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The Journal of Immunology, 2000, 165: 5530-5536.
Copyright © 2000 by The American Association of Immunologists

Inhibition of IgG1 and IgE Production by Stimulation of the B Cell CTLA-4 Receptor1

Claudio Pioli2,*, Lucia Gatta{dagger}, Vanessa Ubaldi* and Gino Doria{dagger}

* Section of Toxicology and Biomedicine, Ente per le Nuove Tecnologie, l’Energia et l’Ambiente C.R. Casaccia; and {dagger} Department of Biology, University of Tor Vergata, Rome, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although a large amount of information is available on the activity of CTLA-4 in T cells, the role of this receptor in B cells has not been previously characterized. Our results show that CD40 or LPS stimulation in the presence of IL-4 induces CTLA-4 expression in purified B cells; the maximum level is reached in both membrane and intracellular compartments after 48–72 h. Engagement of the B cell CTLA-4 by immobilized mAb inhibits IgG1 and IgE production and reduces the frequency of IgG1- and IgE-expressing B cells. C{epsilon} and C{gamma}1 germline mRNA expression as well as NF-{kappa}B and STAT6 activation, events required for isotype switching, are also inhibited by CTLA-4 engagement. Together these findings show the critical role of CTLA-4 in the control of IL-4-driven isotype switching and suggest new approaches for modulating immediate-type hypersensitivity responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The negative role exerted by CTLA-4 on T cell activity has been widely described (1, 2). T cell CTLA-4 engagement by its ligands CD80 and CD86 expressed on APCs inhibits cytokine production (IL-2, IFN-{gamma}, IL-4, and others), IL-2R {alpha}-chain expression, and cell cycle progression (3, 4). CTLA-4 blockade enhances T cell responses to Ags (5) and tumors (6) and exacerbates autoimmune diseases (7, 8). Recently, a role for CTLA-4 in allograft survival (9, 10) and in the maintenance of peripheral tolerance (11, 12) has also been suggested, as well as an association between CTLA-4 polymorphism and predisposition to type I diabetes (13), Grave’s disease (14), and Addison’s disease (15). CTLA-4 knockout (CTLA-4-/-) mice show an elevated frequency of T cells expressing activation markers, massive lymphoproliferative disorders, and autoimmune diseases leading to death at 3–4 wk of age (16, 17). In these mice, the B cell population with activated phenotype is expanded also. All Ig isotypes show higher basal serum levels compared with wild-type mice, ranging from 10-fold for IgM, IgG2a, IgG2b, and IgG3, to 100-fold for IgG1 and IgA, to several thousandfold for IgE (17). The increased Ig serum level may be due to lack of CTLA-4-mediated negative signals delivered to Th cells or B cells. Although it has been reported that CTLA-4 expression can be induced in B cells by activated T cells (18) and that it is constitutively expressed in B non-Hodgkin’s lymphomas (19), the role of B cell CTLA-4 has not been clarified. In the present study we have investigated the effects of B cell CTLA-4 stimulation on the IL-4-driven Ig production, germline mRNA expression, and activation of transcription factors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
B cell purification

B cells were purified by negative selection from spleen cells of specific pathogen-free C57BL/6 mice (Charles River Italia, Milan, Italy). T cells were removed by immunomagnetic cell sorting with anti-Thy1.2 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany, code 491-01), and the remaining cell suspension was further depleted with anti-CD4 and anti-CD8 mAb (culture supernatants from clones RLI724 and 31M, respectively), and complement (Low Tox-M, Cedarlane, Hornby, Ontario, Canada). B cells were then collected at the 1.081–1.086 g/cm3 density interface of a discontinuous Percoll gradient (Amersham-Pharmacia Biotech, Arlington Heights, IL). The resulting cell population contained <2% TCR{alpha}{beta}+ and >95% CD19+ cells, as assessed by cytofluorometric analysis.

