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The Journal of Immunology, 2002, 168: 2676-2682.
Copyright © 2002 by The American Association of Immunologists

Cross-Linking Surface Ig Delays CD40 Ligand- and IL-4-Induced B Cell Ig Class Switching and Reveals Evidence for Independent Regulation of B Cell Proliferation and Differentiation1

James S. Rush2, Jhagvaral Hasbold3 and Philip D. Hodgkin3

Immune Regulation Group, Medical Foundation of the University of Sydney, Centenary Institute of Cell Biology and Cancer Research, Sydney, Australia.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
T cells stimulate B cells to divide and differentiate by providing activating signals in the form of inducible membrane-bound molecules and secreted cytokines. Provision of these signals in vitro reproduces many of the consequences of T-B collaboration in the absence of any form of Ag stimulation. Although clearly not obligatory, Ag signals appear to play an important regulatory role in numerous aspects of the B cell response. To examine directly the effect of an Ag signal, naive B cells were stimulated in the presence of rCD40 ligand, with or without IL-4 in the presence or absence of different anti-Ig mAbs. Anti-Ig mAbs exerted variable effects on the B cell division rate, from enhancement to no effect to inhibition. In contrast, all anti-Ig mAbs tested inhibited division-linked isotype switching to IgG1 and IgE. Thus, B cell Ag receptor ligands could modify the rates of B cell expansion and class switching independently. The ability of anti-Ig reagents to modify class switching suggests the B cell Ag receptor may play an important role in the selection of Ig isotypes during T cell-dependent humoral immune responses to Ags of different physical structure.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The molecular and cellular interactions that occur between T cells and B cells to trigger T cell-dependent humoral immune responses are now relatively well understood (reviewed in Ref. 1). T cells provide two forms of help: 1) cell contact-dependent receptor engagement, and 2) secretion of soluble regulatory cytokines (2, 3, 4). In particular, the interaction of CD40L expressed on the surface of activated T cells (5, 6) with CD40 on the B cell surface can stimulate B cell division, while the provision of cytokines such as IL-4 can enhance B cell proliferation and stimulate Ig class switching (to IgG1 and IgE) and Ab secretion (7, 8, 9, 10). Other cytokines such as IFN-{gamma} and TGF-{beta} can stimulate B cell class switching to the isotypes IgG2a and IgA, respectively (11, 12, 13).

Numerous studies in vitro have demonstrated that CD40L and cytokines delivered to the B cell in the absence of any Ag signal are sufficient to stimulate B cells to divide, class switch, and secrete Ab (reviewed in Ref. 14). Nevertheless, anti-Ig mAbs and specific Ag deliver potent activating signals to B cells, thereby affecting cell viability and the expression of important activation molecules including CD40, B7 molecules, and MHC class II (15, 16). These results suggest that the Ag signal may play an important regulatory role in the rate of T cell-B cell interaction and subsequent division. In contrast, there is limited and somewhat contradictory evidence investigating the ability of anti-Ig reagents to affect both the surface expression and secretion of IgG1 and IgE by B cells in response to T cells or activated T cell products. For example, in one study, B cells secreted more IgG1 and IgM in the presence of activated Th2 clones and the potent surface membrane Ig (sIg)3 stimulus anti-{delta}-dextran than the no anti-Ig control cultures (17). Despite increased secretion of IgG1, the proportion of sIgG1+ cells in these cultures was reduced. In a separate study, the secretion of IgG1 and IgE by B cells stimulated with CD40L and IL-4 was increased by the addition of either anti-{delta}-dextran or anti-µ-dextran (18). However, in contrast to the aforementioned study, the proportion of IgG1+ B cells was increased slightly in cultures containing anti-{delta}-dextran together with CD40L and IL-4. Nevertheless, despite variable findings in studies investigating the effects of an Ag signal on B cell isotype switching and Ig secretion, it is clear that sIg signals are able to affect the differentiation of B cells receiving T cell help.

Recently, fluorescent division-tracking methods using flow cytometry (19, 20) have identified the importance of division number in regulating Ig class switching (10, 21, 22, 23). It was found that Ig switching to all isotypes followed a predictable division-based rate of change that was independent of the time spent in culture. When coupled with quantitative methods for assessing the total number of cells in culture, a useful estimate of cell viability and the rate of proliferation can also be measured (24, 25, 26). In this study, we have used division number as a reference framework for monitoring the effect of anti-Ig reagents on isotype switching, differentiation, and proliferation rate of B cells when stimulated by rCD40 ligand (rCD40L) and IL-4 in vitro. The results revealed that sIg signals delivered by cross-linking the surface receptor with anti-Ig reagents delayed isotype switching, suggesting a novel mechanism by which multivalent Ags might regulate the Ab response.


