Mechanism of CD79A and CD79B Support for IgM+ B Cell Fitness through B Cell Receptor Surface Expression.

The BCR consists of surface-bound Ig and a heterodimeric signaling unit comprised of CD79A and CD79B. Upon cognate Ag recognition, the receptor initiates important signals for B cell development and function. The receptor also conveys Ag-independent survival signals termed tonic signaling. Although the requirement of a CD79A/CD79B heterodimer for BCR complex assembly and surface expression is well established based on mice models, few studies have investigated this in human mature B cells. In this study, we found that human tonsillar B cells with high surface expression of IgM or IgG had potentiated BCR signaling compared with BCRlow cells, and high IgM expression in germinal center B cells was associated with reduced apoptosis. We explored the mechanism for IgM surface expression by CRISPR/Cas9-induced deletion of CD79A or CD79B in four B lymphoma cell lines. Deletion of either CD79 protein caused loss of surface IgM in all cell lines and reduced fitness in three. From two cell lines, we generated stable CD79A or CD79B knockout clones and demonstrated that loss of CD79A or CD79B caused a block in N-glycan maturation and accumulation of immature proteins, compatible with retention of BCR components in the endoplasmic reticulum. Rescue experiments with CD79B wild-type restored surface expression of CD79A and IgM with mature glycosylation, whereas a naturally occurring CD79B G137S mutant disrupting CD79A/CD79B heterodimerization did not. Our study highlights that CD79A and CD79B are required for surface IgM expression in human B cells and illuminates the importance of the IgM expression level for signaling and fitness.

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

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-ß 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 SMAD1 and SMAD5, but reduced levels of the inhibitory SMAD7. 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-ß 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.

Whole-genome integrative analysis reveals expression signatures predicting transformation in follicular lymphoma.

Transformation of follicular lymphoma (FL) to a more aggressive disease is associated with rapid progression and death. Existing molecular markers for transformation are few and their clinical impact is limited. Here, we report on a whole-genome study of DNA copy numbers and gene expression profiles in serial FL biopsies. We identified 698 genes with high correlation between gene expression and copy number, and the molecular network most enriched for these cis-associated genes. This network includes 14 cis-associated genes directly related to the nuclear factor κB (NF-κB) pathway. For each of these 14 genes, the correlated NF-κB target genes were identified and corresponding expression scores were defined. The scores for 6 of the cis-associated NFκB pathway genes (BTK, IGBP1, IRAK1, ROCK1, TMED7-TICAM2, and TRIM37) were significantly associated with transformation. The results suggest that genes regulating B-cell survival and activation are involved in transformation of FL.

Role of Smad proteins in resistance to BMP-induced growth inhibition in B-cell lymphoma.

Bone morphogenetic protein (BMP) expression and signaling are altered in a variety of cancers, but the functional impact of these alterations is uncertain. In this study we investigated the impact of expression of multiple BMPs and their signaling pathway components in human B-cell lymphoma. BMP messages, in particular BMP7, were detected in normal and malignant B cells. Addition of exogenous BMPs inhibited DNA synthesis in most lymphoma cell lines examined, but some cell lines were resistant. Tumor specimens from three out of five lymphoma patients were also resistant to BMPs, as determined by no activation of the BMP effectors Smad1/5/8. We have previously shown that BMP-7 potently induced apoptosis in normal B cells, which was in contrast to no or little inhibitory effect of this BMP in the lymphoma cells tested. BMP-resistance mechanisms were investigated by comparing sensitive and resistant cell lines. While BMP receptors are downregulated in many cancers, we documented similar receptor levels in resistant and sensitive lymphoma cells. We found a positive correlation between activation of Smad1/5/8 and inhibition of DNA synthesis. Gene expression analysis of two independent data sets showed that the levels of inhibitory Smads varied across different B-cell lymphoma. Furthermore, stable overexpression of Smad7 in two different BMP-sensitive cell lines with low endogenous levels of SMAD7, rendered them completely resistant to BMPs. This work highlights the role of Smads in determining the sensitivity to BMPs and shows that upregulation of Smad7 in cancer cells is sufficient to escape the negative effects of BMPs.