Cell culture

B cells (5 x 105/ml) were cultured in 96- or 24-well plates with RPMI 1640 medium, 5% FCS, 2-ME, 2 mM glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml). Anti-CD40 mAb (clone HM-40-3, 1 µg/ml), LPS (1 µg/ml), and IL-4 (200 U/ml) were used at optimal concentrations, as determined in preliminary experiments. Anti-CTLA-4 hamster IgG mAb (clone UC10-4F10-11) was bound to plates at the indicated concentrations. As isotype control for the anti-CTLA-4 mAb, the anti-TNP hamster IgG (clone A19-3) was used. All the Abs used were sodium azide and endotoxin free.

ELISA

IgG1 and IgE concentrations were assessed by sandwich ELISA employing for capture the A85-3 (2 µg/ml) and R35-72 (1 µg/ml) mAb, respectively. Culture supernatants and the Ig standard (mouse IgG1 and mouse IgE, PharMingen, San Diego, CA) were serially diluted and added to wells. To detect bound IgG1 and IgE, the A85-1 (1 µg/ml) and R35-92 (2 µg/ml) biotinylated mAb were used, respectively. Avidin-peroxidase (Sigma, St. Louis, MO) and then 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (Kirkegaard & Perry Laboratories, Gaithersburg, MD) were used to perform the reaction. The reference straight line obtained by plotting the absorbance vs the standard Ig concentrations was used to calculate the IgG1 and IgE concentrations in the supernatants.

Flow cytometric analyses

Cells (5 x 105) were preincubated with Fc Block (rat IgG2b anti-CD16/32, clone 2.4G2, PharMingen, 01241D) to prevent cytophilic binding of labeled Abs and then double stained with FITC-conjugated anti-CD19 (clone 1D3, PharMingen 09654A) and PE-conjugated anti-CTLA-4 (clone UC10-4F10-11). To assess membrane-expressed Ig, PE-conjugated anti-mouse IgM (clone R6-60.2, PharMingen 02085B), FITC-conjugated anti-mouse IgG1 (1075-02, Southern Biotechnology Associates, Birmingham, AL), and biotin-conjugated anti-mouse IgE (clone R35-92, PharMingen 02122D) were used. Only IgM-IgG1+ and IgM-IgE+ cells were defined as switched cells. FITC/PE-conjugated isotype-matched Abs were used as controls. Biotin-conjugated mAb was revealed with streptavidin-CyChrome. After staining, cells were fixed with 0.37% paraformaldehyde and, for intracellular staining, permeabilized with 0.5%saponin. The optimal concentrations of the Abs were assessed in preliminary experiments. Samples of 104 cells were analyzed, and fluorescence signals were collected in log mode using a FACScan (Becton Dickinson, Mountain View, CA).

RT-PCR

Total RNA extracted by the proteinase K protocol (20) was used as template for cDNA synthesis performed by Perkin-Elmer GeneAmp RNA PCR kit. After an initial 5-min denaturation at 94°C, the cDNA was amplified for 40 cycles; each cycle was programmed for denaturation at 94°C for 45 s, annealing at 55°C for 60 s, and elongation at 72°C for 90 s. The following oligonucleotides were used as primers: {beta}-actin forward, 5'-CTGAAGTACC-CATTGAACATGGC; {beta}-actin reverse, 5'-CAGAGCAGTAATCTCCTTCTGCAT; germline {gamma}1 forward, 5'-AGCACGCATCTGTGGCCCTTCCAGATCT; germline {gamma}1 reverse, 5'-CAGGTCA-CTGTCACTGGCTCAGGGAAAT; germline {epsilon} forward, 5'-GCAGAAGATGGCTTCGAATAAGAACAGT; and germline {epsilon} reverse, 5'-TCGTTGAATGATGGAGGATGTGTCACGT.

