Mik-beta 1(Fv)-PE40, a recombinant immunotoxin cytotoxic toward cells bearing the beta-chain of the IL-2 receptor.

Mik-beta 1 is a mAb that binds to the beta subunit of the IL-2R. We have constructed a recombinant single chain immunotoxin Mik-beta 1(Fv)-PE40 by genetically fusing the H and L V domains of Mik-beta 1 to each other via a peptide linker, and then to PE40, a derivative of Pseudomonas exotoxin. Mik-beta 1(Fv)-PE40 was selectively cytotoxic for cells expressing high levels of IL-2R beta (p75) subunit. Mik-beta 1(Fv)-PE40 was cytotoxic to the NK cell line YT-S, which expresses p75 but not p55 subunits, with an IC50 of 6 ng/ml. The ATL line HUT-102 was less sensitive, with an IC50 of 200 ng/ml. However, the IC50 could be lowered to 11 ng/ml when Mik-beta 1(Fv)-PE40 was allowed to bind to HUT-102 cells at 4 degrees C for 4 h before overnight incubation at 37 degrees C. An excess of Mik-beta 1 but not of anti-Tac, the anti-p55 mAb, prevented the cytotoxicity of Mik-beta 1(Fv)-PE40. We constructed a more active version of Mik-beta 1(Fv)-PE40, designated Mik-beta 1(Fv)-PE40KDEL, by converting the carboxyl-terminus of the toxin from -REDLK to -KDEL. Mik-beta 1(Fv)-PE40KDEL showed an IC50 of 2 ng/ml toward YT-S cells and 35 ng/ml toward HUT-102 cells. Binding studies using radioiodinated Mik-beta 1 showed that Mik-beta 1(Fv)-PE40 bound to the p75 receptor subunit with 11% of the affinity of the native Mik-beta 1 antibody. Mik-beta 1(Fv)-PE40 may be a useful reagent to study cells that express IL-2R, and it deserves further study as a possible treatment for cancers in which the malignant cells express high numbers of p75 subunit.     

IL-2-PE40 is cytotoxic for activated T lymphocytes expressing IL-2 receptors

IL-2-PE40 is a chimeric molecule in which IL-2 is attached to the amino end of modified Pseudomonas exotoxin molecule lacking cell recognition domain. This molecule was extremely toxic for Con A-stimulated spleen cells from mice. Moreover, IL-2-PE40 has suppressive effect against Ag-activated cells; it inhibits the generation of cytotoxic T lymphocyte activity in a MLC. IL-2-PE40 could be a useful agent in IL-2R targeting therapy including immunosuppressive therapy for allograft rejection or some autoimmune diseases.     

Single-chain immunotoxins directed at the human transferrin receptor containing Pseudomonas exotoxin A or diphtheria toxin: anti-TFR(Fv)-PE40 and DT388-anti-TFR(Fv).

Two single-chain immunotoxins directed at the human transferrin receptor have been constructed by using polymerase chain reaction-based methods. Anti-TFR(Fv)-PE40 is encoded by a gene fusion between the DNA sequence encoding the antigen-binding portion (Fv) of a monoclonal antibody directed at the human transferrin receptor and that encoding a 40,000-molecular-weight fragment of Pseudomonas exotoxin (PE40). The other fusion protein, DT388-anti-TFR(Fv), is encoded by a gene fusion between the DNA encoding a truncated form of diphtheria toxin and that encoding the antigen-binding portion of antibody to human transferrin receptor. These gene fusions were expressed in Escherichia coli, and fusion proteins were purified by conventional chromatography techniques to near homogeneity. In anti-TFR(Fv)-PE40, the antigen-binding portion is placed at the amino terminus of the toxin, while in DT388-anti-TFR(Fv), it is at the carboxyl end of the toxin. Both these single-chain immunotoxins kill cells bearing the human transferrin receptors. However, anti-TFR(Fv)-PE40 was usually more active than DT388-anti-TFR(Fv), and in some cases it was several-hundred-fold more active. Anti-TFR(Fv)-PE40 was also more active on cell lines than a conjugate made by chemically coupling the native antibody to PE40, and in some cases it was more than 100-fold more active.     

