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 Specific adsorption of IgM antibody onto H-2-activated mouse T lymphocytes.

1976 ◽  
Vol 143 (2) ◽  
pp. 444-449 ◽  
Author(s):  
L Hudson ◽  
J Sprent
Keyword(s):  
T Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Progenitor Cell ◽  
Lymphoid Cells ◽  
Immunofluorescent Staining ◽  
Cell Populations ◽  
Activated T Cells ◽  
Specific Manner

Evidence is presented to support the contention that IgM demonstrable by surface immunofluorescent staining on H-2-activated T cells represents specifically adsorbed B-cell-derived alloantibody. T cells activated to H-2 determinants expressed surface IgM only when the progenitor cell populations contained B lymphocytes. IgM was not detected on T cells activated to determinants which fail to stimulate alloantibody production (e.g., M-locus determinants). In addition, IgM-negative H-2 activated T cells (derived from B-cell-depleted lymphoid cells), unlike M-locus-activated T cells, adsorbed IgM in a specific manner when incubated in vitro with "early bleed" antisera raised against the activating H-2 determinants.

scholarly journals CELL INTERACTIONS IN THE IMMUNE RESPONSE IN VITRO

1972 ◽  
Vol 136 (4) ◽  
pp. 737-760 ◽  
Author(s):  
Marc Feldmann
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Cell Interaction ◽  
T And B Lymphocytes ◽  
Activated T Cells ◽  
Cell Cooperation

The mechanism of interaction of T and B lymphocytes was investigated in an in vitro hapten carrier system using culture chambers with two compartments separated by a cell impermeable nucleopore membrane. Because specific cell interaction occurred efficiently across this membrane, contact of T and B lymphocytes was not essential for cooperation which must have been mediated by a subcellular component or "factor." By using different lymphoid cell populations in the lower culture chamber and activated thymus cells in the upper chamber (with antigen present in both), it was found that the antigen-specific mediator acted indirectly on B cells, through the agency of macrophages. Macrophages which had been cultured in the presence of activated T cells and antigen acquired the capacity to specifically induce antibody responses in B cell-containing lymphoid populations. Trypsinization of these macrophages inhibited their capacity to induce immune responses, indicating that the mediator of cell cooperation is membrane bound. By using antisera to both the haptenic and carrier determinants of the antigen as blocking reagents, it was demonstrated that the whole antigen molecule was present on the surface of macrophages which had been exposed to activated T cells and antigen. Because specifically activated T cells were essential a component of the antigen-specific mediator must be derived from these cells. By using anti-immunoglobulin sera as inhibitors of the binding of the mediator to macrophages, the T cell component was indeed found to contain both κ- and µ-chains and was thus presumably a T cell-derived immunoglobulin. It was proposed that cell cooperation is mediated by complexes of T cell IgM and antigen, bound to the surface of macrophage-like cells, forming a lattice of appropriately spaced antigenic determinants. B cells become immunized by interacting with this surface. With this mechanism of cell cooperation, the actual pattern of antigen-B cell receptor interactions in immunization would be the same with both thymus-dependent and independent antigens. An essential feature of the proposed mechanism of cell cooperation is that macrophage-B cell interaction must occur at an early stage of the antibody response, a concept which is supported by many lines of evidence. Furthermore this mechanism of cell interaction can be elaborated to explain certain phenomena such as the highly immunogenic macrophage-bound antigen, antigenic competition, the distinction between immunity and tolerance in B lymphocytes, and the possible mediation of tolerance by T lymphocytes.

Vav proteins are required for B-lymphocyte responses to LPS

Blood ◽  
2005 ◽  
Vol 106 (2) ◽  
pp. 635-640 ◽  
Author(s):  
Barbara Hebeis ◽  
Elena Vigorito ◽  
Dorottya Kovesdi ◽  
Martin Turner
Keyword(s):  
B Cells ◽  
B Lymphocytes ◽  
B Lymphocyte ◽  
High Dose ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Extracellular Signal ◽  
B Cell Leukemia ◽  
Lymphocyte Responses