Protein extracts and EMSA

To collect cytoplasmic extracts cell membranes were lysed in buffer A (MgCl2 2 mM, KCl 15 mM, HEPES 10 mM, EDTA 0.1 mM, DTT 1 mM, PMSF, 1 mM aprotinin, and 10 µg/ml leupeptin) containing 0.5% Nonidet P-40. To obtain nuclear extracts, nuclei were lysed in buffer B (400 mM NaCl, 20 mM HEPES, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10% (v/v) glycerol). For the EMSA 32P end-labeled double-stranded 5'-ATCAAAAGGGAACTTCCAAGGCTG-3' ({kappa}B1) and 5'-AGGCCGGGGGTTCCCACCCCACTT-3' ({kappa}B2) oligonucleotides were used to detect NF-{kappa}B dimers; 5'-GATCCTTCCCAAGAACAGAACAG-3' was used for STAT6. Nuclear protein-DNA complexes were separated on 4% polyacrylamide gels. Anti-RelA (sc-372X) and anti-p50 (sc-114X) mAb were used for supershift analyses of the NF-{kappa}B complexes; the sc-1698X mAb was used for STAT6. Gels were exposed on a phosphor screen, which was analyzed by the STORM 840 (Molecular Dynamics, Sunnyvale, CA). The intensity of the bands was directly quantified by ImageQuant (Molecular Dynamics) software, which gives rise to a volume report by integrating the area of the band and its density.

Western blot analysis

For Western blot analysis, extracts at equal protein concentrations were subjected to SDS-PAGE using a 7.5% polyacrylamide gel. After electrotransfer onto polyvinylidene difluoride membranes (Amersham), nonspecific sites were blocked by incubation of the membranes with 20 mM Tris-HCl (pH 7.4), 137 mM NaCl, and 0.1% Tween-20 containing 5% BSA (blocking solution). Immunoblots were detected with anti-I{kappa}B{alpha} (sc-371) and anti-{beta}-actin (clone AC-15) mAb according to the Ab producers. The purity of the extracts was verified as previously described (21). An anti-rabbit alkaline phosphatase-conjugated secondary Ab and the enhanced-chemifluorescence (Amersham) substrate were used to develop the reaction (chemifluorescence). Fluorescence was acquired by the phosphorimager/fluorimager STORM 840.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CTLA-4 expression in B cells

Purified murine B cells were stimulated with different combinations of anti-CD40 mAb, LPS, and IL-4 and after 1–6 days were analyzed by flow cytometry for CTLA-4 expression. Since in T cells up to 80% of CTLA-4 is localized in vesicles of the perinuclear Golgi compartment (22), we analyzed the expression of this receptor in B cells both on the cell membrane and in the intracellular compartment after permeabilization. Fig. 1Go, A and B, shows the intracellular staining for CTLA-4. B cell purity was verified by staining the cells with anti-CD19, whereas T cell contamination was ruled out by anti-CD3 staining (Fig. 1GoA). Freshly isolated B cells do not express CTLA-4 in both intracellular (Fig. 1GoA) and membrane (see kinetics in Fig. 1GoC) compartments. After 48 h of stimulation (Fig. 1GoB) with anti-CD40 mAb and IL-4, 28% of B cells express CTLA-4 in the intracellular compartment. When B cells are stimulated with LPS and IL-4 the expression of CTLA-4 is slightly lower (18%). Anti-CD40 or LPS alone does not induce CTLA-4 expression. Nonspecific binding of PE-labeled control Ab is 3% (Fig. 1GoB).



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  		FIGURE 1. CTLA-4 expression in B cells. CTLA-4 expression was evaluated in the intracellular compartment (A–C) and on the cell membrane (C). A, Percentage of B (CD19+) and contaminating T (CD3+) cells after purification and CTLA-4 intracellular expression in unstimulated cells. B, Intracellular CTLA-4 expression after 48-h stimulation with anti-CD40 mAb or LPS in either the presence or the absence of IL-4. The PE-conjugated anti-TNP A19-3 mAb (Ctrl) was used as isotype control for the intracellular staining. Numbers indicate the percentage of cells for each quadrant. C, CTLA-4 mean fluorescence intensity (MFI) for membrane and intracellular staining. Each value represents the difference between the mean fluorescence intensity of the sample stained with the PE-conjugated anti-CTLA-4 mAb and the corresponding sample stained with PE-conjugated anti-TNP A19-3 mAb (Ctrl).

