By
From the * Department of Biochemistry, University of Illinois, Urbana, Illinois 61801; and the Department of Immunology, The Scripps Research Institute, La Jolla, California 92037
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Abstract |
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It has been proposed that the generally low affinities of T cell receptors (TCRs) for their peptide-major histocompatibility complex (pMHC) ligands (Kd ~104 to 10
7 M) are the result of
biological selection rather than an intrinsic affinity limitation imposed by the TCR framework.
Using a soluble version of the 2C TCR, we have used complementarity determining region
(CDR)-directed mutagenesis to investigate whether the affinity of this receptor for its allogeneic pMHC ligand can be improved upon. We report that several mutants at positions lying
within CDR3
and CDR2
showed increased affinities for pMHC compared with the wild-type receptor. Additionally, we have investigated whether V
mutations that have been implicated in the phenomenon of CD8+ repertoire skewing achieve this skewing by means of generalized increases in affinity for MHC-I molecules. Two mutants (S27F and S51P), which each
promote skewing toward a CD8+ phenotype, exhibited significantly reduced affinity for
pMHC-I, consistent with a quantitative-instructional model of CD4/CD8 lineage commitment. This model predicts that CD8 is downregulated on thymocytes that have TCR-ligand
interactions above a minimal energy threshold. Together, the results (a) demonstrate that engineering higher affinity TCRs is feasible, and (b) provide TCR-pMHC energy values associated
with CD4/CD8 repertoire skewing.
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Introduction |
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Antibodies exhibit structural similarity to TCRs and
undergo similar gene rearrangement processes, yet
they generally exhibit much higher affinities for their antigens (Kd ~107 to 10
12 M) than do TCRs for their peptide-MHC complex (pMHC)1 ligands (Kd ~10
4 to 10
7 M)
(1). Although the molecular explanation for these differences could be that somatic mutation does not operate on
the TCR, there are likely functional explanations also. For
example, negative selection acts to eliminate high TCR affinities. In addition, it has been hypothesized that there
may be a ceiling on useful TCR-pMHC affinity (~10
7
M), above which there is no functional advantage (2, 3) (and above which are possibly disadvantages, due to impaired serial triggering of TCRs [4]). Antibodies, which
undergo affinity maturation, may have different functional
ceilings on their affinity optimization (5).
Another possibility is that the framework regions of antibody V regions are more optimally suited as a scaffold on which optimal-affinity CDR regions may be constructed, and that TCR framework regions are handicapped in this regard. Although the three-dimensional structures are similar, important differences have been observed in the overall surface topology of antibodies compared with at least the 2C TCR (6). If the low affinities observed for TCR-pMHC interactions are a result of a selection process rather than inherent structural differences, then it should be possible to improve this affinity through CDR-directed mutagenesis.
Using the 2C TCR system, we have explored the possibility of engineering soluble TCR molecules with improved affinities for the allogeneic ligand QL9/Ld. Previous
work involving alanine scanning mutagenesis of the 2C
CDR regions revealed four residues that yielded moderate
improvements in affinity when changed to alanine (7). In
this report, these mutations have been combined into double and triple mutant TCRs to test whether the affinity of
the 2C TCR for its allogeneic ligand QL9/Ld can be further improved. This interaction has the highest affinity of
TCR-pMHC interactions that have been measured (Kd = 107 M [8]). Engineering a higher affinity TCR-QL9/Ld
interaction would support the notion that a ceiling on
measured TCR-pMHC interactions is imposed by biological processes rather than an intrinsic inability of the TCR
framework to support higher affinities. Additionally, soluble TCR molecules with improved affinities could theoretically serve as antagonists for detrimental T cell responses.
We report here that a two- to threefold increase in TCR
affinity could be achieved through CDR-directed mutagenesis.
