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The Journal of Immunology, 2000, 164: 5605-5614.
Copyright © 2000 by The American Association of Immunologists

B Cell Responses to a Peptide Epitope. IX. The Kinetics of Antigen Binding Differentially Regulates Costimulatory Capacity of Activated B Cells

Lalitha Vijayakrishnan1,*, Krishnamurthy Natarajan1,*, Venkatasamy Manivel*, Sheikh Raisuddin{dagger} and Kanury V. S. Rao2,*

* Immunology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India; and {dagger} Department of Elementology and Toxicology, Jamia Hamdard, New Delhi, India


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We explore the possible mechanism by which association rates of Ag with activated B cells influences the ability of the latter to selectively recruit Th subsets. Our system used cocultures of Ag-activated B and T cells, where the Ag was a synthetic peptide, G41CT3. Restimulation was with either peptide G41CT3 or its analogue, G28CT3. Peptide G28CT3 has been previously shown to display a higher on rate, relative to the homologous peptide G41CT3, of binding to G41CT3-activated B cells. This difference in on rates was eventually exerted at the level of IFN-{gamma}, but not of IL-10, induction from T cells, with peptide G28CT3 proving more effective. However, various treatment regimens rendered peptide G41CT3 as potent as peptide G28CT3 at eliciting IFN-{gamma} responses from the above cultures. This included simultaneous treatment of B cells with peptide G41CT3 and the protein tyrosine kinase inhibitor tyrphostin. Alternatively, pretreatment of B cells with a peptide representing only the B cell epitope constituent of peptide G28CT3 (G28) was also equally effective. Subsequent experiments revealed that IFN-{gamma} production from activated T cells resulted from an engagement of CD28 by B7-1 on the B cell surface. Finally, the extent of cell surface B7-1 up-regulation on activated B cells was dependent on the on rate of Ag binding to the membrane-bound Ig receptor. Thus, cumulative results suggest that the kinetics of Ag binding to activated B cells can differentially regulate intracellular signaling. This influences selective costimulatory molecule expression, with its consequent effects on relative Th subset activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In contrast to naive, resting B cells, it is now accepted that Ag-activated B cells are potent APCs (1, 2, 3, 4, 5). This difference has generally been attributed to the presence of costimulatory molecules on activated B cells, which are absent on naive B cells (1, 2, 3, 4, 5, 6). Activation of B cells is initiated by the B cell Ag receptor (BCR),3 a multimeric receptor complex of membrane-bound Ig (sIg) noncovalently associated with heterodimers of IgA and IgB (7). Engagement of sIg by Ag is translated into intracellular signals by the cytoplasmic domains of IgA and IgB molecules (8, 9, 10). A tyrosine phosphorylation cascade, with subsequent recruitment of second messengers downstream (8, 9, 10, 11), rapidly follows ligation of BCR. Although the intervening pathways remain to be worked out, evidence suggests that at least one culmination point for processes initiated by BCR activation is in the up-regulation of cell surface-costimulatory molecules (12, 13). These latter results are particularly relevant for T-dependent responses, because they reveal the etiological basis by which activated B cells acquire the potential to function as APCs. Thus, in T-dependent responses, engagement of sIg by Ag not only results in its internalization, processing, and presentation of a peptide-MHC class II complex on the cell surface (14, 15, 16, 17) but also delivers the necessary signals for up-regulation of costimulatory molecules, most notably B7-2 (12). Signals delivered by costimulatory molecules are critical in ensuring full activation of T cells in the course of a cognate B-T interaction (18, 19).

Another facet that is now becoming apparent is that a sIg-Ag engagement does not always lead to a productive outcome. Depending on the circumstances and the status of the B cell, BCR ligation may result in stimulation, anergy, or even apoptosis (9, 20). These observations have led to the inference that the nature of the ligand-receptor interaction, among other factors, can potentially direct the physical recruitment of signaling pathways differently inside the lymphocyte and thereby define the nature of the subsequent immune response (9).

Despite the proven capacity of activated B cells to serve as APCs, their role in influencing CD4 T cell responses, particularly with respect to Th1 vs Th2 subsets, is less clear. Although in principle activated B cells have been shown to be capable of interacting with both subsets (21, 22, 23), a variety of studies have suggested a preference for activating Th2 cells (24, 25, 26). However, accumulating evidence now reveals that triggering of CD4 effector subsets is not strictly APC restricted. Rather the dominating influence of a variety of additional factors is being increasingly noted. One such factor appears to be the density of ligand presented, in the context of MHC class II, on APC. Murray et al. (27) have observed that whereas a high ligand density promoted Th1 differentiation, low ligand densities biased toward Th2 responses. In addition to density (27, 28), the affinity (or off rates) of peptide-MHC and peptide-TCR interactions has been identified as another regulatory parameter (29, 30, 31). Perhaps not unrelated to these two factors are the observations that the duration of TCR stimulation also has an important bearing on the outcome (32, 33). Another important regulator of T cell responses is the relative expression of the various costimulatory molecules on APC surface (30). Of particular note here are the B7 molecules, B7-1 and B7-2, which appear to impose selectivity, although in as yet unknown ways, in terms of CD4 T cell subset generation and stimulation (13, 34, 35, 36, 37, 38). Finally, the local cytokine milieu is also known to influence the outcome of a T cell response (39). Collectively, these findings indicate that both generation and regulation of CD4 effector subsets are more complex processes than hitherto suspected. The nuances are only now beginning to be revealed.

The above observations of extraneous influences on APC-mediated T cell responses also appear to extend to B cell APCs. For instance, display of high ligand density on activated B cells has been shown to preferentially stimulate Th1-like immunity (40). More recently, Pasare et al. (41) have demonstrated that increased, targeted delivery of Ag to activated B cells generates a Th1 bias in the immune response. Thus, in light of these findings and given the unique property vis-a-vis other professional APCs of Ag specificity, it can be anticipated that variations in Ag-binding properties of activated B cells may also impose a regulatory effect on subsequently elicited T cell responses.

Our recent studies have identified yet another variable that regulates both quantitative and qualitative aspects of T cells stimulation, in the course of a cognate interaction with activated B cells (42, 43). In these experiments, two single amino acid-substituted analogues of a model peptide Ag PS1CT3, were used , namely, peptides G41CT3 and G28CT3 (42, 43). All three peptides were shown to constitute T-dependent Ags, yielding primary and secondary IgG responses directed exclusively against a common tetrapeptide sequence between positions 4 and 7 (sequence, DPAF) (42). In other words, the substitutions performed in peptides G41CT3 (at position 10) and G28CT3 (at position 1) did not affect the fine specificity of B cells induced in the IgG response (42). Interestingly, however, peptide G28CT3 proved markedly more immunogenic than peptide G41CT3 (42). This difference in immunogenicity resulted from the greater ability of peptide G28CT3 to induce a Th cell recall response by peptide-primed B cells (42). Subsequent investigations revealed that the superior ability of peptide G28CT3 to elicit a B cell-mediated Th recall response was solely attributed to its increased on rate of binding, relative to peptide G41CT3, to B cells primed against the DPAF epitope (42).

More recently, we observed that peptides G28CT3 and G41CT3 also differentially influence the relative extents of Th1 vs Th2 activation by peptide-primed B cells (43). Thus, a coculture of peptide G41CT3-primed B cells and peptide CT3-primed T cells yielded increased levels of the Th1-type cytokines in the presence of peptide G28CT3, relative to that when peptide G41CT3 was included as challenge Ag (43). These differences, again, could be solely ascribed to the increased on rate of binding of peptide G28CT3 (through the DPAF epitope) to the sIg receptor on G41CT3-primed B cells (43). Further, consistent with an on-rate-driven effect, the ratio of IFN-{gamma} to IL-10 induced was dependent on the concentration of peptide G28CT3 used, with an IL-10 dominance at low peptide concentrations (43). Although various possible explanations for these finding were offered, experimental evidence in favor of any of these was lacking (43).

