J Immunol 1997 Vijayakrishnan

B Cell Responses to a Peptide Epitope

IV. Subtle Sequence Changes in Flanking Residues Modulate Immunogenicity

Lalitha Vijayakrishnan,* Surojit Sarkar,* Rajendra P. Roy,' and Kanury V.S. Rao'*

We examined modulation of primary humoral responses to a model synthetic peptide immunogen, peptide PS1 CT3, as a consequence of single amino acid substitutions. Two analogues were employed, one in which the amino-terminal histidine (His', peptide C28CT3) and another in which an internal proline (Prol4, peptide C41CT3) were replaced with glycine residues. Peptide C28CT3 displayed markedly enhanced immunogenicity relative to peptide PS1 CT3 in BALB/c mice, whereas peptide C41 CT3 was only poorly immunogenic. Nevertheless, in all three cases the mature polyclonal IgC response was predominantly directed against a tetrapeptide segment of sequence Asp-Pro-Ala-Phe between positions 4 and 7 of the sequence. While all three peptides proved equally capable of priming Ag-specific Th cells, they, however, displayed significant differences in their abilities to recall T cell responses. Regardless of the priming immunogen, in vitro challenge with either PSlCT3 or its analogues consistently gave a hierarchy of potencies as C28CT3 > PSlCT3 > C41CT3. This could also be correlated with B cell recall responses in which an identical hierarchy was obtained on restimulation of G41CT3-primed B cells in adoptive transfer experiments. Subsequent studies revealed that peptide-mediated modulation of Th cell recruitment by Ag-primed B cells was probably due to differences in on-rates for engagement of B cell Ag receptor by these analogues. This was despite the fact that all three peptides displayed equally randomized conformations in solution. These studies indicate that even subtle variations in the flanking sequences can markedly influence the immunogenicity of B cell

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epitopes. The journal of Immunology, 1997, 159: 1809-1 81 9.

n systemic primary humoral responses, an encounter between Ag and the host preimmune B cell repertoire results in activation of that B cell subset bearing appropriate surface im-

munoglobulin (sIg)* receptors. The principal outcome of such a recognition is the induction of Ag-specific IgM Abs. Subsequent developments include Ab isotype switch (usually predominantly to IgG) and B cell differentiation into either plasma or sIgGt memory B cells (1, 2). In T-dependent responses, progression of Ag- activated B cells through each of these stages is driven by Ag- specific activated Th cells (3-5). B cells bind Ag through their sIg receptors, which is then followed by endocytosis, Ag processing, and presentation of appropriate fragments in the context of MHC class I1 molecules (6). Such Ag-presenting B cells engage relevant Th cells in a cognate interaction, one consequence of which is B cell proliferation and differentiation (4-6). While there appears to be some debate in the literature about the ability of Ag- presenting B cells to prime naive T cells (7-16), a considerable body of evidence suggests that the Ag-presenting function of B cells is critical for sustaining B cell components of humoral responses ( 17-25).

A variety of studies have demonstrated that Ags that bind spe- cifically to Ig receptors on B cells are presented extremely effi-

I

'Immunology Group, lnternatlonal Centre ior Genetic Engineering and Biotech nology, and 'National Institute of Immunology, New Delhi, India

Received ior publication November 4, 1996. Accepted ior publication May 19, 1997.

The costs of publication oi this artlcle were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' Address correspondence and reprint requests to Dr. Kanury V.S. Rao, Immu- Aruna Asaf Ali Marg. New Delhi-110067, India. nology Croup, International Centre ior Genetic Engineering and Biotechnology,

' Abbrevlations used in thrs paper: slg, surface immunoglobulin: BCR, B cell antigen receptor; HRPO, horseradish peroxidase; LNC, lymph node cells; CD, circular dichroism: DPAF, a tetrapeptide segment spanning positions 4 to positions 7 within the antigens used here o i sequence Asp-Pro-Ala-Phe.

Copyright 0 1997 by The Amerlcan Association of Immunologists

ciently (reviewed in Ref. 6). Indeed, under conditions of limiting Ag supply, B cells are probably the most efficient among the various Ag-presenting cell types. The bulk of available evidence sup- ports the view that the Ig receptor functions primarily as a high affinity trapping site for Ag for eventual presentation (6, 26, 27). Thus, the ability of a given B cell to present a specific Ag can be expected to be governed by a variety of factors. For example, avidity of sIg for Ag, or more precisely its epitope on Ag, may be expected to eventually define the ability of the B cell to recruit T cell help. Other parameters such as stability of the receptor-Ag complex, endocytic rate, Ag-dependent induction of costimulatory molecules on the B cell surface, etc., may also be expected to influence the Ag-presenting efficacy of B cells.

In addition to influencing quantitative aspects of humoral responses, our own recent results suggest that the interplay between Ag-activated B and Th cells also plays a crucial role in defining the Ab specificities selected for maturation during a primary response (28,29). Using both recombinant and synthetic peptide model Ags, we have demonstrated, in the mouse model, that while the IgM response initially induced on primary immunization was composed of heterogeneous specificities, class switch to the IgG isotype of Abs was accompanied by a strict selection for only a limited subset of these (28-30). In both of these systems, success or failure of an Ag-activated B cell of a given fine specificity for retention in the response was primarily determined by its ability to access T cell help (28, 29). Thus, it would seem that individual clonotypes that are activated upon initial encounter with Ag are subjected to a selection pressure in which the critical determinant is the efficacy of these B cells to function as APCs. The Ag-presenting ability was in turn found to be regulated by a variety of extraneous parameters that included affinity of B cell Ag receptor (BCR) for Ag (29) and competition between BCR and secreted Ig for the corresponding epitope on Ag (28). It is the culmination of such processes that eventually defines the spectrum of Ab fine specificities obtained in the later stages of a humoral response.

0022-1 767/97/$02.00

1810 MODULATION OF B CELL RESPONSES

We have continued these studies to further explore the regulation of epitope-specific B cell responses to polypeptide immunogens. In this report, we show that single amino acid substitutions outside of the immunodominant domain of a model synthetic peptide immunogen, peptide PSlCT3, can have profound effects on immunogenicity without altering the fine specificity of the IgG Ab response invoked. Interestingly, these differences in immunogenicity did not appear to be due to any alteration in the levels of T cell activation but, rather, are due to varying abilities of these analogues to engage the BCR and thereby differentially influence Ag-specific B cell recruitment of T cell help. It therefore appears that, in addition to T cell determinants, interactions between B cells and their corresponding epitopes also represents an important paran- eter for defining immunogenicity of polypeptide immunogens.