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

Male and female CBA/H inbred mice between 8- and 12-wk old were used for all experiments, and were purchased from the Animal Resource Center (Canning Vale, Perth, Australia) and maintained under specific pathogen-free conditions.

Reagents and Abs

Cell membranes expressing mouse CD40L were prepared, as previously described (27), from the Sf9 insect cell line infected by baculovirus vector containing a murine CD40L cDNA (a gift from M. R. Kehry; (Boehringer-Ingelheim, Danbury, CT). Mouse rIL-4 was a gift from R. Kastelein (DNAX, Palo Alto, CA). The anti-mouse IgD hybridoma 1.19 was provided by G. Klaus (NIMR, Mill Hill, U.K.). Anti-mouse Fc{gamma}RIIb mAb (2.4G2) (28), anti-{kappa} (187.1) (29), anti-mouse CD3 (2C11) (30), anti-mouse IgE (R1E4) (31), anti-mouse IgM (RS3.1) (32), and two anti-mouse IgM mAbs 331.12 (33) and Bet-2 (34) were purified from hybridoma supernatant by protein G affinity chromatography (Pharmacia-LKB, Uppsala, Sweden). Unlabeled and biotin-conjugated goat anti-mouse IgG1 Abs were purchased from Southern Biotechnology Associates (Birmingham, AL). Unlabeled goat anti-mouse IgG1 Abs were conjugated to PE by using succinimidyl 4-(p-maleimidophenyl) butyrate (Pierce, Rockford, IL), according to the manufacturer’s instructions. Streptavidin-Tricolor (SA-TC) was purchased from Caltag Laboratories (Burlingame, CA). Fluorochrome or biotinylated anti-mouse L-selectin, anti-mouse B220, anti-mouse B7-2, anti-mouse CD40L (MR1) (5), anti-mouse MHC class II, and isotype control mAbs were obtained from BD PharMingen (San Diego, CA). Purified Abs were biotinylated using normal human serum biotin (Sigma-Aldrich, St. Louis, MO).

Cell preparation and in vitro culture

Small resting B cells were prepared, as described previously (35), and were routinely >90% B220 and IgD positive and <1% CD4 and CD8 positive, as determined by FACS. Freshly isolated B cells were cultured in B cell medium consisting of RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with 2 mM L-glutamine, 0.1 mM nonessential amino acids, 10 mM HEPES (pH 7.4), 100 µg/ml streptomycin, 60 µg/ml penicillin, 5 x 10-5 M 2-ME (all from Sigma-Aldrich), 1 mM Na-pyruvate (Life Technologies), and 10% heat-inactivated FCS (CSL, Victoria, Australia). Unless otherwise stated, B cells were cultured at 2 x 105 cells/ml in 1- or 0.2-ml volumes in 24- or 96-well, flat-bottom tissue culture plates (Falcon; BD Biosciences, San Jose, CA) at 37°C in a humidified atmosphere of 5% CO2.

Cell proliferation assay

B cell division measured by [3H]TdR ([methyl-3H]; ICN Pharmaceuticals, Irvine, CA). B cell cultures were pulsed for 4 h with 1 µCi/ml [3H]TdR, and scintillation counting was performed on a betaplate counter (Pharmacia-LKB). Results are expressed as a mean counts per minute and standard error of triplicate cultures.

Cell division tracking

Resting B cells were labeled with CFSE (Molecular Probes, Eugene, OR), according to the original method described elsewhere (19, 20). A 5-mM stock solution of CFSE was prepared by dissolving in DMSO, and was stored at -20°C. Before labeling, B cells were washed and resuspended at 1 x 107 cells/ml in PBS containing 0.1% BSA. The CFSE stock was diluted 1/10 in PBS/BSA, and 20 µl was added to 1 ml cell suspension (to a final concentration of 10 µM). Cells were incubated for 10 min at 37°C, then were diluted with cold PBS/BSA solution before being spun down and resuspended in B cell medium.

