BMP-7 induces apoptosis in human germinal center B cells and is influenced by TGF-β receptor type I ALK5

<div><p>Selection and maturation of B cells into plasma cells producing high-affinity antibodies occur in germinal centers (GC). GCs form transiently in secondary lymphoid organs upon antigen challenge, and the GC reaction is a highly regulated process. TGF-β is a potent negative regulator, but the influence of other family members including bone morphogenetic proteins (BMPs) is less known. Studies of human peripheral blood B lymphocytes showed that BMP-6 suppressed plasmablast differentiation, whereas BMP-7 induced apoptosis. Here, we show that human naïve and GC B cells had a strikingly different receptor expression pattern. GC B cells expressed high levels of BMP type I receptor but low levels of type II receptors, whereas naïve B cells had the opposite pattern. Furthermore, GC B cells had elevated levels of downstream signaling components <i>SMAD1</i> and <i>SMAD5</i>, but reduced levels of the inhibitory <i>SMAD7</i>. Functional assays of GC B cells revealed that BMP-7 suppressed the viability-promoting effect of CD40L and IL-21, but had no effect on CD40L- and IL-21-induced differentiation into plasmablasts. BMP-7-induced apoptosis was counteracted by a selective TGF-β type I receptor (ALK4/5/7) inhibitor, but not by a selective BMP receptor type I inhibitor. Furthermore, overexpression of truncated ALK5 in a B-cell line counteracted BMP-7-induced apoptosis, whereas overexpression of truncated ALK4 had no effect. BMP-7 mRNA and protein was readily detected in tonsillar B cells, indicating a physiological relevance of the study. Altogether, we identified BMP-7 as a negative regulator of GC B-cell survival. The effect was counteracted by truncated ALK5, suggesting greater complexity in regulating BMP-7 signaling than previously believed.</p></div

Introduction of truncated ALK5 in the B-cell lymphoma cell line Mino abrogates BMP-7-induced apoptosis.

<p>Mino cells were transduced with truncated ALK5 or truncated ALK4. (A) The cells were stained with biotinylated anti-ALK5, followed by streptavidin PE or by biotinylated anti-ALK4, followed by streptavidin APC, and analyzed by flow cytometry. Receptor expression is compared in GFP⁺ or mCherry⁺ transduced cells vs. GFP^-/mCherry^- non-transduced cells. (B-C) The transduced Mino cells were cultured in serum free media (X-VIVO 15) over night and then left in medium alone (unstim) or stimulated with BMP-2, BMP-7 or TGF-β for 60 min, before detection of phosphorylated (p-) Smad 1/5 or p-Smad 2/3 by flow cytometry. (B) One representative experiment showing p-SMAD1/5 vs. GFP in truncated ALK5-2A-GFP expressing cells. (C) BMP- or TGF-β-induced phosphorylation is shown relative to unstimulated cells, using arcsinh transformation of median fluorescence intensity data. Mean ± SEM, <i>n</i> = 5. (D-E): Transduced Mino cells were cultured in X-VIVO 15 and left unstimulated or stimulated with TGF-β or BMP-7 for 72 hours and stained for active caspase-3 before analysis by flow cytometry. Shown here is active caspase-3 staining of control cells and transduced cells for (D) one representative experiment and (E) mean ± SEM, <i>n</i> = 3. *<i>p</i> < 0.05; two-tailed, paired Student’s <i>t</i>-test.</p

Tonsillar B-cell subsets have differential expression patterns of BMP receptors.