Abstract B lymphocytes respond to bacterial lipopolysaccharide (LPS) through Toll-like receptor 4 (TLR4) and CD180 (previously called RP105). We show here that the responses of B lymphocytes to LPS require the function of the Vav family of guanine nucleotide exchange factors. Vav1-mutant mice generate defective humoral immunoglobulin G (IgG) responses following administration of low doses of LPS but respond normally to higher doses, while mice lacking both Vav1 and Vav2 manifest defective responses even after a high dose of LPS. Vav1/2-mutant B cells fail to divide extensively in vitro in response to LPS or CD180, while deficiency of Vav1 alone impairs CD180-but not LPS-driven proliferation. Likewise, activation of Akt (a PI3K [phosphatidylinositol 3-kinase] target) and phosphorylation of IκBα in response to CD180 or LPS required Vav1 and Vav2, while Vav1 deficiency led to defective responses to CD180. In addition, activation of ERK (extracellular signal regulated kinase) required Vav1 and Vav2 in response to CD180 but was Vav1 and vav2 independent in response to LPS. Induction of CD86 and CD25 by anti-CD180 also required Vav function, as did the induction of the antiapoptotic protein Bcl-xL (B-cell leukemia XL). These data provide evidence for the function for the Vav proteins in regulating the responses of B cells to LPS. (Blood. 2005;106:635-640)

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 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 The autoantigen-binding B cell repertoires of normal and of chronically graft-versus-host-diseased mice.

1987 ◽  
Vol 165 (6) ◽  
pp. 1675-1687 ◽  
Author(s):  
A G Rolink ◽  
T Radaszkiewicz ◽  
F Melchers
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
The Other ◽  
Cell Populations ◽  
Foreign Antigen ◽  
Alloreactive T Cells ◽  
Polyclonal Activator ◽  
Activated B Cells

A quantitative analysis of the frequencies of autoantibody-producing B cells in GVHD and in normal mice has been undertaken by generating collections of hybridomas of activated B cells. These hybridomas secreted sufficient quantities of Ig to allow binding analyses on a panel of autoantigens. B cells have been activated in a variety of ways. In vivo they were activated by injection of alloreactive T cells of one parent, leading to GVHD by a foreign antigen, sheep erythrocytes, in a secondary response, or by the polyclonal activator LPS. B cells from an experimentally unstimulated animal were used for an analysis of the normal background. In vitro B cells were activated by alloreactive T cells or by LPS. The frequencies of hybridomas and, therefore, of activated B cells producing autoantibodies to DNA or to kidney were not significantly different in mice activated by a graft-vs.-host T cell response as compared with B cell populations activated by any of the other procedures. They were found to compose 7.1-17.1% of the total repertoire of activated B cells. Moreover, the frequencies of autoantibody-producing activated B cells does not change with time after induction of the graft-vs.-host reaction. The pattern and frequencies of autoantigen-binding specificities to cytoskeleton, smooth muscle, nuclei, mitochondria, and DNA were not found to be different in any of the groups of hybridomas. The single notable exception, found in GVHD mice, were hybridomas producing autoantibodies to kidney proximal tubular brush border. These results allow the conclusion that autoantigen-binding B cells exist in an activated state in GVHD mice, as well as in mice activated by a foreign antigen or by a polyclonal activator, in B cell populations activated in vitro either by alloreactive T cells or by a polyclonal activator, and even in the background of experimentally unstimulated animals. T cell-mediated graft-vs.-host activation, in large part, does not lead to a selective expansion of autoantigen-binding B cells. The main difference between the graft-vs.-host-activated B cell repertoire and all others is that approximately 90% of teh autoantibodies were of the IgG class, whereas al autoantibodies found in the other groups were IgM.

scholarly journals IMMUNOLOGIC REACTIONS TO HAPTENS ON AUTOLOGOUS CARRIERS

1973 ◽  
Vol 137 (2) ◽  
pp. 411-423 ◽  
Author(s):  
John W. Moorhead ◽  
Curla S. Walters ◽  
Henry N. Claman
Keyword(s):  
T Cells ◽  
B Cells ◽  
Aminocaproic Acid ◽  
Single Amino Acid ◽  
Secondary Response ◽  
Spleen Cells ◽  
Activated T Cells ◽  
Thymus Cells

Both thymus-derived (T) and bone marrow-derived (B) lymphocytes participate in the response to a hapten 4-hydroxy-3-iodo-5-nitrophenylacetic acid (NIP), coupled to a nonimmunogenic isologous carrier, mouse gamma globulin (MGG). Spleen cells from mice immunized with NIP-MGG show increased DNA synthesis in vitro when cultured with NIP-MGG. The participation of and requirement for T cells in the response was demonstrated by treating the spleen cells with anti-θ serum. This treatment resulted in a 77% inhibition of the antigen response. Furthermore, adoptively transferred normal thymus cells could be specifically "activated" by NIP-MGG in vivo and they responded secondarily to the antigen in vitro. The active participation of B cells in the secondary response was demonstrated by passing the immune spleen cells through a column coated with polyvalent anti-MGG serum. Column filtration reduced the number of NIP-specific plaque-forming cells and NIP-specific rosette-forming cells (both functions of B cells) and produced a 47% inhibition of the NIP-MGG response. The ability of the cells to respond to phytohemagglutinin (PHA) was not affected by column filtration showing that T cells were not being selectively removed. The participation of B cells in the in vitro NIP-MGG response was also shown by treatment of the spleen cells with antiserum specific for MGG and MGG determinants. B cells were removed by treatment with anti-IgM or polyvalent anti-MGG serum plus complement, resulting in a respective 46 and 49% inhibition of the response to NIP-MGG. (Treatment with anti-IgM serum had no effect on T cells.) The contribution of the hapten NIP to stimulation of T cells was investigated using NIP-MGG-activated thymus cells. These activated T cells responded in vitro very well to the NIP-MGG complex but not to the MGG carrier alone demonstrating the requirement of the hapten for T cell stimulation. The response was also partially inhibited (41%) by incubating the activated cells with NIP coupled to a single amino acid (epsilon-aminocaproic acid) before addition of NIP-MGG. These results demonstrated that T cells recognize the hapten NIP when it is coupled to the isologous carrier MGG.