Surface staining and flow cytometry

Cultured B cells were harvested at various times and washed twice with PBS/BSA. Cells were stained in 96-well V-bottom plates (Falcon; BD Biosciences) with specific mAbs. Biotinylated mAbs were detected using SA-TC (Caltag Laboratories). Abs and SA-TC were diluted in PBS/0.1% BSA/0.1% NaN3 (PBA) containing 1% normal rat serum. All incubations were conducted on ice, and after each step, cells were washed twice with 200 µl cold PBA. Stained cells transferred to round-bottom tubes (Falcon; BD Biosciences) for flow cytometry analysis using CellQuest software (BD Biosciences) on a FACScan flow cytometer (BD Biosciences).

Total cell number determination

Total cell numbers in culture were determined by flow cytometry by reference to a known number of CaliBRITE beads (BD Biosciences), as described previously (21, 25, 26). Typically, 1 x 104 beads were added to each cell culture immediately before harvest, and the bead and live cell events were discriminated using orthogonal and forward scatter parameters. The cell numbers in culture were calculated from the ratio of beads to live cell events. The number of cells in each division was calculated by estimating the proportion of cells in each division following curve-fitting analysis of each CFSE profile using Profit software (QuantumSoft, Zurich, Switzerland), and by multiplying the proportion of cells in each division, relative to the entire CFSE division profile, by the total cell number.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of anti-IgD mAb on rCD40L-induced B cell division

Anti-Ig reagents have been used to stimulate the Ag signal received by a B cell via its B cell receptor; however, analysis of the effect of anti-Ig mAbs on switching is complicated by the observation that some anti-IgM and anti-IgD mAbs promote B cell division that is further enhanced by the presence of IL-4 (36, 37). Thus, this raises the possibility that an Ag signal could exert variable effects on B cell differentiation by affecting rates of B cell division and/or survival. In this study, we set out to use quantitative division-tracking methods to assess the effect of anti-Ig reagents on B cell activation using division number as a reference for monitoring the effect of anti-Ig on rates of cell proliferation and division-based switching.

To investigate the effect of a representative agonistic anti-IgD mAb (1.19) on rCD40L-induced B cell division, CFSE-labeled naive B cells were stimulated using rCD40L with or without IL-4 in the presence or absence of 1.19 mAb. At days 2, 3, and 4, the total number of viable cells in culture and the absolute number of cells per division were calculated (Fig. 1Go). In combination with IL-4, 1.19 induced significant cell division. Furthermore, this anti-IgD mAb greatly enhanced B cell division stimulated using rCD40L only, leading to a 7- to 8-fold increase in total viable cell numbers in culture by days 3 and 4 (Fig. 1Go). Additionally, the distribution of cells within each division revealed that rCD40L- and anti-IgD-stimulated B cells had undergone approximately one to two divisions more than the rCD40L-only control (Fig. 1Go). Anti-IgD mAb also stimulated an increase in B cell numbers in rCD40L and IL-4 cultures, although the proportional increase in cell number was not as great as cultures containing rCD40L only, with 3-fold higher viable cell numbers by day 3 (Fig. 1Go). Cell numbers began to decline in cultures containing rCD40L, IL-4, and anti-IgD mAb after day 3, and B cells slowed in their progression through division due to nutrient depletion of culture medium (not shown).



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  		FIGURE 1. Anti-IgD mAb enhances rCD40L-induced B cell division. Naive CFSE-labeled B cells were stimulated using rCD40L (1/1000) with or without IL-4 (100 U/ml), or using IL-4 only. Selected cultures were supplemented with 40 µg/ml anti-IgD. Division profiles were acquired at days 2, 3, and 4, and were constructed from the CFSE profiles of viable B cell populations analyzed using Profit, as described in Materials and Methods. The activating stimulus and culture duration are indicated, and the presence or absence of anti-IgD is shown by filled and open squares, respectively. Graphs illustrate the mean and standard error of absolute cell numbers vs division number derived from triplicate cultures (except IL-4-only cultures). Culture replicates often yielded similar cell number values, and thus small standard errors, which are present, but obscured by graphical symbols. Note that the y-axes of some cell division plots are different to enable a meaningful comparison of cell number and division distribution in cultures under different stimulation conditions. Data shown are representative of two experiments.

