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Molecular Genetics Program, Virginia Mason Research Center, Seattle, WA 98101; and Department of Immunology, University of Washington, Seattle, WA 98195
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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various substitutions still allowed for some degree of recognition. One
modest substitution, alanine for glycine (essentially the addition of a
methyl group), significantly broadened the specificity of the TCR.
Transfectants expressing this mutant TCR responded strongly in the
context of multiple HLA-DR alleles and to HA peptide variants with
substitutions at each TCR contact residue. These results suggest that
the conformations of CDR3 loops are crucial to TCR specificity and that
it may not be reliable to extrapolate from primary sequence
similarities in TCRs to similarities in specificity. The ease with
which a broad specificity is induced in this mutant TCR has
implications for the mechanisms and frequency of alloreactivity and
promiscuity in T cell responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The complex structure of the ligand recognized by TCR, consisting of
both an HLA-presenting molecule and a bound peptide, might be expected
to impose restrictions on the orientation with which TCR can interact
with ligands that are not required of Ig. Davis and Bjorkman
(1) first proposed a model of this interaction in which
complementarity-determining regions
(CDRs)3 1 and 2 of the
TCR interacted with the MHC helices while the CDR3 regions
interacted with peptide. A key feature of this model was that it
allowed for the most diverse portions of the TCR (CDR3s) to bind the
most diverse portion of its complex ligand, the peptide. The predicted
interaction of CDR3 residues with peptide has been verified by direct
mutagenesis studies of TCRs (2, 3, 4, 5, 6, 7, 8, 9), by cross-linking with
a photoactivatable peptide (10), and by the in vivo
selection of compensatory TCR mutations with variant peptides
(11, 12). Additional studies have demonstrated that
mutation of CDR1 or CDR2 can abrogate or alter recognition (4, 5, 6, 13, 14, 15, 16, 17). Framework residues must also have a role in recognition
because CDR grafting experiments do not consistently transfer
specificity between TCRs (6, 18).
Based on mutagenesis studies in which class I MHC mutants were selected
with a CTL clone, Sun et al. (19) proposed that TCRs
interacted with the complex of class I MHC and peptide in a standard
conformation. In this conformation, the TCR was oriented parallel to
the ß-pleated strands in the floor of the class I molecule and
diagonal relative to the helices. Several x-ray crystallographic
structures of TCRs bound to peptide-MHC class I complexes have been
solved and yield support for this diagonal orientation of recognition
(20, 21, 22, 23). The emerging picture from these structures is
that peptide interacts with both CDR3s but also with both CDR1s.
CDR3 and CDR3ß form a pocket that accepts a side chain from a
specific, centrally located peptide residue. The CDR2 of the TCR
-chain is located over the MHC 2 helix while the TCRß CDR2
interacts with the class I 1 helix. At present, a class
II-peptide-TCR crystal structure has not been reported. Furthermore,
the static picture afforded by a crystal structure alone cannot resolve
the relative contributions of different elements of the TCR in shaping
its structure, allowing or preventing recognition of its ligand.
We have previously described site-specific mutagenesis of TCRs derived
from two human T cell clones specific for the immunodominant influenza
virus hemagglutinin (HA) peptide, HA307–319
(16). In these studies, mutagenesis of CDR2 residues
altered the HLA-DR restriction of the TCRs while maintaining peptide
specificity. These results are consistent with a TCR orientation in
which CDR2 interacts with the DRß molecule. Further, they point
out an advantage of mutagenesis studies of TCRs from human
HA307–319-specific T cell clones. Because the HA
peptide binds different allelic DR molecules, DR1, DR2, DR4w4, DR5, and
DR7, with similar conformations and affinities (24, 25),
mutagenesis of appropriate TCRs should facilitate the separate
identification of TCR sites that contact specific peptide or HLA
residues. In the current study, this mutagenesis approach has been
applied to the CDR3 regions of
HA307–319-specific TCRs. Most mutations in
CDR3 or CDR3ß resulted in loss of recognition. However, a modest
change of Gly to Ala at one position in CDR3 dramatically broadened
the specificity of the TCR such that HA variant peptides with
substitutions at any TCR contact residue as well as multiple different
DR presenting molecules now elicited responses. The effect of this
mutation on DR recognition was dominant when paired with CDR2
mutations. These results suggest that, despite a standard orientation
of TCR relative to its ligand, there is substantial flexibility in this
interaction such that minor differences in CDR2 residues may have
dramatic effects on TCR specificity and that simple one to one
correspondences between TCR and peptide or MHC residues may be
difficult to generalize across TCRs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The specificities of the TCRs from T cell clones JS515.11 (JS)
and 3BC6.6 (3BC) have been described previously (16). When
transfected into Jurkat cells, the TCR from 3BC recognizes the
HA307–319 peptide only in the context of DR4.