Materials and Methods Materials

Heavy chain-specific horseradish peroxidase (HRPO) labeled secondary Abs and anti-mouse IgG (7-specific) were purchased from Sigma Chemical Co. (St. Louis, MO). Magnetic beads for panning of T (Dynabeads Mouse pan T) and B (Dynabeads Mouse pan B) cells were obtained from Dynal (Oslo, Norway). ['HITdR (specific activity, 5 Ci/mmol) was purchased from Amersham (Arlington Heights, IL). For multipin synthesis of peptides (31), both the noncleavable kits and F-moc amino acid derivatives were purchased from Chiron Mimotopes Inc. (Victoria, Australia).

Peptide synthesis

Peptides were synthesized by the solid phase method (32.33) on a Milligen 9050 automated synthesizer using the F-moc chemistry (34). Crude peptides were purified to at least 95% by reverse phase HPLC on a C,, column (15-pm 6 Pak; (Waters, Milford, MA) 19 X 300 mm) using an aqueous gradient of 0 to 70% acetonitrile in 0.1 % trifluoroacetic acid. Identities of all peptides were ascertained by amino acid analysis.

The overlapping hexapeptide panels were synthesized using a Multipin noncleavable peptide kit (Chiron Mimotopes). strictly adhering to the protocol recommended by the manufacturer. After completing the synthesis, all hexapeptides were routinely acetylated at the amino terminus with a 50/5/1 (v/v/v) mixture of dimethylformamide, acetic anhydride, and trieth- ylamine. Side chain deprotection was accomplished over a 2-h period at room temperature with a 3X/1/1/ (v/v/v) mixture of trifluoracetic acid. ethanedithiol, and anisole.

Conjugation of peptides to anti-mouse IgG

Peptides were conjugated to anti-mouse IgC using a standard procedure (35). Briefly, 1 mg each of peptide and anti-mouse IgC were dissolved in a total volume of 450 p1 of PBS. To this was added, dropwise with mixing, 500 pl of a 0.2% glutaraldehyde solution in PBS. The solution was incubated with gentle mixing at room temperature for 1 h. Then, 240 pI of 1 M glycine in PBS was added to quench the reaction over a further I -h incubation at room temperature. Any unconjugated peptide was removed by overnight dialysis against PBS with a minimum of four changes. Concen- tration of each peptide in the solution of conjugate was estimated by in- cubating with an equal volume of a 1 % solution of trifluoroacetic acid in water for 30 min at room temperature. Released peptide was then estimated by quantitative reverse phase HPLC with dual detection at 254 nm and 215 nm.

Animals and immunization

Female BALB/c mice (6-X wk old) were obtained from the small animal breeding facility at the National Institute of Nutrition (Hyderabad, India). Immunizations were generally given i.p. with a dose of SO pglmouse as an emulsion in CFA (except where indicated). For polyclonal sera, mice were bled from the retroorbital plexus, and sera within a group were usually pooled.

ELlSAs

Plates were coated with 2 pg of peptide/well in 100 pl of PBS, pH 7.2, at 37C for 3.5 h. Subsequently, they were blocked with 300 pl/well of a 5% solution of fat-free dry milk powder in PBS at 37C for 1 h. Then, 100 pI of the appropriate dilution of mouse antiserum was added and incubated at 37C for 1 h. After washing, bound Ab was detected with HRPO-labeled

secondary Ab (37C 1 h), followed by color development with o-phenyl- enediamine as chromogen. Absorbance was measured at 490 nm.

For competitive ELISA experiments, antisera were used at dilutions representing 50% of titer value. Twofold higher concentrations of antiserum and competitor peptide were mixed in equal volume and incubated for 10 min at room temperature. This was then added to duplicate wells at 100 pl/well. The remaining procedure was as described above.

ELlSAs for pin-bound hexapeptides

Hexapeptide sets synthesized on noncleavable pins were also evaluated for Ab cross-reactivity by ELISA. For this, the protocol recommended by the manufacturer was strictly followed. Primary Abs were diluted appropri- ately in PBS containing 2% BSA, 0.1% (v/v) Tween-20, and 0.1% (vlv) Tween-20, and 0.1% sodium azide. Pins were incubated in 200 p l each of Ab solution at 4C overnight with gentle shaking. Subsequently, they were washed and subjected to a second round of incubation with appropriate dilution of HRPO-labeled goat anti-mouse IgM or IgG at room temperature for 1 h, again with gentle shaking. The chromogen used for detecting bound Ab was 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)diam- monium, and the absorbance was measured at 405 nm after subtracting background at 490 nm.

Enrichment of cell populations

For enrichment of T cells, lymph node cells (LNCs) from either immune or nonimmune mice were first depleted of RBCs by lysis with ammonium chloride. and subsequently, adherent cells were removed by panning on plastic plates at 37C for 1 h. Nonadherent cells were collected and diluted to a cell concentration of 5 X 10' cells/ml. B cells were depleted from this population by two rounds of panning with Dynabeads mouse antikB220 (Dynal, 4 X IO* beaddml) following the recommended protocol of the manufacturer.

For obtaining enriched B cells, mice immunized with peptide G41CT3 were boosted 3 wk later with a 50 pg/mouse dose of G41CT3 in PBS given i.v. Three days later, the spleen was removed and depleted of RBCs by ammonium chloride lysis, following which adherent cells were removed by panning as described above. Nonadherent cells were diluted to 5 X IO7 cells and T cells were depleted by two rounds of panning with Dynabeads anti-mouse Thy 1.2 (Dynal, 4 X 10* beads/ml) as recommended by the manufacturer. The resultant enriched B cells were then treated with mitomycin C at a final concentration of 50 pg/ml at 37C for 20 min. Cells were washed thoroughly in culture medium prior to use. When unprimed B cells were required, an identical protocol was followed except splenocytes were taken from nonimmune mice.

Lymphocyte proliferation assays

BALB/c mice were immunized S.C. at the base of the tail with 50 pg/mouse of the relevant peptide as an emulsion in CFA. Seven days later, the mice were killed and inguinal lymph nodes removed. LNCs, at 5 X I O 5 cells/ well, were cultured in quadruplicate in 200 p1 of RPMI 1640 containing 10% FCS, gentamycin (2 mg/L) and 2-ME (0.05 mM) in wells of a 96-well plate, In addition, indicated concentrations of appropriate peptides were also included as challenge Ag. Cultures were incubated at 37C in a hu- midified atmosphere of 5% carbon dioxide for 72 h. They were then pulsed with 1 pCilwell of ['HlTdR for an additional 18 h prior to harvesting for determination of radioactive TdR incorporation by liquid scintillation counting. Values are presented as stimulation index, which represents the ratio of mean counts obtained at a given concentration of Ag over that obtained in the absence of any challenge Ag. Background counts generally varied between 1000 to 2500 cpm.