 
Anti-IgD mAb-mediated enhancement of rCD40L-induced B cell division is not due to B cell-derived CD40L

The above data suggested that anti-IgD mAb could enhance rCD40L-induced B cell proliferation by generating more cells in each division at each harvest time. A mechanism by which anti-IgD mAb may mediate this enhancement could be a result of it directly stimulating B cell proliferation following sIg cross-linking by providing additional activating signals distinct from CD40L and IL-4. Recently, an alternative explanation was suggested for the effect of anti-Ig stimulation. It was proposed that murine B cells synthesize and secrete autocrine CD40L upon stimulation with anti-Ig mAbs (38). To assess whether B cell-derived CD40L was responsible for the enhancement of cell division by the 1.19 Ab, naive B cell cultures containing anti-IgD mAb with or without IL-4 were cultured for 2 days with or without a range of concentrations of the anti-CD40L hamster mAb MR1 (Fig. 2GoA). Control cultures consisted of rCD40L and/or IL-4 cultures with or without MR1, or the anti-hamster mAb 2C11 possessing the irrelevant specificity for CD3 (Fig. 2GoB). The results indicated that 1.19-induced B cell proliferation was not affected by the addition of MR1 at concentrations that completely abrogated rCD40L-induced B cell division in control cultures. This suggested that the mechanism of 1.19-induced enhancement of cell division was not due to the release of B cell-derived CD40L. There was no inhibition of rCD40L-induced proliferation in control cultures containing the control anti-hamster Ab 2C11 (Fig. 2GoB).



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  		FIGURE 2. B cell-derived CD40L does not affect the enhancement of rCD40L-induced B cell proliferation by anti-IgD. A, Naive B cells were stimulated using 10 µg/ml anti-IgD in the presence or absence of IL-4 (100 U/ml). Selected cultures were supplemented with a 2-fold dose titration of anti-CD40L (MR1) from 80 µg/ml over two orders of magnitude. Cell proliferation was assessed by thymidine incorporation at day 2. B cells stimulated using anti-IgD with or without IL-4 are represented by circular and square symbols, respectively. Open symbols indicate cultures containing anti-CD40L mAb, whereas the filled symbols represent the no anti-CD40L controls. Results are presented as the mean ± SE of triplicate cultures, and are representative of two separate experiments. B, B cells were stimulated using 1/1000 rCD40L with or without 100 U/ml IL-4. Selected cultures were supplemented with a 2-fold dose titration of either anti-CD40L mAb or an isotype-matched anti-hamster mAb (2C11) of irrelevant specificity from 80 µg/ml over two orders of magnitude. Square symbols indicate B cells stimulated with rCD40L alone, whereas cultures containing rCD40L and IL-4 are represented by circles. Open symbols indicate cultures containing the dose titration of either anti-CD40L (left graph) or 2C11 (right graph), and the filled symbols represent the no anti-CD40L or anti-CD3 mAb controls. Results are presented as the mean ± SE of triplicate cultures, and are representative of two separate experiments.

 
Anti-IgD mAb delays division-linked isotype switching to IgG1 and IgE

Activating signals provided by CD40L and IL-4 have been shown to induce naive B cells to class switch to IgG1 and IgE (5, 6, 9, 10, 39) in a manner that increases with cell division number while remaining independent of culture duration (10, 22). This behavior has allowed us to use division number as a reference for assessing the effect of various stimuli on the rate of isotype switching independently of any simultaneous effect on division rate. To examine the effect of 1.19 on isotype switching, CFSE-labeled B cells were stimulated using rCD40L in the presence or absence of IL-4, with selected cultures receiving anti-IgD mAb. Cells were harvested on days 3 and 4 and were stained for expression of surface IgG1 and IgE, and the proportion of switched cells was determined per division. Fig. 3Go indicates that anti-IgD mAb delayed the isotype switch to IgG1 by two to three divisions. Furthermore, the presence of anti-IgD in rCD40L and IL-4 cultures also provoked a delay in isotype switching to IgE (one to two divisions), although the degree of inhibition was not as great as that observed with IgG1 (Fig. 3GoA). The delay in isotype switching to IgG1 and IgE was independent of culture duration (Fig. 3GoA), but was dependent on the concentration of anti-IgD mAb (Fig. 3GoB, IgE not shown). Interestingly, anti-IgD dose-response curves were different for division and differentiation, as concentrations of anti-Ig stimulating maximal delay of switching did not optimally enhance rCD40L-induced B cell division. For example, doses of anti-IgD of 40 µg/ml optimally enhanced rCD40L-induced B cell division (Fig. 1Go and data not shown), whereas maximal delay of switching was observed at anti-IgD concentrations of 2.5 µg/ml and above (Fig. 3GoB). Fig. 3GoB also shows that B cells failed to switch to IgG1 in cultures containing only rCD40L and anti-IgD, and despite dividing up to five times, B cells in anti-IgD and IL-4 cultures displayed no evidence of IgG1 acquisition. Furthermore, the anti-IgD-dependent inhibition of IgG1 switching was not due to coligation of Fc{gamma}RIIb, as the inclusion of an Fc{gamma}RIIb-blocking mAb had no effect on the delay of class switching to IgG1 (not shown).



