Augmented Antibody Response with Premature Germinal Center Regression in CD40L Transgenic Mice

The CD40 molecule, a member of the TNFR family, is expressed on various cell types including B cells, macrophages, and dendritic cells (DCs) (1, 2). The ligand for CD40 (CD40L) is a member of the TNF family mainly expressed on activated Th cells as a transmembrane protein. CD40 ligation in vitro promotes survival and proliferation of B cells (3, 4) and induces the maturation of DCs, resulting in enhanced Ag presentation capacity through increased surface expression of MHC class II (MHC-II) and costimulatory molecules, such as CD80 and CD86 (5, 6). Moreover, studies on mice deficient in CD40 or CD40L demonstrated that CD40/CD40L is crucial for T cell-dependent humoral immune responses and cellular immune responses, especially the generation of CTLs (5–8). These findings strongly suggest that CD40 transmits activation and/or differentiation signals crucial for immune responses in B cells and DCs.

In human systemic lupus erythematosus (SLE) patients and SLE-prone BXSB mice, B cells ectopically express CD40L (9–11). CD40L is also expressed in EBV-infected B cells and plays a role in their in vitro transformation (12). To assess in vivo immunological function of CD40L on B cells, we established CD40L transgenic (CD40LTg) mice that constitutively express CD40L on B cells and demon-strated that CD40LTg mice spontaneously develop SLE-like autoimmune disease (13), suggesting that constitutive CD40L expression on B cells has a role in the development of SLE. Recently, Hömig-Hölzel et al. (14) established transgenic mice that express EBV la-tent protein membrane 1/CD40 chimera on B cells that generates constitutive CD40 signaling and demonstrated that these mice develop B cell lymphomas. Thus, CD40 signaling seems to play a crucial role in autoimmunity and leukemogenesis, as well as normal immune responses.

During immune responses, Ag-activated B cells differentiate to short-lived plasma cells that form foci in the extrafollicular region or form germinal centers (GCs) by rapid proliferation, followed by differentiation to memory B cells and long-lived plasma cells (15, 16). Patients with X-linked hyper-IgM syndrome that lack expression of CD40L and mice deficient in CD40 or CD40L do not form GCs nor generate memory responses, clearly demonstrating that CD40/CD40L is crucial for GC formation and the differentiation of memory B cells. However, studies by Erickson et al. (17) on mice in vivo treated with anti-CD40 Ab generated an unexpected result; this treatment augments Ab production but ablates GC reaction. They concluded that CD40 signaling directs B cells to extrafollicular plasma cell fate rather than GC formation. To address the role of in vivo CD40 signaling in B cell differentiation, we analyzed the immune response of CD40LTg mice that express CD40L on B cells. Although GCs rapidly regressed after day 5 and did not generate late-appearing high-affinity Abs, GC forma-tion at the early phase of the immune response was intact histo-logically and functionally, including affinity maturation and generation of long-lived plasma cells and memory B cells. Thus, CD40 signaling does not block GC formation; rather, it blocks the growth and maintenance of GCs, probably by regulating the balance of proliferation versus differentiation. This finding may be important in understanding the role of CD40 signaling in normal immune responses and autoimmune responses in SLE patients.

CD40LTg mice (13), CD40^−/− mice (7), and Bcl-6^−/− mice (18) were described elsewhere. CD40LTg mice with BALB/c background were obtained by backcrossing more than eight generations. Mice were main-tained under specific pathogen-free conditions in the animal facility in the Medical Research Institute, Tokyo Medical and Dental University (Tokyo, Japan). In some experiments, we used mice maintained under cleaner conditions, in which pathogens are more strictly controlled. Eight- to 12-wk-old mice were used in all experiments. Mice were immunized i.p. with chicken γ-globulin (CGG) coupled with the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) precipitated with alum. In some experiments, 100 μg agonistic anti-CD40 mAb (FGK45; kindly provided by Dr. A. Rolink (University of Basel, Basel, Switzerland) (19) was given i.v. to normal mice as a control for CD40 ligation. Bone marrow chimera were established by reconstituting sublethally irradiated CB17-scid/scid mice with 2 × 10⁷ bone marrow cells.