 
Fig. 1GoC shows the kinetics of membrane and intracellular CTLA-4 expression as induced by anti-CD40 or LPS in the presence of IL-4. In both groups mean fluorescence intensity is slightly increased after 24 h, is maximum after 48–72 h, and is decreased after 6 days. Together, these results indicate that CTLA-4 expression can be induced in B cells when two signals are delivered. As in T cells, CTLA-4 expression is transient and mainly localized in intracellular compartments.

CTLA-4 engagement inhibits IL-4-driven IgE and IgG1, but not IgM production

B cells were stimulated with different combinations of IL-4, anti-CD40 mAb, and LPS in the presence of either immobilized hamster mAb with irrelevant binding activity (isotype control) or immobilized anti-CTLA-4 mAb. In preliminary experiments we found the following conditions as optimal to induce IgE production: 200 U/ml IL-4, 1 µg/ml anti-CD40 mAb, or 1 µg/ml LPS and 5 x 105 cells/ml. B cells stimulated with IL-4 (not shown) or anti-CD40 alone (Fig. 2Go, A and B) do not produce IgE as previously described (23). Conversely, anti-CD40 mAb in combination with IL-4 is able to induce IgE production in a dose-response manner. When B cells are stimulated with the higher concentration of anti-CD40 (1 µg/ml), both concentrations of anti-CTLA-4 mAb reduce IgE production by a similar extent (57–63%; Fig. 2GoA). In the groups stimulated with the lower concentration of anti-CD40 (0.1 µg/ml), CTLA-4 inhibits IgE production by 79 and 95% when the anti-CTLA-4 mAb is used at 1 and 10 µg/ml, respectively (Fig. 2GoB). CTLA-4 stimulation also inhibits (25–40%) IgE production induced by LPS and IL-4 (Fig. 2GoC). The isotype control does not affect IgE production.



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  		FIGURE 2. CTLA-4 engagement inhibits IgE and IgG1, but not IgM, production. B cells were stimulated with different combinations of IL-4; 200 U/ml), anti-CD40 mAb (A, 1 µg/ml; B, 0.1 µg/ml) and LPS (C, 1 µg/ml) in the presence of either isotype control hamster mAb at 10 µg/ml (Iso 10) or anti-CTLA-4 mAb at 1 (A4 (1)) and 10 (A4 (10)) µg/ml. Columns represent the IgE concentration in culture supernatants after 10-day stimulation. The kinetics of IgG1 (D) and IgM (E) production as induced by anti-CD40 and IL-4 ({circ}, •), and by LPS and IL-4 ({square}, {blacksquare}) in the presence of the isotype control ({circ}, {square}) or the anti-CTLA-4 mAb (•, {blacksquare}) are shown. Similar results were obtained in three other independent experiments.

 
Since in mice the IL-4-driven isotype switching leads first to IgG1 and then to IgE expression (24), we also investigated the effects of CTLA-4 stimulation on IgG1 production. Fig. 2GoD shows that after 6 days of culture LPS and IL-4 stimulate B cells to produce higher amounts of IgG1 compared with anti-CD40 and IL-4, but on day 10 of culture the same IgG1 concentration is attained in both groups. CTLA-4 stimulation inhibits IgG1 production in LPS- and IL-4-stimulated B cells by 63 and 60% after 6 and 10 days, respectively. When B cells are stimulated by anti-CD40 and IL-4 the CTLA-4-induced inhibition is evident only after 10 days (56%). Conversely, anti-CTLA-4 mAb does not affect IgM production induced by LPS and IL-4 or by anti-CD40 and IL-4 (Fig. 2GoE). This result rules out the possibility that the inhibition of the IL-4-driven Ig production may be due to an effect of CTLA-4 on B cell differentiation to Ab-producing cells.