Another aspect of TCR-pMHC affinity relates to the
process of CD4/CD8 phenotype selection. The 2C TCR
is invariably skewed toward the CD8 phenotype, consistent
with findings that this TCR is positively selected by a class
I MHC product, Kb (9). However, the 2C TCR is somewhat unusual in that it expresses the V3.1 region, which
has been shown by Gascoigne and colleagues to skew polyclonal populations of T cells toward the CD4 phenotype (13). In fact, various TCR V
region genes have now been
associated with CD4/CD8 repertoire selection (10),
presumably because these V
chains preferentially bind to
either an MHC class II or class I restricting element. In the
case of the most thoroughly studied family, V
3, the phenomenon is apparently dependent on two amino acid residues, at position 27 in CDR1 and position 51 in CDR2. Either residue is sufficient to affect the CD4/CD8 balance,
and together they have an additive effect (13, 14). V
3.2 is
strongly skewed toward the CD8 phenotype and has
Phe27
and Pro51
at these positions, whereas V
3.1 is reciprocally skewed toward CD4 and has Ser27
and Ser51
.
Thus, it was proposed that V
3.2 interacts "preferentially" with MHC class I molecules and that the skewing attributed to positions 27 and 51 might result from increased
positive selection (13). It is interesting to note that V
11.3
also contains a proline at position 51 and, like V
3.2, is
skewed toward the CD8 phenotype (15).
Structural analysis of the 2C TCR places both Ser27 and
Ser51
in contact with the
helices of MHC class I. This
has led to the suggestion that they may play a key role in
orienting the TCR-pMHC interaction (16). One of these
interactions (Ser51
with the
2 helix) is also present in the
A6/HLA-A2/Tax structure (17). The binding energetics
associated with the preferential interaction of these two residues with either MHC class I or II molecules has yet to be
elucidated. While the intuitive explanation might predict
that the skewing phenomenon resulted from an increase in
affinity (e.g., of V
3.2 for MHC class I) that favored positive selection, this has not been proven. In this study, we
have tested the hypothesis that the CD8/CD4 repertoire
skewing attributed to these two residues is accomplished by
increases in affinity for MHC class I molecules. Unexpectedly, we found that single or double mutants at these two
positions exhibited affinities that were not consistent with a
simple affinity model of increased positive selection, but
rather support the quantitative-instructional model of lineage commitment.
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Materials and Methods |
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Single-chain TCR Mutagenesis.
CDR mutant single-chain (sc)TCRs were constructed using a PCR-based technique (18). In brief, a short mutagenic primer and a VscTCR Expression and Purification.
Proteins were expressed in E. coli as described previously (7, 19) and purified from inclusion bodies by denaturing metal affinity chromatography and G-200 size exclusion chromatography. Protein purity was assessed by SDS-PAGE as well as by electrospray mass spectrometry for several of the mutants (performed at the University of Illinois Mass Spectrometry Facility).mAbs.
KJ16 (20) is a rat IgG mAb that is specific for the mouse VELISAs.
VPeptide-MHC Binding Assays.
A competition, cell binding assay was used to monitor binding of TCR to QL9/Ld complexes, as described previously (7, 24, 25). To form peptide-Ld complexes on the surfaces of T2-Ld target cells, cells were incubated with ~10 µM of the specific peptide for 3-5 h. For the competitive binding assay, peptide-upregulated cells (3 × 105/ well) were incubated with 0.7 nM 125I-labeled 30-5-7 Fab and various concentrations of scTCR for 1 h on ice in the presence of 0.7% BSA. After incubation, bound and free ligands were separated by centrifugation through dibutyl phthalate/olive oil. All assays were done in triplicate. The cell binding assay was able to detect binding that was 15-20-fold reduced compared with wild-type. All surface plasmon resonance (SPR) measurements were performed on a BIAcore 2000 instrument (BIAcore) at 25°C in PBS. Purified molecules were immobilized on a CM5 chip at pH 5.2 in 10 mM sodium acetate buffer by classical amine coupling chemistry. Blank surfaces were made by ethanolamine deactivation of a dextran surface. Purified Ld-peptide complexes were injected at 6, 3, 1.5, 0.75, and 0.375 µM at a flow rate of 20 µl/ min. Data were normalized and analyzed using the BIAevaluation 2.1 and 3.0 software programs (BIAcore). Single Langmuir binding model was used for curve fitting analysis. Fittings were assessed by K2 function (<5.0). Baseline drift between successive injections was <2%. Scatchard analyses were plotted from subtracted sensorgrams (Ld-QL9 minus Ld-murine CMV [MCMV] traces). Binding studies were performed with immobilized TCR proteins and the reverse orientation with immobilized Ld-peptide complexes.Statistical Analysis of Binding Data.