In this report, we explore the possible mechanistic basis for the variable influence of BCR-Ag association kinetics on CD4 T cell subset stimulation. Evidence presented here suggests that the kinetics of Ag binding to activated B cells can differentially regulate intracellular signaling pathways. This in turn influences selective costimulatory molecule expression on the B cell surface, with its consequent effects on relative Th subset activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

Heavy chain-specific, HRP-labeled secondary Abs, the protein kinase inhibitors tyrphostin 51, genistein, and calphostin were all purchased from Sigma (St. Louis, MO). Rat mAbs against the mouse molecules CD80 (clone 1G10), CD86 (clone GL1), and CD28 (clone 37.51) and isotype controls were obtained from PharMingen (San Diego, CA). ELISA kits for the measurement of cytokines were purchased from Genzyme (Cambridge, MA), and magnetic beads for the purification of B and T cells were purchased from Dynal (Oslo, Norway). Peptides used in this study were synthesized, purified, and characterized as described earlier (42, 43).

Animals and immunizations

Female BALB/c mice (6–8 wk of age) 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 50 µg/mouse in CFA (except where indicated otherwise). For polyclonal sera, mice were bled from the retroorbital plexus at indicated times.

Enrichment of B and T cell populations

This was achieved essentially as described before (43, 44). Briefly, for B cells, mice were immunized with peptide G41CT3 with a 50-µg/mouse dose of peptide G41CT3 in CFA given i.p. Three weeks later, the spleens were removed and depleted of RBCs by lysis with ammonium chloride, and adherent cells were then depleted by panning on plastic plates at 37°C for 1 h. Nonadherent cells were collected and diluted to a cell concentration of 5 x 107 cells/ml. From this, T cells were depleted by two rounds of incubation with Dynabeads anti-mouse Thy-1.2 (4 x 108 beads/ml, Dynal) as recommended by the manufacturer. The resultant enriched B cells were then treated with mitomycin C at a final concentration of 50 µg/ml at 37°C for 20 min. Cells were washed thoroughly in culture medium before use.

For enriched T cells, mice were immunized with peptide CT3 and boosted 7 days later. At 3 days after the boost, inguinal lymph node cells were collected and first depleted of RBCs and adherent cells as described above. Nonadherent cells were collected and diluted to a cell concentration of 5 x 107 cells/ml. B cells were depleted from this population by two rounds of incubation with Dynabeads mouse anti-B220 (4 x 108 beads/ml, Dynal) following the recommended protocol of the manufacturer. As previously described, the above protocol yields a cell purity of 85–90% for both B and T cells, as assessed by flow cytometry.

Treatment of B cells with protein kinase inhibitors

Enriched B cells from G41CT3-primed mice were incubated at 5 x 106 cells/well in wells of a 24-well culture plate in the presence of 37 µM final concentrations of peptide G41CT3, G28CT3, or CT3. These concentrations have been optimized earlier (42, 43). In addition, the protein kinase inhibitors genistein, tyrphostin, or calphostin were also included at the indicated final concentrations. The total volume of each culture was 2 ml and, except where indicated, incubation was for 16 h at 37°C in a humidified atmosphere of 5% CO2. The culture medium was RPMI 1640 containing, in addition to antibiotics, 10% FCS and 50 µM 2-ME. Subsequently, cells were washed extensively in HBSS to free them of residual peptides and inhibitors. Aliquots of 2.5 x 105 B cells were mixed with an equal number of enriched T cells from CT3-primed mice, in a total volume of 200 µl culture medium (see above), and plated in wells of a 96-well culture plate. After 48 h, a time point standardized earlier (43), supernatants were collected for the estimation of cytokines by ELISA.

Pulsing of G41CT3-primed B cells with peptide G28

Enriched B cells (5 x 106 from G41CT3-primed mice were pulsed with a final 37 µM concentration of peptide G28, either in the presence or the absence of 0.3 µM tyrphostin, for 16 h at 37°C in a humidified atmosphere of 5% CO2. At the end of this period, cells were washed in HBSS. Subsequent coculture with CT3-primed T cells and cytokine estimation in culture supernatants was as described above.

Coculture of primed B and T cells in the presence of anti-CD28

Enriched B cells from G41CT3-primed mice (2.5 x 105 cells/well) were cocultured with enriched T cells from CT3-primed mice (2.5 x 105 cells/well) in multiple wells of a 96-well culture plate. The total volume of each culture was 200 µl. Increasing concentrations of either peptide G41CT3 or G28CT3 were also included, along with a final concentration of 2 µg/ml anti-CD28 mAb (clone 37.51, PharMingen). Control wells included cells incubated with either peptide only or peptide with nonspecific rat IgG at 2 µg/ml.

Treatment of G41CT3-primed B cells with anti-B7 mAbs

Enriched B cells from G41CT3-primed mice were pulsed as described above, harvested, and washed in HBSS. Aliquots of 5 x 106 cells each were then incubated with either anti-B7-1 or anti-B7-2 mAbs in a total volume of 2 ml culture medium. Final concentrations of anti-B-7 mAbs used were 10 µg/ml to ensure saturation, and incubation as for 4 h at 4°C. At the end of this period, cells were washed and cultured with CT3 primed T cells and a final concentration of 37 µM peptide G41CT3, followed by cytokine estimation as described above.

Estimation of cytokines

A sandwich ELISA protocol using commercially available kits (Genzyme) measured all cytokines. The recommended protocol of the manufacturer was strictly followed in all cases. Quantitation was against a standard curve obtained for individual cytokine standards provided by the manufacturer. Background values obtained in cultures where no Ag was added was subtracted in all cases.

Inhibition of BCR internalization

Naive B cells were enriched from splenocytes of nonimmune BALB/c mice as described above. These were diluted to a concentration of 5 x 106 cells/ml and chilled on ice, and 1-ml aliquots were incubated with a final concentration of 10 µg/ml FITC-labeled anti-mouse IgG (Fab fragment). Incubation was at 4°C for overnight and in either the presence or the absence of a final concentration of 0.3 µM tyrphostin or 60 µg/ml of genistein. Cells were then washed with ice-cold RPMI containing, where necessary, the indicated concentration of the relevant inhibitor. Subsequently, the cells were warmed to room temperature, and aliquots were monitored for residual surface-bound fluorescence at regular intervals of 5 min, for a total period of 30 min.

Fab anti-mouse IgG, its derivatization, and measurement of binding properties

Monovalent Fab was generated by papain digestion of goat anti-mouse IgG (Sigma), followed by elution over a protein G-Sepharose column to remove Fc fragments. Completeness of digestion was ascertained by PAGE under nonreducing conditions. An aliquot of the resulting Fab fragment, at a final concentration of 2 mg/ml, was incubated with an 80-fold molar excess of trinitrobenzenesulfonic acid in PBS (pH 8.0) in a total volume of 500 µl, with occasional shaking, at room temperature for 45 min. After incubation, excess trinitrobenzenesulfonic acid was removed by exhaustive dialysis against PBS (pH 7.5). The stoichiometry of derivatization, as determined spectrophotometrically, was 8 mol trinitrophenyl (TNP) per mol Fab.

Both derivatized and underivatized Fab preparations were compared, at a final concentration of 5 µg/ml, for binding to an immobilized mouse monoclonal IgG Ab by the surface plasmon resonance technique (IASys, Affinity Sensors, Cambridge, U.K.). Reactions were conducted at 25°C in PBS (pH 7.5) containing 0.05% Tween 20, and both association and dissociation curves were obtained.

Anti-Ig stimulation of splenic B cells and FACS analysis

Splenic B cells were enriched for the sIgG+ subset by treatment with a combination of anti-mouse IgD, anti-mouse IgM, and complement. By this procedure, at least 85% of the B220+ cells were sIgG+ as determined by FACS analysis. A 2.5 x 106 sample of these B cells was plated in 1 ml culture medium which also contained either the Fab or its TNP derivative at appropriate concentrations as well as 100 µl 10x concentrated supernatant from Con A-activated T cells. Cells were cultured for 60 h, at which time they were collected, chilled on ice, and processed for analysis by flow cytometry. To examine the effect of tyrphostin, B cells were cultured as above with a final concentration of 10 µg/ml TNP-derivatized Fab. In addition, the medium also contained tyrphostin at a final concentration of 0.5 µM. After 16 h, the cells were washed to remove tyrphostin and resuspended in medium containing only the TNP-Fab and culture supernatant from Con A-activated T cells. After an additional incubation for 44 h, the cells were then processed for analysis.

