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R Devos

Publications and source records attributed to R Devos.

At least 55 records · Page 3Linked to original sources
PubMed1987

Production of stable cytolytic T-cell clones directed against autologous human melanoma.

We have attempted to optimize the production of stable human cytolytic T lymphocyte clones directed against autologous melanoma cell lines. MLTC were restimulated every week with irradiated melanoma cells in medium containing human serum and IL-2. After 21 to 35 days, in 5 out of 6 patients, these cultures expressed a preferential cytolytic activity against the autologous melanoma cells, as compared to autologous EBV-B cells or NK target K562. Limiting dilution of MLTC responder cells was performed at times varying from days 7 to 28, in medium containing IL-2 and allogeneic EBV-B cells as feeders. Approximately 1% of these responder cells gave rise to CTL clones that lysed the autologous melanoma cells, but did not lyse K562 or autologous B cells. It was possible to maintain in culture for several months a large number of CTL clones that retained this specificity with high activity, and multiplied more than 5-fold every week. Some of these CTL clones were dependent on the presence of the autologous melanoma cells for their growth. With one melanoma, the use of autologous CTL clones made it possible to identify 3 different antigens on the tumor cells.

Antigens, Neoplasm↗
PubMed1986

Structural studies of the carbohydrate chains of human gamma-interferon.

Human gamma-interferon (IFN-gamma) was prepared biotechnologically using Chinese hamster ovary cells. These cells were shown to be able to produce glycosylated IFN-gamma. Sugar analysis revealed the presence of Man, Gal, GlcNAc, NeuAc and Fuc residues in a molar ratio of 3.8:2.0:3.5:0.6:0.4 suggesting the occurrence of N-glycosidically linked N-acetyllactosamine type of carbohydrate chains. For structure determination of these chains, the glycoprotein was subjected to the hydrazinolysis procedure, yielding oligosaccharide-alditols. The latter compounds were analysed by 500-MHz 1H-NMR spectroscopy. The carbohydrate material was found to consist of biantennary structures, exhibiting microheterogeneity as to the terminal sialic acids and the core Fuc residue: (Formula: see text). As similar carbohydrates are present on several human secreted proteins, this glycosyl group is not expected to be immunogenic in man. It remains to be established to what extent the carbohydrate chains of this biotechnologically produced IFN-gamma are identical to those of naturally occurring human IFN-gamma.

Animals↗
PubMed1986

Cloning and structure of a mouse interleukin-2 chromosomal gene.

Using non-stringent hybridization with a human interleukin-2 cDNA probe, we have isolated recombinant phages from a mouse genomic DNA library cloned in the EMBL3 phage. The sequence and organization of the mouse interleukin-2 (IL-2) gene was determined. By comparison with the human IL-2 sequence, three introns can be identified with lengths of 99, +/- 2 400, and +/- 1 900 base pairs, respectively. The mouse IL-2 gene codes for a polypeptide of 169 amino acids and contains a putative signal peptide of 20 amino acids. The homology to the human interleukin-2 is 72% at the nucleotide level in the coding part and 65% at the amino acid level. An extraordinary sequence, consisting of 12 consecutive CAG codons coding for glutamine, is found in the first exon.

Amino Acid Sequence↗
PubMed1985

Recombinant interleukin 2 induces immunoglobulin secretion in Staphylococcus aureus Cowan strain I activated human B-cells.

Human B-cells, exhaustively depleted for T-cells, were activated with Staphylococcus aureus Cowan strain I (SAC) and responded to recombinant human interleukin 2 (rIL2) by secretion of immunoglobulin (Ig), as measured by a protein A hemolytic plaque assay. The rIL2, however, had to be present early, since addition later than 24 h after SAC-activation of the B-cells reduced the response to background levels. No clear dose response was observed and Ig-secreting cells (ISC) could be induced even with rIL2 at 0.5 U/ml. The monoclonal antibody anti-TAC prevented the rIL2-promoted induction of ISC. Ig production could be induced in SAC-activated cultures with supernatants of Xenopus laevis oocytes injected with sucrose-gradient-fractionated poly(A+) RNA derived from a stimulated human spleen cell culture. This activity coincided with the IL2 mRNA activity and was well separated from the interferon-gamma mRNA activity. Our results suggest that IL2 is not only a B-cell growth factor but also promotes the differentiation of activated human B-cells towards Ig secretion.

