scholarly journals Receptors for T cell-replacing factor/interleukin 5. Specificity, quantitation, and its implication.

1988 ◽  
Vol 168 (3) ◽  
pp. 863-878 ◽  
Author(s):  
S Mita ◽  
N Harada ◽  
S Naomi ◽  
Y Hitoshi ◽  
K Sakamoto ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Binding Sites ◽  
B Cell Lymphoma ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Activated B Cells

T cell-replacing factor (TRF)/IL-5 is a glycosylated polypeptide that acts as a key factor for B cell growth and differentiation. Since IL-5 action is probably mediated by specific cell surface receptor(s), we have characterized the binding of IL-5 to cells using biosynthetically [35S]methionine-labeled IL-5 and 125I-IL-5 that had been prepared using Bolton-Hunter reagent. The radiolabeled IL-5 binds specifically to BCL1-B20 (in vitro line) (a murine chronic B cell leukemic cell line previously shown to differentiate into IgM-secreting cells in response to IL-5) within 10 min at 37 degrees C. There are two classes of binding sites with high affinity (Kd = 66 pM) and low affinity (Kd = 12 nM) for IL-5 and an average number of binding sites for high affinity and for low affinity were 400 and 7,500 per cell, respectively. The specificity of binding of radiolabeled IL-5 has been confirmed by demonstrating that only unlabeled IL-5 and anti-IL-5 mAb but not by IL-1, IL-2, IL-3, IFN-gamma, and GM-CSF inhibit radiolabeled IL-5 binding to BCL1-B20 cells. Treatment of surface-bound radiolabeled IL-5 with bivalent crosslinkers identified a membrane polypeptide of Mr 46,500 to which IL-5 is crosslinked. A variety of cell types have been surveyed for the capacity to bind specifically radiolabeled IL-5 with high affinity. BCL1 cells MOPC104E (murine myeloma cell line) expressed IL-5-R, whereas BAL. 17 and L10 A (B cell lymphoma) did not. T cell line, mastocytoma cell line, or macrophage tumor cell line did not display detectable levels of IL-5-R. were hardly detectable on normal resting B cells but were expressed on LPS-activated B cells, fitting the function of IL-5 that acts on activated B cells for their differentiation into Ig-secreting cells. Intriguingly, early B cell lines (J-87 and T-88) that grow in the presence of IL-5 expressed significant but low numbers of high-affinity binding sites for IL-5. The biological effects of IL-5 were mediated by high-affinity binding sites. The identification and characterization of IL-5-R should provide new insight into the apparent diverse biological activities of IL-5.

scholarly journals Interleukin 2 induces antigen-reactive T cell lines to secrete BCGF-I.

1983 ◽  
Vol 158 (6) ◽  
pp. 2024-2039 ◽  
Author(s):  
M Howard ◽  
L Matis ◽  
T R Malek ◽  
E Shevach ◽  
W Kell ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Interleukin 2 ◽  
Activated T Lymphocytes ◽  
T Cell Lines ◽  
Activated B Cells

Antigen-activated T lymphocytes produce within 24 h of stimulation a factor that is indistinguishable biochemically and functionally from the B cell co-stimulating growth factor, BCGF-I, originally identified in induced EL4 supernatants: Supernatants from antigen-stimulated T cell lines are not directly mitogenic for resting B cells, but synergize in an H-2-unrestricted manner with anti-Ig activated B cells to produce polyclonal proliferation but not antibody-forming-cell development; biochemical studies reveal the B cell co-stimulating factor present in antigen-stimulated T cell line supernatants is identical by phenyl Sepharose chromatography and isoelectric focusing (IEF) to EL4 supernatant BCGF-I. We thus conclude that normal T cells produce BCGF-I in response to antigenic stimulation. Analysis of the mechanism of BCGF-I production by antigen-stimulated T cells showed that optimum amounts of BCGF-I were obtained as quickly as 24 h post-stimulation, and that the factor producing cells in the T cell line investigated bore the Lyt-1+2- phenotype. As few as 10(4) T cells produced sufficient BCGF-I to support the proliferation of 5 X 10(4) purified anti-Ig activated B cells. Finally, the activation of normal T cell lines to produce BCGF-I required either antigen presented in the context of syngeneic antigen-presenting cells (APC) or interleukin 2 (IL-2).

scholarly journals Expression of interleukin 2 receptors on activated human B cells.

