scholarly journals Antigen-induced aggregation and modulation of receptors on hapten-specific B lymphocytes.

1976 ◽  
Vol 143 (3) ◽  
pp. 511-528 ◽  
Author(s):  
G J Nossal ◽  
J E Layton
Keyword(s):  
Cell Surface ◽  
B Lymphocytes ◽  
B Lymphocyte ◽  
Lymphocyte Activation ◽  
Fluorescein Isothiocyanate ◽  
Receptor Occupancy ◽  
Surface Receptors ◽  
Continuous Presence ◽  
Induced Aggregation

Mouse spleen cells were subjected to a fractionation procedure designed to enrich for 4-hydroxy-3-iodo-5-nitro-phenylacetyl (NIP)- or DNP-specific B lymphocytes, which depended on adherence of specific cells to a layer of hapten-gelatin at 4 degrees C, recovery of bound cells by melting, and digestion of adherent antigen by collagenase. A population of cells resulted which contained 90% typical B cells and 37% of cells capable of binding a fluorescent, haptenated polymeric protein. Fractionated cells were reacted in vitro with fluorescent conjugates of the specific haptens with polymerized flagellin [NIP-polymerized flagellin (POL)-tetramethylrhodamine isothiocyanate conjugate or DNP-POL-fluorescein isothiocyanate conjugate] under a variety of conditions, with the aim of investigating the behavior of Ig receptors on B lymphocytes after exposure to antigen; Experiments were performed with immunogenic and tolerogenic concentrations of antigen. Furthermore, four experimental designs were used, namely: (a) brief labeling with fluorescent antigen followed by culture without antigen (pulse design); (b) culture in the continuous presence of fluorescent antigen (continuous-labeling design); (c) culture in the continuous presence of nonlabeled antigen followed by labeling of unoccupied receptors by fluorescent antigen (receptor status design); and (d) culture with nonlabeled antigen for 2 h followed by incubation without further antigen for 20 h and labeling with fluorescent antigen (modulation design). Further insight into receptor occupancy and distribution was gained by the use of fluorescent antihapten and antiglobulin reagents. It was found that both immunogenic and tolerogenic antigen concentrations caused rapid patching and capping of the receptors to which they attached, followed by endocytosis and probably some shedding of Ig receptors. However, a proportion of cells continued to bear some cell surface antigen for 24 h. The immunogenic antigen concentration failed to completely remove the receptor coat from the cell surface. At all stages of immunogenesis, plentiful unoccupied receptors could be demonstrated. The tolerogenic concentration nearly saturated available receptors, and in its continuous presence, only few unoccupied or antigen-occupied surface receptors could be detected after 24 h of culture. Experiments of the modulation design showed that brief incubation with the tolerogenic concentration appeared to suppress receptor resynthesis, as few new receptors could be demonstrated after 20 h of further culture without antigen. Experiments were performed to determine whether fractionated cells prepared from spleens of 8-day-old mice showed an unusual tendency for modulation, even with immunogenic antigen concentrations. They were found to behave essentially like adult fractionated cells. The results are discussed in the framework of current theories of B-lymphocyte activation and tolerization.

scholarly journals Measles Virus Nucleocapsid Protein Binds to FcγRII and Inhibits Human B Cell Antibody Production

1997 ◽  
Vol 186 (2) ◽  
pp. 269-278 ◽  
Author(s):  
Kissia Ravanel ◽  
Claire Castelle ◽  
Thierry Defrance ◽  
T. Fabian Wild ◽  
Dominique Charron ◽  
...  
Keyword(s):  
B Cell ◽  
B Lymphocytes ◽  
Mhc Class Ii ◽  
Antibody Production ◽  
Nucleocapsid Protein ◽  
Measles Virus ◽  
B Lymphocyte ◽  
Lymphocyte Activation ◽  
B Lymphocyte Activation

