TY - JOUR
AU1 - Yasui, Teruhito
AU2 - Muraoka, Masaaki
AU3 - Takaoka-Shichijo, Yuko
AU4 - Ishida, Isao
AU5 - Takegahara, Noriko
AU6 - Uchida, Junji
AU7 - Kumanogoh, Atsushi
AU8 - Suematsu, Sachiko
AU9 - Suzuki, Misao
AU1 - Kikutani, Hitoshi
AB - Abstract CD40 is essential for efficient humoral immune responses. CD40 has two cytoplasmic domains required for binding of tumor necrosis factor receptor-associated factors (TRAF). The TRAF6-binding site is within the membrane proximal cytoplasmic (Cmp) region, while a PXQXT motif in the membrane distal cytoplasmic (Cmd) region needs to engage TRAF2/3/5. To dissect CD40 signals necessary for B cell differentiation, we generated transgenic mice expressing wild-type and mutant human CD40 (hCD40) molecules in a mouse CD40-deficient (mCD40−/−) background. The B cell-specific expression of hCD40 in mCD40−/− mice resulted in T-dependent antibody responses including germinal center (GC) formation. Mutant hCD40 molecules that carry either a point mutation of the TRAF2/3/5-binding site or a deletion of the Cmd region rescued extrafollicular B cell differentiation but not GC formation. A mutant hCD40 that comprises of only the TRAF2/3/5-binding site in the cytoplasmic region also rescued low but significant titers of antigen-specific IgG1 without GC formation. These results demonstrated that two distinct signals either from the Cmp or from the Cmd region induced the extrafollicular B cell differentiation and Ig class switching; however, GC formation required both. We conclude that combinations of these two signals determine which of the extrafollicular or the follicular (GC) differentiation pathway B cells enter. B lymphocyte, germinal center, Ig class switching, signal transduction, tumor necrosis factor receptor-associated factor, transgenic mouse AFC antibody-forming cell, APC antigen-presenting cell, CD154-CHO CD154-expressing CHO, CGG chicken γ-globulin, Cmd membrane distal cytoplasmic, Cmp membrane proximal cytoplasmic, DC dendritic cell, GC germinal center, hCD40 human CD40, ICAM-1 intracellular adhesion molecule-1, Jak3 Janus kinase 3, LPS lipopolysaccharide, LMP1 latent membrane protein 1, mCD40 mouse CD40, PALS periarteriolar lymphoid sheath, PNA peanut agglutinin, TD T-dependent, Tg transgenic, TLR Toll-like receptor, TNF tumor necrosis factor, TNFR TNF receptor, TRAF TNFR-associated factor Introduction The development of a high-affinity antibody response depends on the coordinate interaction of B cells, T cells, dendritic cells (DC) and follicular dendritic cells. B cell differentiation requires that B cells first interact with antigen-specific T cells in the extrafollicular area of the lymph nodes or spleen. These antibody-forming cells (AFC) secrete only low-affinity antibodies that have not undergone affinity maturation. A subset of antigen-specific B cells migrates into the lymphoid follicle and forms the germinal center (GC) where T cells and follicular DC provide signals that induce affinity maturation and Ig class switching (1–3). Many members of the tumor necrosis factor (TNF) and receptor (TNFR) superfamily are crucially involved in the adaptive humoral immune response and in the peripheral lymphoid organogenesis (4). Of this superfamily, CD40 is a 45–50-kDa glycoprotein that is preferentially expressed on both B cells and specialized antigen-presenting cells (APC), such as DC and macrophages. In B cells, CD40 ligation leads to proliferation and in combination with IL-4 leads to Ig class switching. Further effects of CD40 ligation include adhesion, preventing IgM- or Fas-mediated apoptosis, and inducing expression of CD23, CD80 (B7-1), CD86 (B7-2), CD95 (Fas), CD54 [intracellular adhesion molecule-1 (ICAM-1)] and MHC class II (5,6). The phenotype of CD40-deficient (mCD40−/−) or CD154-deficient mice and the features of patients with X-linked hyper-IgM syndrome caused by mutations of the CD154 gene have demonstrated a critical role of CD40 in B cell development (7–14). Like other TNFR family members, CD40 utilizes various TNFR-associated factors (TRAFs) such as TRAF2, TRAF3, TRAF5 and TRAF6 for its signal transduction (15). There are two major TRAF-binding sites in the cytoplasmic region of CD40. A PXQXT motif comprising residues 250–254 of CD40 is required for binding to TRAF2, TRAF3 and TRAF5 (16–21). A threonine to alanine substitution at position 254 abolishes TRAF2 and TRAF3 binding, and changes the growth regulation by CD40 (15,22). Furthermore, a 17-amino-acid sequence comprising residues 250–266 restores TRAF2-mediated NF-κB activation (23). On the other hand, TRAF6, which is also known to be involved in signaling of IL-1 and lipopolysaccharide (LPS), can interact with residues 231–238 of CD40 (24–27). Recent gene-targeting experiments have demonstrated the