CTLA-4 engagement reduces the frequency of IgE+ and IgG1+ cells

Whether the inhibitory effects of CTLA-4 stimulation could result from reduced frequency of IgG1- and IgE-producing cells was investigated by flow cytometric analyses. Fig. 3GoA shows the percentage of IgM+, IgG1+, and IgE+ B cells after 6 and 10 days stimulation with anti-CD40 mAb and IL-4. As during culture IgM+ nonswitched B cells might load by their Fc receptors Abs produced by other B cells and thus be detected as IgG1- or IgE-expressing cells, we performed a three-color analysis, considering only IgM- cells as cells expressing IgG1 or IgE B cell receptor. It was found that anti-CTLA-4 mAb does not reduce the percentage of IgM-expressing cells. Conversely, after CTLA-4 stimulation the percentage of IgG1+ cells that reaches its maximum after 6 days of culture, as assessed in previous experiments, is lower. As expected from the sequence of isotype switching (24), the frequency of IgE+ cells reaches a maximum after 10 days. Upon CTLA-4 engagement, the percentage of IgE+ cells is drastically reduced. Thus, CTLA-4 not only inhibits IgG1 and IgE production, but also reduces the percentage of IgG1+ and IgE+ cells.



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  		FIGURE 3. CTLA-4 stimulation inhibits the IL-4-driven differentiation to IgG1+ and IgE+ B cells. A, B cells were stimulated with anti-CD40 and IL-4 in the presence of either isotype control hamster mAb or anti-CTLA-4 mAb and then analyzed for the expression of membrane IgM, IgG1, and IgE. Data represent the percentage of IgM+, IgM-IgG1+ (IgG1+), and IgM-IgE+ (IgE+) cells at the indicated time of culture. Total recovered cells: after 6 days, 5.9 x 106 (isotype control group) and 5.6 x 106 (anti-CTLA-4 group); after 10 days, 7.1 x 106 (isotype control group) and 6.8 x 106 (anti-CTLA-4 group). The number of plated cells was 2 x 106. B, B cells were stimulated as described in A for 3 and 5 days. Equal amounts of cDNA were used to amplify by PCR I{gamma}1-C{gamma}1, I{epsilon}-C{epsilon}, and {beta}-actin transcripts. Amplified products were fractionated in a 2% ethidium bromide-stained agarose gel. C, B cells were stimulated with anti-CD40 and IL-4 in the presence of either isotype control hamster mAb (left graph) or anti-CTLA-4 mAb (right graph) for the indicated days and were analyzed for the expression of CD23. The filled histogram represents the fluorescence (FL2) of cells stained with the isotype control mAb. These findings were confirmed in two other independent experiments.

 
CTLA-4 engagement down-regulates C{epsilon} germline mRNA expression

Switch recombination from µ to a downstream CH gene is preceded by transcriptional activation of that specific CH gene in the form of an mRNA containing a noncoding exon, called I region (25, 26). To verify whether the effect of CTLA-4 on the frequency of IgG1 and IgE cells is due to a negative effect on C{gamma}1 and C{epsilon} germline mRNA expression that would lead to inhibition of isotype switching, RNA was extracted from B cells stimulated with anti-CD40 and IL-4 for 3 and 5 days. RNA was then reverse transcribed, and cDNA was amplified by PCR using appropriate primer pairs specific for I{gamma}-C{gamma}1 and I{epsilon}-C{epsilon} (see Materials and Methods). In a preliminary experiment we found that upon anti-CD40/IL-4 stimulation germline mRNA expression for C{gamma}1 reaches a maximum on day 3 and disappears on day 5, whereas germline mRNA expression for C{epsilon} is undetectable on day 3 and maximum on day 5. Fig. 3GoB shows that after 5 days stimulation C{epsilon} germline mRNA expression is drastically inhibited by CTLA-4 engagement, whereas after 3 days it reduces C{gamma}1 germline mRNA expression only partially.