Cells were treated with anti-CD16/CD32 mAb 2.4G2 to block nonspecific binding of IgG to cell surface via the low-affinity receptors for the Fc portion of IgG. Cells were then reacted by the following reagents: FITC-labeled anti-CD3 mAb, PE- and FITC-labeled anti-B220 mAb, FITC-labeled anti-GL-7 mAb, biotin- and PE-labeled anti-IgG₁ mAb, FITC- and PE-labeled anti-IgM mAb, FITC-labeled anti-CD21, FITC-labeled anti-CD11c, biotin-labeled anti-CD23, PE- and biotin-labeled anti–MHC-II mAb, PE-labeled anti-CD86 mAb, PE-labeled anti-CD80, PE-labeled anti-CD95 mAb, PE- and biotin-labeled anti-CD138, FITC-labeled anti-BrdU mAb, PerCP-labeled streptavidin, and Cy-Chrome-labeled streptavidin (all from BD Pharmingen, San Diego, CA); PE-labeled streptavidin (DakoCytomation, Glostrup, Denmark), and NP-conjugated allophycocyanin. Cells were analyzed by a FACSCalibur or BD-LSR flow cytometer (BD Biosciences, San Jose, CA). Detection of BrdU⁺ cells and measurement of DNA contents by DAPI staining were done as described previously (20).

Plastic-immobilized anti-IgM Ab was prepared as described previously (4), using F(ab′)₂ fragments of goat anti-mouse IgM Ab (Cappel, Aurora, OH). Small dense B cells were purified from mouse spleen, as described previously (4), and were cultured in RPMI 1640 medium (Life Technologies, Rockville, MD) supplemented with 10% FCS, 50 μM 2-ME, and 2 mM l-glutamine, with or without immobilized anti-IgM Ab. The numbers of live and dead cells were counted by trypan blue exclusion.

Spleens from immunized mice were embedded in OCT compound (Sakura Finetechnical, Tokyo, Japan) and rapidly frozen in liquid nitrogen. Frozen sections were stained with biotin-labeled peanut agglutinin (PNA) (Vector Laboratories, Burlingame, CA), followed by staining with alkaline phosphatase-labeled streptavidin (BD Pharmingen) and HRP-labeled anti-IgD mAb (Southern Biotechnology Associates, Birmingham, AL). Sections were washed and incubated with 0.1 mM Tris-HCl (pH 8.2) containing Fast Red (Boehringer Mannheim, Mannheim, Germany), followed by incubation with 0.1 M Tris-HCl (pH 7.2) containing 0.0035% H₂O₂ and diaminobenzidine.

Total amounts of IgM and IgG were measured by standard sandwich ELISA analysis using goat anti-IgM or IgG Ab (Southern Biotechnology Associates). For measuring the amounts of total and high-affinity anti-NP IgG, ELISA plates were coated with NP₁₅-BSA and NP_2.5-BSA, respectively. Ab bound to the plates was detected using alkaline phosphatase-labeled anti-IgG, anti-IgM, anti-IgMa, anti-IgMb, anti-IgG1a, or anti-IgG1b mAb (BD Pharmingen). ELISPOT assay was done as described previously (21).

To induce CTLs against male Ags, female C57BL/6 mice were inoculated i.p. with 2 × 10⁶ splenocytes from male C57BL/6 mice. After 14 d, spleen cells were prepared and depleted of B cells by MACS sorting using anti-IgM and anti-B220 mAbs. B cell-depleted splenocytes (2 × 10⁶) were restimulated with irradiated (2000 rad) spleen cells (2 × 10⁶) from male C57BL/6 mice in 24-well plates for 6 d. Cells were harvested, and cytotoxic activity against LPS blasts from male C57BL/6 mice was measured by the JAM test, as described elsewhere (6).

DCs were purified by sorting CD11c⁺ cells, using AutoMACS, from spleen cells of C57BL/6 mice or CD40LTg mice with C57BL/6 background and treated with 10 mg mitomycin C at 37°C for 30 min. Mitomycin C-treated DCs (2 × 10³) were cultured in round-bottom 96-well plates with T cells purified by nylon wool column from spleens of BALB/c and C57BL/6 mice. After 3 d, proliferation of cells was assessed by the WST-1 Cell Proliferation Assay System (Takara Shuzo, Otsu, Japan).

