Exposure of K562 cells to anti-receptor monoclonal antibody OKT9 results in rapid redistribution and enhanced degradation of the transferrin receptor

When the human erythroleukemia cell line K562 is treated with OKT9, a monoclonal antibody against the transferrin receptor, effects on receptor dynamics and degradation ensue. The apparent half-life of the receptor is decreased by greater than 50% as a result of OKT9 treatment. The transferrin receptor is also rapidly redistributed in response to OKT9 such that a lower percentage of the cellular receptors are displayed on the cell surface. OKT9 treatment also leads to a decrease in the total number of receptors participating in the transferrin cycle for cellular iron uptake. The reduction in iron uptake that results from the loss of receptors from the cycle leads to enhanced biosynthesis of the receptor. Receptors with bound OKT9 continue to participate in multiple cycles of iron uptake. However, OKT9 treatment appears to result in a relatively small increase per cycle in the departure of receptors from participation in iron uptake to a pathway leading to receptor degradation. Radiolabeled OKT9 is itself degraded by K562 cells and this degradation is inhibitable by leupeptin or chloroquine. In the presence of leupeptin, OKT9 treatment results in the enhanced intracellular accumulation of transferrin. Because the time involved in the transferrin cycle is shorter (12.5 min) than the normal half-life of the receptor (8 h), a small change in recycling efficiency caused by OKT9 treatment could account for the marked decrease in receptor half-life. In this paper the implications of these findings are discussed as they relate to systems in which receptor number is regulated by ligand.     

Comparison of the kinetics of cycling of the transferrin receptor in the presence or absence of bound diferric transferrin.

The kinetics of cycling of the transferrin receptor in A431 human epidermoid-carcinoma cells was examined in the presence or absence of bound diferric transferrin. In order to investigate the properties of the receptor in the absence of transferrin, the cells were maintained in defined medium without transferrin. It was demonstrated that Fab fragments of a monoclonal anti-(transferrin receptor) antibody (OKT9) did not alter the binding of diferric 125I-transferrin to the receptor or change the accumulation of [59Fe]diferric transferrin by cells. OKT9 125I-Fab fragments were prepared and used as a probe for the function of the receptor. The first-order rate constants for endocytosis (0.16 +/- 0.02 min-1) and exocytosis (0.056 +/- 0.003 min-1) were found to be significantly lower for control cells than the corresponding rate constants for endocytosis (0.22 +/- 0.02 min-1) and exocytosis (0.065 +/- 0.004 min-1) measured for cells incubated with 1 microM-diferric transferrin (mean +/- S.D., n = 3). The cycling of the transferrin receptor is therefore regulated by diferric transferrin via an increase in both the rate of endocytosis and exocytosis. Examination of the accumulation of OKT9 125I-Fab fragments indicated that diferric transferrin caused a marked decrease in the amount of internalized 125I-Fab fragments associated with the cells after 60 min of incubation at 37 degrees C. Diferric transferrin therefore increases the efficiency of the release of internalized 125I-Fab fragments compared with cells incubated without diferric transferrin. These data indicate that transferrin regulates the sorting of the transferrin receptor at the cell surface and within endosomal membrane compartments.     

Isolation and chemical conversion of two novel prostaglandin endoperoxides: 5(6)-epoxy-PGG1 and 5(6)-epoxy-PGH1

The possible molecular heterogeneity of human transferrin receptors was analyzed using two murine monoclonal antibodies, Tü15 and Tü67. Both reagents precipitated from lysates of 125I-labeled HL-60 cells a major component of 88 kDa which could be identified as the transferrin receptor by comparison with the proteins detected by monoclonal antibody OKT9. Although sequential immunoprecipitations appeared to demonstrate molecular heterogeneity of transferrin receptors, since the Tü15-reactive species were fully included in the Tü67-positive population, but not vice versa, the possible association of Tü15-reactive molecules with transferrin receptor is also discussed.     