Determination of on-rates

Equal volumes of mAb PC286 and relevant peptide in PBS were mixed at room temperature, and time-dependent Ab binding in terms of quenching of tryptophan fluorescence was continuously monitored over a 100-min period in a Shimadzu RF-1501 spectrofluorimeter (Shimadzu Corporation, Tokyo, Japan). The excitation wavelength used was 280 nm. and emission was recorded at 330 nm. The final Ab concentration employed was 300 nM, whereas peptide was maintained at > 10-fold in excess over binding sites (7.5 p,M) to ensure psuedo-first-order conditions. Extent of fluorescence quenching was used to determine unbound Ab concentrations as a function of time. The log of concentration of unbound Ab was plotted vs time, and the slope, which was obtained by linear regression analysis, was used to determine k,,,. The k,,, value was subsequently calculated by dividing k,,, with peptide concentration. Values of k,,, presented are the mean ( i S D ) of three independent determinations.

The Journal of Immunology

Results Peptide PS 7 CT3 and its single-amino acid-substituted analogues

As a follow up to our earlier studies (29), we have continued to use the model synthetic peptide immunogen PSlCT3. Peptide PSICT3 represents a chimeric molecule containing a B cell epitope (segment PSI) derived from the large protein of the hepatitis B virus surface Ag (36) and a well-characterized promiscuous T cell epitope (segment CT3) from the circumsporozoite protein of the malaria parasite P/asmodium falciparum (37). For reasons described earlier (38), the PS1 and CT3 segments were separated by a spacer of two glycine residues. Prior studies have established that the PS 1 segment does not encode a T cell determinant, at least for the murine system (39, 40).

In the course of a study of immunogenicity with a variety of amino acid-substituted analogues of peptide PSlCT3, we noticed that there were two analogues that showed a marked difference in behavior from the parent peptide. Both of these represented single amino acid-substituted analogues of peptide PSlCT3 in which either the histidine residue at position 1 (peptide G28CT3) or the proline residue at position 14 (peptide G41CT3) were substituted by glycines. It must be emphasized here that both substitutions were performed within the B epitope segment of the molecule, whereas the T cell epitope sequence remained unaltered. Figure I A gives the amino acid sequence of peptide PSICT3 and its two analogues.

We have shown earlier that peptide PS ICT3 exists in a predominantly random structure in solution (29). To verify whether glycine substitutions in peptides G28CT3 and G41CT3 had any influence on secondary structural preferences, we examined the circular dichroism (CD) spectra of these peptides. As shown in Figure lB, all three peptides gave virtually identical CD spectra of a negative peak with a minima at 199.3 nm. This finding indicates that the degree of randomness (41) was retained in spite of glycine substitutions, with no new secondary structural constraint imposed.

Immunogenicity of peptide PS 7 CT3 and its analogues

To assess the influence of glycine substitutions on immunogenicity, we immunized separate groups of BALB/c mice with a single dose of either peptide PSICT3, G28CT3, or G41CT3, and serum Ab responses were monitored periodically. Surprisingly, glycine substitutions at both positions were found to have a profound but opposite effect on the immunogenicity of the resultant analogues (Fig. 2). While primary IgG Ab titers to peptide G28CT3 were at least sixfold higher than that obtained against peptide PSlCT3, that against peptide G41CT3 was severely diminished (Fig. 2). In contrast to IgG Ab titers, early primary IgM responses were relatively unaffected with all three peptides eliciting comparable levels of Abs (Fig. 2, inset). Finally, immunization of ndnu mice with either of these peptides did not yield primary IgM or IgG antibodies, suggesting that both IgM and IgG stages of the primary response to these peptides were T dependent.

We also investigated whether Ab responses to these three immunogens were restricted to the B epitope segments only (i.e., either PSI, G28. or G41) or whether they were more broadly dis- tributed. For this determination, we performed competitive inhibition experiments in which binding of day 28 sera from each of the groups represented in Figure 2 to homologous immunogen was examined in the presence of various inhibitors. The inhibitors used were synthetic peptides representing the B epitope only (iz., PSI, G41, or G28), the T epitope only (i.e., CT3), an equimolar mixture of both, or the homologous immunogen. Results from these ex-

A

PEPTIDE

PS l a 3

G28Cr3

G41CT3

6

181 1

SEQUENCE

HQLDPAFGANSTNPDGGDIEKKIAKMEKASSVFNVVNS

GQLDPAFGANSTNPDEDIEKKIAKMEKASSVFNVVNS

HQLDPAFGANSTNGDGGDIEKKIAKMEKASSVFNVVNS

E a Y

0

-3.500E+01 L \L :vi / I 190.0 260.0

WAVELENGTH ( n m )

FIGURE 1 . Single amino acid glycine-substituted analogues of peptide PSlCT3 and their CD spectra. A, The amino acid sequence of peptide PSlCT3 and its analogues, where either His (peptide G28CT3) or Pro14 (peptide G41CT3) were individually substituted with a glycine residue. The substituted glycine is shown in bold and the two-glycine residue spacer is underlined. 5, The CD spectra obtained for these peptides. These were recorded on a JASCO model J710 spectropolarimeter (JASCO Corporation, Tokyo, Japan) over a wavelength range of 190 to 260 n m , with a step resolution of 0.1 n m and a scan speed of 50 nm/min. Peptides were dissolved at a final concentration of 0.25 mdml in PBS before recording the spectra. -, Indicates peptide PSlCT3; --, peptide G41CT3; ---, peptide G28CT3.

periments are shown in Figure 3. For all three antisera, nearly identical inhibition profiles were obtained regardless of whether the competitive inhibitor employed was the B epitope peptide or the total homologous immunogen (Fig. 3). In contrast, no inhibition was observed when peptide CT3 was used as inhibitor, whereas a mixture of B epitope peptide and CT3 yielded an inhibition pattern identical to that with B epitope peptide alone (Fig. 3). Collectively, these results indicate that, for all three analogues, day 28 IgG Ab responses are, at least predominantly, directed against the B epitope segment only. Also notable from Figure 3 is that peptide G41CT3 induces a day 28 IgG Ab response that is between 5- to IO-fold lower in afinity relative to that induced by either peptide PSlCT3 or G28CT3 (compare Fig. 3, panel C with A and B). Thus, substitution of Pro for Gly at position 14 has both a quantitative and a qualitative influence on the humoral response.

