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.

Defective IL-4 signaling in T cells defines severe common variable immunodeficiency.

Common variable immunodeficiency (CVID) is defined by hypogammaglobulinemia and B-cell dysfunction, with significant clinical and immunological heterogeneity. Severe non-infectious complications, such as autoimmunity, granulomatous disease and splenomegaly, constitute a major cause of morbidity in CVID patients. T cells are generally regarded important for development of these clinical features. However, while T-cell abnormalities have been found in CVID patients, functional characteristics of T cells corresponding to well-defined clinical subtypes have not been identified. As common γ-chain cytokines play important roles in survival and differentiation of T cells, characterization of their signaling pathways could reveal functional differences of clinical relevance. We characterized CVID T cells functionally by studies of cytokine-induced signaling, and correlated the findings to defined clinical subtypes. Peripheral blood T cells from 29 CVID patients and 19 healthy donors were analyzed for i) phenotype, ii) cytokine-induced (interleukin (IL)-2, IL-4, IL-7 and IL-21) phosphorylation of signal transducer and activator of transcription (STAT) 3, STAT5 and STAT6, and iii) T-helper (Th)1/Th2 polarization. Expression of IL-4 receptor and downstream signaling molecules was measured. A subgroup of CVID patients (n = 7) was identified by impaired IL-4-induced p-STAT6 in naive and memory CD4 and CD8 T cells. This corresponded to patients with the largest accumulation of severe (non-infectious) complications. The signaling defect persisted over years and was not due to constitutively activated p-STAT6. The CD4 T cells were strongly Th1-skewed, but IL-4 signaling was impaired independently of Th status. However, IL-4Rα and Janus kinase (JAK) 1 mRNA levels were significantly lower than in normal donors, providing a likely mechanism for the defective IL-4-induced p-STAT6 and Th1-bias. In conclusion, we identified a subgroup of CVID patients with defective IL-4 signaling in T cells, with severe clinical features of inflammation and autoimmunity.

A gene panel, including LRP12, is frequently hypermethylated in major types of B-cell lymphoma.

Epigenetic modifications and DNA methylation in particular, have been recognized as important mechanisms to alter gene expression in malignant cells. Here, we identified candidate genes which were upregulated after an epigenetic treatment of B-cell lymphoma cell lines (Burkitt's lymphoma, BL; Follicular lymphoma, FL; Diffuse large B-cell lymphoma, DLBCL activated B-cell like, ABC; and germinal center like, GCB) and simultaneously expressed at low levels in samples from lymphoma patients. Qualitative methylation analysis of 24 candidate genes in cell lines revealed five methylated genes (BMP7, BMPER, CDH1, DUSP4 and LRP12), which were further subjected to quantitative methylation analysis in clinical samples from 59 lymphoma patients (BL, FL, DLBCL ABC and GCB; and primary mediastinal B-cell lymphoma, PMBL). The genes LRP12 and CDH1 showed the highest methylation frequencies (94% and 92%, respectively). BMPER (58%), DUSP4 (32%) and BMP7 (22%), were also frequently methylated in patient samples. Importantly, all gene promoters were unmethylated in various control samples (CD19+ peripheral blood B cells, peripheral blood mononuclear cells and tonsils) as well as in follicular hyperplasia samples, underscoring a high specificity. The combination of LRP12 and CDH1 methylation could successfully discriminate between the vast majority of the lymphoma and control samples, emphasized by receiver operating characteristic analysis with a c-statistic of 0.999. These two genes represent promising epigenetic markers which may be suitable for monitoring of B-cell lymphoma.

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.

Identification of highly methylated genes across various types of B-cell non-hodgkin lymphoma.