Detection of a molecular complex between ras proteins and transferrin receptor

Immunoprecipitation of extracts of human carcinoma cell lines with three different monoclonal antibodies generated against ras proteins revealed the coprecipitation of a 90,000 dalton protein. The coprecipitated protein was identified as the transferrin receptor by comigration in both reducing and nonreducing SDS-polyacrylamide gels, by absorption with a monoclonal antibody directed against transferrin receptor, and by analysis of partial proteolysis products. Coprecipitation of the transferrin receptor with three monoclonal antibodies with differing specificities to ras proteins, as well as the inability to coprecipitate the transferrin receptor from cell extracts from which ras proteins were depleted by preabsorption, indicates that ras proteins and the transferrin receptor form a molecular complex. This complex is disrupted by addition of transferrin to cell extracts. These findings suggest that ras proteins function in regulation of cell growth via interaction with the cell surface receptor for transferrin.     

Coupled cellular trafficking and diffusional limitations in delivery of immunotoxins to multicell tumor spheroids

Immunotoxins have the potential to be powerful tools for selective cell killing, but their lack of clinical success against solid tumors indicates a need to better understand factors which limit immunotoxin transport in three-dimensional systems. In this work, a previously developed model which related immunotoxin toxicity to cellular trafficking in a single cell was coupled with a term accounting for diffusive transport of immunotoxin in a solid tumor sphere. This created a mathematical model which is capable of simulating the biological response of multicell tumor spheroids (MTS) to immunotoxin treatment. The model was used to predict the kinetics of protein synthesis inhibition in MTS treated with transferrin receptor-targeted immunotoxins as a function of immunotoxin concentration and toxin choice. HeLa cells were grown as MTS and treated with immunotoxins constructed from the anti-transferrin receptor antibody OKT9 and the toxins gelonin or CRM107, and the average protein synthesis inhibition and growth rates were measured. With no fitted parameters, the mathematical model quantitatively predicted the experimental observations. Immunotoxins were generally less effective against MTS than monolayer cells at equivalent conditions; for OKT9-gelonin at high concentrations this decrease in efficacy was attributed primarily to heterogeneous receptor distribution in MTS whereas for OKT9-CRM107 the decrease was caused primarily by a large barrier to penetration of the immunotoxin into the spheroid. The experimentally verified model was used to define the conditions which lead to large penetration barriers. In general, transport barriers in MTS become more important as immunotoxins become more effective against cells grown as monolayers. The proposed model is unique in its ability to predict toxicity in MTS directly, and is an important step toward understanding immunotoxin effect on tumors in vivo.     

Monoclonal antibody OKT-9 recognizes the receptor for transferrin on human acute lymphocytic leukemia cells

The monoclonal antibody OKT-9 recognizes a surface protein of human lymphocytes that consists of a disulfide bonded homodimer of m.w. 200,000 intact and 95,000 reduced. A similar protein is precipitated by transferrin-agarose, but not by agarose alone. Peptide mapping by limited proteolysis shows that the proteins precipitated by OKT-9 antibodies and transferrin-agarose are homologous. It is concluded that OKT-9 antibodies recognize the transferrin receptor. Expression of receptors for transferrin may be useful as a marker for activated or dividing cells.     

Characterization of proteins that associate with an unglycosylated form of the transferrin receptor in A431 cells

A protein doublet (Mr = 135,000/130,000) was found to coprecipitate with an unglycosylated form of the transferrin receptor in tunicamycin-treated A431 cells. This doublet is not detected with either a monoclonal or polyclonal antibody to the transferrin receptor on Western blots indicating that these proteins do not interact directly with transferrin receptor antibody. Proteolytic digestion patterns of the individual proteins of the Mr = 135,000/130,000 doublet suggest that they are related to one another and are distinct from the transferrin receptor. Further characterization of these proteins indicates that they form a high molecular weight complex with the unglycosylated but not the glycosylated form of the transferrin receptor. Pulse-chase experiments demonstrate that the proteins post-translationally associate with the receptor.     

Cytotoxic ribonuclease chimeras. Targeted tumoricidal activity in vitro and in vivo.