Staining for B cells was achieved with biotinylated anti-B220 mAb (1 µg/106 cells at 4°C for 1 h), followed by streptavidin-PE (4°C, 1 h). Labeling of cell surface B7-1 and B7-2 was performed with the appropriate FITC-labeled Abs (PharMingen, 1 µg/106 cells, 1h at 4°C). A parallel set was also stained with FITC-labeled rat IgG2a as an isotype-matched staining control. Staining procedures were all conducted in FACS buffer (PBS containing 0.1% BSA and 0.01% azide). Stained cells were fixed in buffer containing 0.1% paraformaldehyde before analysis.

Two-color analysis was performed by using a FACScalibur flow cytometer (Becton Dickinson Immunocytometry Systems, Mountain View, CA). Logarithmically amplified fluorescence data were collected on 1 x 104 viable cells as determined by forward scatter intensity and by exclusion of propidium iodide-staining cells.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Protein kinase inhibitors differentially modulate cytokine production from peptide stimulated B-T cocultures

Both peptide analogues used in these studies, G28CT3 and G41CT3 (Fig. 1Go), have been previously described (42, 43). They represent single-amino acid-substituted analogues of a parent peptide PS1CT3 in which position 1 contains a histidine residue and position 10 contains an aspartic acid residue (42). As described earlier (42), peptide PS1CT3 represents a hybrid peptide containing a hepatitis B virus-derived B cell epitope (residues 1–15, segment PS1) and a promiscuous T cell epitope (residues 18–38, segment CT3) from an Ag of the malaria parasite Plasmodium falciparum. Separating the B and T cell epitopes is a spacer of two glycine residues at positions 16 and 17 (Fig. 1Go). As already discussed, these two peptides differ in their on rates of binding to G41CT3-primed B cells, leading to altered Th subset recruitment profiles from CT3-primed T cells (42, 43).



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  		FIGURE 1. Amino acid sequence of peptides G28CT3 and G41CT3. Their properties have been described earlier (42 43 ). Positions of nonidentity in sequence have been underlined.

 
Our intent was to explore the underlying mechanisms by which BCR-Ag association rates can influence Th subset recruitment (43). For this, we first examined the effect of protein kinase inhibitors on peptide-stimulated recall responses from cocultures of G41CT3-primed B cells and CT3-primed T cells. It may be recalled here that peptide CT3 represents the common T epitope segment (residues 18–38) of the Ags described in Fig. 1Go. We have shown earlier that peptide CT3 elicits comparable recall responses from T cells primed with either G28CT3 or G41CT3 (42). For the present study, three protein kinase inhibitors were selected, tyrphostin, genistein, and calphostin. Whereas tyrphostin and genistein are inhibitors of protein tyrosine kinases (45, 46), calphostin inhibits the serine/threonine kinase protein kinase C (47).

G41CT3-primed B cells were incubated with either peptide G28CT3 or G41CT3 in either the presence or the absence of the protein kinase inhibitors described above. After a 16-h incubation, the cells were washed thoroughly to remove both excess peptide and inhibitor and then cocultured with CT3-primed T cells (Materials and Methods). Supernatants from such cultures were subsequently collected for the measurement of cytokines, and the data for IFN-{gamma} and IL-10 as prototypic cytokines are presented in Fig. 2Go.



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  		FIGURE 2. Effect of various protein kinase inhibitors on cytokine responses elicited in B-T cocultures. B cells from G41CT3-primed mice were incubated with peptide G28CT3 or G41CT3. Each of these sets were also included with 1 µM tyrphostin (T), 0.1 µM calphostin (C), 60 µg/ml genistein (G), or no inhibitor (-). The experimental protocols used for these cultures and for the subsequent determination of IFN-{gamma} (A) and IL-10 (B) are described in Materials and Methods. Results are representative of four separate experiments, and individual values are mean (±SE) of that from quadruplicate wells.

 
As expected (43), in the absence of any inhibitor, the G28CT3 recall response was dominated by IFN-{gamma} relative to that in the presence of peptide G41CT3 (Fig. 2Go). Interestingly, however, nonidentical effects of protein kinase inhibitors on IFN-{gamma} production were observed in cultures challenged with either G28CT3 or G41CT3. Both calphostin and genistein completely inhibited all cytokine induction in response to challenge with peptides G28CT3 and G41CT3 (Fig. 2Go). However, the inclusion of tyrphostin resulted in a selective enhancement of IFN-{gamma}, but not IL-10, in cultures stimulated with peptide G41CT3 (Fig. 2Go). Indeed, the levels obtained were comparable with those seen in G28CT3-stimulated cultures, the latter proving resistant to tyrphostin treatment (Fig. 2Go). These results suggest that, at least in terms of IFN-{gamma} induction, the drawback of a poorer on rate for peptide G41CT3 can be offset by treatment of the B cells with the protein tyrosine kinase (PTK) inhibitor tyrphostin, but not genistein.

Treatment of G41CT3-primed B cells alone with either of the kinase inhibitors, followed by coculture with T cells in the presence of peptide G41CT3, resulted in cytokine levels that were comparable to those obtained when inhibitors were omitted (data not shown). In addition to revealing a concomitant requirement for both Ag and inhibitor, these results also exclude the possibility that inhibitory effects seen in Fig. 2Go may represent carryover effects of trace levels of inhibitors on T cells. Further, omission of CT3-primed T cells in the cocultures did not result in any detectable levels of cytokine production (data not shown), suggesting that the observed cytokines in Fig. 2Go were derivatives of the included T cells. This is in keeping with our earlier findings that cytokine production in peptide-stimulated cultures of G41CT3-primed B cells and CT3-primed T cells is due to CD4+ T cells (43). An examination of the dose-dependent efficacy of tyrphostin in enhancing G41CT3-dependent IFN-{gamma} production indicated that this effect peaks at a dose of 0.1–0.25 µM (Fig. 3Go).



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  		FIGURE 3. Dose dependence of tyrphostin-mediated IFN-{gamma} enhancement. G41CT3-activated B cells were incubated with either peptide G28CT3 ({circ}) or G41CT3 (•) in the presence or absence of the indicated concentrations of tyrphostin before coculture with CT3-primed T cells (Materials and Methods). The resulting levels of IFN-{gamma} in culture supernatants were determined by ELISA. Results are for one of the two independent experiments, and independent values are presented as described for Fig. 2Go.

 
It has been shown previously that PTK inhibitors also inhibit internalization of the sIg receptors on B cells (48). However, because both genistein and tyrphostin inhibit BCR internalization, whereas only tyrphostin was capable of stimulating G41CT3-dependent IFN-{gamma}, it was unlikely that the internalization-inhibitory activity could account for the differences seen in Fig. 2Go. Nevertheless, we reconfirmed that both these PTK inhibitors do inhibit BCR internalization in our hands. Using FITC-derivatized Fab fragment of anti-mouse IgG as the tracer, we observed that both compounds completely blocked sIgG receptor internalization in purified splenic B cells at their respective concentrations used for the experiment shown in Fig. 2Go (data not shown). This reiterates that the selective G41CT3-mediated, IFN-{gamma}-stimulatory effect of tyrphostin (Fig. 2Go) was not due to inhibition of BCR and, consequently, Ag internalization.

