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.

The targeting of human and mouse B lymphocytes by dasatinib.

Dasatinib inhibits B-cell receptor-Abelson murine leukemia viral oncogene homologue 1, Src, and other tyrosine kinases. Few studies have addressed the impact of dasatinib on normal blood cells, especially in vivo. Here we show that dasatinib leads to a reduced number of human CD19+ peripheral B cells owing to a strong induction of apoptosis. In contrast, no similar effect on T-cell viability was observed. However, dasatinib induced a comparable broad inhibition of the early events of B- and T-cell receptor signaling. Furthermore, dasatinib was shown to be a more pronounced inhibitor of both basal and B-cell receptor-induced activity of Bruton's tyrosine kinase and PLCγ2 compared with the more specific Bruton's tyrosine kinase inhibitor ibrutinib. Human progenitor B cells from the pre-B stage were sensitive to dasatinib. In an in vivo murine model, dasatinib reduced B-lineage cells in the bone marrow with a marked effect on the pre-B subpopulation. Dasatinib led to a reduced spleen size, with a loss of large immature transitional immunoglobulin M(+)/immunoglobulin D(-) B cells and a reduction in germinal center B cells. Dasatinib caused a marked loss of thymocytes without affecting myeloid lineage cells or hematopoietic progenitors. This study reveals important side effects of dasatinib with specific loss of activated B and thymocyte populations, which may have an impact during long-term treatment.

Expression of B-cell surface antigens in subpopulations of exosomes released from B-cell lymphoma cells.

PURPOSE: Exosomes are small (30- to 100-nm) vesicles secreted by all cell types in culture and found in most body fluids. A mean of 1 mL of blood serum, derived from healthy donors, contains approximately 10(12) exosomes. Depending on the disease, the number of exosomes can fluctuate. Concentration of exosomes in the bloodstream and all other body fluids is extremely high. Several B-cell surface antigens (CD19, CD20, CD22, CD23, CD24, CD37, CD40, and HLA-DR) and the common leukocyte antigen CD45 are interesting in terms of immunotherapy of hematologic malignant neoplasms. The established standard for exosome isolation is ultracentrifugation. However, this method cannot discriminate between exosome subpopulations and other nanovesicles. The main purpose of this study was to characterize CD81(+) and CD63(+) subpopulations of exosomes in terms of these surface markers after release from various types of B-cell lymphoma cell lines using an easy and reliable method of immunomagnetic separation. METHODS: Western blotting, flow cytometry, and electron microscopy were used to compare the total preenriched extracellular vesicle (EV) pool to each fraction of vesicles after specific isolation, using magnetic beads conjugated with antibodies raised against the exosome markers CD63 and CD81. FINDINGS: Magnetic bead-based isolation is a convenient method to study and compare subpopulations of exosomes released from B-cell lymphoma cells. The data indicated that the specifically isolated vesicles differed from the total preenriched EV pool. CD19, CD20, CD24, CD37, and HLA-DR, but not CD22, CD23, CD40, and CD45, are expressed on exosomes from B-cell lymphoma cell lines with large heterogeneity among the different B-cell lymphoma cell lines. Interestingly, these B-cell lymphoma-derived EVs are able to rescue lymphoma cells from rituximab-induced complement-dependent cytotoxicity. IMPLICATIONS: Distribution of exosomes that contain CD19, CD20, CD24, CD37, and HLA-DR may intercept immunotherapy directed against these antigens, which is important to be aware of for optimal treatment. The use of an immunomagnetic separation platform enables easy isolation and characterization of exosome subpopulations for further studies of the exosome biology to understand the potential for therapeutic and diagnostic use.

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.

Phospho-specific flow cytometry identifies aberrant signaling in indolent B-cell lymphoma.