Monoclonal antibodies to the transferrin receptor or to the T cell antigen, CD5, were chemically linked to mammalian RNase A and found to specifically inhibit protein synthesis in antigen-positive cells. Antibody-mediated specificity of these cytotoxic ribonuclease chimeras (CRCs) was demonstrated in three ways. 1) Toxicity was due to the chemical linkage of RNase to antibody, as the individual components added separately or in combination did not inhibit protein synthesis; 2) the anti-transferrin receptor CRCs inhibited protein synthesis in those cells expressing the human transferrin receptor (K562, U251, Jurkat cells) but had no detectable toxicity to cells lacking the human transferrin receptor (Vero or NIH 3T3 cells); 3) free antibody to either the human transferrin receptor (454A12 or 5E-9) or to the T cell antigen, CD5 (T101), blocked the cytotoxicity of the respective CRC. Two CRC species, designated P1 and P2, that differed in size and stoichiometry of RNase A to antibody, were purified by size-exclusion high performance liquid chromatography. The higher molecular weight P1 conjugate had an IC50 of 20-30 nM, whereas the P2 conjugate had a higher IC50 of 300-500 nM. Bioactivity could be reversibly increased more than 10-fold by freezing. The cytotoxicity of the CRCs was examined in vivo in a solid tumor animal model. Intratumoral injections of an anti-transferrin receptor CRC into established U251 human glioblastoma tumors grown in the flanks of nude mice prevented tumor growth, whereas RNase A mixed with antibody was ineffective. CRCs, therefore, express cytotoxicity in vitro and in vivo. Mammalian nucleases coupled to antibodies may be utilized as cell type-selective cytotoxins and have potential as pharmacologic reagents. The systemic toxicity and immunogenicity observed with mammalian derived cytotoxins may be significantly less than that of the currently employed plant- and bacterial-derived immunotoxins.     

Vitamin D receptor expression in human lymphocytes. Signal requirements and characterization by western blots and DNA sequencing.

The signals controlling the expression of the receptor protein for 1 alpha,25-dihydroxyvitamin D3 in normal human lymphocytes and the relationship of this protein to the classical vitamin D receptor were examined. Lymphocytes activated with the OKT3 antibody to the T-cell antigen receptor expressed fewer binding sites as compared to lymphocytes that were activated by the polyclonal activator phytohemagglutinin (PHA). However, combination of OKT3 and phorbol myristate acetate produced a concentration of binding sites similar to the PHA-activated cells. The receptor from OKT3 and OKT3 + phorbol myristate acetate-activated lymphocytes exhibited decreased binding to DNA-cellulose compared to PHA-activated lymphocytes. In lymphocytes activated either by PHA or OKT3 (but not in resting cells), a 50-kDa species cross-reacting with a monoclonal antibody against the intestinal vitamin D receptor was detected. Finally, RNA from activated lymphocytes was amplified by polymerase chain reaction using oligonucleotide primers flanking the 196 base pair long region encoding the DNA-binding domain of the human intestinal receptor. The amplified product showed an identical nucleotide sequence to the DNA-binding domain of the human intestinal receptor. These findings suggest that expression of the 1,25-(OH)2D3 receptor in lymphocytes is triggered by distinct and contingent signals, and that the protein and the mRNA encoding it are identical to the classical vitamin D receptor.     

The role of transferrin in natural killer cell and IL-2-induced cytotoxic cell function

The growth factor transferrin (Tf) enhanced natural killer (NK) cell cytotoxicity. This enhancement was due to direct effects on NK cell function, and Tf treatment of the K562 target cell had no effect on their sensitivity. NK cells were highly enriched in the low-density large granular lymphocyte population (LGL) by Percoll gradient centrifugation. Despite the direct effect of Tf on NK cells, the number of cells expressing receptors for Tf (TfR) in NK-enriched LGL was the same as the NK-cell-depleted high-density small lymphocyte population (SL). All populations, tested without stimulation, had very few TfR+ cells. Interleukin 2 (IL-2) could induce very high NK-like activity in the LGL but not in SL. Similarly, only LGL could be induced by IL-2 to express TfR. In serum-free cultures, only limited NK-like activity could be developed which was greatly enhanced by supplementing with Tf in the cultures. The importance of Tf in NK-like development was confirmed by modulating the expression of TfR in IL-2 containing cultures with mouse monoclonal antibody OKT9 specific for TfR. OKT9 totally abrogated the induction of cytotoxic activity by IL-2 against K562 and NK-resistant target. OKT9 inhibited the induction of cytotoxicity in both lymphocytes containing active NK cells and in those predepleted of active NK cells, indicating that the development of NK-like activity from both precursor populations requires Tf. The inhibition by OKT9 was only during the induction phase. The same antibody had no effect on the cytotoxicity of fresh NK cells or the mature IL-2-induced NK-like cells. Our data therefore do not support the hypothesis of TfR as the NK recognition structure. Instead, these results indicate that Tf is important for the development of NK and NK-like activities.     