Peptide-dependent differences in IFN-{gamma} production are mediated through BCR activation

We have already proved earlier that the differences in on rates of binding of peptides G28CT3 and G41CT3 to G41CT3-primed B cells are a direct reflection of the rapidity with which the DPAF epitope in these analogues can accommodate within the paratope of the sIg receptor (43). These differences could conceivably translate into altered levels of Ag uptake, eventually leading to differing densities of ligand presented in association with MHC class II. Ligand density differences are known to variably modulate T cell responses (27, 28, 40). Alternatively, as has recently been shown for T-independent responses (49), Ag-binding characteristics may variably influence BCR-mediated intracellular signaling, resulting in different outcomes.

To distinguish between the above possibilities, we devised a pulse-chase strategy where G41CT3-primed B cells were first pulsed with a peptide representing only the B cell epitope segment (residues 1–15, peptide G28) of peptide G28CT3. After this, the B cells were cocultured with CT3-primed T cells in the presence of peptide G41CT3. It was rationalized that a G28 pulse would facilitate BCR triggering, but without leading to any Ag presentation. Indeed, we have earlier confirmed that peptide G28 is devoid of a T cell epitope and that its inclusion as a challenge Ag in coculture of G41CT3-primed B cells and CT3-primed T cells does not lead to either T cell proliferation (42) or cytokine induction (43). Thus, the protocol outlined above was expected to permit a resolution between the relative contributions of BCR triggering alone and Ag presentation to the effect under study.

In initial experiments, G41CT3-primed B cells were incubated with peptide G28, in either the presence or the absence of tyrphostin, for a period of 16 h. Cells were then washed and cultured with CT3-primed T cells along with peptide G41CT3 as challenge Ag (Materials and Methods). Subsequently, supernatants were collected and cytokine levels were measured. The results from such an experiment are presented in Fig. 4Go. As is evident, pulsing of G41CT3-primed B cells with peptide G28 resulted in a markedly enhanced production of IFN-{gamma} in subsequent cocultures (Fig. 4Go). Interestingly, tyrphostin treatment now appeared to be redundant with no further effect on IFN-{gamma} levels in cultures containing G28-pulsed B cells (Fig. 4Go). A pulse with peptide G28 alone, in the absence of peptide G41CT3 challenge, did not yield any detectable levels of cytokines (Fig. 4Go). Similarly, peptide G41CT3 challenge in the absence of a G28 pulse yielded the expected low levels of IFN-{gamma}. Levels of IL-10, however, remained unaffected either on G28 pulsing or tyrphostin treatment of the B cells (Fig. 4Go). Thus, the results in Fig. 4Go indicate that a pretreatment of G41CT3-primed B cells with peptide G28 markedly augments the ability of peptide G41CT3 to subsequently stimulate IFN-{gamma}, but not IL-10, production from cocultures of these B cells with CT3-primed T cells. Indeed, the levels of IFN-{gamma} obtained here were comparable with those elicited by the higher binding on rate peptide G28CT3 (Fig. 4Go). Thus, the differences exerted due to differences in Ag binding kinetics, at least in terms of IFN-{gamma} elicitation from primed T cells, appear to originate from effects on BCR triggering and not on Ag presentation.



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  		FIGURE 4. Prepulsing of B cells with peptide G28 selectively stimulates IFN-{gamma} responses. G41CT3-primed B cells were pretreated with peptide G28 in the presence or absence of tyrphostin (Tyr) as described in Materials and Methods. After pretreatment, these cells were cultured with CT3-primed T cells in the presence of G41CT3 as the recall Ag. Results obtained from a parallel set of cells in which the challenge Ag was peptide G28CT3, in the absence of any pretreatment, are also shown for comparison. Results depicted are from one of four experiments.

 
Anti-CD28 Abs augment IFN-{gamma} production from peptide-stimulated cultures

A productive interaction between Ag-primed B and T cells requires two signals. The first is provided by specific peptide-MHC class II ligand cognately engaged by the TCR, and the second is the range of noncognate costimulatory signals provided by the APC to the T cell during cognate interactions (18, 19). Both the quantity and the quality of costimulatory signals provided play a significant role in determining the outcome of T cell stimulation, as well as biases in favor of either Th1 or Th2 cytokines (30). One such set of costimulatory interactions that regulate both T cell stimulation and cytokine production is that between the B7 family of proteins on APC and CD28/CTLA-4 on T cells (13, 34). Whereas B7-CD28 interactions have been shown to be stimulatory, engagement of CTLA-4 is thought to down-regulate T cell activation (50, 51).

To explore whether the effects of Ag-B cell association rates influence at the level of a cognate B-T interaction, the effect of addition of anti-CD28 mAb was examined on cocultures stimulated with varying doses of both analogue peptides. Levels of IFN-{gamma} and IL-10 thus obtained in supernatants are shown in Fig. 5Go. An interesting effect of anti-CD28 on peptide G28CT3-stimulated IFN-{gamma} induction is evident (Fig. 5GoA). Although maximal levels attained remained unaffected, the efficiency of peptide G28CT3 nevertheless increased significantly in the presence of anti-CD28. Six-fold lower concentrations of peptide G28CT3 were sufficient to achieve half-maximal responses, relative to that when anti-CD28 was absent (Fig. 5GoA). The effect of anti-CD28 addition was markedly more profound, in terms of IFN-{gamma} induction, on G41CT3-stimulated cultures, where both qualitative and quantitative shifts were seen (Fig. 5AGo). Indeed, in the presence of anti-CD28, the IFN-{gamma} induction dose response profile was identical with that observed for anti-CD28-supplemented, G28CT3-stimulated cultures (Fig. 5GoA). Thus, in the presence of anti-CD28, peptides G28CT3 and G41CT3 do not display any differences in their ability to induce IFN-{gamma} secretion from cocultures of G41CT3-primed B cells and CT3-primed T cells. This strongly suggests that, at least at one level, differences in BCR-Ag binding rates exert their influence at the level of T cell costimulation.



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  		FIGURE 5. Anti-CD28 modulates cytokine production from B-T cocultures. Enriched B cells from G41CT3-primed mice were cocultured with CT3-primed T cells in the presence of varying concentrations of either peptide G28CT3 ({circ}, •) or peptide G41CT3 ({triangleup}, {blacktriangleup}). Each of the peptide stimulated groups also consisted of replicates sets where anti-CD28 was either included (•, {blacktriangleup}) or excluded ({circ}, {triangleup}). At the end of the culture period, supernatants were collected and assayed for the levels of IFN-{gamma} (A) or IL-10 (B). Results from one of three experiments are shown, and individual values are presented.

 
In contrast to effects on IFN-{gamma}, no qualitative differences in IL-10 responses could be observed in anti-CD28-supplemented cultures stimulated with either peptide G41CT3 or peptide G28CT3 (Fig. 5GoB). In both cases, addition of anti-CD28 resulted in a modest but comparable enhancement in IL-10 levels and with no significant effect on the dose-response profile (Fig. 5GoB).

B7-1 (CD80) and B7-2 (CD86) differentially regulate cytokine production from CT3-primed T cells

As discussed above, the data in Fig. 5Go implicate differential engagement of CD28 in cultures stimulated with either G28CT3 or G41CT3. The counterreceptor for CD28 can be either B7-1 or B7-2 (34), and it has been suggested earlier that whereas B7-1 promotes Th1 responses, B7-2 directs Th2 responses (35). Further, Agrewala et al. (52) have recently shown that, in a coculture of activated B and T cells, anti-B7-1 selectively inhibited production of the Th1-type cytokines but not those from Th2 cells. Thus, it was likely that the observed differences in the profile of cytokines induced in response to the two analogue peptides may represent a consequence of differential involvement of the two B7 proteins during costimulation.

