Constitutive expression of the B7h ligand for inducible costimulator on naive B cells is extinguished after activation by distinct B cell receptor and interleukin 4 receptor-mediated pathways and can be rescued by CD40 signaling

Abstract

The recently described ligand-receptor pair, B7h-inducible costimulator (ICOS), is critical for germinal center formation and antibody responses. In contrast to the induced expression of the related costimulatory ligands B7.1 and B7.2, B7h is constitutively expressed on naive B cells and is surprisingly extinguished after antigen engagement and interleukin (IL)-4 cytokine signaling. Although signaling through both B cell receptor (BCR) and IL-4 receptor (R) converge on the extinction of B7h mRNA levels, BCR down-regulation occurs through Ca2+ mobilization, whereas IL-4R down-regulation occurs through a distinct Stat6-dependent pathway. During antigen-specific B cell activation, costimulation through CD40 signaling can reverse both BCR- and IL-4R-mediated B7h down-regulation. These data suggest that the CD40-CD40 ligand signaling pathway regulates B7h expression on activated B cells and may control whether antigen-activated B cells can express B7h and costimulate cognate antigen-activated T cells through ICOS.

Figure 2.

Constitutive B7h expression on naive B cells is down-regulated after BCR signaling and by the cytokine, IL-4. (A) B7h is constitutively expressed on naive B cells and mature splenic dendritic cells. Naive splenic B cells, T cells, and CD11c⁺ splenic dendritic cells were stained for B7h expression (solid line) compared with staining with an isotype-matched control antibody (shaded histogram). (B) B7h expressed on B cells costimulates IL-4 production by T cells. Purified B cells were used as APCs to activate DO11.10 T cells in the presence of OVA peptide in cultures containing either a blocking anti-B7h mAb or control hamster IgG for 4 d. T cells were repurified and IL-4 production was assayed 24 and 72 h after rechallenge with splenic APCs presenting OVA peptide in the absence of blocking. (C) Rapid down-regulation of B7h is induced by BCR and IL-4R signaling. Naive B cells, or B cells activated for 24 h with anti-IgM F(ab′)₂, LPS, anti-CD40 mAb, or with IL-4 were stained for B7h or B7.2 expression (solid line) compared with staining with an isotype-matched control antibody (shaded histogram). (D) B7h down-regulation induced by BCR signaling is progressive and occurs in concert with B7.2 up-regulation. Purified B cells, activated with anti-IgM F(ab′)₂, were harvested at the indicated times and stained for B7h and B7.2 expression. Data shown in A and C are representative of at least three independent experiments.

Figure 1.

B7h is required for the formation of germinal centers, but not extrafollicular foci. Mice were immunized with the T cell–dependent antigen, NP-CG in alum, and were treated with daily intraperitoneal injections of 100 μg of either a blocking anti-B7h mAb or a hamster IgG control. Splenic cryosections at day 8 were analyzed by immunohistochemistry to detect λ⁺ NP-specific foci and peanut agglutinin⁺ germinal centers. ×200.

Figure 3.

BCR and IL-4 signaling induces the extinction of B7h mRNA in activated B cells. Total cellular RNA were prepared from B cells activated with PMA and ionomycin for 24 h, anti-IgM F(ab′)₂ or membrane CD40L for 72 h, or IL-4 or anti-CD40 mAb for 48 h. The mRNA levels of B7h and β-actin were determined by RNase protection assay and sample lanes from autoradiographs exposed for the same amount of time are boxed. Cell surface B7h expression as determined by flow cytometry is indicated under each activation condition.

Figure 4.

BCR-mediated B7h down-regulation occurs through Ca²⁺ signaling, whereas IL-4–mediated down-regulation occurs through a distinct Stat6-dependent signaling pathway. (A) B7h down-regulation induced by BCR signaling is Ca²⁺ dependent. Purified B cells were treated for 24h with PMA alone, ionomycin alone, or a combination of both and stained for B7h expression. (B) B7h down-regulation by BCR signaling is CsA sensitive. Purified B cells were activated with PMA and ionomycin, anti-IgM F(ab′)₂, IL-4, or untreated either in the presence (solid lines) or absence (solid filled) of CsA. After 24 h of stimulation, cells were stained for B7h expression compared with control staining (shaded histogram). (C) IL-4–mediated B7h down-regulation is Stat-6 dependent. Purified B cells from Stat6^−/− and wild-type control mice were either untreated or activated with IL-4, anti-IgM F(ab′)₂ and IL-4, or PMA and ionomycin. Cells were stained for B7h expression after 24 h of culture.

Figure 5.

CD40 signaling rescues BCR-mediated B7h down-regulation. (A) Soluble HEL induces B7h down-regulation on naive transgenic B cells. Purified B cells from IgHEL transgenic or nontransgenic littermates were activated with HEL or PMA and ionomycin, or left untreated, and stained for B7h and B7.2 expression (solid lines) compared with staining by an isotype-matched control antibody (shaded histogram) after 48 h of stimulation. Data are representative of five similar experiments. (B) CD40 signaling rescues HEL-induced B7h down-regulation. B cells from transgenic or nontransgenic mice were activated with HEL and costimulated through CD40 using an activating anti-CD40 mAb or mCD40L. CD40 stimulation was provided either simultaneously with HEL 0 or 24 h after HEL activation. At the indicated times, cells were stained for B7h expression (solid line) compared with staining with an isotype-matched control antibody (shaded histogram). The level of B7h expression on unstimulated B cells is indicated by a dashed line. Data are representative of two similar experiments. (C) Costimulation through CD40 rescues IL-4–mediated B7h down-regulation. Purified B cells were untreated or stimulated with IL-4 or IL-4 and anti-CD40 mAb. After 48 h of activation, cells were stained for B7h (solid lines) compared with control staining (shaded histogram). (D) IL-4 signaling enhances HEL-mediated B7h down-regulation and costimulation through CD40 rescues B7h down-regulation by the combination of antigen and IL-4. Purified B cells from IgHEL transgenic and nontransgenic animals were activated with soluble HEL in the presence or absence of IL-4 and costimulation with anti-CD40 mAb. After 48 h of activation, cells were stained for B7h (solid lines) compared to control staining (shaded histogram).

Figure 6.

Reversal of antigen-induced B7h down-regulation is specific to CD40 signaling. (A) Cytokines and LPS do not prevent antigen-induced B7h down-regulation. Purified B cells were activated with soluble HEL antigen and the indicated treatments. After 48 h of stimulation, cells were stained for B7h expression. Relative B7h expression was determined as the ratio of mean B7h fluorescence intensity on experimental to unstimulated B cells. (B) CD40 signaling does not prevent B7h down-regulation by anti-IgM cross-linking. B cells from transgenic and nontransgenic mice were activated with anti-IgM F(ab′)₂ in the presence or absence of anti-CD40 mAb. After 48 h, cells were stained for B7h expression.

Figure 7.

Costimulation by B7.2 is more effective than B7h in stabilizing CD40L expression on activated T cells. (A) Expression of B7h, B7.2, and I-A^d (filled histograms) on CHO cell lines compared with control staining (shaded histograms). (B) Representative expression of CD40L on naive compared with activated T cells. (C) Induction of CD40L expression after the activation of naive or rechallenged DO11.10 transgenic T cells by B7h, B7.2, or control CHO cells prepulsed with different concentrations of OVA peptide in the presence or absence of blocking anti-B7h or anti-B7.2 mAb. CD40L expression was measured and plotted as the percentage of CD4⁺ T cells expressing CD40L. Error bars indicate the standard deviation of duplicate samples. Where not observed, the error is less than the symbol size.