration is generally observed in TCR
-pMHC class II complexes awaits the
elucidation of the corresponding X-ray structures.
Current models of T cell activation support a role for the affinity/avidity of the TCR-pMHC as being a key parameter in the outcome of the T cell-antigen-presenting cell (APC) interaction (17). In particular, the off-rate of the TCR-pMHC complex appears to play a central role (17, 18). The coreceptors CD4 or CD8 can also increase the avidity of the interaction and/or affect the signaling efficiency via associated proteins such as p56lck (19). Using binding assays with living CTLs (24) and soluble molecules in surface plasmon resonance experiments (25), CD8 has been demonstrated to increase the affinity of the TCR for cognate pMHC ligand. In contrast to affinity based models, for effective signaling the need for a conformational change in the TCR post-ligand binding without a major role for avidity has also been proposed (26, 27). Furthermore, data have been presented in support of a requirement for ordered oligomerization or aggregation of TCRs (28), which for optimal signaling may be followed by coreceptor recruitment (31). Conformational and affinity/avidity models are therefore not mutually exclusive; both may be relevant in "sequential engagement" models (31, 32, 34) where there is a need for a threshold time of TCR occupancy to be reached to induce the necessary configuration of TCR-pMHC complexes of a sufficient half-life to allow coreceptor association. This concept is supported by recent data demonstrating that CD4 can enhance responses to agonist ligands but not to antagonist ligands due to the shorter half-life of the TCR antagonist-MHC complex (31). This is also consistent with the proposed role of kinetic proofreading (35, 36) in increasing the fidelity of T cell recognition.
Toward the aim of better understanding the molecular
nature of T cell recognition, in the current study we have
analyzed the effect of alteration of amino acids in the fourth
hypervariable loop of the TCR chain (HV4
) on T cell
responsiveness. The crystal structure of a TCR V
domain
indicated that residues in HV4
are almost coplanar with
the CDR loops, which led to the suggestion that they have
the potential to interact with the pMHC complex (4).
More recently, the structure of a human TCR complexed with an HIV Tax peptide bound to HLA-A2 (11) shows
that the highly conserved residue K68 of HV4
contacts
HLA-A2 by forming hydrogen bonds to residues T163 and
E166 of this MHC class I molecule. These amino acids
are conserved in MHC class I but not in class II molecules
(37). However, in other structural studies describing class
I-restricted TCRs, HV4
residues do not contact pMHC
ligand (10, 12, 13). The functional role of HV4
residues
in antigen responsiveness has not to date been elucidated,
although recent in vitro binding studies with the soluble
2C (murine) TCR and pMHC have indicated a minor but
significant role for HV4
residues in contributing to the
binding energy of this interaction (38).
In this study, we have analyzed the role of HV4 residues of a TCR derived from the encephalitogenic T cell
hybridoma 1934.4 (39) in antigen responsiveness. Using
the X-ray crystallographic structure of the 1934.4 TCR V
domain as a guide (4), amino acid substitutions of the two
central, exposed residues of HV4
(Lysine 68 [K68],
glutamic acid 69 [E69]) have been introduced. Wild-type
(WT) and mutated TCRs have been expressed in T cell transfectants and IL-2 production following antigen exposure analyzed in the presence and absence of CD4 coexpression. Our results indicate that in the absence of CD4
coexpression, mutation of K68 to alanine (K68A) results in
greatly reduced IL-2 production, whereas mutation of E69
to alanine (E69A) has a comparatively minor effect. Furthermore, expression of the coreceptor CD4 can compensate for the reduced responsiveness of transfectants bearing
the K68A mutant TCR. The data indicate that HV4
residues play an important role in modulating T cell responsiveness and support models for T cell activation in which
both affinity of TCR for pMHC ligand and coreceptor
density determine the outcome of the T cell-APC interaction (31, 32, 34).
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell Lines, Antibodies, and Peptides.
The CD4Expression Plasmids.
TheGeneration of Transfectants.
For transfections, the 1934.4Analysis of Cell Surface Expression of TCR and CD4 by Flow Cytometry.
For analysis of cell surface expression of TCR and CD4, transfectants (1 × 105/well in a 200 µl volume) were incubated with 10 µg/ml of anti-VStimulation by Cross-Linking Antibodies or PMA/Ionomycin.
Transfectants (1 × 105/well in 96-well plates) were stimulated either with 10 ng/ml PMA + 500 ng/ml ionomycin, plate-bound anti-VStimulation by Cognate Peptide:MHC Class II.
Transfectants (1 × 105/well in 96-well plates) were incubated with graded doses of the peptides Ac1-11 or Ac1-11[4Y] together with PL-8 cells (1 × 105/well) as APCs. No peptide was added to the control wells. For anti-CD4 or anti-I-Au inhibition, graded doses of anti-CD4 (GK1.5) or anti-I-A (Y3P, 10.2.16) mAbs were used. Appropriate isotype matched antibodies were used as controls. The responses of transfectants were measured by IL-2 production that was detected by the proliferative response of CTLL-2 cells as above. All stimulation assays were done in triplicates and data expressed as percent of response to the plate-bound anti-CD3Circular Dichroism (CD) Analyses.
