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.

Assessment of myocardial viability by dynamic tomographic iodine 123 iodophenylpentadecanoic acid imaging: comparison with rest-redistribution thallium 201 imaging.

BACKGROUND: This study examined the ability of dynamic 123I-labeled iodophenylpentadecanoic acid (IPPA) imaging to detect myocardial viability in patients with left ventricular (LV) dysfunction caused by coronary artery disease. METHODS AND RESULTS: Serial 180-degree single-photon emission computed tomographic (SPECT) images (five sets, 8 minutes each) were obtained starting 4 minutes after injection of 2 to 6 mCi 123I at rest in 21 patients with LV dysfunction (ejection fraction [EF] 34% +/- 11%). The segmental uptake was compared with that of rest-redistribution 201Tl images (20 segments/study). The number of perfusion defects (reversible and fixed) was similar by IPPA and thallium (11 +/- 5 vs 10 +/- 5 segments/patient; difference not significant). There was agreement between IPPA and thallium for presence or absence (kappa = 0.78 +/- 0.03) and nature (reversible, mild fixed, or severe fixed) of perfusion defects (kappa = 0.54 +/- 0.04). However, there were more reversible IPPA defects than reversible thallium defects (7 +/- 4 vs 3 +/- 4 segments/patient; p = 0.001). In 14 patients the EF (by gated pool imaging) improved after coronary revascularization from 33% +/- 11% to 39% +/- 12% (p = 0.002). The number of reversible IPPA defects was greater in the seven patients who had improvement in EF than in the patients without such improvement (10 +/- 4 vs 5 +/- 4 segments/patient; p = 0.075). CONCLUSIONS: 123I-labeled IPPA SPECT imaging is a promising new technique for assessment of viability. Reversible defects predict recovery of LV dysfunction after coronary revascularization.

Serial changes in left ventricular function after coronary artery bypass: implications in viability assessment.

Improvement in left ventricular (LV) performance after coronary artery bypass surgery remains the gold standard in myocardial viability assessment. The time-related changes, however, are not well known. This study examined the LV ejection fraction (EF) by gated blood pool imaging early (6 +/- 4 days) and late (62 +/- 24 days) after surgery in patients with normal preoperative EF (group 1, n = 12) and those with LV dysfunction (group 2, n = 15). There were no changes in the clinical status between the early and late studies, and all patients had normal sinus rhythm. Group 1 had no significant change in EF (preoperatively 62%, early postoperatively 64%, late postoperatively 63%; p = NS). In group 2, EF was 26% +/- 8% preoperatively; 30% +/- 10% early postoperatively; and 34% +/- 8% late postoperatively (p < 0.05). Postoperatively there was > or = 5% improvement in EF in 4 patients early and 11 patients late (p < 0.05). Patients who showed early improvement continued to do so in the late study but, additionally, 7 patients showed improvement only in the late study. Thus the timing of EF measurement after surgery is important in patients with LV dysfunction but not in patients with normal LV function. Early assessment may underestimate the prevalence and degree of recovery.

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.

Effects of three colors in an office interior on mood and performance.

This study examined the effects of three hues on subjects' performance and mood while in an office work environment for 1 hour. Pretest/posttest measurements were completed. Work performance was measured using words typed, typing errors, and a ratio of errors to words typed. Anxiety, depression, and arousal were measured by the Eight State Questionnaire of Curran and Cattell. A total of 45 women, ages 18 to 24 years, were tested individually in a single office space: 15 when the office walls were painted red/warm, 15 when walls were blue-green/cool, and 15 when walls were white/neutral. Analysis of covariance of posttest measurements with the pretest as a covariate showed no significant differences among the three groups on performance or scores on anxiety, depression, and arousal. If color of the environment has an effect on work performance or mood, either the effect was too small to be detected with samples of 15 subjects or longer participation than one hour was required.

Two roles for CD4 cells in the control of the generation of cytotoxic T lymphocytes.

The generation of CTL against Qa-1 Ag in C57BL/6 (B6) (Qa-1b) and B6.Tlaa (Qa-1a) congenic strains requires in vivo priming with the Qa-1 alloantigen together with a helper Ag, such as H-Y. The primed precursors obtained from these female mice generate Qa-1-specific CTL activity upon culture in vitro. Although the presence of the H-Y helper Ag is not required for the in vitro sensitization, no response occurs in the absence of CD4 cells. Addition of unprimed B6.Tlaa CD4 cells from Qa-1 incompatible radiation bone marrow chimeras (B6.Tlaa----B6), that are presumably tolerant to Qa-1b, provide helper activity for Qa-1b-specific CTL. This indicates that although CD4 cells are obligatory for the Qa-1 response, they need not be specific for alloantigens on the APC to generate helper activity in in vitro cultures. Addition of unirradiated B6 CD8-depleted spleen cells to CD4-depleted B6.Tlaa anti-B6 cultures in the presence of either B6.Tlaa CD4 cells or rIL-2 prevents the generation of Qa-1 specific CTL. This inhibition is not due to an anti-idiotypic Ts cell since B6.Tlaa----B6 chimeric cells do not suppress an anti-Qa-1b response. Rather, this finding is consistent with that of a veto cell mechanism. To determine whether CD4 cells themselves exhibit veto activity, highly purified CD4 populations were tested for their ability to inhibit the generation of Qa-1-specific CTL. CD4 cells precultured for 2 to 3 days with Con A and rIL-2 specifically inhibit CTL activity whereas resting cells do not, similar to that noted for CD8 veto cells. The relative efficiency of activated CD4 cells is greater than that of resting NK cells but is less than that of activated CD8 or NK cells. Thus, CD4 cells not only provide helper activity for CTL precursors, but also act as veto cells to prevent the generation of CTL activity.

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.

Linked recognition of helper and cytotoxic antigenic determinants for the generation of cytotoxic T lymphocytes.

These studies present a model for T-T collaboration for the generation of cytotoxic T lymphocytes. The in vitro CTL response against Qa-1 alloantigens in B6 Qa-1 congenic mice requires that animals are first primed in vivo with Qa-1 together with a second (helper) antigen. Both the antigen recognized by CTL (Qa-1) and Th (H-Y) must be presented on the same APC for successful priming. This findings is consistent with a linked recognition model whereby both molecules are presented on the same APC in order to accomplish close proximity between the two T cells. To further test this model, we demonstrated that in the absence of the helper antigen, H-Y, mice could be successfully primed against Qa-1 if coinoculated with a product of a Th, IL-2. We further showed that mice treated with anti-L3T4 antibodies could not be primed to Qa-1 even though the cells used for immunization expressed the H-Y helper antigen. Taken together, these results lend further support to a model of linked recognition between Th and CTL.P where close proximity allows for the lymphokine IL-2 to bind to its receptor on the CTL allowing for the successful induction of CTL.P.

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.

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.

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.

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.

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.

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.

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.