physiological roles of TRAF molecules. TRAF2−/−, TRAF3−/− and TRAF6−/− mice are runted at birth, and died within several weeks (26–29). Reconstitution in mice with TRAF3−/− fetal liver cells has shown that TRAF3 is required for T-dependent (TD) humoral responses; however, these defects are CD40 independent (28). In vitro responses of B lymphocytes from TRAF2−/−, TRAF5−/−, TRAF6−/− or TRAF2-dominant-negative transgenic (Tg) mice have revealed that these molecules are required for CD40 signaling (26,30–32). However, it remains to be clarified how TRAF molecules and their respective binding sites of CD40 are involved in in vivo B cell differentiation and humoral immune responses. Here we describe Tg mice expressing human CD40 (hCD40) or the mutants that fail to interact with TRAFs to assess their role in B cell differentiation and effector function. Methods Mice and immunizations mCD40−/− mice were maintained as described previously (11). For generating Tg mice, hCD40 cDNA and the mutant hCD40 cDNA were prepared as described previously (22). Tg expression vector (pEmVhp) was constructed by first inserting the mouse rearranged H chain promoter derived from pMO-μ4C8 clone into the XbaI–SmaI site of pBluescript II KS(+) (Stratagene, La Jolla, CA) and then ligating the mouse H chain enhancer (Eμ) into the SacI–XbaI site (33). Finally, the SalI-splicing cassette of rabbit γ-globin from the BCMGneo vector was inserted into the XhoI site of this construct (34). The fragment of wild-type or mutant hCD40 cDNA was cloned into the XhoI site of pEmVhp. Purified transgenes were microinjected into BDF1 or C57BL/6 mice-derived fertilized eggs as described previously (35). Mice were backcrossed to C57BL/6 mice or mCD40−/− mice of a C57BL/6 background and the offspring were screened for the transgenes by PCR with the tail DNA. Mice (10 weeks old) were immunized i.p. with 100 μg of DNP-ovalbumin (OVA) in complete Freund's adjuvant or with 150 μg of NP-conjugated chicken γ-globulin (NP-CGG) in alum-precipitated suspension and analyzed 7–14 days later as indicated in the text. Flow cytometric analysis Cells were prepared from spleens and red blood cells were lysed before staining. Multi-color flow cytometry of mononuclear cells was performed on a FACS Vantage or on a FACSCalibur with CellQuest software (Becton Dickinson, San Jose, CA). The following antibodies and reagents used in a flow cytometric analysis were purchased from PharMingen (San Diego, CA): FITC–anti-hCD40 (5C3), biotin–anti-mCD40 (3/23), FITC– or biotin–anti-CD45R/B220 (RA3-6B2), phycoerythrin–anti-CD138 (Syndecan-1; 281-2), FITC–anti-CD90. 2 (Thy1.2; 53-2.1), FITC–anti-CD11b (anti-Mac-1; M1/70), FITC–Gr-1 (RB6-8C8) and biotin–anti-IgG1 (A85-1). Cell preparation and cell culture To isolate small resting B cells, B cells were purified from spleens of normal and Tg mice by in vitro treatment with anti-Thy 1.2 mAb (F7D5; Serotec, Oxford, UK) followed by complement treatment (rabbit Low-Tox-M; Cedarlane, Hornby, Ontario, Canada). Surviving cells were placed on a Percoll step gradient and cells on the 66/70% interface were recovered as the small resting B cells. B cells were cultured in RPMI 1640 medium supplemented with 10% FCS, 50 μM 2-mercaptoethanol, 2 mM l-glutamine and antibiotics. Determination of Ig concentration Antigen-specific serum Ig levels or in vitro Ig production were determined by ELISA as described previously (36). Briefly, to determine the level of in vitro Ig production, high-density B cells (1×105 cells) stimulated by 100 U/ml IL-4 (Genzyme, Cambridge, MA) + CD154-expressing CHO (CD154-CHO) cells (1×105 cells) were cultured in a 96-well plate for 7 days. Isotypic Ig concentration of the cultured supernatant was measured by ELISA. Ninety-six-well flat-bottomed plates were coated with purified goat polyclonal anti-mouse IgM and IgG1 antibodies (Southern Biotechnology Associates, Birmingham, AL) respectively. Plates treated with blocking buffer were incubated with a series of dilutions of supernatants and purified Ig as standards. After washing, plates were incubated with alkaline phosphatase-conjugated, affinity-purified polyclonal anti-mouse IgM and IgG1 (Southern Biotechnology Associates). For measurement of NP-specific antibody from sera, Total and high-affinity NP-specific antibodies were determined employing an ELISA with either 12 NP-haptenated BSA (NP12-BSA) or 2 NP-haptenated BSA (NP2-BSA)-conjugated 96-well plates. Isotype-specific antibodies were determined as described above. Standard antibodies for anti-NP-specific IgM and IgG1 were kindly provided by Dr Azuma (Science University of Tokyo, Japan) Immunohistochemistry Spleens were embedded in OCT compound (Sakura, Tokyo, Japan) and were cut on a cryostat as 6-μm thick longitudinal sections. For detection of GC in