Methylene Blue Selectively Inhibits B Cell Development and E2A Activity

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4848-4848
Author(s):  
Eroica Soans ◽  
John K Choi
Keyword(s):  
Flow Cytometry ◽  
Methylene Blue ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Bone Marrow Cells ◽  
Myeloid Cell ◽  
Luciferase Reporter ◽  
Flow Cytometry Analysis ◽  
Murine Bone Marrow

Abstract Introduction E2A, a basic helix loop helix transcription factor, is essential for B lymphocyte proliferation, survival and differentiation. Mutations in E2A are associated with B cell leukemia and lymphomas. Our preliminary data suggests that E2A activity is altered by HSP70 and since HSP70 ATPase activity is inhibited by methylene blue (MB), we hypothesized that MB will alter E2A activity and thus alter B cell development. Methods We studied the effects of MB on normal and leukemic B cells compared to its effect on myeloid cells. The cell lines used were REH, NALM-6 and BAF3-p185 cells compared to myeloid cell lines, CCRF and CMK for the proliferation assay. To study MB effect on normal B and myeloid progenitors, methylcellulose colony formation assays with murine bone marrow cells (strain 129S1/SVImJ, 8-12 weeks old mice) were performed. We also treated mice with MB in their drinking water (1000 μM) up to 16 weeks to study the in-vivo effect on B cells as compared to granulocyte, macrophages, T cells and NK cells in the peripheral blood by flow cytometry analysis. To understand the mechanisms by which MB alters B cell proliferation, we examined the effects of MB on cell cycle progression, apoptosis and E2A activity in B cells.  For cell progression NALM-6 cells were treated with 4 μM MB for 0, 6, 12 and 24 hrs and assayed using BRDU staining by flow cytometry analysis. Further, apoptosis was measured using Annexin V staining by flow cytometry analysis in NALM-6 cells up to 48hrs post treatment with MB. Lastly, E2A activity was measured using an E2A-specific luciferase reporter assay in 293T cells. Results MB decreased the proliferation of precursor B cell lines, REH, NALM-6 and BAF3-p185 cells compared to myeloid cell lines, CCRF and CMK. Similar effects of MB were also seen with normal B and myeloid progenitors as assayed by methylcellulose colony formation assays with murine bone marrow cells. Similarly, there is a significant decrease in peripheral B cells as compared to granulocytes, macrophages, T cells and NK cells when the mice were treated with 1000 μM of methylene blue in their drinking water as compared to those that remained untreated. MB caused apoptosis and decreased the S phase fraction of treated NALM-6 cells. In addition, we found that MB inhibited E2A activity using an E2A-specific luciferase reporter assay. Conclusion Our findings suggest that MB is a specific inhibitor of B cell proliferation, survival and development. This effect may be mediated by the inhibition of E2A activity by methylene blue. Disclosures: No relevant conflicts of interest to declare.

Transformation of murine bone marrow cells with combined v-raf-v-myc oncogenes yields clonally related mature B cells and macrophages

1990 ◽  
Vol 10 (7) ◽  
pp. 3562-3568
Author(s):  
M Principato ◽  
J L Cleveland ◽  
U R Rapp ◽  
K L Holmes ◽  
J H Pierce ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Bone Marrow Cells ◽  
Hematopoietic Differentiation ◽  
Developmental Relationships ◽  
Murine Bone Marrow ◽  
B Lineage ◽  
Marrow Cells

Murine bone marrow cells infected with replication-defective retroviruses containing v-raf alone or v-myc alone yielded transformed pre-B cell lines, while a retroviral construct containing both v-raf and v-myc oncogenes produced clonally related populations of mature B cells and mature macrophages. The genealogy of these transformants demonstrates that mature myeloid cells were derived from cells with apparent B-lineage commitment and functional immunoglobulin rearrangements. This system should facilitate studies of developmental relationships in hematopoietic differentiation and analysis of lineage determination.

scholarly journals Transformation of murine bone marrow cells with combined v-raf-v-myc oncogenes yields clonally related mature B cells and macrophages.