The percent inhibition of T2-Ld binding was corrected for the binding of 125I-labeled 30-5-7 Fab fragments to Ld molecules which do not contain the peptide of interest, as follows: % inhibition = (cpmQL9, no TCRStructural Analysis.
Structural analysis was performed on an Indigo 2 workstation (Silicon Graphics) using the Quanta software package (Molecular Simulations). The refined structure of the 2C/dEV8/Kb complex (16) was used to assign pMHC contacts for those 2C residues tested by mutagenesis. ![]() |
Results |
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Recently, we have mapped the energy of interactions of
2C TCR residues by performing alanine scanning mutagenesis of the 2C TCR and analyzing the mutants for
binding to QL9/Ld (7). The mutagenesis study revealed
four residues (Glu56, Thr55
, Ser76
, Ser102
) that, when
changed to alanine, exhibited increases (up to twofold) in
affinity for QL9/Ld. Two other residues (Ser27
and Ser51
)
are also thought to play a significant role in the binding of the class I ligand. This prediction is based both on the contact of these residues with conserved residues in the MHC
helices (16) and on their effects on skewing of T cells to a
CD4 or CD8 phenotype (13, 14). These six residues are
shown in Fig. 1, where the 2C TCR is modeled onto the
QL9/Ld ligand based on coordinates of the 2C/dEV8/Kb
complex (26). To explore various issues that involve the
binding contributions of these residues, mutagenesis was
performed and the two classes of mutants (alanine-substituent affinity mutants and CD4/CD8 repertoire-skewing
mutants) were analyzed for binding to anti-TCR antibodies and to the pMHC ligand QL9/Ld.
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TCR Mutagenesis, Expression, and Purification
The V3.1/V
8.2 TCR from CTL clone 2C has been
produced as a single-chain thioredoxin fusion protein in E.
coli for use in binding and mutagenesis studies (7, 24, 25, 27).
Various single, double, and triple mutants involving six residues (Glu56
, Thr55
, Ser76
, Ser102
, Ser27
, and Ser51
) were
generated using a two-step PCR-based approach. Mutants were subjected to automated DNA sequencing to confirm
mutations. TCRs were expressed in E. coli and purified by
metal affinity and size exclusion chromatography to
95%
purity. Electrospray mass spectrometry was used to confirm
the expected mass difference relative to wild-type for several
of the mutant TCRs (Fig. 2). The wild-type scTCR is evident as a single peak of mass 40,538 ± 4 daltons (0.01% accuracy), whereas the various mutant TCRs exhibit mass differences equivalent to those predicted based on their amino
acid differences. This sensitive method is able to confirm the
mass difference attributable to the loss of as little as a single
oxygen atom in an ~40,000-dalton protein, as demonstrated with the
S27A mutant (Fig. 2).
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Alanine-substituent Affinity Mutants
The four single-site alanine mutants of TCR residues
(Glu56, Thr55
, Ser76
, Ser102
) that exhibited modest increases
in affinity for QL9/Ld (7) were examined as double and triple
alanine mutants to test whether synergistic affinity increases
might be achieved. In addition, residue 56
was changed to a
positive charge (Lys56
) to test the possibility that this glutamic
acid side chain in the wild-type is near a negatively charged
residue in the ligand and thus that a positive charge at residue
56
may yield even higher affinity for QL9/Ld.
The degree to which each mutant
was properly refolded was analyzed using the mAbs KJ16
(V8.1/8.2 specific) and F23.1 (V
8.1/8.2/8.3 specific).
