Lymphokine regulation of activated (G1) lymphocytes. II. Glucocorticoid and anti-Tac-induced inhibition of human T lymphocyte proliferation

The regulation of the first cell cycle of human, activated (G1) PBL was analyzed by flow cytometry and [3H]thymidine incorporation. Endogenous IL 2 production was blocked in situ by pharmacologic concentration of DEX (100 to 1000 nM), resulting in an 80 to 90% reduction of thymidine uptake. Although T lymphocyte activation (G0-G1a transition) by PHA was unaltered, cells remained in the G1a phase of the cell cycle due to insufficient RNA synthesis for proliferation. The addition of IL 2-containing supernatants reversed this inhibitory effect of DEX by allowing the cells to synthesize more RNA (G1a-G1b transition). Such cells could enter the S phase and proliferate. Similar studies were performed on cells treated with a monoclonal antibody (anti-Tac) against the IL 2 receptor. In these studies, IL 2-induced RNA synthesis, and subsequent proliferation of DEX-treated and PHA-stimulated cells was inhibited by anti-Tac. Anti-Tac did not, however, inhibit the effect of endogenous IL 2 (PHA-stimulated PBL without DEX treatment), although it did bind equally well to such cells. Thus, IL 2 directly or indirectly regulates human T cell proliferation at the level of RNA synthesis. Furthermore, anti-Tac can inhibit the mitogenic signal given by endogenous IL 2, but not by in situ produced IL 2, an observation of importance to further investigations of the mechanisms by which IL 2 interacts with specific receptors to elicit proliferation.     

Differential effect of cyclosporin A on the expression of T and B lymphocyte activation antigens

We have used a monoclonal antibody (Mab) to the interleukin 2 (IL 2) receptor as well as a Mab to the transferrin receptor to analyze the effects of cyclosporin A (CsA) on the induction and expression of these activation antigens on mitogen-stimulated murine T and B lymphocytes. The same concentration (0.25 microgram/ml) of CsA that produced optimal inhibition of the T cell proliferative response to concanavalin A (Con A) was also very effective at inhibiting IL 2 production and the induction of IL 2 responsiveness, as well as the expression of the IL 2 and transferrin receptors when measured 72 hr after mitogen activation. Surprisingly, CsA only minimally inhibited expression of these receptors 24 hr after the addition of mitogen; however, T cell blasts recovered from these cultures failed to respond to IL 2 even though IL 2 receptor expression was only modestly decreased. These results suggest that inhibition of the maturation of receptor expression is secondary to an early effect of CsA in blocking the induction of IL 2 responsiveness or to an arrest in the sequence of events required for maturation of T cells that bear high densities of these receptors. In contrast to the results observed with T lymphocytes, CsA had no effect on the B cell proliferative response to lipopolysaccharide (LPS) or on the induction of the IL 2 and transferrin receptors on activated B lymphocytes.     

Comparison of the expression of IL 2 receptors by human T and B cells: induction by the polyclonal mitogens, phorbol myristate acetate, and anti-mu antibody

It is well established that IL 2 plays an important role in the proliferative response of T cells. Activated B cells were also recently found to express IL 2 receptors. The present studies were designed to compare qualitative, quantitative, and functional aspects of IL 2 receptor expression by activated T and B cells. Phorbol myristate acetate (PMA)-activated human T and small resting B cells and enhanced the expression of HLA-DR, HLA-DC/DS, and transferrin receptors while reducing Leu-4 antigen expression by T cells and IgM and IgD expression on B cells. PMA induced both T and B cells to express functional IL 2 receptors before cellular proliferation. Immune interferon did not participate in this induction. The m.w. of the IL 2 receptors expressed by activated T and B cells was identical: 54,000 to 59,000. Several differences were noted in the expression of IL 2 receptors by activated T and B cells on stimulation with PMA; T cells expressed IL 2 receptors sooner than B cells and in higher density, and the enhanced proliferative response of T cells to IL 2 was more difficult to inhibit with antibody to IL 2 receptors. In addition, IL 2 enhanced the expression of transferrin receptors by activated T cells but did not have a similar effect on activated B cells. Small B cells from the blood could also be induced by a mitogenic monoclonal anti-IgM antibody to express functional IL 2 receptors. Relatively large B cells in fresh blood samples were found to express functional IL 2 receptors and were capable of a modest proliferative response to IL 2. The intensity of the IL 2 receptor expression and the proliferative response by large B cells were enhanced by PMA stimulation. The data suggest that IL 2 receptors may play an auxiliary role in the B cell proliferative response and that IL 2 may exert its effect at a late phase in the B cell activation process.     