Antibody-Producing Cells↗
PubMed1985

Characterization of antibodies against recombinant HuIFN-gamma produced by hybridoma cells.

Balbc/c mice were immunized with purified recombinant E. coli-derived human gamma-interferon (HuIFN-gamma). Their spleen cells were fused with a mouse myeloma cell line (Sp2/0). Hybridomas producing antibodies reacting with HuIFN-gamma were screened by a soluble-phase radioimmunoassay using pure 125I-labeled cloned IFN-gamma as antigen, and tested for their ability to neutralize the antiviral activity of IFN. Three hybridomas S1-1, S1-2, and S1-3, were cloned and subcloned and remained stable. Although the antibodies produced by clones S1-1 and S1-2 were both able to neutralize specifically the antiviral activity of natural and recombinant HuIFN-gamma, they appeared to recognize different epitopes on the HuIFN-gamma molecule. The antibodies produced by the S1-3 clone failed to neutralize the antiviral activity of either interferon. The antibodies from all three clones were characterized as IgG1 subclass. Their affinity constants were determined from competitive inhibition curves and ranged from 1 to 4.3 X 10(8) M-1.

Animals↗
PubMed1985

Binding of unglycosylated and glycosylated human recombinant interferon-gamma to cellular receptors.

Recombinant human interferons (IFNs), either unglycosylated produced in E. coli (rIFN-gamma) or glycosylated produced in CHO cells (g-rIFN-gamma), were labeled with 125I to similar specific activities to study their interaction with cell-surface receptors. When analyzed by gel electrophoresis, rIFN-gamma run as a single polypeptide of Mr 15,000-17,000, whereas g-rIFN-gamma separated into three components of Mr 20,000, 22,000, and 43,000, which corresponded to the known size of the two monomeric and one dimeric forms of glycosylated natural IFN-gamma. The binding of the two species of 125I-IFN-gamma was competed equally by rIFN-gamma in competition displacement experiments with Daudi cells, indicating that these IFNs bind with similar high affinity to the same receptors. KD values of 1.25 X 10(-10) and 2.5 X 10(-10) M were determined for g-rIFN-gamma and rIFN-gamma, respectively. This relatively small difference in KD does not apparently result in a detectable difference in biological activity, as measured by the increase in 2',5'-oligo(A) synthetase activity in IFN-treated HeLa and A549 cells. These results indicate that glycosylation of IFN-gamma does not play a significant role in its interaction with cellular receptors and in the induction of a biological response.

2',5'-Oligoadenylate Synthetase↗
PubMed1985

Interferon-gamma modulates HLA class II antigen expression on cultured human thymic epithelial cells.