To confirm whether the two B7 molecules do, in fact, differentially regulate cytokine production in our system, we used the earlier described protocol of prepulsing the B cells with peptide G28. G41CT3-primed B cells were pulsed with peptide G28, following which they were treated with anti-B7-1, anti-B7-2, or nonspecific rat IgG (Materials and Methods). Finally, these cells were washed and then cocultured with CT3-primed T cells in the presence of an optimal concentration of peptide G41CT3 (Materials and Methods). Levels of cytokines produced in culture supernatants were then measured, and the results are presented in Fig. 6Go. As shown, treatment of B cells with anti-B7-2 selectively inhibited IL-10 production, but with no effect on IFN-{gamma} levels (Fig. 6Go). In contrast, anti-B7-1 was found to completely inhibit IFN-{gamma} secretion, but not that of IL-10 (Fig. 6Go). Similar results were also obtained for cocultures challenged with peptide G28CT3 in either the presence or the absence of G28 pulsing (data not shown). Thus, the data in Fig. 6Go reveal that at least for the present system, whereas B7-1 preferentially activated Th1 responses, B7-2 promoted Th2 stimulation. Further, the fact that enhancement of G41CT3-dependent IFN-{gamma} responses by G28 pulsing is also neutralized by anti-B7-1 (Fig. 6GoA) reveals the possibility of a causal relationship between G28 pulsing and the extent of B7-1 involvement during costimulation.



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  		FIGURE 6. Anti-B7 Abs selectively inhibit Th subset-specific responses. Untreated G41CT3-primed B cells (-) or those first treated with peptide G28 (+) were incubated with either anti-B7-1, anti-B7-2, or nonspecific (NS) rat IgG before their inclusion in culture with CT3-primed T cells (Materials and Methods). Levels of IFN-{gamma} and IL-10 that resulted in the culture supernatants are shown as mean (±SE) of at least triplicate wells. Data represent one of three independent experiments.

 
Differential BCR triggering thresholds for up-regulation of cell surface B7-1 and B7-2

Our findings that peptide-dependent B cell-mediated differences in cytokine induction from CT3-primed cells may reflect differential engagement of CD28 by the B7 molecules prompted us to examine the effect of Ag-binding rates on relative B7-1 and B7-2 expression on B cells. Unfortunately, however, the extremely low frequency of Ag-specific B cells precluded a direct analysis of the effects of G28CT3 vs G41CT3 binding. To circumvent this problem, we adopted an alternate system wherein splenic B cells were triggered with the Fab fragment of anti-mouse IgG. The utility of this system was greatly facilitated by our finding that TNP derivatization of the Fab led to a near 4-fold reduction in association rates of binding to mouse IgG, but with no concomitant effect on dissociation rates of the resultant complexes (Fig. 7Go).



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  		FIGURE 7. TNP derivation of Fab anti-mouse IgG reduces its rate of association with mouse IgG. Binding of either Fab anti-mouse IgG (Ig) or its TNP derivative (T-Ig) to immobilized mouse IgG was monitored by the surface plasmon resonance technique as described in Materials and Methods and Ref. 53 . Both association and dissociation curves are shown. The arrow marked b indicates start of the binding process whereas the arrow d indicates start of dissociation. The table summarizes the association and dissociation rates for the two preparations, the former being estimated as the averaged increment in response signal per second.

 
Splenocytes enriched for sIgG+ B cells were incubated with varying concentrations of either Fab anti-mouse IgG, or its TNP derivative along with a constant concentration of culture supernatant from activated T cells (Materials and Methods). Subsequently, the levels of induced surface B7-1 and B7-2 were analyzed by flow cytometry (Materials and Methods). As shown in Fig. 8Go, both Fab and its TNP derivative were equally proficient at up-regulating B7-2 in a dose-dependent manner (Fig. 8Go, A and B). However, B7-1 induction was severely attenuated in the presence of TNP-derivatized Fab, relative to that obtained in the presence of the unmodified molecule (Fig. 8Go, C and D). Because the only distinction in the two Ab preparations resides in their nonidentical association rates with mouse IgG, we infer that differences in the extent of B7-1 up-regulation seen in Fig. 8Go, C and D, is a direct consequence of this.



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  		FIGURE 8. Differential effects of BCR occupancy rates on B7-1 and B7-2 up-regulation. Induction of cell surface B7-2 (A and B) and B7-1 (C and D) in response to splenic B cell stimulation with either Fab or TNP-Fab was determined by flow cytometry as described in Materials and Methods. A and C depict B7-2 and B7-1 staining, respectively, obtained on stimulation of cells with a final concentration of 10 µg/ml either Fab (thin line) or TNP-Fab (dashed line). Histograms are for a gated B220+ population of cells. The thick line represents cells treated with T cell supernatant alone (Materials and Methods), which was superimposable with the profile obtained for unstimulated cells. Additional controls also included isotype-matched staining controls for B7 (Materials and Methods). Also shown are the dose-dependent increases in B7-2 (panel B) and B7-1 (D) levels with indicated doses of either Fab ({circ}) or TNP-Fab (•). Values for the B7 molecules are given in terms of modal fluorescence intensity (MFI) for each experimental group. Data in all panels are from one of three separate experiments. FL1-H, fluorescence.

 
We also examined the effect of tyrphostin on B cells stimulated with TNP-derivatized Fab (Materials and Methods). As shown in Fig. 9GoA, no potentiating effect of tyrphostin treatment on B7-2 expression was detected. In contrast, treatment with tyrphostin resulted in a further, although modest, increase in B7-1 levels.



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  		FIGURE 9. Tyrphostin treatment enhances B7-1 but not B7-2 levels in TNP-Fab stimulated B cells. For experimental details, refer to Materials and Methods. Histograms depict staining for B7-2 (A) and B7-1 (B) in B220+ gated cells stimulated with TNP-derivatized Fab in the presence (thin line) or absence (dashed line) of tyrphostin. The thick line represents staining of cells treated with tyrphostin, but in the absence of TNP-Fab. The latter was superimposable with B7 staining obtained for a parallel culture of cells that were not treated with with either tyrphostin or TNP-Fab. Although modest, the tyrphostin-mediated enhancement in B7-1 levels in B was, nevertheless, significant. A Kolmogorov-Smirnov statistical analysis (Cell Quest, Becton Dickinson Immunocytometry Systems) yielded a value for D of 0.25–0.30 in three separate experiments. In contrast, the mean value of D for the tyrphostin-untreated groups between the three experiments was 0.10 ± 0.03.

 
Extent of cell surface B7-1 expression correlates with ability to elicit IFN-{gamma} responses from T cells

The ability to differentially modulate B7-1 levels on B cells with chemically derivatized Fab anti-IgG also facilitated a direct examination of the role of B7-1 in induction of IFN-{gamma} responses from T cells. For this, enriched sIgG+ B cells were incubated with varying concentrations of either Fab anti-mouse IgG, or its TNP derivative and subsequently cocultured with allogeneic T cells derived from C57BL/6 mice. Production of IL-10 and IFN-{gamma} in culture supernatants was then determined; the results are presented in Fig. 10Go. As is clear, dose-dependent induction of IL-10 was equally sensitive to both analogues (Fig. 10GoA). In contrast, however, TNP-derivatized Fab was markedly less proficient at inducing IFN-{gamma} responses where the dose-response profile obtained (Fig. 10GoB) was consistent with that for B7-1 up-regulation seen in Fig. 8GoD. Coincubation with tyrphostin augmented TNP-Fab-mediated IFN-{gamma} elicitation from allogeneic T cells, but not IL-10 (Fig. 10Go).



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  		FIGURE 10. Fab anti-mouse IgG and its TNP derivative differentially induce IFN-{gamma} production from cocultures of allogeneic B and T cells. Enriched sIgG+ splenic B cells from BALB/c mice were activated with indicated doses of Fab anti-mouse IgG ({circ}), TNP-Fab (•), or a combination of TNP-Fab and 0.3 µM tyrphostin (x) for 16 h as described in Materials and Methods. Subsequently, cells were washed, and aliquots of 2.5 x 105 B cells were mixed with an equal number of enriched T cells obtained from C57BL/6 mice, in a total volume of 200 µl culture medium (Materials and Methods). Production of IL-10 (A) and IFN-{gamma} (B) was monitored in culture supernatants collected 48 h later. Results shown are from one of two separate experiments, and values are mean (±SE) of triplicate determinations. Cocultures where untreated B cells were used gave values for IL-10 and IFN-{gamma} that were <120 pg/ml.