Activation of resting T lymphocytes by anti-CD3 (T3) antibodies in the absence of monocytes

The antigen receptor molecules on human T lymphocytes are noncovalently associated on the cell surface with the CD3 (T3) molecular complex. Perturbation of this complex with anti-CD3 monoclonal antibodies induces T cell activation. Previous studies have demonstrated that this process requires the participation of monocytes. In the present report, we demonstrate that purified, resting (G0 phase) T cells incubated with monoclonal anti-CD3 antibodies proliferate in response to purified interleukin 2 (IL 2), in a lymphokine dose-dependent fashion. Anti-CD3 antibody or IL 2 alone did not trigger cell division. The effect was specific for anti-CD3 antibodies because monoclonal antibodies reactive with other surface molecules (OKT4, OKT8, L368) were inactive. Furthermore, the same phenomenon was observed when anti-CD3 antibody Leu-4 (IgG1) was incubated with cells of individuals whose monocytes cannot process antibodies of the IgG1 subclass (Leu-4 nonresponders). In addition, both F(ab')2 and Fab fragments of anti-CD3 antibody OKT3 were also capable of rendering T cells receptive to the IL 2 growth signal. These data indicate that neither monocytes nor CD3 receptor cross-linking are required absolutely for resting T cell activation, provided that IL 2 is supplied exogenously. T lymphocytes treated with anti-CD3 antibodies proliferated in response to both purified mitogen-induced and recombinant IL 2. Antibodies to the IL 2 receptor (anti-Tac) inhibited the proliferation. Thus, the most likely mechanism for anti-CD3 antibody-mediated triggering is induction of IL 2 receptors.     

The role of transferrin receptors and iron delivery in mouse embryonic morphogenesis

The iron-carrying serum protein transferrin is required for the proliferation and differentiation of embryonic tissues in culture. We studied the expression and role of transferrin receptors in two model systems using a monoclonal antibody against the transferrin receptor of mice. The addition of 20-100 micrograms/ml antibody to a chemically defined culture medium containing transferrin (10 micrograms/ml) inhibited morphogenesis and cell proliferation in kidneys and teeth. However, the antibody did not inhibit development when iron was delivered to the cells by a lipophilic iron chelator i.e., by-passing the receptor-mediated pathway. Hence, the binding of the receptor antibody to the receptor apparently did not affect cell proliferation, and the antibody was not toxic to the tissues. Our results suggest that the antibody to the transferrin receptor inhibits development by blocking the normal endocytotic route of iron delivery. Cells derived from embryonic kidneys and teeth expressed the transferrin receptor when cultured as monolayers. However, using immunofluorescent techniques, we were unable to detect the receptor in frozen tissue sections. It is possible that the seeding of cells in monolayer cultures affects the expression of the transferrin receptor, since it is known that all types of cells require transferrin for continued proliferation in culture. Organ-cultured kidney mesenchymal cells are not initially responsive to transferrin, but they acquire responsiveness as a consequence of an inductive tissue interaction. Although it remains unknown as to whether the acquisition of transferrin responsiveness is directly related to the expression of transferrin receptors, our results suggest that transferrin and its receptors play a role in embryonic morphogenesis.     

