scholarly journals THE MECHANISM OF ANTIGENIC STIMULATION OF PRIMARY AND SECONDARY CLONAL PRECURSOR CELLS

1972 ◽  
Vol 136 (2) ◽  
pp. 241-260 ◽  
Author(s):  
Norman R. Klinman
Keyword(s):  
Serum Antibody ◽  
Precursor Cells ◽  
Antigenic Stimulation ◽  
Soluble Antigen ◽  
Antigen Receptors ◽  
Antigen Concentration ◽  
High Affinity ◽  
Specific Enhancement ◽  
Stimulation Of

Cell transfers to carrier-immunized irradiated mice have permitted an analysis of the in vitro stimulation of clonal precursors of anti-2,4-dinitrophenyl (DNP) antibody-producing cells derived from both immune and nonimmune mice. The results indicate that: (a) carrier-specific enhancement is obligatory for stimulation of primary precursor cells and increases both the size and number of detectable foci derived from secondary precursors. (b) This carrier-specific enhancement is most apparent in the stimulation of precursors of high-affinity antibody producer cells. (c) The antibody produced by primary foci, like that of secondary foci, appears homogeneous. (d) The frequency of clonal precursors in normal spleens is 38% that in spleens from mice 4–8 months after immunization, and the number of such precursors in normal spleens can be reduced fivefold by specific suppression of donor mice with soluble antigen. (e) The average of association constants of primary monofocal antibodies, like that of primary serum antibody produced in carrier-primed mice, is less than 10-fold lower than that of secondary clonal or serum antibody. (f) The affinity of primary monofocal antibodies shows a slight dependence on stimulating antigen concentration; however, a minimum threshold affinity consonant with stimulation is apparent. (g) Free hapten inhibits antigenic stimulation of primary precursor cells at a much lower concentration than is required for the inhibition of secondary precursors. These results are interpreted as indicating that (a) primary stimulation, like secondary stimulation, results from the selective stimulation by antigen of a population of cells differing from one another in their potential antibody product but each having only a single such product; (b) the antigen receptors of primary cells interact with antigen as if they are monovalent while receptors of secondary cells evidence multivalence; (c) antigenic stimulation appears to require both a relatively high affinity of receptors for bound antigen and an interlinking of receptors through such antigen; stimulation is thus seen as resulting from a stabilization of receptors within antigen-receptor aggregates to the cell surface; (d) T-cells appear to serve both in cross-linking antigens and in amplifying the size of stimulated clones.

scholarly journals Modification of the 85-kilodalton subunit of phosphatidylinositol-3 kinase in platelet-derived growth factor-stimulated cells.

1992 ◽  
Vol 12 (8) ◽  
pp. 3415-3424 ◽  
Author(s):  
W M Kavanaugh ◽  
A Klippel ◽  
J A Escobedo ◽  
L T Williams
Keyword(s):  
Tyrosine Phosphorylation ◽  
Platelet Derived Growth Factor ◽  
Affinity Binding ◽  
Pdgf Receptor ◽  
Phosphatidylinositol 3 Kinase ◽  
High Affinity Binding ◽  
High Affinity ◽  
Pdgf Stimulation ◽  
Stimulation Of

The activated platelet-derived growth factor (PDGF) receptor physically associates with p85, a subunit of phosphatidylinositol-3 kinase. Although this interaction may activate phosphatidylinositol-kinase and is crucial for PDGF-induced mitogenesis, it has not been shown whether p85 is modified in the process. p85 contains two SH2 (Src homology) domains, designated SH2-N and SH2-C. Recent experiments have shown that the SH2-C domain alone determines high-affinity binding of p85 to the PDGF receptor. The function of SH2-N, which binds receptors with lower affinity, is unknown. In this study, using a receptor-blotting technique, we find that p85 is modified by PDGF stimulation of intact cells. This modification involves inhibition of binding of the SH2-N region of p85 to the PDGF receptor. Studies with vanadate suggest that tyrosine phosphorylation of p85 is responsible for the modification of p85 detected by receptor blotting. Furthermore, recombinant p85 is modified in a similar manner when it is tyrosine phosphorylated in vitro by PDGF receptors. Tyrosine phosphorylation of p85 does not block binding of the SH2-C domain and therefore does not release p85 from high-affinity binding sites on the receptor in vitro. Instead, phosphorylation may regulate the ability of the SH2-N of p85 to bind to a different portion of the PDGF receptor or to another molecule in the signaling complex. This study provides the first evidence that p85 is tyrosine phosphorylated upon PDGF stimulation of cells and suggests that tyrosine phosphorylation of p85 regulates its activity or its interaction with other proteins.

