Hideki Tozawa

Distinct reactivities of four monoclonal antibodies with human interleukin 2 receptor

Two new murine monoclonal IgG1 antibodies, H‐31 and H‐A26, were characterized in comparison with two previously obtained monoclonal antibodies against human interleukin 2 (IL‐2) receptor (IL‐2 R), anti‐Tac and HIEI. In immunofluorescence assays with various human hematopoietic cells, H‐31 and H‐A26 antibodies both reacted with only IL‐2 R‐positive cells, and they precipitated IL‐2 R molecules, glycoproteins with molecular weights of 60K and 53K daltons (gp60/gp53), from human T‐cell leukemia virus type I (HTLV‐I)‐carrying MT‐2 cells, as demonstrated by sequential immunoprecipitation after absorption of IL‐2 R with anti‐Tac. Antibody‐binding competition assays showed that H‐31 and anti‐Tac, and H‐A26 and HIEI, respectively, competed reciprocally in binding to the cells, and that anti‐Tac also inhibited the binding of HIEI but not vice versa. H‐31, like anti‐Tac, strongly inhibited the IL‐2‐dependent proliferation of normal activated T‐cells, absorption of IL‐2 and direct binding of IL‐2 to the cells, while H‐A26, like HIEI, inhibited those processes only weakly. The spectra of reactivities of these antibodies with various simian cell lines derived by HTLV‐I infection were different, as revealed by immunofluorescence studies. Human IL‐2 R was shown to express a unique antigenic determinant, detected with HIEI, that was not detectable in IL‐2 R molecules of Old and New World monkeys, and also to express determinants common to simian IL‐2 R molecules. These observations indicate that H‐31 and H‐A26 recognize human IL‐2 R molecules and that the antigenic sites on the IL‐2 R molecule defined by H‐31, H‐A26, anti‐Tac, and HIEI are different.

Response of human glioblastoma cells to recombinant interleukin-2.

We investigated the ability of glioma cells to respond to T cell-derived lymphokines. The growth of astrocytoma and mixed glioblastoma cell lines, as assessed by DNA synthesis, was inhibited in the presence of supernatants derived from mitogen-stimulated human T cells, an HTLV-II-transformed human T cell line, Mo, and human interleukin-2 (IL-2). The mixed glioblastoma cell line, 138-MG-C, was subjected to limiting dilution analysis, and two cell lines (5D7, 5C5) were derived which were homogeneous with respect to staining for galactocerebroside (GalC) (100%). These two GalC+ glioblastoma cell lines proliferated in the presence of high concentrations of recombinant human interleukin-2 (RIL-2). Additionally, these cell lines bear receptors for the IL-2 molecule as determined by immunofluorescent staining with various anti-IL-2 receptor antibodies.

New Monoclonal Antibodies That Define Multiple Epitopes and a Human-Specific Marker on the Interleukin 2 Receptor Molecules of Primates

Twelve hybridoma cell lines producing monoclonal antibodies to the human interleukin 2 (IL‐2) receptor (IL‐2R) molecule were prepared. These antibodies were characterized by competitive antibody‐binding assay and sequential immunoprecipitation assay with four known monoclonal antibodies to the human IL‐2R molecule. The twelve new monoclonal antibodies were divided among the four known antibody types, the HIEI‐, H‐A26‐, H‐31‐, and anti‐Tac‐type, and an additional new type, the H‐48‐type. The H‐48 antibody did not compete with any other antibodies in the competitive binding assay. The binding of 125I‐IL‐2 to MT‐2 cells and the IL‐2‐dependent growth of normal activated T‐cells were both strongly inhibited by all the H‐31‐ and anti‐Tac‐type antibodies, and partially or slightly inhibited by HIEI‐ and H‐A26‐type antibodies, but were not inhibited by the H‐48 antibody. Thus, the same type of monoclonal antibodies had a similar effect on the function of IL‐2R. These results suggest that epitopes for the same type of antibodies could be single identical epitopes or epitopes closely associated with each other. On the other hand, these antibodies also reacted variously with a panel of various human and simian lymphoid cell lines immortalized with human T‐cell leukemia virus type‐I (HTLV‐I): the H‐45 antibody reacted only with the human cell lines, the H‐Cl and H‐44 and H‐47 antibodies reacted with human and ape cell lines, and the other antibodies reacted with cell lines of humans, apes and Old and New World monkeys. These differences in the reactivity of the antibodies with the primate cell lines suggest that the antigenic structure of the IL‐2R molecule changed during evolutionary divergence of the primates.

