DCs induce CD40-independent immunoglobulin class switching through BLyS and APRIL

Abstract

Immunoglobulin (Ig) class-switch DNA recombination (CSR) is thought to be highly dependent upon engagement of CD40 on B cells by CD40 ligand on T cells. We show here that dendritic cells up-regulate BLyS and APRIL upon exposure to interferon-alpha, interferon-gamma or CD40 ligand. In the presence of interleukin 10 (IL-10) or transforming growth factor-beta, BLyS and APRIL induce CSR from C(mu) to C(gamma) and/or C(alpha) genes in B cells, whereas CSR to C(epsilon) requires IL-4. Secretion of class-switched antibodies requires additional stimulation by B cell antigen receptor engagement and IL-15. By eliciting CD40-independent Ig class switching and plasmacytoid differentiation, BLyS and APRIL critically link the innate and adaptive immune responses.

Figure 1. BLyS, APRIL, IL-4 and/or IL-10 induce CSR from C_μ to C_γ, C_α or C_ε

(a) Schematics of CSR from C_μ to C_γ3. Ovals indicate S regions; rectangles are I_H exons and C_H gene exons; iE_μ is the IgH intronic enhancer; V-shaped lines indicate splicing; arrow-heads indicate the positions of the primers used to amplify SCs, CTs, germline I_H-C_H transcripts and mature V_HD_H-C_H transcripts, (b) Expression of SCs and β-actin (genomic DNA-loading control) in B cells incubated for 4 days with medium (control), IL-10, IL-4, BLyS or APRIL alone, BLyS + IL-10, BLyS + IL-4, APRIL + IL-10 or APRIL + IL-4 was assessed, (c) Expression of CTs, AID and β-actin transcripts; cells were treated as in b. One of three separate experiments that gave similar results is shown.

Figure 2. BLyS or APRIL and TGF-β induce CSR from C_μ to C_α

(a) IgD⁺ B cells were incubated with medium (control), TGF-β1, BLyS + TGF-β1 or APRIL + TGF-β1. Expression of S_α1/2Sμ and genomic β-actin after 4 days was assessed. (b) Expression of I_α1/2-C_μ, AID and β-actin transcripts; cells were treated as in a. One of three separate experiments that gave similar results is shown.

Figure 3. CSR is induced by BLyS, APRIL or CD40L but not by TNF-α or LT-α

(a) IgD⁺ B cells were incubated with medium alone (control), APRIL + IL-4, BLyS + IL-4, CD40L + IL-4, TNF-α + IL-4 or LT-α + IL-4. Expression of total S_γ-S_μ SCs, S_α1/2-S_μ SCs and β-actin after 4 days was assessed, (b) IgD⁺ B cells were incubated with IL-4 and 100, 10, 1 and 0.1 ng/ml of BLyS or APRIL. Expression of total S_γ-S_μ, after 4 days was assessed. One of three separate experiments that gave similar results is shown.

Figure 4. BLyS or APRIL up-regulate multiple germline I_H-C_H and mature V_HD_H-C_H transcripts

(a) Germline I_H-C_H, mature V_HD_H-C_H and β-actin transcripts from IgD⁺ B cells incubated for 4 days with IL-10, IL-4, BLyS or APRIL alone, BLyS + IL-10, BLyS + IL-4, APRIL+ IL-10 or APRIL + IL-4 were analyzed. The first lane of each panel shows a 100-bp DNA ladder, (b–d) NF-κB binding to DNA after incubation of IgD⁺ B cells with medium alone (control), BLyS or APRIL in the presence or absence of anti-BCR (b) and with or without a mouse control IgG1, TACI-Ig, BCMA-Ig or an antibody to soluble BLyS (sBLyS) (d). Arrows indicate p50–p65 and p50–c-Rel complexes, as identified by inhibition assays in which nuclear proteins from BLyS- or APRIL-activated B cells were first exposed to antibodies to p65, c-Rel, RelB and p50 or p52 and then incubated with the radiolabeled NF-κB–binding oligonucleotide (c). An irrelevant goat IgG was used as control. One of three separate experiments that gave similar results is shown.