Delayed in Vitro Immunoglobulin Production by Cord Lymphocytes

PEDIATRICS ◽  
1980 ◽  
Vol 65 (3) ◽  
pp. 497-500
Author(s):  
Yukiaki Miyagawa ◽  
Kenichi Sugita ◽  
Atsushi Komiyama ◽  
Taro Akabane
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Mononuclear Cells ◽  
Pokeweed Mitogen ◽  
Immunoglobulin Production ◽  
Mitogen Stimulation ◽  
Functional Immaturity ◽  
Adult Cells

Pokeweed mitogen-induced immunoglobulin (Ig) production by cord lymphocytes was studied in vitro by Ig-secreting plaque-forming cell (Ig-PFC) assay. Although adult mononuclear cells generated all of IgM-, IgG-, and IgA-PFC, cord mononuclear cells generated only IgM-PFC when cultured for seven days. The number of cord IgM-PFC was 102 ± 26/104 mononuclear cells, being about one fourth of that of adult IgM-PFC. When cultured for 14 days, cord mononuclear cells formed increased numbers of IgM-PFC in contrast to adult cells, and yielded IgG-PFC as well, indicating delayed Ig production. Cord T cells were much less effective at helping adult B cells to differentiate into Ig-PFC as compared with adult T cells. Substitution of adult T cells for cord T cell markedly improved the response of cord B cells. The present study demonstrates Ig secretion by cord lymphocytes in response to pokeweed mitogen stimulation. The results further indicate that the delayed Ig production by cord lymphocytes is largely due to functional immaturity of the T cells.

scholarly journals Interleukin-13 in Combination With CD40 Ligand Potently Inhibits Apoptosis in Human B Lymphocytes: Upregulation of Bcl-xL and Mcl-1

Blood ◽  
1997 ◽  
Vol 89 (12) ◽  
pp. 4415-4424 ◽  
Author(s):  
Jon Lømo ◽  
Heidi Kiil Blomhoff ◽  
Sten Eirik Jacobsen ◽  
Stanislaw Krajewski ◽  
John C. Reed ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Peripheral Blood ◽  
Cell Apoptosis ◽  
Cell Types ◽  
Cd40 Ligand ◽  
Interleukin 13

Abstract Interleukin-13 (IL-13) is a novel T-cell–derived cytokine with IL-4–like effects on many cell types. In human B lymphocytes, IL-13 induces activation, stimulates proliferation in combination with anti-IgM or anti-CD40 antibodies, and directs Ig isotype switching towards IgE and IgG4 isotypes. We show here that IL-13 also regulates human B-cell apoptosis. IL-13 reduced spontaneous apoptosis of peripheral blood B cells in vitro, as shown by measurement of DNA fragmentation using the TUNEL and Nicoletti assays. The inhibition of cell death by IL-13 alone was significant but modest, but was potently enhanced in combination with CD40 ligand (CD40L), a survival stimulus for B cells by itself. Interestingly, IL-13 increased the expression of CD40 on peripheral blood B cells, providing a possible mechanism for the observed synergy. IL-13 alone was a less potent inhibitor of apoptosis than IL-4. Moreover, there was no additive effect of combining IL-4 and IL-13 at supraoptimal concentrations, which is consistent with the notion that the IL-4 and IL-13 binding sites share a common signaling subunit. The combination of IL-13 with CD40L augmented the expression of the Bcl-2 homologues Bcl-xL and Mcl-1, suggesting this as a possible intracellular mechanism of induced survival. By contrast, levels of Bcl-2, and two other Bcl-2 family members, Bax and Bak, remained unaltered. Given the importance of the CD40-CD40L interaction in B-cell responses, these results suggest a significant role of IL-13 in the regulation of B-cell apoptosis.

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