Far-ultraviolet CD analyses were performed as previously described (44), using an AVIV model 60DS circular dichroism spectrophotometer at 25°C and a cell of 0.2 cm path length. ![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To
analyze the effects of mutating HV4 residues 68 and 69 of
the 1934.4 TCR on antigen responsiveness, WT and mutated
chain genes were transfected with the WT
(V
8.2-J
2.3) chain into a TCR
thymoma, 58
(40). This murine T cell thymoma line lacks an endogenous TCR, but has a functional CD3-TCR
complex that
can be expressed on the cell surface with the transfected
TCR
and
chains. Fig. 1 shows the location of the residues that were targeted for mutagenesis on the X-ray crystallographic structure of the 1934.4 V
domain (4).
|
Mycophenolic acid-resistant transfectants were analyzed
for expression levels of surface TCR by flow cytometry using a V8-specific mAb, F23.1. Since the expression levels
varied between transfectants, transfectants with comparable
levels of surface TCR were chosen for further analysis (Fig.
2). These transfectants were analyzed for responsiveness (assessed by quantitating IL-2 production) to PMA in addition
to anti-V
8 (F23.1) and anti-CD3
(145-2C11) antibody-mediated cross-linking. Responses to all three types of
stimulation did not differ markedly, indicating that the signaling machinery of the transfectants was intact and that the
V
mutations do not affect signaling via antibody-mediated
chain cross-linking (Fig. 2 D). Differences observed upon
145-2C11 (Fig. 2 C) stimulation probably reflect minor
differences in the surface TCR levels observed in Fig. 2 A.
To control for variability, responses to cognate pMHC (below) have been normalized with respect to those obtained from stimulation with 145-2C11 and expressed as percentages of 145-2C11 responses. A similar approach was taken
by Patten and colleagues for the analysis of anti-cytochrome
c-I-Ek responses by transfectants expressing the WT 2B4
TCR and its mutated derivatives (43).
|
The 1934.4 TCR recognizes the NH2-terminal 11-mer (or nonamer) of myelin basic protein (MBP) associated with I-Au and the acetylation of the NH2 terminus of this peptide (abbreviated to Ac1-11) is essential for T cell recognition (39). Position 4 analogs (position 4 substituted by alanine and tyrosine, designated Ac1-11(4A) and Ac1-11(4Y), respectively) of this peptide bind with higher affinity to I-Au (47, 48), resulting in shifts of dose response curves of 1934.4 hybridoma cells (47). We tested the ability of the WT versus mutant transfectants to recognize the Ac1-11 peptide presented in the context of the MHC class II molecule I-Au. When Ac1-11 was used for stimulation (Fig. 3 A), the WT transfectants respond, albeit poorly, whereas there is an almost undetectable response observed for the three types of mutant transfectants (K68A, E69A, and K68AE69A). This poor responsiveness is most likely due to the lack of CD4 expression by the transfectants, which in other systems is known to decrease antigen responsiveness (31, 49). Therefore, to make quantitative comparisons between the WT and mutant transfectants in the absence of CD4, we tested their responses to the higher affinity peptide Ac1-11(4Y). Fig. 3 B shows that the response of the WT transfectants to Ac1-11(4Y) is significantly better compared with their response to Ac1-11 (Fig. 3 A), and this is reminiscent of the data of others for the parent 1934.4 hybridoma (47, 48, 50). The mutant transfectants also show higher and detectable levels of responses to the higher affinity peptide. Replacement of glutamic acid at position 69 with alanine (E69A) results in a slight, but significant, reduction in response as compared with WT; however, the double mutants (K68AE69A) and the K68A mutants show substantial decreases in their responses relative to WT transfectants.
|
The data indicate that lysine at position 68 of HV4
plays a crucial role in the TCR-pMHC interaction, most
likely by either directly contacting Ac1-11-I-Au or indirectly by affecting the conformation of neighboring CDRs in the V
domain. Importantly, several observations indicate that the mutations analyzed in this study do not have
significant effects on the conformation of the TCR V
domain. First, the WT and mutant transfectants express
similar levels of surface TCR, and aberrant folding of the
TCR
chain due to mutation would be expected to affect
this. Second, CD spectroscopic analysis of the WT and mutant V
domains, expressed as recombinant proteins in Escherichia coli, indicate that the V
domains are folded into
structures of high
-sheet content (data not shown). Unfortunately, the lack of an anti-V
4.2 antibody that is conformationally dependent precludes analyses using such a reagent.