Anti-Tac(Fv)-PE40, a single chain antibody Pseudomonas fusion protein directed at interleukin 2 receptor bearing cells

Anti-Tac(Fv)-PE40 is a chimeric single chain immunotoxin in which anti-Tac variable heavy and light chains held together by a peptide linker are attached to PE40, a truncated form of Pseudomonas exotoxin. This molecule was shown to be extremely cytotoxic for interleukin 2 (IL2) receptor bearing cells in tissue culture (Chaudhary, V. K., Queen, C., Junghans, R. P., Waldmann, T. A., FitzGerald, D. J., and Pastan, I. (1989) Nature 339, 394-397). Here we describe various forms of anti-Tac(Fv)-PE40 protein in which the order of the variable domains of anti-Tac has been switched and also three different types of peptide linkers have been used. All these proteins were purified to near homogeneity and were found to have similar cytotoxic activities against various human cells expressing the p55 subunit of the IL2 receptor. Anti-Tac(Fv)-PE40 was also found to have a very potent suppressive activity against phytohemagglutinin-activated human lymphoblasts and in a human mixed lymphocyte reaction. Anti-Tac(Fv)-PE40 appeared in the blood rapidly in mice after intraperitoneal administration and could be detected in the blood for up to 8 h. Anti-Tac(Fv)-PE40 warrants evaluation as an anti-tumor and immunosuppressive agent in humans.     

Interleukin 2 (IL2) PE40 is cytotoxic to cells displaying either the p55 or p70 subunit of the IL2 receptor

IL2-PE40 is a chimeric protein composed of human interleukin 2 (IL2) genetically fused to the amino terminus of a modified form of pseudomonas exotoxin (PE). Internalization of IL2 via the individual p55 and p70 subunits of the IL2 receptor was studied using IL2-PE40 on several mouse and human cell lines expressing either the p55, the p70, or both IL2 receptor subunits. Internalization was assessed by measuring inhibition of protein synthesis caused by the toxin moiety of IL2-PE40. The results demonstrate that IL2 internalization is mediated by either the p55 receptor subunit or by the p70 subunit but is much more efficient when high affinity receptors composed of both subunits are present. IL2-PE40 is a powerful reagent for studying IL2 receptor interactions and for analyzing pathways of the immune response and its regulation.     

Physiological effects of TGF(alpha)-PE40 expression in recombinant Escherichia coli JM109

Physiological effects of isopropyl-thiogalactopyranoside (IPTG) induction were examined in Escherichia coli strain JM109 expressing a fusion protein composed of transforming growth factor alpha and a 40-kD portion of Pseudomonas aeruginosa exotoxin A (TGF(alpha)-PE40) under control of the tac promoter. Fermentations at the 15-L scale in complex medium compared growth and metabolite profiles of the untransformed JM109 host strain, the strain transformed with the vector lacking the TGF(alpha)-PE40 open reading frame (JM109[pKK2.7]), and the strain with the complete plasmid for TGF(alpha)-PE40 expression (JM109[pTAC-TGF57-PE40]). Metabolite and growth profiles of JM109 (pTAC-TGF57-PE40) cultures changed significantly in IPTG-induced versus uninduced cultures. Prior to induction, glucose was metabolized to acetate or completely oxidized to CO(2). Following induction, pyruvate was also excreted in addition to acetate. In the absence of inducer, pyruvate was excreted by JM109 (pTAC-TGF57-PE40) only when dissolved oxygen levels fell to less than 10% of saturation (microaerobic rather than anaerobic conditions). The untransformed JM109 host strain or JM109 (pKK2.7) did not excrete pyruvate in the presence or absence of inducer, although JM109 (pKK2.7) exhibited a pattern of growth following addition of IPTG that closely resembled JM109 (pTAC-TFG57-PE40). Fermentations of JM109 (pTAC-TFG57-PE40) in a synthetic medium supported lower expression levels, but resulted in similar alterations in metabolite profiles. Induction in synthetic medium resulted in pyruvate excretion without further acetate accumulation. Taken together, these data suggest that one consequence of TGF(alpha)-PE40 expression in JM109 is altered patterns of pyruvate oxidation. (c) 1992 John Wiley & Sons, Inc.     

