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Immunology Group, International Centre for Genetic Engineering and Biotechnology, and
National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the above notion represents a logical derivative of our understanding of parameters regulating B cell responses, it, however, appears to be inconsistent with experimental observations. Characterization of immune responses to a variety of protein Ags has revealed that only a fraction of the repertoire of B cell epitopes presented by such Ags is functionally capable of eliciting Abs (4, 5, 6, 7, 8, 9, 10, 11, 12, 13). Some insight into this apparent discrepancy was provided by our own studies employing synthetic peptides as model T-dependent Ags (13, 14, 15). These investigations revealed that the primary IgM response was indeed consistent with expectations in that Abs were produced against all accessible domains on Ag (14). Interestingly however, these early activated clonotypes were soon subjected to a stringent selection process which ensured continued survival of only those induced B cells with a high enough affinity for Ag (14). This selection process preceded initiation of the germinal center (GC)3 reaction, and was enforced by the need for the diverse Ag-activated B cells to compete for a limiting pool of Ag-primed Th cells (14). Although the precise mechanism is unclear, affinity for Ag was found to be crucial in defining ability of the individual clonotypes to recruit T cell help in a competitive milieu (14). Only those Ag-activated B cells selected at this stage were shown to be permitted to populate GCs, thereby influencing the character of secondary responses (16, 17). These observations could also be complemented, at the cellular level, by our demonstration that differential Th cell thresholds operate during Ag-driven activation of preimmune B cells and the subsequent selection step that restricts clonotype seeding of GCs (17, 18).
It is obvious, therefore, that the pre-GC phase selection process outlined above constitutes the first active step initiating maturation of T-dependent humoral responses to multideterminant Ags. However, although cellular interactions that drive this process were becoming apparent, there was one important facet that remained unresolved. This related to the question of whether there were any underlying factors regulating the affinity of Abs recruited from a pool of stochastically generated receptors. This issue became particularly relevant in light of our observations of an intriguing parallel wherein Ag-affinity dependent pre-GC phase selection also simultaneously resulted in a "filtering" of epitopes recognized on Ag (14, 17). Furthermore, relative immunodominance of an individual epitope subsequently proved to be resistant to a variety of predictable variables such as mouse H-2 haplotype, nature of flanking domains, positional effects, varying degrees of conformational flexibility, as well as any restriction at the level of B cell repertoires induced against it (13, 14, 15, 19). These and related results suggested, to us, that the affinity of early primary responses may be epitope dependent. In other words, the chemical composition of a given epitope may constitute at least one parameter that dictates the affinity of primary Ab responses elicited against it.
The present set of investigations was, therefore, undertaken with the objective of verifying the above possibility. Results presented here, indeed, argue in favor of such a correlation. Furthermore, they also establish the functional significance of such distinctions in epitope-specific early primary Ab affinities, by demonstrating that they impinge upon the hierarchy of epitopic dominance observed in the later stages of humoral responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Heavy chain-specific, HRP-labeled, goat anti-mouse IgM and IgG were purchased from Sigma (St. Louis, MO). Anti-B7-2 (clone GL1) was purchased from PharMingen (San Diego, CA). For multipin synthesis of peptides, both the noncleavable kits and F-moc amino acid derivatives were obtained from Chiron Mimotopes (Victoria, Australia).
Peptide synthesis
Peptide PS1CT3 and all its analogues used in this study were
synthesized by the solid-phase method (20, 21) using F-moc
chemistry (22) on a Milligen 9050 automated peptide
synthesizer (Millipore, Bedford, MA). Crude peptides were purified to
at least 95% purity by reverse-phase HPLC on a
C18 column (15µ 
pak, 19 x 300 mm;
Waters, Milford, MA) using an aqueous gradient of 0–70% acetonitrile
in 0.1% trifluoroacetic acid. The identities of all peptides were
ascertained by mass spectrometry.
Overlapping hexapeptide panels were synthesized according to the method of Geysen et al. (23) on noncleavable multipin kits. For synthesis, the standard protocol recommended by the manufacturer was adhered to. After completion of synthesis, all peptides were routinely acetylated at the amino terminus with a 50:5:1 (v/v/v) mixture of dimethylformamide, acetic anhydride, and triethylamine. Side chain deprotection was accomplished over a 2-h period at room temperature with a 38:1:1 (v/v/v) mixture of trifluoroacetic acid, ethanedithiol, and anisole.
