Impact of tofacitinib treatment on human B-cells in vitro and in vivo

ABSTRACT We have shown previously that rotavirus (RV) can infect murine intestinal B220+ cells in vivo (M. Fenaux, M. A. Cuadras, N. Feng, M. Jaimes, and H. B. Greenberg, J. Virol. 80:5219-5232, 2006) and human blood B cells in vitro (M. C. Mesa, L. S. Rodriguez, M. A. Franco, and J. Angel, Virology 366:174-184, 2007). However, the effect of RV on B cells, especially those present in the human intestine, the primary site of RV infection, is unknown. Here, we compared the effects of the in vitro RV infection of human circulating (CBC) and intestinal B cells (IBC). RV infected four times more IBC than CBC, and in both types of B cells the viral replication was highly restricted to the memory subset. RV induced cell death in 30 and 3% of infected CBC and IBC, respectively. Moreover, RV induced activation and differentiation into antibody-secreting cells (ASC) of CBC but not IBC when the B cells were present with other mononuclear cells. However, RV did not induce these effects in purified CBC or IBC, suggesting the participation of other cells in activating and differentiating CBC. RV infection was associated with enhanced interleukin-6 (IL-6) production by CBC independent of viral replication. The infection of the anti-B-cell receptor, lipopolysaccharide, or CpG-stimulated CBC reduced the secretion of IL-6 and IL-8 and decreased the number of ASC. These inhibitory effects were associated with an increase in viral replication and cell death and were observed in polyclonally stimulated CBC but not in IBC. Thus, RV differentially interacts with primary human B cells depending on their tissue of origin and differentiation stage, and it affects their capacity to modulate the local and systemic immune responses.

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RANTES and macrophage inflammatory protein 1 alpha selectively enhance immunoglobulin (IgE) and IgG4 production by human B cells.

Journal of Experimental Medicine ◽

10.1084/jem.183.5.2397 ◽

1996 ◽

Vol 183 (5) ◽

pp. 2397-2402 ◽

Cited By ~ 70

Author(s):

H Kimata ◽

A Yoshida ◽

C Ishioka ◽

M Fujimoto ◽

I Lindley ◽

...

Keyword(s):

B Cells ◽

Necrosis Factor Alpha ◽

Platelet Factor ◽

Human B Cells ◽

Inflammatory Protein ◽

Chemotactic Protein ◽

Macrophage Inflammatory Protein ◽

Do So

We studied the effects of various chemokines including neutrophil-activating peptide 2 (NAP-2), beta-thromboglobulin (beta-TG), platelet factor 4 (PF-4), melanoma growth stimulating activity (GRO), gamma interferon-induced protein (IP-10), regulated on activation, normal T expressed and secreted (RANTES), macrophage inflammatory protein 1 alpha (MIP-1 alpha), MIP-1 beta, and monocyte chemotactic protein 1 (MCP-1) on Immunoglobulin (IgE) and IgG4 production by human B cells. None of these chemokines with or without interleukin (IL-4), anti-CD40 or -CD58 monoclonal antibody (mAb), induced IgE and IgG4 production by B cells from nonatopic donors. However, RANTES and MIP-1 alpha selectively enhanced IgE and IgG4 production induced by IL-4 plus anti-CD40 or -CD58 mAb without affecting production of IgM, IgG1, IgG2, IgG3, IgA1, or IgA2, whereas other chemokines failed to do so. Enhancement of IgE and IgG4 production by RANTES and MIP-1 alpha was specifically blocked by anti-RANTES mAb and anti-MIP-1 alpha antibody (Ab), respectively, whereas anti-IL-5 mAb, anti-IL-6 mAb, anti-IL-10 Ab, anti-IL-13 Ab, and anti-tumor necrosis factor-alpha mAb failed to do so. Purified surface IgE positive (slgE4) and slgG4+ B cells generated either in vitro or in vivo spontaneously produced IgE and IgG4, respectively, whereas sIgE- and sIgG4- B cells failed to do so. RANTES and MIP-1 alpha enhanced spontaneous IgE and IgG4 production in slgE+ and slgG4- B cells, respectively, whereas neither RANTES nor MIP-1 alpha did so in sIgE- or sIgG4- B cells. Purified sIgE4+ and sIgG4+, but not sIgE- or sIgG4- B cells, generated in vitro and in vivo expressed receptors for RANTES and MIP-1 alpha, whereas they failed to express receptors for other chemokines. These findings indicate that RANTES and MIP-1 alpha enhance IgE and IgG4 production by directly stimulating sIgE+ and sIgG4+ B cells.