1990 ◽  
Vol 10 (7) ◽  
pp. 3562-3568 ◽  
Author(s):  
M Principato ◽  
J L Cleveland ◽  
U R Rapp ◽  
K L Holmes ◽  
J H Pierce ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Bone Marrow Cells ◽  
Hematopoietic Differentiation ◽  
Developmental Relationships ◽  
Murine Bone Marrow ◽  
B Lineage ◽  
Marrow Cells

Murine bone marrow cells infected with replication-defective retroviruses containing v-raf alone or v-myc alone yielded transformed pre-B cell lines, while a retroviral construct containing both v-raf and v-myc oncogenes produced clonally related populations of mature B cells and mature macrophages. The genealogy of these transformants demonstrates that mature myeloid cells were derived from cells with apparent B-lineage commitment and functional immunoglobulin rearrangements. This system should facilitate studies of developmental relationships in hematopoietic differentiation and analysis of lineage determination.

TOSO-Deficient B Cells Show An Impaired NF-κB and Migration Signaling In Vivo

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1779-1779
Author(s):  
Alexandra Schulz ◽  
Christian P. Pallasch ◽  
Michael Hallek ◽  
Lukas P. Frenzel ◽  
Clemens Wendtner
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
B Cells ◽  
Mouse Model ◽  
B Cell ◽  
Promoter Region ◽  
Targeted Mutagenesis ◽  
Luciferase Reporter ◽  
And Migration

Abstract Abstract 1779 Background: Our group firstly demonstrated that TOSO (FAIM3) is over-expressed in CLL compared to healthy B cell subsets as well as other B cell lymphomas. Furthermore, we detected a significant correlation of high TOSO expression to high lymphocyte count, unmutated IgVH status and Binet C, which are all markers for poor prognosis. TOSO has been described as pro-survival gene also in other settings. However, its mode of action is discussed controversially. Therefore, we aimed to elucidate the role of TOSO in B-cell specific gene expression by creating a knockdown mouse model. CD40 ligation and B cell receptor (BCR) activation influences TOSO expression and the fact that transcriptional regulation of TOSO is still unknown, we were eager to determine transcriptional factors that are directly responsible for the alterable TOSO levels. Methods: Faim3-floxed C57BL/6 FLP deleter mice were crossbred with CD19 specific Cre recombinase expressing mice. B-cells from the TOSOCD19−/− (KO) mice were isolated and gene expression was analyzed via mRNA based Illumina microchip array. Convincing results were verified by flow cytometry and blood count was carried out in addition. To determine the promoter region of TOSO, three overlapping DNA fragments (containing either NF-κB, Bcl-6 or both binding sites) upstream of the transcription start site of the first TOSO exon were cloned into a luciferase reporter vector lacking a promoter. Those constructs were transfected into HeLa cells. After 24 hours luciferase assays were performed. The involvement of NF-κB in the regulation of TOSO transcription was measured by TNFα stimulation of transfected cells prior to luminescence measurement. Targeted mutagenesis of the NF-κB binding site was performed to confirm the data. In addition, Bcl-6 expression vector was co-transfected for evaluation of repressing influence on TOSO expression. Results: In order to cover the functional part of TOSO, we generated a B-cell specific TOSOCD19−/− mouse model. Downstream effects of TOSO were validated via microarray-based gene expression analysis. Results displayed a clear clustering of deregulated genes compared to control mice. Nearly 400 genes showed expression alterations; genes involved in the NF-κB pathway and migration processes were downregulated in TOSOCD19−/−. These results were confirmed by flow cytometry analysis. The TOSO KO displayed also relevant effects on the hematopoietic system. Lymphocyte (p=0,0048), neutrophil (p=0,0007) and red blood cell counts (p=0,0051) were significantly decreased in the TOSOCD19−/− mice. Most important, the B-cell count was significantly reduced in TOSO-deficient settings (n=9; p=0,032). Since TOSO level seems to be so important for such fundamental pathways, investigation of gene expression regulation is mandatory. In situ analysis of the TOSO promoter region revealed NF-κB and Bcl-6 as promising results. Luciferase reporter assays including targeted mutagenesis confirmed the positive regulation of NF-κB and the repressing influence of Bcl-6 on TOSO expression significantly. Conclusions: We reveal for the first time a TOSO-dependent expression profile. We identified TOSO-dependent deregulated genes, which were involved in NF-κB signaling and migration, suggesting that TOSO represents an important factor in these pathways. Additionally, TOSO KO caused a decrease of peripheral B-cells in vivo. Furthermore, we identified NF-κB and Bcl-6 to regulate the TOSO expression in an opposite manner. Disclosures: No relevant conflicts of interest to declare.

