CD30 is a CD40-inducible molecule that negatively regulates CD40-mediated immunoglobulin class switching in non-antigen-selected human B cells

Abstract

We used our monoclonal model of germinal center maturation, CL-01 B cells, to investigate the role of CD30 in human B cell differentiation. CL-01 cells are IgM+ IgD+ CD30+ and switch to IgG, IgA, and IgE when exposed to CD40L and IL-4. Switching is hampered by CD30 coengagement, possibly through interference with the CD40-mediated NF-kappaB-dependent transcriptional activation of downstream C(H) genes. The physiological relevance of this phenomenon is emphasized by similar CD30-mediated effects in naive B cells. Expression of CD30 by these cells is induced by CD40L but is inhibited by B cell receptor coengagement and/or exposure to IL-6 and IL-12. Our data suggest that CD30 critically regulates the CD40-mediated differentiation of non-antigen-selected human B cells.

Figure 1. sIgM⁺sIgD⁺ CL-01 B Cells Express Surface CD30 Whose Engagement Negatively Modulates CD40L and IL-4-Induced Switching to IgG, IgA, and IgE but Not Surface CD40 Expression

(A and B) Upon incubation for 2 days in medium only or in medium containing 0.5 μg/ ml of htCD40L, 5 μg/ml of control MOPC-21 MAb, or 5 μg/ml of MAb to human CD30, CL-01 cells were analyzed for expression of surface CD30 or CD40. (C and D) CL-01 cells were cultured for 5 days in medium only or in medium containing IL-4 alone (100 U/ml), htCD40L alone (0.5 μg/ml), or htCD40L and IL-4 in the presence of either MOPC-21 or anti-CD30 MAbs (5 μg/ml). Equal amounts of template cDNA were used to PCR amplify I_H-C_H (C), V_HDJ_H-C_H (D), and β-actin transcripts (C and D). Amplified products were then fractionated into 2% ethidium bromide–stained agarose gel. These findings were derived from one of three experiments yielding comparable results.

Figure 2. Normal sIgM⁺sIgD⁺ B Cells Express CD30 mRNA, Surface CD30, and GC Markers upon CD40 Engagement by CD40L; BCR Coengagement Down-Regulates Surface CD30 Expression

Naive B cells were cultured for 3 days in medium only (A) or in medium containing 0.5 μg/ml of htCD40L (B) or htCD40L and 2 μg/ml of anti-κ and anti-λ chain (anti-BCR) MAbs (C) and then analyzed for expression of surface CD19, CD30, CD71, CD80, CD86, and CD95. β-actin and CD30 cDNAs from the same cells were PCR amplified and fractioned into a 2% ethidium bromide–stained agarose gel. These findings were derived from one of five experiments yielding comparable results.

Figure 3. Stimuli Other than CD40L Do Not Induce CD30 Expression in Normal sIgM⁺sIgD⁺ B Cells

Naive B cells were cultured for 3 days in medium only or in medium containing IL-1β (10 ng/ml), IL-2 (200 U/ml), IL-4 (200 U/ml), IL-7 (100 U/ml), IL-10 (200 ng/ml), IL-13 (5 ng/ml), IFNγ (200 U/ml), IFNα (200 U/ml), TNFα (2.5 ng/ml), LTβ (2.5 ng/ml), TGFβ (0.5 ng/ml), IL-6 (200 ng/ml), IL-12 (1 ng/ml), immobilized anti-κ and anti-λ (anti-BCR) MAbs (2 μg/ml each), and a combination of IL-6, IL-12, and anti-BCR Ab in the presence (full histograms) or in the absence (open histograms) of htCD40L (0.5 μg/ml). Cells were then reacted with FITC-conjugated anti-CD30 MAb and PE-conjugated anti-CD19 MAbs and analyzed by flow cytometric analysis. The percentage of CD19⁺CD30⁺ B cells corresponds to the proportion of cells reacted with the above MAbs that display a fluorescence intensity higher than that displayed by similar cells reacted with control FITC- and PE-conjugated isotype matched MAbs. Values are mean ± SD of three determinations from three independent experiments.