Epigenetic alterations of gene expression are important in the development of cancer. In this study, we identified genes which are epigenetically altered in major lymphoma types. We used DNA microarray technology to assess changes in gene expression after treatment of 11 lymphoma cell lines with epigenetic drugs. We identified 233 genes with upregulated expression in treated cell lines and with downregulated expression in B-cell lymphoma patient samples (n = 480) when compared to normal B cells (n = 5). The top 30 genes were further analyzed by methylation specific PCR (MSP) in 18 lymphoma cell lines. Seven of the genes were methylated in more than 70% of the cell lines and were further subjected to quantitative MSP in 37 B-cell lymphoma patient samples (diffuse large B-cell lymphoma (activated B-cell like and germinal center B-cell like subtypes), follicular lymphoma and Burkitt`s lymphoma) and normal B lymphocytes from 10 healthy donors. The promoters of DSP, FZD8, KCNH2, and PPP1R14A were methylated in 28%, 67%, 22%, and 78% of the 36 tumor samples, respectively, but not in control samples. Validation using a second series of healthy donor controls (n = 42; normal B cells, peripheral blood mononuclear cells, bone marrow, tonsils and follicular hyperplasia) and fresh-frozen lymphoma biopsies (n = 25), confirmed the results. The DNA methylation biomarker panel consisting of DSP, FZD8, KCNH2, and PPP1R14A was positive in 89% (54/61) of all lymphomas. Receiver operating characteristic analysis to determine the discriminative power between lymphoma and healthy control samples showed a c-statistic of 0.96, indicating a possible role for the biomarker panel in monitoring of lymphoma patients.

Influence of polymorphisms in genes encoding immunoregulatory proteins and metabolizing enzymes on susceptibility and outcome in patients with diffuse large B-cell lymphoma treated with rituximab.

We analyzed the allelic distribution of 12 candidate polymorphisms in a large retrospective study of 486 patients with diffuse large B-cell lymphoma (DLBCL) treated at Oslo University Hospital with 1056 blood donors serving as controls. Variants in TNFα (rs1800629) (GG vs. AG/AA, p < 0.001) and LTA (rs909253) (AA vs. AG/GG, p = 0.02) and deletions in GSTM1 and GSTT1 (undeleted vs. deleted, p = 0.01 and p = 0.01, respectively) were associated with increased susceptibility of developing DLBCL. IL-10 (rs1800896) variants (GG vs. AG/AA, p = 0.03) were associated with decreased susceptibility. In line with several previous reports, patients carrying the TNFα (rs1800629) A allele treated in the pre-rituximab era had inferior outcome compared to patients carrying the homozygous GG genotype (p = 0.004, n = 33). However, patients receiving at least one dose of rituximab had equal outcome regardless of their TNFα genotype (HR = 0.94, p = 0.79, n = 307). Deletion in GSTM1 was associated with inferior outcome for patients with low International Prognostic Index (IPI) score (p = 0.04). Our findings support the suggestions that polymorphisms in genes encoding immunoregulatory proteins and enzymes that metabolize carcinogens and chemotherapeutic drugs influence DLBCL susceptibility and possibly treatment outcome. The influence of polymorphisms in immunoregulatory genes on outcome in DLBCL should be reevaluated in the rituximab era.

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.

Polymorphisms in genes encoding interleukin-10 and drug metabolizing enzymes GSTP1, GSTT1, GSTA1 and UGT1A1 influence risk and outcome in Hodgkin lymphoma.

We genotyped 224 patients with Hodgkin lymphoma (HL) and 1056 healthy controls and related the risk for HL and outcome of chemotherapy treatment to polymorphisms in genes encoding interleukins and metabolizing enzymes by capillary electrophoresis. Patients with the UGT1A1 TA tandem repeat TA6/6 genotype had a poorer overall survival (OS) (relative risk [RR] 3.63, p = 0.004), and patients above 40 years with the GSTA1 AA genotype had poorer event-free survival (EFS) (RR 4.38, p = 0.003) after chemotherapy. In patients above 40 years, the IL-10 rs1800890 T-allele was associated with lower risk for HL (TT genotype vs. AA, odds ratio [OR] 0.38 [95% confidence interval 0.21-0.69], p = 0.001; AT/TT combined genotypes vs. AA, OR 0.45 [0.27-0.74], p = 0.001). The GSTP1 rs1695 A-allele reduced the risk for HL (GG vs. AG, OR 0.64 [0.42-0.99], p = 0.04; GG vs. AG/AA combined genotypes, OR 0.70 [0.47-1.04], p = 0.07), and the GSTT1 deleted genotype increased the risk for HL (OR 3.17 [1.97-5.09], p < 0.001) regardless of age.

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.