The TCR from JS responds strongly to the HA peptide in the context of
DR7 but also displays a weaker, promiscuous response to the peptide
presented by DR1, DR4, and DR5. Both clones use products of the TCR
BV3S1 gene and allelic forms of the TCR AV1S2 gene, which differ by
only two residues in CDR2 (16). J.RT3-T3.5 (JRT3), a
Jurkat cell line derivative defective in endogenous TCRß expression
(26), was obtained from the American Type Culture
Collection (Manassas, VA). B lymphoblastoid cell lines (B-LCL) that
were homozygous for HLA-DR alleles, HOM2 (DR1), MGAR (DR2), Priess
(DR4), Sweig (DR5), and DBB (DR7), were used as APCs. Cells were grown
in RPMI 1640 supplemented with sodium pyruvate, penicillin,
streptomycin, and 10% calf serum.
Mutagenesis
Chimeric TCR and TCRß constructs were created by PCR
amplification using primers that spanned the positions where sequences
exchanged from one receptor to the other. Point mutations were
introduced by making the desired change in the appropriate primer.
Constructs were cloned initially in Bluescript (Stratagene, San Diego,
CA), and their nucleotide sequences were confirmed. Subsequently, the
mutant constructs were transferred to the episomal expression vectors
pREP8 and pREP9 (Invitrogen, San Diego, CA).
Electroporation and selection
Episomal vectors containing in vitro mutagenized TCR genes were introduced into JRT3 cells by electroporation as previously described (16). Briefly, electroporation was performed at 250 V, 960 µF using a Gene Pulser (Bio-Rad, Hercules, CA). Cells were allowed to recover for 24 h, the medium was changed, and cells were grown for a further 3 days under nonselective conditions. Cells were then transferred to medium containing 600 µg/ml active geneticin (Life Technologies, Gaithersburg, MD) and 0.5 mM histidinol (Sigma, St. Louis, MO). After 2–3 wk of selection, cells were stained with an Ab to Vß3 (ßV3a; T Cell Diagnostics, Cambridge, MA) and analyzed on a FACSort (Becton Dickinson, San Jose, CA). All transfectants were strongly positive for Vß3 expression. For experiments in which the responses of different mutagenized TCR constructs were compared, surface Vß3 expression levels on all transfectants were measured by FACS to ensure that there were no significant differences. Retrospective examination of the expression data revealed no cases in which increased or decreased responsiveness of a given transfectant correlated with high or low expression, respectively, of the TCR. Cells were always stained within 48 h of their use in stimulation assays.
T cell stimulations
Transfected T cells were stimulated with the influenza virus
(A/PR/8/34) HA peptide 307–319 (PKYVKQNTLKLAT) and single position
variants presented by B-LCL. Transfected JRT3 cells (5 x 104)
and APCs (5 x 104) were cultured in the presence of various
concentrations of peptide and 3 ng/ml PMA in triplicate in volumes of
150 µl in 96-well plates. At 16 h, 1 µCi of
[3H]thymidine (5 Ci/mmol) was added. Cells were
harvested 8 h later, and [3H]thymidine
incorporation was measured by scintillation counting. Controls for all
experiments included stimulation of T cells with 3 ng/ml PMA and
stimulation in microtiter wells coated with -CD3.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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necessary for peptide recognition and HLA restriction
To evaluate the effect of differences in the CDR3 regions on
peptide recognition and/or HLA restriction, the CDR3 and ß regions
of the TCRs from the T cell clones JS and 3BC were systematically
mutagenized by exchanging analogous sequences between the two
receptors. The J regions of the 3BC and JS TCRs are of identical
length and vary at 12 of 20 residues between positions 95 and 114.