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  		FIGURE 3. Anti-IgD delays the IL-4-dependent isotype switch to IgG1 and IgE. A, Naive, CFSE-labeled B cells were cultured with 1/1000 rCD40L and 100 U/ml IL-4, and selected cultures were also supplemented with 10 µg/ml anti-IgD mAb. At day 3, 4, or 5, B cells were harvested and examined for expression of surface IgG1 and IgE. The differentiation plots show the percentage of positive IgG1 or IgE cells per division on a linear scale and are compared with the proportion of live cells within each division at each time point. Square, circle, and triangle symbols indicate day 3, 4, or 5, and cultures with or without supplementary anti-IgD mAb are shown as filled or open symbols, respectively. Data shown are representative of three independent experiments. B, The anti-IgD-induced delay in isotype switching to IgG1 is concentration dependent. Naive, CFSE-labeled B cells were cultured with 100 U/ml IL-4, 1/1000 rCD40L, or rCD40L and IL-4, and selected cultures were supplemented with one of three doses of anti-IgD mAb (10, 2.5, or 0.5 µg/ml). At day 4, B cells were harvested and stained for surface IgG1 expression. Each differentiation plot shows the percentage of IgG1+ cells per division. Open squares, diamonds, and circles represent the three concentrations of anti-IgD mAb used, with the filled triangles illustrating the no anti-IgD control. Data shown are representative of two independent experiments.

 
Other anti-Ig mAbs have variable effects on CD40L-induced B cell division, but all delay isotype switching to IgG1 and IgE

It was of interest to determine whether other anti-Ig reagents were also able to inhibit the division-based rate of isotype switching. Two other anti-Ig mAbs directed to surface IgM (Bet-2 and 331.1) and an anti-{kappa} (187.1) were examined. Cells cultured with rCD40L, IL-4, and various concentrations of each Ab were harvested on day 3 and stained for surface IgG1. Fig. 4Go, A and B, indicates that all anti-Ig reagents tested delayed division-linked isotype switching to IgG1 in a concentration-dependent manner. Thus, the delay in class switching induced by 1.19 was not unique to that Ab. Furthermore, repeat experiments indicated that the delay in switching induced by anti-IgD and anti-{kappa} was highly reproducible (Fig. 4GoB). Interestingly, the degree of class switch inhibition differed depending on the anti-Ig mAb, with the anti-IgM reagents stimulating a more profound delay in the IgG1 isotype switch than anti-{kappa} (Fig. 4GoA). In a separate experiment, anti-{kappa} and anti-IgM (331.12) also delayed the isotype switch to IgE to a similar extent to that observed using anti-IgD (Fig. 4GoC).



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  		FIGURE 4. Other anti-Ig mAbs inhibit the division-dependent isotype switch to IgG1 and IgE. A, Naive, CFSE-labeled B cells were cultured with 1/1000 rCD40L and 100 U/ml IL-4. Selected cultures were supplemented with one of three doses of anti-{kappa}, anti-IgM (331.12), or anti-IgM (Bet-2) (10, 0.1, or 0.001 µg/ml). At day 3, B cells were harvested, and their levels of surface IgG1 were examined. Each differentiation plot shows the percentage of positive IgG1 cells per division. Open squares, circles, and triangles represent the three concentrations of anti-Ig, and the filled squares illustrate the no anti-Ig control. B, Naive, CFSE-labeled B cells were cultured with 1/1000 rCD40L and IL-4 (100 U/ml) (filled squares), with selected cultures supplemented with either anti-{kappa} (open triangles) or anti-IgD (open circles) at 10 µg/ml. Surface IgG1 expression was measured by FACS at day 4. Data shown represent the mean and SE of three independent experiments. C, Naive CFSE-labeled B cells were cultured with 1/1000 rCD40L and IL-4 (100 U/ml). Selected cultures were supplemented with either anti-{kappa}, anti-IgM (331.12), or anti-IgD at 10 µg/ml. At days 3.5 and 4, B cells were harvested and stained for intracellular IgE expression. Each differentiation plot shows the percentage of IgE-positive cells per division at each harvest time in cultures supplemented with different anti-Ig mAbs. Open circles, triangles, and diamonds represent anti-IgD, anti-{kappa}, or anti-IgM, respectively, with filled squares illustrating the no anti-Ig control. Data shown are representative of two independent experiments.

