scholarly journals Analysis of the 5'UTR of HCV genotype 3 grown in vitro in human B cells, T cells, and macrophages

2010 ◽  
Vol 7 (1) ◽  
pp. 155 ◽  
Author(s):  
Dennis Revie ◽  
Michael O Alberti ◽  
John G Prichard ◽  
Ann S Kelley ◽  
S Zaki Salahuddin
Keyword(s):  
T Cells ◽  
B Cells ◽  
Genotype 3 ◽  
Hcv Genotype ◽  
Human B Cells ◽  
Hcv Genotype 3

scholarly journals Production and characterization of a bispecific IgG capable of inducing T-cell-mediated lysis of malignant B cells

Blood ◽  
1993 ◽  
Vol 81 (12) ◽  
pp. 3343-3349 ◽  
Author(s):  
BK Link ◽  
GJ Weiner
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Hybridoma Cells ◽  
Activated T Cells ◽  
Human B Cells ◽  
B Cell Malignancy ◽  
Cell Malignancy

Abstract Bispecific monoclonal antibodies (bsabs) recognizing both CD3 and a tumor antigen can redirect T-cell-mediated cytotoxicity toward cells bearing that antigen. Such bsabs have been shown to be more effective than monospecific monoclonal antibodies (MoAbs) at preventing tumor growth in animal models of B-cell malignancy. The current studies describe the production and preliminary evaluation of a bsab designed to induce the lysis of malignant human B cells by human T cells. The bsab was obtained from a hybrid-hybridoma cell line produced by fusing OKT3-secreting hybridoma cells with hybridoma cells that secrete 1D10. 1D10 is an MoAb that recognizes an antigen found on a majority of malignant human B cells that has not been detected to a significant degree on normal resting or activated lymphocytes. High performance liquid chromatography (HPLC) was used to separate bsab from monospecific antibodies that were also present in the hybrid-hybridoma antibody product. The bsab was then evaluated in vitro for its ability to induce lysis of malignant B cells by activated T cells. The bsab consistently induced extensive lysis in vitro of 1D10 (+) cells, including both cell lines and cells obtained from patients with a variety of B-cell malignancies. No such effect was seen with activated T cells alone or activated T cells with monospecific antibody. No increased lysis was seen with 1D10 (-) cell lines. The bsab also mediated lysis of malignant B cells by autologous T cells. We conclude bsab containing an OKT3 arm and a 1D10 arm can induce T-cell-mediated lysis in a manner that is both potent and specific. This supports further evaluation of this bsab as a potential immunotherapy of B-cell malignancy.

The Molecular Pathway of Lysosomal Antigen Processing in Peripheral Blood Dendritic Cells (DC) Significantly Differs from That in Monocyte-Derived DC Generated Ex Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3449-3449
Author(s):  
Christoph Driessen ◽  
Alexander Beck ◽  
Ekkehard Weber ◽  
Hans U. Haering ◽  
Hubert Kalbacher ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
B Cells ◽  
Western Blot ◽  
Peripheral Blood ◽  
Antigen Processing ◽  
Ex Vivo ◽  
Proteolytic Processing ◽  
Human B Cells