Expression of transferrin receptors and intracellular ferritin during terminal differentiation of human monocytes

Human blood monocytes when cultured on hydrophobic Teflon membranes differentiate into mature macrophages. The expression of transferrin receptors was monitored by monoclonal antibody (OKT9) binding as detected by immunoperoxidase staining. Whereas monocytes were negative, an increasing percentage of macrophages, starting from day 2 in culture, labelled with the antitransferrin receptor antibody as these cells undergo differentiation. After completion of maturation more than 90% of macrophages expressed transferrin receptors. While 90-95% of macrophages from broncho-alveolar lavage fluids labelled with the OKT9 antibody, only a minor portion of macrophages obtained from peritoneal and pleural cavities did so. In parallel, intracellular ferritin in cells of the monocyte-macrophage lineage increased from 10 ng/10(6) cells to 350-1,500 ng/10(6) cells during maturation in vitro. Alveolar macrophages proved to have the highest ferritin content which ranged from 355-8,400 ng/10(6). The results may indicate that iron uptake and storage is a function of cells at late stages of macrophage maturation and that the occurrence of surface receptors for transferrin can be regarded as differentiation dependent marker.     

Immunological and biochemical characterization of an epitope of the transferrin receptor involved in the production of interferon-gamma and B-cell growth factor

We have investigated the effect of a newly developed monoclonal antibody, MoAb NDA9, on human lymphocyte function. This MoAb inhibits the capacity of peripheral blood lymphocytes to display blastogenic responses and to produce immunoglobulins when stimulated in vitro with PWM or with soluble antigens. The inhibitory effect seems to result from the decreased ability of T lymphocytes to produce B cell growth factors (BCGF) in the presence of MoAb NDA9. This antibody also blocks the capacity of polyclonal or monoclonal populations of activated human T cells to produce immune interferon (gamma) but has no direct effect on B cell activation and growth in T-cell-independent systems. Immunochemical studies of the antigen recognized by MoAb NDA9 showed that it is an epitope of the transferrin receptor molecule which is distinct from that recognized by the MoAb OKT9.     

Characterization of the transferrin receptor in tunicamycin-treated A431 cells

The transferrin receptor undergoes extensive co- and post-translational modifications during its biosynthesis. In this study, the functional and structural properties of the transferrin receptor from tunicamycin-treated A431 cells were examined. Incubation of A431 cells with this inhibitor of asparagine-linked glycosylation results in a shift of the apparent molecular weight of the transferrin receptor from 94,000 to 79,000. The electrophoretic mobility of the receptor from treated cells is that of a monomer under nonreducing conditions, whereas the transferrin receptor in untreated cells has the mobility of a dimer under identical conditions. This result indicates a lack of disulfide bond formation between subunits of the receptor from tunicamycin-treated cells. In solution no dimers can be detected with cross-linking studies. This unglycosylated receptor does not appear to stably bind transferrin as demonstrated by a lack of isolation of this form of the receptor with transferrin-linked Sepharose. It is not transported to the surface of A431 cells.     