Since the differences in response to peptide were
most striking between the WT and the K68A mutant,
transfectants expressing these TCRs were analyzed further.
The T cell coreceptor, CD4, has been shown to enhance
the capability of thymocytes and mature T cells to recognize antigen (reviewed in references 19 and 51). To analyze
whether CD4 can compensate for the effect of the K68A
mutation, cotransfections of the 1934.4 WT or K68A mutant chain together with WT
chain and CD4 expression constructs using the 58
thymoma cell line as recipient were carried out. The surface TCR and CD4 levels
are comparable between the WT and K68A transfectants (Fig. 4 A), although the TCR levels for the transfectant
K68A/CD4-32 are slightly lower. These CD4+ transfectants show similar responses to 145-2C11 and F23.1 stimulation (Fig. 4, B and C). In addition to shifting the dose response curve of the WT transfectants, cotransfection of
CD4 almost completely (K68A/CD4-12 mutant) or partially (K68A/CD4-32 mutant) restores the K68A mutant
responses to cognate pMHC to the levels seen with the
WT transfectant, WT/CD4-43 (Fig. 5). The WT transfectant WT/CD4-33 is, however, still more responsive than
the two mutant transfectants and this is particularly so for
the Ac1-11 peptide (Fig. 5 A). This significant gain of
function for the K68A mutants suggests that either the increased avidity and/or enhancing the efficiency of TCR
signaling by CD4 coexpression can compensate, in part at least, for the suboptimal nature of the K68A TCR-pMHC
interaction. In addition, the enhancing effect of CD4 is
much greater for the K68A mutants than for the WT transfectants (Figs. 3 and 5).
|
|
To demonstrate that the increased IL-2 production by the K68A mutants is due to CD4 coexpression, the sensitivity of the WT and K68A transfectant responses following Ac1-11(4Y)-I-Au exposure to blockade by the anti-CD4 mAb GK1.5 was investigated. K68A transfectants are significantly more sensitive to the effects of GK1.5 than the WT transfectants (Fig. 6 A). Importantly, this difference is observed for K68A/CD4-12 relative to WT/CD4-43, and these two transfectants show essentially the same dose response to Ac1-11(4Y)-I-Au. The effects of GK1.5 are consistent with the observation that WT transfectants without CD4 coexpression are responsive to antigen, in contrast to the K68A transfectants (Fig. 3). Finally, the sensitivity of the WT and K68A transfectants to blockade by anti-I-A antibodies was also investigated, because this was expected to reveal differences in affinities of the corresponding TCR-pMHC complexes. Two anti-class II mAbs (10-2.16 and Y3P) that recognize I-Au were used, and for both anti-MHC class II antibodies the K68A transfectants are more sensitive to inhibition than the WT transfectants (Fig. 6 B; data shown only for 10.2.16). As with the GK1.5 inhibition, this difference in sensitivity to anti-MHC class II inhibition is seen when the WT/CD4-43 and K68A/ CD4-12 transfectants are compared. Taken together with the similarity of the dose responses to cognate antigen in the absence of antibodies for these two transfectants (Figs. 5 and 6), this indicates that the affinity of the K68A TCR for cognate antigen is lower than that of the WT TCR.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To date, there are no high-resolution structural data
available for a TCR-pMHC class II complex, but functional studies (14, 15) of class II-restricted TCRs indicate
that the orientation of the TCR is similar to the diagonal
configuration observed in the X-ray structures of several
class I-restricted TCRs (10). The crystal stuctures indicate that HV4 residues sometimes (11), but not always
(10, 12, 13), make contact with cognate ligand. In addition,
binding studies using a recombinant class I-restricted TCR
indicate a minor contribution for these residues to the
binding energy of the interaction (38). However, the functional relevance of HV4
residues in T cell activation have
not, to our knowledge, been investigated for either class I-
or class II-restricted TCRs, and the current study addresses
this issue for an autoreactive TCR that recognizes MBP
Ac1-11 bound to I-Au. In contrast to amino acids located in
HV4
, the functional significance of HV4
residues have
been more extensively analyzed, and this TCR region is
known to play a role in contacting several bacterial and endogenous superantigens (52). In addition, recent modeling/X-ray structural studies for the interaction of the murine 2C TCR with allo-ligand have indicated that the
HV4
residue R69 might contribute toward the binding
energy through electrostatic effects (55).