Given the relatively lower affinity of anti-G41CT3 IgG Abs, we also performed a comparative evaluation of its affinity for the other two peptide analogues. As shown in Figure 4, nearly identical competitive inhibition profiles were obtained with all three peptides, indicating that day 28 anti-G41CT3 polyclonal antiserum

1812

0 m I 5 10 15 20 2 5 30

DAYS AFTER PRIMARY IMMUNIZATION

FIGURE 2. Relative immunogenicity of peptides PSlCT3 and its analogues in BALB/c mice. Groups of four BALB/c mice were each immunized with either peptide PS1 CT3 (O), peptide C41 CT3 (0) or peptide C28CT3 (A), and pooled sera obtained at indicated time points were taken for determination of peptide-specific IgC titers by ELISA (see Materials and Methods). /met shows peptide-specific IgM response at day 5 after primary immunization. a, Peptide PSlCT3; 0, peptide G41 CT3; B, peptide G28CT3. This figure is a representative of five independent experiments.

does not discriminate significantly between peptide G41CT3 and its analogues. From this we also infer that at least a predominant component of the Ab fine specificities that comprise an anti- G41CT3 response is directed against determinants common to all three analogues and does not include the substituted positions.

Polyclonal IgG responses to all three immunogens are monospecific

In our previous report, we showed that immunization of BALB/c mice with peptide PS 1CT3 induced an early primary IgM response that was composed of multiple fine specificities collectively spanning the entire PSI domain (29). However, subsequent class switch to IgG Abs was accompanied by stringent selection for a predominantly monospecific population directed against a tetrapeptide segment spanning positions 4 to 7 (sequence: DPAF) (29). It was of interest, therefore, to ascertain the situation with respect to peptides G28CT3 and G41CT3. For this we used a panel of overlapping hexapeptides representing single amino acid dis- placements over either the PSI or its analogue sequences. Poly- clonal Ab cross-reactivities with this hexapeptide panel was determined by ELISA. As shown in Figure 5 , qualitatively similar results were obtained for primary IgM and IgG responses to all three immunogens. While the early primary IgM response displayed multiple specificities, cross-reactivities of the mature IgG response were restricted to within three overlapping hexapeptides (sequences: QLDPAF, LDPAFG, and DPAFGA) between residues 2 and 9 in all three cases (Fig. 5). Thus, similar to the situation with PSICT3 (29), both peptides G28CT3 and G41CT3 also induce a primary IgG response in BALB/c mice that is at least predominantly directed against the tetrapeptide sequence DPAF between positions 4 and 7. It therefore appears that enhancement or sup-

MODULATION OF B CELL RESPONSES

pression of immunogenicity seen as a consequence of glycine substitution at either position I or position 14 simply represents quantitative alterations in humoral recognition of an identical determinant within the PS1 segment of peptide PSlCT3.

Modulation of PSlCT3 immunogenicity by glycine substitution is not due to altered Th cell activation

A simple explanation for the observed effects of glycine substitution on immunogenicity could be that the analogues are altered in their ability to activate Ag-specific Th cells. Although both substitutions performed were not within the T epitope of peptide PSlCT3, prior studies have shown that amino acid changes outside of the T cell determinant nevertheless have dramatic effects on the ability to activate T cells (42-44). We therefore conducted a series of experiments to ascertain whether this could account for our observations. As a first step, we examined the ability of these individual immunogens to prime Th cells specific for the determinant(s) encoded within the CT3 segment of PSlCT3; for this, individual groups of BALB/c mice were primed with either peptide PSICT3, G28CT3 or G41CT3. For comparative purposes, an additional group was primed with a peptide representing the CT3 segment of peptide PSlCT3 (peptide CT3). Seven days later, inguinal lymph nodes were removed and the resulting cells (LNCs) challenged with peptide CT3 in vitro. As shown in Figure 6A, recall responses were generally poorer in LNCs from mice primed with peptide PSlCT3 or its analogues relative to CT3 priming. Nevertheless, priming with either PSlCT3 or its analogues yielded comparable recall responses to peptide CT3 challenge, suggesting that all three of these immunogens are equally proficient at priming CT3-specific Th cells (Fig. 6A). The poorer priming ability of these peptides relative to peptide CT3 may be indicative of a re- quirement for processing prior to presentation for Th cell recruitment.

We also evaluated the ability of peptides PSlCT3 and its analogues to elicit recall responses in LNCs from mice primed with peptide CT3. As shown in Figure 6B, all Ags tested gave nearly identical proliferative responses, suggesting that both of the glycine substitutions performed in the PSI sequence did not alter the ability of PS 1 CT3 to recall a preprimed population of CT3-specific T cells. Also, if processing of PSICT3 is indeed required for optimal presentation of the CT3 fragment, then the data shown in Figure 6B may also be taken to indicate that peptides G28CT3 and G41CT3 do not differ significantly, in terms of processability, from peptide PSlCT3. Finally, the data shown in Figure 6 also indicate that while peptide CT3 is more potent relative to peptide PS 1 CT3 and its analogues in terms of priming for a CT3-specific T cell response, they are all equally proficient at restimulating a CT3- primed T cell population.

To further confirm the absence of altered T cell immunogenicity as a consequence of glycine substitution, we performed an additional experiment in which LNCs from mice primed with each of these immunogens were challenged in vitro with a common peptide. Figure 7A shows the results of an experiment in which LNCs from mice primed with either PSlCT3, G28CT3, or G41CT3 were challenged with peptide PSlCT3. As is evident, very similar lymphocyte proliferative responses were obtained in all three groups. This was also true in parallel experiments in which the challenge Ag was either G28CT3 (Fig. 7 B ) or G41CT3 (Fig. 7 0 . We interpret these data to suggest that priming with either of these Ags activates T cell subsets sharing, at least predominantly, a common spectrum of epitope specificities. Further, this result also extends the observation made in Figure 6A that these three immunogens prime Ag-specific T cells equally well.