1984 ◽  
Vol 160 (5) ◽  
pp. 1450-1466 ◽  
Author(s):  
T A Waldmann ◽  
C K Goldman ◽  
R J Robb ◽  
J M Depper ◽  
W J Leonard ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Interleukin 2 ◽  
Antigen Expression ◽  
Pokeweed Mitogen ◽  
Binding Studies ◽  
High Affinity ◽  
Activated B Cells

Using anti-Tac, a monoclonal anti-interleukin 2 (IL-2) receptor antibody, we have explored the possibility that certain activated B cells display receptors for IL-2. Resting normal B cells and unselected B cell lines established from normal individuals were Tac antigen negative. In contrast, the cell surface Tac antigen expression was demonstrable on 6 of 10 B cell lines from patients with Burkitt's lymphoma, 5 of 6 B cell lines derived from patients with HTLV-I-associated adult T cell leukemia (including all four that had integrated HTLV-I into their genome), and on certain normal B cells activated with pokeweed mitogen. Furthermore, cloned Epstein-Barr virus-transformed B cell lines derived from Tac-positive normal B cells continued to express the Tac antigen in long-term cultures and manifested high affinity IL-2 receptors identified in binding studies with purified radiolabeled IL-2. The line 5B4 developed in the present study could be induced with purified JURKAT-derived or recombinant IL-2 to express a larger number of IL-2 receptors. Furthermore, the addition of IL-2 to the 5B4 B cell line augmented IgM synthesis, which could be blocked by the addition of anti-Tac. The size of the IL-2 receptors expressed on the cloned normal B cell lines was similar (53,000-57,000 daltons) to that of receptors on phytohemagglutinin-stimulated T cell lymphoblasts. Thus, certain malignant and activated normal B cells display the Tac antigen and manifest high affinity receptors for IL-2. These data suggest that IL-2 may play a role in the differentiation of activated B cells into immunoglobulin-synthesizing and -secreting cells.

scholarly journals New Molecular and Therapeutic Insights into Canine Diffuse Large B Cell Lymphoma Elucidates the Role of the Dog As a Model for Human Disease

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4173-4173
Author(s):  
Luca Aresu ◽  
Serena Ferraresso ◽  
Laura Marconato ◽  
Luciano Cascione ◽  
Sara Napoli ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
Clinical Outcome ◽  
B Cell ◽  
Cell Line ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Rna Seq ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Background. Diffuse large B-cell lymphoma (DLBCL) is the commonest lymphoma in both humans and dogs. Canine DLBCL (cDLBCL) is considered an ideal comparative model for drug development, but a complete genomic characterization of this tumor is still lacking. In this study, we report an integrated analysis to comprehensively define the molecular mechanisms of cDLBCL and possible associations with clinical outcome. Methods. Fifty cDLBCLs were analyzed by RNA-Seq, methyl-CpG-binding sequencing and array comparative genomic hybridization. Normal B-cells derived from lymph nodes of 11 healthy dogs were used as controls.Additionally, immunohistochemistry, in vitroand in vivoexperiments were performed as validation analyses. Results.Compared to normal B-cells, cDLBCL showed a marked up-regulation of genes involved in the PI3K/mTOR and NF-κB pathways, including several TLRs in association with MYD88, indicating mechanisms similar to the human activated B cell-like subtype DLBCL. Both RNA-Seq and methylation sequencing led to the identification of two groups of cDLBCLs bearing different clinical outcome. The two groups did not overlap with the human germinal center B-cell (GCB) and the activated B-cell-like (ABC) DLBCL subtypes or the human DLBCL consensus clusters. The dogs with the poorest outcome presented a signature largely defined by markers of T-cell-mediated immune responses, with a high expression of PDL-1, PD-1 and CTLA-4, also validated in an independent cohort of cDLBCL by immunohistochemistry. These data provide a strong rationale for the use of cDLBCL to study immune checkpoint modulators. The observed high expression of PI3K/mTOR pathway genes was confirmed and validated achieving a clear anti-tumor activity with the use of the PI3K-delta inhibitor idelalisib and of the novel dual PI3K/mTOR inhibitor bimiralisib in the cDLBCL cell line CLBL-1. The cDLBCLs showed an up-regulation of MYC and of its targets, sustained by recurrent gains in the chromosome 13, where the oncogene is located, in approximately half of the cases. Thus, we have exposed the cDLBCL cell line CLBL-1 to the BET inhibitor birabresib (OTX015) and to the BRD4 degrader MZ1. Both compounds caused a significant reduction in the proliferation of tumor cells, and this effect was stronger especially with the second compound. Exposure to MZ1 determined an important downregulation of MYC and also of LIN28B, the most overexpressed transcript in cDLBCL when compared to controls. While LIN28B does not seem to be a relevant gene for human DLBCL, its overexpression causes murine T-cell lymphomas (Beachy et al, Blood 2011), and there is a direct association of MYC with LIN28B promoter resulting in transcriptional transactivation (Chang et al, PNAS 2009). Here, LIN28B genetic silencing in the CLBL-1 lead to a reduction in cell growth, opening new therapeutic target perspectives in canine lymphoma. Conclusions. We have reported the first large next generation sequencing study investigating the cDLBCL transcriptome, methylome and the genome-wide CNVs. We identified deregulated pathways and individual transcripts providing therapeutic targets, including an immune-related signature affecting the outcome of a subgroup of cDLBCL. Our data sustain the use of cDLBCL as comparative models for human DLBCL but also highlight differences that must be kept in consideration. Disclosures Hillmann: PIQUR Therapeutics AG: Employment. Wymann:PIQUR Therapeutics AG: Employment, Equity Ownership, Patents & Royalties.