Chimeric TCR -chain genes were created in which all sequences to the
N-terminal side (left in Fig. 1
A) were derived from one TCR
-chain while all sequences to the C-terminal side (right in Fig. 1A) were derived from the other chain. TCR chimeras in
which the "crossover" point was located in the middle of CDR3,
between positions 98 and 99 (constructs JS98 and 3BC98), could
not be activated when paired with either the 3BC TCRß or JS TCRß,
regardless of whether the HA307–319 peptide was
presented by DR4 or DR7 (Figs. 2A and
3A). Similarly, no response
was observed when the crossover point was between positions 96 and 97
(JS96 and 3BC96). In these latter constructs, only the two
N-terminal residues of CDR3 are altered relative to the cognate
receptor. In TCR chimeras where the crossover point was between
positions 94 and 95 (constructs JS94 and 3BC94), the expected
peptide recognition and HLA restriction was maintained, as previously
described (16).
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are outside of the structurally defined CDR3 region.
Nevertheless, there is significant sequence variation at these
positions. For example, the two TCR -chains studied here differ in
five of these 12 residues. Therefore, TCR chimeras were made with
the crossover point between residues 104 and 105, immediately
N-terminal to the nonconserved residue in the FGXG sequence. In
contrast to results obtained from chimeras with breakpoints within the
structurally defined CDR3 region, both of these constructs (JS104
and 3BC104), when paired with the cognate ß-chain, had responses
characteristic of the TCR from which the -chain sequences N-terminal
to the FGXG sequence were derived (Figs. 2
B and
3B). In addition, single point mutations at the
nonconserved, "X", position in the FGXG sequence had no effect on
TCR recognition (constructs JS105P and 3BC105K). TCR ß-chain chimeras with breakpoints in CDR3 do not allow recognition
Chimeric TCR ß-chain constructs analogous to those made for
TCR were also generated, paired with each normal TCR -chain, and
tested for recognition of the HA peptide presented by HLA-DR4 and by
DR7. Breakpoints in the TCRß constructs were located in the middle of
CDR3ß (constructs JSß98 and 3BCß98, Fig. 1B). When
these chimeric TCRßs were introduced into cells, no activation was
observed regardless of which TCR -chain they were paired with or
whether the HA peptide was presented by DR4 or DR7 (Figs. 2C
and 3C). As was observed for CDR3, sequences within or
C-terminal to the conserved FGXG sequence in Jß could be exchanged
without loss of responsiveness or specificity (constructs JSß105 and
3BCß105).
Mutations at position 95 of TCR have differential effects on
peptide recognition and HLA restriction
Because chimeric TCR -chains with crossover points between
positions 94 and 95 functioned normally while those with crossover
points between positions 96 and 97 were nonresponsive, the role of
specific residues between these crossover points was further explored
by site-specific mutagenesis. The most significant difference between
the JS and 3BC TCR -chains at these sites is at position 95 where
the JS TCR has a small neutral residue, glycine, while the 3BC
TCR has an aspartic acid residue. Site-directed mutagenesis was used
to create TCR -chains in which the D residue at position 95 in the
3BC TCR was changed to A, E, and G. Substitution of the 95D residue
in the 3BC TCR with either A or even the similarly charged E residue
abrogated recognition (data not shown). The substitution of residue 95D
with G, the analogous amino acid from the JS TCR, retained the 3BC
receptor’s response to peptide presented by DR4 (data not shown).
Mutations were also made at position 95 of the JS TCR -chain where
the G residue was changed to A, E, and D. The JS TCR mutant in which
the 95G residue was substituted with D was unresponsive to peptide
presented by either DR4 or DR7. The JS TCR 95G to E mutant responded
to higher concentrations of peptide presented by DR7, but the
promiscuous response in the presence of DR4 was lost (Fig. 4A). The JS TCR 95G to A
mutant, which differed only by the addition of a single methyl group to
the wild-type JS receptor, had a significantly enhanced response to the
HA peptide presented by DR4 such that this promiscuous response was as
sensitive as the primary response in the context of DR7. Thus, four
different residues at position 95 of the TCR -chain of the JS
receptor yield four very different responses to peptide and
MHC.