spleen, sections were doubly stained with anti-mouse IgM antibody conjugated with FITC (PharMingen) and biotinylated peanut agglutinin (PNA) (Honen, Tokyo, Japan), followed by streptavidin–Texas Red (Gibco, Carlsbad, CA). For detection of periarteriolar lymphoid sheath (PALS)-associated foci, FITC–anti-λ1 L chain (Southern Biotechnology Associates) and biotin–anti-IgG1 antibodies (PharMingen) were used, followed by streptavidin–Texas Red. Results Production of mice expressing either wild-type or mutant hCD40 on B cells Tg mice were generated expressing wild-type hCD40 or mutants directed by the mouse Ig enhancer and promoter (Fig. 1A). CD40del221 is a deletion mutant of CD40 lacking the whole cytoplasmic region. CD40del252 preserves the Janus kinase 3 (Jak3)- and TRAF6-binding site in the membrane proximal cytoplasmic (Cmp) region but deletes the TRAF2/3/5-binding site in the membrane distal cytoplasmic (Cmd) region (Fig. 1B). Tg mice were maintained in a CD40 null background by crossing with mCD40−/− mice (CD40tg/mCD40−/−mice). Flow cytometric analysis of spleen cells prepared from the Tg mice showed that hCD40 molecules were expressed specifically on B220+ B cells and the expression levels of Tg CD40 were comparable to that of endogenous mouse CD40 (mCD40) on normal B cells (Fig. 1C). However, Tg hCD40 molecules were not expressed on other APC such as DC and macrophages where endogenous mCD40 is expressed (data not shown). In addition, unlike endogenous mCD40, whose expression is lost or suppressed upon plasma cell differentiation, Tg hCD40 was abundantly expressed on CD138 (Syndecan-1)+ AFC that were induced in vitro by LPS (data not shown). Such expression patterns of Tg hCD40 are probably due to the fact that they were driven by the Ig promoter/enhancer. Regulation of B cell response by Cmp region of CD40 Small resting B cells were prepared from mice expressing wild-type hCD40 or its mutant molecules and stimulated with CD154-CHO cells in vitro. B cells from CD40wt/mCD40−/− and CD40del252/mCD40−/− mice as well as normal mice could proliferate in response to CD154 stimulation, while those from CD40del221/mCD40−/− B cells and mCD40−/− B cells could not (Fig. 2A). B cells were further cultured with CD154-CHO cells in the presence of IL-4 to assess their ability to produce Ig. As shown in Fig. 2(B), comparable levels of IgM and IgG1 were produced by CD40wt/mCD40−/− B cells, CD40del252/mCD40−/− B cells and normal B cells. CD40del221/mCD40−/− B cells could produce neither IgM nor IgG1 in response to CD154 stimulation as expected. These results indicated that hCD40 could induce proliferation, Ig production and Ig class switching in mouse B cells in vitro. Furthermore, signals through Cmp region of CD40 including the TRAF6- and Jak3-binding site was sufficient for CD154-induced proliferation and production of IgM and IgG (Fig. 2B). CD40 stimulation enhances expression of various surface molecules such as CD23, CD54/ICAM-1, CD80/B7-1, CD86/B7-2 and CD95/Fas in B cells. Expression levels of these activation markers on Tg B cells were investigated after ligation of CD40. The analysis of cell surface expression also indicated that CD40wt and CD40del252 as well as mCD40 could induce comparable expression levels of activation markers on B cells. Thus, induction of cell surface markers by CD40wt or CD40del252 was indistinguishable from mCD40 (Fig. 2C). These results indicated that hCD40 signaling could induce B cell proliferation, Ig production and Ig class switching in a manner indistinguishable from those signals transduced via mCD40. Restoration of TD antigen-driven Ig production accompanied by Ig class switching in CD40del252/mCD40−/−mice The humoral immune response was evaluated in mCD40−/−, CD40wt/mCD40−/−, CD40del252/mCD40−/− and CD40del221/mCD40−/− mice by immunizing with TD antigen, NP-CGG. Mice were bled and anti-NP-specific serum antibody titers were assayed. Total and high-affinity anti-NP antibody titers were determined by binding to antigens with either a high (NP12-BSA) or low (NP2-BSA) haptenation ratio respectively. Anti-NP IgM titers in serum at 14 days after immunization were observed in all immunized mice including mCD40−/− mice (Fig. 3A). CD40wt/mCD40−/− mice produced similar levels of total anti-NP IgG1 compared with mCD40+/+ mice. In contrast, no anti-NP IgG1 was detected in either CD40del221/mCD40−/−or mCD40−/− mice. CD40del252/mCD40−/− mice produced anti-NP IgG1 although their titers were significantly lower than those of CD40wt/mCD40−/− mice (Fig. 3B). The affinity of anti-NP antibodies in CD40wt/mCD40−/− mice was comparable to that in mCD40+/+ mice. CD40del252/mCD40−/− mice produced antibodies of relatively low affinity (Fig. 3C). These results suggest that CD40del252 could rescue Ig class switching but