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The Journal of Immunology, 1998, 161: 4618-4626.
Copyright © 1998 by The American Association of Immunologists

Evidence for a Critical Role for IL-2 in CD40-Mediated Activation of Naive B Cells by Primary CD4 T Cells

Caroline Johnson-Léger, Jillian R. Christenson, Mary Holman and Gerry G. B. Klaus1

Division of Cellular Immunology, National Institute for Medical Research, London, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The interaction of CD40 on B cells with the CD40 ligand (CD40L) on preactivated CD4 T cells is critical for the initiation of T-dependent Ab responses. It is believed that signals via CD40 synergize with cytokines (e.g., IL-4 and IL-5) to drive B cell activation. However, primary T cells preactivated via CD3 alone cannot induce B cell proliferation; we have shown previously that costimulation of T cells via CD3 and CD28 stabilizes the expression of the CD40L, which we propose contributes to their capacity to act as competent helper-effector cells. Here we show that an additional, critical reason why CD3-stimulated CD40L-bearing T cells are incompetent helper cells is because they secrete insufficient IL-2. In contrast, CD28/CD3-activated T cells induce B cells to become IL-2 responsive via a combination of CD40L and IL-2-mediated signals, and these two stimuli subsequently drive B cell proliferation and IgM secretion. We therefore propose that T cells must first encounter Ag in conjunction with CD80/86 on APCs. This leads to the stable expression of CD40L and maximal secretion of IL-2, which together render primary T cells competent to activate B cells in an IL-2-dependent fashion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The activation of B cells in response to T cell-dependent (TD)2 Ags requires a combination of CD4 T cell-derived contact-mediated signals plus a variety of cytokines (reviewed in Refs. 1 and 2). Although several receptor-ligand pairs have been reported to play a role in this process, it is now clear that the key molecule on B cells for the reception of contact-mediated helper signals is CD40. Activation of B cells occurs when this surface receptor is engaged by its ligand (CD40L or gp39), a protein expressed on activated CD4 T cells (reviewed in 2 . Evidence for this conclusion has come from several sources. Firstly, mutations in the gene encoding CD40L are responsible for the X-linked hyper-IgM syndrome in man (reviewed in 3 . Although these patients can produce IgM-secreting plasma cells, they cannot form germinal centers in response to TD Ags or switched Ig isotypes. Secondly, mice with targeted mutations in the genes for CD40 or CD40L have a similar phenotype (4, 5, 6). Finally, Abs against CD40L block the development of both primary and secondary Ab responses in mice (7).

Interactions between T and B cells must be tightly controlled to minimize the activation of self-reactive or bystander cells. This is particularly cogent, since it is known that preactivated T cells, which express CD40L protein, activate B cells in a polyclonal, MHC-unrestricted, Ag-independent fashion (8, 9, 10, 11, 12). It is therefore not surprising that the expression of the CD40L is tightly regulated. The protein is rapidly induced following stimulation of primary CD4 T cells or T cell clones via the TCR/CD3 complex, and then wanes within 16 to 24 h (8, 13, 14). In addition, contact of CD40L-bearing T cells with CD40-positive cells (such as B cells, macrophages, or dendritic cells) leads to very rapid down-regulation of the protein, initially as a result of receptor-mediated endocytosis, followed subsequently by down-regulation of CD40L mRNA (15, 16).

Much of our current knowledge about the role of CD40/CD40L interactions in B cell activation has come from studies with recombinant CD40L, CD40L-transfected cell lines, or mAb against CD40 either in solution or presented on CD32-transfected fibroblasts (the CD40 system) (17). Studies with T cells have employed mostly preactivated T cell clones. The results from these models have led to the conclusion that signals via CD40 alone can induce B cell proliferation, which is enhanced by costimuli such as anti-Ig or certain cytokines (principally IL-4 and IL-5 in the mouse, and IL-4, IL-10, and IL-13 in man). In vitro, Ig production by CD40-activated B cells, especially of switched isotypes such as IgE, is dependent on the presence of appropriate cytokines, in this case IL-4 (reviewed in 18 , although in vivo mice can produce IgE in the absence of IL-4 (19). Such in vitro studies have clearly been useful for elucidating the nature of the signals delivered through CD40 on B cells and for providing a platform for understanding the role of CD40 in immunoregulation. However, it is uncertain whether these systems represent valid models to mimic the events that occur when naive B cells first encounter CD40L expressed at physiologic levels on activated primary T cells, especially since the latter are known to be extremely poor helper-effector cells (8, 9).

The present study was prompted by experiments that demonstrated that B cells require 36-h exposure to CD40L-transfected fibroblasts before becoming committed to DNA synthesis (20). Since B cells rapidly induce down-regulation of CD40L on preactivated T cells (15), this observation raised fundamental questions about how signals are generated for sufficient periods via CD40 to induce B cell activation during the initiation of TD Ab responses. We showed that coligation of CD3 and CD28 on primary T cells stabilizes the expression of the CD40L (20). CD28 is known to be an important coreceptor on T cells, which interacts with the counter-receptors B7-1 and B7-2 (CD80 and CD86), molecules that are found on APCs such as dendritic cells and activated B cells (reviewed in 21 . Ligation of CD28 on CD3-activated T cells markedly increases their proliferation and the secretion of various cytokines (most notably IL-2). In addition, it is clear that CD28/B7 interactions are crucial for the induction of responses to TD Ags, since mice lacking CD28 are grossly immunodeficient (22, 23). In our earlier study we found that T cells preactivated through CD3 alone in the absence of a CD28 costimulus were unable to induce B cell proliferation. In the present communication we demonstrate that such T cells nevertheless re-express CD40L following restimulation via CD3 and are not rendered anergic by this prestimulation. These findings therefore indicate that CD40L alone when expressed at physiologic levels on primary T cells is not sufficient to induce full-blown B cell activation. This implies that in addition to stabilizing the expression of CD40L, CD28 costimulation of primary T cells must induce the expression of an additional signal(s) that synergizes with CD40L to activate B cells. The results presented here indicate that this additional signal is IL-2. These findings therefore provide an explanation for the many earlier studies that demonstrated a requirement for IL-2 in the induction of primary Ab responses to TD Ags (24, 25, 26, 27).


