Biomedical subjects

D J Cassell

Publications and source records attributed to D J Cassell.

10 recordsLinked to original sources

PubMed2001

Validity of the two-signal model for activation of CD28-deficient T lymphocytes: quantitative characterization of an alternative costimulatory function of dendritic cells.

The observation that primary T-dependent immune responses are generated in mice lacking CD28, the only receptor definitively shown to costimulate naive T cells, has led to ambiguity as to whether costimulation is absolutely required for initiation of T-cell responses. In this report, in vitro analysis of the relationship between cell density and proliferation demonstrates that activation of CD28-/- T cells to immobilized T-cell receptor (TCR)-alpha monoclonal antibodies (MoAb) depends on costimulatory signals provided by other cells in culture and occurs only at cell densities sufficient to permit these intercellular interactions. These signals are necessary even under TCR triggering conditions that obviate the CD28 requirement. Dendritic cells (DCs) provide the necessary costimulation in vitro and prime T cells in vivo in CD28-/- mice. Single-cell and limiting dilution analyses indicate that individual T cells from normal and CD28-/- mice produce equivalent interleukin (IL)-2 in response to DCs. However, half as many T cells produce IL-2 when only the CD28-independent pathway is used. Nonetheless, CD28-/- T cells produce sufficient IL-2 to support clonal expansion comparable to that of CD28+/+ T cells, which may account for the equally robust in vivo responses initiated by DCs in normal and CD28-deficient animals.

Animals↗

PubMed1994

A quantitative analysis of antigen-presenting cell function: activated B cells stimulate naive CD4 T cells but are inferior to dendritic cells in providing costimulation.

Ligation of CD28 on CD4 Th1 clones and freshly isolated mixtures of naive and memory CD4 T cells triggered their T cell receptors (TCR) is sufficient to induce the costimulatory signals necessary for interleukin 2 (IL-2) production by these cells. CTLA-4-reactive ligands expressed on antigen-presenting cells (APC) are critical in providing costimulatory signals to these T cell populations. We demonstrate that these activation characteristics apply equally to purified naive CD4 T cells. Because B cell blasts express CTLA-4-reactive ligands and high levels of adhesion and major histocompatibility complex class II molecules, they would be expected to engage both the TCR and CD28 and consequently stimulate IL-2 production by naive CD4 T cells. Using purified populations of cells in limiting dilution cultures, we have carried out a quantitative analysis of the interaction between naive CD4 T cells and either activated B or dendritic cells. We demonstrate that B cell blasts stimulate a high frequency of naive CD4 T cells. Slight differences in TCR signaling efficiency between the two APC types were observed. Even at optimal peptide concentrations, however, the amount of IL-2 made by individual T cells was fourfold lower in response to B cell blasts than to dendritic cells. This relative deficiency of activated B cells was due to their inability to optimally costimulate naive CD4 T cells.

Abatacept↗

PubMed1990

Regulation of the cytotoxic T lymphocyte response against Qa-1 alloantigens.

Spleen cells from B6.Tlaa (Qa-1a) mice primed against C57BL/6 (Qa-1b) splenocytes in vivo generate Qa-1-specific CTL when rechallenged with Qa-1b Ag in vitro. The addition of unirradiated Qa-1b splenocytes to these cultures inhibits the generation of Qa-1-specific CTL. By using highly purified cell populations, we demonstrate that the only cell population in resting spleen capable of causing this inhibition is NK1.1+. Although resting CD8 cells lack inhibitory activity, purified CD8 cells precultured with Con A and IL-2 inhibit anti-Qa-1 CTL. This inhibition is specific for the Qa-1b Ag expressed on the inhibitor cells, is not due to cold target competition, and is thus similar to that ascribed to veto cells. Although NK cells from resting spleen inhibit the generation of Qa-1-specific CTL, NK cells precultured in the presence of Con A and IL-2 show an approximate 30-fold increase in veto activity. Thus, NK cells represent the most likely cell population for down-regulating anti-self class I-reactive CTL.

Animals↗

PubMed1985

Characterization of an insulin degrading enzyme from cultured human lymphocytes.

An insulin degrading enzyme from cultured human lymphocytes, IM-9 cells, has been purified and characterized. The biochemical, enzymatic and immunological characteristics of this enzyme were all found to be similar to the characteristics of insulin degrading enzymes previously isolated from rat and pig skeletal muscle. Furthermore, this insulin degrading enzyme was found to have no effect on the structure of the insulin receptor nor to be linked to the insulin receptor either on the plasma membrane of cells or when they are shed into the media. The present studies suggest that the IM-9 lymphocytes, which have been extensively used to study the human insulin receptor, may also be a good system for studying human insulin degrading enzymes.

Animals↗

PubMed1984

Effects of covalently linked insulin dimers on receptor kinase activity and receptor down regulation.

Certain covalently linked insulin dimers have previously been found to have a greater ability to bind to the insulin receptor than to stimulate lipogenesis in adipocytes. The present report presents data indicating that the same insulin dimers also have a greater ability to bind to the receptor than to stimulate the kinase activity of the insulin receptor. In particular, one such covalently linked insulin dimer had less than 1% the potency of native insulin in stimulating the receptor kinase although it could bind to the solubilized receptor with 30% the potency of native insulin. In contrast, this dimer could down regulate the insulin receptor with approximately 30% the potency of native insulin. These results suggest that stimulation of the receptor kinase may require more than simple occupancy of the receptor binding site whereas down regulation of the receptor may require only the binding of ligand to the receptor.