 
Although the above data strongly support that the extent of expression of B7-1 on B cells correlates with their ability to recruit IFN-{gamma} responses from T cells, the formal possibility that these effects derived from alterations in B cell Ag-presenting capacities could not be excluded. We therefore compared the effects of treatment of B cells with Fab, its TNP derivative, or a combination of TNP-Fab and tyrphostin on cell surface expression of MHC class II molecules. As may be expected for an activated subset, sIgG+ B cells displayed high basal levels of class II expression. The various treatments described above resulted in only a marginal enhancement of class II levels (Fig. 11Go). This enhancement was essentially indistinguishable between the three groups (Fig. 11Go).



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  		FIGURE 11. Fab anti-mouse IgG and its TNP derivative do not differ in their ability to enhance MHC class II expression on activated B cells. Enriched sIgG+ B cells were cultured with varying doses of either Fab anti-mouse IgG, TNP-Fab, or a combination of TNP-Fab and 0.3 µM tyrphostin as described in Materials and Methods for a period of 48 h. Under these conditions, resting sIgD+ B cells yield a 5- to 10-fold enhancement in surface I-A levels on activation with anti-IgM. Following this, cells were washed and surface levels of I-A were determined by flow cytometry. Although the figure shows data obtained for treatment with 20 µg/ml Ab, essentially similar results were obtained over a dose range of 5–40 µg/ml. Histograms are for a gated B220+ population of cells. In both panels, the dashed line represents anti-I-A staining in unstimulated cells, whereas the thick line represents that for cells treated with either Fab (A) or TNP-Fab (B). In addition, the thin line in B depicts staining for cells treated with TNP-Fab plus tyrphostin. Data are from one of two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present report, we examined the basis of our earlier findings (42, 43) that the kinetics of Ag recognition by activated B cells can profoundly influence, both quantitatively (42) and qualitatively (43), the outcome of a cognate B-T interaction. Our findings that a pulse of G41CT3-primed B cells with peptide G28 rendered them more sensitive to peptide G41CT3-stimulated induction of IFN-{gamma} responses from CT3-primed T cells proved particularly revealing. We have shown earlier that peptide G28, but not its analogue of scrambled sequence, competed effectively with both peptides G28CT3 and G41CT3 for binding to G41CT3-primed B cells (42, 43). However, lacking a T cell epitope, it was unable to induce either proliferation (42) or cytokine responses (43) from CT3-primed T cells. In other words, peptide G28 binds G41CT3-primed B cells, but without leading to Ag presentation. Thus, the positive effect of a G28 pulse of G41CT3-primed T cells in potentiating IFN-{gamma} production from CT3-primed T cells clearly implies a regulatory role for BCR triggering as opposed to effects resulting from any alterations in Ag presentation. Additional evidence supporting the absence of a role for Ag presentation could also be gleaned from experiments with tyrphostin-treated B cells. Although tyrphostin inhibited BCR internalization and, consequently, Ag uptake it nevertheless promoted IFN-{gamma} responses from peptide G41CT3-stimulated cocultures. A notable effect of prepulsing B cells with G28 was that it eliminated the subsequent on-rate-dependent differences in IFN-{gamma} induction due to G41CT3 and G28CT3 challenge, rendering the former analogue as potent as the latter. These cumulative results, therefore, potentially localize the origin of these on-rate-dependent differences to derive from altered BCR activation.

An immediate consequence of BCR ligation by Ag is the initiation of the intracellular signal transduction machinery mediated, at least in part, by cellular PTKs (8, 9, 10). Although some of the intermediates involved have now been identified (8, 9, 10, 11), the biochemical pathways that eventually confer the "activated" phenotype to these B cells remains to be elucidated. Our initial observations that tyrphostin potentiates IFN-{gamma} induction from G41CT3-stimulated cultures, whereas genistein inhibits, was somewhat puzzling. Both of these compounds are known inhibitors of PTKs, and also block BCR internalization (48). However, some resolution of this paradox is afforded by our more recent results revealing that tyrphostin and genistein do not display identical specificities with respect to inhibition of intracellular, BCR-stimulated PTKs (K. Natarajan and K. V. S. Rao, unpublished results). Whereas BCR-stimulated tyrosine phosphorylation of syk was inhibited by both genestein and tyrphostin, that of phospholipase C{gamma} was inhibited only by genistein, but not by tyrphostin (K. Natarajan and K. V. S. Rao, unpublished results). Thus, the differences in results with genistein and tyrphostin, in terms of peptide-inducible IFN-{gamma} production, may reflect differences at the level of PTKs that eventually participate in BCR-initiated intracellular signaling. Consequently, whereas inhibition by genistein implicates the involvement of BCR-stimulated PTKs, results with tyrphostin suggest that selective modulation of PTKs recruited on BCR activation may influence the outcome.

Also significant were our findings that, in similarity with G28 pulsing, tyrphostin treatment of G41CT3-primed B cells also rendered peptide G41CT3 as competent as peptide G28CT3 at eliciting IFN-{gamma} responses from B-T cocultures. Further, there was no evidence for either synergistic, additive, or competitive influences when both treatments were simultaneously included. This implies, but does not confirm, that these two treatments probably act on a common biochemical pathway. These collective results may, therefore, be interpreted to suggest that the peptide on rate-mediated differences, which are initially exerted on relative BCR activation, translate into differences at the level of intracellular signals that are generated, at least with respect to PTK involvement.

That the BCR is in fact susceptible to Ag-regulated differential signaling has been recently demonstrated, albeit for T-independent responses (49). Using a panel of M13-phage-displayed peptide ligands with varying affinities for B cells bearing a transgenic sIg receptor, Kouskoff et al. (49) reported that BCR-mediated activation, as judged by a variety of parameters, is variably influenced by the affinity of Ag for BCR. Indeed some, but not all, of the early BCR-dependent signaling such as Ca2+ mobilization and tyrosine phosphorylation of Syk and IgA, but not Lyn, were highly dependent on affinity of BCR for peptide (49). These latter results are particularly significant in the present context. They reveal that the spectrum of PTKs recruited on BCR activation is dependent on its Ag-binding properties. This lends credence to our own argument, made above, that differences in the kinetics of Ag association by B cells may variably modulate intracellular signaling. Indeed, in their study, Kouskoff et al. have noted that the correlation between response and affinity of BCR for Ag was not perfect (49). They have consequently suggested that, rather than equilibrium affinity, the kinetics of BCR-ligand interaction may constitute the principal determinant of the outcome of activation responses (49).

Activation of T cells requires two signals to be provided by an APC. Whereas one derives from the peptide-MHC class II complex, a range of accessory cell surface molecules, also known as costimulatory molecules, provides the second. Of the latter, the most prominent of the currently identified interactions, particularly with respect to cytokine choices, is that between the B7 molecules on APC and CD28 on T cells (13, 34, 38). Although subject to debate (34, 38), it has been suggested that B7-1 directs Th1 responses whereas B7-2 promotes a bias in favor of Th2 responses (35). In the context of B-T interactions, Agrewala et al. (52) have recently demonstrated that production of IFN-{gamma} by T cells was dependent on the presence of B7-1 on B cells. As shown, the present system examined here was also subject to a similar categorization. Thus, whereas anti-B7-2 blocked IL-10 production from peptide-stimulated cocultures of G41CT3-activated B cells and CT3-primed T cells, anti-B7-1 selectively abrogated IFN-{gamma} responses. Interestingly, results from such experiments also suggested that differences in on rates of peptide G28CT3 and G41CT3 association with G41CT3-activated B cells are eventually exerted at the level of costimulation resulting from B7-CD28 interactions. Two lines of evidence supported such an inference. First was the fact that inclusion of anti-CD28 in the cocultures rendered the efficiency of peptide G41CT3 comparable with that of peptide G28CT3 with respect to elicitation of T-dependent IFN-{gamma}. Parity was established not only in terms of peak IFN-{gamma} levels induced but also at the level of the respective dose-response profiles. This influence of anti-CD28 in establishing equipotency between the two peptides implies that differences in peptide-B cell association rates eventually reflect at the level of CD28 activation on T cells. Indirect substantiation for such an inference was also afforded by our findings that the potentiatory effect of G28, vis-a-vis G41CT3-inducible IFN-{gamma} secretion, could be neutralized by treatment of B cells with anti-B7-1. Thus, collectively, these results strongly suggested the possibility that differences in BCR-ligand association rates may eventually regulate at the level of relative involvement of B7-1 vs B7-2 in CD28 engagement, leading to altered T cell-elicited cytokine profiles. In this connection, it has been shown that binding of a T-dependent Ag to activated B cells eventually leads to up-regulation, albeit with different kinetics, of the two B7 molecules on the cell surface (12, 13). Thus, our deduction that binding properties of Ag with BCR could variably influence at the level of costimulation was not altogether an improbable one.