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

Specific pathogen-free (CBAxC57BL/10)F1 mice bred at the National Institute for Medical Research (London, U.K.) were used at the age of 3–4 mo.

Reagents

The following mAb were used: hamster anti-CD3 (145-2C11), hamster anti-CD28 (37.51.1, from J. Allison), hamster anti-CD40L (MR1, a gift from R. Noelle), rat anti-Thy 1 (NIMR-1), rat anti-CD8 (YTS 169.4.2.1) and anti-CD4 (YTS 191.1.1.2; both obtained from H. Waldmann), rat anti-µ (b.7.6), rat anti IL-2 (1A12 or S4B6), rat anti-IL-2R {alpha}-chain (7D4), rat anti-IL-2R ß-chain (TMß1), and rat anti-ovine placental lactogen (Mac-193, from G. Butcher). These were purified on protein G-Sepharose (Pharmacia, Piscataway, NJ) and coupled with biotin or FITC by standard methods. Biotinylated or FITC-coupled rat anti-B220 and biotinylated anti-CD69 were purchased from PharMingen (San Diego, CA). AffiniPure goat anti-mouse IgG and IgM (H+L) was obtained from Stratech (London, U.K.), and phycoerythrin-conjugated streptavidin (PE-SA) was obtained from Southern Biotechnology Associates (Birmingham, AL). Mouse IL-2 was obtained from supernatants of a stably transfected cell line kindly provided by F. Melchers (28). IL-2 bioactivity was titrated using the CTLL indicator cell line by standard procedures.

Preparation of T cells and B cells

Small, dense B cells were prepared from mouse spleen by a modification of a method previously described (29). Briefly, T cells were killed by incubation with a mixture of anti-Thy 1, anti-CD4, and anti-CD8 mAbs and guinea pig complement for 40 min at 37°C. The remaining cells were washed and layered onto a 50–75-85% Percoll gradient. After centrifugation, the cells banding at the 75 to 85% interface were recovered. These were typically >90% B220+, with <1% T cell contamination. Splenic CD4+ T cells were prepared as follows. Single cell suspensions were loaded onto a discontinuous (50–75-85%) Percoll gradient. After centrifugation, cells at the 75 to 85% interface were harvested and resuspended in medium with a saturating concentration of anti-CD8. After 40 min at 4°C, the cells were washed, resuspended in PBS/3% FCS to give between 4 and 5 x 107 cells in 3 ml, and plated onto washed bacteriologic petri dishes previously coated overnight at 4°C with 10 µg/ml affinity-purified goat anti-mouse Ig in 0.05 M Tris-HCl, pH 9.5. Plates were swirled after 40 min to redistribute unattached cells and were left for a further 30 min. Nonadherent cells were recovered and subjected to a second round of panning as before. The nonadherent cells were typically >90% CD4 positive, with <5% B cell contamination.

Induction and re-expression of CD40L

T cells were cultured (at 106/ml) in supplemented RPMI 1640 medium plus 5% FCS in flasks coated with anti-CD3 (in PBS (10 µg/ml) for 24 h at 4°C). Some cultures received in addition soluble anti-CD28 (or normal hamster IgG) at 1 to 5 µg/ml. CD40L expression was first detectable by 4 h, reached maximal levels by 12 to 16 h, and subsequently waned. Hence, in most experiments T cells were stimulated for 12 to 16 h as indicated. These cells were then replated in anti-CD3-coated flasks or microtiter plates.

Flow cytometric (FCM) analyses and electronic cell sorting

T cells (or T and B mixtures) were suspended in PBS/0.2% BSA/0.1% NaN3 and stained with the appropriate combinations of mAbs by conventional methods. Appearance of CD40L was assessed by two-color FCM analyses, using a combination of FITC-anti-CD4 and biotinylated anti-CD40L, revealed by PE-SA. Appearance of the IL-2R on B cells was assessed by two-color FCM analyses using a combination of biotinylated or FITC-anti-B220 and FITC-anti-IL-2R{alpha} or biotinylated anti-IL-2Rß, respectively, revealed by PE-SA. Flow cytometric analyses were performed on either a FACStarPlus or a FACSVantage (Becton Dickinson, Mountain View, CA). For some experiments whole spleen (WSC) cultures were cultured with immobilized anti-CD3 as indicated. Then T cells were depleted (as above), the remaining cells were stained with FITC-anti-B220, and B cells were purified by electronic cell sorting on a FACStarPlus cytometer.

Coculture of T cells and B cells

CD4 T cells that had been preactivated with anti-CD3, with or without anti-CD28 were harvested, irradiated (3000 rad), and then plated into 96-well microtiter wells (105/well) that were uncoated or had been coated with anti-CD3 as described above together with an equal number of resting B cells and appropriate mAb as indicated. These cultures were labeled with [3H]TdR (0.5 µCi/well) generally after 68 h of culture and harvested 4 h later, and incorporation of radiolabel into DNA was determined by standard methods.