Bone marrow cells were stained with PE-labeled anti-CD138 mAb. CD138⁺ cells were sorted using a FACSVantage (BD Biosciences). V_H genes containing a V_H186.2 segment were amplified by RT-PCR, as described elsewhere (22), except that 10 and 20 amplification cycles were applied to the first and second rounds of PCR, respectively. Amplified fragments were cloned using the pGEM-T easy vector system (Promega, Madison, WI), and nucleotide sequences were determined by an ABI 310 DNA sequencer (Applied Biosystems, Foster City, CA). The error rate of Taq polymerase in our experimental conditions was <1 in 5 × 10³ bp. Cluster analysis of amino acid sequences of V_H genes was done as described elsewhere (23).

We examined the cell numbers and activation status of various lymphocyte subsets from thymus, spleen, bone marrow, and lymph nodes by flow cytometry. The number of T cells was normal in spleen (Fig. 1A, 1B), lymph nodes, and thymus (data not shown) in CD40LTg mice. Mice also exhibited normal numbers of pre-B cells, immature B cells, and transitional B cells, but they showed 2-fold increase in the number of follicular B cells in spleen (Fig. 1A, 1B) and lymph nodes (data not shown). In CD40LTg mice, CD40L was constitutively expressed on B cells, but it was not detected on other cell types (data not shown). CD40LTg B cells were mostly quiescent and expressed normal levels of activation markers, such as CD86, CD23, and CD95, except that expression of the MHC-II molecule was slightly enhanced (Fig. 1C, 1D). MHC-II expression was increased in follicular and marginal zone B cells (data not shown). These results indicate that B cells in CD40LTg mice do not spontaneously undergo activation or proliferation. In contrast, purified spleen B cells from CD40LTg mice exhibited reduced levels of spontaneous and BCR-mediated cell death in vitro (Fig. 1E), indicating that survival of CD40LTg B cells is enhanced probably as a result of constitutive survival signaling through CD40 (3, 4). These results suggest that the increased number of follicular B cells in these mice is not due to their proliferation but probably is due to prolonged survival by constitutive CD40 signaling.

We next assessed Ab production in CD40LTg mice. In unimmunized mice, serum IgM and IgG levels were increased by 4–6-fold in CD40LTg mice (Fig. 2A). In contrast, the mice maintained under cleaner conditions exhibited a significant, but weaker, increase in serum Ig levels and the numbers of bone marrow Ab-forming cells (AFCs) of IgM class but no increase in those of IgG class (Fig. 2B, 2C), although these mice showed a 2-fold increase in the number of follicular B cells (data not shown), as was the case for the mice maintained under less clean conditions. These results suggested that CD40LTg mice respond to microbial stimulation, leading to augmented Ab production.

After immunization with 10 μg NP-CGG, CD40LTg mice produced 5–10 times more anti-NP Abs of IgM and IgG classes than the normal littermates (Fig. 2D). Enhancement of the Ab production in CD40LTg mice was less prominent when mice were immunized with 100 μg of Ag (Fig. 2D), probably because stronger endogenous CD40L expression induced by a larger amount of the Ag may reduce the effect of transgenic CD40L expression. Moreover, CD40LTg mice exhibited increased AFC generation at day 7 of immunization (Fig. 2E), suggesting enhanced differentiation of Ag-activated B cells to extrafollicular plasma cells.

To determine whether CD40L on B cells stimulates neighboring B cells, irradiated CB17-scid/scid mice were transferred with bone marrow cells from WT or CD40LTg BALB/c mice (IghA), together with the same number of bone marrow cells from CB17 mice (IghB), resulting in CB17+BALB/c and CB17+BALB/c-CD40L mixed bone marrow chimera, respectively. These chimera contain roughly equal numbers of B cells from CB17 mice and from WT or CD40LTg BALB/c mice (Fig. 2F). We immunized these chimera with NP-CGG and examined Ab production from CB17 B cells and from BALB/c B cells separately by allotype-specific ELISA. The levels of anti-NP IgMA and IgG1A produced by CD40LTg BALB/c B cells in CB17+BALB/c-CD40L chimera was three to five times greater than those derived from nontransgenic BALB/c B cells in CB17+BALB/c chimera (Fig. 2G). In contrast, the production of NP-specific IgMB and IgG1B derived from CB17 B cells was nearly equivalent between CB17+BALB/c and CB17+BALB/c-CD40L chimera, indicating that Ab production from CD40L⁻CB17 B cells was not enhanced, even in the presence of coexisting CD40L⁺ B cells. Thus, CD40L on B cells appears to stimulate B cells preferentially by an autocrine pathway in CD40LTg mice, and it is unlikely that CD40L on B cells enhances Ab production by stimulating neighboring B cells or through enhancement of Ag presentation by CD40⁺ APCs.