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Cultivation and Immortalization of Human B-Cells Producing a Human Monoclonal IgM Antibody Binding to MDA-LDL: Further Evidence for Formation of Atherogenic MDA-LDL Adducts in HumansIn Vivo

Oxidative Medicine and Cellular Longevity ◽

10.1155/2017/6047142 ◽

2017 ◽

Vol 2017 ◽

pp. 1-7 ◽

Cited By ~ 1

Author(s):

Franz Tatzber ◽

Edith Pursch ◽

Ulrike Resch ◽

Roswitha Pfragner ◽

Sandra Holasek ◽

...

Keyword(s):

B Cells ◽

Cell Line ◽

Oxidized Ldl ◽

Malonic Dialdehyde ◽

Density Lipoprotein ◽

Ldl Oxidation ◽

Human Donor ◽

Human B Cells

Oxidatively modified low-density lipoprotein (oLDL) is firmly believed to play an important role in the initiation and development of atherosclerosis, and malonic dialdehyde (MDA) is one of the major lipid peroxidation breakdown products involved in this process. In recent decades, antibodies against MDA-LDL have been detected in human and animal sera. In our study, human B-cells from the peripheral blood of a healthy female donor were fused with the SP2/0 mouse myeloma cell line. Antibody-producing hybridomas were detected by MDA-LDL-IgG/IgM enzyme-linked immunosorbent assays (ELISA) and Cu++-oxidized LDL IgG/IgM (oLAb) ELISA. Cells with supernatants emitting positive signals for antibodies were then cloned and after sufficient multiplication frozen and stored under liquid nitrogen. Due to the loss of antibody-producing ability, we established an MDA-LDL-IgM-producing cell line by recloning. This allowed isolation and immortalization of several human B-cells. The human donor had not been immunized with MDA-modified proteins, thus obviously producing MDA-LDL antibodiesin vivo. Furthermore, using these antibodies forin vitroexperiments, we were able to demonstrate that MDA epitopes are among the epitopes generated during Cu++-LDL oxidation as well. Finally, these antibodies compete in ELISA and cell culture experiments with MDA as a challenging toxin or ligand.

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Genetic modification of primary human B cells to model high-grade lymphoma

Nature Communications ◽

10.1038/s41467-019-12494-x ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 8

Author(s):

Rebecca Caeser ◽

Miriam Di Re ◽

Joanna A. Krupka ◽

Jie Gao ◽

Maribel Lara-Chica ◽

...

Keyword(s):

B Cells ◽

Ex Vivo ◽

B Cell Lymphoma ◽

Genetic Alterations ◽

Cancer Genes ◽

Full Transformation ◽

Human B Cells ◽

Sequencing Studies

Abstract Sequencing studies of diffuse large B cell lymphoma (DLBCL) have identified hundreds of recurrently altered genes. However, it remains largely unknown whether and how these mutations may contribute to lymphomagenesis, either individually or in combination. Existing strategies to address this problem predominantly utilize cell lines, which are limited by their initial characteristics and subsequent adaptions to prolonged in vitro culture. Here, we describe a co-culture system that enables the ex vivo expansion and viral transduction of primary human germinal center B cells. Incorporation of CRISPR/Cas9 technology enables high-throughput functional interrogation of genes recurrently mutated in DLBCL. Using a backbone of BCL2 with either BCL6 or MYC, we identify co-operating genetic alterations that promote growth or even full transformation into synthetically engineered DLBCL models. The resulting tumors can be expanded and sequentially transplanted in vivo, providing a scalable platform to test putative cancer genes and to create mutation-directed, bespoke lymphoma models.