Animals and immunizations
Female BALB/c mice (6–8 wk old) were obtained from the small animal facility at the National Institute of Nutrition (Hyderabad, India). Except where stated, immunizations were generally given i.p. at a dose of 50 µg/mouse as an emulsion in CFA. For polyclonal sera, mice were bled from the retro-orbital plexus, and sera within a group were pooled. In all cases, the IgM and IgG fractions were separated by passing over a protein G-Sepharose column (Pharmacia, Uppsala, Sweden). The flow through contained IgM, whereas bound IgG, after thorough washing of the column, was eluted with glycine-HCl buffer (pH 2.7). The eluate was immediately neutralized, concentrated, and then dialyzed against PBS before use.
Affinity purification of Abs
All IgM mAbs described were obtained as ascites from hybridomas that had been generated earlier (14, 15). These mAbs represent primary IgM responses to the PS1 segment at 4 days after a primary immunization of BALB/c mice (14, 15). Each of the IgM preparations was purified by affinity chromatography over an Affi-Gel 10 Gel (Bio-Rad, Hercules, CA) column coupled to peptide PS1CT3 at 10 mg/ml of gel. The ascites preparation was incubated with the affinity matrix overnight at 4°C on a rotating shaker. After this, the flow through was removed and the column was washed with 10 column volumes of PBS (pH 7.2), followed by 10 column volumes of phosphate buffer containing 1 M NaCl, and finally again with 5 column volumes of PBS. Bound IgM was then eluted in glycine-HCl buffer (pH 2.7) and then immediately neutralized with 1 M Tris buffer (pH 8.0). The neutralized eluate was subjected to buffer exchange into 0.5 M Tris buffer containing 2 mM EDTA (pH 8.0) and concentrated to a final IgM concentration of 10 mg/ml. Purity of the IgM fraction was ascertained by SDS-PAGE.
For peptide-specific polyclonal IgM, sera were first depleted of IgG Abs by two rounds of incubation with protein G-Sepharose. The flow through fraction was further enriched for IgM by chromatography over DEAE-cellulose (Sigma). The IgM-containing fraction was then purified by affinity chromatography for Abs specific either for peptide PS1CT3 or, as required, its analogues as described above.
Preparation of monomeric IgMs
This was essentially achieved using a standard protocol (24). Briefly, affinity-purified IgM Abs obtained above were reduced with a final concentration of 1 mM DTT at room temperature for 1 h with gentle shaking. After this, reduced IgM was alkylated with a final concentration (added in one-tenth the volume) of 2.1 mM iodoacetamide in the dark at 4°C for 30 min. At the end of this period, the mixture was resolved over a Sephadex G-200 column (Sigma; column dimensions, 25 cm x 1.5 cm; load volume, 0.5 ml) using blue dextran as a marker for the void volume. Fractions containing monomeric IgM, which elutes immediately following the void volume, were pooled and concentrated to between 10 and 40 mg/ml in PBS containing 0.05% Tween 20. Ab concentrations were determined spectrophotometrically by measuring absorbance at 280 nm. An average molecular mass of 200 kDa was taken for monomeric IgM (24).
Analysis of monomeric IgM binding to peptide PS1CT3
Monomeric mAb binding to peptide PS1CT3 was examined according to the technique of surface plasmon resonance on an Iasys Auto+ instrument (Affinity Sensors, Cambridge, U.K.). Peptide was immobilized onto carboxymethyl dextran cuvettes using the protocol recommended by the manufacturer. Ab binding to immobilized peptide was monitored at multiple Ab concentrations, ranging from 5-fold below to at least 5-fold above preliminary estimates of equilibrium dissociation constants (Kd) for each mAb. All reactions were conducted at 25°C. Association was monitored over a period of 7–10 min, whereas dissociation was monitored for 5 min. Kinetic analysis was performed using the FASTfit software (Affinity Sensors, Cambridge, U.K.) provided by the manufacturer, which provided the values of kon and kdiss at each Ab concentration. Second-order association rate constants (kass) were then obtained from a linear regression plot of kon vs Ab concentration, again using the FASTfit software. Equilibrium affinity constants (Ka) were then derived, for each mAb, from the equation: Ka = kass/kdiss.
For a thermodynamic analysis of peptide-mAb association, monomer Abs were used at a single concentration that was 5-fold above its Kd value. The association reaction for each mAb was studied, as described above, at four different temperatures of 20, 25, 30, and 35°C. The corresponding kon values were obtained, from which the apparent association rate constant for each Ab at each temperature was obtained by dividing it with Ab concentration.
ELISAs
Plates were coated with 2 µg/ml of peptide per well in 100 µl of PBS (pH 7.2) at 37°C for 3.5 h. Subsequently, they were blocked with 300 µl/well of a 5% solution of fat-free dry milk powder in PBS at 37°C for 1 h. Then 100 µl of the appropriate dilution of mouse antiserum was added and incubated at 37°C for 1 h. After washing, bound Ab was detected with HRP-labeled secondary Ab (37°C, 1 h), followed by color development with o-phenylenediamine as chromogen. Absorbance was measured at 490 nm, and background absorbance obtained for preimmune serum at the corresponding dilution was subtracted.