BACKGROUND: Knowledge about signaling pathways in malignant cells may provide prognostic and diagnostic information in addition to identify potential molecular targets for therapy. B-cell receptor (BCR) and co-receptor CD40 signaling is essential for normal B cells, and there is increasing evidence that signaling via BCR and CD40 plays an important role in the pathogenesis of B-cell lymphoma. The aim of this study was to investigate basal and induced signaling in lymphoma B cells and infiltrating T cells in single-cell suspensions of biopsies from small cell lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL) and marginal zone lymphoma (MZL) patients. METHODS: Samples from untreated SLL/CLL and MZL patients were examined for basal and activation induced signaling by phospho-specific flow cytometry. A panel of 9 stimulation conditions targeting B and T cells, including crosslinking of the B cell receptor (BCR), CD40 ligand and interleukins in combination with 12 matching phospho-protein readouts was used to study signaling. RESULTS: Malignant B cells from SLL/CLL patients had higher basal levels of phosphorylated (p)-SFKs, p-PLCγ, p-ERK, p-p38, p-p65 (NF-κB), p-STAT5 and p-STAT6, compared to healthy donor B cells. In contrast, anti-BCR induced signaling was highly impaired in SLL/CLL and MZL B cells as determined by low p-SFK, p-SYK and p-PLCγ levels. Impaired anti-BCR-induced p-PLCγ was associated with reduced surface expression of IgM and CD79b. Similarly, CD40L-induced p-ERK and p-p38 were also significantly reduced in lymphoma B cells, whereas p-p65 (NF-κB) was equal to that of normal B cells. In contrast, IL-2, IL-7 and IL-15 induced p-STAT5 in tumor-infiltrating T cells were not different from normal T cells. CONCLUSIONS: BCR signaling and CD40L-induced p-p38 was suppressed in malignant B cells from SLL/CLL and MZL patients. Single-cell phospho-specific flow cytometry for detection of basal as well as activation-induced phosphorylation of signaling proteins in distinct cell populations can be used to identify aberrant signaling pathways.

Effect of cycloheximide on epidermal growth factor receptor trafficking and signaling.

Cycloheximide is the most common protein synthesis inhibitor, and is believed to specifically inhibit the cytoplasmic protein synthesis. Here we demonstrate that cycloheximide induces internalization and redistribution of EGF receptor to early endosomes in HeLa cells independent of receptor tyrosine phosphorylation, but dependent on p38 MAPK activity. Degradation of EGF receptor or its downstream effectors was not observed. EGF-induced activation of ERK1/2 was inhibited upon pre-treatment with cycloheximide, but did not activate JNK. The observed effects of treatment with cycloheximide alone are significant and therefore results involving the use of cycloheximide for inhibition of protein synthesis must be interpreted with caution.

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.

Side population cells in highly enriched CD34-positive cells from peripheral blood progenitor cells identify an immature subtype of hematopoietic progenitor cells but do not predict time to engraftment in patients treated with high-dose therapy.

OBJECTIVE: A Hoechst 33342 dye efflux assay can be used to define a population of immature hematopoietic progenitor cells (HPC) that are called side population (SP) cells. Previously, SP cells examined from bone marrow (BM) and peripheral blood progenitor cells (PBPC) were found to be predominantly CD34 negative. METHODS AND RESULTS: In this study, we show that the level of CD34+ cells within the SP fraction increases from 2% in BM to 15% in mobilized PBPC. Furthermore, SP cells are found in highly enriched CD34+ cells from both BM and PBPC, and these cells define an immature phenotype of HPC. We also observed a higher level of CD133+ cells within the SPCD34+ cell population. Moreover, the frequency of long-term culture-initiating cells (LTC-IC) was markedly increased in SPCD34+ cells. To further investigate whether variations in the level of SP cells in the CD34+ cell fraction influenced short-term engraftment, we studied 20 patients with Hodgkin lymphoma that were autotransplanted with highly enriched CD34+ cells from PB. The percentage of SP cells in the PBCD34+ cell fraction was highly variable, ranging from 0.3 to 22%. No correlation was found between the content of SP cells in the autotransplanted CD34+ cells and time to short-term engraftment. CONCLUSION: SPCD34+ cells in PBPC define an immature phenotype of HPC with increased numbers of LTC-IC, and they are more frequently found in PBPC than in BM. The number of SP cells does not predict time to engraftment.