IL2-PE664Glu, a new chimeric protein cytotoxic to human-activated T lymphocytes

To produce a molecule that will kill activated T cells as well as lymphomas and leukemias expressing interleukin 2 (IL2) receptors, we have created a recombinant chimeric protein in which IL2 is attached in peptide linkage to a truncated mutant form of Pseudomonas exotoxin (PE) (Lorberboum-Galski, H., FitzGerald, D.J.P., Chandhary, V.K., Adhya, S., and Pastan, I. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 1922-1926). Although this molecule was very active on rodent cells, it had lower activity on some human cell types. A new chimeric protein termed IL2-PE664Glu has been constructed that is extremely toxic to both phytohemagglutinin blasts and mixed leukocyte reaction blasts prepared from monkey and human lymphocytes. The chimeric gene encoding this protein was constructed by fusing a cDNA clone for human interleukin 2 to the 5' end of a mutated cDNA encoding a full-length PE molecule. Four amino acids in domain I of PE were changed thus decreasing its nonspecific toxicity. IL2-PE664Glu is a much more active cytotoxic molecule for primate and human-activated T cells than IL2-PE40 which is a chimeric protein that was found to be an effective immunosuppressive agent in rodent models. Our results indicate that IL2-PE664Glu should be evaluated as an immunosuppressive agent for the treatment of human immune disorders in which activated T cells expressing the IL2 receptor are prominent.     

Cell-specific toxicity of a chimeric protein composed of interleukin-6 and Pseudomonas exotoxin (IL6-PE40) on tumor cells.

IL6-PE40 is a chimeric toxin composed of human interleukin-6 (IL6) linked by a peptide bond to PE40, a form of Pseudomonas exotoxin (PE) devoid of its cell recognition domain. To identify cancer cell lines with high numbers of IL6 receptors and to assess the usefulness of IL6-PE40 as a possible anticancer agent, we evaluated the toxicity of IL6-PE40 on a variety of tumor cell lines and demonstrated that certain human myeloma and hepatoma cell lines were particularly sensitive. IL6 binding to selected hepatoma and myeloma cell lines were determined by using [125I]IL6. IL6 receptor mRNA levels were measured by polymerase chain reactions. When comparisons were made among different hepatoma cell lines, the sensitivity to IL6-PE40 correlated with the number of IL6 receptors. However, the hepatoma line PLC/PRF/5, which contains 2,300 IL6 receptors, was more sensitive to IL6-PE40 (amount of protein required to inhibit protein synthesis by 50% was 5 ng/ml) than both the myeloma cell lines U266 and H929 (for both cell lines, the 50% inhibitory dose was 8 ng/ml), which contain 15,500 and 16,500 IL6 receptors, respectively. RNA analysis confirmed that the sensitivity of these cells to IL6-PE40 and the amount of IL6 receptor RNA detected did not correlate. These data suggest that factors in addition to the number of IL6-binding sites contribute to the sensitivity of cells to IL6-PE40.     

Selective immunosuppression of activated T cells with the chimeric toxin IL-2-PE40. Inhibition of experimental autoimmune uveoretinitis

A characteristic of activated T lymphocytes is the expression of high affinity IL-2R. We studied a new method of selective immunosuppression directed against activated T cells by using a chimeric recombinant protein (IL-2-PE40) composed of IL-2 fused to a modified Pseudomonas exotoxin lacking its cell recognition domain. As a model of T cell-mediated disease, we used experimental autoimmune uveoretinitis (EAU) produced in Lewis rats by active immunization with the retinal S-Ag. The treatment protocol consisted of i.p. injection of IL-2-PE40 at 0.25 micrograms/g every 12 h. Controls were PBS, PE40, or IL-2-PE40asp553 a mutant form of the molecule with reduced activity. Treatment with IL-2-PE40 resulted in a significant reduction of the incidence and severity of EAU over controls. The analysis of the effect of i.p. injection of IL-2-PE40 on the popliteal draining lymph nodes of immunized animals showed a marked reduction in the lymphocytes content. Transfer experiments demonstrated that IL-2-PE40 prevented the development of EAU effector T cells. Interestingly, although activated B cells were reported to express IL-2R, there was no significant reduction of antibody production against the immunizing Ag under IL-2-PE40 treatment, suggesting sparing of the B cells.     