could not completely rescue affinity maturation in the mCD40−/− background. PALS-associated AFC differentiation with Ig class switching in CD40tg/mCD40−/−mice It is known that AFC positive for λ1 L chain, some of which express γ H chains, can be generated and form clusters along T cell-rich PALS of spleen during early primary responses to NP-CGG in mice of the IgHb allotype (37–41). We analyzed the ability of these mice to form PALS-associated B cell foci in spleen. Histological analysis showed that splenic architecture was normal in CD40tg/mCD40−/− mice including mCD40−/− mice (data not shown). Seven days after NP-CGG immunization, sections from spleen were doubly stained with antibody to the λ1 L chain characteristic of an anti-NP response. Typical λ1+ foci were formed at the outer margin of PALS of spleens of mCD40+/+ mice and some of λ1+ AFC were also stained with anti-γ1. Only λ1+ γ1− AFC were found to form small foci along the PALS in spleens of CD40del221/mCD40−/− as well as mCD40−/− mice. As expected from the results of anti-NP antibody titers in serum, λ1+γ1+ foci in spleens from both CD40wt/mCD40−/− and CD40del252/mCD40−/− mice were rescued, revealing that extrafollicular AFC differentiation in spleen is driven by signals through the CD40 Cmp region (Fig. 4A). Defective GC formation in CD40del252/mCD40−/−mice Efficient humoral immune responses are totally dependent on GC where selection and proliferation of high-affinity B cells and the generation of memory B cells take place. Therefore, GC formation was examined in these mutant mice. Ten days after DNP-OVA immunization, sections from spleen were prepared and doubly stained with PNA and anti-IgM. GC indistinguishable from those of immunized normal mice were observed in spleens of CD40wt/mCD40−/− mice. On the other hand, such a characteristic GC was not observed in spleen sections of CD40del252/mCD40−/− mice although a small number of PNA+ cells were scattered in follicles. No GC or PNA+ cells were seen in follicles of CD40del221/mCD40−/− and mCD40−/− mice (Fig. 4B). The generation of antigen-specific memory-like or GC B cells was quantitatively analyzed. Splenocytes from each mutant mouse were prepared 14 days after immunization and analyzed for binding to NP-hapten or PNA and expression of membrane IgG1. Similar frequencies of NP-binding and membrane IgG1+ (NP+IgG1+) cells were clearly observed in CD40wt/mCD40−/− (0. 6%) as well as mCD40+/+ mice (0.7%). Furthermore, NP+PNA+ B cells were also detected in these mice. In contrast, such cells were not detected in the splenocytes of mCD40−/−, CD40del221/mCD40−/− and CD40del252/mCD40−/− mice (Fig. 5). TRAF2-, TRAF3- and TRAF5-binding site on CD40 Cmd region is necessary but not sufficient for induction of GC formation To further pinpoint a site in the CD40 Cmd region required for GC formation, two more Tg lines expressing mutant CD40 molecules were generated. One expressed the mutant CD40 carrying the threonine to alanine substitution at position 254 (CD40T254A). The other line expressed a mutant CD40 comprised of amino acids 250–266 in the cytoplasmic region (CD40IC17). These mice were maintained in the mCD40−/− background (CD40T254A/mCD40−/− mice and CD40IC17/mCD40−/−mice). The expression levels of mutant molecules in these two Tg lines were comparable to those in other Tg lines such as CD40wt, CD40del252 and CD40del221 Tg mice (data not shown). As shown in Fig. 6 these two mutants of CD40 but not endogenous mCD40 were expressed specifically in B220+ B cells of CD40 Tg mice respectively. Analysis of the immune response to NP-CGG showed that CD40T254A/mCD40−/− mice produced 10-fold less anti-NP IgG1 antibodies compared to CD40+/+ mice (Fig. 7A). CD40T254A expression failed to rescue production of high-affinity antibodies in the mCD40−/− background (Fig. 7B). This pattern of Ig responses is very similar to that observed in CD40del252/mCD40−/− mice (Fig. 3). CD40IC17/mCD40−/− mice produced 100-fold less anti-NP IgG1 than control mice (Fig. 7A and B). These mice were also defective in the generation of memory-type (NP+IgG1+) or GC-type (NP+PNA+) B cells (Fig. 8). GC formation was not detected in either CD4T254A/mCD40−/− or CD40IC17/mCD40−/− mice (data not shown). Discussion We attempted in this study to rescue humoral immune responses of mCD40−/− mice by Tg expression of hCD40 molecules and to analyze the role of CD40 signals in B cells for in vivo antibody responses. We chose human cDNA as the transgene for the following reasons. (i) Mouse CD154 equally binds both mCD40 and hCD40 to transduce signals (42). (ii) Immunological phenotypes of mCD40−/− or CD154-deficient mice and clinical characteristics of CD154-deficient patients are almost identical, suggesting that signals via both mCD40 and hCD40 exert similar or identical