Measurements of Ig secretion

Resting B cells were cultured (at 3.3 x 105/ml) with an equal number of CD3-primed or CD28/CD3-primed, irradiated T cells in wells coated with anti-CD3 for 7 days, at which time supernatants were collected. Levels of different isotypes were determined by standard ELISA methodology. In brief, microwells were coated with goat anti-mouse Ig, and the individual isotypes were determined by subsequent addition of isotype-specific biotinylated goat Ab, revealed by horseradish peroxidase-conjugated streptavidin, followed by ABTS substrate (Sigma, St. Louis, MO). All immunologic reagents were obtained from Southern Biotechnology Associates.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CD3-primed and CD28/CD3-primed T cells re-express similar levels of CD40L upon restimulation via CD3

In an attempt to mimic the sequence of events that may occur in vivo during the initiation of a TD Ab response, we developed the following two-stage culture system. Purified CD4 T cells were activated overnight on immobilized anti-CD3 in the presence or the absence of anti-CD28 (to mimic the effects of CD28/B7 interactions). These (primed) T cells were then irradiated and recultured (with or without immobilized anti-CD3) with B cells for measurements of proliferation or Ig secretion. We have previously shown that CD3-preactivated primary CD4 T cells cannot induce DNA synthesis in B cells when they are restimulated via CD3, whereas CD3/CD28-preactivated T cells are able to do so (20). This prompted us to compare the levels of CD40L on these two populations of T cells following restimulation with immobilized anti-CD3. As we previously reported, CD3/CD28-primed cells re-expressed CD40L more rapidly than CD3-primed cells. However, after 16 h of restimulation the two populations of T cells expressed essentially identical levels of CD40L on their surface (Fig. 1GoA). CD28-derived signals are known to play an important role in the prevention of anergy in T cells (reviewed in 30 . However, Figure 1GoB shows that these CD3-primed T cells were not anergic, as demonstrated by their capacity to proliferate when restimulated with immobilized anti-CD3. This is presumably because under these conditions, they secrete sufficient IL-2 (20–40 U/ml; see below) to drive their own proliferation.



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  		FIGURE 1. CD3-primed CD4 T cells re-express similar levels of CD40L following restimulation and are not anergic. Purified CD4 T cells were primed on immobilized anti-CD3 in the presence or the absence of anti-CD28 (5 µg/ml) for 24 h. They were then rested for 15 h to allow spontaneous decay of CD40L. In Expt. 1 (A) these two populations were then restimulated with anti-CD3 for 16 h, when they were stained with anti-CD4 and anti-CD40L for FCM analyses. The histograms illustrate levels of CD40L on CD4+ cells primed with anti-CD3 alone (thick histogram; 34% positive; MFI, 48) or with anti-CD28/CD3 (thin histogram; 42% positive; MFI, 50); the dashed profile represents background staining. In Expt. 2 (B) CD4 T cells primed as described above were immediately replated into wells that were coated with anti-CD3 (hatched bars) or were uncoated (open bars), and their proliferation was measured after 3 days in culture. Results are from one of two experiments that gave comparable results.

 
The available data (8, 9, 20) therefore suggested that expression of CD40L is insufficient for primary T cells to induce B cell proliferation. This, in turn, implied that CD40L acts in concert with a second cell-bound or soluble factor to induce B cell proliferation, and that it is this second signal that is lacking in T cells prestimulated through CD3 alone. An alternative possibility was that the CD40L on such T cells is nonfunctional for unknown reasons.

CD40L expressed by CD3-primed restimulated T cells is functional

To address the latter possibility we cultured CD3-primed, irradiated T cells on immobilized anti-CD3 with B cells in the presence or the absence of anti-µ (which synergizes with anti-CD40 mAbs to induce B cell activation). The results showed clearly that anti-µ induced a synergistic proliferative response with CD3-primed, restimulated T cells (and modestly enhanced the response to CD28/CD3-primed T cells; Fig. 2Go). Furthermore, this synergistic effect was completely abrogated by the inclusion of anti-CD40L, thereby establishing the critical role of this protein in the observed effects and confirming its functional presence on CD3-primed T cells. Although not shown in this experiment, the addition of anti-CD40L had no effect on the responses of purified B cells to anti-µ (not shown).



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  		FIGURE 2. Effects of CD3-primed T cells on B cell proliferation induced by anti-µ. CD4 T cells were primed with immobilized anti-CD3 in the presence or the absence of anti-CD28 for 24 h. These cells were irradiated and replated with B cells (1:1 ratio) in the presence or the absence of anti-CD3 and together with the indicated concentrations of anti-µ ({alpha}µ; in micrograms per milliliter). Some cultures also received anti-CD40L (1 µg/ml). Cultures were harvested after 3 days for measurements of B cell DNA synthesis. The data shown are the mean ± SEM (n = 3), where the open bars are B cells alone, the hatched bars are B cells plus CD3-primed T cells, and the closed bars are B cells plus CD28/CD3-primed T cells. B cells cultured with T cells in the absence of anti-CD3 gave background incorporation of [3H]TdR (not shown). T cells cultured in medium alone or on anti-CD3 incorporated no more than 100 cpm (not shown). The data shown are representative of two experiments that gave comparable results.