These two mAbs recognize determinants on the TCR distal from the CDR regions. Each of the mutants exhibited
essentially the same reactivity as wild-type (Fig. 3 A, and
data not shown), indicating that the mutant TCRs have the
same degree of refolding as wild-type, and confirming that
mutations have not globally disrupted the protein's tertiary
conformation. Thus, any differences observed in reactivities of the mutants toward other antigens can be attributable to the role of the substituted amino acid and not to a
global destabilization of the receptor.
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The mAb F23.2 is specific for V8.2, and its epitope has
been shown to include residue Glu56
, which is critical for
binding (7). The reactivity of each mutant was analyzed in
F23.2 competition ELISA experiments where it was confirmed that those mutants containing the
E56A mutation (i.e., 102/56, 102/56/76, 56/76, E56A, or E56K) reduced
binding to F23.2 by at least 100-fold (
2.5 kcal/mol). Alanine substitution at Thr55
exerts only a moderate effect
(three- to fourfold reduction) on F23.2 binding (7). As expected, this moderate reduction in affinity is also observed
in the T55A-containing mutants (i.e., 102/55, 55/76, 102/
55/76, and T55A; Fig. 3 B, and Fig. 4). Thus, mAb F23.2
serves as an additional probe of the tertiary conformation of
the scTCR mutants.
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The clonotypic antibody 1B2 recognizes an epitope on
the 2C TCR which overlaps with the pMHC epitope (7).
Therefore, it was of interest to determine the effect of double and triple alanine mutations on 1B2 binding. Similar to
the effect observed with F23.2, each of the mutants that
contained an E56 mutation had at least a 20-fold reduction
in 1B2 activity (Fig. 3 C, and Fig. 4). The S102A mutant
exhibited a more moderate fivefold reduced 1B2 activity,
whereas T55A had threefold reduced activity. The combination of these two mutants in a single protein resulted in
an approximately additive effect on binding (i.e., 11-fold reduced binding activity for either 102/55 or 102/55/76).
Consistent with the observation that Ser76 is not involved
in either the F23.2 or 1B2 epitopes (7), no additional effect
on reactivity was observed with double or triple mutants
that involved this position.
To assess the effect of alanine-substituent mutations on pMHC recognition, mutants
were tested for the ability to inhibit binding of 125I-labeled
anti-Ld Fab fragments to the QL9/Ld alloantigen. In contrast to the significant impairments seen in F23.2 and 1B2
antibody reactivity, all of the alanine-substituent mutants
bound to pMHC at least as well as the wild-type scTCR (Fig. 3 D, and Fig. 4). The greatest improvement in
pMHC recognition was achieved with the 102/55 double
mutant, which was 2.3-fold better than wild-type (free energy change [G] =
0.44 kcal/mol). The effect observed with 102/55 appears to have been at least partially
additive, in that S102A and T55A had
G values of
0.31 and
0.23 kcal/mol, respectively. In contrast, the
effect seen with 102/56 (
G =
0.32 kcal/mol) is only a
slight improvement on the binding energy of the S102A
single mutant (S102A and E56A have
Gs of
0.31 and
0.11 kcal/mol, respectively).
Residue Ser76 is within the HV4 loop and does not interact with antigen in the refined 2C/dEV8/Kb structure
(16). Consequently, the modest improvement in pMHC affinity initially observed with S76A was attributed to indirect effects (7). In this study, the effects of the S76A mutation when paired with other mutations was variable. The
55/76 mutant appeared to bind pMHC marginally better
than T55A, but incorporation of an S76A mutation reduced the affinity of 102/55, 102/56, and E56A (Fig. 4).
Thus, whatever indirect conformational effects S76A
makes on its own to increase affinity do not appear to be readily accommodated in the presence of other mutations.
In the model of the 2C TCR-QL9/Ld complex, residue
Glu56 is positioned 9 Å opposite Glu 71 of the Ld
1 helix.
We suggested that this pairing might represent an unfavorable electrostatic interaction (7). We reasoned that introduction of a positively charged lysine residue at position
56
might create a more favorable electrostatic pairing of
the 2C and QL9/Ld surfaces. In contradiction to this notion, the E56K mutant exhibited significantly decreased
binding to QL9/Ld (
G = 1.06 kcal/mol). Thus, simple
introduction of a positive charge at this position does not
create a more favorable electrostatic interaction between
the TCR and pMHC.