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Genetic modification of primary human B cells generates translationally-relevant models of high-grade lymphoma

10.1101/618835 ◽

2019 ◽

Cited By ~ 1

Author(s):

Rebecca Caeser ◽

Miriam Di Re ◽

Joanna A Krupka ◽

Jie Gao ◽

Maribel Lara-Chica ◽

...

Keyword(s):

B Cells ◽

Ex Vivo ◽

B Cell Lymphoma ◽

Cancer Genes ◽

Full Transformation ◽

Growth And Survival ◽

Human B Cells ◽

Sequencing Studies

AbstractSequencing studies of Diffuse Large B Cell Lymphoma (DLBCL) have identified hundreds of recurrently altered genes. However, it remains largely unknown whether and how these mutations may contribute to lymphomagenesis, either individually or in combination. Existing strategies to address this problem predominantly utilize cell lines, which are limited by their initial characteristics and subsequent adaptions to prolongedin vitroculture. Here, we describe a novel co-culture system that enables theex vivoexpansion and viral transduction of primary human germinal center B cells. The incorporation of CRISPR/Cas9 technology enables high-throughput functional interrogation of genes recurrently mutated in DLBCL. Using a backbone ofBCL2with eitherBCL6orMYCwe have identified co-operating oncogenes that promote growth and survival, or even full transformation into synthetically engineered models of DLBCL. The resulting tumors can be expanded and sequentially transplantedin vivo, providing a scalable platform to test putative cancer genes and for the creation of mutation-directed, bespoke lymphoma models.

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Anti-Proliferative and Pro-Apoptotic Effects of Short-Term Inhibition of Telomerase In Vivo and in Human Malignant B Cells Xenografted in Zebrafish

Cancers ◽

10.3390/cancers12082052 ◽

2020 ◽

Vol 12 (8) ◽

pp. 2052

Author(s):

Silvia Giunco ◽

Manuela Zangrossi ◽

Francesca Dal Pozzolo ◽

Andrea Celeghin ◽

Giovanni Ballin ◽

...

Keyword(s):

Cell Proliferation ◽

B Cells ◽

Zebrafish Embryos ◽

Short Term ◽

Post Transplantation ◽

Telomerase Inhibition ◽

Human B Cells ◽

Apoptotic Effects

Besides its canonical role in stabilizing telomeres, telomerase reverse transcriptase (TERT) may promote tumor growth/progression through extra-telomeric functions. Our previous in vitro studies demonstrated that short-term TERT inhibition by BIBR1532 (BIBR), an inhibitor of TERT catalytic activity, negatively impacts cell proliferation and viability via telomeres’ length-independent mechanism. Here we evaluate the anti-proliferative and pro-apoptotic effects of short-term telomerase inhibition in vivo in wild-type (wt) and tert mutant (terthu3430/hu3430; tert−/−) zebrafish embryos, and in malignant human B cells xenografted in casper zebrafish embryos. Short-term Tert inhibition by BIBR in wt embryos reduced cell proliferation, induced an accumulation of cells in S-phase and ultimately led to apoptosis associated with the activation of DNA damage response; all these effects were unrelated to telomere shortening/dysfunction. BIBR treatment showed no effects in tert−/− embryos. Xenografted untreated malignant B cells proliferated in zebrafish embryos, while BIBR pretreated cells constantly decreased and were significantly less than those in the controls from 24 to up to 72 h after xenotransplantation. Additionally, xenografted tumor cells, treated with BIBR prior- or post-transplantation, displayed a significant higher apoptotic rate compared to untreated control cells. In conclusion, our data demonstrate that short-term telomerase inhibition impairs proliferation and viability in vivo and in human malignant B cells xenografted in zebrafish, thus supporting therapeutic applications of TERT inhibitors in human malignancies.