ZAP-70 Enhances Migration of Malignant B Cells towards Lymphoid Organs in a Burkitt Lymphoma Xenograft Model

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2844-2844
Author(s):  
Noelia Purroy ◽  
Eva Calpe ◽  
Pau Abrisqueta ◽  
Cecilia Carpio ◽  
Carles Palacio ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
B Cells ◽  
Tumor Growth ◽  
B Cell ◽  
Cell Lines ◽  
Ectopic Expression ◽  
Xenograft Model ◽  
Lymphoid Organs ◽  
Transfected Cells

Abstract Abstract 2844 Introduction. ZAP-70 (ξ-associated protein) is a protein tyrosine kinase of the Syk/ZAP family that plays a crucial role in cellular activation in T and NK cells. High expression of ZAP-70 protein in malignant cells from Chronic Lymphocytic Leukemia (CLL) correlates with adverse clinical prognostic features, such as unmutated IgHV genes, short time to progression, and short survival. Moreover, ZAP-70 protein has been related to aggressive features of the CLL cells, such as enhanced B-cell receptor (BCR) signaling and higher migration capacity. To further investigate into the mechanisms by which ZAP-70 protein influences the clinical outcome of patients with CLL, we analyzed the functional consequences of ZAP-70 ectopic expression in malignant B-cells. For this, Ramos and Raji (Burkitt) B-cell lines were stably transfected with a ZAP-70 expressing vector (pEGFP-N2ZAP-70). Raji transfectant showed constitutively phosphorylated ZAP-70 protein, whilst Ramos cells required stimulation with 5 μg/ml F(ab') 2 anti-IgM to get ZAP-70 activated. ZAP-70 expression induced the upregulation of the chemokine receptor CCR7, thus giving the cells the ability to better respond and migrate towards CCL21 (own data, Blood 2011 pre-published). CCR7 ligands (chemokines CCL21 and CCL19) are mainly expressed in high endothelial venules and the T zones from secondary lymphoid organs. The aims of this study were firstly to evaluate in vivo the migratory/invasive capability of pEGFP-N2ZAP-70 transfected Raji and Ramos cell lines compared to pEGFP Raji and Ramos cell lines; and later, to compare the overall survival (OS) of mice injected with pEGFP-N2ZAP-70 transfected cells to those injected with only pEGFP transfected cells. Methods. For this, a total of 27 7- to 8-week old SCID (CB17Crl) mice were used. Mice were inoculated intravenously with 5×106 cells of each cell line (6 mice with Raji-GFP, 5 mice with Raji-GFP-ZAP-70, 5 mice with Ramos-GFP and 10 mice with Ramos-GFP-ZAP-70). Mice were observed for the onset of hind legs paralysis, dyspnea, or evidence of tumor growth, once symptoms appeared, mice were euthanized and lymphoid and non-lymphoid organs were obtained for further analysis of the presence of GFP-positive cells by flow cytometry and immunohistochemistry. Results. Twenty-six out of twenty-seven injected mice were included in the analysis. The excluded mouse was found dead before it could be euthanized to obtain the organs. In the Raji xenograft model, 11/11 (100%) of mice had hind legs paralysis as the first symptom to appear. The median survival was 19 days for GFP-ZAP-70 and 16 days for GFP injected mice. There were no statistically significant differences between survival of GFP-ZAP-70 and GFP injected mice (OS was 66.7% [95% CI 38.4–100] vs 33.3% [95% CI 0–71.1], p=0.784, at 19 and 16 days, respectively). In the Ramos xenograft model, 6/15 (40%) of mice showed hind legs paralysis as the first symptom to appear, as well as evidence of abdominal tumor growth in 6/15 (40%), whereas in 3/15 (20%) the established event was dyspnea. The median survival in Ramos xenograft model was 40 days for GFP-ZAP-70 and 38 days for GFP injected mice. Again there were no statistically significant differences between survival of GFP-ZAP-70 and GFP Ramos injected mice (OS was 50% [95% CI 18.4–81.6] vs 40% [95% CI 0–83.8], p=0.180, at 40 and 38 days, respectively). By flow cytometry analysis of GFP cells we found that in the Raji xenograft model there were statistically significant differences between the migration of GFP-ZAP-70 and GFP injected cells towards bone marrow (21.5% vs 5.17, p=0.011), spleen (0.08% vs 0.01%, p=0.006) and thymus (0.00% vs 0.02%, p=0.037). The highest percentages of GFP positive cells were found in bone marrow samples (mean, 9.85%), whereas in spleen and thymus the percentages of GFP positive cells were all below 0, 1%. There was no statistically significant difference between the cellular migration in the Ramos xenograft model in any of the organs analyzed. Conclusion. In conclusion, malignant B-lymphocytes with ectopic expression of activated ZAP-70 protein show enhanced ability to migrate towards and infiltrate lymphoid organs in a xenograft model, specially the bone marrow, although it does not translate into a worse survival of the animals. Further specific immunohistochemical assays to determine infiltrated areas by ZAP-70 expressing lymphocytes are in process. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Epstein-Barr virus transformation of human pre-B cells.