scholarly journals Rapid Affinity Maturation of Novel Anti-PD-L1 Antibodies by a Fast Drop of the Antigen Concentration and FACS Selection of Yeast Libraries

2019 ◽  
Vol 2019 ◽  
pp. 1-22 ◽  
Author(s):  
Biancamaria Cembrola ◽  
Valentino Ruzza ◽  
Fulvia Troise ◽  
Maria Luisa Esposito ◽  
Emanuele Sasso ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Surface Display ◽  
Affinity Maturation ◽  
Yeast Surface Display ◽  
Antigen Concentration ◽  
Selection Scheme ◽  
High Affinity ◽  
Novel Approach ◽  
Yeast Surface

The affinity engineering is a key step to increase the efficacy of therapeutic monoclonal antibodies and yeast surface display is the most widely used and powerful affinity maturation approach, achieving picomolar binding affinities. In this study, we provide an optimization of the yeast surface display methodology, applied to the generation of potentially therapeutic high affinity antibodies targeting the immune checkpoint PD-L1. In this approach, we coupled a 10-cycle error-prone mutagenesis of heavy chain complementarity determining region 3 of an anti‐PD-L1 scFv, previously identified by phage display, with high-throughput sequencing, to generate scFv-yeast libraries with high mutant frequency and diversity. In addition, we set up a novel, faster and effective selection scheme by fluorescence-activated cell sorting, based on a fast drop of the antigen concentration between the first and the last selection cycles, unlike the gradual decrease typical of current selection protocols. In this way we isolated 6 enriched mutated scFv-yeast clones overall, showing an affinity improvement for soluble PD-L1 protein compared to the parental scFv. As a proof of the potency of the novel approach, we confirmed that the antibodies converted from all the mutated scFvs retained the affinity improvement. Remarkably, the best PD-L1 binder among them also bound with a higher affinity to PD-L1 expressed in its native conformation on human-activated lymphocytes, and it was able to stimulate lymphocyte proliferation in vitro more efficiently than its parental antibody. This optimized technology, besides the identification of a new potential checkpoint inhibitor, provides a tool for the quick isolation of high affinity binders.

An innovative flow cytometric approach for small-size platelet microparticles: Influence of calcium

2012 ◽  
Vol 108 (08) ◽  
pp. 373-383 ◽  
Author(s):  
Silvia Montoro-García ◽  
Eduard Shantsila ◽  
Esteban Orenes-Piñero ◽  
María Lozano ◽  
Gregory Y. H. Lip
Keyword(s):  
Annexin V ◽  
Prolonged Storage ◽  
Moderate Increase ◽  
High Affinity ◽  
Flow Cytometric ◽  
Initial Plasma ◽  
In Vitro Assessment ◽  
Free Plasma ◽  
Stimulation Of