Although the low frequency of Ag-specific B cells in splenocytes precluded a direct verification of our inference above we could, nevertheless, demonstrate "proof of principle" by comparing between Fab anti-IgG and its TNP derivative as surrogate Ags for sIgG+ splenic B cells. Whereas the native Fab was fully competent at inducing enhanced cell surface expression of both B7-1 and B7-2, its TNP-derivatized analogue proved significantly less effective at inducing B7-1 although B7-2 levels remained unchanged. In light of the fact that these two Ab preparations principally differ in their rates of association with IgG, these results confirm the existence of differential BCR-triggering thresholds for induction of B7-1 and B7-2, with the former being highly sensitive to BCR-Ag association rates. Although modest, the positive effect of tyrphostin treatment on B7-1 levels in B-cells activated with TNP-derivatized Fab also supports our earlier inference of on-rate-dependent differences in engagement of intracellular PTKs. Finally, this system also permitted a direct verification of our proposal that BCR-Ag association rates directly regulate expression of B7-1 on B cells which, in turn, impacts on Th subset recruitment.

The intracellular biochemical pathways, which mediate between BCR activation and B7 expression are currently unknown and await delineation. Also of interest would be a resolution of how Ag-binding rates modulate BCR-initiated signaling. Here, one plausible explanation derives from the "strength of signal" hypothesis proposed for T cell activation (13). At high on rates of association, it can be anticipated that, at any given time, a large proportion of BCRs would be ligand occupied. This would result in simultaneous triggering of multiple BCRs, generating a high intensity intracellular signal. In contrast, low on rates would also imply low BCR occupancy rates, leading to the generation of a low intensity signal. Again in similarity with the proposal for T cells (13), the intensity of intracellular signal produced may dictate different outcomes, particularly in the present context, with respect to B7 regulation. Based on our prior estimates of peptides G41CT3 and G28CT3 binding to anti-G41CT3 mAbs (43), it would appear, at least in the context of G41CT3-activated B cells, that kassoc values of >106 M-1 s-1 are necessary to elicit significant levels of Th1-type responses from CT3-primed T cells. Needless to stress, however, that this is only a hypothesis and requires experimental verification. Also, potential contributions from a possible effect of binding on rates on intracellular Ag-targeting pathways, for subsequent processing and presentation, may also have to be considered. Nonetheless, our results do emphasize the important role played by Ag-binding properties of activated B cells in influencing the outcome of a T-dependent response.


   Footnotes
 
1 The contributions of L.V. and K.N. should be considered equal. Back

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. E-mail address: Back

3 Abbreviations used in this paper: BCR, B cell Ag receptor; sIg, membrane-bound surface Ig; PTK, protein tyrosine kinase; TNP, trinitrophenyl. Back