Abstract Dendritic cells (DC) initiatiate immunity and maintain tolerance. They internalize exogenous antigen and convert it into immunogenic peptides by lysosomal proteolytic degradation, ultimately followed by presentation to CD4 T cells. Monocyte-derived DC (MO-DC) generated in vitro with GM-CSF and IL-4 serve as prototype DC to analyse the cellular biology and biochemistry of DC. However, different types of primary DC, whose functional role in vivo and relationship to MO-DC generated in vitro is unclear, reside in human tissue as well as peripheral blood. The composition of lysosomal proteases in these primary human DC1 and DC2-cells and the way they handle a clinically relevant antigen are unknown, and there is no comparison of the lysosomal processing of antigen by these primary DC to that in primary human B cells or MO-DC generated ex vivo. We have isolated human peripheral blood (PB) DC1 and DC2 cells as well as primary B lymphocytes by magnetic separation and isolated lysosomal compartments from these cells, as well as from MO-DC. Expression and activity of endocytic proteases were assessed by western blot and active site-restricted affinity labelling using a synthetic probe that selectively binds to the active centre of cysteine proteases and allows a simultaneous semiquantitaive assessment and identification of multiple active protease species. In this analysis, PB-DC1 and DC2-cells lacked significant active Cathepsins (Cat) S, L and B as well as asparagine-specific endoprotease AEP, the major enzymes involved in antigen processing in the MHC II-compartment. Surprisingly, lysosomal extracts from PB-DC1 were by far more effective than MO-DC in processing the muliple sclerosis-associated autoantigen myelin basic protein (MBP) in vitro. When analyzed on a molecular scale using mass spectrometry, MBP processing was dominated by CatS, CatD and AEP in MO-DC, as expected, similar to B-lymphoblastoid cells (BLC). PB-DC, however, did not generate proteolytic processing intermediates indicative of CatS or AEP activity but showed the same pattern as primary B-lymphocyte-derived lysosomes, i.e. processing was performed by two cleavage sites that can be reproduced by purified CatG in vitro, suggesting a CatG like dominant lysosomal protease. While active CatG was present in primary human B cells, PB-DC1 cells lacked CatG protein by western blot, suggesting the presence of an as yet unknown dominant endoprotease with CatG-like activity in PB-DC1. By cleaving MBP after pos F90 and F114, this protease directly eliminates the integrity of the major immunodominant MBP epitope MBP85-99. This might lead to poor presentation of this epitope to regulatory T cells resulting in inefficient silencing of MBP-autoreactive T cells during the development of autoimmunity. Our results emphasize the need to apply state-of-the-art biochemical tools to primary human types of APC for the understanding of antigen processing and the rational design of tolerogenic or immunotherapy approaches towards human malignant and autoimmune disorders.

scholarly journals Production and characterization of a bispecific IgG capable of inducing T-cell-mediated lysis of malignant B cells

Blood ◽  
1993 ◽  
Vol 81 (12) ◽  
pp. 3343-3349 ◽  
Author(s):  
BK Link ◽  
GJ Weiner
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Hybridoma Cells ◽  
Activated T Cells ◽  
Human B Cells ◽  
B Cell Malignancy ◽  
Cell Malignancy

Bispecific monoclonal antibodies (bsabs) recognizing both CD3 and a tumor antigen can redirect T-cell-mediated cytotoxicity toward cells bearing that antigen. Such bsabs have been shown to be more effective than monospecific monoclonal antibodies (MoAbs) at preventing tumor growth in animal models of B-cell malignancy. The current studies describe the production and preliminary evaluation of a bsab designed to induce the lysis of malignant human B cells by human T cells. The bsab was obtained from a hybrid-hybridoma cell line produced by fusing OKT3-secreting hybridoma cells with hybridoma cells that secrete 1D10. 1D10 is an MoAb that recognizes an antigen found on a majority of malignant human B cells that has not been detected to a significant degree on normal resting or activated lymphocytes. High performance liquid chromatography (HPLC) was used to separate bsab from monospecific antibodies that were also present in the hybrid-hybridoma antibody product. The bsab was then evaluated in vitro for its ability to induce lysis of malignant B cells by activated T cells. The bsab consistently induced extensive lysis in vitro of 1D10 (+) cells, including both cell lines and cells obtained from patients with a variety of B-cell malignancies. No such effect was seen with activated T cells alone or activated T cells with monospecific antibody. No increased lysis was seen with 1D10 (-) cell lines. The bsab also mediated lysis of malignant B cells by autologous T cells. We conclude bsab containing an OKT3 arm and a 1D10 arm can induce T-cell-mediated lysis in a manner that is both potent and specific. This supports further evaluation of this bsab as a potential immunotherapy of B-cell malignancy.