Inhibition of IL 2-driven proliferation of murine T lymphocyte clones by supraoptimal levels of immobilized anti-T cell receptor monoclonal antibody

Both cloned murine helper T lymphocytes (HTL) and cytolytic T lymphocytes (CTL) proliferated and secreted lymphokines when stimulated with immobilized anti-T cell receptor monoclonal antibody (anti-TCR mAb). However, although proliferation of CTL increased and reached plateau levels as concentrations of anti-TCR mAb were increased, the proliferation of HTL decreased with high concentrations of anti-TCR mAb. A reduction of IL 2-dependent proliferation by CTL was observed when IL 2 was added to cultures of CTL in the presence of high concentrations of anti-TCR mAb, whereas IL 2-independent proliferation appeared to be unaffected by these concentrations of anti-TCR mAb. Inhibition of IL 2-driven proliferation caused by high concentrations of immobilized anti-TCR mAb did not seem to be mediated by soluble factors. Cells continued to express cell surface receptors for IL 2 and transferrin after treatment with immobilized anti-TCR mAb. Inhibition of IL 2-driven proliferation by high concentrations of immobilized anti-TCR mAb may represent a mechanism for regulating the proliferation of T lymphocytes. This inhibitory mechanism is initiated by stimulation of the T cell receptor, in this case by immobilized anti-TCR mAb, and is independent of other cells and factors.     

Modulation of interleukin 2 internalization and interleukin 2-dependent cell growth by antireceptor antibodies

The growth factor interleukin 2 (IL2) binds to and is internalized together with high-affinity surface receptors present on lymphoid cells. This endocytosis thus results in down-regulation of the receptors. However, it is not known if the internalization is relevant to the induction of cell growth. In the present study a rat monoclonal antibody to the P55 chain of the IL2 receptor was used to examine the role of receptor internalization in the IL2-dependent autocrine human tumor T cell line IARC 301. When given alone, this antibody did not inhibit IL2 binding, internalization, or IL2-dependent cell proliferation. However, crosslinking by anti-rat immunoglobulins, which did not affect binding of the growth factor, inhibited both IL2 internalization and cell proliferation. Besides offering a novel means for the specific inhibition of the uptake of IL2 bound to IL2 high-affinity receptors, the results are compatible with the association of this receptor-ligand uptake to the growth stimulation by IL2.     

Changes in glycosylation alter the affinity of the human transferrin receptor for its ligand

When transferrin receptors of human erythroleukemic cells were pulse-labeled with [35S]methionine and then chased in the absence of radioactive precursor, the first detectable immunoprecipitable form of the receptor had a molecular mass of 85 kDa. This form of the receptor was converted to the mature form of 93 kDa with a half-time of about 40-60 min. Both the immature (85 kDa) and mature (93 kDa) receptors associated as dimers, the native form of the receptor. The 85-kDa, as well as the 93-kDa, receptors bound to a monoclonal antibody raised against the transferrin receptor or to transferrin-Sepharose. In order to determine whether glycosylation was necessary for ligand binding, purified receptors were isolated from cells grown in the presence of tunicamycin. When K562 cells were grown in the presence of tunicamycin, an 80-kDa nonglycosylated form of the receptor was synthesized. This nonglycosylated receptor was also capable of dimer formation; however, much less of it reached the cell surface than the fully glycosylated form, although both untreated and tunicamycin-grown cells appeared to synthesize transferrin receptors at similar rates. Although the number of receptor molecules/cell was similar in control and tunicamycin-treated cells, the nonglycosylated receptors exhibited a much lower affinity for transferrin than those of untreated cells; in contrast, when receptors were purified by immunoprecipitation and digested with bacterial alkaline phosphatase, no difference was observed between the affinity of these receptors and undigested immunoprecipitated receptors. These results suggest that glycosylation is not necessary for specific binding of transferrin to its receptor, but the affinity of this binding can be influenced greatly by the presence or absence of carbohydrate residues.     

Induction of proliferation in vitro of resting human natural killer cells: expression of surface activation antigens

In this report, we show that resting human peripheral blood NK cells, positively enriched with antibody B73.1, can be induced to proliferate in vitro in short-term cultures, and newly express membrane activation antigens. B73.1+ NK cells, cultured for 6 days in the presence of conditioned medium containing IL 2 and irradiated B lymphoblastoid cells, show significant [3H]TdR incorporation beginning on day 4. At that time, a large proportion of the cells express HLA-DR and 4F2 antigens, and transferrin and IL 2 receptors, all detectable at high density by indirect immunofluorescence with the use of monoclonal antibodies. These cells maintain their ability to lyse target cells spontaneously or in the presence of antibodies. By two-color immunofluorescence and cell cycle analysis combined with indirect immunofluorescence, we demonstrate directly that the activation antigens are expressed on all NK (B73.1+) cells in the S/G2/M phases of the cell cycle, and on only a proportion of those in the G0/G1 phases.     