CTLA-4 does not inhibit CD23 expression

Since anti-CD40 (or LPS) with IL-4 up-regulates the expression of CD23 (Fc{epsilon}RII), the low affinity IgE receptor (27), we investigated whether CTLA-4 engagement also affects the expression of this receptor. Barely expressed after 24 h, CD23 is appreciable after 3 days (not shown), maximum at 6 days, and down-regulated after 10 days of culture (Fig. 3GoC). CTLA-4 engagement not only does not inhibit the expression of this receptor at all times of culture, but seems to sustain CD23 expression, as after 10 days of culture the level of this receptor is higher than that in the control group.

CTLA-4 inhibits NF-{kappa}B activation

Germline CH mRNA expression is required for both isotype switching and Ig secretion (28). The {epsilon} mRNA expression requires activation of NF-{kappa}B, a transcription factor that can be activated by CD40 and LPS and binds two sites belonging to the {epsilon} promoter (23, 27). We investigated whether CTLA-4 engagement could inhibit Ig production by affecting NF-{kappa}B activity. Nuclear proteins from B cells were analyzed by EMSA for binding to the {kappa}B1 (Fig. 4GoA) and {kappa}B2 (Fig. 4GoB) sites. Although single stimuli alone slightly increase NF-{kappa}B DNA binding, stimulation in the presence of IL-4 drastically increases DNA binding of the p50/p65 NF-{kappa}B complex for both {kappa}B1 and {kappa}B2 sequences. The specificity of such a complex has been revealed by supershifting the band with anti-p50 and anti-p65 mAb (Fig. 4GoC). CTLA-4 engagement counteracts the NF-{kappa}B DNA binding induced by CD40 or LPS stimulation in the presence of IL-4. This inhibition is evident for both {kappa}B1 and {kappa}B2 sites (Fig. 4Go, A and B). Densitometric analyses revealed that CTLA-4 engagement reduces the DNA binding, as induced by anti-CD40 and IL-4, by 61 and 56% for {kappa}B1 and {kappa}B2 sequences, respectively (lanes 7 vs 5). Also, the LPS- and IL-4-induced NF-{kappa}B DNA binding is inhibited by CTLA-4 stimulation (Fig. 4Go, A and B, lanes 8 vs 6) by 59 and 47% for {kappa}B1 and {kappa}B2 sequences, respectively.



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  		FIGURE 4. CTLA-4 inhibits NF-{kappa}B activation by preventing the I{kappa}B{alpha} degradation. Nuclear extracts were analyzed by EMSA with the radiolabeled double-stranded {kappa}B1 (A) and {kappa}B2 (B) sequences. B cells were stimulated with IL-4 (lanes 2), anti-CD40 (lanes 3), or LPS (lanes 4), alone or in combination with IL-4/anti-CD40 (lanes 5), IL-4/LPS (lanes 6), IL-4/anti-CD40/anti-CTLA-4 (lanes 7), IL-4/LPS/anti-CTLA-4 (lanes 8). C, Supershift analyses. Before undergoing EMSA, nuclear extracts were incubated with anti-p50 or anti-p65 (RelA) or the isotype control (ctrl) mAb to assess the specificity of the band. D, Western blot analysis for I{kappa}B{alpha} on cytoplasmic extracts from the experimental groups of A and B. {beta}-Actin was used as a loading control. The reproducibility of these results was assessed in three other independent experiments.