The journal of Immuno logy 1813

2 4 6 8 1 0 20 2 4 6 8 1 0 20 2 4 6 8 1 0 20 COMPETITOR CONCENTRATION OJM)

FIGURE 3. Anti-peptide polyclonal IgG responses are exclusively directed against the B epitope segment. Polyclonal day 28 antisera from each of the groups listed in Figure 2 were diluted to 50% of titer value and incubated with indicated final concentrations of either a peptide representing only the homologous B cell epitope (i.e., either PS1, C41, or G28 (0), only the T cell epitope (CT3, A), a mixture of the two (A), or the homologous immunogen (i.e., either PS1 CT3, G41 CT3, or G28CT3 (0)) as described in Materials and Methods. Subsequently, 1 00-pl aliquots were added in duplicate wells coated with homologous Ag and bound Abs determined by ELlSA (Materials and Methods). A, Anti-PSlCT3 antiserum; B, anti- G28CT.3 antiserum; c, anti-C41CT3 antiserum. Dilutions of individual sera used were 1 : lo00 for anti-PS1 CT3,l :3000 for antiLG28CT3, and 1 : l o0 for anti-G41 CT3. Results are representative of three separate experiments

L I I I 1 10 20 30 40

COMPETITOR CONCENTRATION(lJM1

FIGURE 4. Relative affinity of antiLC41CT3 antiserum for peptide PSlCT3 and its analogues. Diluted day 28 antiLG41CT3 polyclonal serum was incubated with indicated concentrations of either peptide PSlCT3 (O), G28CT3 (A), or G41 CT3 (0) as described in the legend of Figure 3. Residual Ab binding to wells coated with peptide G41CT3 was determined by ELSA (Materials and Methods). Data are presented in terms of bound Ab obtained at each competitor peptide concentration expressed as a percentage of that obtained in the absence of any competitor.

Thus, the data given in Figures 6 and 7 collectively rule out the possibility that the differences in humoral responses to peptides PSICT3, G28CT3, and G41CT3 seen in Figure 2 arise due to altered immunogenicity at the T cell level as a consequence of glycine substitution.

Peptides PS1 CT3, G28CT3, and G4 I CT3 differ in their abilities to restimulate I cells primed with the whole immunogen

An interesting feature of the data given in Figure 7 was that, while T cell proliferative responses to a common challenge Ag were independent of priming immunogen, there were nevertheless quantitative differences in the ability of a given peptide to induce recall responses (compare panels A , B, and C in Fig. 7). This, again, was true regardless of the priming Ag. To confirm this, LNCs from mice primed with either peptide PS1CT3, G28CT3, or G41CT3 were individually challenged with each of these immunogens and lymphocyte proliferation measured. These results are depicted in

A 1.21 PSlCT s 0.4 %Il P S I C T 3 0.4

0.8

0.6

0.4

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HEXAPEPTIDE SEQUENCE

FIGURE 5. Hexapeptide mapping of IgM and IgG responses elicited by peptide PSlCT3 and its analogues in BALB/c mice. Day 5 (A) or day 28 (6) sera from mice immunized with either peptide PSlCT3, G41 CT3, or G28CT3 were screened for either IgM (A) or IgG (6) cross- reactivity against a panel of overlapping hexapeptides derived from the homologous B cell epitope segment (i.e., either PS1, C41, or C28), as described in Materials and Methods. The x-axis denotes each hexapeptide as its N-terminal residue; only the PS1 sequence is given for the sake of convenience. Dilutions of individual sera used were: 1/1000 for PSlCT3; 1/4000 for G28CT3; and 1/100 for G41CT3.

Figure 8. It is notable that, regardless of the Ag with which the LNCs were primed, proliferation was always maximal with peptide G28CT3 as recall Ag. The next most potent was peptide PSICT3, followed by peptide G41CT3, in which responses were barely significant. This hierarchy was common to LNCs primed with all three Ags. For example, G41CT3-primed LNCs were more responsive to in vitro challenge with G28CT3 relative to challenge with peptide G41CT3, a situation identical to that observed with PSICT3- and G28CT3-primed LNCs.

Thus, combining these results with those given in Figure 7, i t would appear that while peptide PSICT3 and its analogues are


FIGURE 6. Peptides PSlCT3, G41CT3, and G28CT3 are equipotent at both priming CT3-specific T cells and eliciting recall responses from CT3 primed LNCs. A, Priming of CT3-specific T cells. LNCs from mice immunized with either peptide PSlCT3 (GI, G4lCT3 (01, C28CT3 [A), or CT3 (A) were cultured in vitro in the presence of indicated concentrations of peptide CT3 and lymphocyte proliferation measured as described in Materials and Methods. Values are presented as stimulation index (Materials and Methods) and are representative of three independent experiments. Mean background counts obtained in this experiment were 1680 cpm. B, Recall of a CT3-primed T cell response. LNCs from mice immunized with peptide CT3 were CUI- tured in vitro in the presence of indicated concentrations of either peptide PS1 CT3 [G), G41 CT3 (Oj, or G28CT3 (A) as challenge Ag. Measurement of lymphocyte proliferation and data presentation are as described in Maferials and Methods. Mean background counts obtained were 1460 cpm; data are from one of three separate experiments.

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5 1

2 4 c I- d

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f 2 4 c a A 3

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2 Z

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b- z 3 ul

2

25 50 75 100 125 25 50 75 100 125 2 5 50 75 100 125 ANTIGEN CONCENTRATION (uM)

FIGURE 7. T cell recall response to either peptide PSlCT3 or its analogues is independent of the priming immunogen. LNCs from mice primed with either PSI CT3 (O), C41 CT3 (O), or C28CT3 (A) were challenged in vitro with indicated concentrations of either peptide PSI CT3 (A), G28CT3 (B), or G41CT3 (0. Further protocol and data presentation is as described in Figure 6. Mean background counts obtained were 201 0 cpm. Results are representative of four independent experiments.

equally proficient at priming Ag-specific T cells, they nevertheless differ in their ability to elicit T cell recall responses, at least in this system. Also of interest is the fact that the relative efficacies of these peptides to recall Ag-specific T cell responses parallels the trend of the relative immunogenicity of these Ags seen in Figure 2.

Differential recall responses to peptide PS7 CT3 and its analogues are due to Ag presentation by activated B cells Figure 8 demonstrates that when T cells are primed with either PS 1CT3, G28CT3, or G41CT3, maximal proliferative responses are obtained on in vitro challenge with peptide G28CT3, followed by peptide PSlCT3, and finally by peptide G41CT3, which was only weakly stimulatory. However, this was in contrast to the earlier experiment with CT3-primed LNCs in which comparable proliferative responses were obtained with all three peptides (Fig. 6B) . Since the principal difference in these two experiments was either the inclusion or exclusion of the B cell epitope segment in the priming Ag, we reasoned that the hierarchy seen in Figure 8 could represent a consequence of Ag-activated B cells functioning as APCs.