3'-Azido-3'-deoxythymidine reduces the rate of transferrin receptor endocytosis in K562 cells.

K562 cells, exposed for at least 24 h to 5 microM 3'-azido-3'-deoxythymidine (AZT), gave rise to an overall increase in the number of cell surface transferrin binding receptors (18-20%). This effect was ascertained either with binding experiments by using 125I-transferrin and with immunoprecipitation by using a specific monoclonal antibody against the transferrin receptor. At higher AZT concentrations (20 and 40 microM), a further increase was found, that is, up to 23% by binding experiments and up to 110% by immunoprecipitation. However, Scatchard analysis of the binding data indicated that although the number of cell surface transferrin receptors increased, the affinity of transferrin for its receptor did not change (Ka=4.0x108 M). Surprisingly, immunoprecipitation of total receptor molecules showed that the synthesis of receptor was not enhanced by the drug treatment. The effect of AZT on transferrin internalization and receptor recycling was also investigated. In this case, data indicated that the increase in the number of receptors at the cell surface was probably due to a slowing down of endocytosis rate rather than to an increased recycling rate of the receptor to cell surface. In fact, the time during which half the saturated amount of transferrin had been endocytosed (t1/2) was 2.15 min for control cells and 3.41, 3.04, and 3.74 min for 5, 20, and 40 microM AZT-treated cells, respectively. Conversely, recycling experiments did not show any significant differences between control and treated cells. A likely mechanism through which AZT could interfere with the transferrin receptor trafficking, together with the relevance of our findings, is extensively discussed.     

Centrifugal elutriation of human lymphoid cells: synchronization and separation of aneuploid cells into diploid and tetraploid populations

Both normal and leukemic human lymphoid cell lines were separated into populations corresponding to different positions in the cell cycle by centrifugal elutriation. Each population was analyzed for cell concentration, cell volume, [3H]thymidine incorporation, percentage S phase by autoradiography, and percent G1, S, and G2/M phases by flow cytometry. The smallest cells, collected at the lowest flow rate, were in G1 phase. Cells collected at increasing flow rates progressively increased in volume and represented distinct positions in the cell cycle transition from G1 phase, through S phase, and into G2/M phase. The purity of the G1 population varied according to cell load. One hundred percent of cells were recovered and cells collected in G1- and S-phase populations proliferated in culture with patterns characteristic of synchronized cells. An aneuploidy leukemia cell line, CEM, was separated into near-diploid and near-tetraploid populations by centrifugal elutriation. This method of cell separation provides large numbers of human lymphoid cells at different positions in the cell cycle for investigating the relationship between the cell cycle and various surface membrane and metabolic properties of cells. Aneuploid leukemia and lymphoma cells can be separated by centrifugal elutriation into populations which contain different numbers of chromosomes for comparisons of their biologic properties.     

Murine cell surface transferrin receptor: Studies with an anti-receptor monoclonal antibody

A rat monoclonal antibody against the murine transferrin receptor has been identified. The receptor is a 95,000 molecular weight species that exists in the cell membrane as a disulphide-bonded dimer. Whereas 29 of 29 murine hematopoietic tumor cell lines express detectable numbers of transferrin receptors, less than 1% of adult thymocytes or spleen cells and only 5% of bone marrow cells are positive. However, fetal liver and neonatal spleen contain substantial numbers of transferrin receptor-positive cells. Induction of Friend cells in vitro with dimethyl-sulphoxide leads to an overall increase in the expression of transferrin receptors on the cell surface. The anti-transferin receptor antibody we have obtained partially blocks iron uptake from ⁵⁹Fe-transferrin by a variety of murine cell lines and inhibits the growth of a murine myeloma cell line in vitro.     