Figure 5. BLyS or APRIL up-regulate both membrane-bound and secreted IgG and IgA

(a) IgD⁺ B cells were incubated with medium alone or BLyS and IL-4. Expression of surface IgM (sIgM), sIgD, sIgG and sIgA after 10 days was assessed. An isotype control is shown. Numbers indicate the percentages of class-switched B cells, (b) IgG and IgA secreted by IgD⁺ B cells incubated for 10 days with BLyS or APRIL + anti-BCR or with BLyS or APRIL + anti-BCR and IL-15 and in the presence or absence of TACI-Ig or BCMA-Ig was assessed. Mean ± s.d. data of one of three separate experiments that gave similar results are shown.

Figure 6. Activated DCs induce CSR through BLyS and APRIL

(a) DCs were incubated with medium alone, CD40L, IFN-α or IFN-γ. Expression of BLyS, APRIL and β-actin transcripts, and total BLyS, APRIL and actin proteins were assessed after 3 days. (b) Mean ± s.d. expression of sBLyS. (c) Expression of membrane-bound BLyS. Dotted and thick profiles depict control and BLyS antibodies, respectively, (d) IgD⁺ B cells were cocultured with unstimulated or stimulated DCs or epithelial cells (EPCs) in the presence or absence of a control mouse antibody, TACI-Ig, BCMA-Ig, CD40-Ig or an antibody to sBLyS. I_γ1/2-C_μ, I_γ3-C_μ and I_α1/2-C_μ CTs as well as AID and Igβ (CD79b, B cell–specific loading control) transcripts were amplified after 4 days.

Figure 7. Activated monocytes induce CSR through BLyS and APRIL

(a) Monocytes (Mos) were incubated with medium alone, IFN-γ, IFN-α or LPS for 3 days. Soluble and membrane-bound BLyS were measured after 3 days, (b) IgD⁺ B cells were incubated with resting or IFN-γ–activated monocytes in the presence or absence of a neutralizing antibodies to sBLyS, BCMA-Ig, TACI-Ig or CD40-Ig. Expression of I_α1/2-C_μ CTs and β-actin was assessed after 4 days, (c) IgD⁺ B cells were incubated with supernatants from monocytes (MoSup) cultured for 3 days alone or with IFN-γ in the presence or absence of a control mouse antibody, BCMA-Ig, TACI-Ig, CD40-Ig or an antibody to sBLyS. I_α1/2-C_μ CTs and β-actin were amplified after 4 days. One of three separate experiments that gave similar results is shown.

Figure 8. Activated DCs induce plasmacytoid differentiation through BLyS and APRIL

(a) BCR-activated IgD⁺ B cells were incubated with resting or CD40L-, IFN-α–or IFN-γ–activated DCs or epithelial cells in the presence or absence of a control antibody, TACI-Ig, BCMA-Ig, CD40-Ig or a neutralizing antibody to sBLyS or IL-15. Secreted IgG and IgA were measured after 10 days, (b–d), BCR-activated IgD⁺ B cells were incubated with IFN-α–activated epithelial cells, IL-15 and CD40-Ig (b), IFN-α–activated DCs, IL-15 and CD40-Ig (c), or IFN-α–activated DCs, IL-15 and BCMA-Ig (d). After 10 days, cells were stained with FITC-conjugated antibodies to IgA (green). Nuclear counterstaining was done with DAPI (blue, magnification:×1,000) and numbers indicate the percentages of cells expressing cytoplasmic IgA (e–g) IgD⁺ B cells in e–g were cultured for 10 days as in b–d, respectively. Numbers indicate percentages of CD38^hiCD20^lo and CD38^hiCD 138⁺ plasmacytoid cells. Data are mean ± s.d. of one of three separate experiments that gave similar results.