 
Investigation of the effects of the two anti-IgM mAbs and anti-{kappa} on rCD40L- and IL-4-induced B cell proliferation found that, unlike anti-IgD, they had little effect on either total cell numbers, or the progression of cells through division (Fig. 5GoA). In the presence of rCD40L alone, neither anti-IgM mAb had a significant effect on the progression of B cells through division. In contrast, anti-{kappa} acted to inhibit the division of rCD40L-only stimulated B cells. Thus, the ability of different anti-Ig mAbs to retard class switching was independent of their effects on rCD40L-induced B cell proliferation.



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  		FIGURE 5. Anti-IgM and anti-{kappa} have little effect on B cell division stimulated by CD40L and IL-4. Naive CFSE-labeled B cells were stimulated using 1/1000 rCD40L with or without 100 U/ml IL-4. Selected cultures contained one of two anti-IgM reagents (331.12, left panel; or Bet-2, right panel) at 10 µg/ml. Proliferation was measured in 12 hourly increments from 60 to 96 h, and total cell numbers were estimated using beads. Cultures with or without anti-IgM are indicated by filled or open squares, respectively. Note that the y-axes of some cell division plots are different to enable a meaningful comparison of cell number and division distribution in cultures under different stimulation conditions. Results are representative of two experiments.

 
Anti-Ig mAbs have little effect on the division-linked expression of other B cell surface molecules

As all the anti-Ig mAbs tested inhibited B cell class switching to IgG1 and IgE, it was possible that these Abs might affect division-based expression of other B cell surface molecules. Consequently, the effect of anti-IgD and anti-{kappa} mAbs on the expression of pan-B cell markers, or various B cell surface molecules known to be involved in T cell-B cell collaboration was investigated. Fig. 6Go shows the effect of anti-IgD on the expression of IgM, B220, B7-2, and MHC class II on B cells cultured with rCD40L and IL-4. For comparison, the anti-IgD-induced delay in the division-dependent IgG1 switch is also shown. In a separate experiment, the effect of anti-{kappa} mAb on the expression of B220, L-selectin, and class II on B cells cultured with IL-4, rCD40L, or rCD40L and IL-4 was evaluated (Fig. 7Go). Both figures indicated that the expression profiles of B220 and MHC class II were similar, irrespective of the presence of anti-IgD or anti-{kappa}. In addition, the division-linked expression of class II, B220, and B7-2 in response to rCD40L with or without IL-4 remained unaffected by inclusion of the anti-IgM mAbs (not shown). Collectively, these results raised the possibility that the anti-Ig mAbs used might be specifically affecting the expression of B cell isotypes without significantly affecting the division-dependent expression of other B cell molecules involved in T cell-B cell collaboration. An exception to this observation was the effect of anti-IgD on B7-2 expression, with levels of this molecule ~5-fold lower than the no anti-IgD control by divisions seven and eight (Fig. 6Go). Interestingly, none of the other anti-Ig mAbs used in this study induced the same down-regulation of B7-2 (not shown).



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  		FIGURE 6. Anti-IgD has little effect on the IL-4-dependent differentiation of MHC class II, B220, or B7-2. Naive CFSE-labeled B cells were cultured with 1/1000 rCD40L and 100 U/ml IL-4, with selected cultures supplemented with 10 µg/ml anti-IgD mAb. At day 4, B cells were harvested, and their expression of surface IgM, IgG1, MHC class II, B220, and B7-2 was examined. A, Two-dimensional log-log contour plots illustrating division-dependent expression of selected surface molecules of B cells. Division profiles are shown in the top panel, and the undivided peak is indicated by the hashed line in the rCD40L and IL-4 histogram. B, Division-linked expression of selected B cell surface molecules. rCD40L and IL-4 cultures with or without anti-IgD mAb are shown as filled and open symbols, respectively, and days 3, 4, and 5 are represented by squares, triangles, and circles. The distribution of cells with division over three time points is illustrated in the top graph. The differentiation plots show the percentage of positive IgG1 cells per division on a linear scale, whereas the mean fluorescence intensity (MFI) of the surface expression of MHC class II and B7-2 is presented using a logarithmic y-axis. The MFI of B220 is plotted using a linear scale. Note that the y-axes of each differentiation plot are different. Data shown are representative of three independent experiments.