1983 ◽  
Vol 158 (2) ◽  
pp. 616-622 ◽  
Author(s):  
M Hansson ◽  
K Falk ◽  
I Ernberg
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Bone Marrow Cells ◽  
Fetal Bone ◽  
Barr Virus ◽  
Epstein Barr

In vitro infection of human B lymphocytes with Epstein-Barr virus (EBV) results in establishment of B lymphoblastoid cell lines that reflect normal B cell phenotypes. In this study we have investigated whether immature B cells from fetal bone marrow and liver can serve as targets for EBV. The fetal bone marrow cells were readily transformed by EBV. Among the resulting cell lines, five were surface Ig (sIg)-negative. Three B cell-associated antigens defined by monoclonal antibodies were expressed to the same extent on the fetal cell lines, whether they belonged to the sIg- or sIg+ group. The various differentiation stages that these cell lines may represent are discussed.

scholarly journals T-cell growth factor P40 promotes the proliferation of myeloid cell lines and enhances erythroid burst formation by normal murine bone marrow cells in vitro

Blood ◽  
1990 ◽  
Vol 76 (5) ◽  
pp. 906-911 ◽  
Author(s):  
DE Williams ◽  
PJ Morrissey ◽  
DY Mochizuki ◽  
P de Vries ◽  
D Anderson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Growth Factor ◽  
Cell Growth ◽  
Cell Lines ◽  
Bone Marrow Cells ◽  
Myeloid Cell ◽  
Murine Bone Marrow ◽  
T Cell Growth Factor ◽  
Marrow Cells