 
IL-2 synergizes with CD40L in whole spleen cell cultures to induce B cells to become IL-2 responsive

The foregoing results indicated that CD28 costimulation of naive T cells induced the production of an essential costimulus, necessary for CD40L-driven B cell activation. We suspected that this cofactor was IL-2 for several reasons. Firstly, we had observed that B cells stimulated with anti-CD40 mAbs in the presence of IL-2 become IL-2 responsive (data not shown). In addition, IL-2 is rapidly produced by activated T cells, and it is well established that CD3/CD28 costimulation of T cells stabilizes IL-2 mRNA and markedly enhances IL-2 secretion (reviewed in 21 . We therefore hypothesized that in the present system B cells first become IL-2 responsive by exposure to IL-2 and signaling via CD40. They then proliferate in response to IL-2 in a CD40-dependent or independent manner.

Consequently, a set of two-step culture experiments was conducted to examine the capacity of activated primary T cells to induce B cells to become IL-2 responsive in a physiologic context. In these, WSC were cultured in anti-CD3-coated flasks. Following 18 to 24 h of culture T cells were depleted by mAb/complement-mediated lysis. The enriched B cell population (>85% B cells and <10% T cells) was then replated together with varying concentrations of IL-2. Figure 3GoA shows that these activated B cells proliferated strongly in response to IL-2 in a concentration-dependent fashion, whereas freshly prepared B cells only gave a modest response to very high concentrations of IL-2. In addition, purified B cells cultured on immobilized anti-CD3 did not become IL-2 responsive (not shown). The possibility that contaminating T cells were responsible for this effect was addressed in subsequent experiments; in these, the B cell-enriched population was stained with anti-B220 and further purified by electronic cell sorting to yield a population of cells that contained >95% B220+ cells and <1% T cells. These cells again responded vigorously when recultured with IL-2 for 3 days (Fig. 3GoB).



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  		FIGURE 3. B cells activated in CD3-stimulated whole spleen cell cultures become IL-2 responsive. WSC were cultured on immobilized anti-CD3 for 24 h. Then, in Expt. 1 (A) T cells in these cultures were killed (see Materials and Methods), the remaining B cells (>85% B220+, with <10% CD3+ cells; {circ}) or fresh B cells ({square}) were recultured (at 105 cells/well) with the indicated concentrations of IL-2, and B cell proliferation was measured as before after a further 72-h culture. In Expt. 2 (B) activated WSC cultures were T cell depleted as described above, but the remaining cells were stained with anti-B220, and B cells were further purified by electronic cell sorting. These cells (>95% B220+, <1% CD3+) were recultured with IL-2 as described above. The data shown are from one of two experiments that gave comparable results.

 
These results indicate that B cells rapidly become IL-2 responsive when exposed to CD3-stimulated T cells in WSC cultures, suggesting that this could be mediated via CD40L/CD40 interaction. To test this, WSC cultures were primed with anti-CD3 in the presence or the absence of anti-CD40L or anti-IL-2, and T cell-depleted B cells were restimulated with IL-2 as before. Interestingly, blocking CD40L/CD40 interactions or neutralization of IL-2 in the priming cultures substantially reduced the subsequent responses of B cells to IL-2, and the two mAb together produced almost complete inhibition (Fig. 4Go, A and B). In contrast, addition of neutralizing mAb against IL-4 or IFN-{gamma} had no effect, nor did the addition of normal hamster Ig or irrelevant isotype-matched rat IgG2a (Mac-193; data not shown). In further control experiments, addition of anti-CD40L did not affect the proliferation of anti-CD3/CD28-stimulated T cells or the levels of IL-2 they produced (not shown). We believe that the modest proliferative responses given by resting B cells cultured with 500 to 1000 U/ml IL-2 are explicable by the following factors. Firstly, B cells cultured in medium alone express (mostly low) levels of IL-2R (see Fig. 6Go). Secondly, such high concentrations of IL-2 alone induce a modest level of IL-2 responsiveness in B cells (not shown). Nevertheless, the above results clearly show that when B cells are cultured with preactivated T cells, both CD40L/CD40 interactions and IL-2 are required for the optimal induction of IL-2 responsiveness in B cells.



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  		FIGURE 4. Effects of blocking CD40L/CD40 interactions or neutralizing IL-2 on the induction of IL-2 responsiveness in B cells. In Expt. 1 (A) WSC cultures were primed with immobilized anti-CD3 in the presence ({circ}) or the absence (•) of anti-CD40L (1 µg/ml) for 24 h. when T cells were depleted as described in Figure 3GoA. The resulting B cells or fresh B cells ({square}) were recultured with the indicated concentrations of IL-2 as before. In Expt. 2 (B) WSC were primed as described above in the presence ({circ}) or the absence (•) of anti-CD40L (1 µg/ml), anti-IL-2 ({triangleup}; S4B6; 20 µg/ml), or the two mAb together ({diamond}), and B cell proliferation in response to IL-2 was measured as before. The data shown are the mean ± SEM (n = 3). The results are representative of those obtained in two or three experiments.