 


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  		FIGURE 7. Anti-{kappa} has little effect on the division-dependent expression of B220, L-selectin, and MHC class II. Naive CFSE-labeled B cells were cultured with IL-4 (100 U/ml IL-4), CD40L (1/1000), or CD40L and IL-4. Selected cultures were supplemented with one of three concentrations of anti-{kappa} (10, 2.5, 0.5 µg/ml). At day 4, B cells were harvested, and levels of surface MHC class II, B220, and L-selectin were measured by FACS. Graphs show the percentage of positive expression of each of these molecules with division, with open squares, diamonds, and circles representing the three concentrations of anti-{kappa}, while the filled triangles illustrate the no anti-{kappa} control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A common feature of many in vitro models of T-B cell collaboration is that polyclonal B cell proliferation and differentiation can be induced in the absence of an Ag signal (2, 3, 5, 6, 8, 10, 14, 40, 41, 42). Although sIg signals may not be essential for T cell-dependent B cell activation, it is clear that they modulate the behavior of responding B cells. For example, specific Ag can alter the migration pathway of a recirculating B cell by stimulating it to migrate to the edge of a T cell zone within secondary lymphoid organs (43, 44, 45, 46). In addition, sIg signals have been shown to stimulate B cells to increase surface expression levels of a number of activation molecules (15, 16), and to promote B cell division and differentiation to Ig-secreting cells both during and following the provision of T cell-independent stimuli (47, 48, 49) or T cell-dependent B cell stimuli (17, 18, 39, 50). These effects of Ag will clearly play an enhancing role in promoting Ab secretion by increasing the rate by which B cells are stimulated by T cells. Less well understood is the effect of sIg-mediated signals on isotype switching. A series of studies have found that despite increasing the rate of IgG1 Ab secretion, multivalent anti-Ig mAb had variable effects on the proportion of B cells switching to IgG1 (17, 18). However, these results were obtained in bulk culture in vitro studies in which separation of cell death and cell division on the rate of isotype switching and Ab-forming cell generation were difficult to assess. Monitoring the effect of Ag on switching by measuring secreted Ab and Ig expression of cells is complicated by the recent observations that B cell proliferation and class switching are linked to cell division number (10, 21, 22, 23, 25). As a result, increasing the rate of proliferation could indirectly increase the number of switched cells. Thus, an Ag signal could exert varying effects on B cell division and cell death, which could affect the rates of Ig secretion and isotype switching. This study sought to resolve these possibilities by using division number as a reference frame for quantitative comparisons of isotype switching (10, 23, 24, 25).

Anti-IgD stimulated an increase in the total number of viable cells in B cell cultures containing rCD40L in the presence and absence of IL-4, consistent with previous reports (17, 18, 39, 50). By tracking cell division, we noted that anti-IgD-treated B cells had progressed through further divisions than controls, particularly at early time points. There was also a reduction in the total number of undivided cells in cultures containing anti-IgD mAb. Thus, a sIg-mediated signal transmitted following engagement of anti-IgD mAb provided additional activating signals to B cells receiving T cell help, which reduced the average division time. The possibility that this enhancement of proliferation was mediated by autocrine production of CD40L, as previously observed (38), was discounted, as the presence of an anti-CD40L mAb had no effect on proliferation induced by anti-IgD.

In contrast to its effects on B cell division, inclusion of anti-IgD in B cell cultures containing rCD40L and IL-4 reduced the proportion of B cells switching to IgG1 and IgE in a dose-dependent manner. Anti-IgD mAb appeared to act specifically on the ability of B cells to undergo Ig class switching, as it had little effect on the division-linked expression of other non-Ig B cell surface molecules involved in T-B cell collaboration. Interestingly, inhibition of IgE switching was less than that seen for IgG1, suggesting that there might be differential regulation of the expression of these two isotypes, despite both being induced following CD40L and IL-4 signals. Another not necessarily exclusive explanation of differential effects of anti-IgD on IgG1 and IgE levels is that the signaling pathways controlling {gamma}1 germline transcript production and/or class switch recombination may be more sensitive to signals delivered by anti-Ig mAbs than the {epsilon} class switch pathway.

