Engagement of CD153 (CD30 ligand) by CD30+ T cells inhibits class switch DNA recombination and antibody production in human IgD+ IgM+ B cells - PubMed (original) (raw)

Engagement of CD153 (CD30 ligand) by CD30+ T cells inhibits class switch DNA recombination and antibody production in human IgD+ IgM+ B cells

A Cerutti et al. J Immunol. 2000.

Abstract

CD153 (CD30 ligand) is a member of the TNF ligand/cytokine family expressed on the surface of human B cells. Upon exposure to IL-4, a critical Ig class switch-inducing cytokine, Ag-activated T cells express CD30, the CD153 receptor. The observation that dysregulated IgG, IgA, and/or IgE production is often associated with up-regulation of T cell CD30 prompted us to test the hypothesis that engagement of B cell CD153 by T cell CD30 modulates Ig class switching. In this study, we show that IgD+ IgM+ B cells up-regulate CD153 in the presence of CD154 (CD40 ligand), IL-4, and B cell Ag receptor engagement. In these cells, CD153 engagement by an agonistic anti-CD153 mAb or T cell CD30 inhibits S mu-->Sgamma, Smu-->Salpha, and S mu-->Sepsilon class switch DNA recombination (CSR). This inhibition is associated with decreased TNFR-associated factor-2 binding to CD40, decreased NF-kappaB binding to the CD40-responsive element of the Cgamma3 promoter, decreased Igamma3-Cgamma3 germline gene transcription, and decreased expression of Ku70, Ku80, DNA protein kinase, switch-associated protein-70, and Msh2 CSR-associated transcripts. In addition, CD153 engagement inhibits IgG, IgA, and IgE production, and this effect is associated with reduced levels of B lymphocyte maturation protein-1 transcripts, and increased binding of B cell-specific activation protein to the Ig 3' enhancer. These findings suggest that CD30+ T cells modulate CSR as well as IgG, IgA, and IgE production by inducing reverse signaling through B cell CD153.

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Figures

FIGURE 1

FIGURE 1

Human B cells up-regulate CD153 in the GC of secondary lymphoid organs. A, CD153 expression was analyzed by flow cytometry on electronically gated IgD+ CD38− (B1, naive), IgD+ CD38+ (B2, founder GC), IgD− CD38+ (B3, GC), and CD38− IgD− (B4, memory) B cells. Filled and shaded histograms correspond to control and anti-CD153 mAbs, respectively. β_-actin (593-bp), I_γ_3-C_γ_3 (670-bp), and VDJ-C_γ_3 (416-bp) transcripts were PCR amplified (35 cycles) from each B cell fraction. B, IgD, CD153, and CD79a (Ig_α) were visualized (brown cells) in serial sections from the same lymphoid follicle by immunohistochemistry (×400). C, Expression of CD153 was analyzed on freshly isolated tonsil IgD+ B cells (day 0) or on IgD+ B cells exposed to htCD154 and IL-4 with or without anti-BCR Abs.

FIGURE 2

FIGURE 2

CD153 cross-linking inhibits germline I_γ_3-C_γ_3 transcription in CD154- and IL-4-induced IgD+ B cells. PB IgD+ B cells were cultured for 2 days with or without htCD154 and IL-4, and in the presence or absence of MOPC-21 mAb, anti-CD44 mAb, anti-CD134L mAb, anti-CD153 mAb, human CD192:Fc IgG1, or human CD30:Fc IgG1 immobilized on irradiated CD32 L cells. _β_-actin (593-bp) and I_γ_3-C_γ_3 (670-bp) transcripts were PCR amplified (18 cycles) from equal amounts of cDNA in the presence of [_α_-32P]dCTP. PCR products were then fractionated on a 6% polyacrylamide gel. These findings were derived from one of three experiments yielding comparable results.

FIGURE 3

FIGURE 3

CD153 cross-linking inhibits S_μ_ →S_γ_3 CSR in CD154-and IL-4-induced IgD+ B cells. PB IgD+ B cells were cultured for 4 days with or without htCD154 and/or IL-4, and in the presence or absence of MOPC-21 or anti-CD153 mAbs immobilized on CD32 L cells. A, B cell apoptosis and proliferation measured by propidium iodide plus annexin V staining and [3H]TdR incorporation assays, respectively. B, S_γ_3-S_μ_ DNAs (0.5–3 kb) PCR amplified (30 cycles) from 500 ng of genomic DNA, and hybridized with S_μ_ and S_γ_ probes. C, _β_-actin (593 bp), VHDJH-C_γ_3 (416 bp), Ku70 (518 bp), Ku80 (492 bp), DNA-PK (578 bp), SWAP-70 (548 bp), Msh2 (449 bp), and TdT (276 bp) PCR amplified (25 cycles; _β_-actin 20 cycles) from equal amounts of cDNAs. D, Percentage of CD19+ IgD+ B cells expressing surface IgG (arrows indicate <0.05). These findings were derived from one of five experiments yielding comparable results (error bars indicate ± SD).