To examine this possibility, we performed an experiment in which G41CT3-primed LNCs were cultured in vitro with peptide

G28CT3 either in the presence or absence of varying concentrations of peptides representing the B cell epitope segments of either peptide G28CT3 (peptide G28) or peptide G41CT3 (peptide G41). A parallel set of wells containing a peptide of scrambled G28 sequence was also included as control. Our objective was to assess T cell proliferation in a situation in which B cell sIg receptor recognition of peptide G28CT3 was competitively inhibited by the presence of B cell epitope-only peptides. The results of such an experiment are shown in Figure 9. Peptide G28CT3 induced proliferation of G41CT3-primed LNCs was clearly inhibited, in a dose-dependent manner, by both peptide G41 and G28 but not by the scrambled peptide. Maximal inhibition obtained was between 85 and 95% with an eightfold molar excess of the B epitope peptide (Fig. 9). We interpret these results as suggesting that the principal APCs in this system are the Ag-activated B cells. This is also consistent with the data in Figure 6 showing that PSlCT3 and its analogues were more efficient as recall Ags when LNCs were primed with total immunogen rather than with the T cell determinant peptide CT3 only.

If, indeed, Ag-specific T cell recall responses to PS 1 CT3 or its analogue peptide-primed LNCs were being mediated by Ag-specific B cells functioning as APCs, then it was logical to expect that

The Journal of Immunology 1815

ANTIGEN CONCENTRATION OJM)

FIGURE 8. Peptide PSlCT3 and its analogues differ in their ability to recall immunogen-primed T cell responses. LNCs from mice primed with either PS1CT3 (A), G28CT3 (g), or G41CT3 (0 were each cultured in vitro with the indicated concentrations of either peptide PSlCT3 (O), G41CT3 (Oj, or G28CT3 (A), and lymphocyte proliferation was subsequently measured as described in Materials and Methods. Mean background counts were 1750 cpm. Values presented are mean stimulation indices of quadruplicates (2SEj ; results are representative of four independent experiments.

COMPETlTOR CONCENTRATION (pM)

FIGURE 9. Differential T cell recall abilities of peptide analogues is a consequence of B cell-mediated Ag presentation. G41CT3-primed LNCs were cultured in vitro with a 30 pM concentration of peptide G28CT3 either in the presence or absence of indicated concentrations of either peptide G28 (A), G41 (O), or a control peptide of scrambled C28 sequence (x). Lymphocyte proliferation obtained in these three groups was determined as described in Materials and Mefhods, and data were presented as stimulation index obtained at each competitor concentration expressed as a percentage of that obtained in the absence of any competitor peptide. A stimulation index of 6.5 was obtained in the absence of competitor peptides (100%) against a background of 1400 cpm. Results presented are representative of three separate experiments.

differences in Ag recognition by B cells may account for the differential T cell recall abilities of these peptides. We examined this possibility in a relatively more defined experiment in which purified CT3-primed T cells were cocultured with mitomycin C-treated enriched splenic B cells from mice primed with peptide G41CT3. This culture was subjected to antigenic challenge with optimal concentrations of either peptide PSlCT3, G28CT3 or G41CT3 and CT3-specific T cell proliferation measured as counts of [3H]TdR incorporated. As shown in Figure 10, restimulation of CT3-primed T cells was by far the most efficient when peptide G28CT3 was present. This was followed by peptide PSlCT3, whereas the homologous immunogen, peptide G41CT3, was the least effective.

T

T

CHALLENGE ANTIGEN FIGURE 10. Differential restimulation of CT3-primed T cells by G41CT3-activated B cells In the presence the peptide analogues. MI- tomycin C-treated enriched splenic B cells derived from either G41CT3-immunized (0) or naive (0) mice were cocultured at 2.5 X 10' cells/welI along with 2.5 X 10' enriched T cells from LNCs of mice primed with peptide CT3. In addition, a 50 pM concentration of either peptide PSlCT3, C41CT3, or G28CT3 was also included in quadruplicate wells in a total culture volume of 200 PI. After 72 h, the cells were pulsed with ['HITdR and radioactivity incorporated determined as described in Materials and Methods. Mean background counts obtained were 1 a40 cprn for primed and 1630 for unprimed B cells. This figure is representative of two separate experiments; data are the mean stimulation indices (+SE) of quadruplicate sets.

Such differences were not observed when unprimed B cells were used (Fig. lo), suggesting that Ag priming of B cells was a nec- essary prerequisite for differential T cell recall.


L

CHALLENGE ANTIGEN

FIGURE 11. Conjugation of peptides to anti-mouse IgG abolishes their differential abilities to restimulate CT3-specific T cells. Mitomycin C-treated enriched splenic B cells derived from G41CT3 immune mice were cocultured with enriched T cells from CT3-primed mice as described in Figure 10. In addition, 50 FM concentrations of the indicated peptides were added either in free form (O), or as conjugates with anti-mouse IgC (0). For the purposes of control, a 32-amino acid residue irrelevant peptide derived from the ORF-2 Ag of hepatitis E virus (sequence: AGAGPRVRQPARPLCSAWRDQAQRPAVASRRR) was also included. Further protocol and data presentation is as described in Figure 10. Mean background counts obtained were 2250 cpm. Data are representative of three separate experiments.

Differential recall abilities of peptide PSlCT3 and its analogues are a consequence of differential BCR-Ag interactions

The data shown in Figure 10 indicate that CT3-primed T cells are optimally restimulated by G41CT3-primed B cells in the presence of peptide G28CT3 as challenge Ag, whereas the homologous Ag, G41CT3, was the least effective. Given that these differences seem to arrive as a consequence of Ag-primed B cells functioning as APCs, we thought it likely that they may reflect differences at the level of BCR-Ag interactions. To verify this, we prepared individual conjugates of the peptides with anti-mouse IgG. It was anticipated that such a conjugation would provide for an alternate targeting pathway to B cells, bypassing the need for peptide-specific recognition to enable uptake and subsequent presentation to Th cells.

Mitomycin C-treated enriched splenic B cells and enriched lymph node-derived T cells, both from G41CT3-primed mice, were cocultured in vitro in the presence of either free peptides or peptides conjugated to anti-mouse IgG, and lymphocyte proliferation was measured subsequently. As shown in Figure 11, proliferative responses to the free peptides were essentially similar to those obtained earlier. However, conjugation of either peptide PSlCT3 or its analogues to anti-mouse IgG abolished these differences in their B cell-dependent abilities to recall T cell responses. All three analogues were now equipotent (Fig. 11).