Our findings identify a role for HV4 residues of the
1934.4 TCR in pMHC recognition. This is consistent
with the X-ray crystallographic structure of the 1934.4 V
domain (V
4.2), in which HV4
forms part of a relatively
flat continuous surface with CDR1, 2, and 3, leading to the
earlier suggestion that it might be involved in interactions
with cognate pMHC ligand (4). In the current study, the
role of the two central exposed residues (K68, E69) of this
loop in pMHC recognition have been investigated by expressing 1934.4-derived TCRs with mutations to alanine at
these positions in T cell transfectants. K68 is highly conserved in murine and human V
sequences, whereas the
variability at position 69 is higher (37). Antigen responsiveness of the TCR transfectants has been analyzed in the
presence and absence of CD4 coexpression and compared
with that of 1934.4 WT transfectants. These studies have
shown that K68 plays an important role in antigen responsiveness, as assessed by quantitating IL-2 secretion, whereas
E69 has a lesser role. In addition, mutation of both K68
and E69 to alanine within the same TCR results in responsiveness to Ac1-11(4Y)-I-Au complexes that is intermediate between that of the transfectants expressing K68A and
E69A TCRs. However, transfectants expressing this double
mutant show an almost undetectable response similar to
that of the K68A mutants when presented with the lower
affinity peptide Ac1-11 in the context of I-Au. Thus, the
effect of the double mutation appears to be analogous to
that of the K68A mutation, reiterating the importance of
K68 in the interaction.
The role of K68 in pMHC responsiveness of the 1934.4 TCR could be through several possible mechanisms that
are not mutually exclusive. First, K68 may contact either
peptide or I-Au (or both) in the pMHC complex either directly or via ordered water, which has been shown to play
a role in stabilizing antibody-antigen interactions (56, 57).
Electrostatic interactions, which can occur over distances
up to 20 Å (58), may also occur between K68 and cognate
pMHC. Consistent with this possibility and assuming that
the I-Au structure is similar to that of the recent I-Ak/I-Ad
structures (59, 60), several acidic I-Au residues (E84, E85;
numbering as in reference 37) would contact/be in proximity to HV4
if the orientation of 1934.4 TCR binding resembles that in TCR-pMHC class I complexes (10).
Alternatively, the effects of mutation of K68 may be indirect through destabilization of other CDR loops of V
4.2
that are involved in pMHC binding. However, we favor a
more direct effect of the mutation, since the X-ray crystallographic structure of V
4.2 (4) indicates that K68 is exposed and does not make stabilizing H-bonds with CDR
residues. Furthermore, it is unlikely that the mutations analyzed in this study have resulted in gross structural perturbations since the CD spectra of the corresponding recombinant V
domains indicate that they are correctly folded,
and the WT and mutant transfectants express similar levels
of surface TCR.
Regardless of the mechanism by which the effect of mutation of K68 occurs, the almost total abrogation of responsiveness of the CD4 mutant transfectants to Ac1-11-I-Au
complexes is unexpected since K68 would be predicted to
be at the periphery of the interacting surfaces of TCR and
pMHC. However, the absence of CD4 together with the
low affinity of this peptide for I-Au most likely contribute
toward the unresponsiveness, and in this context CD4
WT transfectants also respond relatively poorly to this
ligand. In contrast, when the density of cognate pMHC is
increased by using Ac1-11(4Y) which has a ~1000-fold
higher affinity for I-Au (61), responsiveness of the K68A
and K68AE69A transfectants is observed albeit at considerably lower levels than that of the WT transfectants. This
supports the concept that the K68A mutant TCR binds to
cognate antigen (Ac1-11-I-Au complexes) with an affinity/
avidity which, in the absence of CD4, falls below the
threshold needed for effective signaling (IL-2 production in
the current study). Consistent with this, coexpression of CD4
partially compensates for the defective signaling of transfectants expressing K68A. The enhancing effect of CD4 could
be through one or more mechanisms. First, CD4 may increase the affinity/avidity of the TCR for pMHC by stabilizing the trimeric complex via CD4-MHC interactions
(19, 33), or by increasing the affinity of the TCR-pMHC interaction in an analogous way to that demonstrated for CD8 (24, 25). Second, CD4 is able to recruit the
phosphotyrosine kinase p56lck to the TCR-CD3 complex
increasing the strength of the signal delivered postantigen
recognition by the TCR (22, 23). In the current study,
this could compensate for the loss in affinity of the K68A
mutant. Consistent with this, several studies have shown a
significant loss of CD4 effects on T cell activation in the
absence of p56lck-CD4 interaction (62). However,
CD4 lacking its cytoplasmic lck-binding domain can still
augment T cell reactivity (65), and in one study the extracellular domains of CD4 and not its cytoplasmic tail are responsible for restoration of IL-2 secretion (66).