To exclude the possibility that CD40L on B cells stimulates DCs, we examined the Ag-presentation capacity of DCs from CD40LTg mice. Although DCs from anti-CD40 mAb-treated mice showed an increased expression of MHC-II and costimulatory molecules (CD80 and CD86) and enhanced MLR, these were not enhanced in DCs from CD40LTg mice (Fig. 3A, 3B). To assess the in vivo activation of DCs, we examined CTL generation against male Ags, because activated DCs efficiently generate CTLs in vivo (5, 24, 25). Indeed, mice treated with agonistic anti-CD40 mAb showed enhanced CTL induction. In contrast, in vivo CTL induction was not enhanced in CD40LTg mice (Fig. 3C). These results indicated that the Ag-presentation capacity of DCs is not augmented in CD40LTg mice and suggested that CD40L on B cells does not stimulate DCs. Taken together, CD40L on B cells enhances Ab response by stimulating B cells through an autocrine pathway, but it does not stimulate neighboring B cells or APCs. The increased number of follicular B cells might be involved in enhanced Ab production in CD40LTg mice. However, the finding that the bone marrow chimera carrying roughly the same number of CD40LTg and CD40L⁻ B cells (Fig. 2F) exhibit augmented Ab production from CD40LTg B cells (Fig. 2G) strongly suggests that CD40L enhances Ab production by augmenting B cell responses to Ags.

To address the GC reaction in CD40LTg mice, we immunized them and their normal littermates with 100 μg NP-CGG in alum and examined GC formation immunohistologically. At day 5 of immunization, CD40LTg spleen formed small but detectable GCs, and their size was almost equivalent to GCs in normal spleen at the same time point (Fig. 4A). However, spleens of CD40LTg mice showed only rudimentary GCs at days 8 and 14 after immunization, whereas the same treatment generated large GCs in normal spleens. Thus, GCs rapidly regressed after day 5 of immuni-zation in CD40LTg mice. Flow cytometry showed that the percentage and absolute number of GL-7⁺B220⁺ GC B cells in spleen increased from days 5–14 in the wild-type (WT) littermates (Fig. 4B, 4C), but they decreased from days 5–8 in CD40LTg mice. Essentially, the same results were obtained by immunization with 10 μg of NP-CGG (Fig. 4A, 4B). Taken together, GCs are generated at the early phase of immune responses but regress rapidly thereafter in CD40LTg mice.

To address how much the GC reaction contributes to Ab response in CD40LTg mice, we crossed CD40LTg mice with Bcl-6–deficient mice, because they completely lack GCs in peripheral lymphoid organs but show normal extrafollicular B cell responses (18, 26). When immunized with 10 μg NP-CGG, Bcl-6–deficient CD40LTg mice produced a much smaller amount of Ab than did CD40LTg mice (Fig. 4D), indicating that a major part of the primary Ab re-sponse in CD40LTg mice requires Bcl-6, a key inducer of GC reaction. Essentially the same result was obtained when they were immunized with 100 μg NP-CGG (data not shown). Thus, aug-mented Ab responses in CD40LTg mice appear to largely depend on small GCs.

GC reaction is involved in the generation of memory B cells and long-lived AFCs (15, 16). CD40LTg mice exhibited a normal number of NP-specific bone marrow plasma cells at day 45 after immunization (Fig. 5A, 5B). When we treated mice with BrdU from days 7–14 after immunization, ~60% of the NP-binding CD138⁺IgG⁺ plasma cells were BrdU⁺ in bone marrow from CD40LTg mice and their WT littermates (Fig. 5B, 5C). This indicated that the majority of AFCs in CD40LTg mice at day 45 is generated at the early phase of immunization and, thus, is long-lived and suggests that generation of long-lived AFCs is normal in these mice. Moreover, CD40LTg mice exhibited a normal secondary Ab response to NP-CGG (Fig. 5D), indicating that generation of memory B cells is not impaired in CD40LTg mice. Taken together, GCs do not grow after day 5 of immunization, but they do generate plasma cells and memory B cells in a normal fashion.