The data shown in Figure 11 suggest to us that the observed hierarchy of G28CT3 > PSlCT3 > G41CT3 in recruiting T cell help is probably a consequence of differential engagement of either PSlCT3 or its analogue peptides by the DPAF-specific Ig receptor on Ag-primed B cells. Where DPAF is a tetrapeptide segment spanning position 4 to position 7 within the antigene used here of sequence Asp-Pro-Ala-Phe.

e e "a"

10 e

FIGURE 12. Peptide PS1 CT3 and its analogues also induce differential E cell recall responses from G41CT3-primed splenocytes. Day 21 splenocytes (5 X 107/mouse) from C41CT3-primed mice were in- jected i.v. in 0.5 ml of PES into the tail vein of irradiated (550 rad) BALBlc recipients. At 16 h after transfer, the hosts were challenged i.v. with soluble forms of either peptide PSlCT3, C41CT3, or C28CT3 in sterile PES. A n additional control Ag, V3MNCT3, was also included; V3MN represents a 15-amino acid residue sequence derived from the V3 loop of the M N isolate of HIV-1 (sequence: RKRIHICPGRAFYTT). Blood was collected 6 days after antigenic challenge and peptide- specific IgC Abs determined by ELlSA (Materials and Methods). A parallel set of experiments in which naive splenocytes were transferred did not yield any IgG responses at this time point. Data from individual mice are shown and the mean is indicated. Results are representative of two separate experiments.

Peptide PSI CT3 and its analogues also vary in their ability to recall B cell responses

The results described thus far indicate that recruitment of activated Th cells by Ag-primed B cells is most efficient in the presence of peptide G28CT3 and least efficient in the presence of peptide G41CT3. Furthermore, this trend was maintained regardless of whether the B cells were primed with peptide PSlCT3, G28CT3, or G41CT3, implying differential B cell recognition of a common determinant within these peptides. In addition, we also demonstrated earlier that murine humoral responses to all three peptides are T dependent. It was therefore highly probable that Ag-dependent differences in T cell recruitment by B cells might account for the differences in IgG Ab titers elicited by each of these three analogous immunogens. To test this probability, we immunized BALB/c mice with a single dose of peptide G41CT3, and the spleens were collected 3 wk later. Primed splenocytes were adoptively transferred into irradiated BALB/c recipients, which were then challenged with soluble forms of either peptide PSlCT3, G28CT3, or G41CT3. Anamnestic IgG responses were monitored 6 days later, and the results are presented in Figure 12. In accordance with our results on T cell recall, anamnestic Ab titers were maximal with peptide G28CT3 challenge, followed by PSlCT3, whereas G41CT3, although it is the homologous immunogen, was extremely inefficient. Thus, there is a direct correlation between individual T and B cell recall propensities of peptide PS 1CT3 and its two analogues.

The journal of Immunology ?817

COMPETITOR CONCENTRATION (pM)

FIGURE 13. Relative binding of mAb PC286 to peptide PS1 CT3 and its analogues. mAb PC286 at a final concentration of 120 ndml was incubated with indicated concentrations of either peptide PSlCT3 (O), G41CT3 (O), or G28CT3 (A) as described in Materials and Methods. Subsequently, 1OO-pl aliquots were added in duplicate wells coated with peptide PSlCT3, and bound Ab detected was by ELlSA as described in the legend to Figure 3. Results presented are a representative of three separate experiments.

Peptides PSlCT3 and its analogues differ in their on-rates for Ab binding

The cumulative evidence described thus far indicates that the hi- erarchical ability of peptides PSlCT3, G28CT3, and G41CT3 to induce Ag-primed recruitment of T cell help may account for the differential immunogenicities of these molecules. However, the basis for differences in recognition of these Ag by primed B cells remained unclear. This was particularly true given that there was no evidence for sequence-dependent structural alterations in these peptides as revealed by their CD spectra. Furthermore, we were also unable to detect any heteroclitic binding of these peptides by anti-G41CT3 antisera. We therefore decided to investigate whether the single amino acid substitutions performed on peptide PSlCT3 had any influence on the kinetics of Ag-Ab interactions.

Our prior observation that the hierarchy of efficacies as G28CT3 > PSICT3 G41CT3 was independent of priming immunogen suggested to us that the relative potencies of these analogues was an inherent property. For purposes of the present study, therefore, we used a mAb that had previously been generated against peptide PSlCT3 and found to be directed against the DPAF determinant (mAb PC286 (29)) for determination of on- rates for binding to either peptide PSICT3 or its analogues. Prior to this, however, we first established that this mAb bound all three peptides with comparable affinities (shown in Fig. 13). Subse- quently, on-rates for peptide binding were determined in a fluorescence quenching assay under pseudo-first-order conditions, as described in Materials and Methods. The results of such an experiment are presented in Figure 14 and the calculated k,, values were 2.7 2 0.1 X lo7 "Is" for peptide G41CT3,4.2 ? 0.2 X

peptide G28CT3. As is evident, the on-rate for individual peptide binding by mAb PC286 follows the trend of G28CT3 > PSICT3 > G41CT3. This is consistent with the observed efficacies of these peptides in terms of B cell-mediated Th cell recruitment and relative immunogenicities, implying a direct causal relationship.

I O ~ M - ~ ~ - ~ for peptide PSlCT3, and 9.0 ? 0.4 X lo7 "'s" for

Discussion The preimmune B cell repertoire is generally thought to be composed of such a diverse array of Ab specificities that virtually

2.4

2.2

2

Time (seconds)

FIGURE 14. On-rates of peptide binding to mAb PC286. The exper- imental protocol employed for determination of on-rates is described in Materials and Methods; data analysis was as previously described (54). This figure shows linear regression plots of log of unbound Ab vs time for peptides PSlCT3 (O), C41CT3 (O), and C28CT3 (A). Data presented are from one of three independent runs.

any Ag can be recognized (45, 46). However, for generation of effective Ab responses, Ag recognition must necessarily be followed by clonal amplification of responder B cells, somatic mutations in Ig variable region genes, with subsequent selection for higher affinity clonotypes and eventual differentiation into Ab-secreting plasma or memory B cells (1, 2). In T-dependent responses, the drive for each of these stages is provided by Ag-activated Th cells that are recruited by the relevant B cells in an Ag-presenting mode (17-25).

In the case of multideterminant Ags such as polypeptides, the situation is somewhat more complex, with activation of B cells directed against the range of epitopes presented by Ag. This, however, is probably true only for the early stages of a primary humoral response. Subsequent progression appears to be accompanied by a combination of selection processes that serve to restrict both heterogeneity and fine specificity of the B cells that are eventually retained (28, 29). While reducing levels of Ag supply has been suggested as the driving force for competitive B cell selection (47), our own results have identified at least two additional mechanisms. One of these is the competition between secreted Ig and B cell mIg receptor for Ag binding (28). This, in turn, limits the amount of available Ag for B cell binding and eventual presentation for T help (28). Additionally, we have also recently demonstrated that the limiting pool of activated Ag-specific Th cells in the early stages of a primary response enforces a competitive interaction between Ag-activated B cells, resulting in an Ag affinity- driven selection (29). Indeed, the latter mechanism appears to be the principal one accounting for dominance of anti-DPAF Abs over alternate specificities during maturation of a murine primary humoral response to PSlCT3 (29).