T-cell growth factor P40 was examined for possible effects on murine interleukin-3 (IL-3)-dependent myeloid cell lines and freshly isolated murine bone marrow cells. The results showed that P40 stimulated the proliferation of some IL-3-dependent myeloid cell lines of both early myeloid and mast cell phenotype and synergized with IL-3. P40 did not promote proliferation of fresh bone marrow cells, bone marrow enriched for early myeloid cells by 5-fluorouracil treatment, or bone marrow derived mast cells as assessed in 3H-TdR incorporation assays. P40 did not influence the growth of murine colony-forming unit granulocyte- macrophage in agar cultures, either alone or in the presence of optimal or sub-optimal concentrations of CSF-1, GM-colony-stimulating factor, or IL-3. P40 did potentiate burst-forming unit-erythroid (BFU-E) formation in the presence of erythropoietin; however, this was dependent on the cell plating density, suggesting an indirect stimulation of BFU-E by P40. The indirect nature of P40 action on BFU-E was further demonstrated in cell separation experiments and indicated that the effect was mediated by T cells. These data expand the repertoire of cells that P40 influences.

The BLyS Survival Pathway in NHL-B Cells: Constitutive NF-kB and NFAT Lead to Activation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2279-2279
Author(s):  
Lingchen Fu ◽  
Tamayo Archito ◽  
Yen-Chiu Lin-Lee ◽  
Lan Pham ◽  
Linda Yoshimura ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Low Grade ◽  
Rt Pcr ◽  
Protein Levels ◽  
Realtime Pcr ◽  
Survival Pathway ◽  
Hodgkin's Lymphomas

Abstract B-cell non-Hodgkin’s Lymphomas (NHL-B), neoplasms of the immune system have shown a significant increase in incidence in the USA over the last three decades. While the pathophysiology of the NHL-B is still unclear, the need to identify the relevant genes and critical signaling pathways, and their involvement in the disease processes in NHL-B have begun to be elucidated. Recently, B Lymphocyte Stimulator (BLyS) has been described as a relatively new member of the TNF ligand family, as a potent cell survival factor that is expressed in many hematopoietic cells, including neoplastic B cells. BLyS can bind to three receptors: TACI, BCMA, BAFF-R, and plays a critical role in B cell maturation, differentiation and proliferation. Relatively high levels of BLyS has been found in the serum of NHL-B patients as well as of the patients with autoimmune disease. The mechanisms of BLyS gene expression and regulation is still unclear, but we have recently found that BLyS is constitutively expressed in several NHL-B cell lines and patient tumor samples by RT-PCR, confocal microscopy, realtime PCR and flow cytometry (FCM). We detected high levels and differential expression of BLyS receptors (TACI, BCMA, BAFF-R) in several NHL-B cell lines by flow cytometry, RT-PCR and realtime PCR in both NHL-B cell lines and patient tumor samples. We have identified a single binding site for NF-kB and two binding sites for NFAT in the BLyS promoter. We also show in aggressive lymphoma B cells that constitutive NF-kB and NFAT binds to the BLyS promoter constitutively. Inhibiting NF-kB/NFAT activity levels, using the NF-kB inhibitors, BAY-11 or Velcade (PS-341), can decrease NF-kB binding activity in the BLyS promotor by EMSA. These inhibitors also decrease BLyS and BAFF-R mRNA and protein levels by realtime PCR and flow cytometry. Similarly, when NHL-B cells were transfected with dominant negative NFAT or NF-kB constructs, there is a 50% decrease in BLyS and BAFF-R expressions, demonstrating that both the ligand (BLyS) and the receptor (BAFF-R) expression are regulated by NFAT and NF-kB. Interestingly, follicular (low grade) lymphoma cells do not express constitutive NF-kB/NFAT activation, and barely detectable mRNA and protein levels of BlyS, but can be activated with exogenous CD154/anti IgM in vitro, activating NF-kB/NFAT and promoting binding to the BLyS promoter by EMSA. This results in a significant increase of BLyS protein level by flow cytometry. Our studies indicate that constitutive NF-kB and NFAT are critical transcriptional regulators of the BLyS survival pathway in malignant B cells that may provide a future therapeutic target in the aggressive NHL-B.

scholarly journals Expression of T cell receptor genes in human B cells.