Cytotoxicity of IL6-PE40 and derivatives on tumor cells expressing a range of interleukin 6 receptor levels

The chimeric toxin IL6-PE40, which is composed of interleukin 6 (IL6) fused to a mutant form of Pseudomonas exotoxin (PE) devoid of its native cell recognition domain, can kill myeloma and hepatoma cells which express high levels of IL6 receptors. To enhance the usefulness of IL6-PE40 on potential target cells, we have attempted to develop more potent IL6-PE derivatives. We have developed nine new IL6-PE derivatives and assessed their cytotoxicity on human myeloma cells. Two of these new forms, IL6-domain II-PE40 and IL6-PE664Glu were more toxic to myeloma cells bearing IL6 receptors than was IL6-PE40. These two chimeric toxins were compared with IL6-PE40 for cytotoxicity toward a variety of tumor cell lines. We found that most tumor cell lines which are sensitive to IL6-PE40 are more sensitive to IL6-domain II-PE40 and IL6-PE664Glu. Cells with as few as 200-600 IL6 receptors/cell could be killed. The specificity of these chimeric toxins was shown through competition with recombinant IL6. Toxicity studies in mice demonstrated that the two new molecules had an LD50 of 10-20 micrograms/mouse. This compares to an IL6-PE40 LD50 of 20 micrograms/mouse. The new IL6-toxins could be detected in the serum up to 8 h after intraperitoneal administration with a peak at 1 h. These data suggest that IL6-domain II-PE40 and IL6-PE664Glu may be more useful than IL6-PE40 in killing IL6 receptor-bearing tumor cells in animals.     

Immunoreactivities for epidermal growth factor (EGF) and for EGF receptors in rats with gastric ulcers

Summary The present study was aimed at assessing whether epidermal growth factor (EGF) and its receptors are present in the gastric mucosa during the healing of gastric ulcers. Immunohistochemical, immunochemical and functional studies were performed in rats after induction of ulcers in the oxyntic mucosa. Controls, which included non-operated and sham-operated animals, displayed only rare cells in the bottom of the oxyntic glands showing EGF-like immunoreactivity. Within one day after ulcer induction, a markedly increased number of chief cells in undamaged mucosa showed intense staining. Concomitantly, there was an increased immunoreactivity for EGF receptors in the mucous neck cells. Maximal immunostaining for both compounds was observed at 3 days after ulcer induction; augmented staining was still demonstrable after 3 weeks. RIA revealed significantly increased EGF concentration in the oxyntic mucosa three days after ulcer induction, and at this stage stimulated gastric acid secretion, measured in a parallel group of chronic fistula rats, indicated significant inhibition. The transient increases in EGF-like and EGF receptor immunoreactivities may stimulate gland cell proliferation. The local release of EGF-like substances may also serve to reduce gastric acidity and thereby promote ulcer healing.     

Inhibition of TNF-alpha produced by Kupffer cells protects against the nonspecific liver toxicity of immunotoxin anti-Tac(Fv)-PE38, LMB-2

LMB-2 (anti-Tac(Fv)-PE38) is a recombinant immunotoxin composed of the Fv fragment of the anti-Tac Ab fused to a 38-kDa form of Pseudomonas: exotoxin A. Recent clinical trials showed that LMB-2 is a promising agent for the treatment of patients with Tac-positive leukemia or lymphoma. One major side effect that needs to be overcome is nonspecific liver toxicity. In the current study, we have analyzed the mechanism of this toxicity using a mouse model. Mice that were injected with a lethal dose of LMB-2 showed severe hepatic necrosis. Immunohistochemistry revealed that LMB-2 accumulated in Kupffer cells in the liver, suggesting that the damage to the hepatocytes was indirect. When we examined the effects of LMB-2 on peritoneal macrophages, cells in the same lineage as Kupffer cells, we found that LMB-2 induced the production of TNF-alpha by these cells. Following LMB-2 administration to mice, the levels of TNF-alpha in the liver increased to very high levels, whereas the rise in serum levels was modest. In addition, the LMB-2-induced liver toxicity was blocked by a specific TNF binding protein (TNFsRp55). Liver toxicity was also blocked by indomethacin, which also blocked the rise of TNF-alpha in the liver. Both TNFsRp55 and indomethacin treatment protected mice against a lethal dose of LMB-2. These data indicate that TNF-alpha produced in the liver by Kupffer cells has an important causal role in the nonspecific liver toxicity of LMB-2. These findings have important clinical implications for the use of immunotoxins in the therapy of patients with cancer.     