Structural analyses of progesterone receptors

Progesterone receptors of T47Dco human breast cancer cells consist of two equimolar hormone-binding proteins of mol. wt approximately 85,000 (A protein) and 115,000 (B protein). Both proteins can be demonstrated in intact cells by in situ photoaffinity labeling; that is, in cells treated with the synthetic progestin [3H]R5020, irradiated 2 min with 300 nm u.v., solubilized directly in SDS and subjected to electrophoresis under denaturing conditions. These proteins are 6000-10,000 dalton heavier than the corresponding proteins of chick oviducts. This difference has been measured by direct comparison of photolabeled chick and human receptors on SDS-PAGE and by immunoblotting with the 9G10 antibody prepared against chick protein B. The antibody binds to a protein of mol. wt approximately 106,000 in human cells that is smaller than the hormone-bound B protein and larger than the hormone-bound A. In T47Dco cells, in situ photolabeled, untransformed receptors, as well as transformed nuclear-bound receptors, have equimolar amounts of A and B proteins. This ratio remains stable during a 1 h 37 degrees C in vitro incubation. Analysis of the in situ labeled receptors on gradient gels shows that the untransformed B protein exists as a doublet of mol. wt approximately 115,000 and 119,000 while the A protein is a singlet. After [3H]R5020 treatment, nuclear receptors change further: during the first 30 min in the nucleus the B protein shifts entirely to the heavier, mol. wt = 119,000 form. Between 30 and 60 min after nuclear binding, the A protein first becomes a doublet of 85,000 and 89,000 dalton then shifts entirely to the 4000 dalton heavier form. Later, nuclear processing leads to the simultaneous disappearance of both proteins without generation of smaller molecular weight fragments. Cleveland mapping studies show that the A and B proteins are closely related; despite the initial difference in the molecular weight of A and B, digestion with S. aureaus V8 protease yields identical fragmentation patterns for each, with sequential peptides of mol. wt approximately 49,000, 39,000, 26,000 and 14,000.(ABSTRACT TRUNCATED AT 400 WORDS)     

Plasma membrane and intracellular pools of transferrin receptors decline during in vitro cultivation of U937 cells

Transferrin receptor expression in the monocyte-like cell line U937 was investigated during in vitro cultivation. U937 cells expressed a single class of high affinity surface transferrin receptors (KD approximately 4 nM), with apparent subunit Mr of 90-95,000 Da as determined by SDS-reducing PAGE. [125I]-transferrin binding studies on detergent-solubilized cells revealed that half to two-thirds of the total functional binding sites were located intracellularly. Radioligand binding, immunofluorescence and flow cytometry studies were performed on intact, detergent-solubilized, or saponin-permeabilized cells, using either transferrin or the anti-transferrin receptor monoclonal antibody OKT9 IgG. These studies demonstrated that functional and antigenic transferrin receptor levels were maximal on cells 24 h after subculture at low density and declined during the culture period. Scatchard analysis of radioligand binding data suggested that the decline in functional transferrin binding sites resulted from a decline in the number of available receptors. These results demonstrate that in U937 cells there is a density-dependent regulation of transferrin receptor expression, resulting in a loss of functional and antigenic receptors from both plasma membrane and intracellular locations.     

Early events in lymphocyte activation as defined by three new monoclonal antibodies

Three new lymphocyte activation antigens are described whose kinetics of appearance place them very early in the activation pathway. The 78,000 dalton early antigen (Ea) 1 is present at low levels on resting lymphocytes, and its expression is enhanced twofold to threefold within 3 hr of stimulation. Ea2, a nondisulfide-bonded 86,000 and 73,000 dalton heterodimer, is first detectable 3 hr after activation and peaks by 9 hr. Its presence on all but a few cell lines, plus the variable association with a lower m.w. (28,000) structure, suggest that it may serve as a receptor for a growth factor. Neither Ea1 nor Ea2 are restricted to lymphocytes. The 31,000 dalton Ea3 antigen is induced only by PHA but not by other means of activation, and may pre-exist within the cell. The Ea3 antibody blocks PHA-induced but not OKT3-induced mitogenesis, suggesting differences in the pathways of activation by these two stimuli. These reagents, and OKT3, were used to define the cyclosporine A (CSA)-sensitive stage of lymphocyte activation. CSA blocks at a point before the biosynthesis of Ea1 and after that of T3/T cell receptor loss from the cell surface, at a point close to Ea2 biosynthesis.     