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Direct contact between Plasmodium falciparum and human B-cells in a novel co-culture increases parasite growth and affects B-cell growth

Malaria Journal ◽

10.1186/s12936-021-03831-x ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Sreenivasulu B. Reddy ◽

Noemi Nagy ◽

Caroline Rönnberg ◽

Francesca Chiodi ◽

Allan Lugaajju ◽

...

Keyword(s):

Plasmodium Falciparum ◽

B Cells ◽

Direct Contact ◽

Culture Medium ◽

Parasite Growth ◽

Memory B Cells ◽

Human B Cells ◽

Long Time

Abstract Background Plasmodium falciparum parasites cause malaria and co-exist in humans together with B-cells for long periods of time. Immunity is only achieved after repeated exposure. There has been a lack of methods to mimic the in vivo co-occurrence, where cells and parasites can be grown together for many days, and it has been difficult with long time in vitro studies. Methods and results A new method for growing P. falciparum in 5% CO2 with a specially formulated culture medium is described. This knowledge was used to establish the co-culture of live P. falciparum together with human B-cells in vitro for 10 days. The presence of B-cells clearly enhanced parasite growth, but less so when Transwell inserts were used (not allowing passage of cells or merozoites), showing that direct contact is advantageous. B-cells also proliferated more in presence of parasites. Symbiotic parasitic growth was verified using CESS cell-line and it showed similar results, indicating that B-cells are indeed the cells responsible for the effect. In malaria endemic areas, people often have increased levels of atypical memory B-cells in the blood, and in this assay it was demonstrated that when parasites were present there was an increase in the proportion of CD19 + CD20 + CD27 − FCRL4 + B-cells, and a contraction of classical memory B-cells. This effect was most clearly seen when direct contact between B-cells and parasites was allowed. Conclusions These results demonstrate that P. falciparum and B-cells undoubtedly can affect each other when allowed to multiply together, which is valuable information for future vaccine studies.

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AB0024 IN VITRO CHARACTERIZATION OF CENERIMOD, A POTENT AND SELECTIVE SPHINGOSINE 1-PHOSPHATE RECEPTOR 1 (S1P1) MODULATOR IN PREVENTING MIGRATION OF NON-ACTIVATED AND ACTIVATED PRIMARY HUMAN B CELLS IN THE PRESENCE OR ABSENCE OF GLUCOCORTICOIDS

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-eular.2482 ◽

2021 ◽

Vol 80 (Suppl 1) ◽

pp. 1046.1-1046

Author(s):

L. Schlicher ◽

P. Kulig ◽

M. Murphy ◽

M. Keller

Keyword(s):