Evidence for epidermal growth factor (EGF)-induced intermolecular autophosphorylation of the EGF receptors in living cells.

In response to epidermal growth factor (EGF) stimulation, the intrinsic protein tyrosine kinase of EGF receptor is activated, leading to tyrosine phosphorylation of several cellular substrate proteins, including the EGF receptor molecule itself. To test the mechanism of EGF receptor autophosphorylation in living cells, we established transfected cell lines coexpressing a kinase-negative point mutant of EGF receptor (K721A) with an active EGF receptor mutant lacking 63 amino acids from its carboxy terminus. The addition of EGF to these cells caused tyrosine phosphorylation of the kinase-negative mutant by the active receptor molecule, demonstrating EGF receptor cross-phosphorylation in living cells. After internalization the kinase-negative mutant and CD63 have separate trafficking pathways. This limits their association and the extent of cross-phosphorylation of K721A by CD63. The coexpression of the kinase-negative mutant together with active EGF receptors in the same cells suppressed the mitogenic response toward EGF as compared with that in cells that express active receptors alone. The presence of the kinase-negative mutant functions as a negative dominant mutation suppressing the response of active EGF receptors, probably by interfering with EGF-induced signal transduction. It appears, therefore, that crucial events of signal transduction occur before K721A and active EGF receptors are separated by their different endocytic itineraries.     

Preparation and properties of monoclonal and polyclonal antibodies to mouse epidermal growth factor (EGF) receptors: evidence for cryptic EGF receptors in embryonal carcinoma cells

Monoclonal antibodies to mouse epidermal growth factor (EGF) receptor were prepared by the immunization of rats with receptor glycoprotein purified from mouse liver by affinity chromatography on immobilized EGF. Purified mouse EGF receptor retained EGF-inducible autophosphorylating activity and was antigenic in rats and rabbits. The monoclonal antibodies cross react very poorly with human EGF receptor, while polyclonal rabbit antibodies immunoprecipitate human, rat and mouse EGF receptor equally well. The rabbit antibody blocks EGF binding to mouse fibroblast cells and, at 20-fold higher concentrations, stimulates uptake of tritiated thymidine into DNA. This indicates that antibodies bind at or close to the EGF-binding site and can mimic the effects of the growth factor. None of the monoclonals bind at the EGF site of the receptor. Immunoprecipitation, immunoblotting, 125I-EGF cross linking, 125I-surface labelling, immunohistochemistry and autophosphorylation techniques were used to delineate the basis for the induction of EGF receptors when OC15 embryonal carcinoma (EC) cells differentiate into endodermal derivatives (END). EGF-stimulated autophosphorylation of a 170 X 10(3) Mr protein in solubilized OC15 EC cells is readily detectable, although intact EC cells do not bind or respond to EGF by all other tests. The results suggest that cryptic EGF receptors are present in EC stem cells, a finding with implications in development.     

Loss of cytotoxic effect of epidermal growth factor (EGF) on EGF receptor overexpressing cells is associated with attenuation of EGF receptor tyrosine kinase activity

The biological activity of epidermal growth factor (EGF) is mediated through the intrinsic tyrosine kinase activity of the EGF receptor (EGFR). In numerous cell types, binding of EGF to the EGFR stimulates the tyrosine kinase activity of the receptor eventually leading to cell proliferation. In tumor-derived cell lines, which overexpress the EGFR, however, growth inhibition is often seen in response to EGF. The mechanism for growth inhibition is unclear. To study the relationship between growth inhibition and EGFR kinase activity, we have used a cell line (PC-10) derived from a human squamous cell carcinoma that overexpresses EGFR. When exposed to 25 ng/ml EGF at low cell densities (1,300 cells/cm²), PC-10 cells exhibit cell death. In contrast, if EGF is added to high density cultures, no EGF mediated cell death is seen. When PC-10 cells were maintained at confluency in the presence of 25 ng/ml EGF for a period of 1 month, they were subsequently found competent to proliferate at low density in the presence of EGF. We designate these cells APC-10. The APC-10 cells exhibited a unique response to EGF, and no concentration of EGF tested could produce cell death. By ¹²⁵I-EGF binding analysis and [³⁵S]methionine labeling of EGFR, it was found that the total number of EGFR on the cell surface of APC-10 was not decreased relative to PC-10. No difference between PC-10 and APC-10 was seen in EGF binding affinity to the EGFR. Significantly, EGF stimulated autophosphorylation of the EGFR of APC-10 was 8–10-fold lower than that of PC-10. This reduced kinase activity was also seen in vitro in membrane preparations for EGFR autophosphorylation as well as phosphorylation of an exogenously added substrate. No difference between PC-10 and APC-10 in the overall pattern of EGFR phosphorylation in the presence or absence of EGF was detectable. However, the serine and threonine phosphorylation of the EGFR of APC-10 cells was consistently 2–3-fold lower than that seen in PC-10 cells. These results suggest a novel mechanism for EGFR overexpressing cells to survive EGF exposure, one that involves an attenuation of the tyrosine kinase activity of the EGFR in the absence of a change in receptor levels or receptor affinity. © 1994 Wiley-Liss, Inc.     