A new compound which reversibly arrests T lymphocyte cell cycle near the G1/S boundary

A novel cell cycle blocking agent profoundly suppressed the proliferation of mitogen-stimulated T lymphocytes. The carboxythiazole derivative arrested cells in the G1 phase of the cell cycle but did not inhibit the induction of cell surface receptors for either interleukin-2 or transferrin. The uncoupling of transferrin receptor expression from DNA synthesis indicated that a previously undefined restriction point in the cell cycle has been identified which occurs after transferrin receptor expression in late G1 and just prior to the initiation of DNA replication in S phase. T cells incubated in an inhibitory dose of the carboxythiazole derivative resumed cell cycle progression subsequent to its removal, indicating that the compound reversibly arrests cells at the late G1 restriction point. In contrast to other techniques which have been inefficient in achieving T cell synchronization, T cells released from the block mediated by the carboxythiazole compound progress through S phase with a considerable degree of synchrony.     

Development of a cell with dendritic morphology from a precursor of B lymphocyte lineage

Cells developing dendritic morphology were detected in cultures of highly purified human B cells incubated with 4 beta-phorbol 12-myristate 13-acetate (PMA). After 72 hr of culture, 2 to 7% of the cells had assumed a dendritic shape provided that contact with a plastic or glass surface also occurred. Dendritic cells developed in cultures of B cells prepared by positively selecting cells that stained with the B cell-specific monoclonal antibody B1 with the fluorescence-activated cell sorter. By contrast, dendritic cells could not be detected in cultures of cells obtained from patients with Bruton's type agammaglobulinemia that lacked B cells. Cells with dendritic morphology were nonspecific esterase negative and not phagocytic. They expressed HLA-DR, DQ, and DP antigens, receptors for interleukin 2 and transferrin, and were stained by B1 and 60.3, an antibody that identifies the beta-chain common to lymphocyte function associated antigen-1, complement receptor 3, and the p150,95 antigen, but not by monoclonal antibodies to monocytes, complement receptors 2 or 3, NK cells, T cells, or Langerhans' cells. Formation of dendritic cells was inhibited by microtubule poisons (vinblastine, colchicine), a microfilament inhibitor (cytochalasin B), and the 60.3 monoclonal antibody, but not by inhibition of DNA synthesis. These data indicate that a subset of B cells is capable of assuming dendritic morphology after stimulation with phorbol esters and attachment to a surface. These dendritic cells exhibit characteristics that are quite similar to the interdigitating cells found in T cell-dependent areas of lymph nodes.     

Monoclonal antibodies that inhibit activation and proliferation of lymphocytes. I. Expression of the antigen on monocytes and activated lymphocytes

It has been shown previously that HBJ127 and HBJ98 monoclonal antibodies raised against a human bladder cancer cell line, and B3 monoclonal antibody against a rat bladder cancer cell line recognized unique cell surface antigens abundant in proliferating cells of the corresponding species. Distribution of the antigens and kinetics of the appearance on human and rat lymphoid cells were examined by means of flow cytometry. Rat macrophages and human peripheral blood monocytes were stained strongly with the B3 and HBJ127 monoclonal antibodies, respectively. With regard to lymphocytes, the expression of the B3-defined antigen on rat lymphocytes was found to have a negative correlation with the maturation of the lymphocytes; the antigen was most abundant in bone marrow cells, less abundant in thymocytes, and least abundant in spleen, lymph node, and peripheral blood lymphocytes. Similarly, the HBJ127-defined antigen on human peripheral lymphocytes was negligible. On activation with Con A or alloantigens, however, both rat and human T lymphocytes did strongly express these antigens. Activation of human or rat B cells with lipopolysaccharide also resulted in the augmented expression of these antigens. Kinetics studies revealed that the antigen expression was readily manifested within 12 hr on activation of rat or human T cells with Con A, was augmented progressively with culture time, and reached a plateau within 36 hr. This somewhat earlier appearance of these antigens apparently preceded the manifestations of the IL 2 receptor (Tac antigen) and the augmented DNA synthesis. The B3-defined antigen on Con A-stimulated T cells was more rich on the lymphocytes in S and G2/M phases than those in G1 phase, and the expression was not significantly affected by the addition of hydroxyurea, but was moderately inhibited by the addition of sodium butylate. These results suggest that the appearance and expression of the B3-defined antigen and probably also those of the HBJ127/HBJ98-defined antigen are correlated with lymphocyte activation and subsequent progression through the cell cycle.     