In mice with the IghB haplotype, anti-NP response is dominated by λ-bearing Abs, whose V_H region is encoded by the V_H186.2 gene (27). To examine the V gene repertoire in CD40LTg mice in response to NP, CD138⁺ plasma cells were purified from bone marrow of CD40LTg (C57BL/6 background) mice and their littermates 45 d after immunization with NP-CGG in alum (Fig. 6A). We isolated 14 and 13 V_H genes that use the V_H186.2 segment and have the unique CDR3 sequences from WT and CD40LTg mice, respectively. In V_H genes from normal mice and those from CD40LTg mice, somatic mutations were present in all of the V_H genes, except for one each (Fig. 6B), confirming that bone marrow AFCs at day 45 are generated through GC reaction. There was no significant difference between CD40LTg mice and WT littermates in the frequency of the V_H genes in which tryptophan at position 33 is replaced by leucine (Trp³³ → Leu) (Fig. 6C), a key mutation that increases the affinity to NP (28). This result suggests that high-affinity V_H genes are generated and positively selected in CD40LTg GCs. Indeed, CD40LTg mice showed normal Ag-driven affinity maturation (Fig. 6E). However, the average number of mutations per V_H gene was drastically reduced in CD40LTg mice (Fig. 6C), regardless of whether they contained the Trp³³ → Leu mutation. Thus, CD40LTg mice generate high-affinity Abs without accumulating many somatic mutations.

Furukawa et al. (23) demonstrated that at the late stage of anti-NP response, high-affinity Abs containing V_H186.2 with the Trp³³ → Leu mutation are replaced by V_H186.2-containing high-affinity Abs that lack the key mutation. These late-appearing Abs acquire high affinity by accumulating many somatic mutations in V_H in conjunction with distinct CDR3. The presence of distinct CDR3 indicates that these V_H mutants are developed by a distinct pathway from high-affinity V_H genes containing the Trp³³ → Leu mutation. The pathways through which Abs accumulate somatic mutations can be determined by the pattern of amino acid substitutions using cluster analysis and be expressed as a phylogenic tree (23). Thus, we performed cluster analysis of amino acid sequences of the V_H genes containing the V_H186.2 segment obtained from CD40LTg mice and the normal littermates, as well as those obtained from hybridomas generated from normal mice, at various time points after immunization with NP. Late-appearing V_H mutants were clustered in a distinct branch, probably because of their distinct sequences, especially those in CDR3 (Fig. 6D). This branch included 4 of 14 V_H genes from WT mice, whereas none of the V_H genes from CD40LTg mice were present in this branch. This result indicates that CD40LTg mice fail to generate late-appearing high-affinity Abs. Taken together, CD40LTg mice show normal-affinity maturation by positively selecting early-appearing high-affinity B cells containing the Trp³³ → Leu mutation, but they fail to accumulate somatic mutations in Ig V genes or to generate late-appearing high-affinity Abs that require many somatic mutations.

We demonstrated, using CD40LTg mice, that CD40L expression on B cells increases the number of mature B cells and enhances Ab response. However, CD40L on B cells does not enhance Ab pro-duction from coexisting CD40L⁻ B cells in mixed bone marrow chimera. This indicates that CD40L on a B cell stimulates the same B cell via an autocrine pathway, but it fails to enhance Ab production from other B cells by directly ligating their CD40 or by enhancing APC activity to Th cells through ligation of CD40 on DCs. The inability of CD40L on B cells to stimulate DCs is confirmed by our finding that APC activity is not enhanced in DCs in CD40LTg mice in vivo and in vitro. Taken together, CD40L on B cells enhances Ab production by an autocrine pathway. This is the first demonstration that a membrane-bound ligand preferentially generates autocrine signaling via its receptor on the same cell. In our transgenic mice, CD40 signaling was preferentially generated in B cells, whereas treatment with agonistic anti-CD40 Ab activated B cells and APCs. Therefore, our transgenic mice are useful for analyzing the roles of CD40 signaling in B cells in in vivo immune function, such as maturation and selection of B cells.