To (TGF)beta or not to (TGF)beta: fine-tuning of Smad signaling via post-translational modifications

Smad proteins are key signal transducers for the TGF-beta superfamily and are frequently inactivated in human cancers, yet the molecular basis of how their levels and activities are regulated remains unclear. Recent progress, discussed herein, illustrates the critical roles of Smad post-translational modifications in the cellular outcome to TGF-beta signaling.     

Monoclonal antibody OKT11A inhibits and recombinant interleukin 2 (IL 2) augments expression of IL 2 receptors at a pretranslational level

The expression of receptors for interleukin 2 (IL 2) represents a critical event regulating the growth of normal T lymphocytes. We investigated the effects of the inhibitory monoclonal antibody OKT11A (anti-sheep erythrocyte receptor) and of purified recombinant IL 2 (rIL 2) on the expression of IL 2 receptors by activated T cells at both the protein and the mRNA levels. Adding OKT11A antibody (0.5 microgram/ml) to phytohemagglutinin (PHA)-stimulated cultures of human peripheral blood mononuclear cells (PBMC) markedly suppressed cellular proliferation (assessed by [3H]thymidine incorporation) and IL 2 receptor expression (determined by immunofluorescence assay by using the anti-IL 2-receptor antibody, anti-Tac). Northern blot analysis performed with the use of a cDNA probe specific for the human IL 2 receptor gene demonstrated that OKT11A antibody also decreased the accumulation of IL 2 receptor mRNA induced by PHA in PBMC. Purified rIL 2 (10 U/ml) alone had little effect on the expression of IL 2 receptors in unstimulated PBMC cultures. In combination with PHA or with PHA plus OKT11A, however, rIL 2 augmented both the expression of IL 2 receptor protein on PBMC and the accumulation of IL 2 receptor mRNA in PBMC. Adding anti-Tac antibody to PBMC cultures to block the interaction of IL 2 with its receptor diminished the accumulation of IL 2 receptor mRNA induced by PHA. Taken together, these data demonstrate that OKT11A antibody inhibits and IL 2 augments expression of IL 2 receptors on PHA-stimulated T cells, at least in part, at a pretranslational level.     

Epidermal growth factor (EGF)-stimulated tyrosine phosphorylation and EGF receptor degradation in cells expressing EGF receptors truncated at residue 973.

Epidermal growth factor (EGF)-stimulated tyrosine phosphorylation of proteins was examined in cells expressing wild-type (WT-EGFR) EGF receptors or EGF receptors truncated at residue 973 (973-EGFR). A much broader spectrum of tyrosine phosphorylated proteins was found following EGF treatment of 973-EGFR expressing cells compared with cells expressing wild-type receptors. Several additional ras GTPase activating protein-associated tyrosine phosphorylated proteins were found in EGF-treated 973-EGFR cells relative to WT-EGFR cells. Additional tyrosine-phosphorylated proteins were also found to co-immunoprecipitate with phospholipase C gamma 1 (PLC gamma 1) following EGF treatment of cells expressing 973-EGFR relative to cells expressing WT-EGFR. EGF-stimulated tyrosine phosphorylation of PLC gamma 1 was found in cells expressing WT-EGFR, but not in cells expressing 973-EGFR. WT-EGF receptor from EGF-treated cells bound well to bacterially expressed src homology (SH) regions of PLC gamma 1 and to a lesser extent to bacterially expressed GTPase activating protein SH regions. No binding of 973-EGF receptor to SH regions of either protein could be detected. EGF treatment greatly reduced the half-life of WT-EGFR, but had relatively little effect on the half-life of 973-EGFR. EGF induced internalization of 973-EGFR at a slower rate than WT-EGFR and caused the appearance of discrete receptor degradation products for both cell types. The data indicate that truncation of the EGF receptor at residue 973 alters receptor substrate specificity, decreases the rate of receptor internalization, and has an inhibitory effect on receptor degradation.     