Monoclonal antibody that inhibits infection of HeLa and rhabdomyosarcoma cells by selected enteroviruses through receptor blockade.

BALB/c mice were immunized with HeLa cells, and their spleen cells were fused with myeloma cells to produce hybridomas. Initial screening of culture fluids from 800 fusion products in a cell protection assay against coxsackievirus B3 (CB3) and the CB3-RD virus variant yielded five presumptive monoclonal antibodies with three specificities: protection against CB3 on HeLa, protection against CB3-RD on rhabdomyosarcoma (RD) cells, and protection against both viruses on the respective cells. Only one of the monoclonal antibodies (with dual specificity) survived two subclonings and was studied in detail. The antibody was determined to have an immunoglobulin G2a isotype and protected cells by blockade of cellular receptors, since attachment of [35S]methionine-labeled CB3 was inhibited by greater than 90%. The monoclonal antibody protected HeLa cells against infection by CB1, CB3, CB5, echovirus 6, and coxsackievirus A21 and RD cells against CB1-RD, CB3-RD, and CB5-RD virus variants. The monoclonal antibody did not protect either cell type against 16 other immunotypes of picornaviruses. The monoclonal antibody produced only positive fluorescence on those cells which were protected against infection, and 125I-labeled antibody confirmed the specific binding to HeLa and RD cells. The results suggest that this monoclonal antibody possesses some of the receptor specificity of the group B coxsackieviruses.     

Human T cell antigen expression during the early stages of fetal thymic maturation

Human thymus tissue was examined from 7 wk of gestation through birth for the expression of antigens reacting with a panel of anti-T cell monoclonal antibodies. Additionally, the reactivities of reagents against the transferrin receptor, against leukocytes, against low m. w. keratins, and against major histocompatibility complex antigens were studied on human fetal thymic tissue. Frozen tissue sections were evaluated by using indirect immunofluorescence assays. At 7 wk of gestation, no lymphoid cells were identified within the epithelial thymic rudiment; however, lymphoid cells reacting with both antibody 3A1, a pan T cell marker, and antibody T200, a pan leukocyte reagent, were identified in perithymic mesenchyme. After lymphoid colonization of the thymic rudiment at 10 wk of fetal gestation, fetal thymic tissue reacted with antibodies T1, T4, and T8. At 12 wk of gestation, antibodies T3, T6, A1G3 (anti-p80, a marker of mature thymocytes), and 35.1 (anti-E rosette receptor) all reacted with thymic tissue. Our findings indicate that T cell antigens were acquired sequentially on thymocytes at discrete stages during the first trimester of human fetal development. The 3A1 antigen was present on fetal lymphocytes before lymphoid cell colonization of thymic epithelium, suggesting that passage through the thymus was not required for the expression of the 3A1 antigen by T cell precursors. The appearance of mature T cell antigens, T3 and p80, on thymocytes by 12 wk of gestation implies that the T cell antigen repertoire may be established in the thymus during the first trimester. Thus, a critical period of T cell maturation appears to occur between 7 and 12 wk of human fetal gestation.     

Insulin stimulates cellular iron uptake and causes the redistribution of intracellular transferrin receptors to the plasma membrane