biological functions (7–14). (iii) The cytoplasmic region of hCD40 has been well characterized in terms of signal transduction (22). (iv) The expression of Tg hCD40 can be readily distinguished from that of endogenous mCD40 by anti-hCD40 mAb. Like many other members of the TNFR family, major signal transducers of CD40 are TRAF molecules. CD40 recruits TRAF2, TRAF3 and TRAF5 to the Cmd region, and TRAF6 to the Cmp region respectively. Several in vitro studies with cell lines expressing mutant CD40 have revealed two distinct pathways in the CD40 signaling: one dependent on TRAF2 and TRAF5, and the other on TRAF6 (16–21,24,43–45). It was unknown how these two distinct signals were involved in activation of normal primary B cells or in vivo humoral immune responses. However, targeted disruption of either TRAF2 or TRAF6 severely affected CD40-mediated proliferation and NF-κB activation in B cells (26,31). Humoral immune responses were also impaired in TRAF2−/− mice (31). Although these data suggested that both TRAF2 and TRAF6 are necessary for CD40 signaling, TRAF molecules are involved in signals not only of CD40 but also of many other TNFR family members (15,26,31). Toll-like receptors (TLR) and IL-1R also utilize TRAF6 as their signal transducer (25–27). Therefore, all the observed defects of immune responses in these mutant mice cannot be simply attributed to CD40 signals that were hampered by deficiency of each TRAF molecule. In the present study, we attempted to reconstitute CD40–CD154 interactions in the mCD40−/− mice by Tg expression of wild-type hCD40 and various mutant hCD40 molecules on B cells to dissect signals of CD40. Reconstitution of wild-type hCD40 on B cells resulted in normal B cell responses including Ig production, the formation of GC and extrafollicular foci in spleen against TD antigen in mCD40−/− mice. We have further showed that GC formation requires signals through both Cmp and Cmd regions of CD40, although the Cmd region was not necessary for the induction of PALS-associated foci and Ig class switching. These results have clearly dissected CD40 signals required for both extrafollicular and follicular B cell differentiation pathways. Either deletion of the Cmd region of CD40 or mutation of the TRAF2/3/5-binding site attenuated the GC formation without affecting the extrafollicular B cell differentiation. It has been reported that inactivation of some transcription factors results in the impairment of GC formation and reduced IgG responses, which is similar to immunological phenotypes of CD40del252/mCD40−/− and CD40T254A/mCD40−/− mice. OBF-1/OCA-B/Bob1, a B cell-specific transcription co-activator of Oct-1 and Oct-2, is known to be involved in the transcription of Ig genes (46–48). OBF-1/OCA-B/Bob1 can be induced by combinations of CD40 and other stimuli such as IL-4 or anti-IgM and deficiency of this protein in mice leads to loss of GC formation but intact extrafollicular B cell differentiation (49–51). Therefore, OBF-1/OCA-B/Bob1 may play a role in downstream events of signals via the TRAF2/3/5-binding site of CD40. A transcription repressor factor, Bcl-6, is preferentially expressed in the GC B cells. Like OBF-1/OCA-B/Bob1−/− mice, mice with the disrupted Bcl-6 gene have no GC cells and severely impaired humoral immune response (52–54). Interestingly, it has been reported that Bcl-6 blocks the transcriptional activation mediated by CD40 and IL-4 (55). Considering the fact that CD40 tranduces signals for both extrafollicular and follicular B cell differentiation, Bcl-6 may be involved in counteracting the signals to induce the former. However, unlike OBF-1/OCA-B/Bob1, Bcl-6 is not induced in B cells by CD40 stimulation (51). Thus, it remains to be clarified whether CD40 signals are directly responsible for induction of Bcl-6 in GC B cells in vivo. Although the Cmd region of CD40, particularly the TRAF2/3/5-binding site, was necessary for induction of GC, the TRAF2/3/5-binding site alone was not sufficient for the development of GC since CD40IC17/mCD40−/− mice failed to form GC in response to TD antigen. It thus appears that a combination of signals from both Cmp and Cmd regions is critical for the effective GC formation. In our preliminary experiments, double Tg mice expressing both CD40del252 and CD40IC17 in the mCD40−/− background failed to form GC even though immunized with high doses of TD antigens (data not shown). Taking all these results into account, both Cmp and Cmd regions may cooperate on the same CD40 molecule to synthesize a signal required for GC formation. Both CD40del252 and CD40T254A rescued Ig class switching in mCD40−/− mice. More importantly, CD40IC17/mCD40−/− mice also produced antigen-specific IgG antibodies. The collected results indicate that Ig class switching can be mediated