A New Monoclonal Antibody Recognizing an Antigen of Human Lymphocytes Similar or Identical to Tac Antigen

A new mouse monoclonal antibody (HIEI, IgG1 type) that reacts with a cell surface glycoprotein of human lymphocytes was isolated. Membrane immunofluorescence assay showed that HIEI, like the anti‐Tac monoclonal antibody, reacted preferentially with activated normal human T‐cells and adult T‐cell leukemia (ATL) virus (ATLV)‐carrying human T‐ and B‐cell lines. However, an interesting difference between HIEI and anti‐Tac antibody was that HIEI did not react with ATLV‐transformed simian cell lines or those cultured with interleukin‐2 (IL‐2), whereas the anti‐Tac antibody did. The immunoprecipitation assay showed that both HIEI and anti‐Tac antibody precipitated a glycoprotein with a molecular weight of 60,000 daltons (gp60) from activated normal T‐cells and ATLV‐positive T‐ and B‐cells, and also gp53 from MT‐2 and MT‐2‐related T‐cell lines transformed with ATLV in vitro by the MT‐2 cocultivation method. HIEI inhibited the Independent proliferation of normal T‐cells, but its inhibitory effect was much weaker than that of the anti‐Tac antibody. The anti‐Tac antibody interfered with the binding of HIEI to target cells, but HIEI did not block binding of the anti‐Tac antibody to the cells. These observations indicate that HIEI antibody recognizes a new antigenic determinant of the human Tac antigen.

Flourescence sandwich enzyme-linked immunosorbent assay for detecting human interleukin-2 receptors☆

A very sensitive fluorescence sandwich enzyme-linked immunosorbent assay (FS-ELISA) for the detection of soluble and cell-associated human interleukin-2 receptors (IL-2R) has been developed. For use in this assay system, two anti-human IL-2R monoclonal antibodies, H 48 and biotinylated HA 26, were selected from six monoclonal antibodies directed against different epitopes on the IL-2R molecule. The FS-ELISA specifically detected human IL-2R in both the culture supernatants and cell lysates of human IL-2R-bearing cells and the assay displayed a 10(3)-fold increase in sensitivity over the usual colorimetric IL-2R ELISA. The IL-2R molecules in supernatants and lysates of the PHA-stimulated mononuclear cells were of 50,000 and 60,000 molecular weight, respectively, as estimated by size exclusion high performance liquid chromatography.

Expression of Human T‐cell Lymphotropic Virus Type‐1 Gene Products in the Short‐term Cultured Skin Tissues of an Adult T‐cell Leukemia/Lymphoma Patient with Cutaneous Manifestations

Adult T‐cell leukemia/lymphoma (ATLL) is recognized as a disease etiologically associated with human T lymphotropic virus type‐1 (HTLV‐1) infection, but, neither viral replication nor specific virus antigen expression have been detected on ATLL cells distributed in organs, including skin. To examine the latent expression of HTLV‐1 in the cutaneous lesions of ATLL patients, we cultured the lesional skin tissues in vitro and applied immunofluorescence staining with mouse monoclonal antibodies Lt‐4, GIN‐14, and F10, which react with p40tax, p19 and gp21, respectively.