scholarly journals Cholera Toxin and Escherichia coli Heat-Labile Enterotoxin, but Not Their Nontoxic Counterparts, Improve the Antigen-Presenting Cell Function of Human B Lymphocytes

2009 ◽  
Vol 77 (5) ◽  
pp. 1924-1935 ◽  
Author(s):  
Donatella R. M. Negri ◽  
Dora Pinto ◽  
Silvia Vendetti ◽  
Mario Patrizio ◽  
Massimo Sanchez ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
T Cells ◽  
B Cells ◽  
Cholera Toxin ◽  
B Lymphocytes ◽  
Antigen Presenting Cell ◽  
Heat Labile ◽  
Human B Cells ◽  
Antigen Presenting

ABSTRACT B lymphocytes play an important role in the immune response induced by mucosal adjuvants. In this study we investigated the in vitro antigen-presenting cell (APC) properties of human B cells upon treatment with cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) and nontoxic counterparts of these toxins, such as the B subunit of CT (CT-B) and the mutant of LT lacking ADP ribosyltransferase activity (LTK63). Furthermore, forskolin (FSK), a direct activator of adenylate cyclase, and cyclic AMP (cAMP) analogues were used to investigate the role of the increase in intracellular cAMP caused by the A subunit of CT and LT. B lymphocytes were cultured with adjuvants and polyclonal stimuli necessary for activation of B cells in the absence of CD4 T cells. Data indicated that treatment with CT, LT, FSK, or cAMP analogues, but not treatment with CT-B or LTK63, upregulated surface activation markers on B cells, such as CD86 and HLA-DR, and induced inhibition of the proliferation of B cells at early time points, while it increased cell death in long-term cultures. Importantly, B cells treated with CT, LT, or FSK were able to induce pronounced proliferation of both CD4+ and CD8+ allogeneic T cells compared with untreated B cells and B cells treated with CT-B and LTK63. Finally, only treatment with toxins or FSK induced antigen-specific T-cell proliferation in Mycobacterium tuberculosis purified protein derivative or tetanus toxoid responder donors. Taken together, these results indicated that the in vitro effects of CT and LT on human B cells are mediated by cAMP.

Induction of CD4+ T-cell anergy and apoptosis by activated human B cells

Blood ◽  
2008 ◽  
Vol 112 (12) ◽  
pp. 4555-4564 ◽  
Author(s):  
Theresa Tretter ◽  
Ram K. C. Venigalla ◽  
Volker Eckstein ◽  
Rainer Saffrich ◽  
Serkan Sertel ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Cell Contact ◽  
Activated T Cells ◽  
Cell Responses ◽  
T Cell Anergy ◽  
Human B Cells ◽  
Cell Anergy

Abstract B cells are well-known mediators of humoral immunity and serve as costimulators in the generation of T cell–mediated responses. In several mouse models, however, it was observed that B cells can also down-regulate immune reactions, suggesting a dual role for B cells. Due to this discrepancy and so far limited data, we directly tested the effects of primary human B cells on activated CD4+ T helper cells in vitro. We found that under optimal costimulation large, activated CD25+ B cells but not small CD25− B cells induced temporary T-cell anergy, determined by cell division arrest and down-regulation of cytokine production. In addition, large CD25+ B cells directly induced CD95-independent apoptosis in a subpopulation of activated T cells. Suppression required direct B-T-cell contact and was not transferable from T to T cell, excluding potential involvement of regulatory T cells. Moreover, inhibitory effects involved an IL-2–dependent mechanism, since decreasing concentrations of IL-2 led to a shift from inhibitory toward costimulatory effects triggered by B cells. We conclude that activated CD25+ B cells are able to costimulate or down-regulate T-cell responses, depending on activation status and environmental conditions that might also influence their pathophysiological impact.

scholarly journals 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

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

Sign in / Sign up

Export Citation Format

Share Document