Cultures of human thymic epithelial cells (TEC) were tested for the expression of HLA class I (A, B, C) and class II (DR and DC) antigens by indirect immunofluorescence. The epithelial nature of the cells was proven by using an antikeratin antiserum. A high level of expression (close to 100% positive cells) of HLA class I antigens was observed on TEC at the beginning of the culture and remained unchanged for up to 12 days. In contrast, HLA class II antigen expression (85% DR+ and 75% DC+ cells on day 2) decreased gradually and reached very low levels (less than 5% DR+ or DC+) by day 7 of culture. This loss of class II antigen expression was not seen when cultures were performed in the presence of supernatants from activated T cells containing interferon-gamma (IFN-gamma). Furthermore, the presence of recombinant IFN-gamma (rIFN-gamma) in the medium from the onset of culture maintained HLA-DR and DC antigen expression on a high number of cells (comparable to that observed on day 2 of culture). A large percentage of rIFN-gamma-treated cells also showed intracytoplasmic HLA-DR antigen expression. Addition of rIFN-gamma at various times after the onset of the culture led to a reinduction of DR and DC antigen expression. This effect of rIFN-gamma was observed in 48 hr with concentrations as low as 10 IU/ml and was apparently specific for this IFN species, in that rIFN-alpha was unable to modify HLA class II antigen expression at concentrations up to 1000 IU/ml. The increased expression of HLA class II antigen was truly due to induction in individual TEC, rather than selection of class II-positive cells, because induction under the influence of IFN-gamma was reversible and occurred in the absence of proliferation in mitomycin-treated or gamma-irradiated cultures. Our results indicate that synthesis and membrane expression of class II HLA antigens are enhanced by IFN-gamma in TEC cultures. This finding raises the possibility that IFN-gamma participates in the mechanisms that assure the permanent expression of DR and DC antigens observed in TEC in vivo, with potentially important functional consequences in terms of education for self recognition.

Cell Survival↗
PubMed1984

Activated B cells express receptors for, and proliferate in response to, pure interleukin 2.

In this study we investigated whether interleukin 2 (IL-2) acts on B cell proliferation and whether activated B cells express IL-2 receptors. First, the functional activity of immunoaffinity-purified or recombinant human IL-2 was studied in a B blast assay using positively selected murine surface Ig-positive cells that had been activated by lipopolysaccharide (LPS) plus anti-Ig antibodies (anti-Ig). In this assay, T cells were not detected by fluorescence-activated cell sorter analysis. It was found that both IL-2 preparations led to optimal B cell proliferation compared with supernatants obtained from murine or human spleen cells or murine cloned T helper cells. Second, we observed that the IL-2 requirement in this assay was about the same as in a proliferation assay using lectin-activated polyclonal murine Lyt-2-positive T cells. Third, analysis of the binding of radiolabeled immunoaffinity-purified IL-2 to B cells indicated that LPS plus anti-Ig-activated B cells expressed a mean of 3,500 IL-2 receptors per cell with an apparent dissociation constant of 150 pM. However, neither nonactivated B cells nor B cells activated by LPS alone exhibited significant specific IL-2 binding. The functional and the receptor data are consistent with the conclusion that IL-2 is a growth factor not only for T cells but also for B cells.

Animals↗
PubMed1984

Pure interferon gamma enhances class II HLA antigens on human monocyte cell lines.

Human recombinant interferon gamma (IFN-gamma) with a chemical purity of over 90% was shown to enhance membrane expression of HLA-DR antigens on cells from 3 human myelomonocytic lines (HL-60, U-937 and THP-1). Immunofluorescence techniques using a series of anti-HLA-DR monoclonal antibodies showed that the low, although variable, levels of DR-positive cells were clearly enhanced as soon as 24 h after incubation with IFN-gamma. Only IFN-gamma was able to exert this effect, since incubation with high concentrations of IFN-alpha or -beta did not induce any significant modification of the percentage of HLA-DR-positive cells. In contrast, doses of IFN-gamma as low as 2 units were effective, indicating a highly preferential, apparently selective effect of IFN-gamma for enhancement of HLA-DR expression. Private class II antigen expression was also enhanced by IFN-gamma on the U-937 cell line. Through its enhancing effect on class II public and private HLA antigens on the membrane of human monocytes, the IFN-gamma lymphokine may have a critical role in the modulation of antigen presentation by monocytes and on the regulation of HLA-DR-restricted cell cooperation.

Antibodies, Monoclonal↗
PubMed1984

Induction of cytolytic cells by pure recombinant human interleukin 2.