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In addition to its normally sensitive response to the
HA307–319 peptide presented by DR7, the TCR from
the JS T cell clone also exhibits a modest response to high
concentrations of the HA peptide (
10 µg/ml) when presented by DR1,
DR4, and DR5. No response is seen with the DR2 homozygous APC, where
the HA peptide is presented by the DRB5 molecule rather than DRB1. To
look more broadly at the effect of the 95G to A mutation on the HLA
restriction of the JS TCR, the mutant TCR was transfected into cells
and challenged with the HA peptide presented by DR1, DR2, DR4, DR5, and
DR7 (Fig. 4B). The JS TCR with the alanine substitution
at position 95 was significantly more sensitive (>2 orders of
magnitude) to peptide presented by DR1, DR4, and DR5. Response to
peptide presented by DR2 was largely unaffected except for a slight
increase in responsiveness at the highest peptide concentration.
Despite the increased sensitivity in the promiscuous response seen with
this mutant TCR, the dose-response curve for DR7 was unchanged.
The position 95G to A mutation in the JS TCR -chain enhances
responsiveness to multiple peptide variants
Five amino acids in the HA307–319 peptide
have been demonstrated to act as T cell contact residues when presented
by the various DR alleles studied here (25). A panel of HA
variant peptides incorporating residues varying in charge and size at
each of these T cell contact positions was tested for recognition by
the JS TCR with the 95A mutation (Fig. 5). There was no clear one to one
correspondence between substitutions at a specific position in the
peptide and either increased or decreased responsiveness seen with the
TCR mutant. Although cell-surface expression of the normal and the
mutant TCR was similar, the mutant TCR consistently exhibited stronger
responses to peptide variants at every T cell contact position. In some
cases (e.g., V310L or N313T), robust responses were seen with the
mutant TCR but were undetectable with the normal receptor. In no case
did a peptide variant elicit a significantly weaker response with the
95A mutant TCR.
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mutation, JS95A, dominantly increases receptor
affinity when paired with CDR2 mutations
In previous studies, we have demonstrated that amino acid
substitutions at one or both of two positions in the CDR2 region of
the JS TCR still allows recognition of the HA peptide when presented by
DR7, but eliminates the promiscuous response seen in the context of DR4
(16). These CDR2 mutations, which depress
responsiveness in the context of DR4, and the CDR3 mutant, which
increases responsiveness in the context of multiple DR alleles,
including DR4, were incorporated into a single JS TCR construct and
introduced into cells with the cognate JS TCRß. The resulting
transfected cells, along with appropriate transfectants of the single
mutants and the wild-type JS TCR, were challenged with the HA
peptide presented by DR4 and by DR7 (Fig. 6). The single mutant transfectants
reproduced their previously demonstrated specificities. The double
mutant responded similarly to the JS95A single mutant. The
dose-response curve for DR7 was shifted to the left relative to the
CDR2 single mutant, and the DR4-restricted response was
significantly increased relative to the wild-type JS TCR.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, CDR3, and
CDR3ß). Previously, we have shown that changes in CDR2 of the
HLA-promiscuous receptor, JS, modulate promiscuity in this system
(16). This current study examines the specificity of TCRs
chimeric in the CDR3 region and compares the relative strength of the
effects of substitutions in CDR2 and CDR3 through the construction of
double mutants.