B Cells ◽

B Cell ◽

B Lymphocytes ◽

Cell Activation ◽

Surface Expression ◽

Cell Surface Expression ◽

B Cell Activation ◽

Human B Cells ◽

Circulating Lymphocytes

Background:Cenerimod is a potent, selective, and orally active sphingosine 1-phosphate receptor 1 (S1P1) modulator that is currently being evaluated in a Phase 2b study in patients with systemic lupus erythematosus (SLE) (NCT03742037). S1P1 receptor modulators sequester circulating lymphocytes within lymph nodes, thereby reducing pathogenic autoimmune cells (including B lymphocytes) in the blood stream and in inflamed tissues. Extensive clinical experience has become available for the nonselective S1P receptor modulator fingolimod in relapsing forms of multiple sclerosis, supporting this therapeutic concept for the treatment of autoimmune disorders.Objectives:Although the effect of S1P-receptor modulators in reducing peripheral B cells is well documented1,2, the role of the S1P1 receptor on this cell type is only incompletely understood. In this study, the mode of action of cenerimod on primary human B cells was investigated in a series of in vitro experiments, including S1P1 receptor cell surface expression and chemotaxis towards S1P. Moreover, S1P1 expression following B cell activation in vitro was studied. As glucocorticoids (GC) are frequently used in the treatment of patients with autoimmune disorders including SLE, the potential influence of GC on the mode of action of cenerimod was evaluated.Methods:Primary human B lymphocytes from healthy donors were isolated from whole blood. In one set of experiments, cells were treated with different concentrations of cenerimod to measure S1P1 receptor internalization by flow cytometry. In a second set of experiments, isolated B cells were activated using different stimuli or left untreated. Cells were then analysed for S1P1 and CD69 cell surface expression and tested in a novel real-time S1P-mediated migration assay. In addition, the effect of physiological concentrations of GCs (prednisolone and prednisone) on cenerimod activity in preventing S1P mediated migration was tested.Results:In vitro, cenerimod led to a dose-dependent internalization of the S1P1 receptor on primary human B lymphocytes. Cenerimod also blocked migration of nonactivated and activated B lymphocytes towards S1P in a concentration-dependent manner, which is in line with the retention of lymphocytes in the lymph node and the reduction of circulating lymphocytes observed in the clinical setting. Upon B cell activation, which was monitored by CD69 upregulation, a simultaneous downregulation of S1P1 expression was detected, leading to less efficient S1P-directed cell migration. Importantly, physiological concentrations of GC did not affect the inhibitory activity of cenerimod on B cell migration.Conclusion:These results show that cenerimod, by modulating S1P1, blocks B lymphocyte migration towards its natural chemoattractant S1P and demonstrate compatibility of cenerimod with GC. These results are consistent with results of comparable experiments done previously using primary human T lymphocytes.References:[1]Nakamura M et al., Mult Scler. 2014 Sep; 20(10):1371-80.[2]Strasser DS et al., RMD Open 2020;6:e001261.Disclosure of Interests:None declared

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Ginkgolic acid induces apoptosis and autophagy of endometrial carcinoma cells via inhibiting PI3K/Akt/mTOR pathway in vivo and in vitro

Human & Experimental Toxicology ◽

10.1177/09603271211023789 ◽

2021 ◽

pp. 096032712110237

Author(s):

L Zhou ◽

S Li ◽

J Sun

Keyword(s):

Endometrial Cancer ◽

B Cells ◽

Western Blot ◽

Signal Pathway ◽

Mtor Pathway ◽

Related Protein ◽

Western Blot Assay ◽

Ginkgolic Acid

Endometrial cancer (EC) is the fourth most common malignancy in women in developed countries. The prognosis of EC is extremely poor, and it is an important factor that contributes to the death of patients. Therefore, studying EC pathogenesis and therapeutic targets, and exploring effective drugs are the primary tasks to improve the prognosis of EC. In the present study, we aimed to explore the function of ginkgolic acid (GA) in EC cell apoptosis and autophagy through PI3K/Akt/mTOR signal pathway in vitro and in vivo. Firstly, MTT assay and clone formation assay were employed to analyze the Ishikawa and HEC-1-B cell viabilities and proliferation after treatment with GA. The results showed that GA inhibited endometrial cancer cell survival. Flow cytometry assay and western blot assay were applied to examine the apoptosis and apoptosis related protein Bcl-2, Bax, Cleaved caspase-3 expression levels of Ishikawa and HEC-1-B cells after treatment with GA. Next, we applied western blot assay to analyze the autophagy associated proteins LC3I, LC3II, p62 and Beclin-1 in GA treated Ishikawa and HEC-1-B cells. We found that GA promoted apoptosis and induced autophagy of endometrial cancer cells. Meanwhile, western blot assay was also used to determine the expression levels of the PI3K/Akt/mTOR signal pathway related protein and the results revealed that GA inhibited the activity of PI3K/Akt/mTOR pathway. Finally, we found that GA inhibited tumor growth in vivo through immunohistochemistry assay. In conclusion, GA induces apoptosis and autophagy of EC cells via inhibiting PI3K/Akt/mTOR pathway in vivo and vitro.

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