scholarly journals A fluorescent probe for STED microscopy to study NIP-specific B cells

The Analyst ◽  
2021 ◽  
Author(s):  
Selda Glowacki ◽  
Maria Angela Gomes de Castro ◽  
Ka Man Yip ◽  
Ommolbanin Assadpour ◽  
Matthias Muenchhalfen ◽  
...  
Keyword(s):  
B Cells ◽  
Fluorescent Probe ◽  
B Lymphocytes ◽  
Sted Microscopy ◽  
Affinity Probes

We have developed a series of monovalent fluorophore-conjugated affinity probes based on the hapten 3-nitro-4-hydroxy-5-iodophenylacetyl (NIP), which is widely used as a model antigen to study B lymphocytes and the...

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

scholarly journals SELECTIVE TRIGGERING OF HUMAN T AND B LYMPHOCYTES IN VITRO BY POLYCLONAL MITOGENS

1974 ◽  
Vol 140 (1) ◽  
pp. 1-18 ◽  
Author(s):  
Melvyn Greaves ◽  
George Janossy ◽  
Michael Doenhoff
Keyword(s):  
B Cells ◽  
B Lymphocytes ◽  
Human Lymphocytes ◽  
Culture Conditions ◽  
Pokeweed Mitogen ◽  
Cell Populations ◽  
T And B Cells ◽  
Activated T Cells ◽  
Lymphocyte Responses

Human lymphocytes from spleen and tonsils have been cultured with a variety of polyclonal mitogens. Cultures consisted of either unseparated T and B cells or alternatively purified T or B lymphocytes. The purity of the starting cell populations and the origin of activated lymphoblasts was analyzed with a panel of seven markers which discriminate between T and B cells. The selectivity of the lymphocyte responses was influenced by cell populations in a given culture, the mitogen used, and to a limited extent on culture conditions. Purified T lymphocytes from tonsil and spleen responded to phytohemagglutinin (PHA), pokeweed mitogen (PWM), and staphylococcal enterotoxin B (SEB). Purified B cells from spleen responded well to PWM, weakly to SEB and lipopolysaccharide, but not at all to PHA. Tonsil B cells responded weakly to PWM and SEB but not to PHA. Some B lymphocytes do respond to PHA in the presence of activated T cells. These results are discussed in relation to previously reported selective responses of human cells and parallel studies in animal species.

scholarly journals Interleukin 4 induces selective production of interleukin 6 from normal human B lymphocytes.

1989 ◽  
Vol 170 (4) ◽  
pp. 1463-1468 ◽  
Author(s):  
E B Smeland ◽  
H K Blomhoff ◽  
S Funderud ◽  
M R Shalaby ◽  
T Espevik
Keyword(s):  
B Cells ◽  
B Lymphocytes ◽  
Interleukin 6 ◽  
Specific Effect ◽  
Interleukin 4 ◽  
Tnf Alpha ◽  
Ifn Gamma ◽  
Normal Human ◽  
Tnf Secretion

In this paper we have shown that extensively purified human B lymphocytes respond to IL-4 treatment with a marked production of IL-6. Addition of anti-mu potentiated the effect of IL-4 on IL-6 production. Other cytokines tested like TNF-alpha and-beta, IFN-gamma, IL-1, IL-2, and IL-5 did not induce IL-6 secretion when given to resting B cells. Although B cells generally also produced TNF-alpha and TNF-beta upon stimulation, IL-4 did not induce TNF secretion and seemingly had a specific effect on IL-6 production.

CD100/Plexin-B1 interactions sustain proliferation and survival of normal and leukemic CD5+ B lymphocytes

Blood ◽  
2003 ◽  
Vol 101 (5) ◽  
pp. 1962-1969 ◽  
Author(s):  
Luisa Granziero ◽  
Paola Circosta ◽  
Cristina Scielzo ◽  
Elisa Frisaldi ◽  
Stefania Stella ◽  
...  
Keyword(s):  
B Cells ◽  
B Lymphocytes ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Follicular Dendritic Cells ◽  
Growth And Survival ◽  
High Affinity Receptor ◽  
Activated T Lymphocytes ◽  
Affinity Receptor