by signals either from the Cmp or from the Cmd region. A putative major signal transducer from the Cmp region, TRAF6 is also utilized by TLR4 that is critical for LPS responses. LPS is well known to induce both in vitro and in vivo antibody responses accompanied by Ig class switching. TRAF2 binds not only to the Cmp region of CD40 but also to the cytoplasmic domain of latent membrane protein 1 (LMP1) that is one of the transforming gene products of Epstein–Barr virus (15). We have recently shown that the Tg expression of LMP1 in B cells rescues IgG production upon immunization with TD antigen to a certain extent in mCD40−/− mice (36). These observations suggest that either TRAF6 or TRAF2 as signal transducers of CD40 can mediate a signal for Ig class switching. Analysis of in vivo antibody responses both from CD40T254A/mCD40−/− and from CD40IC17/mCD40−/− mice also indicates that the Cmp region mediates a more potent inducing signal for IgG production than the Cmd region. There are several possible explanations for this. CD40 has been reported to recruit Jak3 and Ku70 through its Cmp region (56,57). These molecules may be also involved in differentiation of B cells. Alternatively, TRAF6 may be a more potent inducer toward the differentiation of B cells to AFC than TRAF2, because a binding site of TRAF6 but not TRAF2/3/5 in the CD40 cytoplasmic region has been recently shown to be necessary for CD40-induced IL-6 and Ig production in mouse B cell lines (45). It is known that defective CD40–CD154 interaction between APC and T cells results in impairment of T cell priming and Th cell function (58). In the present study, the Tg expression of hCD40 on B cells could rescue TD humoral immune responses, which includes the generation of GC in mCD40−/− mice, although CD40 is not expressed on the professional APC like as DC in these mice. Furthermore, transfer of B220+CD40+ B cells into mCD40−/− mice could restore both IgG production and PNA+ B cells bearing IgG (data not shown). It appears that the CD40-expressing B cells can compensate defective functions of CD40−/− professional APC particularly for in vivo antibody responses. In summary, the reconstitution of CD40 by Tg expression in the gene-knockout background has allowed us to dissect CD40 signals for GC formation and Ig class switching. Several CD40-inducible gene products such as OBF-1/OCA-B/Bob1, CD100 and activation-induced cytidine deaminase have been shown to be critically involved in GC formation, Ig class switching or somatic hypermutation (49–51,59–62). The mice generated in the present study will be good tools not only to delineate the extrafollicular and follicular B cell differentiation, but also to identify which of CD40 signals are responsible for the activation of these genes. Fig. 1. View largeDownload slide Tg expression of hCD40 in mice. (A) Schematic structure of Tg for the expression of hCD40 under the control of the mouse H chain promoter and intronic enhancer. (B) Schematic representation and characterization of hCD40 variants and the molecules associated with the cytoplasmic region of CD40. The Cmp region can interact with Jak3 and TRAF6; in contrast, the Cmd region can interact with TRAF2/3/5. (C) B cell-specific expression of hCD40 in mCD40−/− mice. Splenocytes from CD40tg/mCD40−/−, mCD40−/− and mCD40+/+ mice were stained with mAb and analyzed by flow cytometry. The data shown are for an individual mouse and are representative of results obtained from the littermates. Fig. 1. View largeDownload slide Tg expression of hCD40 in mice. (A) Schematic structure of Tg for the expression of hCD40 under the control of the mouse H chain promoter and intronic enhancer. (B) Schematic representation and characterization of hCD40 variants and the molecules associated with the cytoplasmic region of CD40. The Cmp region can interact with Jak3 and TRAF6; in contrast, the Cmd region can interact with TRAF2/3/5. (C) B cell-specific expression of hCD40 in mCD40−/− mice. Splenocytes from CD40tg/mCD40−/−, mCD40−/− and mCD40+/+ mice were stained with mAb and analyzed by flow cytometry. The data shown are for an individual mouse and are representative of results obtained from the littermates. Fig. 2. View largeDownload slide Responsiveness of CD40tg/mCD40−/− B cells to CD40 stimulation. (A) In vitro proliferative responses of B cells from mCD40−/− mice bearing hCD40 transgenes. Small resting B cells from each mouse spleen were cultured in the presence (black bar) or absence (open bar) of CD154-CHO cells for 72 h. Subsequent proliferation was measured. (B) CD40wt/mCD40−/− B cells produce both IgM and IgG1, and CD40del252/mCD40−/− B cells also cause CD40-dependent Ig class switching. Small resting B cells from each spleen were cultured without (open bar) or with either IL-4 (black bar), CD154-CHO cells (gray bar) or IL-4 plus CD154-CHO (hatched bar). Isotype-specific Ig production was measured by ELISA. (C) Surface expression of B cell activation markers on CD40tg/mCD40−/− B cells after CD40 stimulation. Splenic B cells were stimulated with 10 μg/ml 5C3, anti-hCD40 (closed histogram) or control Ig (dotted histogram) for 48 h, followed by surface staining with fluorescence-conjugated antibodies against CD23, CD54, CD80, CD86 and CD95. The x-axis indicates the fluorescence intensity in arbitrary units and the y-axis indicates the relative number of cells. Fig. 2. View largeDownload slide Responsiveness of CD40tg/mCD40−/− B cells to CD40 stimulation. (A) In vitro proliferative responses of B cells from mCD40−/− mice bearing hCD40 transgenes. Small resting B cells from each mouse spleen were cultured in the presence (black bar) or absence (open bar) of CD154-CHO cells for 72 h. Subsequent proliferation was measured. (B) CD40wt/mCD40−/− B cells produce both IgM and IgG1, and CD40del252/mCD40−/− B cells also cause CD40-dependent Ig class switching. Small resting B cells from each spleen were cultured without (open bar) or with either IL-4 (black bar), CD154-CHO cells (gray bar) or IL-4 plus CD154-CHO (hatched bar). Isotype-specific Ig production was measured by ELISA. (C) Surface expression of B cell activation markers on CD40tg/mCD40−/− B cells after CD40 stimulation. Splenic B cells were stimulated with 10 μg/ml 5C3, anti-hCD40 (closed histogram) or control Ig (dotted histogram) for 48 h, followed by surface staining with fluorescence-conjugated antibodies against CD23, CD54, CD80, CD86 and CD95. The x-axis indicates the fluorescence intensity in arbitrary units and the y-axis indicates the relative number of cells. Fig. 3. View largeDownload slide Serum antibody responses of CD40tg/mCD40−/− mice. Immunization was performed with either NP-CGG (A, B and C) as described in Methods. mCD40+/+ (filled circles), mCD40−/− (open circles), CD40wt/mCD40−/− (filled triangles), CD40del221/mCD40−/− (open triangles) and CD40del252/mCD40−/− (filled diamonds) mice were bled 14 days after immunization. Levels of antigen-specific antibodies of a particular isotype were determined by ELISA. (A) NP-specific IgM responses. (B) NP-specific total IgG1 responses. (C) Affinity of IgG1 antibody to NP. Because Anti-NP IgG1 production was not detectable in both mCD40−/− and CD40del221/mCD40−/− mice, the ratio is defined as zero in (C). Individual symbols indicate data from each mouse. Bars show the mean of each experimental group. Fig. 3. View largeDownload slide Serum antibody responses of CD40tg/mCD40−/− mice. Immunization was performed with either NP-CGG (A, B and C) as described in Methods. mCD40+/+ (filled circles), mCD40−/− (open circles), CD40wt/mCD40−/− (filled triangles), CD40del221/mCD40−/− (open triangles) and CD40del252/mCD40−/− (filled diamonds) mice were bled 14 days after immunization. Levels of antigen-specific antibodies of a particular isotype were determined by ELISA. (A) NP-specific IgM responses. (B) NP-specific total IgG1 responses. (C) Affinity of IgG1 antibody to NP. Because Anti-NP IgG1 production was not detectable in both mCD40−/− and CD40del221/mCD40−/− mice, the ratio is defined as zero in (C). Individual symbols indicate data from each mouse. Bars show the mean of each experimental group. Fig. 4. View largeDownload slide PALS-associated focus and GC response in the spleen of CD40tg/mCD40−/− mice after TD antigen stimulation. (A) CD40wt/mCD40−/− and CD40del252/mCD40−/− mice show antigen-specific PALS-associated focus formation accompanied with Ig class switching. Mice were immunized with NP-CGG, and spleens were taken at 7 days after immunization and sectioned for immunohistochemistry. Sections were doubly stained with anti-λ1 (green) and anti-Thy1.2 (red) as shown in the left panels or with anti-λ1 (green) and anti-γ1 (red) in the right panels. (B) GC was present in spleen from CD40wt/mCD40−/− mice but not from CD40del252/mCD40−/− mice and CD40del221/mCD40−/− mice at 10 days after DNP-OVA immunization. Immunofluorescence microscopy was performed to detect GC formation by staining with both PNA (red) and anti-IgM (green). Scale bar represents 100 μm. Fig. 4. View largeDownload slide PALS-associated focus and GC response in the spleen of CD40tg/mCD40−/− mice after TD antigen stimulation. (A) CD40wt/mCD40−/− and CD40del252/mCD40−/− mice show antigen-specific PALS-associated focus formation accompanied with Ig class switching. Mice were immunized with NP-CGG, and spleens were taken at 7 days after immunization and sectioned for immunohistochemistry. Sections were doubly stained with anti-λ1 (green) and anti-Thy1.2 (red) as shown in the left panels or with anti-λ1 (green) and anti-γ1 (red) in the right