For competitive ELISA experiments, the procedure described earlier was followed (14, 19). Briefly, the purified IgG fraction, at concentrations 2-fold that of the 50% titer value, was mixed with appropriated concentrations of competitor peptide in equal volumes and incubated for 10 min at room temperature. This was then added to duplicate wells at 100 µl/well. The remaining procedure is as described above.
ELISAs for pin-bound hexapeptides
Ab cross-reactivity with pin-bound hexapeptides was also evaluated by ELISA. For this, the protocol recommended by the manufacturer was strictly followed. Primary Abs were diluted appropriately in PBS containing 2% BSA, 0.1% Tween 29, and 0.1% sodium azide. Pins were incubated in 200 µl each of the Ab solution at 4°C overnight with gentle shaking. Subsequently, they were washed and subjected to a second round of incubation with the appropriate dilution of HRPO-labeled goat anti-mouse IgG at room temperature for 1 h, again with gentle shaking. The chromogen used for detecting bound Ab was 2,2'-azino-bis-(3-ethyl-benzthiazoline-6-sulfonic acid)diammonium, and the absorbance was measured at 405 nm after subtracting the background at 490 nm.
Calculation of amino acid side chain 
G values
Specific side chain contribution from residues in interacting
domains derives from two distinct types of interactions: hydrophobic
and electrostatic. If one assumes that side chain entropy changes, when
engaged in an interaction, are negligible, the Gibbs-free energy
changes (G) accompanying idealized interactions for a given residue
side chain can be described as, Gtotal =
GHB + GEL
(25). Here, GHB represents the
energy released through hydrophobic interactions and has been estimated
to be -25 cal/ mol/A2 of surface area
(25, 26). Since side chain area for all individual
residues are known (26), GHB is
easily calculated. GEL represents the enthalpy
change due to electrostatic interactions. For the present case, where
we have assumed idealized interactions, the maximal value of -5.0
kcal/mol was taken for each salt-bridging interaction
(27). Thus, from the values of
GHB and GEL,
Gtotal was easily calculated for each
individual amino acid residue side chain.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We continued to employ the model peptide PS1CT3 for our current
studies. As previously described (14), this peptide
represents a T-dependent immunogen and is comprised of a
well-characterized B (residues 1–15, segment PS1) and a T (residues
18–38, segment CT3) cell epitope. Separating these two domains is a
spacer of two glycine residues at positions 16 and 17 (Fig. 1
). Our earlier results have demonstrated
that the murine primary IgM response to this peptide was composed of
individual Ab specificities that collectively spanned the entire PS1
segment (14). Interestingly, however, subsequent class
switch to IgG was restricted to only those Ab subsets directed against
a unique tetrapeptide epitope between positions 4 and 7 of the PS1
segment (sequence: DPAF, Fig. 1) (14). This apparent
monospecificity, which derived from an oligoclonal B cell population,
was also shown to be retained in the secondary IgG response (14, 19, 28).
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. As an initial
exploration, we asked whether these individual mAbs displayed any
qualitative differences in binding to their respective epitopes on
peptide PS1CT3. To do this, however, we first performed a reduction
reaction on each of these IgM mAbs to obtain monomeric IgMs (see
Materials and Methods). The objective in doing this was
2-fold. At one level it was expected that a bivalent IgM molecule would
allow for a greater degree of resolution of differences in Ag-binding
properties that might normally be masked due to the increased valency
of pentameric IgM. More importantly though, we reasoned that monomeric
IgM would also be reminiscent of the surface (s) IgM receptor on
preimmune B cells, thereby providing an insight into Ag recognition by
preimmune B cells.
Each of the individual monomeric IgM preparations obtained were
subsequently analyzed for their concentration-dependent binding to
peptide PS1CT3. For this, we employed the technique of surface plasmon
resonance (see Materials and Methods), and representative
data for one such monomeric IgM preparation is shown in Fig. 2. Analyses of these data yielded values
for the association (kass) and
dissociation (kdiss) rate constants,
from which the equilibrium affinity constant
(Ka) could readily be determined for
each mAb as described in Materials and Methods. The
monomeric IgM preparations analyzed, their corresponding epitope
specificities, as well as their Ag-binding properties are listed in
Table I. At the first glance, it is
interesting to note that this monomer mAb panel represents a wide
distribution of affinities with Ka
values ranging from 104 to
106 M-1 (Table I).
Considering that these mAbs were derived from B cells actively
participating in a primary response (14, 15), these
results would then suggest that Ag-binding affinity constants of as low
as 104 M-1 are sufficient
to induce preimmune B cells into a primary Ag-specific IgM response.