In the class I-restricted TCRs whose structures when complexed with
ligand have been solved, CDR3 and CDR3ß pair to form a pocket that
accepts protruding amino acid side chains from the MHC-bound peptide
(20, 21, 22, 23). Despite the degree of sequence divergence in the
CDR3 regions in the two TCRs studied here, there are enough general
similarities to suggest that one receptor might be capable of
functionally substituting for the other. The CDR3 sequences from
these two TCRs are exactly the same length. As in the TCR motif
described by Kieber-Emmons et al. (27), both of these
CDR3 regions have the same negatively and positively charged
residues at positions 5 and 11 (counting from the conserved C residue
in the V), which potentially can form an ionic pair and stabilize
the CDR3 loop. Nevertheless, chimeras with a breakpoint in the
middle of CDR3 (constructs JS98 and 3BC98), whether paired
with cognate or the alternative ß-chain, did not respond to the HA
peptide presented by DR4 or DR7. Indeed, chimeras with as little as two
amino acids of the "incorrect" CDR3 region (JS96 and
3BC96) were not able to recognize ligand. In contrast, chimeras with
a breakpoint at position 94 still supported recognition; the TCR
constructed from JS94 and 3BCß behaves like the 3BC receptor and a
3BC94, JSß TCR retained recognition of peptide in the context of
DR7, although the promiscuous recognition in the context of other DRs,
characteristic of the native JS receptor, was lost. These mutants
differ from the native JS or 3BC -chains at only two residues in
CDR2.
CDR3 loops are formally considered to be confined to the N-terminal
side of the conserved FGXG sequence. Nevertheless, the positions in
J and Jß between FGXG and the constant region are highly variable
in TCRs. The -chains of the JS and 3BC receptors differ at five
positions in this region. Sequences to the C-terminal side of the
conserved F of the FGXG sequence could be exchanged between these
receptors without loss of recognition (JS104, 3BCß104). Similar
results were obtained with ß-chain chimeras. Breakpoints in the
middle of CDR3ß, JSß98, and 3BCß98, did not allow recognition
regardless of the -chain they were paired with or the class II
allele presenting the HA peptide. However, sequences to the C-terminal
side of the conserved F residue in CDR3ß could, again, be exchanged
without loss of recognition (constructs JSß105 and 3BCß105). A
previous study of the TCRß peripheral repertoire in humans concluded
that the identity of the "X" residue in the conserved FGXG sequence
played a crucial role in thymic selection of T cells in the context of
DR4 (28). Although the in vitro specificity of only two
TCRs was examined here, no evidence of a role for this residue was
observed in either DR4- or DR7-restricted recognition. Given the
frequency with which site-specific amino acid substitutions in this
region, and, indeed, in TCRs in general, result in loss of recognition,
the maintenance of function in the presence of these nonconservative
substitutions argues against a significant role for residues in this
position in TCR recognition.
The exchange of as few as the first two amino-terminal residues in
CDR3 (3BC96 or JS96) in the TCRs studied led to a loss of
recognition. Site-specific mutants were constructed to look more
closely at requirements for recognition in this region. Substitutions
at position 95 of the JS -chain yielded a diverse array of outcomes
when stimulated with the HA peptide in the context of DR4 or DR7. The
JS receptor normally has G at position 95 of the -chain. Change to
either negatively charged residue reduced responsiveness; 95D did not
allow recognition while 95E responded only at higher peptide
concentrations. Even then, a response was only observed in the context
of DR7; the promiscuous response of the wild-type receptor in the
context of DR4 was lost. In contrast, the modest change of 95G to 95A
created a more sensitive receptor in which the response to the
"promiscuous" alleles was as sensitive as the response to
DR7.
If the enhanced responsiveness observed with the JS TCR 95G to A
mutant is mediated by a specific interaction with a residue in the HA
peptide, then it should be possible to augment or dampen this response
by substitutions at TCR contact residues in the peptide sequence.
However, of 22 variant peptides with substitutions at one of the five T
cell contact residues in the HA peptide, none evoked a significantly
weaker response from the 95A mutant. Some variant peptides (V310L,
N313T) elicited strong responses in 95A-transfected cells while
responses in 95G-transfected cells were undetectable.
The broader specificity observed with TCRs incorporating the 95A mutant
is inconsistent with any simple one to one correspondence between the
side chain at position 95 of the TCR -chain and a side chain of
either peptide or HLA DRß. The 95G to A change appears to affect
receptor affinity globally and allows variants at many positions on the
compound ligand of DR and peptide to be recognized. One possible
explanation for this observation is that the 95A mutation facilitates a
TCR conformation in which the threshold for activation is significantly
lower than in the wild-type JS receptor and allows for more flexibility
in the acceptable amino acids at TCR contact positions in the peptide
or HLA presenting molecule.