Insulin stimulates the accumulation of iron by isolated fat cells by increasing the uptake of diferric transferrin. Analysis of the cell-surface binding of diferric 125I-transferrin indicated that insulin caused a 3-fold increase in the cell surface number of transferrin receptors. This result was confirmed by the demonstration that insulin increases the binding of an anti-rat transferrin receptor monoclonal antibody (OX-26) to the surface of fat cells. The basis of this effect of insulin was examined by investigating the number of transferrin receptors in membrane fractions isolated from disrupted fat cells. Two methods were employed. First the binding isotherm of diferric 125I-transferrin to the isolated membranes was studied. Second, the membranes were solubilized with detergent, and the number of transferrin receptors was measured by immunoblotting using the monoclonal antibody OX-26. It was observed that insulin treatment of intact fat cells resulted in an increase in the number of transferrin receptors located in the isolated plasma membrane fraction of the disrupted fat cells. Furthermore, the increase in the number of plasma membrane transferrin receptors was associated with a concomitant decrease in the transferrin receptor number in a low density microsome fraction previously shown to consist of intracellular membranes. This redistribution of transferrin receptors between cellular membrane fractions in response to insulin is remarkably similar to the regulation by insulin of glucose transporters and type II insulin-like growth factor receptors. We conclude that insulin stimulates fat cell iron uptake by a mechanism that may involve the redistribution of transferrin receptors from an internal membrane compartment (low density microsomes) to the cell surface (plasma membrane).     

Characterization of transferrin receptor in an immortalized cell line of rat brain endothelial cells, RBE4

The content and distribution of transferrin receptors in an immortalized cell line, RBE4, derived from rat cerebral capillary endothelial cells was investigated using the monoclonal antibody MRC OX-26 (OX-26 mAb) specific for the rat transferrin receptor. An ELISA assay was developed with which the OX-26 mAb can be determined quantiatively. The detection limit of the assay was 10 pg or 0.07 fmol of murine antibody. With this technique accurate measurement of native antibody is now possible without the need for isotope labeling (iodination). Immunostaining of confluent monolayers of RBE4 cells using an antibody directed against the tight junction associated protein ZO-1 was indicative for structural intactness of RBE4 cell monolayers. OX-26 immunostaining demonstrated localization of the transferrin receptor at the plasma membrane and/or in the cytosol. Binding studies showed saturation of OX-26 mAb binding. The antibody binding analysis gave a dissociation constant (KD) of 17.1 +/- 1.2 nmol/l. The total amount of transferrin receptors present per cell was 70,800 +/- 17,000. Our results indicate that receptor binding of OX-26 mAb can be studied using an in vitro cell culture model of rat brain mircrovessel endothelium in conjunction with an ELISA technique for detection of native antibody. This approach will be used to investigate mechanisms of transendothelial transport of OX-26 in vitro.     

Regulation of interleukin 2 receptor expression on a human cytotoxic T lymphocyte clone, synergism between alloantigenic stimulation and interleukin 2

A human T cell clone (termed 40.2.6) established from a rejected human kidney allograft has been studied for its ability to express membrane IL 2 receptors in response to antigen (irradiated cells from the graft's donor) and recombinant IL 2 (rec-IL 2). On antigenic stimulation, the 40.2.6 clone produced low levels (0.15 U/ml) of IL 2 (peak at 24 hr) and incorporated (3H)thymidine (peak at 48 hr). This incorporation was strongly enhanced on addition of rec-IL 2 and was inhibited by the 33B31 antibody, an anti-human IL 2 receptor monoclonal antibody (Mab). The 125I-labeled 33B31 Mab has been used to quantify the density of IL 2 receptors on 40.2.6 cells. Cells not re-exposed to antigen or rec-IL 2 had a level of 33B31-binding sites which declined rapidly (10% of starting value after 2 days). This level remained much more stable when rec-IL 2 (1 U/ml) was present in the medium (80% at day 2). Antigen induced a three- to eightfold increase in the level of 33B31-binding sites which peaked at 24 hr and then declined. When a similar antigenic stimulation was performed in the presence of rec-IL 2 (1 U/ml), the level of 33B31-binding sites peaked at a higher value (eight- to 20-fold increase at day 2), and its subsequent decline was slower. These potentiating effects of rec-IL 2 were dose-dependent and occurred at low concentrations corresponding to the saturation by rec-IL 2 of high affinity IL 2 receptor sites. Finally, high affinity IL 2 receptors, as measured by the binding of 35S-labeled rec-IL 2, were found to be similarly up-regulated by antigen and rec-IL 2. Together, our results obtained on a monoclonal human T cell population with highly purified rec-IL 2 demonstrate that rec-IL 2 and antigen act in synergy to induce the expression of both high and low affinity membrane IL 2 receptors.