 


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  		FIGURE 6. Effects of CD28 costimulation on the expression of IL-2R{alpha} on B cells. CD28/CD3-primed or CD3-primed T cells were generated as described in Figure 2Go. These cells were irradiated, replated on anti-CD3, and mixed with fresh B cells (1:1 ratio) in the presence or the absence of anti-CD40L (1 µg/ml) or anti-IL-2 (1A12; 10 µg/ml) alone or together. After a further 24 h these cultures were stained with anti-B220 and anti-IL-2R{alpha} and subjected to FCM analyses. The histograms show the levels of IL-2R{alpha} on B220+ cells cultured with CD28/CD3-primed T cells (A and C) or with CD3-primed T cells (B and D). As indicated in A and B, the dashed histograms are B cells cultured alone, the hatched histograms are T cells plus B cells, and the thick histograms are T cells, B cells, and anti-CD40L. In C and D the hatched histograms are T cells and B cells; the thick histograms are T cells, B cells, and anti-IL-2; and the dashed histograms are T cells, B cells, anti-CD40L, and anti-IL-2. Events to the left of the marker represent background staining. Percentages of positive cells and MFI values are presented in Table IGo. Data are representative of results obtained from two experiments.

 
CD3-primed T cells are defective in their capacity to induce IL-2 responsiveness in B cells

We next compared the capacity of CD3-primed or CD28/CD3-primed T cells to induce B cells to become IL-2 responsive. Hence, fresh B cells were cultured for 24 h with these two populations of primed T cells, which were then killed. The resulting preparations (95% B220+) were recultured with various concentrations of IL-2 (Fig. 5Go). Interestingly, B cells that had been preactivated by CD3-primed T cells gave far lower responses to IL-2 than B cells preactivated by CD28/CD3-primed T cells, but gave comparable responses to anti-µ. Comparable results were obtained after 96-h culture, when the responses of B cells exposed to costimulated T cells had begun to wane (not shown). FCM analyses of these B cells cultured for 3 days in 200 U/ml IL-2 revealed that at least 90% of the cells were B220+, and only 7 to 10% of them were CD4+ at the end of the culture period (not shown). These data therefore suggest that B cells require both a critical level of IL-2 as well as signals via CD40 to become IL-2 responsive, implying that CD3-primed restimulated T cells produce insufficient amounts of IL-2 to activate B cells, unlike CD28/CD3-primed T cells. This would be consistent with the known effects of CD28 costimulation on cytokine secretion reported by others (reviewed in 21 . To study this in the present system, T cells that had been previously activated via CD3 or CD28/CD3 were restimulated on immobilized anti-CD3 for 48 h. Supernatants were collected at this point and assayed for IL-2. The results showed that CD28/CD3-primed T cells secreted between 320 and 400 U/ml of IL-2, while their CD3-primed counterparts only produced between 20 and 40 U/ml of IL-2 (data not shown).



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  		FIGURE 5. Effects of CD28 costimulation of T cells on their capacity to induce IL-2 responsiveness in B cells. Small dense B cells were cultured for 24 h with CD3-primed or CD28/CD3-primed T cells on immobilized anti-CD3, when T cells were depleted as described in Figure 3GoA. The remaining B cells were recultured with the indicated concentrations of IL-2 (in units per milliliter) or with anti-µ (10 µg/ml). Data represent the proliferative responses of B cells derived from CD28/CD3-stimulated cultures (closed bars) or from CD3-primed cultures (open bars), or responses of fresh B cells (hatched bars). Similar results were obtained in a second experiment.

 
Both CD40/CD40L interaction and IL-2 are required for maximal expression of the IL-2R {alpha}-chain on B cells

The acquisition of responsiveness to IL-2 in T cells or B cells depends on the expression of components of the heterotrimeric IL-2R. The individual components of the IL-2R ({alpha}-, ß-, and {gamma}-chains) bind IL-2 with low to moderate affinity, while the full heterotrimer exhibits the highest affinity for IL-2 (reviewed in 31 . Previous work has shown that resting mouse B cells constitutively express the IL-2R ß- and {gamma}-chains and up-regulate IL-2R{alpha} when activated (32). We therefore examined the levels of IL-2R{alpha} on B cells cultured for 24 h with CD3- or CD28/CD3-primed T cells (Fig. 6Go and Table IGo). Resting B cells do not express IL-2R{alpha}, in agreement with published data (not shown), whereas about 70% of B cells cultured in medium alone expressed (mostly) very low levels of the receptor. Essentially all B cells cultured with CD28/CD3-primed T cells became uniformly and strongly IL-2R{alpha} positive (Fig. 6GoA), whereas significantly fewer B cells cultured with CD3-primed T cells expressed high levels of IL-2R (Fig. 6GoB). In both instances, the inclusion of anti-CD40L mAb markedly inhibited the up-regulation of IL-2R{alpha} (Table IGo). Addition of anti-IL-2 also significantly reduced the levels of IL-2R{alpha} on a proportion B cells incubated with CD28/CD3-primed T cells (Fig. 6GoC), but had no impact on the levels of the receptor induced by CD3-primed T cells (Fig. 6GoD). Finally, addition of both anti-CD40L and anti-IL-2 completely abrogated the capacity of CD28/CD3-primed T cells to up-regulate IL-2R{alpha} expression on B cells (Fig. 6GoC). These data therefore indicate that both IL-2 and CD40/CD40L interaction are required for maximal expression of the IL-2R {alpha}-chain on activated B cells. Hence, the failure of CD3-primed T cells to induce uniformly high levels of the receptor on B cells is because they produce insufficient IL-2. These results were reproducible. In a second experiment 90% of B cells cultured with costimulated T cells expressed IL-2R{alpha} (MFI, 126), whereas only 71% (MFI 81) of those cultured with CD3-primed T cells did so (not shown). We have attempted to perform similar studies on the expression of IL-2Rß, but have failed to obtain satisfactory staining on B cells with the only mAb (TMß-1) available to us (although this mAb detects IL-2Rß on T cells effectively).