Recent studies have shown that reduced CD4 availability can convert the functional and biochemical effects of agonist peptides into those characteristic of partial agonists (32) or partial agonists into antagonists (67, 68). Reciprocally, CD4 coexpression can convert partial agonists into agonists, but has no effect on antagonist activity (31). The lack of effect of CD4 on antagonists which form short-lived TCR-pMHC complexes (17) supports the hypothesis that CD4 engagement follows TCR-pMHC complex formation, and that the latter complex needs to be sufficiently long lived to allow CD4 recruitment (31, 32). By extension, in the current study, the enhancing effect of CD4 coexpression on the responsiveness of the K68A mutant suggests that the half life of the K68A TCR-pMHC interaction is long enough to allow CD4 recruitment to the ternary TCR-pMHC complex. However, without CD4 coexpression, the interaction of this TCR with Ac1-11-I-Au complexes appears to be below the threshold needed for T cell activation. In contrast, the WT 1934.4 TCR-pMHC interaction appears to be sufficiently long lived in the absence of CD4 to result in signaling. This is also consistent with the relative resistance of this latter interaction to blockade by the anti-CD4 antibody, GK1.5. In addition, the enhancement in responsiveness by CD4 coexpression is much greater for the K68A mutant than the WT TCR. The effect of CD4 therefore appears to be maximal for suboptimal TCR-pMHC interactions that attain a threshold affinity, and this is consistent with observations using other antigen recognition systems (31, 69). However, in these other systems, the effects of CD4 on responses to agonists/partial agonists/antagonists were analyzed, and this is in contrast to the current study where the affinity of the TCR-pMHC (agonist) interaction has been affected by mutating the TCR. Taken together, our data provide further support for sequential engagement models of T cell signaling in which both the affinity (off-rate) of the TCR- pMHC complex and coreceptor involvement affect the outcome of T cell contact with APCs bearing cognate ligands (31, 32, 34).
In summary, a single amino acid substitution of the exposed, highly conserved residue K68, which is located outside the CDRs of the TCR, appears to have a significant
impact on the outcome of the interaction between a TCR
and its pMHC ligand. Given the indications that the diagonal orientation observed for the binding of class I-restricted
TCRs to pMHC complexes is general (10), it is likely
that the functional role of this HV4 amino acid that we
have defined will be observed for other TCR-pMHC interactions.
![]() |
Footnotes |
---|
Address correspondence to E. Sally Ward, Center for Immunology and Department of Microbiology, University of Texas Southwestern Medical Center at Dallas, 6000 Harry Hines Blvd., Dallas, TX 75235-8576. Phone: 214-648-1260; Fax: 214-648-1259; E-mail: sally{at}skylab.swmed.edu
Received for publication 5 August 1998 and in revised form 20 October 1998.
A. Qadri's present address is National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.We are indebted to Drs. Bertram Ober and Mark Mummert for providing the wild-type TCR transfection constructs and advice with CD analyses, respectively. We also thank Drs. Nicolai van Oers and Stephen Thompson for critical review of the manuscript and Dr. Mischa Machius for assistance with Fig. 1.
This work was supported by grants from the National Multiple Sclerosis Society (RG-2411), National Institutes of Health (AI/NS 42949) and Yellow Rose Gala. E.S.Ward is an Established Investigator of the American Heart Association (Grant 9640277N).
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Wange, R.L., and L.E. Samelson. 1996. Complex complexes: signaling at the TCR. Immunity. 5: 197-205 [Medline]. |
2. | Iwashima, M., B.A. Irving, N.S. van Oers, A.C. Chan, and A. Weiss. 1994. Sequential interactions of the TCR with two distinct cytoplasmic tyrosine kinases. Science. 263: 1136-1139 [Medline]. |
3. |
van Oers, N.S.,
W. Tao,
J.D. Watts,
P. Johnson,
R. Aebersold, and
H.S. Teh.
1993.
Constitutive tyrosine phosphorylation of the T cell receptor (TCR) ![]() ![]() ![]() |
4. |
Fields, B.A.,
B. Ober,
E.L. Malchiodi,
M.I. Lebedeva,
B.C. Braden,
X. Ysern,
J.K. Kim,
X. Shao,
E.S. Ward, and
R.A. Mariuzza.
1995.
Crystal structure of the V![]() |
5. | Bentley, G.A., G. Boulot, K. Karjalainen, and R.A. Mariuzza. 1995. Crystal structure of the beta chain of a T cell antigen receptor. Science. 267: 1984-1987 [Medline]. |
6. | Bentley, G.A., and R.A. Mariuzza. 1996. The structure of the T cell antigen receptor. Annu. Rev. Immunol. 14: 563-590 [Medline]. |
7. | Jorgensen, J.L., U. Esser, B.F. de St. Groth, P.A. Reay, and M.M. Davis. 1992. Mapping T-cell receptor-peptide contacts by variant peptide immunization of single-chain transgenics. Nature. 355: 224-230 [Medline]. |
8. | Davis, M.M., and P.J. Bjorkman. 1988. T-cell antigen receptor genes and T-cell recognition. Nature. 334: 395-398 [Medline]. |
9. | Claverie, A., A. Prochnicka-Chalufour, and L. Bougueleret. 1989. Implications of a Fab-like structure for the T-cell receptor. Immunol. Today. 10: 10-13 [Medline]. |
10. |
Garcia, K.C.,
M. Degano,
R.L. Stanfield,
A. Brunmark,
M.R. Jackson,
P.A. Peterson,
L. Teyton, and
I.A. Wilson.