This is entirely consistent with our previous demonstration that
induction of a primary IgM response involves low activation thresholds
(18).
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is the fact
that Ab affinities were not randomly distributed across the various
epitopes but, rather, were segregated around the epitopes recognized.
Thus, for example, all anti-DPAF monomer IgM mAbs displayed
comparable affinities for epitopes, which was markedly higher than that
of those directed against alternate determinants within the PS1 segment
(Table I). Similarly, the various NSTN-specific mAbs were also
comparable in their affinity for the peptide (Table I). It must be
noted here that the anti-DPAF mAbs 7bM, 7cM, and 604 derive from
clonally distinct B cell precursors as revealed by nucleotide
sequencing of the Ig heavy chain variable region genes. Thus, the
predicted amino acid sequences of the heavy chain CDR3 regions for mAbs
7bM, 7cM, and 604, respectively, were as follows: QRTIGTPGAY, WAWFAY,
and IPITTATNWYDFV (Ref. 15 and B. P. Nayak and
K. V. S. Rao, unpublished results). This was also true of the
anti-NSTN mAbs 42M, 49M, and 8aM where, again, nonidentical IgH
CDR3 sequences were obtained (B. P. Nayak and K. V. S.
Rao, unpublished results). Thus, the comparable affinities displayed by
mAbs directed against a common epitope is not necessarily a consequence
of clonal relatedness of the B cells from which they were derived. Association between Ag and monomeric IgMs is driven by favorable enthalpy changes
The overall energetics and, therefore, the affinity of a binding
interaction is modulated by net changes in two thermodynamic
parameters, namely, enthalpy and entropy. Although the enthalpy term
generally describes heat changes that take place as a consequence of
interactions which occur at the binding interface, entropy changes
largely represent net conformational/stereochemical/structural
perturbations that occur either within the interacting entities or in
the surrounding solvent molecules (29, 30). Therefore, to
understand the basis of epitope-dependent distinction in monomer IgM
affinities seen in Table I, we examined the relative roles of enthalpy
and entropy in regulating association between Ab and Ag.
All mAbs listed in Table I were extensively analyzed for contribution
of enthalpic and entropic parameters in binding to peptide PS1CT3. Even
though the detailed results will be presented elsewhere (V. Manivel, P.
Nakra, N. Sahoo, and K. V. S. Rao, manuscript in
preparation), we discuss here results obtained for the association
phase of the reaction with four representative monomeric IgM mAbs. For
this, Ab association with peptide was studied at various temperatures
and the kass value at each temperature
was determined (see Materials and Methods). Fig. 3 gives the Arrhenius plot for the
temperature dependence of kass for the
four individual Ab preparations. The slope of each plot yielded the
activation energy (Ea) for each of the mAbs. This then permitted
calculation of the individual activation parameters for association
using equations 1–3 given below (see Ref. 31):
 | (1) |
 | (2) |
 | (3) |
. It is evident from these data that,
in all cases, association of monomeric IgM with peptide is accompanied
by large unfavorable changes in entropy as indicated by the large
negative values obtained for Sass (Table II).
In contrast, the negative Hass values clearly
identify that enthalpy changes accompanying Ab-Ag association were
highly favorable in all cases (Table II). Similar results were obtained
for all of the monomeric IgM mAbs listed in Table I (data not shown).
Consequently, the driving force for Ag-monomeric IgM association
appears to derive from stabilizing interactions at the paratope-epitope
interface, and can occur only if these forces can compensate for the
unfavorable entropy changes that such an interaction necessitates.
Because these studies employed monomeric IgM mAbs, this interpretation
could also, in principle, be extended to apply to the sIgM receptor on
preimmune B cells.
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reveal the existence of a
relationship between affinity of primary recognition of Ag and the
chemical nature of the epitope recognized, results summarized in Table II rationalize this observation by implying that this may derive from
differential epitope-dependent enthalpic contributions. Distinctions in affinity of preimmune recognition influence relative immunodominance in the later stages of the response
The influence of affinity of primary epitope-specific humoral responses on the eventual hierarchy of relative immunodominance, in the context of multideterminancy, was suggested from our earlier studies employing single residue glycine substitutions with the DPAF epitope of peptide PS1CT3 (14). To further confirm that the former does, indeed, impact upon the latter, we synthesized three additional analogues of peptide PS1CT3. In one, Asp (4) was replaced with an Asn residue (peptide PS1CT3N31), whereas Phe (7) was substituted for Leu in another (peptide PS1CT3L34). The third analogue represented a doubly substituted peptide where both Asp (4) and Phe (7) were substituted with Asn and Leu residues, respectively (peptide PS1CT3N31L34). The rationale for such substitutions was to specifically alter the chemical nature of the corresponding residue side chains, but without significantly affecting their steric bulk. The side chain areas for Asp and Asn are 106 A2 and 113 A2, respectively, whereas those for Phe and Leu are 175 A2 and 137 A2 (25).