SARA is dispensable for functional TGF-ß signaling.

Smad anchor for receptor activation (SARA or ZFYVE9) has been proposed to mediate transforming growth factor ß (TGF-ß) signaling by direct interaction with the non-activated Smad proteins and the TGF-ß receptors; however, these findings are controversial. We demonstrate no correlation between SARA expression and the levels of TGF-ß-induced phosphorylation of Smads in various B-cell lymphomas. Moreover, knockdown of SARA in HeLa cells did not interfere with TGF-ß-induced Smad activation, Smad nuclear translocation, or induction of TGF-ß target genes. Various R-Smads and TGF-ß receptors did not co-immunoprecipitate with SARA. Collectively, our results demonstrate that SARA is dispensable for functional TGF-ß-mediated signaling.

Bone morphogenetic proteins inhibit CD40L/IL-21-induced Ig production in human B cells: differential effects of BMP-6 and BMP-7.

Bone morphogenetic proteins (BMPs) are members of the TGF-ß superfamily. TGF-ß can affect class switch recombination in human B cells, but whether BMPs also play a role have not been tested. We investigated the functional effects of exogenously added BMPs on CD27(-) naive and CD27(+) memory B cells from healthy donors. BMP-2, -4, -6 and -7 inhibited CD40L/IL-21-induced production of IgM, IgG and IgA. BMP-6 reduced Ig production by 70% in memory B cells and more than 55% in naive B cells, whereas the other BMPs were slightly less potent. We observed a striking difference in functional effects between the structurally similar BMP-6 and BMP-7, as BMP-6 mainly inhibited plasmablast differentiation, and BMP-7 mainly induced apoptosis. In memory B cells, BMP-6 upregulated expression of DNA-binding protein inhibitor genes, but potently inhibited CD40L/IL-21-induced upregulation of the transcription factor XBP1, necessary for the late stages of plasmacytic differentiation. Expression of transcription factors regulating earlier stages (IRF4, PRDM1) was not affected by BMP-6. Taken together, these results show that BMPs are potent suppressors of naive and memory B cells.

TGF-ß-induced growth inhibition in B-cell lymphoma correlates with Smad1/5 signalling and constitutively active p38 MAPK.

BACKGROUND: Cytokines of the transforming growth factor ß (TGF-ß) superfamily exert effects on proliferation, apoptosis and differentiation in various cell types. Cancer cells frequently acquire resistance to the anti-proliferative signals of TGF-ß, which can be due to mutations in proteins of the signalling cascade. We compared the TGF-ß-related signalling properties in B-cell lymphoma cell lines that were sensitive or resistant to TGF-ß-induced anti-proliferative effects. RESULTS: TGF-ß sensitive cell lines expressed higher cell surface levels of the activin receptor-like kinase 5 (Alk-5), a TGF-ß receptor type 1. The expression levels of the other TGF-ß and bone morphogenetic protein receptors were comparable in the different cell lines. TGF-ß-induced phosphorylation of Smad2 was similar in TGF-ß sensitive and resistant cell lines. In contrast, activation of Smad1/5 was restricted to cells that were sensitive to growth inhibition by TGF-ß. Moreover, with activin A we detected limited anti-proliferative effects, strong phosphorylation of Smad2, but no Smad1/5 phosphorylation. Up-regulation of the TGF-ß target genes Id1 and Pai-1 was identified in the TGF-ß sensitive cell lines. Constitutive phosphorylation of MAPK p38 was restricted to the TGF-ß sensitive cell lines. Inhibition of p38 MAPK led to reduced sensitivity to TGF-ß. CONCLUSIONS: We suggest that phosphorylation of Smad1/5 is important for the anti-proliferative effects of TGF-ß in B-cell lymphoma. Alk-5 was highly expressed in the sensitive cell lines, and might be important for signalling through Smad1/5. Our results indicate a role for p38 MAPK in the regulation of TGF-ß-induced anti-proliferative effects.