Dose-Response Relationship, Drug↗

PubMed1983

Preferential degradation of the beta subunit of purified insulin receptor. Effect on insulin binding and protein kinase activities of the receptor.

Collagenase preparations (a mixture of enzymes including collagenase, clostripain, and a casein-degrading protease) degraded the beta subunit (Mr = 95,000) of the purified insulin receptor into fragments of Mr less than 15,000, without degrading the alpha subunit. The resulting beta-digested insulin receptor preparations were found to bind insulin as well as control insulin receptor, as assessed by either cross-linking of 125I-insulin to the digested receptor or by separating insulin bound to receptor from free insulin by high performance liquid chromatography. Moreover, the beta-digested insulin receptor preparations were still precipitated by a monoclonal antibody directed against the insulin-binding site. In contrast, the beta-digested insulin receptor lacked protein kinase activity since it no longer phosphorylated either itself, or an exogenous substrate, calf thymus histone. These results support the identification of the beta subunit of the insulin receptor as a protein kinase.

Cell Line↗

PubMed1983

Regulation of the insulin receptor by a monoclonal anti-receptor antibody. Evidence that receptor down regulation can be independent of insulin action.

In the present study, we investigated the ability of a monoclonal antibody to the insulin receptor to regulate the expression of the insulin receptor of IM-9 lymphocytes. Previously, this antibody was shown to be a competitive antagonist of insulin action on severe metabolic functions. In the present study, we report that preincubation of IM-9 cells with the monoclonal antibody caused a dose- and time-dependent decrease in the subsequent ability of these cells to bind 125I-insulin, a phenomenon termed down regulation. The antibody was approximately 100 times more potent than insulin at down regulating the receptor. In contrast, the antibody was 5 times less potent than insulin in competing for binding to insulin receptors and dissociated 4 times more rapidly than insulin from IM-9 cells. Three lines of evidence suggested that the mechanism of down regulation by the antibody was the same as the one used by insulin. First, both agents caused a rapid initial decrease in insulin binding to cells followed by a slower, gradual decrease in binding. Second, the down regulation caused by both was reversible, and this reversibility required new protein synthesis. Third, the antibody, like insulin, accelerated receptor degradation. Since the antibody does not mimic the other effects of insulin on metabolic processes, these results suggest that the mechanism of insulin receptor down regulation is different from the mechanism of insulin action on other cellular functions.

Antibodies, Monoclonal↗

PubMed1983

Regulation of insulin receptor kinase activity by insulin mimickers and an insulin antagonist.

Three agents which mimic insulin action in intact cells (concanavalin A, wheat germ agglutinin, and polyclonal insulin receptor antibody), mimicked insulin's ability to stimulate the kinase activity of purified insulin receptors. In contrast, monoclonal insulin receptor antibody, an antagonist of insulin action, did not stimulate the phosphorylation of the insulin receptor either in intact IM-9 cells or in purified receptor preparations. This antibody, however, antagonized the ability of insulin to stimulate the phosphorylation of the receptor both in intact cells and in the purified receptor. These studies with insulin mimickers and an insulin antagonist are consistent with a role for the kinase activity of the receptor mediating the actions of insulin.

Antibodies↗

PubMed1983

Insulin receptor: evidence that it is a protein kinase.

Highly purified preparations of insulin receptor catalyzed the phosphorylation of the 95,000-dalton subunit of the insulin receptor. This subunit of the insulin receptor was also labeled with [alpha-32P]8-azidoadenosine 5'-triphosphate, a photoaffinity label for adenosine triphosphate binding sites. The identity of the 95,000-dalton band was confirmed in both cases by precipitation with a monoclonal antibody to the insulin receptor. These results suggest that the insulin receptor is itself a protein kinase.

Adenosine Triphosphate↗

PubMed1982

Monoclonal antibodies to the human insulin receptor block insulin binding and inhibit insulin action.

Antibodies to the insulin receptor were prepared in BALB/c mice by immunization with IM-9 human lymphocytes, a cell type that has a large number of plasma membrane insulin receptors. The spleens of these mice were then removed, and their lymphocytes were fused to a mouse myeloma cell line, FO cells. After screening over 1,200 resulting hybrids, one stable hybrid was obtained that produced IgG1 antibodies directed towards the insulin receptor. This antibody blocked 125I-labeled insulin binding to its receptor by more than 90% in three human tissues: IM-9 cultured lymphocytes, freshly isolated adipocytes, and placenta membranes. In contrast, the antibody did not inhibit insulin binding to rat adipocytes and rat liver plasma membranes, suggesting that the antibody was species specific. In IM-9 cells, which had their proteins prelabeled with [35S]methionine, the antibody precipitated two polypeptides with molecular weights of 135,000 and 95,000; these molecular weights are identical to those previously identified as the alpha and beta subunits of the insulin receptor. The monoclonal antibody inhibited the actions of insulin on both human adipocytes and fibroblasts, suggesting that the antibody was an antagonist of insulin action. The present studies suggest, therefore, that monoclonal antibodies to the insulin receptor may provide new insights into the structure of the insulin receptor and its interaction with insulin.

Adipose Tissue↗