SummaryMicroparticles (MPs) are small submicron membrane-derived vesicles shed from a variety of cells and they have been implicated in different disorders. Accordingly, understanding of physiological characteristics of MPs and improvement of methods of their quantification are important for further advance in the field. Although flow cytometry is the most widely applied technique for MP analysis, it is limited by lack of adequate standardisation. Annexin V (AnV), which binds surface phos-phatidylserine (PS) with high affinity, has been long regarded as a marker of MPs, but AnV binding is Ca2+-dependent and it is unclear how [Ca2+] concentrations could affect AnV binding to MPs and its enumeration. MPs from citrated and heparinised plasma were labelled with AnV, anti-CD42b and quantified using an Apogee A50 flow cytometer. The small-size MP gate was defined with the use of size beads (from 0.1 to 0.5 μm) and confirmed with an in vitro assessment of platelet stimulation. Biotinylated anti-CD42b antibodies were then bound to streptavidin conjugated with different fluorochromes, leading to an amplified signal of platelet MPs (PMPs). Moderate increase of [Ca2+] concentrations in the annexin V staining buffer allows initial plasma recalcification and more accurate MP quantification in citrated plasma. Thrombin stimulation of platelet-free plasma containing only MPs did not produce any changes in the concentration of AnV+ MPs, but decreased the anti-CD42b binding. The results also indicate that prolonged storage and thrombin induce the release of AnV+ MPs whereas PS exposure in pre-existent MPs is not affected by thrombin. In conclusion, we present a sensitive protocol for the analysis of circulating and in vitro induced small-size PMPs that might contribute to future cardiovascular and clinical research.Note: The editorial process for this article was fully handled by Prof. Christian Weber, Editor-in-Chief.

scholarly journals Epigenetic conversion of conventional T cells into regulatory T cells by CD28 signal deprivation

2020 ◽  
Vol 117 (22) ◽  
pp. 12258-12268 ◽  
Author(s):  
Norihisa Mikami ◽  
Ryoji Kawakami ◽  
Kelvin Y. Chen ◽  
Atsushi Sugimoto ◽  
Naganari Ohkura ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
De Novo ◽  
Early Stage ◽  
Antigenic Stimulation ◽  
Binding Motif ◽  
Dna Hypomethylation ◽  
Signature Genes ◽  
Stimulation Of

Foxp3-expressing regulatory T cells (Tregs) can be generated in vitro by antigenic stimulation of conventional T cells (Tconvs) in the presence of TGF-β and IL-2. However, unlike Foxp3+naturally occurring Tregs, such in vitro induced Tregs (iTregs) are functionally unstable mainly because of incomplete Treg-type epigenetic changes at Treg signature genes such asFoxp3. Here we show that deprivation of CD28 costimulatory signal at an early stage of iTreg generation is able to establish Treg-specific DNA hypomethylation at Treg signature genes. It was achieved, for example, by TCR/TGF-β/IL-2 stimulation of CD28-deficient Tconvs or CD28-intact Tconvs without anti-CD28 agonistic mAb or with CD80/CD86-blocked or -deficient antigen-presenting cells. The signal abrogation could induce Treg-type hypomethylation in memory/effector as well as naive Tconvs, while hindering Tconv differentiation into effector T cells. Among various cytokines and signal activators/inhibitors, TNF-α and PKC agonists inhibited the hypomethylation. Furthermore, CD28 signal deprivation significantly reduced c-Rel expression in iTregs; and the specific genomic perturbation of a NF-κB binding motif at the Foxp3 CNS2 locus enhanced the locus-specific DNA hypomethylation even in CD28 signaling-intact iTregs. In addition, in vitro maintenance of such epigenome-installed iTregs with IL-2 alone, without additional TGF-β or antigenic stimulation, enabled their expansion and stabilization of Treg-specific DNA hypomethylation. These iTregs indeed stably expressed Foxp3 after in vivo transfer and effectively suppressed antigen-specific immune responses. Taken together, inhibition of the CD28-PKC-NF-κB signaling pathway in iTreg generation enables de novo acquisition of Treg-specific DNA hypomethylation at Treg signature genes and abundant production of functionally stable antigen-specific iTregs for therapeutic purposes.

scholarly journals HAPTEN-SPECIFIC STIMULATION OF SECONDARY B CELLS INDEPENDENT OF T CELLS

1973 ◽  
Vol 138 (2) ◽  
pp. 473-478 ◽  
Author(s):  
Norman R. Klinman ◽  
Robert A. Doughty
Keyword(s):  
T Cells ◽  
B Cells ◽  
Spleen Cell ◽  
Precursor Cells ◽  
Cell Suspensions ◽  
Low Concentrations ◽  
Specific Stimulation ◽  
Stimulation Of