Despite the development of an efficient specific immune response during measles virus (MV) infection, an immunosuppression occurs contributing to secondary infections. To study the role of nucleocapsid protein (NP) in MV-induced immunosuppression, we produced recombinant MV NP. Purified recombinant NP exhibited biochemical, antigenic, and tridimensional structure similar to viral NP. By flow cytometry, we showed that viral or recombinant NP bound to human and murine B lymphocytes, but not to T lymphocytes. This binding was specific, independent of MHC class II expression, and dependent of the B lymphocyte activation state. The murine IIA1.6 B cell line, deficient in the Fc receptor for IgG (FcγRII) expression, did not bind NP efficiently. Transfected IIA1.6 cells expressing either murine FcγRIIb1 or b2, or human FcγRIIa, b1*, or b2 isoforms efficiently bound NP. Furthermore, this binding was inhibited up to 90% by monoclonal antibodies 2.4G2 or KB61 specific for murine and human FcγRII, respectively. Finally, the in vitro Ig synthesis of CD40- or Ig-activated human B lymphocytes in the presence of interleukin (IL)-2 and IL-10 was reduced by 50% in the presence of recombinant NP. These data demonstrate that MV NP binds to human and murine FcγRII and inhibits in vitro antibody production, and therefore suggests a role for NP in MV-induced immunosuppression.

scholarly journals A Trypanosoma cruzi membrane protein shares an epitope with a lymphocyte activation antigen and induces crossreactive antibodies.

1992 ◽  
Vol 175 (6) ◽  
pp. 1473-1482 ◽  
Author(s):  
C Hernández-Munaín ◽  
J L De Diego ◽  
A Alcina ◽  
M Fresno
Keyword(s):  
Trypanosoma Cruzi ◽  
B Lymphocytes ◽  
B Lymphocyte ◽  
Lymphocyte Activation ◽  
Protozoan Parasite ◽  
Host Cells ◽  
T And B Lymphocytes ◽  
Alternative Mechanism ◽  
Humoral Responses

Chagas' disease results from the infection of the protozoan parasite Trypanosoma cruzi and affects several million people in South America. Several alterations of the immune response have been described in this disease, such as severe immunosuppression of both cellular and humoral responses and massive polyclonal stimulation with the generation of autoantibodies crossreacting with host cells and tissues. We have obtained monoclonal antibodies (mAbs) from T. cruzi-infected mice that recognized a 50/55-kD antigen (GP50/55) on the T. cruzi membrane, but not in other parasites of the family Trypanosomatidae. One of these GP50/55-specific mAbs (C10) crossreacts with a 28-kD antigen (p28) expressed on the membrane of greater than 85% of activated mouse T and B lymphocytes, after in vitro activation with concanavalin A, Salmonella typhosa lipopolysaccharide, phorbol dibutyrate ester, or antigen, and on several murine T and B lymphocyte cell lines. Human T and B lymphocytes also express upon activation with phytohemagglutinin or Staphylococcus aureus Cowan I (SAC) a similar antigen recognized by mAb C10, although in a lower proportion of cells (30-40%). Furthermore, this mAb was able to suppress mouse and human T and B cell proliferation to any of those stimuli. In addition, sera from chagasic patients and T. cruzi-infected mice, but not from control patients or littermates, contain antibodies that recognize a similar p28 antigen on B lymphocytes. Furthermore, the immunoglobulin fractions of some chagasic sera also suppress the proliferation of human T lymphocytes. These results suggest a possible pathological role of autoantibodies as an alternative mechanism for T. cruzi-associated immunosuppression.

scholarly journals Requirement for persistent extracellular antigen in cultures of antigen-binding B lymphocytes.

1976 ◽  
Vol 144 (2) ◽  
pp. 568-571 ◽  
Author(s):  
B L Pike ◽  
G J Nossal
Keyword(s):  
B Lymphocytes ◽  
B Lymphocyte ◽  
Tolerance Induction ◽  
Lymphocyte Activation ◽  
Antigen Binding ◽  
Spleen Cells ◽  
High Concentration ◽  
Clone Size ◽  
B Lymphocyte Activation ◽  
Continuous Presence