1996.
An ![]() ![]() |
11. | Garboczi, D.N., P. Ghosh, U. Utz, Q.R. Fan, W.E. Biddison, and D.C. Wiley. 1996. Structure of the complex between human T-cell receptor, viral peptide and HLA-A2. Nature. 384: 134-141 [Medline]. |
12. |
Garcia, K.C.,
M. Degano,
L.R. Pease,
M. Huang,
P.A. Peterson,
L. Teyton, and
I.A. Wilson.
1998.
Structural basis
of plasticity in T cell receptor recognition of a self peptide-MHC antigen.
Science.
279:
1166-1172
|
13. | Ding, Y.H., K.J. Smith, D.N. Garboczi, U. Utz, W.E. Biddison, and D.C. Wiley. 1998. Two human T cell receptors bind in a similar diagonal mode to the HLA-A2/Tax peptide complex using different TCR amino acids. Immunity. 8: 403-411 [Medline]. |
14. | Hong, S.C., A. Chelouche, R.H. Lin, D. Shaywitz, N.S. Braunstein, L. Glimcher, and C.A. Janeway. 1992. An MHC interaction site maps to the amino-terminal half of the T cell receptor alpha chain variable domain. Cell. 69: 999-1009 [Medline]. |
15. | Sant'Angelo, D.B., and G.Waterbury, P. Preston-Hurlburt, S.T. Yoon, R. Medzhitov, S.C. Hong, and C.A. Janeway. 1996. The specificity and orientation of a TCR to its peptide-MHC class II ligands. Immunity. 4: 367-376 [Medline]. |
16. | Sun, R., S.E. Shepherd, S.S. Geier, C.T. Thomson, J.M. Sheil, and S.G. Nathenson. 1995. Evidence that the antigen receptors of cytotoxic T lymphocytes interact with a common recognition pattern on the H-2Kb molecule. Immunity. 3: 573-582 [Medline]. |
17. | 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: 53-61 [Medline]. |
18. | Alam, S.M., P.J. Travers, and J.L.Wung, W. Nasholds, S. Redpath, S.C. Jameson, and N.R. Gascoigne. 1996. T-cell- receptor affinity and thymocyte positive selection. Nature. 381: 616-620 [Medline]. |
19. | 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]. |
20. | Clayton, L.K., M. Sieh, D.A. Pious, and E.L. Reinherz. 1989. Identification of human CD4 residues affecting class II MHC versus HIV-1 gp120 binding. Nature. 339: 548-551 [Medline]. |
21. | Doyle, C., and J.L. Strominger. 1987. Interaction between CD4 and class II MHC molecules mediates cell adhesion. Nature. 330: 256-259 [Medline]. |
22. | 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]. |
23. | Turner, J.M., M.H. Brodsky, B.A. Irving, S.D. Levin, R.M. Perlmutter, and D.R. Littman. 1990. Interaction of the unique N-terminal region of tyrosine kinase p56lck with cytoplasmic domains of CD4 and CD8 is mediated by cysteine motifs. Cell. 60: 755-765 [Medline]. |
24. | Luescher, I., E. Vivier, A. Layer, J. Mahieu, F. Godeau, B. Malissen, and P. Romero. 1995. CD8 modulation of T-cell antigen receptor-ligand interactions on living cytotoxic T lymphocytes. Nature. 373: 353-356 [Medline]. |
25. | Garcia, K., C. Scott, A. Brunmark, F. Carbone, P. Peterson, I. Wilson, and L. Teyton. 1996. CD8 enhances formation of stable T-cell receptor/MHC class I complexes. Nature. 384: 577-581 [Medline]. |
26. | Janeway, C.A. Jr.. 1995. Ligands for T cell receptors: hard times for avidity models. Immunol. Today. 16: 223-225 [Medline]. |
27. | Yoon, S.T., U. Dianzani, K. Bottomly, and C.A. Janeway. 1994. Both high and low avidity antibodies to the T cell receptor can have agonist or antagonist activity. Immunity. 1: 563-569 [Medline]. |
28. | Reich, Z., J.J. Boniface, D.S. Lyons, N. Borochov, E.J. Wachtel, and M.M. Davis. 1997. Ligand-specific oligomerization of T-cell receptor molecules. Nature. 387: 617-620 [Medline]. |
29. | Symer, D.E., R.Z. Dintzis, D.J. Ciamond, and H.M. Dintzis. 1992. Inhibition or activation of human T cell receptor transfectants is controlled by defined, soluble antigen arrays. J. Exp. Med. 176: 1421-1430 [Abstract]. |
30. | Monks, C.R., B.A. Freiberg, H. Kupfer, N. Sciaky, and A. Kupfer. 1998. Three-dimensional segregation of supramolecular activation clusters in T cells. Nature. 395: 82-86 [Medline]. |
31. | Hampl, J., Y. Chien, and M.M. Davis. 1997. CD4 augments the response of a T cell to agonist but not to antagonist ligands. Immunity. 7: 379-385 [Medline]. |
32. |
Madrenas, J.,
L.A. Chau,
J. Smith,
J.A. Bluestone, and
R.N. Germain.