The above results suggested that in addition to enhancing B cell division, anti-IgD provided additional signals that delayed division-linked Ig class switching. Although it was possible that these effects were intrinsic to the particular anti-IgD mAb, three other anti-Ig mAbs stimulated a delay in the division-linked expression of IgG1 and IgE. These additional Abs further highlighted the potential for signals to independently regulate B cell division compared with differentiation, as they had little effect on rCD40L-induced B cell division despite delaying isotype switching. The varied effects of the different anti-Ig mAbs on cell division suggested that these reagents might differ qualitatively in their ability to signal via sIg. Different mitogenic properties of various anti-Ig reagents have been noted previously and attributed to qualitatively different signals delivered to the B cell as a result of different sIg cross-linking capacities (18, 51, 52, 53), their binding of different sIg epitopes (53, 54), or coligation of Fc{gamma}RIIb (55, 56). This latter possibility was eliminated in this study by the inclusion of an anti-Fc{gamma}RIIb mAb in selected experiments.

Each of the anti-Ig mAbs tested acted as partial antagonists of class switching to IgG1 and IgE, as they were unable to completely abrogate switching to IgG1 and IgE. It is possible that these anti-Ig mAbs reduced the probability of B cells switching to IgG1 and IgE across all divisions; however, it is unclear whether all B cells that continued to divide beyond divisions eight and nine would eventually switch to IgG1 or IgE at a similar level to CD40L and IL-4 cultures. If so, this would reflect a shift in the mean of the division-based probability distributions describing isotype switching to IgG1 and IgE. In contrast to their effects on class switching, none of the anti-Ig reagents tested significantly affected the division-linked differentiation of selected B cell surface molecules involved in T-B cell collaboration, suggesting that these reagents might be acting specifically on the isotype switching mechanism. Therefore, it is possible that a B cell may regulate its effector functions following sIg signals delivered by different types of Ags, or structural variants of the same Ag. Such regulation based on Ag structure may have evolved to facilitate the efficient clearance of Ag. As anti-Ig reagents mimic signals delivered by multivalent Ags (53), the delay in the class switch to IgG1 suggests that B cells responding to these multivalent Ags would continue to produce IgM, which, in a secreted form, maintains a multimeric structure adapted to efficiently deal with multivalent Ags (57). The increased efficiency of clearance of multivalent Ags by multimeric Abs is due to the much longer t1/2 of attachment to Ag compared with dimeric Abs (58), allowing very low affinity binding to contribute to Ag clearance if three, four, or more Ag-binding sites can be engaged. Therefore, if an Ag is multivalent, a large number of low affinity B cells may be recruited into effective Ab production for rapid clearance (59). A delay in the IgG1 switch would prolong the period of IgM production, and thereby facilitate the clearance of this Ag in the shortest time. By inference, it would be predicted that monovalent Ag would not delay the isotype switch to IgG1, as there would be no advantage gained from inhibiting class switching. This prediction is amenable to further research.


   Acknowledgments
 
We thank Charles A. Janeway for valuable manuscript reviews and comments, Paul Lalor and Andrew Heath for mAbs, Marilyn Kehry and Brian Castle for the baculovirus-expressed murine CD40L, and Robert Kastelein for rIL-4.


   Footnotes
 
1 This work was supported by grants from the National Health and Medical Research Council and the Medical Foundation of the University of Sydney. J.S.R. was supported by a Postgraduate Scholarship from the Medical Foundation of the University of Sydney. Back

2 Address correspondence and reprint requests to Dr. James S. Rush at the current address: Howard Hughes Medical Institute and Section of Immunobiology, Yale Medical School, 310 Cedar Street/Box 208011, New Haven, CT 06510-8011. E-mail address: james.rush{at}yale.edu Back

3 Current address: Division of Immunology, Walter and Eliza Hall Institute, Parkville, Victoria, Australia. Back

4 Abbreviations used in this paper: sIg, surface membrane Ig; rCD40L, rCD40 ligand; SA-TC, streptavidin-Tricolor. Back

Received for publication September 19, 2001. Accepted for publication January 9, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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