A system was established to assess the requirement for continuous presence of antigen in B-lymphocyte activation to antibody formation. Mouse spleen B lymphocytes, enriched for cells bearing anti-NIP (hapten 4-hydroxy-3-iodo-5-nitrophenylacetic acid) receptors, were pretreated briefly with NIP-POL (polymerized flagellin) antigen, washed, and added in small numbers to microcultures. The behaviour of these cells was compared with that of cells cultured in the continuous presence of antigen. Unfractionated spleen cells were studied under similar conditions. In contrast to unfractionated cells, enriched cells could not be triggered effectively by brief contact with antigen at any concentration tested. Fewer cells were activated, and clone size was smaller after brief contact with antigen than when antigen was present continuously. Furthermore, brief contact at high concentration did not cause tolerance induction.

scholarly journals T lymphocyte-dependent B lymphocyte proliferative response to antigen. I Genetic restriction of the stimulation of B lymphocyte proliferation.

1981 ◽  
Vol 153 (4) ◽  
pp. 871-882 ◽  
Author(s):  
H Y Tse ◽  
J J Mond ◽  
W E Paul
Keyword(s):  
T Lymphocytes ◽  
B Lymphocytes ◽  
T Lymphocyte ◽  
Lymphocyte Proliferation ◽  
B Lymphocyte ◽  
Antigen Specificity ◽  
Apparent Lack ◽  
The Face ◽  
Stimulation Of

For the purpose of examining more closely the interaction between T and B lymphocytes, we have developed an in vitro T lymphocyte-dependent B lymphocyte proliferation assay. Proliferation of B lymphocytes in response to antigen was found to depend on the presence of primed T lymphocytes; the B lymphocytes could be derived from nonprimed animals. It appears that these B cells were nonspecifically recruited to proliferate. This nonspecific recruitment, however, was found to be Ir-gene restricted in that B lymphocytes from B10.S mice, which are genetic nonresponders to the polymer Glu60-Ala30-Tyr10 (GAT), could not be stimulated by GAT-primed (responder X nonresponder) F1 T cells. The apparent lack of antigen specificity in the face of Ir gene-restricted T-B interaction may have important implications in our understanding of the recognition unit(s) on T lymphocytes.

Fetal liver pro-B and pre-B lymphocyte clones: expression of lymphoid-specific genes, surface markers, growth requirements, colonization of the bone marrow, and generation of B lymphocytes in vivo and in vitro

1992 ◽  
Vol 12 (2) ◽  
pp. 518-530
Author(s):  
R Palacios ◽  
J Samaridis
Keyword(s):  
B Cell ◽  
B Lymphocytes ◽  
Stromal Cells ◽  
B Lymphocyte ◽  
Germ Line ◽  
Fetal Liver ◽  
Rag 1 ◽  
B Cell Precursors

We describe here the development and characterization of the FLS4.1 stromal line derived from 15-day fetal liver of BALB/c embryos and defined culture conditions that efficiently support the cloning and long-term growth of nontransformed B-220+ 14-day fetal liver cells at two stages of B-cell development, namely, pro-B lymphocytes (immunoglobulin [Ig] genes in germ line configuration) and pre-B cells (JH-rearranged genes with both light-chain Ig genes in the germ line state). All B-cell precursor clones require recombinant interleukin-7 (rIL-7) and FLS4.1 stromal cells for continuous growth in culture, but pro-B lymphocyte clones can also proliferate in rIL-3. None proliferate in rIL-1, rIL-2, rIL-4, rIL-5, rIL-6, or leukemia inhibitory factor. FLS4.1 stromal cells synthesize mRNA for Steel factor but not for IL-1 to IL-7; all pro-B and pre-B clones express c-Kit, the receptor for Steel factor, and a c-Kit-specific antibody inhibits the enhanced proliferative response of fetal liver B-220+ B-cell precursors supported by FLS4.1 stromal cells and exogenous rIL-7 but does not affect that promoted by rIL-7 alone. Northern (RNA) blot analysis of the expression of the MB-1, lambda 5, Vpre-B, c mu, RAG-1, and RAG-2 genes in pro-B and pre-B clones show that transcription of the MB-1 gene precedes IgH gene rearrangement and RNA synthesis from c mu, RAG-1, RAG-2, lambda 5, and Vpre-B genes. All clones at the pre-B-cell stage synthesize mRNA for c mu, RAG-1, and RAG-2 genes; transcription of the lambda 5 and Vpre-B genes seems to start after D-to-JH rearrangement in B-cell precursors, indicating that the proteins encoded by either gene are not required for B-cell progenitors to undergo D-to-JH gene rearrangement. These findings mark transcription of the MB-1 gene as one of the earliest molecular events in commitment to develop along the B-lymphocyte pathway. Indeed, both pro-B and pre-B clones can generate in vitro and in vivo B lymphocytes but not T lymphocytes; moreover, these clones do not express the CD3-gamma T-cell-specific gene, nor do they have rearranged gamma, delta, or beta T-cell antigen receptor genes.