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  		Table I. Effects of blocking CD40L/CD40 interactions or neutralization of IL-2 on the expression of IL-2R{alpha} on B cells cultured with activated T cells

 
Addition of IL-2 or costimulation via CD28 restores the helper-effector function of primary T cells preactivated through CD3 alone

The data obtained to date suggested that CD3-primed T cells secrete insufficient IL-2 to induce B cells to become IL-2 responsive and also, perhaps, to drive the proliferation of activated B cells. To confirm the essential role of IL-2 in the present system we added IL-2 or anti-IL-2 to cultures containing CD3-primed or CD28/CD3-primed T cells and resting B cells (Fig. 7GoA). B cell proliferation induced by CD28/CD3-primed T cells was almost completely abrogated by anti-CD40L and was substantially blocked by anti-IL-2, thereby confirming that both these stimuli contribute to the response. Conversely, the addition of IL-2 substantially (but not completely) restored the capacity of CD3-primed T cells to induce B cell proliferation, while the inclusion of anti-CD28 completely restored their helper-effector function (an effect that was abrogated by the inclusion of anti-IL-2; not shown). In contrast, neither of these additions affected the level of B cell proliferation induced by CD28/CD3-primed T cells. These data therefore confirm that a major reason for the ineffective helper-effector function of CD3-primed T cells is their failure to produce sufficient IL-2.



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  		FIGURE 7. A, Effects of exogenous IL-2 or CD28 costimulation on the capacity of CD3-primed T cells to induce B cell activation. CD3-primed or CD28/CD3-primed T cells were generated as described in Figure 2Go. Following irradiation these cells were replated with B cells on immobilized anti-CD3. Some groups received in addition anti-CD40L (2 µg/ml), anti-IL-2 (1A12, 20 µg/ml), anti-CD28 (5 µg/ml), or IL-2 (100 U/ml), as indicated. DNA synthesis was assayed after 72 h. Data represent the responses of B cells alone (open bars), B cells plus CD3-primed T cells (hatched bars), and B cells plus CD28/CD3-primed T cells (closed bars). T cells plus B cells cultured in uncoated wells gave background levels of [3H]TdR uptake (not shown). T cells in medium alone or on anti-CD3 incorporated 100 cpm (not shown). B, Effects of blocking the CD40L/CD40 interaction or neutralizing IL-2 on the effector function of CD28/CD3-primed T cells. T cells were primed with anti-CD28/CD3 as before, irradiated, and replated with B cells and anti-CD3. Some groups received anti-CD40L (2 µg/ml) or anti-IL-2 (1A12; 30 µg/ml) at the times indicated. Background [3H]TdR uptake of B cells alone was 1800 ± 150 cpm. In each case, the data are representative of two experiments.

 
Clearly, B cells activated via CD40L-bearing, IL-2-secreting T cells can proliferate autonomously in response to IL-2 alone. However, these results do not rule out the possibility that when T cells and B cells remain in contact, continuing stimulation via CD40 might contribute to IL-2-induced B cell proliferation. To address this possibility we mixed CD28/CD3-primed T cells with B cells and added anti-CD40L or anti-IL-2 to these cultures at varying times (Fig. 7GoB). Addition of anti-IL-2 at the initiation of the cultures suppressed B cell proliferation by about 50% and caused significant suppression even when added as late as 48 h. Significantly, addition of anti-CD40L at time zero had an even greater effect, and this mAb also caused significant suppression when added after 48 h. These data therefore indicate that when preactivated T cells and B cells are cultured together, continuing stimulation via IL-2R and CD40 contributes to driving B cell activation.

Secretion of IgM induced by activated primary T cells is dependent on both CD40L and IL-2

Previous studies have implicated IL-4 and IL-5 as the major cytokines that regulate CD40-mediated proliferation and Ig secretion in murine B cells (reviewed in 33 . It was therefore of interest to determine whether IL-2 played any role in the induction of Ig secretion in the present system. Accordingly, resting B cells were cultured with CD28/CD3-preactivated T cells in the presence or the absence of anti-CD40L, anti-IL-2, or both. Supernatants were collected after 7 days and assayed for Ig by ELISA (Table IIGo). As expected, B cells cultured on their own or in the presence of IL-2 did not secrete significant levels of IgM, whereas those cultured with primed T cells secreted substantial levels, which were not affected by the addition of IL-2. The addition of either anti-IL-2 or anti-CD40L abrogated IgM secretion. These results therefore demonstrate that the combination of CD40L/CD40 interactions plus IL-2 is required for the differentiation of activated B cells to high level IgM-secreting cells. We also obtained comparable data with some switched isotypes (IgG2b and IgG2a; data not shown), but these studies were hampered by cross-reactions of the isotype-specific sera used in the ELISA with the mAb included in the cultures. A detailed analysis of the influence of IL-2 on isotype switching will therefore be the subject of future investigations.