1997.
The efficiency of CD4 recruitment to
ligand-engaged TCR controls the agonist/partial agonist
properties of peptide-MHC molecule ligands.
J. Exp. Med.
185:
219-229
|
33. |
Konig, R.,
X. Shen, and
R.N. Germain.
1995.
Involvement
of both major histocompatibility complex class II ![]() ![]() |
34. | 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]. |
35. | McKeithan, T.. 1995. Kinetic proof reading in T-cell receptor signal transduction. Proc. Natl. Acad. Sci. USA. 92: 5042-5046 [Abstract]. |
36. |
Rabinowitz, J.D.,
C. Beeson,
D.S. Lyons,
M.M. Davis, and
H.M. McConnell.
1996.
Kinetic discrimination in T-cell activation.
Proc. Natl. Acad. Sci. USA.
93:
1401-1405
|
37. | Kabat, E.A., T.T. Wu, H.M. Perry, K.S. Gottesman, and C. Foeller. 1991. Sequences of Proteins of Immunological Interest. Vol. 1, 5th Ed. U.S. Department of Health and Human Services, Washington, DC. |
38. |
Manning, T.C.,
C.J. Schlueter,
T.C. Brodnicki,
E.A. Parke,
J.A. Speir,
K.C. Garcia,
L. Teyton,
I.A. Wilson, and
D.M. Kranz.
1998.
Alanine scanning mutagenesis of an ![]() ![]() |
39. | Wraith, D.C., D.E. Smilek, D.J. Mitchell, L. Steinman, and H.O. McDevitt. 1989. Antigen recognition in autoimmune encephalomyelitis and the potential for peptide-mediated immunotherapy. Cell. 59: 247-255 [Medline]. |
40. |
Letourner, F., and
B. Malissen.
1989.
Derivation of a T cell
hybridoma variant deprived of functional T cell receptor ![]() ![]() ![]() |
41. | Wraith, D.C., D.E. Smilek, and S. Webb. 1991. MHC-binding peptides for immunotherapy of experimental autoimmune disease. J. Autoimmun. 5 (Suppl A):103-113. |
42. |
Staerz, U.,
H.G. Rammensee,
J.D. Benedetto, and
M.J. Bevan.
1985.
Characterisation of a murine mAb specific for
an allotypic determinant on T cell antigen receptor.
J. Immunol.
134:
3994-4000
|
43. |
Patten, P.A.,
E.P. Rock,
T. Sonoda,
B. Fazekas de St. Groth,
J.L. Jorgenson, and
M.M. Davis.
1993.
Transfer of putative
complementarity determining region loops of T cell receptor
V domains confers toxin reactivity but not peptide/MHC
specificity.
J. Immunol.
150:
2281-2294
|
44. | Ward, E.S.. 1992. Secretion of T cell receptor fragments from recombinant Escherichia coli cells. J. Mol. Biol. 224: 885-890 [Medline]. |
45. | Kunkel, T.A., J.D. Roberts, and R.A. Zakour. 1987. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 154: 367-382 [Medline]. |
46. | Horton, R.M., H.D. Hunt, S.N. Ho, J.K. Pullen, and L.R. Pease. 1989. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene. 77: 61-68 [Medline]. |
47. | Fairchild, P.J., R. Wildgoose, E. Atherton, S. Webb, and D.C. Wraith. 1993. An autoantigenic T cell epitope forms unstable complexes with class II MHC molecules: a novel route for escape from tolerance induction. Int. Immunol. 5: 1151-1158 [Abstract]. |
48. | Mason, K., D.D. Denney, and H. McConnell. 1995. Kinetics of the reaction of a myelin basic protein peptide with soluble I-Au. Biochemistry. 34: 4874-4878 . |
49. | Marrack, P., R. Endres, R. Shimonkevitz, A. Zlotnik, D. Dialynas, F.W. Fitch, and J. Kappler. 1983. The major histocompatibility complex-restricted antigen receptor on T cells. II. Role of the L3T4 product. J. Exp. Med. 158: 1077-1091 [Abstract]. |
50. |
Wraith, D.C.,
B. Bruun, and
P.J. Fairchild.
1992.
Cross-
reactive antigen recognition by an encephalitogenic T cell
receptor.
J. Immunol.
149:
3765-3770
|
51. | Rothenberg, E.V.. 1994. Signaling mechanisms in thymocyte selection. Curr. Opin. Immunol. 6: 257-265 [Medline]. |
52. |
Pullen, A.M.,
T. Wade,
P. Marrack, and
J.W. Kappler.