Characterization of early stages of human B cell development by gene expression profiling.

We have characterized several stages of normal human B cell development in adult bone marrow by gene expression profiling of hemopoietic stem cells, early B (E-B), pro-B, pre-B, and immature B cells, using RNA amplification and Lymphochip cDNA microarrays (n = 6). Hierarchical clustering of 758 differentially expressed genes clearly separated the five populations. We used gene sets to investigate the functional assignment of the differentially expressed genes. Genes involved in VDJ recombination as well as B lineage-associated transcription factors (TCF3 (E2A), EBF, BCL11A, and PAX5) were turned on in E-B cells, before acquisition of CD19. Several transcription factors with unknown roles in B lymphoid cells demonstrated interesting expression patterns, including ZCCHC7 and ZHX2. Compared with hemopoietic stem cells and pro-B cells, E-B cells had increased expression of 18 genes, and these included IGJ, IL1RAP, BCL2, and CD62L. In addition, E-B cells expressed T/NK lineage and myeloid-associated genes including CD2, NOTCH1, CD99, PECAM1, TNFSF13B, and MPO. Expression of key genes was confirmed at the protein level by FACS analysis. Several of these Ags were heterogeneously expressed, providing a basis for further subdivision of E-B cells. Altogether, these results provide new information regarding expression of genes in early stages of human B cell development.

Signaling through toll-like receptor 7/8 induces the differentiation of human bone marrow CD34+ progenitor cells along the myeloid lineage.

Toll-like receptors (TLRs) play a key role in pathogen recognition and regulation of the innate and adaptive immune responses. Although TLR expression and signaling have been investigated in blood cells, it is currently unknown whether their bone marrow ancestors express TLRs and respond to their ligands. Here we found that TLRs (e.g. TLR4, TLR7 and TLR8) were expressed by freshly isolated human bone marrow (BM) hematopoietic CD34+ progenitor cells. Incubation of these primitive cells with TLR ligands such as immunostimulatory small interfering RNAs and R848, a specific ligand for TLR7/8, induced cytokine production (e.g. IL1-beta, IL6, IL8, TNF-alpha, GM-CSF). Moreover, TLR7/8 signaling induced the differentiation of BM CD34+ progenitors into cells with the morphology of macrophages and monocytic dendritic precursors characterized by the expression of CD13, CD14 and/or CD11c markers. By contrast, R848 ligand did not induce the expression of glycophorin A, an early marker for erythropoiesis. Collectively, the data indicate for the first time that human BM CD34+ progenitor cells constitutively express functional TLR7/TLR8, whose ligation can induce leukopoiesis without the addition of any exogenous cytokines. Thus, TLR signaling may regulate BM cell development in humans.

BMP-6 inhibits growth of mature human B cells; induction of Smad phosphorylation and upregulation of Id1.

BACKGROUND: Bone morphogenetic proteins (BMPs) belong to the TGF-beta superfamily and are secreted proteins with pleiotropic roles in many different cell types. A potential role of BMP-6 in the immune system has been implied by various studies of malignant and rheumatoid diseases. In the present study, we explored the role of BMP-6 in normal human peripheral blood B cells. RESULTS: The B cells were found to express BMP type I and type II receptors and BMP-6 rapidly induced phosphorylation of Smad1/5/8. Furthermore, Smad-phosphorylation was followed by upregulation of Id1 mRNA and Id1 protein, whereas Id2 and Id3 expression was not affected. Furthermore, we found that BMP-6 had an antiproliferative effect both in naive (CD19+CD27-) and memory B cells (CD19+CD27+) stimulated with anti-IgM alone or the combined action of anti-IgM and CD40L. Additionally, BMP-6 induced cell death in activated memory B cells. Importantly, the antiproliferative effect of BMP-6 in B-cells was completely neutralized by the natural antagonist, noggin. Furthermore, B cells were demonstrated to upregulate BMP-6 mRNA upon stimulation with anti-IgM. CONCLUSION: In mature human B cells, BMP-6 inhibited cell growth, and rapidly induced phosphorylation of Smad1/5/8 followed by an upregulation of Id1.