Since the greatest improvement in binding affinity was achieved with the 102/55 double mutant, we sought to investigate the kinetic parameters involved in its binding to pMHC. Using immobilized mutant TCRs, binding to soluble pMHC complexes was analyzed by BIAcore SPR. The 102/55 double mutant was found to have on and off rates which were both slowed compared with wild-type (Fig. 5). The on and off rates for wild-type were 2.4- and 3.2-fold faster, respectively, than the rates for 102/55 (Fig. 5). The relatively larger difference observed for the off rates translates into a 23% lower overall equilibrium dissociation constant for 102/55 compared with wild-type, consistent with results from the Fab inhibition experiments. The reverse orientation, with biotinylated Ld-peptide complexes immobilized on the chip, yielded similar kinetic differences between the wild-type and 102/55 TCR (data not shown). In addition, the Kd values of the two proteins differed by 1.3-2.7-fold using three different methods of BIAcore analysis: (a) by kinetic measurements with immobilized TCR, Kd of wild-type TCR = 3.2 µM and of 102/55 TCR = 2.4 µM (Fig. 5); (b) by kinetic measurements with immobilized Ld-peptide, Kd of wild-type TCR = 1.9 µM and of 102/55 TCR = 0.7 µM (data not shown); and (c) by Scatchard analyses of binding at various Ld-peptide concentrations, Kd of wild-type TCR = 1.9 µM and of 102/55 TCR = 1.2 µM (data not shown). These results, together with data from the cell binding assay described above, indicate that the 102/55 mutant has a higher affinity that the wild-type.
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CD4/CD8 Repertoire-skewing Mutants
Previous work has suggested that a phenylalanine at position 27 (in CDR1) and a proline at position 51 (in CDR2)
within the V3.2 region may each interact with MHC class
I, thereby accounting for the tendency of V
3.2+ T cells to
skew toward a CD8 phenotype (13). To test the hypothesis that this preferential interaction involves an increase in affinity for MHC class I, we constructed mutant 2C scTCRs incorporating these "V
3.2-like" substitutions into the 2C
V
3.1 region (see Table I). Additionally, we also analyzed
alanine-substituent mutants at these two positions.
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Each of these V3.2-like mutants
exhibited equivalent reactivity to wild-type in KJ16, F23.1,
and F23.2 ELISAs, indicating the same degree of refolding
(Fig. 6 A, and data not shown). Alanine substitution at either
position did not affect recognition by 1B2, but S27F exhibited a slight increase in reactivity toward 1B2 and S51P had
~60-fold reduced 1B2 reactivity (data not shown). The failure of these mutations to affect F23.2, KJ16, and F23.1 reactivity confirms that there has not been a global effect on TCR conformation, beyond the
chain CDR regions.
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In both the 2C/dEV8/Kb structure and the 2C/QL9/Ld model, serines 27 and 51 are each
involved in hydrogen bonding to conserved residues on the
helices (Fig. 1). We have previously reported that elimination of the hydroxyl moiety of Ser51
does not have an
effect on recognition of the allogeneic pMHC ligand (7).
Elimination of the hydroxyl at Ser27
similarly failed to disrupt binding to QL9/Ld (Fig. 6, B and C). Together, these
data suggest that the amount of binding energy contributed
by these two hydrogen bonds is negligible in the interaction of 2C with the Ld alloantigen.
In contrast to our expectations, substitution of a phenylalanine for the serine at position 27 in CDR1 resulted in a
mutant that had 1.6-fold reduced binding to pMHC (G = 0.24 kcal/mol; Fig. 6, B and C). Furthermore, the
S51P
mutant in CDR2 exhibited ~15-fold reduced binding to
pMHC (
G = 1.5 kcal/mol). Combination of both
mutations yielded approximately additive effects on both QL9/Ld binding (Fig. 6 C) and 1B2 binding (data not
shown). The pMHC affinity of the S27F/S51P V
3.2-like
double mutant was below the detection limits of the cellular binding assay. Together, these results illustrate that the
V
3.2 mutations do not result in a general enhancement of
binding to MHC class I molecules.