Growth and survival of chronic B-cell tumors are favored by the malignant cell's capacity to respond to selected microenvironmental stimuli provided by nontumoral bystander cells. To investigate which mechanisms operate in these crosstalks and whether they are malignancy-related or reproduce the mechanisms used by normal B cells we have studied the expression and functional role of semaphorin CD100 (now called Sema4D) in chronic lymphocytic leukemia (CLL) cells and normal CD5+ B cells. We demonstrate here that (1) leukemic and normal CD5+ B lymphocytes uniformly express CD100; (2) the CD100 high-affinity receptor Plexin-B1 is expressed by bone marrow stromal cells, follicular dendritic cells, and activated T lymphocytes, and is thus available to CD100+ lymphocytes in different specific microenvironments; and (3) upon interaction between CD100 and Plexin-B1 both CLL and normal CD5+ B cells increase their proliferative activity and extend their life span. These findings establish that Plexin-B1 is an easily accessible receptor for CD100 within the immune system. The encounter of CD100+ leukemic cells with Plexin-B1 may promote the proliferation and survival of malignant cells. The crosstalk operated by the CD100/Plexin-B1 interaction is not malignancy related but reproduces a mechanism used by normal CD5+ B cells.

scholarly journals Autologous stimulation of human lymphocyte subpopulation.

1975 ◽  
Vol 142 (5) ◽  
pp. 1327-1333 ◽  
Author(s):  
G Opelz ◽  
M Kiuchi ◽  
M Takasugi ◽  
P I Terasaki
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Lymphocytes ◽  
Human Lymphocyte ◽  
Lymphocyte Subpopulation ◽  
High Background ◽  
Background Stimulation ◽  
Most Probable Explanation ◽  
Stimulation Of

The background stimulation universally seen when lymphocytes are cultured in vitro has been shown to be markedly lowered by reducing the proportion of B lymphocytes. B-rich fractions of lymphocytes had extremely high background stimulation. It is concluded that stimulation of T cells, probably by autologous B cells, provides the most probable explanation for the findings described.

scholarly journals Mitogen-induced phosphorylation of human B-lymphocyte proteins. Relationship to protein kinase C activation

1989 ◽  
Vol 263 (1) ◽  
pp. 57-64 ◽  
Author(s):  
G R Guy ◽  
M Finney ◽  
R H Michell ◽  
J Gordon
Keyword(s):  
Protein Kinase C ◽  
B Cells ◽  
Protein Kinase ◽  
B Lymphocytes ◽  
Dose Response ◽  
Response Curve ◽  
Dose Response Curve ◽  
Beta Form ◽  
Kinase C ◽  
Hydrolysis Of

We have investigated the rapid phosphorylation of proteins in B-lymphocytes incubated with the tumour-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), anti-Ig and combinations of TPA and the Ca2+ ionophore ionomycin. Two-dimensional electrophoretic analysis was used to identify the proteins phosphorylated in cells preincubated with [32P]Pi. TPA induced a characteristic pattern of labelled proteins, four of which (pp85, pp76, pp66 and pp63) showed a dose-dependent incorporation of 32P on serine residues. The phosphorylation of pp63 and pp66, in particular, correlated with the mitogenic dose-response curve. Addition of the Ca2+ ionophore ionomycin to B-cells also stimulated a characteristic incorporation of 32P into proteins, which included pp63 and pp66. With combined doses of TPA and ionomycin, these two proteins show an enhanced phosphorylation, which correlated well with the synergistic enhancement of proliferation shown by this combination of agents. Protein kinase C (PKC) was partially purified from B-cells and separated into alpha and beta subtypes. The activation of both PKCs was assessed with increasing doses of TPA and concentrations of Ca2+ of 0.1 microM and 2 microM. For both forms of PKC, in particular the beta form, higher concentrations of Ca2+ shifted the dose-response curve for TPA to the left and increased the maximum activation. Anti-Ig, which stimulated B-cells by cross-linking surface immunoglobulin and causing hydrolysis of PtdIns(4,5)P2, also caused increased phosphorylation of several proteins, which again included pp63 and pp66. These data suggest that PKC, particularly the beta form, is involved in the early part of the proliferation cascade for human B-lymphocytes. It is most probably activated in a synergistic manner by the increased Ca2+ and diacylglycerol levels which result from the earlier hydrolysis of PtdIns(4,5)P2.