1986 ◽  
Vol 164 (6) ◽  
pp. 1940-1957 ◽  
Author(s):  
A F Calman ◽  
B M Peterlin
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Transcriptional Activity ◽  
Myeloid Cell ◽  
Cdna Clones ◽  
Gene Rearrangements ◽  
Beta Gene

We analyzed the transcription and rearrangement of the T cell antigen receptor (Ti) genes Ti alpha and Ti beta in human B cell, T cell, and myeloid cell lines, as well as in purified tonsillar B and T cells. All four B cell lines examined, as well as one of two myeloid cell lines, expressed low levels of truncated Ti beta transcripts, as did freshly purified tonsillar B cells. Two of the B cell lines and one of the myeloid lines also expressed truncated Ti alpha transcripts, while tonsillar B cells did not. Sequence analysis of cDNA clones from a B cell line demonstrated that these truncated Ti alpha and Ti beta transcripts were composed of unrearranged J and C gene segments. Comparison of cDNA clones from T and B cells suggests that D alpha genes or N regions contribute to the formation of Ti alpha transcripts in T cells but not in B cells. None of the B cell or myeloid cell lines in this study showed evidence of Ti beta gene rearrangements by Southern blotting. Our data, and other studies of gene rearrangements in human tumors, demonstrate that the level of Ti beta transcriptional activity and the frequency of Ti beta gene rearrangements are correlated in all cell types examined. Thus, our data support the accessibility model of antigen receptor gene rearrangement, whereby the susceptibility of gene segments to recombination enzymes is correlated with their transcriptional activity.

scholarly journals The Complex Role of KDM6A in B-Cell Development and Function

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 513-513
Author(s):  
Ling Tian ◽  
Monique Chavez ◽  
Lukas D Wartman
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Bone Marrow Cells ◽  
Cell Development ◽  
Young Female ◽  
B Cell Development ◽  
And Function