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Discussion |
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This study addressed two questions of immunological
significance regarding the binding of a TCR to its class I
ligand. First, is it possible to use site-directed mutagenesis
of CDR residues to increase the affinity of a TCR? Second, do various TCR mutants in V residues involved in
repertoire skewing of a TCR toward the CD8 phenotype
exhibit correlative affinities for the class I ligand?
The TCR from the alloreactive clone 2C could be used
to address each of these questions. The 2C-QL9/Ld interaction represents the highest affinity TCR-pMHC interaction that has been measured (8), and our recent alanine
scanning study identified several residues that might serve
as the starting point for the rational design of higher affinity
TCR mutants (7). To examine if TCR affinity could be
improved, we concentrated on the four CDR positions
initially found to have very modest increases in affinity
when mutated to alanine. Thus, alanine mutants at positions Ser102, Thr55
, Glu56
, and Ser76
were combined as
either double or triple mutants. Since Thr55
and Glu56
are
adjacent to each other in the refined 2C/dEV8/Kb structure, it was thought that they might be achieving their effect via similar mechanisms, and the 55/56 double mutant
was not generated. Additionally, since elimination of the
negative charge at position 56 seemed energetically favorable, the charge reversal mutant E56K was analyzed. Results of antibody reactivity of the various multiple alanine
mutants were entirely consistent with the results from single mutants (e.g., each of the position 56 mutants had
greatly reduced F23.2 binding), and also demonstrated that
the mutants had equivalent refolding as measured by KJ16 and F23.1 (Figs. 3 A and Fig. 4, and data not shown).
The greatest increase in affinity for pMHC was observed
with combined alanine substitution at positions Ser102 and
Thr55
(i.e., 102/55), which exhibited an approximately
twofold increase in affinity over wild-type and had slower
association and dissociation kinetics than wild-type. The
mechanism behind this increase in affinity is not clear, but
based on structural analysis several possibilities exist. First,
modeling of the 2C/QL9/Ld complex places Ser102
in a
position of steric clash with ProP6 (26). Elimination of the
hydroxyl moiety at this position might serve to eliminate or
lessen this steric effect. Second, in the 2C/dEV8/Kb structure (16), Glu56
lies 9 Å opposite Glu71 of the
1 helix, a
negatively charged residue common to both Kb and Ld that
may represent a somewhat unfavorable electrostatic interaction surface. Alanine mutation of adjacent residue Thr55
might allow Glu56
to adopt a less electrostatically impaired
conformation. However, the failure of E56K to improve
pMHC affinity illustrates that the interactions that contribute to the binding energy of TCR-pMHC are subtle, context dependent, and not necessarily amenable to techniques
such as charge swapping. Previous studies with other protein-protein interactions have shown similar difficulties in predicting whether charge complementation pairs might be
successful in this regard.
The potential additivity of the various double and triple mutants analyzed in this study could not be predicted. This contrasts with the alanine scanning analysis of growth hormone and its receptor, in which additivity was the rule (28). This has led many to assume that additivity will be observed when noninteracting mutations are combined. However, consistent with our results with the TCR, a recent study to improve the affinity of an anti-HIV gp120 antibody observed unpredictable additivity (only one of six combinations was additive) when mutations were recombined (29). There is some evidence that a rigorous accounting of bound water molecules in protein-protein interactions can account for certain cases when double mutants behave in a less than additive manner (30). The reasons underlying the unpredictable additivity observed in this study are unclear, but could be related to the presence of water molecules and poor complementarity of fit observed in the refined structure (16).