 
Our previous finding that CTLA-4 engagement on CD4+ T cells inhibits NF-{kappa}B activation by preventing I{kappa}B{alpha} degradation (21) prompted us to examine the levels of I{kappa}B{alpha} protein in B cell cytoplasmic extracts (Fig. 4GoD). Although single stimuli induce weak effects, CD40 and IL-4 stimulation induces a considerable decrease in I{kappa}B{alpha} level. This effect is drastically counteracted by CTLA-4 stimulation, which restores the I{kappa}B{alpha} concentration. CTLA-4 also inhibits the I{kappa}B{alpha} degradation induced by LPS and IL-4. Thus, as in T cells, CTLA-4 stimulation negatively affects NF-{kappa}B activation in B cells by counteracting the signals leading to I{kappa}B{alpha} degradation.

CTLA-4 inhibits STAT6 activation

Upstream of the two NF-{kappa}B binding sites, the {epsilon} promoter contains a binding site for STAT6, a transcription factor activated by the IL-4R {alpha}-chain signaling (27). We, therefore, investigated the effect of CTLA-4 stimulation on the activation of STAT6. Stimulation of B cells with IL-4 alone induces STAT6 DNA binding, which is synergistically increased by the addition of anti-CD40 mAb (Fig. 5Go, lanes 2 and 4), although this mAb alone is ineffective (lane 3). CTLA-4 engagement inhibits DNA binding of STAT6 induced by CD40 stimulation and IL-4 (lane 5 vs 4) by 68% (densitometric analysis). Supershift analysis demonstrated the specificity of the band (not shown).



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  		FIGURE 5. CTLA-4 inhibits STAT6 activation. Nuclear extracts from B cells stimulated with IL-4 (lane 2), anti-CD40 (lane 3), or LPS (lane 4), alone or in combinations with IL-4/anti-CD40 (lane 5) and IL-4/anti-CD40/anti-CTLA-4 (lane 6), were analyzed by EMSA with the radiolabeled double-stranded STAT6 binding sequence belonging to the {epsilon} promoter. Similar results were obtained in two other independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Expression of CTLA-4 on B cells has been previously described (18, 19, 29), but its function has not been characterized. Kuiper et al. (18) reported that CTLA-4 expression on B cells requires direct cell-cell contact between B and activated T cells. At variance, in the present study we found that in purified B cells anti-CD40 or LPS stimulation in the presence of IL-4 induces CTLA-4 expression at a level comparable to that described for T cells in both membrane and intracellular compartments (22), whereas none of these stimuli alone is effective. As in T cells, most of the CTLA-4 receptors are localized in intracellular compartments, while its membrane expression is maximal after 48 to 72 h stimulation. The finding that CTLA-4 expression is induced only when both anti-CD40 and IL-4 or LPS and IL-4 are present implies that full B cell activation is required.

CTLA-4 engagement inhibits IgG1 and IgE production and affects the frequency of both IgG1+ and IgE+ cells (Fig. 3Go). These results are consistent with the hypothesis that CTLA-4 may inhibit IL-4-driven isotype switching. This possibility is supported by the finding that CTLA-4 engagement drastically inhibits the expression of C{epsilon} germline mRNA and partially affects that of C{gamma}1 germline mRNA. As switch recombination from µ to a downstream CH gene requires germline transcription of that specific CH gene, these data indicate that CTLA-4 can affect isotype switching.

C{gamma}1 and C{epsilon} mRNA expression requires both NF-{kappa}B, activated by CD40 or LPS (23, 30), and STAT6, activated by IL-4R {alpha}-chain signaling (31). Our data demonstrate that CTLA-4 engagement inhibits both NF-{kappa}B and STAT6 activation. As the cooperative action of NF-{kappa}B and STAT6 has been shown to be critical for the transcriptional activation of the {epsilon} (32), it is conceivable that CTLA-4 affects isotype switching through this mechanism.