Abstract Loss-of-function mutations in KDM6A, an X-linked H3K27 demethylase, occur recurrently in B-cell lymphoid malignancies, including B-cell acute lymphoblastic leukemia and non-Hodgkin lymphoma. Germline inactivating mutations in KDM6A cause a neurodevelopmental disorder called Kabuki syndrome that is associated with recurrent infections and hypogammaglobulinemia.1 The role of KDM6A in normal B-cell development and function, as well as how the somatic loss of KDM6A contributes to B-cell malignancies, has not been completely defined. To address this issue, we generated a conditional knockout mouse of the KDM6A gene (with LoxP sites flanking the 3rd exon) and crossed these mice with Vav1-Cre transgenic mice to selectively inactivate KDM6A in hematopoietic stem/progenitor cells. We characterized normal hematopoiesis from young (6 to 8 week old) and aged (50 to 55 week old) male and female KDM6A conditional KO mice. We found a significant shift from lymphoid to myeloid differentiation in the bone marrow and peripheral blood of these mice. Young, female KDM6A-null mice had mild splenomegaly. Their spleens had an increased number of neutrophils (Gr-1+CD11b+ cells) and erythrocyte progenitors (CD71+Ter119+ cells) and a decreased number of B-cells (B220+ cells). These changes became more pronounced with age and were specific to the female, homozygous KDM6A knockout mice. Furthermore, analysis of B-cell maturation showed that the loss of KDM6A was associated with decreased immature (B220+IgM+ cells) and mature, resting B-cells (B220+IgD+ cells) in the spleen. Similar changes were present in the bone marrow (decreased B220+IgM+ cells and B220+CD19+ cells) and peripheral blood (decreased B220+IgM+, B220+IgD+ and B220+CD19+ cells). Early B-cell development is also altered in KDM6A-null mice. Flow cytometry showed a decrease in multipotent progenitor cells (MPPs) with a decrease in both common lymphoid progenitors (CLPs) and B cell-biased lymphoid progenitors (BLPs) in young, female KDM6A-null mice bone marrow. Next, we performed flow cytometry to catergorize the Hardy fractions of early B-cell development on bone marrow isolated from young, female KDM6A-null mice. B-cell progenitor analysis (Hardy profiles) showed an increase in Fraction A with a concomitant decrease in Fraction B/C and Fraction D, which was likely indicative of an incomplete block in B-cell differentiation after the Fraction A stage. When bulk bone marrow cells isolated from young, female KDM6A-null mice were plated in methylcellulose supplemented with interleukin-7, we observed a significantly decreased colony formation compared with bone marrow cells isolated from wildtype littermates. This pre-B lymphoid progenitor cell plating phenotype was expected given the flow cytometry results of decreased B-cell progenitors outlined above. We examined the effect of the loss of KDM6A expression on germinal center (GC) formation in the spleen following immunization with NP-CGG (4-Hydroxy-3-nitrophenylacetyl-Chicken Gamma Globulin, Ratio 16). Two weeks after NP-CGG immunization, we observed a significant decrease in follicular B-cells (FO) and a significant increase in GC B-cells as compared to wildtype littermates (Figure 1). The result is significant as GC B-cells are thought to be the cell-of-origin of follicular and DLBCL. To determine if inactivation of KDM6A affected antibody production, we measured IgM, IgG, IgE and IgA levels by ELISA from serum isolated from young, female KDM6A-null mice. Results revealed higher levels of IgM and lower levels of IgG in serum from KDM6A-null mice, which is suggestive of a class switch recombination (CSR) defect. Concordant with this result, we observed that the loss of KDM6A impaired CSR to IgG1 in splenic B cells after in vitro stimulation for three days with lipopolysaccharide (LPS), an anti-CD180 antibody and interleukin-4. Moreover, we observed a striking defect in the production of plasma cells from KDM6A-null B-cells after LPS stimulation. Taken together, our data shows that KDM6A plays an important, but complex, role in B-cell development and that loss of KDM6A impedes the B-cell immune response in a specific manner that may contribute to infection and B-cell malignancies.Stagi S, et al. Epigenetic control of the immune system: a lesson from Kabuki syndrome. Immunol Res. 2016; 64(2):345-359. Disclosures No relevant conflicts of interest to declare.

scholarly journals B Cells of the Proliferative Fraction of CLL Clones Exhibit Activated B-Cell and Myeloid-Cell Signatures Suggesting Enhanced Antigen-Presentation, Integrin Responsiveness, and IL-4 Receptiveness: Additional Targets for CLL Therapy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 302-302
Author(s):  
Xiao-Jie Yan ◽  
Florencia Palacios ◽  
Wentian Li ◽  
Sophia Yancopoulos ◽  
Carlo Calissano ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Antigen Presentation ◽  
B Lymphocytes ◽  
Research Funding ◽  
Myeloid Cell ◽  
Integrin Signaling ◽  
Gene Sets