The Ku70 autoantigen interacts with p40phox in B lymphocytes

1999 ◽  
Vol 112 (4) ◽  
pp. 503-513
Author(s):  
N. Grandvaux ◽  
S. Grizot ◽  
P.V. Vignais ◽  
M.C. Dagher
Keyword(s):  
Nadph Oxidase ◽  
B Lymphocytes ◽  
B Lymphocyte ◽  
Cell Extract ◽  
Dependent Protein ◽  
Terminal Amino ◽  
Flavocytochrome B ◽  
Lymphocyte Cell ◽  
Two Hybrid

Ku70, a regulatory component of the DNA-dependent protein kinase, was identified by a yeast two-hybrid screen of a B lymphocyte cDNA library as a partner of p40phox, a regulatory component of the O2--producing NADPH oxidase. Truncated constructs of p40phox and Ku70 were used to map the interacting sites. The 186 C-terminal amino acids (aa) of Ku70 were found to interact with two distinct regions of p40phox, the central core region (aa 50–260) and the C-terminal extremity (aa 260–339). In complementary experiments, it was observed that Ku70 binds to immobilized recombinant p40phox fusion protein and that p40phox and Ku70 from a B lymphocyte cell extract comigrate in successive chromatographies on Q Separose, Superose 12 and hydroxylapatite columns. Moreover, we report that Ku70 and p40phox colocalize in B lymphocytes and in transfected Cos-7 cells. We also show that the two NADPH oxidase activating factors, p47phox and p67phox are substrates for DNA-PK in vitro and that they are present together with p40phox in the nucleus of B cells. These results may help solve the paradox that the phox protein triad, p40phox, p47phox and p67phox, is expressed equally in B lymphocytes and neutrophils, whereas the redox component of the NADPH oxidase, a flavocytochrome b, which is well expressed in neutrophils, is barely detectable in B lymphocytes.

Laminin E8 alveolarization site: heparin sensitivity, cell surface receptors, and role in cell spreading

1997 ◽  
Vol 272 (3) ◽  
pp. L494-L503
Author(s):  
L. Chen ◽  
V. Shick ◽  
M. L. Matter ◽  
S. M. Laurie ◽  
R. C. Ogle ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Cell Surface ◽  
Surface Proteins ◽  
Cell Surface Proteins ◽  
Surface Receptors ◽  
Beta1 Integrin ◽  
Alpha Dystroglycan ◽  
Alveolar Formation ◽  
Laminin 1

Cell adhesion to amino acids 2179-2198 (SN-peptide) of the laminin-1 alpha1-chain is required for lung alveolar formation in vitro (M. L. Matter and G. W. Laurie. J. Cell Biol. 124: 1083-1090, 1994). The nature of the SN-peptide receptor(s) was probed with neutralizing anti-integrin monoclonal antibodies (MAb), cells lacking integrin subunits, soluble heparin, and SN-peptide columns. Cell adhesion and spreading studies confirmed the specificity of SN-peptide and revealed adhesion to be unaffected by inclusion of anti-beta1-, anti-alpha(2-6)- or anti-alpha(V)beta5-integrin MAb. Cells lacking beta1- or alpha6-integrin subunits were fully adherent. Adhesion was heparin, but not chondroitin sulfate or heparinase, sensitive, much as is alpha-dystroglycan-laminin-1 binding. Heparin eluted approximately 155- and 180-kDa cell-surface proteins from SN-peptide columns. An additional approximately 91-kDa protein was eluted by EDTA. All were unrecognized by anti-beta1-integrin MAb. SN-peptide therefore interacts with three cell-surface proteins for which the identity remains to be determined.