In preliminary experiments, separate groups of mice were immunized with the individual peptides and the consequent peptide-specific IgM Abs were isolated from sera collected 7 days later. Monomeric derivatives were then prepared (see Materials and Methods), and relative avidity for the homologous peptide was determined by competitive inhibition ELISA experiments. These experiments yielded IC50 values of 14 ± 3 µM, 72 ± 8 µM, 64 ± 5 µM, and 88 ± 9 µM for anti-PS1CT3, anti-PS1CT3N31, anti-PS1CT3L34, and anti-PS1CT3N31L34 monomeric IgM preparations, respectively. Thus, the avidity of primary recognition is indeed influenced by the amino acid composition of Ag.
We next immunized mice with an equimolar mixture of peptide PS1CT3 and
the three analogues described above. Since all of these peptides encode
a common T cell epitope (segment CT3), immunization with the mixture
was expected to result in activation of diverse B cells directed
against determinants presented by the individual peptides, but all
requiring help from a common pool of CT3-specific Th cells. In other
words, such a system would simulate the situation obtained with more
complex multideterminant Ags presenting a wide array of B cell
epitopes. For comparative purposes, additional groups of mice were also
immunized with the individual peptides. The subsequent IgG responses,
obtained 4 wk later, were then analyzed and the data are presented in
Fig. 4. An intriguing dichotomy can be
noted in immunogenicity of the analogue peptides when immunized
individually, as opposed to immunization in a mixture (Fig. 4).
Individual immunizations yielded comparable IgG levels for all four
peptides (Fig. 4a). In contrast, immunogenicity of these
analogues was markedly suppressed, relative to that of PS1CT3, in the
mixture (Fig. 4b). This attenuation of immunogenicity of the
analogues, in a mixture, is well in keeping with the poorer avidity of
monomeric IgM responses elicited by them in comparison to that against
the parent peptide PS1CT3.
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Hierarchical contribution of individual amino acid residues toward epitopic dominance
We also adopted an alternate strategy to further confirm the
existence of a relationship between chemical composition of an epitope
on a multideterminant Ag and its position in the hierarchy of relative
immunodominance in a humoral response. For this, we synthesized
analogues of peptide PS1CT3 where the amino-terminal His residue was
independently replaced with each of the 19 remaining naturally
occurring 
amino acid residues. As shown earlier (14, 19, 28), the amino-terminal PS1-derived hexapeptide normally
represents a cryptic determinant in the primary anti-PS1CT3 IgG
response. Our objective, therefore, was to assess the efficacy of each
of the various substitutions in creating a novel amino-terminal
epitope. It was anticipated that such an approach would also provide
information as to whether individual residues do in fact contribute
variably toward immunogenicity of a given epitope on a
multideterminant Ag.
Subsequent to synthesis of these N-terminal substituted analogues of
peptide PS1CT3, we first ensured that these changes did not lead to any
secondary structural changes in the resulting molecule. This was
achieved by circular dichroism (CD) spectroscopy. As earlier described,
peptide PS1CT3 exists in a random distribution of conformations in
solution (14, 27). This "randomness" was found to be
unaffected in all analogues synthesized, and CD spectra of
representatives are shown in Fig. 5.
Although CD studies do not provide information on subtle changes in
structure, these results nevertheless confirm that the substitutions
performed did not lead to imposition of any gross structural
constraint.
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A representative analysis for three separate substitutions is shown in
Fig. 6. It is evident from the data
presented that the proportion of amino-terminal reactivity obtained is,
indeed, dependent on the nature of substitution performed at the
N-terminal position. Thus, the presence of an Arg residue at this
position was most conducive for a maximal shift in immunodominance away
from the DPAF epitope and toward the amino terminus (Fig. 6). In
contrast, substitution for a Gly residue at this position had no
significant effect (Fig. 6).