<p>Single-cell suspensions from human tonsils were stained with lineage markers and anti-BMP receptor antibodies, and analyzed by flow cytometry. (A) Gating strategy to identify B-cell subsets in tonsils: Naïve B cells were defined as CD3^-CD20⁺CD38^-IgD⁺CD27^-, GC B cells as CD3^-CD20⁺CD38⁺IgD^- and plasmablasts as CD3^-CD38^hi. Memory B cells were separated into three subsets: class switched CD20⁺CD3^-CD38^-IgD^-CD27⁺, non-switched CD20⁺CD3^- CD38^-IgD⁺CD27⁺ and class switched CD20⁺CD3^- CD38^-IgD^-CD27^- memory B cells. (B) Histogram overlays of receptor expression in GC and naïve B cells in donor T4. (C) Heatmaps of relative protein expression of BMP type I and type II receptors in tonsils from 4 different donors, denoted T1 –T4. The expression levels were normalized to irrelevant Ab control in each donor, using arcsinh transformation.</p

GC B cells have higher levels of r-Smads and lower levels of i-Smads as compared to naïve or memory B cells.

<p>mRNA expression was determined by qPCR of FACS-sorted B-cell subsets from tonsillar specimens. (A) <i>SMAD</i> mRNA expression and (B) <i>ID</i> mRNA expression. Relative mRNA expression is shown relative to PGK-1 as endogenous control and normalized to total RNA from human fetal brain. Shown is mean values ± SEM, <i>n</i> = 3. (C) Protein levels of Smads were determined by western blotting of B-cell subsets obtained by immunomagnetic bead separation of tonsil single-cell suspensions. Sudhl-6 cells were included as positive control and β-actin was used as loading control. N/M denotes naïve/memory B cells. *<i>p</i> < 0.05, paired two-tailed Student’s <i>t</i>-test.</p

BMP-7 is present in tonsillar B cells.

<p>Primary tonsillar B- and T cells were obtained by immunomagnetic bead isolation, by using positive selection for T cells (CD3 Dynabeads) and the remaining negatively selected cells are mainly B cells. (A) Gene expression of BMP7 was determined by qPCR duplicates and is shown relative to PGK-1 and GAPDH endogenous control and normalized to Sudhl-6. Median ± SD, <i>n</i> = 2. (B) Protein levels of BMP-7 were determined by western blotting. Included were cell lines Sudhl-6, Mino and Jurkat, CD4⁺ T cells from peripheral blood, tonsillar B cells from four donors (denoted T32-35).</p

BMP-7 induces apoptosis in GC B cells and the effect is abrogated by selective inhibition of TGF-β type I receptor.

<p>(A-C) GC B cells were obtained by immunomagnetic bead separation and co-cultured with HK cells in the presence of CD40L/IL-21 with or without BMP-7 and apoptosis was measured by TUNEL assay. (A) Cells were cultured for up to 3 days and analyzed by flow cytometry. Shown is one representative of 2 donors. (B) After 2 days in culture, TUNEL staining (green) and Hoechst staining (blue) was detected by confocal microscopy. Representative images from one of three independent experiments are presented. Scale bar represents 30 μm. (C) The ALK 2/3 and ALK 4/5/7 selective inhibitors, LDN193189 and SB431542, respectively, were added to the cultures as specified and apoptosis was measured by flow cytometry at day 2. Mean ±SEM, <i>n</i> = 4, (D) Single cell suspensions from human tonsils were stained with lineage markers and anti-ALK4, anti-ALK5 or anti-ALK7 antibodies, and analyzed by flow cytometry. Shown are histogram overlays of receptor expression as compared to an irrelevant control. All experiments were repeated at least twice. Statistical testing was performed against CD40L/IL-21 condition. *<i>p</i> < 0.05; two-tailed, paired Student’s <i>t</i>-test.</p

BMPs do not affect plasmablast differentiation in GC B cells.

<p>GC, naïve and memory B cells were isolated by FACS-sorting and cultured on a feeder layer of HK cells for four days in the presence of CD40L/IL-21 and different BMPs before analysis by flow cytometry. Plasmablasts are gated as CD38⁺CD27^hi. Shown here is percentage of plasmablasts in (A) one representative experiment or as (B) mean ± SEM, <i>n</i> = 4 (GC), <i>n</i> = 5 (memory) <i>n</i> = 6 (naïve). Statistical testing was performed against CD40L/IL-21 condition. *<i>p</i> < 0.05; two-tailed Student’s t-test, unequal variance.</p