panels. (B) GC was present in spleen from CD40wt/mCD40−/− mice but not from CD40del252/mCD40−/− mice and CD40del221/mCD40−/− mice at 10 days after DNP-OVA immunization. Immunofluorescence microscopy was performed to detect GC formation by staining with both PNA (red) and anti-IgM (green). Scale bar represents 100 μm. Fig. 5. View largeDownload slide Differentiation of antigen-specific GC B cells in CD40tg/mCD40−/− mice. Mice were immunized i.p. with 100 μg NP-CGG and spleens were subjected to the flow cytometric analysis at 14 days after immunization. Data shown as dot-plots in the population of lymphocytes as defined by forward and side scatters, and also by surface expression in the absence of IgM, IgD, Thy1.2, Mac-1 and Gr-1. Percentages of either NP-binding+ or IgG1+ in the quadrants or NP-binding+ and PNA+ B cells in the squares relative to total spleen cells are given. Fig. 5. View largeDownload slide Differentiation of antigen-specific GC B cells in CD40tg/mCD40−/− mice. Mice were immunized i.p. with 100 μg NP-CGG and spleens were subjected to the flow cytometric analysis at 14 days after immunization. Data shown as dot-plots in the population of lymphocytes as defined by forward and side scatters, and also by surface expression in the absence of IgM, IgD, Thy1.2, Mac-1 and Gr-1. Percentages of either NP-binding+ or IgG1+ in the quadrants or NP-binding+ and PNA+ B cells in the squares relative to total spleen cells are given. Fig. 6. View largeDownload slide Tg expression of the mutant CD40 on B cells in both CD40T254A/mCD40−/− and CD40IC17/mCD40−/− mice. Splenocytes from each mouse were stained with anti-hCD40/B220 or anti-hCD40/anti-mCD40. Cells were analyzed by flow cytometry. Data shown as dot-plots in the population of the lymphocytes gated by forward and side scatters. Fig. 6. View largeDownload slide Tg expression of the mutant CD40 on B cells in both CD40T254A/mCD40−/− and CD40IC17/mCD40−/− mice. Splenocytes from each mouse were stained with anti-hCD40/B220 or anti-hCD40/anti-mCD40. Cells were analyzed by flow cytometry. Data shown as dot-plots in the population of the lymphocytes gated by forward and side scatters. Fig. 7. View largeDownload slide Antigen-specific Ig response in CD40T254A/mCD40−/− and CD40IC17/mCD40−/− mice. Control mCD40+/+ (filled circles), mCD40−/− (open circles), CD40T254A/mCD40−/− (filled triangles) or CD40IC17/mCD40−/− (open triangles) mice were immunized i.p. with 100 μg NP-CGG. Blood from each mouse was bled 14 days after immunization and ELISA was performed to determine the concentration of total anti-NP IgG1 (A) and anti-NP affinity ratios (B). Individual symbols indicate data from each mouse. Bars show the mean of each experimental group. Fig. 7. View largeDownload slide Antigen-specific Ig response in CD40T254A/mCD40−/− and CD40IC17/mCD40−/− mice. Control mCD40+/+ (filled circles), mCD40−/− (open circles), CD40T254A/mCD40−/− (filled triangles) or CD40IC17/mCD40−/− (open triangles) mice were immunized i.p. with 100 μg NP-CGG. Blood from each mouse was bled 14 days after immunization and ELISA was performed to determine the concentration of total anti-NP IgG1 (A) and anti-NP affinity ratios (B). Individual symbols indicate data from each mouse. Bars show the mean of each experimental group. Fig. 8. View largeDownload slide Defective differentiation of antigen-specific GC B cells in both CD40T254A/mCD40−/− and CD40IC17/mCD40−/− mice. Mice were immunized i.p. with 100 μg NP-CGG and splenocytes were subjected to the flow cytometric analysis at 7 days after immunization. Methods are according to Fig. 4. Fig. 8. View largeDownload slide Defective differentiation of antigen-specific GC B cells in both CD40T254A/mCD40−/− and CD40IC17/mCD40−/− mice. Mice were immunized i.p. with 100 μg NP-CGG and splenocytes were subjected to the flow cytometric analysis at 7 days after immunization. Methods are according to Fig. 4. Transmitting editor: T. Watanabe We would like to thank Takachika Azuma (Science University of Tokyo, Japan) for the generous gift of anti-NP mAb, Tasuku Honjo (Kyoto University, Japan) for mouse H chain genomic DNA clone, and Micah A. Luftig and Ellen Cahir-McFarland (Harvard Medical School, MA) for helpful discussion and critical reading of the manuscript. We also thank K. Kubota for excellent secretarial assistance and K. Shiozaki for technical assistance. 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TI - Dissection of B cell differentiation during primary immune responses in mice with altered CD40 signals
JF - International Immunology
DO - 10.1093/intimm/14.3.319
DA - 2002-03-01
UR - https://www.deepdyve.com/lp/oxford-university-press/dissection-of-b-cell-differentiation-during-primary-immune-responses-t0xPYz73bK
SP - 319
EP - 329
VL - 14
IS - 3
DP - DeepDyve
ER -