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, and in light of the
observations in Tables I and II, we reasoned that any variable effects
of individual amino acid residues would probably emanate from
nonidentical contributions of their side chains, toward energetically
stabilizing complexation with the corresponding Ab. In principle, this
argument could also be extended to binding of an epitope with the sIg
receptors on specific B cells. Therefore, to explore for such a
possible correlation, we also calculated the total potential free
energy that each of the 20 various amino acid residue side chains can
contribute in a binding interaction (see Materials and
Methods). The cumulative comparison of values thus obtained for
each residue, with its effect on relative immunodominance of the
amino-terminal hexapeptide segment is shown in Table III. A general correlation between the
potential energetic contribution of side chain of a given residue
toward binding and the proportion of shift in immune recognition toward
the amino terminus is distinctly evident (Table III). All residues with
side chain G values of negativity 
5.0 induced a significant shift
in Ab specificity toward amino-terminal recognition. In contrast, those
with G values of negativity 
4.0 proved ineffective in this regard
(Table III).
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do correlate with
differences in avidity of primary recognition, primary IgM responses
were obtained against a representative set of peptides described in
Table III. These preparations were then resolved for Abs specific for
the amino-terminal segment vs those directed against the remainder of
the sequence by sequential affinity chromatography. Resulting IgM
fractions were reduced to their monomeric forms, and the relative
avidity of these preparations for the homologous peptide was determined
by competitive inhibition ELISA. Results from such experiments are
given in Table IV. It is clear that those
substitutions which yielded a significant shift in epitopic
immunodominance toward the amino terminus in Table III were also those
that produced amino-terminal-specific monomeric IgM Abs of an avidity
that was significantly higher than that obtained against the rest of
the sequence (Table IV). In contrast, substitutions that had a minimal
effect on epitope specificity of the IgG response elicited cognate
monomeric IgM Abs of avidities that were indistinguishable from that
generated against the rest of the sequence (Table IV).
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A notable discrepancy with the hierarchy shown in Table III was
that of the parent peptide PS1CT3. Although results presented both here
and earlier (14, 19, 28) demonstrated that the
amino-terminal segment constituted a cryptic determinant in primary
responses, this was incompatible with the estimated value of
Gtotal of -6.275 for the His side chain
(Table III). To explore the basis for this, we examined the
1H-nuclear magnetic resonance (NMR) spectrum of
peptide PS1CT3. Intriguingly, we observed here that signals for the
protons at C2 and C4 positions of the imidazole ring of His were
shifted significantly downfield from the expected values
(32). As shown in Fig. 7A, signal for the proton at
the C2 position appears at 8.73 ppm, whereas that for the C4 proton
appears at 7.36 ppm. These signals are downfield of the corresponding
shifts obtained for these same protons even when His residues are
present in a highly acidic environment (32). Thus, the
deshielding of ring protons seen in Fig. 7A reveals a
significantly decreased electronegative character for the side chain
imidazole ring of His in peptide PS1CT3.
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amine group of the N-terminal His and the
nitrogen atoms of the imidazole ring. We tested this possibility by
synthesizing a derivative of peptide PS1CT3 where the terminal amino
group was acetylated. This capping of the amino functionality of
the terminal His residue was expected to disrupt any such
intramolecular interactions.
Amino-terminal acetylation of peptide PS1CT3 did, in fact, result in an
upfield shift of both the imidazole protons at the C2 and C4 positions,
as revealed by 1H-NMR spectroscopy (Fig. 7, C and D). Thus, although the precise mode remains
unclear, amino-terminal acetylation of peptide PS1CT3 results in an
increased electronegative character for the His side chain. Parallel
groups of mice were then immunized either with the parent peptide
PS1CT3 or its acetylated derivative. Sera obtained 6 wk later were
subsequently analyzed for both distribution of epitope specificities
and relative proportion of N-terminal-reactive Abs as described for
Fig. 6. The native peptide, as expected (14, 19, 28),
elicited an anti-DPAF monospecific response. In contrast, however,
the polyclonal IgG derived against the acetylated derivative displayed
significant amino-terminal reactivity, both by epitope-mapping and
saturation inhibition protocols (Fig. 8).
As shown in Table III, the relative proportion of N-terminal reactivity
in the acetylated peptide was well in keeping with the calculated G
value for the His side chain.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An intriguing corollary noted in these above studies was that clonotype selection in the pre-GC phase also translates into restriction of epitope specificities. Thus, for example, the early primary response to peptide PS1CT3 always resulted in exclusive selection for B cells directed against a single tetrapeptide epitope of sequence DPAF (14). The consequences of this selection was exercised upon subsequent stages where both late primary IgG and the Ag-specific memory B cell pool were monospecific for the DPAF epitope (17). At least in the context of peptide PS1CT3, these findings suggested that the affinity/avidity of preimmune B cell responses to individual epitopes may not be stochastically generated but, perhaps, could be associated with at least some degree of determinism. However, the factors, if any, that controlled such an outcome proved especially enigmatic since a variety of predictable parameters such as surface accessibility, mouse H-2 haplotype, secondary structural propensities, and positional influences could be ruled out as probable causes (13, 14, 15, 19). Interestingly though, single residue glycine substitutions within the DPAF epitope was found to result in a redistribution of epitope specificities in favor of alternate determinants within the PS1 segment, although the avidity of the resulting responses were markedly reduced. These latter results provided a possible clue by implicating that the affinity/avidity of a primary response to a multideterminant Ag may be epitope dependent.