This report extends our previous observations by demonstrating that subtle sequence variations outside of the immunodominant B cell domain can dramatically influence immunogenicity, without affecting fine specificities of the IgG Abs invoked. An obvious explanation, which could account for such a finding, is that these changes produced an alteration in the ability of the resulting peptides to stimulate/activate Ag-specific T cells. Indeed, a number of prior studies have documented such influences in which sequence changes distal to T cell determinants within polypeptide Ags have been shown to modulate T cell activation potencies (42-44). Such effects are thought to be mediated by an alteration in either peptide processing or in the affinity of the resulting fragment for MHC class I1 binding (48). This explanation, however, did not appear to apply in our case, since all three peptides were found to be equally proficient at both priming for a CT3-specific response and restimulating CT3-primed T cells. Furthermore, if processing of either


peptide PS 1 CT3 or its analogues is indeed a prerequisite for CT3 presentation, then the latter result would also suggest that there is no significant difference in processing of these Ags, at least in the context of presentation via nonspecific uptake of Ag.

Interestingly, however, peptide PSlCT3 and its analogues did reveal differences in their abilities to induce ex vivo recall responses in LNCs primed with the whole immunogen. This hierarchy of relative potencies was independent of whether the priming immunogen was peptide PSlCT3, G28CT3, or G41CT3; suggesting that the observed relative potencies of G28CT3 > PS 1CT3 > G41CT3 were inherent properties of the peptides. These results were in stark contrast to those obtained with CT3-primed LNCs, implicating a role for the B cell epitope in Ag for differential behavior. Subsequent experiments verified this assumption by indicating that these differences in recall abilities were a consequence of Ag-primed B cells functioning as APCs. Particularly notable here was our finding that enriched G41CT3-primed B cells were most efficient at restimulating CT3-primed T cells in the presence of peptide G28CT3 as Ag. The observed hierarchy in T cell recall experiments with G41CT3-primed LNCs was also reproducible in B cell recall experiments in irradiated mice adoptively transferred with G41 CT3-primed splenocytes, indicating a direct correlation between the two findings. Collectively, we take these data to suggest that Ag-primed B cells are most efficient at recruiting T cell help in the presence of peptide G28CT3 and least efficient with peptide G41CT3. This, in turn, may explain the differences in immunogenicity obtained between these three analogue peptides in Figure 2.

Since it was the APC activity of primed B cells that was being differentially influenced depending on challenge Ag, we also characterized serum Ab responses to these three peptides. We hoped that such studies might provide insights that could be extended to B cell sIg receptor interactions with Ag. One interesting finding was that, regardless of immunogenicity, immunization with either peptide PSICT3, G28CT3, or G41CT3 induced, in all cases, a primary IgG response that was at least predominantly directed against the tetrapeptide segment between positions 4 and 7 (sequence DPAF). This could be taken to indicate that, for all three immunogens, the dominant responder B cell populations during the IgG phase of the response were those with sIg receptors for the DPAF sequence within Ag. In other words, regardless of priming or recall Ag used, the dominant B cell APC activity would be that resulting from DPAF-mediated Ag capture by sIg receptors on Ag-activated B cells. Consequently, it was reasonable to infer that Ag-dependent modulation of the efficiency of primed B cells to recruit T help may actually reflect differences in interaction of sIg receptor with the DPAF segment in the context of the various substitutions performed. Evidence in support of our inference was obtained in experiments with anti-mouse IgG conjugates of the peptides, which seemed to indicate differences in interaction of G41CT3-primed B cells with the three analogues. It is possible that such differences in BCR engagement may eventually influence the density of the ligand presented and, consequently, Th cell recruitment. In this connection, it was also of interest that peptide G41CT3 elicited a lower affinity anti-DPAF IgG response in com- parison with that against either peptide PSlCT3 or G28CT3, implying a slower rate of affinity maturation. Thus, substitution of a distal proline residue at position 14 for glycine does exert a qualitative influence, in addition to a quantitative one, on anti-DPAF B cell responses.

How such minor changes in primary amino acid sequence could so markedly influence cognate interactions between immunocom- petent cells was an intriguing issue that remained to be resolved. Our CD studies revealed that all three peptides were equally ran-

domized with respect to conformation in solution. This finding ruled out induction/disruption of a secondary structural feature as the causative factor. However, since it is well known that Ag-Ab binding involves conformational accomodation at the level of both entities (49, 50), it was possible that the substitutions performed somehow altered the inducability of peptide (or DPAF) fit within the paratope of Ab. Even if this were true, i t was not however reflected at the level of affinity of Ab for the three analogues. For instance, anti-G41CT3 Abs were found to bind all three peptides equally well, implying the absence of a heteroclitic effect.

In contrast to equilibrium binding, the kinetics of peptide binding to a representative Ab proved to be markedly different. The obtained k,, values were in the order of G28CT3 > PS 1 CT3 > G41CT3. This hierarchy was consistent with that observed for quantitative differences in Th cell recruitment by Ag-primed B cells, recall of G41CT3-primed B cell responses, and relative immunogenicities at the level of a humoral response. We, therefore, infer that these differences in binding on-rates directly influence the ability of Ag-primed B cells to recruit T cell help, which in turn determines the extent of B cell proliferation and levels of secreted Ig. It is expected that kinetic differences in Ag binding would be reflected at the level of internalized Ag within B cells, with its consequences on density of Ag presented for TCR recognition. The density of surface MHC class 11-associated ligand on an APC is likely to be important in defining the rate at which that threshold number of TCRs is triggered as a prerequisite to T cell activation (51, 52). Prior studies have already established that on-rates represent a regulatory parameter for B cell selection during affinity maturation of an Ab response (53, 54). Results presented here suggest that on-rates of Ag recognition may also quantitatively influence amplification of the responder B cells during the course of an immune response.

In summary, the results presented in this report indicate that minor changes in sequences flanking an immunodominant epitope can markedly influence immunogenicity even in the absence of any obvious structural alterations. Such changes seem to operate by modulating the kinetics of B cell recognition of its corresponding epitope and thereby regulating its ability to access T cell help. In addition, studies of the kind described here may also help provide further insights into the basis the underlying generation of autoimmune B cell responses as a consequence of antigenic mimicry.

Acknowledgments We are grateful to Dr. V. Manivel and Mr. B. Ganesan for peptide synthesis and amino acid analysis. We also thank Mr. S. Kumaran (National Institute of Immunology) for assistance with the on-rate measurements.

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J Immunol 1997 Vijayakrishnan

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