Abstract CLL results from the accumulation of monoclonal B lymphocytes that derive from a small fraction of cells with proliferative activity. Because expression of the DNA mutator, activation-induced cytidine deaminase (AID) is restricted to these dividing cells, they can develop new DNA abnormalities leading to more lethal disease. Hence, such cells are important targets for therapy. Our previous study indicated that the B-cell subset with low levels of CXCR4 and high levels of CD5 (CXCR4DimCD5Bright) is enriched in these cells ("proliferative fraction", PF), whereas the less vital, resting cells exhibit a CXCR4BrightCD5Dim phenotype ("resting fraction", RF). In this study, we focused on analyzing the significantly differentially expressed genes (DEGs) between PF and RF. PF and RF were isolated from 26 CLL (13 U-CLL and 13 M-CLL) and microarrays (llumina HT12) were performed. Selected DEGs between PF and RF were confirmed by rtQ-PCR and/or by flow cytometry. Array data were interpreted using Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA). First, focusing on the Immunologic Signature of B lymphocytes with GSEA, we found the PF was enriched in gene sets shared with IgM memory cells and pre-germinal center B cells, whereas RF was enriched in gene sets in common with naïve, IgD+ B cells. Notably, the PF also shared gene sets with myeloid dendritic cells and monocytes. Protein expression of 10 myeloid markers (CD68, CD1c, CD11a, CD11b, CD11c, CD13, CD31, CD205, CXCR3 and CLECL1) was documented in PF B cells from 11 U-CLL and 11 M-CLL patients, and each was more highly expressed in the PF than RF. No difference in myeloid markers was observed between U-CLL and M-CLL, suggesting that this expression is independent of IGHV mutation status. DEGs were also determined based on expression ratios for PF and RF for each patient; t tests were performed using R. With a fold change cutoff of > 1.5 or < -1.5 and a level of significance of P < 0.01, we identified 198 genes significantly upregulated in PF and 88 in RF. The top biological-function categories identified using IPA indicated that these genes related to cellular development (n=37), cellular growth and proliferation (n=49), cellular movement (n=42) and cell survival and death (n=50). In addition, these DEGs mapped to 8 canonical pathways with Z-scores ≥2, the highest being the integrin signaling pathway (Z-score = 2.887). Twelve of 13 genes were upregulated in the PF and correlated significantly with the integrin pathway: ACTG, ARPC5, ARPC1B, BCAR3, CAPNS1, ITGAX, ITGB1, ITGB2, ITGB7, PFN1, RAC2 and RHOC. Upregulation of integrin subunits was confirmed by Q-PCR and cell surface staining by flow cytometry. Preliminary cellular adhesion experiments suggest PF bind to fibronectin coated plates, and those cells that bind survive better. Next, comparing PF and RF expression ratios for U-CLL vs. M-CLL revealed 502 DEGs in U-CLL and 179 in M-CLL; 144 genes were shared by both U-CLL and M-CLL. IPA analysis of the latter genes correlated best with integrin signaling, and the potential upstream regulators of these were IFNg, IL1, F2 and IFNa. The Rho Family GTPases signaling pathway was significant (10 genes) for DEGs unique to U-CLL. No significant pathway or bio-function relationship was observed in DEGs only in M-CLL. Finally, IPA analysis showed IL4 as an upstream regulator of DEGs unique to the PF of U-CLL based on upregulation of 12 IL4 target genes (APRT, CCL5, CDKN1A, DECR1, IL17RB, ITGAL, ITGB1, LTA, MAPKAPK3, PIGR, SAMSN1, TIMP1). Three other IL-4 targeted genes were paradoxically under-represented in the U-CLL PF (BCL2, CCR7 and VIPR1). The upregulated findings are consistent with PF B cells inducing T cells to produce IL-4 via co-receptors on B/myeloid cells that foster a Th2 response (e.g., CLECL1). In conclusion, gene expression profiling indicates that cells of the PF display a dual activated B cell/myeloid cell phenotype suggesting enhanced antigen-presentation capacities. Integrin signaling appears to be a key pathway for these cells which could foster cell proliferation, survival, and migration, especially in U-CLL clones where integrin activation can lead to Rho GTPases activation. Finally, genes regulated by IL-4 in the PF could be induced by interactions of autologous T cells with CLL B cells. These findings suggest antigen-presentation and integrin and IL-4 signaling pathways as therapeutic targets in CLL, particularly for U-CLL. Disclosures Barrientos: AbbVie: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Janssen: Consultancy.

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