Treatment of spleen cell suspensions from immunized mice with anti-theta serum and complement before transfer to nonimmune irradiated recipients reduced the degree of in vitro stimulation by hapten-homologous carrier complexes by 90%, but did not decrease at all the number of isolated precursor cells stimulated by hapten on heterologous carriers. Thus, secondary B cells can be stimulated by low concentrations of multiply substituted hapten-carrier complexes in the apparent complete absence of specific T cells.

scholarly journals Antibodies to Factor VIII. I. Variations in Stability of Antigen-Antibody Complexes in Hemophilia A

Blood ◽  
1973 ◽  
Vol 42 (3) ◽  
pp. 437-444 ◽  
Author(s):  
J.-P. Allain ◽  
D. Frommel
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Hemophilia A ◽  
Direct Consequence ◽  
Antigenic Stimulation ◽  
Factor Viii Activity ◽  
Human Factor Viii ◽  
Antigen Antibody ◽  
Stimulation Of

Abstract Human factor VIII-anti-factor VIII complexes were formed in vitro in slight antigen excess, using plasma of hemophiliacs who were found to have antibodies neutralizing AHF activity. These complexes, stable at +37°C and pH 7.4, were submitted to classical procedures known to favor dissociation of antibody from antigen. The methods used to obtain dissociation, incubations at +56°C and at pH 4.2, inactivated both unbound factor VIII and that released as a consequence of dissociation. The extent of dissociation was measured by the recovery of anti-factor VIII activity. Increasing resistance of complexes towards dissociation was observed in the plasma of the patients whose titer of inhibitor was increasing after recent transfusions. These observations suggested the emergence, as a direct consequence of renewed antigenic stimulation, of a population of different antibodies characterized by higher combining strength.

Effects of vanadate on MgATP stimulation of Na-Ca exchange support kinase-phosphatase modulation in squid axons

1994 ◽  
Vol 266 (5) ◽  
pp. C1382-C1391 ◽  
Author(s):  
R. DiPolo ◽  
L. Beauge
Keyword(s):  
Phosphatase Activity ◽  
Response Curve ◽  
Maximum Velocity ◽  
Apparent Affinity ◽  
High Affinity ◽  
Intracellular Ca ◽  
Phosphatase System ◽  
Stimulation Of ◽  
Do So

We have proposed that in squid axons MgATP stimulation of Na-Ca exchange involves a phosphorylation-dephosphorylation process catalyzed by a kinase-phosphatase system. In the present work, we used vanadate as a tool to gather further evidence about the mechanism of metabolic control of the Na-Ca exchanger in internally dialyzed and voltage-clamped squid axons. Vanadate, at concentrations up to 100 microM, stimulated extracellular Na (Nao)-dependent Ca efflux only in the presence of MgATP but failed to do so when the axons were dialyzed with the nonhydrolyzable ATP analogue beta, gamma-methyleneadenosine 5'-triphosphate or with CrATP, a MgATP analogue that completely abolishes MgATP stimulation of the Na-Ca exchange. In axons fully activated by Mg-adenosine 5'-O-(3-thiotriphosphate), vanadate had no effect on Na-Ca exchange. The dose-response curve for vanadate stimulation followed Michaelian kinetics with a Km of 5.6 +/- 0.4 microM and a maximum velocity of 216 +/- 10 fmol.cm-2.s-1 (intracellular Ca concentration = 0.8 microM). This coincides with the high affinity of vanadate in inhibiting the in vitro phosphatase activity of an alkaline phosphatase extracted from rat liver. In addition, vanadate increased fivefold the apparent affinity for MgATP (Km from 220 +/- 14 to 40 +/- 4 microM). Concentrations of vanadate in the millimolar range inhibited the MgATP-stimulated Na-Ca exchange (apparent Ki of 5.7 +/- 0.3 mM) and the in vitro phosphorylation by the catalytic subunit of a adenosine 3',5'-cyclic monophosphate protein kinase (apparent Ki 2.64 +/- 0.04 mM). We conclude that MgATP stimulation of Na-Ca exchange is proportional to the levels of phosphorylation that result from the balance of the activity of a kinase and a phosphatase activity.