A study of the energy of binding for peptide/MHC recognition by
single-chain TCRs suggests that CDRs 1 and 2 contribute significantly
more energy to binding than CDR3 (29). We have previously
identified substitutions in CDR2 of the JS TCR that depress the
promiscuous response of this TCR to HA peptide presented by DR4, while
in this current study we have identified a mutation in CDR3 (95A)
that enhances this response. When both of these mutations were paired
together in the same TCR -chain, the response of this double mutant
to the HA peptide presented by either DR7 or DR4 was significantly more
sensitive than that for the CDR2 single mutant. These results
suggest that, at least in the context of this particular TCR,
interactions involving CDR3 predominate in determining specificity.
In previous studies of TCR/MHC/peptide interactions by systematic
mutagenesis of CDR residues, most mutations resulted in nonresponsive
TCRs. Where success has been achieved in identifying contact residues
in CDR3, it has been through an elegant genetic approach in which
single-chain TCR transgenic mice are challenged with peptides that have
substitutions at putative TCR contact residues. The powerful selection
afforded by this approach allows the isolation and identification of
TCRs with "compensating" amino acid substitutions in CDR3 and hence
the definition of one to one side-chain interactions between specific
residues in peptide and TCR. If such localized interactions are a
common feature of TCR recognition, then it should be possible to
identify analogous examples by the reverse approach of mutagenizing
CDR3 residues and testing for compensation either with variant peptides
or by presentation on different allelic DR molecules. Further, it might
be expected that CDRs would tolerate at least conservative amino acid
substitutions at some positions without loss of responsiveness.
However, our results, and those from previous TCR mutagenesis studies
(4, 5, 9, 13), indicate that TCR recognition is
exquisitely sensitive to even modest substitutions in CDR3 and suggest
caution in correlating CDR sequences with similarity in recognition in
the absence of a known specificity for a TCR. Many CDR3 substitutions
may have significant effects that are not localized to interaction with
a specific side chain on a specific ligand. These effects could include
alterations in the shape of the CDR3 loop or abrogation of the
interaction with the cognate CDR3 of the other TCR chain. Where we have
been able to identify substitutions that retain some responsiveness, as
in the 95A mutation described in this study or previous CDR2
mutations (16), the nature of the substituted amino acids
does not suggest obvious side-chain interactions, and the effect of the
substitution appears to be global, altering interactions with multiple
DR alleles and/or multiple peptide variants. Thus, while the general
orientation of the interaction between TCRs and their peptide/MHC
ligands appears to be standard, the specific details of side-chain
interactions involving TCR CDR3 residues may not be generalizable. The
ability of a single CDR3 substitution, such as the JS 95A variant,
to govern a broad specificity phenotype has implications for the
selection and maintenance of specific T cells capable of promiscuous
and alloreactive recognition.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Patrick Concannon, Molecular Genetics Program, Virginia Mason Research Center, 1201 Ninth Avenue, Seattle, WA 98101-2795. E-mail address:
3 Abbreviations used in this paper: CDR, complementarity-determining region; HA, hemagglutinin; JS, JS515.11; 3BC, 3BC6.6; JRT3, J.RT3-T3.5; B-LCL, B lymphoblastoid cell line.
Received for publication May 20, 1999. Accepted for publication August 19, 1999.
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References |
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-repressor: identification of residues critical for antigen recognition. J. Immunol. 144:4851.[Abstract]
chain. J. Exp. Med. 175:1553. chain affect antigen-major histocompatibility complex recognition. J. Biol. Chem. 265:88.
chain variable domain. Cell 69:999.[Medline]
CDR1 and CDR2. Science 273:963.[Abstract]
ß T cell receptor at 2.5
and its orientation in the TCR-MHC complex. Science 274:209.ß T cell receptor: mapping the energy of antigen recognition. Immunity 8:413.[Medline]
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