As previously found in CD4+ T cells (21), CTLA-4 inhibits NF-{kappa}B activation by counteracting the degradation of I{kappa}B{alpha} in B cells also. The initial trigger for I{kappa}B{alpha} degradation is a signal-induced phosphorylation of the Ser32 and Ser36 residues (33). Only a few events leading to I{kappa}B kinase activation have been identified (34), and all the available data on the CTLA-4 inhibitory pathways are limited to T cells (35, 36). In T cells, the Src homology 2 domain tyrosine phosphatase (SHP2)3 binds to phosphorylated Tyr201 of the CTLA-4 cytoplasmic tail (36, 37) and dephosphorylates members of the Ras-activating pathway, which, in turn, affect mitogen-activated protein kinases involved in I{kappa}B kinase activation (38). Although the specific roles of SHP2 (and SHP1) phosphatases in IL-4 signaling have not been delineated, inhibition of phosphatase activity may result in STAT6 activation, indicating a possible modulatory role of these enzymes (39). Whether these mechanisms are also involved in the effects induced by B cell CTLA-4 stimulation requires further investigations.

It is noteworthy that CTLA-4 engagement does not affect CD23 expression, whereas it inhibits IgE production under the same experimental conditions. Despite the high degree of sequence homology between the CD23 and {epsilon} gene promoters (27), CD23 expression, unlike IgE production, can be induced by stimulation with IL-4, LPS, or anti-CD40 alone (23, 27). Thus, CD23 expression requires less stringent conditions, and the signals leading to its expression could be differently regulated and not affected by CTLA-4 stimulation. Moreover, as CTLA-4 expression reaches a maximum after 48–72 h, its negative signaling may not affect the expression of CD23 but may affect later events, such as isotype switching.

The idea that CTLA-4 counteracts the effect of CD40 stimulation on Ig production suggests that it might play a role in the regulation of B cell activities in the germinal centers. Maturation of the humoral response, indeed, occurs in germinal centers, where CD40 can be engaged by its ligand CD154, leading B cells to isotype switching under the control of IFN-{gamma} and IL-4 produced by Th1 and Th2 cells, respectively. Interestingly, it has been reported that CTLA-4 plays a role in the regulation of Th cell subpopulations by inhibiting the differentiation to Th2 cells (40) and that its blockade accelerates a typical Th2-type immune response against nematodes (41). Thus, CTLA-4 could inhibit B cell differentiation to IgE-secreting cells not only indirectly by dampening the IL-4-secreting Th2 cell population but also, as supported by the present results, by a direct action counteracting the effects of IL-4 on anti-CD40- or LPS-stimulated B cells.

B cell CTLA-4 could be engaged by CD80 or CD86 expressed on cognate B cells or other APCs. Functional interactions among B cells have been described in the lymph node dark zone, where T cells are absent, and in the light zone, where they are infrequent. In the dark and light zones, activated CD154-expressing B cells can provide the CD40-mediated signals required for B cell activation (42). In this context, activated B cells could receive a modulatory signal by the interaction of CTLA-4 with CD80 or CD86.

In conclusion, our results demonstrate that CTLA-4 receptor expressed on B cells regulates Ig production by inhibiting IgG1 and IgE production. The inhibition of IgG1+ and IgE+ cell differentiation by CTLA-4 could be mediated by the inhibitory effects on germline mRNA expression, which, in turn, is compromised by the negative effects on NF-{kappa}B and STAT6 activation. These mechanisms induced by engagement of the B cell CTLA-4 receptor are relevant to the regulation of immediate-type hypersensitive responses.


   Acknowledgments
 
We thank Dr. M. M. Rosado for skillful suggestions on FACS analyses and for helpful discussion of the data.


   Footnotes
 
1 This work was supported in part by Consiglio Nazionale delle Ricerche, Progetto Finalirrato Biotecnologie. Back

2 Address correspondence and reprint requests to Dr. Claudio Pioli, Section of Toxicology and Biomedicine, Ente per lo Nuove Tecnologie, l’Energia et l’Ambiente, C.R. Casaccia, Via Anguillarese 301, 00060 Rome, Italy. Back

3 Abbreviation used in this paper: SHP, Src homology 2 domain tyrosine phosphatase. Back

Received for publication February 22, 2000. Accepted for publication August 21, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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