High affinity binding sites on plasma membrane obtained from the lymphoblastoid cultured 1301 cell line for highly radioactive serum thymic factor

1982 ◽  
Vol 684 (1) ◽  
pp. 117-126 ◽  
Author(s):  
Louis Noël Gastinel ◽  
Jean-Marie Pleau ◽  
Mireille Dardenne ◽  
André Sasaki ◽  
Evanghelos Bricas ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Line ◽  
Binding Sites ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Serum Thymic Factor ◽  
Thymic Factor

High Affinity Binding Sites for Activated Protein C and Protein C on Cultured Human Umbilical Vein Endothelial Cells

1994 ◽  
Vol 72 (03) ◽  
pp. 465-474 ◽  
Author(s):  
Neelesh Bangalore ◽  
William N Drohan ◽  
Carolyn L Orthner
Keyword(s):  
Binding Sites ◽  
Protein C ◽  
Umbilical Vein ◽  
Activated Protein C ◽  
Affinity Binding ◽  
Cell Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Gla Domain ◽  
Umbilical Vein Endothelial Cells

SummaryActivated protein C (APC) is an antithrombotic serine proteinase having anticoagulant, profibrinolytic and anti-inflammatory activities. Despite its potential clinical utility, relatively little is known about its clearance mechanisms. In the present study we have characterized the interaction of APC and its active site blocked forms with human umbilical vein endothelial cells (HUVEC). At 4° C 125I-APC bound to HUVEC in a specific, time dependent, saturable and reversible manner. Scatchard analysis of the binding isotherm demonstrated a Kd value of 6.8 nM and total number of binding sites per cell of 359,000. Similar binding isotherms were obtained using radiolabeled protein C (PC) zymogen as well as D-phe-pro-arg-chloromethylketone (PPACK) inhibited APC indicating that a functional active site was not required. Competition studies showed that the binding of APC, PPACK-APC and PC were mutually exclusive suggesting that they bound to the same site(s). Proteolytic removal of the N-terminal γ-carboxyglutamic acid (gla) domain of PC abolished its ability to compete indicating that the gla-domain was essential for cell binding. Surprisingly, APC binding to these cells appeared to be independent of protein S, a cofactor of APC generally thought to be required for its high affinity binding to cell surfaces. The identity of the cell binding site(s), for the most part, appeared to be distinct from other known APC ligands which are associated with cell membranes or extracellular matrix including phospholipid, thrombomodulin, factor V, plasminogen activator inhibitor type 1 (PAI-1) and heparin. Pretreatment of HUVEC with antifactor VIII antibody caused partial inhibition of 125I-APC binding indicating that factor VIII or a homolog accounted for ∼30% of APC binding. Studies of the properties of surface bound 125I-APC or 125I-PC and their fate at 4°C compared to 37 °C were consistent with association of ∼25% of the initially bound radioligand with an endocytic receptor. However, most of the radioligand appeared not to be bound to an endocytic receptor and dissociated rapidly at 37° C in an intact and functional state. These data indicate the presence of specific, high affinity binding sites for APC and PC on the surface of HUVEC. While a minor proportion of binding sites may be involved in endocytosis, the identity and function of the major proportion is presently unknown. It is speculated that this putative receptor may be a further mechanisms of localizing the PC antithrombotic system to the vascular endothelium.

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