Although somewhat unusual, the above proposal is not altogether
untenable if one considers the fact that the affinity of interaction of
Ag with either Ab or sIgM receptor on B cells directly relates to the
Gibbs-free energy of complexation (G) by the equation, G =
-RTlnKd. Here
Kd represents the equilibrium
dissociation constant. In this context, it is pertinent to recall that,
as demonstrated for a variety of systems (17, 42, 43, 44),
Ag-Ab interactions in the early primary response do not involve
idealized surface complementarity. Rather, this is subsequently
achieved by the combined processes of somatic mutation and positive
selection within GCs (45, 46, 47). Consequently, the role of
the chemical composition of an epitope in defining the energetics and
thereby the affinity of its interaction with a stochastically generated
receptor on preimmune B cells could not be excluded.
Support for such a possibility was afforded by our examination of monomeric IgM mAbs derived against the PS1 segment of peptide PS1CT3. As already indicated, our choice of monomeric IgMs was based on the expectation that they would be reminiscent of the sIgM receptor on preimmune B cells from which they were derived. At one level, it was interesting to note that a diverse range of affinity constants were obtained, with some Abs yielding values for Ka as low as 104 M-1. Although this confirms our earlier proposal that Ag-driven preimmune B cell activation involves low thresholds (18), they further add by revealing that Ag-binding affinity constants of as low as 104 M-1 suffice to induce preimmune B cells into a primary response. Another important highlight of these results was the comparable affinities displayed by Abs directed against a given epitope that, at least in some instances, was independent of their clonal origins. Consequently, the hierarchy of Ab affinities obtained also distinguished between independent epitopes recognized on the PS1 segment; implicating epitope dependency of monomeric IgM Ab affinity.
A rationale for the above findings was provided by our subsequent thermodynamic analysis of the association process between Ag and monomeric IgMs. Regardless of either clonal origin or epitope specificity of Ab, these studies identified that the driving force for this process derived exclusively from net favorable changes in enthalpy and occurred at the expense of an overall decrease in the entropy of the system. Although the entropy changes probably reflect conformational readjustments within the paratope of Ab, the negative enthalpy changes clearly point to the dominant contributions from epitope-paratope interface interactions in rendering this association permissible. Thus, a synthesis of these two lines of evidence permits the inference that, by influencing the nature of interface interactions, the chemical composition of an epitope represents at least one parameter that defines the affinity of its binding to either its corresponding monomeric IgM mAb or, by extension, the sIgM receptor on cognate preimmune B cells.
Although these studies provided an insight into the physicochemical forces driving Ag recognition by preimmune B cells and distinctions arising therein, the functional relevance of these distinctions remained to be validated. One approach to resolving this employed isosteric substitutions for chemically dissimilar residues within the DPAF epitope of peptide PS1CT3. This permitted generation of analogues that, upon immunization, yielded primary monomeric IgM Abs of markedly reduced avidities relative to that obtained against the native peptide. Interestingly, immunization with equimolar mixtures of these analogue peptides and peptide PS1CT3 resulted in a marked suppression in immunogenicity of the analogues relative to the parent peptide. These results provided empirical evidence in favor of a causal link between avidity of primary recognition and immunogenicity in the later stages of the response.
The dependence of immunogenicity of an epitope on its amino acid
composition could also be unequivocally demonstrated with analogues of
peptide PS1CT3 incorporating various substitutions at the amino
terminus. Amino-terminal substitutions were preferred on the basis that
alteration at this position was least likely to impose any structural
influences. Thus, depending upon whether, for example, the
amino-terminal residue was Gly or Arg, the resulting primary IgG
response displayed the extreme situations from a cryptic amino-terminal
segment to one where this domain was the most immunodominant.
Furthermore, by using the anti-DPAF response as an internal
comparative standard, we were also able to delineate a hierarchy for
individual amino acid residues in terms of their relative ability to
render the amino-terminal segment immunogenic. Interestingly, in
addition to underscoring the nonidentical contributions from individual
residues, this hierarchy was found to correlate well with the potential
energetic contribution of each amino acid side chain, defined as
calculated Gibbs-free energy changes, toward a binding interaction.