Binding properties of human immunodeficiency virus type‐2 (HIV‐2) rna corresponding to the packaging signal to its nucleocapsid protein

The nucleocapsid (NC) protein of human immunodeficiency virus type‐1 (HIV‐1) contains two zinc finger motifs (ZFMs), and binds specifically to the packaging signal which is located in the 5′ leader sequence of the viral genomic RNA between the first splice donor and the gag initiator codon (AUG). In this study, we analyzed the specificity of the binding of the corresponding region of HIV‐2 (Region 3) to its NC protein (NCp8), by performing a competitive ultraviolet (UV) cross‐linking assay using in vitro‐synthesized 32P‐labeled and unlabeled RNAs corresponding to a sequence between the primer binding site and the gag AUG (Region 1). Binding of 32P‐labeled Region 1 RNA to NCp8 was inhibited specifically by adding unlabeled Region 1 and 3 RNAs and no specific binding was detected using deletion mutant peptides of NCp8. These findings suggest that the region(s) which bind(s) specifically to HIV‐2 NCp8 lie(s) between the first splice donor and the gag AUG in the 5′ leader sequence and that NCp8 is the minimum binding region responsible for the specific binding of the region downstream of the first splice donor site of HIV‐2 RNA.

Neutralization of Sendai Virus by the IgG Subclass Antibodies of the Guinea Pig

A comparative study was made of the neutralizing activities of IgG subclasses IgG1 and IgG2, fractionated from guinea pig antisera against Sendai virus. The yields of IgG2 from the antisera were about 16 times as much as those of IgG1. The neutralizing activity of IgG2 per unit weight was four times as high as that of IgG1. This neutralizing activity of both IgG subclasses was enhanced about 10 times by addition of antibodies to the L‐chain of guinea pig immunoglobulin. It is suggested that, in the complement‐dependent neutralization of the virus, IgG1 and IgG2 activate the complement through the alternative and the classical pathway, respectively.

Viral RNA-binding activity of HIV-2 nucleocapsid protein is inhibited by a synthetic peptide containing the first zinc finger motif of HIV-2.

Objective:To analyze the antigenic structure and viral RNA-binding activity of HIV-2GH-1 nucleocapsid protein (NC). Methods:Five synthetic peptides corresponding to hydrophilic regions of HIV-2GH-1 NC were prepared. The reactivity of rabbit antisera directed against these synthetic peptides was examined by Western blot (WB) assay, using lysates of purified HIV-2GH-1 virus and HIV-2GH-1-infected cells as antigen. The binding activity of NC to viral RNA synthesized in vitro, was assessed by Northwestern blot (NWB) assay using 32P-labeled RNA as a probe. Results:One of five antisera against the peptides reacted with a protein of 8 kD (p8) of HIV-2GH-1 in WB. p8 was analyzed for the amino-terminal amino-acid sequence and identified as a portion of NC including two zinc finger motifs (ZFM), comprising the DNA-binding region in the molecule. The binding of the RNA probe to p8 was observed by NWB and did not always depend on zinc. In competition experiments, the reactivity of p8 with the RNA probe ceased to be evident following preincubation of the probe with a peptide containing the first ZFM in the molecule. Conclusion:p8 of HIV-2GH-1 NC binds to viral RNA in vitro. The first ZFM in the p8 molecule appears to be essential to binding activity. Functional analysis of synthetic peptides corresponding to ZFM of HIV p8 may provide important information on the development of antiviral agents.

Adsorption of LLCMK2 Cell‐Grown Sendai Virus onto Human Red Blood Cells and Its Release from the Virus Adsorbed Cells

An early stage of virus adsorption was studied in a system of Sendai virus metabolically labeled with [3H] leucine in LLCMK2 cells and of human red blood cells (RBCs). The efficiency of viral release from the virus‐bound RBCs by incubation at 37 C depended on the number of virus particles which had been used for adsorption onto the RBC at 4 C. When 7.8 × 102 virus particles were previously adsorbed onto the RBC at 4 C, most of the viruses were dissociated from the RBC at 37 C. In the case of adsorption of 3 to 12 virus particles per RBC, however, most of the viruses were not dissociated from the RBC by incubation at 37 C. Such RBC‐bound viruses were released by incubation with various bacterial neuraminidases (Clostridium perfringens, etc.) or with a large number of LLCMK2 cell‐grown Sendai virus (LLCMK2‐Sendai) particles, but not released by treatment with hemagglutinin‐neuraminidase protein (Sendai‐gp) isolated from egg‐grown Sendai virus.