scholarly journals Development of suppressor T lymphocytes for Epstein-Barr virus-induced B-lymphocyte outgrowth during acute infectious mononucleosis: assessment by two quantitative systems

Blood ◽  
1981 ◽  
Vol 57 (3) ◽  
pp. 510-517 ◽  
Author(s):  
RT Schooley ◽  
BF Haynes ◽  
J Grouse ◽  
C Payling-Wright ◽  
AS Fauci ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
B Lymphocytes ◽  
Epstein Barr Virus ◽  
B Lymphocyte ◽  
Acute Stage ◽  
Cell Mediated Immunity ◽  
Barr Virus ◽  
Epstein Barr

Abstract A system of 3H-thymidine incorporation by lymphocytes in culture for 3 wk has been utilized for quantitative assessment of the ability of T lymphocytes to inhibit outgrowth of autologous Epstein-Barr virus (EBV) transformed B lymphocytes. Lymphocytes from EBV-seronegative individuals lack the ability to suppress outgrowth of autologous EBV- transformed B lymphocytes. This capability appears during the course of primary EBV-induced infectious mononucleases (IM) as the atypical lymphocytosis is subsiding and persists for years after recovery from primary EBV infection. The ability of T lymphocytes from EBV- seropositive subjects or convalescent IM patients to inhibit B- lymphocyte outgrowth is not HLA restricted. Thus, T lymphocytes capable of inhibition of in vitro EBV-induced B-cell outgrowth emerge during the acute stage of IM and may represent an important control mechanism of EBV-induced B-lymphocyte proliferation in vivo. The system provides a highly sensitive quantitative means for in vitro assessment of cell- mediated immunity to EBV.

scholarly journals INDEPENDENCE OF H-2K AND H-2D ANTIGENIC DETERMINANTS ON THE SURFACE OF MOUSE LYMPHOCYTES

1973 ◽  
Vol 137 (2) ◽  
pp. 511-526 ◽  
Author(s):  
Catherine Neauport-Sautes ◽  
Frank Lilly ◽  
Danielle Silvestre ◽  
François M. Kourilsky
Keyword(s):  
Cell Surface ◽  
Fluorescein Isothiocyanate ◽  
Antigenic Determinants ◽  
Membrane Structures ◽  
F1 Hybrid ◽  
Living Mouse ◽  
Lymph Node Cells ◽  
In Cis ◽  
Induced Aggregation ◽  
Mouse Lymphocytes

At 37°C, fluorescein-conjugated anti-H-2 alloantibodies specifically induce, at the surface of living mouse lymphocytes, the redistribution of the corresponding H-2 antigens, which cluster as patches and sometimes single caps at one pole of the cell. This aggregation is inhibited at 0°C and the H-2 antigens, stained by fluorescent antibodies in the cold, appear evenly spread over the cell surface. This phenomenon was used to define the relationships between the membrane structures bearing the antigens coded by the H-2K and the H-2D genes of the H-2 region. Monospecific anti-H-2 antibodies coupled to either tetramethyl rhodamine isothiocyanate or fluorescein isothiocyanate were used to induce the redistribution of H-2D and H-2K antigens of the H-2b and H-2k haplotype at the surface of lymph node cells from homozygous and F1 hybrid mice. It was observed that the diffuse distribution of H-2K antigens labeled at 0°C was not affected by the prior antibody-induced aggregation of H-2D antigens and vice versa. The results were the same for H-2 antigens governed by genes located either in cis or in trans position. These data indicate that the H-2K and H-2D antigens migrate independently at the cell surface, and suggest that the gene products from the D and the K end of the H-2 region are expressed on independent molecules or structures at the cell membrane.

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