Despite the only twofold increase in affinity achieved by directed site-specific mutagenesis, these results indicate the promise in using more sophisticated techniques to obtain even greater improvements in TCR affinity. In fact, the earliest efforts to engineer higher affinity antibodies yielded a similar magnitude of increase (31, 32). More recent approaches have greatly enhanced these efforts (33), and we predict the same will now be possible with the TCR. This expectation is also based on the observation that the TCR- pMHC interaction is marked by poor complementarity of fit (16). According to an algorithm used to calculate complementarity of fit, the 2C/dEV8/Kb and A6/HLA-A2/ Tax complexes gave values of 0.45 and 0.47, respectively, whereas most antigen-antibody interactions have values in the range of 0.66-0.68, and oligomeric proteins in the range of 0.68-0.75 (16). Clearly, there are regions of the TCR-pMHC interface where the fit can be improved through the introduction of amino acid substitutions. Thus, mutagenesis methods that explore the effects of amino acids other than alanine could yield still higher affinity soluble TCRs.
The second aspect of this report concerns the role of
TCR affinity in repertoire skewing of a TCR toward the
CD8 phenotype. The observation that V3.1 transgenic
mice expressing the mutations S27F and S51P have a repertoire skewed toward a CD8+ phenotype suggested the
notion that these positions contacted the class I selecting element, and the interaction was described as being preferential toward class I (13, 14). Structural analysis of the 2C
TCR showed that the serines at positions 27 and 51 of the
chain contact conserved residues on the class I helices
(16; depicted in Fig. 1). Together, these studies might lead
one to predict (a) that the serines at positions 27 and 51 would contribute some energy to the TCR-pMHC interaction, and (b) that substitution of phenylalanine (at position 27) and proline (at position 51) would yield even
higher affinity. Surprisingly, neither of these predictions
was borne out in the measurements of binding affinities
described here. Both alanine mutants at position 27 and 51 had negligible effects on the binding of the QL9/Ld ligand.
That is, elimination of either hydroxyl group and the disruption of that hydrogen bonding capability had no effect
on the equilibrium affinity of the TCR for pMHC. Substitution of the V
3.2 residues (Phe and Pro) yielded not
higher but lower affinities for the QL9/Ld ligand.
What, then, accounts for the apparent discrepancy between these results and the observed skewing? Serine is the
most frequently occurring amino acid at both positions 27 and 51 in murine V genes (34). Threonine is also a frequent residue at both these positions, but Phe27
and Pro51
are found much less often. At position 27, the relative frequency at which these residues are found is as follows:
serine 43%, threonine 22%, phenylalanine 1%. At position
51, serine occurs at 54% frequency, threonine at 25%, and
proline at only 7% (34). Thus, there is conservation of hydroxyl-containing amino acids at these positions. Based on
these findings and the 2C/dEV8/Kb structure, it has been
suggested that these serines may help to orient the TCR
diagonally on the pMHC (16). Serine residues at both positions are also found contacting MHC helices in the recent
B7/Tax/HLA-A2 structure (35). Nevertheless, elimination of the hydroxyl group at either position failed to significantly effect the overall affinity of the 2C-pMHC interaction (Fig. 6). Two possible explanations may account for
these observations. First, although the equilibrium affinity
of the S27A and S51A mutants are unchanged from wild-type (perhaps due to compensation by bridging water molecules), it is possible that these hydroxyl residues have a favorable effect on binding kinetics (e.g., by increasing both
on and off rates). This would be consistent with the kinetic
differences observed for the 102/55 double alanine mutant,
where elimination of two hydroxyl moieties at Ser102
and
Thr55
slowed both the on and off rates. Second, it is possible that the interaction of the 2C TCR with the QL9/Ld
ligand may differ fundamentally from the interaction of the
2C TCR with the dEV8/Kb ligand. Arguing against this
explanation is the fact that those residues within the Kb
helices which contact Ser27
and Ser51
(Glu 58 and Arg 62 contacting Ser27
, and Ala 152, Gly 162, and Glu 166 contacting Ser51
) are identical in Ld. If the interaction with Kb
and Ld was in fact fundamentally different, it is conceivable
that V
3.1 and V
3.2 would differ in skewing toward a
CD4 or CD8 phenotype if the selecting environment contained Ld as the only class I molecule.