Stimulation and binding of myocardial phospholipase C by phosphatidic acid

1995 ◽  
Vol 269 (2) ◽  
pp. C349-C358 ◽  
Author(s):  
R. A. Henry ◽  
S. Y. Boyce ◽  
T. Kurz ◽  
R. A. Wolf
Keyword(s):  
Phosphatidic Acid ◽  
Phospholipase C ◽  
Ventricular Myocytes ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Adult Rabbit ◽  
High Affinity ◽  
Rabbit Ventricular Myocytes ◽  
Stimulation Of

Exposure of adult ventricular myocytes to exogenous natural phosphatidic acid results in the production of inositol phosphates by unknown mechanism(s). We characterized stimulation of myocytic phosphoinositide-specific phospholipase C (PLC) by synthetic dioleoyl phosphatidic acid (PA) as a potential mechanism for modulation of inositol phosphate production. Our data demonstrate that exogenous PA, at 10(-8)-10(-5) M, caused a concentration-dependent increase in inositol 1,4,5-trisphosphate in adult rabbit ventricular myocytes. PA also caused a concentration-dependent increase in in vitro activity of myocytic PLC in the presence or absence of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). PLC-delta 1, the predominant isozyme of PLC expressed in adult rabbit ventricular myocytes, bound to liposomes of PA with high affinity in the presence of EGTA. The phosphomonoester group of PA was critical to in vitro stimulation of myocytic PLC activity and high-affinity binding of PLC-delta 1. We propose that binding of PLC-delta 1 to phosphatidic acid may be a novel mechanism for dynamic membrane association and modulation of PLC in adult ventricular myocytes.

Modification of the 85-kilodalton subunit of phosphatidylinositol-3 kinase in platelet-derived growth factor-stimulated cells

1992 ◽  
Vol 12 (8) ◽  
pp. 3415-3424
Author(s):  
W M Kavanaugh ◽  
A Klippel ◽  
J A Escobedo ◽  
L T Williams
Keyword(s):  
Tyrosine Phosphorylation ◽  
Platelet Derived Growth Factor ◽  
Affinity Binding ◽  
Pdgf Receptor ◽  
Phosphatidylinositol 3 Kinase ◽  
High Affinity Binding ◽  
High Affinity ◽  
Pdgf Stimulation ◽  
Stimulation Of

The activated platelet-derived growth factor (PDGF) receptor physically associates with p85, a subunit of phosphatidylinositol-3 kinase. Although this interaction may activate phosphatidylinositol-kinase and is crucial for PDGF-induced mitogenesis, it has not been shown whether p85 is modified in the process. p85 contains two SH2 (Src homology) domains, designated SH2-N and SH2-C. Recent experiments have shown that the SH2-C domain alone determines high-affinity binding of p85 to the PDGF receptor. The function of SH2-N, which binds receptors with lower affinity, is unknown. In this study, using a receptor-blotting technique, we find that p85 is modified by PDGF stimulation of intact cells. This modification involves inhibition of binding of the SH2-N region of p85 to the PDGF receptor. Studies with vanadate suggest that tyrosine phosphorylation of p85 is responsible for the modification of p85 detected by receptor blotting. Furthermore, recombinant p85 is modified in a similar manner when it is tyrosine phosphorylated in vitro by PDGF receptors. Tyrosine phosphorylation of p85 does not block binding of the SH2-C domain and therefore does not release p85 from high-affinity binding sites on the receptor in vitro. Instead, phosphorylation may regulate the ability of the SH2-N of p85 to bind to a different portion of the PDGF receptor or to another molecule in the signaling complex. This study provides the first evidence that p85 is tyrosine phosphorylated upon PDGF stimulation of cells and suggests that tyrosine phosphorylation of p85 regulates its activity or its interaction with other proteins.

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