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  		Table II. Effects of blocking CD40L/CD40 interaction, or neutralizing IL-2 on the capacity of activated T cells to induce IgM secretion by B cells

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is now clear that CD4 T cells first encounter Ag on dendritic cells, such as those in the T cell areas of peripheral lymphoid organs, which constitutively express B7-1 and 2 (CD80 and CD86) and other accessory molecules and hence provide essential costimulatory signals to T cells via CD28 (reviewed in Refs. 34 and 35). This interaction is believed to result in the expression of CD40L, so that subsequent engagement of these T cells with B cells bearing the cognate peptide induces CD40-mediated B cell activation. Signals via CD40, in conjunction with appropriate cytokines, then direct the complex program of B cell proliferation, Ig secretion, isotype switching, and the generation of B memory cells (reviewed in Refs. 2 and 33). In the present study we have used a polyclonal two-stage culture system to investigate some of the events that occur during the initial phases of this process. We show that primary T cells require CD28 costimulation to become competent helper-effector cells, in agreement with published data (20, 36). These cells can be induced to express CD40L in the absence of CD28/B7 interactions (36, 37, 38). However, we have previously demonstrated that CD28 costimulation stabilizes the expression of the CD40L and results in the more rapid re-expression of the protein after the restimulation of primed T cells (20). We now show that T cells primed via CD3 alone eventually reexpress as much CD40L as CD28/CD3-primed T cells, are not anergic, and yet still fail to induce B cell proliferation (Fig. 1Go). Clearly, CD3-primed T cells lack an essential costimulus (or stimuli) necessary for B cell activation. We provide evidence that the principal costimulus in this system is IL-2, which synergizes with CD40-mediated signals to drive both B cell proliferation and IgM secretion. The evidence for this conclusion is as follows. Firstly, highly purified B cells harvested from WSC cultures stimulated with anti-CD3 responded to IL-2 in a dose-dependent fashion (Fig. 3Go), and the induction of IL-2 responsiveness was substantially blocked by including either anti-IL-2 or anti-CD40L in the priming cultures (Fig. 4Go). Similarly, anti-IL-2 or anti-CD40L dramatically inhibited the secretion of IgM by B cells cocultured with preactivated T cells (Table IIGo). T cells primed via CD3 alone secrete minimal levels of IL-2 and are less effective than CD28/CD3-primed T cells in inducing B cells to become IL-2 responsive (Fig. 5Go). The capacity of CD3-primed T cells to activate B cells can be substantially restored by the inclusion of IL-2 in the readout cultures and can be completely restored by restimulation via CD28 (Fig. 7Go). Contact of naive B cells with CD28/CD3-primed T cells induces B cells to express high levels of IL-2R{alpha}, while CD3-primed T cells were less effective. However, in both instances the induction of IL-2R was substantially blocked by inclusion of anti-CD40L, and in the case of CD28/CD3-primed T cells, neutralization of IL-2 also reduced their capacity to up-regulate IL-2R{alpha} on B cells (Fig. 6Go). Taken together, these results indicate that CD40L/CD40 interactions plus IL-2 rapidly induce naive B cells to become IL-2 responsive and that this cytokine then plays a key role in driving B cell proliferation and IgM secretion. To our knowledge this is the first demonstration that the CD40L on activated primary T cells, in conjunction with IL-2, results in the induction of IL-2 responsiveness in naive mouse B cells.

It has been known for many years that IL-2 is somehow involved in T-dependent B cell activation (24, 25, 26, 39, 40, 41). However, there have been no systematic studies that have attempted to place the role of IL-2 into a physiologic context, given our current understanding of the central importance of CD40/CD40L interactions in the above process. It is clear that B cells need to be activated to become IL-2 responsive and that not all activating stimuli will induce responsiveness. Hence, Mond et al. (42) demonstrated that large (i.e., preactivated) B cells proliferate in response to IL-2, while B cells activated by soluble anti-Ig do not. Similarly, Zubler et al. (40) showed that induction of IL-2 responsiveness of mouse B cells required preactivation by LPS plus anti-Ig; the latter was not sufficient to induce responsiveness. Of particular relevance to our observations is the study by Forman and Pure (26), who showed that B cells activated by alloreactive Th cells become IL-2 responsive. Induction of responsiveness to this growth factor has also been attributed to various cytokines and stimuli, including IL-4, IL-5, or IL-2 and anti-Ig (32, 39, 43, 44). In one study, IL-2 itself was reported to induce responsiveness via up-regulation of the IL-2R ß-chain (45). More recently, Moreau and co-workers (32) have produced clear evidence that murine B cells constitutively express IL-2R ß- and {gamma}-chains, whereas the IL-2R {alpha}-chain is inducible. They demonstrated that priming with soluble anti-Ig and IL-2 was required for B cell acquisition of IL-2 responsiveness, presumably via the induction of IL-2 R{alpha}, thereby resulting in the expression of a fully functional, high affinity IL-2R. In the present study we confirm earlier observations (27) that ligation of CD40 induces IL-2R{alpha} expression on B cells. However, blocking either CD40L/CD40 interactions or IL-2 during the induction phase of the present culture system substantially diminished the ability of B cells to respond to this cytokine and reduced their levels of IL-2R{alpha} (Figs. 4Go and 6Go). This indicates that IL-2 is itself required for the optimal induction of IL-2 responsiveness of B cells in addition to signaling via CD40. We therefore conclude that CD28 costimulation of primary CD4 T cells has at least two important consequences: 1) it stabilizes the expression of CD40L (20); and 2) it optimizes the secretion of IL-2, which acts in concert with CD40L to induce the B cells to up-regulate IL-2R and subsequently proliferate and differentiate to IgM-secreting cells in response to IL-2.