Alterations in the transferrin receptor of human erythroleukemic cells after induction of hemoglobin synthesis

When K562 human erythroleukemic cells are induced to differentiate by addition of hemin to their medium, the number of binding sites for transferrin on the cell surface is substantially reduced. This reflects an internalization of receptors since no such reduction is observed when the total binding sites in soluble extracts of uninduced and differentiating cells are compared. The internalization of transferrin receptors has also been shown using lactoperoxidase-mediated radioiodination of cell surfaces and by immune precipitation of total and surface labeled receptors using an anti-receptor monoclonal antibody. Transferrin receptors from uninduced and differentiating cells were partially purified by affinity chromatography on transferrin-Sepharose and shown to be disulfide-bridged homodimers of a polypeptide with an apparent molecular weight of approximately 90,000. This protein is a phosphoprotein that can be resolved by isoelectric focusing into three major and two minor forms. By digestion with bacterial alkaline phosphatase, it was shown that at least four of these forms are probably phosphorylation variants of a single polypeptide. As differentiation proceeds, the proportions of the individual forms of the receptor change with a shift to the more phosphorylated polypeptides.     

Suppression of interleukin 2 receptor acquisition by monoclonal antibodies recognizing the 50 KD protein associated with the sheep erythrocyte receptor on human T lymphocytes

Monoclonal antibodies OKT11A, 9.6, and 35.1 recognize epitopes on a 50000 dalton surface molecule (p50) identical to or closely associated with the sheep erythrocyte receptor (E receptor) on human T lymphocytes. These three antibodies were investigated for ability to inhibit T cell proliferation and interleukin 2 (IL 2) receptor acquisition (determined with anti-Tac antibody in an immunofluorescence assay) induced by the lectin mitogen phytohemagglutinin (PHA) or by the phorbol ester 12-O-tetradecanoyl-phorbol-13 acetate (TPA). OKT11A, 9.6, and 35.1 were found to suppress [3H]thymidine incorporation and IL 2 receptor acquisition stimulated by PHA but not by TPA. This inhibition was not attributable to a lag in kinetics, but was sustained throughout 4 to 5 days of culture. Because OKT11A and 9.6 have been reported to suppress lectin mitogen-induced IL 2 production, we attempted to overcome inhibition of proliferation with exogenous IL 2 (MLA144 supernatants or immunoaffinity-purified human IL 2). Adding IL 2 at the initiation of culture abrogated the suppressive effect of all three anti-p50 antibodies on proliferation and on the acquisition of IL 2 receptors, raising the possibility that IL 2 may up-regulate expression of its cellular receptor on human T lymphocytes. These data, together with previous reports, indicate that OKT11A, 9.6, and 35.1 suppress lectin mitogen-induced T cell proliferation by impairing both IL 2 elaboration and IL 2 receptor acquisition, and suggest that IL 2 may be capable, at least under some conditions, of increasing expression of IL 2 receptors on human T lymphocytes.     

Activated lymphocytes in patients with newly diagnosed type 1 (insulin-dependent) diabetes mellitus

The expression of activation antigens (transferrin receptor, IL-2 receptor and Ia antigen) on circulating T lymphocytes from Japanese children with Type 1 diabetes was studied using five monoclonal antibodies (Ab), OKT9, anti-Tac Ab, OKIa1, anti-human HLA-DR Ab and OKT3. For detecting Ia positive T cells, the dual staining technique using OKT3 and anti-Ia antibody was employed. Four out of six patients (67%) with newly diagnosed Type 1 diabetes showed a raised level of either OKT9 or Tac positive cells when examined at diagnosis. These patients, however, rapidly lost these activation antigens after the insulin therapy was started. In contrast, in 32 long-standing patients, only 2 (6%) had a high percentage of OKT9 positive cells and none of them demonstrated Tac positive cells. One out of six newly diagnosed patients or three out of 21 long-standing patients had a significantly high percentage of Ia-positive T cells compared with normal subjects. In poorly controlled long-standing patients whose HbA1 value was higher than 14%, none of them had an increased number of activated lymphocytes. Therefore, it is unlikely that insulin deficiency and hyperglycemia were responsible for the changes observed in these studies. Activated lymphocytes might be related to activation of the immune system involved in pathogenesis of Type 1 diabetes.