Purified recombinant human interleukin 2, produced in Escherichia coli, was sufficient to generate cytolytic cells in concanavalin A-stimulated, T helper cell-depleted (Lyt-1.1-) or accessory cell-depleted (Ia-) murine spleen cell cultures. Moreover, recombinant interleukin 2 (rIL2) was able (even at 0.5 ng/ml) to induce cytolytic cells in undepleted murine spleen cell cultures and undepleted human peripheral blood lymphocyte cultures in the absence of a mitogen. Purified recombinant human interferon-gamma, produced in Chinese hamster ovary cells, did not induce either cytolytic activity or IL2 responsiveness in human peripheral blood lymphocyte cultures. Possibly, the rIL2-induced cytolytic cells are formed as a result of nonspecific secondary cytotoxic T lymphocyte activation.

Animals↗
PubMed1984

High-level expression of human interferon gamma in Escherichia coli under control of the pL promoter of bacteriophage lambda.

Several recombinant plasmids have been constructed which direct high-level synthesis of mature human interferon gamma (IFN-gamma) in Escherichia coli using the inducible leftward promoter pL of phage lambda followed by a translational initiator region derived either from the phage MS2 replicase gene or the E. coli tryptophan attenuator region. Under these conditions, IFN levels of up to 25% of the total cellular protein can be achieved. The highest levels were obtained when a terminator of transcription was cloned downstream from the IFN-gamma sequence. IFN-gamma was almost entirely found in the initial pellet fraction and not in soluble extracts. Co-induction of the lysis genes derived from phage MS2 or from phage lambda, inserted downstream from the IFN-gamma sequence, did not enhance the biological activity present in the supernatant fraction.

Bacteriophage lambda↗
PubMed1984

Purification of recombinant glycosylated human gamma interferon expressed in transformed Chinese hamster ovary cells.

Human IFN-gamma was produced in cultures of a Chinese hamster ovary (CHO) cell line transformed with a combination of plasmids encoding HuIFN-gamma cDNA and mouse DHFR cDNA and subsequently selected for growth in the presence of methotrexate. Confluent monolayers of these cells constitutively secrete HuIFN-gamma into the medium reaching a concentration of 2-5 micrograms/ml; the supernatant of the monolayer could be harvested daily for a period of more than 10 days. IFN-gamma was purified by passing the filtered CHO cell culture medium directly through a phosphocellulose column followed by elution and adsorption on a Con A-Sepharose column. Further concentration on an AMICON PM 10 filter and removal of high mw contaminating proteins with DEAE-Sephacel resulted in a IFN-gamma preparation of more than 99% purity (specific activity of about 10(8) International units per mg of protein). Each liter of CHO conditioned culture medium yielded 1-2 mg pure HuIFN-gamma. Its molecular weight, as determined by gel filtration, is about 50 kD and corresponds to a dimer structure. SDS-polyacrylamide gel electrophoresis indicated the presence of a 21 kD and a 25 kD polypeptide as compared with 17 kD for unglycosylated, bacterially made HuIFN-gamma and consistent with the two glycosylated forms of HuIFN-gamma produced in mitogen-stimulated human lymphocyte cultures.

Animals↗
PubMed1983

Molecular cloning of human interleukin 2 cDNA and its expression in E. coli.

A recombinant plasmid containing human interleukin 2 (IL2) cDNA was identified in a cDNA library constructed from mRNA derived from PHA-TPA induced splenocytes. Using this cDNA as a hybridization probe, a DNA fragment containing the IL2 gene was isolated from a collection of hybrid phages derived from human genomic DNA. A unique reading frame was identified from the nucleotide sequence derived from these plasmids coding for a polypeptide of 153 amino acids and containing a putative signal sequence of 20 amino acids. A mature polypeptide starting with either Met-Ala-Pro or Met-Pro was expressed in E. coli under control of the E. coli trp promoter or using a combination of the phage lambda PL promoter and a ribosome binding site derived from phage Mu. The bacterial IL2 polypeptide had a molecular weight of 15,000 daltons and accounted for more than 10% of the total E. coli proteins in fully induced cells; it was biologically active in the T-cell specific DNA synthesis assay, even after recovery from a SDS-containing polyacrylamide gel.