Earlier studies on the nature of the cytokines that regulate CD40-generated B cell activation in the mouse demonstrated a major role for IL-4 and IL-5, while IL-2 had no discernible impact on the response elicited by mAb against CD40, T cell clones, or CD40L-transfected cell lines (46, 47, 48). A discrepancy between the effects of anti-CD40 mAb and CD40L-bearing T cells was first identified by Blanchard et al. (49), who reported that CD3-preactivated T cell clones induced human B cells to become IL-2 responsive, whereas IL-2 played no discernible role in B cell activation induced by anti-CD40 presented on CD32-transfected fibroblasts. This may reflect the prolonged signaling via CD40 induced by mAb or transfectants, in contrast to short lived signaling initiated via the CD40L, which is rapidly down-regulated following contact with CD40 (15). Whatever the explanation, such discrepancies do raise fundamental questions about the physiologic relevance of using anti-CD40 or CD40L transfectants to study physiologic T cell-B cell collaboration, especially responses involving primary T cells, which express much lower levels of CD40L than preactivated T cell clones (8, 9). Indeed, CD40L transfectants and some anti-CD40 mAbs are powerfully mitogenic for B cells in the absence of cytokines, whereas we show here that CD3-primed CD40L+ T cells are not, unless the cultures are supplemented with exogenous IL-2 (Fig. 7GoA).

The effects of IL-2 in the present system are clearly dependent on the presence of CD40L, indicating that while the CD40L is not sufficient to drive B cell proliferation (when expressed at physiologic levels on primary T cells), it is required for the induction of IL-2 responsiveness. Furthermore, the experiments with B cells purified from WSC cultures suggest that a key consequence of CD40L/CD40 interactions is the induction of IL-2 responsiveness in B cells, since these cells responded extremely well to IL-2 alone (Figs. 3Go and 4Go). However, when preactivated T cells were cultured with resting B cells, B cell proliferation was significantly inhibited by adding either anti-CD40L or anti-IL-2, even as late as 48 h (Fig. 7GoB). This suggests that when T cells and B cells remain in contact, continuing stimulation via CD40 also contributes to IL-2-mediated B cell proliferation. In additional experiments (not shown) we observed that B cells that had been preactivated by CD28/CD3-primed T cells gave additive proliferative responses when restimulated with anti-CD40 plus IL-2. Interestingly, these cells were remarkably hyper-responsive to restimulation by anti-CD40 alone, suggesting that priming via CD40 initiates some type of feedforward effect, which deserves further study.

The physiologic relevance of these findings is supported by the fact that failure to produce IL-2 is a (rare) cause of severe combined immunodeficiency in man (50, 51, 52). In addition to the expected T cell proliferative defects, these patients were also hypogammaglobulinemic and failed to mount normal Ab responses when exposed to naturally occurring infections. This is in contrast to the phenotype of IL-2-deficient mice, which have elevated levels of TD Ig isotypes such as IgG1 both before and after immunization with TD Ag (53) (A. Schimpl, unpublished observation). This may be because of the lymphoproliferative disorder and overproduction of Th2-type cytokines in these animals. However, it is noteworthy that their CD4 T cells were also defective as helper cells, as judged by their capacity to induce IgM secretion by anti-Ig-stimulated B cells, and this defect was corrected by the addition of IL-2 (53).

We therefore propose the following hypothesis for the sequence of events that may occur during the initiation of a TD Ab response. T cells first recognize Ag on CD80/CD86-bearing interdigitating/dendritic cells in the T cell areas of lymphoid tissues (reviewed in 34 . This encounter, by engaging CD28 (and other accessory molecules, such as LFA-1/ICAM (38, 54)), should therefore lead to stable expression of CD40L and maximize the production of IL-2. T cells first interact with B cells in the T cell areas as well (55), and we propose that a key event at this stage is the up-regulation of functional IL-2R on B cells via a combination of CD40L- and IL-2-mediated signals, and subsequent CD40- and IL-2-mediated B cell proliferation and differentiation to (at least) IgM-secreting cells. These conclusions are consistent with immunohistochemical data showing the colocalization of IL-2-producing T cells and activated B cells in the T cell areas of lymphoid tissues (56, 57). In addition, CD40-activated dendritic cells may play a crucial role at this stage, based on the recent demonstration that these cells induce naive B cells to secrete large amounts of IgM in the presence of IL-2 (58). The present results also provide an explanation for several earlier studies that invoked an essential role for IL-2 in the induction of primary TD Ab responses in vitro (24, 25, 26, 27, 41). The scenario we propose obviously does not preclude an important role for other cytokines, especially in the process of isotype switching to IgG1 or IgE (in the case of IL-4) and IgG2a and IgG2b (in the case of IFN-{gamma}) (reviewed in 59 . However, we believe that these cytokines are involved in later stages of the response. In addition, in the current study we have not addressed how signals generated through the B cell Ag receptor may impinge upon the process of naive B cell activation by primary T cells. It will obviously be interesting to ascertain how CD40L- and IL-2-generated activating signals interact with the above costimuli to regulate the complex processes of isotype switching and the formation of B memory cells.


   Acknowledgments
 
We are grateful to Chris Atkins for performing many of the FCM analyses during this study. We are also indebted to Drs. J. Abrams, J. Allison, G. Butcher, R. Noelle, H. Waldmann, and F.Melchers for gifts of reagents.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Gerry G. B. Klaus, Division of Cellular Immunology, National Institute for Medical Research, Mill Hill, London NW7 1AA, U.K. Back

2 Abbreviations used in this paper: TD, T cell-dependent; CD40L, CD40 ligand; PE-SA, phycoerythrin-conjugated streptavidin; FCM, flow cytometric; WSC, whole spleen cells; MFI, median fluorescence intensity. Back

Received for publication March 25, 1998. Accepted for publication June 29, 1998.


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