Received for publication September 29, 1999. Accepted for publication March 14, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Chestnut, R. W., H. M. Grey. 1981. Studies on the capacity of B cells to serve as antigen presenting cells. J. Immunol. 126:1075.[Medline]
  2. Janeway, C. A., J. Ron, M. E. Katz. 1987. The B cell is the initiating antigen presenting cell in the lymph nodes. J. Immunol. 138:1051.[Abstract/Free Full Text]
  3. Hayglass, K. T., S. J. Naides, C. F. Scott, B. Benacerraf, M. S. Sy. 1986. T cell developement in B cell deficient mice. IV. The role of B cells as antigen presenting in vivo. J. Immunol. 136:823.[Abstract]
  4. Jelachich, M. L., E. K. Lakey, L. Casten, S. K. Pierce. 1986. Antigen presentation is a function of all B cell subpopulations separated on the basis of size. Eur. J. Immunol. 16:411.[Medline]
  5. Lanzavecchia, A.. 1985. Antigen-specific interactions between T and B cells. Nature 314:537.[Medline]
  6. Clark, E. A., J. A. Ledbetter. 1994. How B and T cells talk to each other. Nature 367:425.[Medline]
  7. Venkitaraman, A. R., G.T. Williams, P. Dariaich, M. S. Neuberger. 1991. The B-cell antigen receptor of the five immunoglobulin classes. Nature 352:777.[Medline]
  8. Cambier, J. C., C. M. Pleiman, M. R. Clark. 1994. Signal transduction by the B cell antigen receptor and its coreceptors. Annu. Rev. Immunol. 12:457.[Medline]
  9. Alberola-Ila, J., S. Takaki, J. D. Kerner, R. M. Perlmutter. 1997. Differential signaling by lymphocyte antigen receptors. Annu. Rev. Immunol. 15:125.[Medline]
  10. Reth, M., J. Weinands. 1997. Initiation of processing of signals from the B cell antigen receptor. Annu. Rev. Immunol. 15:453.[Medline]
  11. Fu, C., C. W. Turck, T. Kurosaki, A. W. Chan. 1998. BLNK: a central linker protein in B cell activation. Immunity 9:93.[Medline]
  12. Lenschow, D. J., A. I. Sperling, M. P. Cooke, G. Freeman, L. Rhee, D. C. Decker, G. Gray, L. M. Nadler, C. C. Goodnow, J. A. Bluestone. 1994. Differential up-regulation of the B7-1 and B7-2 costimulatory molecules after Ig receptor engagement. J. Immunol. 153:1990.[Abstract]
  13. Lenschow, D. J., T. L. Walunas, J. A. Bluestone. 1996. CD28/B7 system of T cell costimulation. Annu. Rev. Immunol. 14:233.[Medline]
  14. Lanzavecchia, A.. 1990. Receptor-mediated antigen uptake and its effect on antigen presentation to class II-restricted T lymphocytes. Annu. Rev. Immunol. 8:773.[Medline]
  15. Watts, C.. 1997. Capture and processing of exogenous antigens for presentation on MHC molecules. Annu. Rev. Immunl. 15:821.[Medline]
  16. Drake, J. R., P. Webster, J. C. Cambier, I. Mellman. 1997. Delivery of B cell receptor-internalized antigen to endosomes and class II vesicles. J. Exp. Med. 186:1299.[Abstract/Free Full Text]
  17. Siemasko, K., B. J. Eisfelder, E. Williamson, S. Kabak, M. R. Cark. 1998. Signals from the B lymphocyte antigen receptor regulated MHC class II containing late endosomes. J. Immunol. 160:5203.[Abstract/Free Full Text]
  18. Schwartz, R. H., D. L. Meulleer, M. K. Jenkins, H. Quill. 1989. T-cell clonal anergy. Cold Spring Harbor Symp. Quant. Biol. 54:605.
  19. Schwartz, R. H.. 1992. Costimulation of T lymphocytes: the role of CD28, CTLA-4, and B7/BB1 in interleukin-2 production and immunotherapy. Cell 71:1085.
  20. Goodnow, C. C.. 1996. Balancing immunity and tolerance: deleting and tuning lymphocyte repertoires. Proc. Natl. Acad. Sci. USA 93:2264.[Abstract/Free Full Text]
  21. Chang, T. L., M. Shea, S. Urioste, R. C. Thompson, W. H. Bloom, A. K. Abbas. 1990. Heterogeneity of helper/inducer T lymphocytes. III. Response of IL-2 and IL-4 producing (Th1 and Th2) clones to antigens presented by different accessory cells. J. Immunol. 145:2803.[Abstract]
  22. Noelle, R. J., J. Daum, W. C. Bartlett, D. M. Shepherd. 1991. Cognate interactions between helper T cells and B cells. V. Reconstitution of T helper/inducer cell function using purified plasma membranes from activated Th1 and Th2 cells and lymphokines. J. Immunol. 146:1118.[Abstract]
  23. Hodgkin, P.A., A. Basten. 1995. B cell activation, tolerance and antigen presenting functions. Curr. Opin. Immunol. 7:121.[Medline]
  24. Gajewski, T. F., M. Pinnas, T. Wong, F. W. Fitch. 1991. Murine Th1 and Th2 clones proliferate optimally in response to distinct antigen presenting cell populations. J. Immunol. 146:1750.[Abstract]
  25. Saoudi, A., S. Simmonds, I. Huitinga, D. Mason. 1995. Prevention of experimental allergic encephalomyelitis in rats by targeting auto-antigen to B cells: evidence that the protective mechanism depends on changes in cytokine responses and migrating properties of auto-antigen specific to cells. J. Exp. Med. 182:335.[Abstract/Free Full Text]
  26. Stockinger, B., T. Zal, A. Zal, D. Gray. 1996. B cells solicit their own help from T cells. J. Exp. Med. 183:891.[Abstract/Free Full Text]
  27. Murray, J. S., C. Pfieffer, J. Madri, K. Bottomly. 1992. Major histocompatibility complex (MHC) control of CD4 T cell subset activation. II. A single peptide induces either humoral or cell-mediated responses in mice of distinct genotypes. Eur. J. Immunol. 22:559.[Medline]
  28. Rogers, P. R., G. Huston, S. L. Swain. 1998. High antigen density and IL-2 are required for generation of CD4 effectors secreting Th1 rather than Th0 cytokines. J. Immunol. 161:3844.[Abstract/Free Full Text]
  29. Tao, X., S. Constant, P. Jorritsma, K. Bottomly. 1997. Strength of TCR signal determines the costimulatory requirements for Th1 and Th2 CD4 T cell differentiation. J. Immunol. 159:5956.[Abstract]
  30. Constant, S. L., K. Bottomly. 1997. Induction of Th1 and Th2 CD4 T cell responses: the alternative approaches. Annu. Rev. Immunol. 15:297.[Medline]
  31. Shaw, A. S., M. L. Dustin. 1997. Making the T cell receptor go the distance: a topological view of T cell activation. Immunity 6:361.[Medline]
  32. Kundig, T. M., A. Shahinian, K. Kawai, H.-W. Mittrucker, E. Sebzda, M. F. Bachmann, T. W. Mak, P. S. Ohashi. 1996. Duration of TCR stimulation determines costimulatory requirements of T cells. Immunity 5:41.[Medline]
  33. Lezzi, G., K. Karjalainen, A. Lanzavecchia. 1998. The duration of antigenic stimulation determines the fate of naive and effector T cells. Immunity 8:89.[Medline]
  34. Bluestone, J. A.. 1995. New perspectives of CD28–B7-mediated T cell costimulation. Immunity 2:555.[Medline]
  35. Kuchroo, V. K., M. P. Das, J. A. Brown, A. M. Ranger, S. S. Zamvil, R. A. Sobel, H. L. Weiner, N. Nabavi, L. H. Glimcher. 1995. B7-1 and B7-2 costimulatory molecules activate differentially the Th1/Th2 developmental pathways: application to autoimmune disease therapy. Cell 80:707.[Medline]
  36. Borrieolo, F., M. P. Sethna, S. D. Boyd, A. N. Schweitzer, E. A. Tivol, D. Jacoby, T. B. Strom, E. M. Simpson, G. J. Freeman, A. H. Sharpe. 1997. B7-1 and B7-2 have overlapping, critical roles in immunoglobulin class switching and germinal center formation. Immunity 6:303.[Medline]
  37. Schweiter, A. N., A. H. Sharpe. 1998. Studies using antigen presenting cells lacking expression of both B7-1 (CD80) and B7-2 (CD86) show distinct requirements for B7 molecules during priming versus restimulation of Th2 but not Th1 cytokine production. J. Immunol. 161:2762.[Abstract/Free Full Text]
  38. McAdam, A. J., A. N. Schweitzer, A. H. Sharpe. 1998. The role of B7 costimulation in activation and differentiation of CD4 and CD8 T cells. Immunol. Rev. 165:231.[Medline]
  39. O’Garra, A.. 1998. Cytokines induce the development of functionally heterogenous T helper subsets. Immunity 8:275.[Medline]
  40. Murray, J. S., J. P. Kasselman, L. T. Schountz. 1995. High density antigen presentation of an immunodominant minimal peptide on B cells is MHC-linked to Th1 like immunity. Cell. Immunol. 166:9.[Medline]
  41. Pasare, C., V. Morafo, M. Entringer, P. Bansal, A. George, V. Bal, S. Rath, J. M. Durdik. 1998. Presence of activated-binding B cells during immunization enhances relative levels of IFN-{gamma} in T cell responses. J. Immunol. 160:778.[Abstract/Free Full Text]
  42. Vijayakrishnan, L., S. Sarkear, R. P. Roy, K. V. S. Rao. 1997. B cell responses to a peptide epitope. IV. Subtle sequence changes in flanking residues modulate immungenicity. J. Immunol. 159:1809.[Abstract]
  43. Vijayakrishnan, L., V. Manivel, K. V. S. Rao. 1998. B cell responses to a peptide epitope. VI. The kinetics of antigen recognition modulates B cell-mediated recruitment of T helper subsets. J. Immunol. 161:4661.[Abstract/Free Full Text]
  44. Agarwal, A., B. P. Nayak, K. V. S. Rao. 1998. B cell responses to a peptide epitope. VII. Antigen-dependent modulation of the germinal center reaction. J. Immunol. 161:5832.[Abstract/Free Full Text]
  45. Levitzki, A., A. Gazit, N. Osherov, I. Posner, C. Gillon. 1991. Inhibition of protein-tyrosine kinases by tyrophostins. Methods Enzymol. 201:345.
  46. Akiyama, T., H. Ogawara. 1991. Use and specificity of genistein as inhibitor of protein tyrosine kinases. Methods Enzymol. 201:360.
  47. Hidaka, H., M. Watanabe, R. Kobayashi. 1991. Properties and use of H-series compounds as protein kinase inhibitors. Methods Enzymol. 201:328.[Medline]
  48. Pure, E., L. Tardelli. 1991. 2. Tyrosine phosphorylation is required for ligand-induced internalization of the antigen receptor on B lymphocytes. Proc. Natl. Acad. Sci. USA 89:114.[Abstract/Free Full Text]
  49. Kouskoff, V., S. Famiglietti, G. Lacaud, P. Lang, J. E. Rider, B. K. Kay, J. C. Cambier, D. Nemazee. 1998. Antigens varying in affinity for the B cell receptor induce differential B lymphocyte responses. J. Exp. Med. 188:1453.[Abstract/Free Full Text]
  50. Ward, S. G.. 1996. CD28: a signaling perspective. Biochem. J. 318:361.
  51. Thompson, C. B., J. P. Allison. 1997. The emerging role of CTLA-4 as an immune attenuator. Immunity 7:445.[Medline]
  52. Agrewala, J. N., S. Suvas, R. K. Verma, G. C. Mishra. 1998. Differential effect of anti-B7-1 and anti-M150 antibodies in restricting delivery of costimulatory signals form B cells and macrophages. J. Immunol. 160:1067.[Abstract/Free Full Text]
  53. George, A. J. T., R. R. French, M. J. Glennie. 1995. Measurement of kinetic binding constants of a panel of anti-saporin antibodies using a resonant mirror biosensor. J. Immunol. Methods 183:51.[Medline]



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