Thus, while the experimental data in Table III clarify a role for amino
acid constituents of epitope in defining its relative immunogenicity,
the correlation with side chain G values supports that this role
derives from energetic considerations. Experimental support for this
correlation could be provided, at least for some of the analogues, by
comparing avidities of primary monomeric IgMs obtained against the
amino-terminal epitope vs that obtained against the remainder of the
sequence. Whereas those substitutions that enhanced immunogenicity of
the amino-terminal epitope also yielded amino-terminal-specific monomer
IgMs of higher relative avidity, this was not true of those that had no
significant effect.
More recently, we have also examined the frequency of occurrence of
individual amino acid residues as anchor residues within epitopes
described in the literature (V. Manivel and K. V. S. Rao,
unpublished results). Such a frequency analysis revealed an
overwhelming preference, as anchor residues, for amino acid residues
with side chain G values of negativity >5.0 (as defined in Table III) (V. Manivel and K. V. S. Rao, unpublished results).
These results further support the proposal that delineation of
functional B cell epitopes from the array presented by a
multideterminant Ag is driven by thermodynamic considerations.
Thus, in summary, divergent lines of evidence presented in this report highlight some important operating principles governing the early stages of a humoral response to T-dependent multideterminant Ags. First, the initial encounter with Ag results in activation of an array of B cell clonotypes against diverse epitopes and with varying affinities for Ag. Although this was not unexpected, what was surprising, however, was the near complete dependence of affinity on epitope recognized, as also its independence from the clonal origins of Ab. This dependence appears to derive from thermodynamic considerations, an analysis of which revealed yet another interesting facet of Ag recognition by preimmune B cells. At least for peptide PS1CT3, binding to the B cell receptor on preimmune B cells was found to involve large conformational changes within the paratope of receptor. As a result, association was allowed only if the gain resulting from paratope-epitope interface interactions could sufficiently compensate for this. It is pertinent to recall here that Ag recognition by preimmune B cells differs from bindings observed for classical receptor-ligand pairs that regulate biological responses. Although the latter generally represents systems where both partners have coevolved for idealized complementarity over biological time, the former constitutes a search for the best available fit by scanning through a library of "imperfect" receptors. Similarly, Ag recognition in a primary response is also distinct from that in a secondary IgG response, the latter not requiring unfavorable changes in paratope conformation (V. Manivel, N. Samoo, and K. V. S. Rao, manuscript in preparation). Thus, in a situation where the feasibility of an induced fit is defined by the enthalpy of the reaction, it is not surprising that the chemical composition of an epitope plays an important role.
Epitope-dependent distinctions in Ab affinity in the early primary response are functionally significant in that they impact upon the hierarchy of epitopic dominance observed in the later stages of the response. This is presumably achieved by mechanisms elucidated earlier (14, 16, 17, 18), wherein seeding of GCs is restricted to the highest affinity B cell subsets which, consequently, also leads to a restriction in epitope specificities. We emphasize here that these studies are not intended to permit prediction of functional B cell epitopes on multideterminant Ags. Indeed, given that the three-dimensional structure of most Ags is unknown, as also the fact that the influence of chemical composition of epitope is limited by accessibility of its side chains for interaction with Ab, any prediction is clearly not possible at this stage. Nonetheless, the relevance of our present investigations lie in elucidating mechanisms that govern the initiating step in Ag-driven optimization of T-dependent humoral responses.
In previous studies, we had shown that, at least in the context of peptide epitopes, Ab optimization for epitope within GCs was driven in favor of increased on-rates of Ag binding (19). Interestingly, this kinetic control of clonotype selection in GCs was found to correlate with increased Ag specificity because it facilitated discrimination between conformational variants of the same epitope (19). These earlier results, in conjunction with the present data, therefore reveal that maturation of Ag-specific T-dependent humoral responses occurs in two discrete sequential stages. The first, which is under thermodynamic control, constitutes a selection for the best of available fits between the spectrum of epitopes on Ag and the repertoire of receptors on preimmune B cells. This is then followed by a further optimization, within GCs, of this selected subset for the kinetics of Ag/epitope binding. It is probably due to the concerted action of these two processes, that the twin imposing demands of high affinity and exquisite fidelity are so rapidly achieved in T-dependent humoral responses.
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2 Address correspondence and reprint requests to Dr. Kanury V. S. Rao, Immunology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110 067, India.
3 Abbreviations used in this paper: GC, germinal centers; s, surface; CD, circular dichroism; NMR, nuclear magnetic resonance.
Received for publication November 8, 1999. Accepted for publication March 14, 2000.
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), and 19M (
) at various temperatures (see Materials
and Methods) are plotted against 1/T x 10-3.
T represents the temperature in degrees Kelvin. All rate constants
are the mean values of three independent runs. Slopes yield the Ea
for the association step for each monomeric IgM mAb.