1990.
Identification of a T cell receptor ![]() |
53. |
Choi, Y.,
A. Herman,
D. DiGiusto,
T. Wade,
P. Marrack, and
J. Kappler.
1990.
Residues of the variable region of the
T-cell-receptor ![]() |
54. |
Fields, B.A.,
E.L. Malchiodi,
H. Li,
X. Ysern,
C.V. Stauffacher,
P.M. Schlievert,
K. Karjalainen, and
R.A. Mariuzza.
1996.
Crystal structure of a T-cell receptor ![]() |
55. | Speir, J.A., K.C. Garcia, A. Brunmark, M. Degano, P.A. Peterson, L. Teyton, and I.A. Wilson. 1998. Structural basis of 2C TCR allorecognition of H-2Ld peptide complexes. Immunity. 8: 553-562 [Medline]. |
56. |
Fischmann, T.O.,
G.A. Bentley,
T.N. Bhat,
G. Boulot,
R.A. Mariuzza,
S.E.V. Phillips,
D. Tello, and
R.J. Poljak.
1991.
Crystallographic refinement of the three-dimensional structure of the FabD1.3-lysozyme complex at 2.5 Å resolution.
J.
Biol. Chem.
266:
12915-12920
|
57. | Tulip, W.R., J.N. Varghese, W.G. Laver, R.G. Webster, and P.M. Colman. 1992. Refined crystal structure of the influenza virus N9 neuraminidase-NC41 Fab complex. J. Mol. Biol. 227: 122-148 [Medline]. |
58. | McCoy, A.J., V. Chandana-Epa, and P.M. Colman. 1997. Electrostatic complementarity at protein/protein interfaces. J. Mol. Biol. 268: 570-584 [Medline]. |
59. | Scott, C.A., P.A. Peterson, L. Teyton, and I.A. Wilson. 1998. Crystal structures of two I-Ad-peptide complexes reveal that high affinity can be achieved without large anchor residues. Immunity. 8: 319-329 [Medline]. |
60. | Fremont, D.H., D. Monnaie, C.A. Nelson, W.A. Hendrickson, and E.A. Unanue. 1998. Crystal structure of I-Ak in complex with a dominant epitope of lysozyme. Immunity. 8: 305-317 [Medline]. |
61. | Fugger, L., J. Liang, A. Gautam, J. Rothbard, and H. McDevitt. 1996. Quantitative analysis of peptides from myelin basic protein binding to the MHC class II protein, I-Au, which confers susceptibility to experimental allergic encephalomyelitis. Mol. Med. 2: 181-188 [Medline]. |
62. | Miceli, M.C., P. von Hoegen, and J.R. Parnes. 1990. Adhesion versus coreceptor function of CD4 and CD8: role of the cytoplasmic tail in coreceptor activity. Proc. Natl. Acad. Sci. USA. 88: 2623-2627 [Abstract]. |
63. |
Collins, T.L.,
S. Uniyal,
J. Shin,
J.L. Strominger,
R.S. Mittler, and
S.J. Burakoff.
1992.
p56lck association with CD4 is
required for the interaction between CD4 and the TCR/
CD3 complex for optimal antigen stimulation.
J. Immunol.
148:
2159-2162
|
64. | Glaichenhaus, N., N. Shastri, D.R. Littman, and J.M. Turner. 1991. Requirement for association of p56lck with CD4 in antigen specific signal transduction in T cells. Cell. 64: 511-520 [Medline]. |
65. | Xu, H., and D.R. Littman. 1993. A kinase-independent function of Lck in potentiating antigen-specific T cell activation. Cell. 74: 633-643 [Medline]. |
66. | Vignali, D.A.A., R.T. Carson, B. Chang, R.S. Mittler, and J.L. Strominger. 1996. The two membrane proximal domains of CD4 interact with the T cell receptor. J. Exp. Med. 183: 2097-2107 [Abstract]. |
67. |
Mannie, M.D.,
J.M. Rossier, 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 non-efficacious signals
during T cell antigen recognition.
J. Immunol.
154:
2642-2645
|
68. | 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]. |
69. |
Viola, A.,
M. Salio,
L. Tuosto,
S. Linkert,
O. Acuto, and
A. Lanzavecchia.
1997.
Quantitative contribution of CD4 and
CD8 to T cell antigen receptor serial triggering.
J. Exp. Med.
186:
1775-1779
|
70. | Esnouf, R.M.. 1997. An extensively modified version of MolScript that includes greatly enhanced coloring capabilities. J. Mol. Graph. 15: 133-138 . |
71. | Meritt, E.A., and M.E.P. Murphy. 1994. Raster3D Version 2.0: a program for photorealistic molecular graphics. Acta Cryst. D. 50: 869-873 . [Medline] |