Natural killer (NK) cells as a responder to interleukin 2 (IL 2). II. IL 2-induced interferon gamma production

In the accompanying paper, we showed that natural killer (NK) cells were a major population in the naive spleens of normal mice that responded directly to a T cell growth factor, interleukin 2 (IL 2), and clonally replicated without other stimulating agents. The cloned cells growing in IL 2 showed a potent NK activity against several NK targets without addition of an NK-activating agent, interferon (IFN). In the present study, therefore, we examined whether these cloned NK cells on their own produced IFN. It was found that all NK clones growing in IL 2 produced IFN in the culture fluids. The titers of IFN produced in the IL 2-containing media correlated well with the number of growing cells. With the culture in the absence of IL 2, neither cell growth nor IFN production could be detected. Addition of Con A into the culture in the IL 2-free media showed no IFN production. The antiserum neutralizing IFN alpha and IFN beta failed to significantly neutralize IFN produced by NK clones. Treatment with either a pH of 2.0 or antiserum neutralizing mouse IFN gamma resulted in a marked reduction of IL 2-induced NK IFN, indicating that a major part of IFN produced was IFN gamma. These results indicate that IL 2 stimulates NK clones to proliferate, accompanied by IFN gamma production. The results also show that an NK clone, when stimulated with Sendai virus, produced a type 1 IFN (IFN alpha and/or IFN beta), suggesting that murine NK cells can produce both type 1 (alpha and/or beta) and type 2 (gamma) IFN, depending on inducers.     

TCGF (IL 2)-receptor inducing factor(s). I. Regulation of IL 2 receptor on a natural killer-like cell line (YT cells)

A continuous cell line (YT cells) with inducible receptor for T cell growth factor (TCGF)/interleukin 2 (IL 2) was established from a 15-yr-old boy with acute lymphoblastic lymphoma and thymoma. YT cells were tetraploid, having 4q+ chromosomal markers, and proliferated continuously in vitro without conditioned medium (CM) or IL 2. They were weakly positive for OKT9, OKT11, and Tac antigen (Ag), a determinant closely associated with the receptor for IL 2 (IL 2-R), and were negative for OKT1, OKT3, OKT4, and OKT8 Ag. YT cells also expressed HNK-1 Ag and Fc receptors for IgG, which are expressed on natural killer (NK) cells. They retained a killing activity against human cell lines, including K562 (myeloid), T, and B cell lines. Unlike Tac Ag/IL 2-R(+) cell lines derived from adult T cell leukemia (ATL), YT cells were negative for HTLV, as proved by Southern blotting with cDNA for viral DNA. The expression of Tac Ag was markedly enhanced in 18 hr, when YT cells were incubated with CM from PHA-stimulated peripheral blood leukocytes (PBL) or spleen cells, as determined by immunofluorescence by using flow cytometry and binding assay with 125I-anti-Tac antibody (Ab). The binding study with 125I-labeled recombinant IL 2 showed 3.2 X 10(4) IL 2 receptor sites on YT cells precultured with CM. PHA-P and Con A neither agglutinate nor enhance the expression of IL 2-R/Tac antigen on these non-T cell line cells. Furthermore, neither recombinant IL 2 nor gamma-interferon could induce IL 2-R on YT cells, suggesting the presence of a unique IL 2-R inducing factor in PBL or spleen CM. Unlike Tac Ag on HTLV(+), ATL-derived cell lines (Hut-102, MT-1, ATL-2), the expression of Tac Ag on YT cells was down-regulated by anti-Tac Ab. The induction of Tac Ag/IL 2-R on YT cells seemed specific, because the enhancement of Tac Ag expression was not associated with that of Ia Ag and T9/transferrin receptor.