By analyzing immunized CD40LTg mice, we demonstrated that GCs grow only up to day 5 in these mice, followed by rapid regression. Early regression of GCs by CD40 signaling is consistent with the previous observation that GC formation is ablated by stimulation with agonistic anti-CD40 Ab or constitutive CD40 signaling (14, 17). Although previous studies did not examine the early GC responses, we demonstrated that generation of early GCs up to day 5 is not defective in CD40LTg mice and presented evidence strongly suggesting that early GCs in CD40LTg mice are functional. First, these mice show normal or even enhanced Ab response in a manner dependent on Bcl-6. Because Bcl-6 is required for GC formation but not extrafollicular responses (18, 26), Ab production is mostly mediated by AFCs derived from GCs in CD40LTg mice. Second, B cell responses requiring GCs, such as memory B cell response and generation of high-affinity long-lived AFCs (15, 16), are not impaired in CD40LTg mice. In contrast, the frequencies of somatic mutations of V_H genes are reduced, and late-appearing high-affinity Abs that accumulate somatic mutations (23) are not generated in these mice, suggesting that the long-term function, such as generation of late-appearing high-affinity Abs, is defective. Interestingly, the VH sequences of CD40LTg mice 45 d after immunization (Fig. 6B, 6C) are similar to those of early bone marrow plasma cells (22); both carry a disproportionately high frequency of the Trp³³ → Leu mutation in V_H186.2, with relatively few overall mutations, supporting early termination of GC reaction in CD40LTg mice. Taken together, functional GCs are generated at the early phase of immune responses but are prematurely regressed in the presence of constitutive CD40 signaling.

Based on the present findings, the effect of CD40 signaling in GC reaction needs to be revisited. Erickson et al. (17) suggested that CD40 signaling ablates GC formation by blocking differentiation of Ag-activated B cells into GC B cells and directs Ag-stimulated B cells into extrafollicular response. However, we found that early GC formation is normal, suggesting that CD40 signaling does not block B cell differentiation to GC B cells. Rather, CD40 signaling may block expansion of GC B cells during immune responses. This assumption is supported by the recent findings by Shimoda et al. (29). They established IAB mice in which the MHC-II iab gene is conditionally deleted. In these mice, a small number of B cells express MHC-II because of incomplete cd19-cre–driven deletion, and these B cells, but not MHC-II⁻ B cells, respond to T cell-dependent Ags, thereby generating GCs and producing Abs at the usual level. However, the kinetics of the response is delayed, probably because extensive B cell proliferation is required to compensate for the reduced MHC-II⁺ B cell number. Interestingly, generation of GC B cells and Ab production were markedly re-duced in IAB/CD40LTg mice compared with CD40LTg mice (30). Thus, B cell response requiring extensive B cell proliferation seems to be defective in the presence of constitutive CD40 sig-naling, suggesting that CD40 signaling blocks sustained expansion of GC B cells.

Our finding fits with the previous one that CD40 signaling downregulates Bcl-6 (31–33), a transcriptional repressor regula-ting genes, such as PRDM1 encoding Blimp1, which is essential for plasma cell differentiation, and p53 and ATM involved in inhibition of the genotoxic stress response (34–36). Inhibition of the genotoxic stress response is thought to be crucial for proliferation of GC B cells in the presence of genotoxic stress, induced by rapid cell division, and DNA breaks induced by somatic hypermutation and class-switch recombination (15). Thus, downregulation of Bcl-6 may play an important role in CD40-mediated downmodulation of B cell expansion and augmentation of B cell differentiation. The dark zone of GCs is largely devoid of T cells or other accessory cells that express CD40L (37), and GC B cells mostly lack CD40 gene-expression signature (33). Thus, lack of CD40 signaling in the dark zone may allow centroblasts to extensively expand without differentiation, whereas CD40-mediated cell cycle inhibition and differentiation may take place in the light zone when centrocytes exit GCs and differentiate to plasma cells and memory B cells.

We thank Dr. A. Rolink for reagent, Dr. H. Yagita (Juntendo University, Tokyo) for discussions, Dr. N. Ohtsuki (Tokyo Medical and Dental University) for technical advice, and Dr. T. Kojima, S. Fujimoto, and S. Irie for technical assistance.

Disclosures The authors have no financial conflicts of interest.