The possibility that some class I molecules could interact
with the V3 region in a different manner remains to be
examined, and in this respect it is important to recognize
that our findings involve measurements with a single
TCR-pMHC system. Other TCR-pMHC interactions
will no doubt need to be analyzed to support a particular model of CD4/CD8 skewing. Nevertheless, we believe
our results with the alanine and V
3.2-like substitutions at
positions 27 and 51 are most consistent with the recent
quantitative-instructional model for CD4/CD8 lineage
commitment (36). According to this model, gradations
in the intensity of signaling through the TCR complex of
CD4+/CD8+ thymocytes determines lineage commitment. Signals of strong intensity promote CD4 differentiation, whereas weaker intensity signals promote a CD8 fate.
Consistent with this model, V
3.1 2C (Ser27
and Ser51
)
binds class I with ~15-fold higher affinity than V
3.2-like 2C (Phe27
and Pro51
). Consequently, V
3.1 is skewed
toward CD4, whereas V
3.2 is skewed toward CD8.
Thus, it appears that energy changes amounting to as little
as 0.25 kcal/mol (S27F) can skew the lineage commitment of developing thymocytes. Since 2C is a class I-restricted
TCR, we are unable to address the effect of these mutations on binding to MHC class II. According to the quantitative-instructional model, one might expect that V
3.1
(Ser27
and Ser51
) would contribute to increased affinity
for MHC class II as well as class I. In this regard, the high
frequency of serine residues at these positions (43 and 54%)
is not surprising, since it would then act to promote general
MHC reactivity. However, it is possible that interaction of
thymocytes with class I molecules alone may explain V
3
skewing, since commitment to the CD4+ lineage can occur in the absence of MHC class II molecules and does not
require MHC class II-restricted TCRs (39).
A second, not mutually exclusive, possibility is that differential negative selection could also be involved in the
observed CD4/CD8 skewing. According to this mechanism, CD8+ thymocytes which express V3.1 TCRs (i.e.,
possessing Ser27
and Ser51
) could bind selectively to
MHC class I molecules, predisposing them to elimination
by negative selection. CD8+ thymocytes which express
V
3.2 TCRs (i.e., Phe27
and Pro51
) would have an inherently lower affinity for MHC class I, thereby allowing
them to escape negative selection. Presumably, this process would operate at the stage of the semimature single-positive heat stable antigen (HSA)hi thymocyte, a cell type that
remains susceptible to negative selection within the thymic
medulla (40). It has recently been estimated that 20-30% of
thymocytes in mice are susceptible to negative selection
(41), a figure which could easily accommodate a degree of
repertoire skewing by differential negative selection.
In conclusion, we have provided direct evidence that the TCR framework can be engineered to support higher affinities for pMHC than are selected for during thymic development. Additionally, we have shown that the mechanism whereby two CDR mutants skew the T cell repertoire toward a CD8+ phenotype is not attributable to a general increase in affinity for MHC class I, but rather is consistent with predictions from the quantitative-instructional model of lineage commitment.
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Footnotes |
---|
Address correspondence to David M. Kranz, Department of Biochemistry, University of Illinois, 600 S. Mathews, Urbana, IL 61801. Phone: 217-244-2821; Fax: 217-244-5858; E-mail: d-kranz{at}uiuc.edu
Received for publication 2 June 1998 and in revised form 25 November 1998.
We wish to thank Drs. Carol Schlueter and Thomas Brodnicki for cloning several of the single-site mutants, Dr. Ted Hansen for providing the 30-5-7 hybridoma line, and Dr. Peter Cresswell for providing the T2-Ld cell line. We are grateful to Dr. Nicholas Gascoigne for critical reading of the manuscript. The expertise and assistance of the Mass Spectrometry and Genetic Engineering Facilities at the University of Illinois were greatly appreciated.
This work was supported by National Institutes of Health R01 grants GM55767 (to D.M. Kranz) and AI42267 (to L. Teyton).
Abbreviations used in this paper
G, free energy change;
MCMV, murine CMV;
pMHC, peptide-MHC complex;
scTCR, single-chain TCR;
SPR, surface plasmon resonance.
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