FIGURE 4

FIGURE 4

CD153 cross-linking inhibits activation of the C_γ_3 germline gene promoter, nuclear translocation of NF-_κ_B, and CD40:TRAF-2 association in CD154- and IL-4-induced IgD+ B cells. CL-01 B cells were cultured with or without htCD154 and/or IL-4, and in the presence of MOPC-21 or anti-CD153 mAbs immobilized on the plastic plate. A, _β_-actin (593-bp), RAG-2 (1,105 bp), and I_γ_3-C_γ_3 (670-bp) transcripts were PCR amplified (25 cycles) from equal amounts of cDNA. B, CL-01 cells were transfected with an ECS-I_γ_3-pGL3 construct, and the luciferase activity was measured after 24 h. C, The binding of NF-_κ_B and STAT-6 to the CD40 and IL-4R REs of the C_γ_3 promoter was assessed by EMSA. D, Cytoplasmic proteins obtained from 4-h-stimulated B cells were transferred to nitrocellulose membranes, and immunoblotted for TRAF-2 and actin. In additional experiments, total proteins immunoprecipitated with an anti-CD40 mAb were immunoblotted for TRAF-2. These findings were derived from one of three experiments yielding comparable results (error bars indicate ± SD).

FIGURE 5

FIGURE 5

CD153 cross-linking down-regulates IgG secretion and Blimp-1 transcripts while up-regulating BSAP nuclear activity in CD154- and cytokine-induced IgD+ B cells. A, Tonsil IgD+ (■) or IgG+ (□) B cells were cultured with or without htCD154 and cytokines (IL-4 and IL-10), and in the presence of MOPC-21 or anti-CD153 mAbs immobilized on CD32 L cells. IgG concentration and CD38++ CD138+ plasmacytoid B cells were analyzed after 8 days (arrows indicate IgG and plasma cell values <100 ng/ml and <0.1%, respectively). B, Tonsil IgD+ B cells were cultured as above. After 4 days, IL-6 was measured by ELISA; Blimp-1 (355-bp) and _β_-actin (593-bp) transcripts were PCR amplified (25 cycles) from equal amounts of cDNA; and BSAP binding to the BSAP site 1 of the mouse Ig 3′ enhancer was determined by EMSA. These findings were derived from one of three experiments yielding comparable results (error bars indicate ± SD).

FIGURE 6

FIGURE 6

CD4+ T cells express CD154 and CD30 4 days after TCR, CD28, and CD134 stimulation. A and B, PBMCs were incubated with IL-4 and mAbs to CD3, CD28, and CD134 immobilized on CD32 L cells. After 8 h (A) or 4 days (B), CD4, CD154, and CD30 were analyzed on gated CD3+ T cells. C, IgD+ (■) or IgG+ (□) B cells were cultured with or without cytokines (IL-4 and IL-10), anti-BCR Abs, and CD4+ T cells isolated from 4-day-stimulated PBMCs. Before culture, CD4+ T cells were fixed and preincubated with MOPC-21 or blocking mAbs to CD154, CD27, or CD30. IgG were measured after 8 days. These findings were derived from one of three experiments yielding comparable results (error bars indicate ± SD).

FIGURE 7

FIGURE 7

CD30+ T cells inhibit CSR in CD154- and cytokine-induced IgD+ B cells. IgD+ B cells were cultured with htCD154, cytokines (IL-4 and IL-10), and anti-BCR Abs in the presence or absence of CD4+ CD30− or CD4+ CD30+ T cells sorted from 4-day-stimulated PBMCs. Before culture, all CD4+ T cells were fixed, washed, incubated with a blocking anti-CD154 mAb, and washed. CD30+ T cells were also preincubated with blocking anti-CD30 or anti-CD27 mAbs. S_γ_-S_μ_ (γ), S_α_-S_μ_ (α), and S_ε_-S_μ_ (ε) junction DNAs (A) as well as IgG, IgA, and IgE secretion (B) were assessed after 8 days. These findings were derived from one of three experiments yielding comparable results (error bars indicate ± SD).

FIGURE 8

FIGURE 8

Proposed role of CD30+ T cells in the regulation of CSR and IgG, IgA, and IgE production. Upon TCR, CD28, and CD134 engagement by Ag and costimulatory molecules on APCs, CD4+ Th cells express CD154 and secrete IL-4. In the presence of Ag, CD154, and IL-4, IgM+ IgD+ CD30− B cells undergo clonal expansion and up-regulate CD153. Within 4 days, Ag-selected B cells complete CSR to IgG, IgA, or IgE and down-regulate CD153. At this time, CD4+ T cells induce CD30 while still expressing significant levels of CD154. These CD30+ T cells inhibit CSR in newly CD154:CD40-activated IgD+ and/or IgM+ B cells by inducing reverse signaling through CD153.

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