Figure 4. CD30 Engagement in the Absence of BCR Coengagement Inhibits Ig Class Switching but Not Proliferation in Normal sIgM⁺sIgD⁺ B Cells Exposed to CD40L and Appropriate Cytokines

Naive B cells were preincubated for 2 days with medium only, htCD40L (0.5 μg/ml), or htCD40L and immobilized anti-κ and anti-λ chain (anti-BCR) MAbs (2 μg/ml each). Cells were then harvested, washed, and seeded in culture plates coated with either MOPC-21 (open histograms) or anti-CD30 (full histograms) MAbs (5 μg/ml) in the presence of medium only or medium containing IL-4 (100 U/ml) and IL-10 (100 ng/ml); htCD40L; or htCD40L, IL-4, and IL-10. In selected experiments, TGFβ1 (0.5 ng/ml) was added to htCD40L, IL-4, and IL-10 to optimize IgA secretion. After 8 days, the concentration of IgM, IgG, IgA, and IgE in the culture fluids was measured by ELISA. To measure DNA synthesis, B cells (1 × 10⁵/well) were cultured as above for 5 days and then pulsed for 18 hr with [³H]TdR. B cells were harvested at day 3 and counted (200 cells) by trypan blue dye exclusion to measure cell viability. Values are mean ± SD of four determinations from three independent experiments.

Figure 5

CD30 Engagement Down-Regulates the CD40-Mediated NF-κB-Dependent Activation of the Human Cγ3 Germline Gene Promoter CL-01 cells were transiently cotransfected with a pRL-CMV control luciferase plasmid and either a wild-type (wt) ECS-Iγ3-pGL3 (LUC) plasmid (A) or an ECS-Iγ3 CD40RE-SV40-pGL3 (LUC) minimal promoter plasmid (B). The top graphics depict the organization of these constructs. The initiation of transcription within the wt ECS-Iγ3 promoter is indicated by an arrow, and numbering is such that +1 corresponds to the first residue after this initiation site. After transfection, B cells were seeded in culture plates coated with either MOPC-21 (open histograms) or agonistic anti-CD30 MAbs (full histograms) in medium only or in medium containing IL-4 (500 U/ml); htCD40L (0.5 μg/ml); htCD40L and IL-4; or htCD40L, IL-4, and anti-IFNγ Abs (1:500). Luciferase activity was measured after 24 hr. Values are mean ± SD of three independent experiments.

Figure 6. CD30 Engagement Down-Regulates the CD40-Mediated Binding of NF-κB to the CD40RE of the Human Cγ3 Germline Gene Promoter

(A) Sequence of the ECS-Iγ3 CD40RE encompassed by the κB1 probe. The two κB sites within the CD40RE are boxed. (B) CL-01 cells were cultured with MOPC-21 MAb (5 μg/ml), anti-CD30 MAb (5 μg/ml), MOPC-21 MAb and htCD40L (0.5 μg/ml), anti-CD30 MAb and htCD40L, or anti-CD27 MAb (5 μg/ml) and htCD40L in the presence or in the absence of 100 μg/ml of cycloheximide (CHX). Nuclear proteins were extracted after 6 hr of culture, incubated with a radiolabeled κB1 oligonucleotide for 30 min, and then fractionated through a 6% polyacrylamide gel (lanes 2–10; lane 1 corresponds to the protein-free probe). The specificity of shifted complexes A, B, and C, and their identity to NF-κB complexes were established by pre-incubating nuclear proteins from CD40L-induced cells with competitors 1 (unlabeled κB1 probe), 2 (unlabeled κB1 mutant), and 3 (palindromic NF-κB binding sequence from the Ig κ promoter) (lanes 11–13). The composition of complexes A, B, and C was determined by preincubating nuclear proteins with rabbit Abs to human thyreoglobulin (control), p50, p52, p65, c-Rel, or Rel B proteins (lanes 14–19).

Figure 7. Role of CD30 in the Regulation of Human CD40-Dependent B Cell Differentiation

In the upper limb of the scheme, a naive CD30⁻ B cell differentiates into a GC B cell and undergoes switching to IgG, IgA, or IgE upon CD40 engagement by CD40L expressed by an activated CD4⁺ T cell and exposure to T cell–derived cytokines. BCR coengagement by Ag as well as IL-6 and IL-12 secreted by an activated dendritic cell inhibit the expression of CD30 by this GC B cell. In the lower limb, a naive B cell is activated by a CD4⁺CD40L⁺ T cell in the absence of BCR coengagement by Ag complexes on the surface of a dendritic cell and, therefore, expresses CD30. In this non-Ag-selected B cell, CD40L-induced switching to IgG, IgA, or IgE is negatively modulated through CD30 engagement by CD30L expressed on either a CD4⁺ or a CD8⁺ T cell.