Amino Acid Sequence↗
PubMed1983

Cloning and structure of the human interleukin 2 chromosomal gene.

Southern hybridization using 32P-labelled human interleukin 2 (IL2) cDNA probes revealed the existence of a single human IL2 gene. Five clones containing the human IL2 chromosomal gene were isolated from two different human DNA libraries cloned in either lambda Charon 4A or L47 phages. Analysis of the clones showed that they contained different, overlapping portions of human DNA which were derived from the same chromosomal segment. Restriction fragments which hybridized with labelled IL2 cDNA probes were subcloned into plasmid pUR250 and the sequence and organization of the IL2 gene was determined. It contains three introns, 90 bp, +/- 2400 bp and +/- 1900 bp in length, respectively. The organization of the genomic clone resembles that of another lymphokine, interferon-gamma, but no clear homology was found by comparing either the coding sequence or the 5'- and 3'-flanking sequences of the two genes.

Amino Acid Sequence↗
PubMed1983

Expression and characterization of the product of a human immune interferon cDNA gene in Chinese hamster ovary cells.

Cotransformation with two plasmids, one [pSV2-IFN-gamma] encoding human immune interferon (Hu IFN-gamma) and the other [pAdD26SV(A)-3] encoding mouse dihydrofolate reductase, has been used to establish Chinese hamster ovary (CHO) cell lines that secrete high levels of Hu IFN-gamma. Hu IFN-gamma production by the transformed CHO cell lines E-10B and E-10C reached approximately 50,000 units/ml of culture medium, which compares favorably with that of stimulated lymphocytes. Furthermore, as the Hu IFN-gamma cDNA gene used in these studies is under the transcriptional control of the simian virus 40 early promoter, Hu IFN-gamma production is constitutive and thus does not require induction. CHO-produced Hu IFN-gamma migrates as two bands corresponding to molecular weights of 25,000 and 21,000 on NaDodSO4/polyacrylamide gels. These two species are shown to be the products of a single gene. As the molecular weight of native Hu IFN-gamma is around 55,000, it is likely to be a dimer. We have shown that the subunits of such a dimer cannot be linked by a disulfide bridge(s). Hu IFN-gamma from CHO cells is likely to be glycosylated and this should now permit comparison of the biological activities of glycosylated and nonglycosylated (bacterially produced) Hu IFN-gamma in animal studies.

Animals↗
PubMed1983

Depletion of lymphocytes with membrane markers of helper phenotype: a feature of acute and chronic drug-induced immunosuppression.

T cell subsets tested with markers for Fc receptors for Ig (TM, TG and EAhu rosettes) or monoclonal antibodies (T4 and T8 lymphocytes) were investigated both in normal volunteers and in kidney transplant recipients with a well functioning graft and receiving low immunosuppressive therapy, before and 4 hr after administration of 100 mg of hydrocortisone. Hydrocortisone induced a redistribution which was characterized by a decrease in the percentages of TM (38 +/- 2.4 before; 22 +/- 2.9 after; P less than 0.0005) and T4 (48 +/- 2.6 before; 35.8 +/- 2.7 after; P less than 0.0025) lymphocytes. Transplanted patients under chronic immunosuppression already disclosed a reduction of the percentages of TM (19.4 +/- 2.6; P less than 0.005) and T4 (41.1 +/- 3.6; P less than 0.05) lymphocytes before the administration of hydrocortisone when compared to the values obtained in normals. Moreover, significant decrease of percentages of TM lymphocytes (19.4 +/- 2.6 before; 12.8 +/- 2.6 after; P less than 0.01) were obtained after hydrocortisone injection. In contrast, T8, TG and EAhu rosettes percentages were characterized by a relative resistance to immunosuppressive agents--the only exception being TG lymphocytes in transplant recipients. It is concluded that TM and T4 depletion is a common feature of acute and chronic drug-induced immunosuppression, suggesting that helper-inducer cells are important targets for immunosuppressive therapy.

Adult↗