Synergistic Cooperation Between T and B Lymphocytes from Old Mice in the Production of Auto-Anti-Idiotypic Antibody Regulation in Adult Irradiated Hosts

1982 ◽  
Vol 1 (1-2) ◽  
pp. 3-10 ◽  
Author(s):  
Myron R. Szewczuk
Keyword(s):  
B Cells ◽  
B Lymphocytes ◽  
B Lymphocyte ◽  
Intrinsic Property ◽  
Adoptive Cell Transfer ◽  
Cell Transfer ◽  
Lymphocyte Population ◽  
Thymus Cells ◽  
Old Mice ◽  
Idiotypic Antibody

ABSTRACTThe effect of age on the ability of B lymphocytes and thymus cells from donors of various ages to be capable of producing an anti-idiotype-blocked, hapten-augmentable PFC was studied by adoptive cell transfer techniques. Lethally irradiated mice were reconstituted with syngeneic B lymphocytes and thymus cells from donors of various ages. Recipients were immunized with trinitrophenylated bovine gamma globulin (TNP-BGG) one or seven days after cell transfer. Splenic IgG anti-TNP plaque-forming cell (PFC) responses were assayed in the absence and presence of hapten for anti-idiotype (Id)-blocked, hapten-augmentable PFC, 14 days after immunization. It was found that the B lymphocyte population from 2 month old donors together with thymus cells from donors of various ages (2 to 19 months) were incapable of reconstituting mice to produce anti-Id-blocked, hapten-augmentable PFC. Similar results were obtained when mice were reconstituted with thymus cells from 2-month-old donors together with B cells from donors of various ages (2 to 14 months). In contrast, mice reconstituted with B cells plus thymus cells from the same 8-month or older donors produced a significantly high percentage of anti-Id-block, hapten augmentable PFC. Mice reconstituted with B cells from 8 months or older donors plus thymus cells from donors of various ages (8 to 19) months also produced a significantly high percentage of hapten-augmentable PFC. Experiments with B cells and thymus cells from 2-or 8-month old donors parked in lethally irradiated 2-or 8-months old recipients for 7 days revealed that neither lymphocytes from old donors or old recipients were capable of inducing the appearance of anti-Id-blocked, hapten-augmentable PFC in the lymphocyte population from 2-month-old donors. Thus, the results of this study indicate syner-gistic co-operation between B lymphocytes and thymus cells from old donors for the production of auto-anti-idiotypic antibody regulation with age. This production of auto-anti-Id antibody with age seems not to be an induced maturation event but perhaps an intrinsic property unique to lymphocytes from old donors.

scholarly journals Complement and virus-specific antibody-dependent infection of normal B lymphocytes by human immunodeficiency virus type 1

Blood ◽  
1993 ◽  
Vol 81 (7) ◽  
pp. 1808-1818
Author(s):  
G Gras ◽  
Y Richard ◽  
P Roques ◽  
R Olivier ◽  
D Dormont
Keyword(s):  
Human Immunodeficiency Virus ◽  
B Cells ◽  
Hiv Infection ◽  
B Lymphocytes ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Virus Envelope ◽  
Immunodeficiency Virus ◽  
Virus Specific Antibody

We tested the susceptibility of human purified, normal B lymphocytes to human immunodeficiency virus type 1 (HIV-1) infection, in the presence or absence of complement-sufficient serum and of virus-specific antibodies. Virus replication was detected when cells were infected in the presence of both complement and anti-HIV antibodies (C'-ADE conditions), by day 2 postinfection. Similar results were obtained when B lymphocytes were purified either from peripheral blood (three healthy donors) or from tonsils (four individuals with chronic tonsillitis). HIV infection was shown by polymerase chain reaction (PCR) detection of proviral sequences (gag and pol genes), by p24 antigen synthesis, and by cocultivation assay with MT2 cells. The higher p24 production was obtained when B cells were preactivated for 2 days by phorbol 12- myristate 13-acetate (PMA) before infection and then cultured in the presence of low-molecular weight B-cell growth factor (LMW-BCGF). Expression of virus envelope glycoprotein (gp) 120 could also be detected on a subpopulation of B cells (CD19+, CD22+) by flow cytometry. Blocking experiments with monoclonal antibodies (MoAbs) against CD4, CD21 (complement receptor 2 [CR2]), CD35 (CR1), CD19, and CD5 surface molecules indicated that infection of B cells involves CD4, CD21, and CD35 antigens. Indeed, blocking of CD4 receptor inhibited 10% of p24 production, and blocking of both CD21 and CD35 led to extinction of p24 signal. CR-dependent pathway is thus a major route for C'-ADE of HIV infection in normal B cells. Our results emphasize the importance of studying interactions between HIV and the complement system for better understanding infection mechanisms and the major dysfunctions of B cells in HIV-infected individuals.

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