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A new mitogen for tumor infiltrating lymphocytes (TILS) derived from a tumor cell line
146 / SECOND INTERNATIONAL
WORKSHOP
ON CYTOKINES
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A NOVEL HUMAN MONOCYTE-DERIVED CHEMOTACTIC PEPTIDE WITH APPARENT MONOCYTE SPECIFICITY: PURIFICATION AND PARTIAL BIOLOGIC CHARACTERIZATION. v, Mrowietz, J.-M. SchrBder, E. ChristoDhers, Dept. of Dermatology, University of Kiel, Kiel, FRG. Accumulation of monocytes (MO) and MO-derived macrophages during some inflammatory processes indicate the existence of MO-specific chemotaxins. In this study MO-chemotactic activity was detected in the culture supernatants of lipopolysaccharide stimulated purified human MO. purification of MOchemotactic factors by different reversed phase HPLC-procedures revealed three peaks of MOwhich were distinct from chemotactic activity, other cytokines like IL-1 or TNF. One of these MOchemotaxins could be purified to apparent homogeneity giving upon SDS-PAGE a single band corresponding to a ML. of 10 kD. Dose response studies revealed a chemotaxis maximum at about 100 rig/ml. Significant MO chemotactic activity could still be seen at concentrations below 1 rig/ml. At concentrations higher than 30 rig/ml weak chemotactic activity was detectable using human neutrophils. From our results we conclude that human MO secrete at least three MO-specific chemotaxins, one being a 10 kD polypeptide, which appears to be different from known MO-chemotaxins.
TETRANECTIN: A NOVEL SECRETORY PROTEIN FROM HUMAN MONOCYTES. and I. Clemmensen. Statens Seruminstitut, Dept Clin --H. Nielsen Microbiolmigshospitaret, Copenhagen, OK-2200 Denmark. Tetranectin (TN) is a recently characterized protein in human plasma composed of four 181 amino acid polypeptide chains. TN has been demonstrated in many types of human cells including neutrophils (Christensen & Clemm?lisen. Histochemistry, in press). We now extend these findings and report that human monocytes are diffusely stained for TN by immunocytochemical methods. By immunoblotting the serum-free culture supernatant of human monocyte-derived macrophages #as confirmed to contain a single 20 kD band identical to plasma TN. Stimulation of macrophages with PMLP, C5a or PMA induced6release of TN in small but reproducible quantities (20 ng/h/lO cells). The biological role of TN is at present unknown, but preliminary experiments suggest that preincubation with highly purified TN may modulate monocyte function; i.e. increase spontaneous migration (150% of controls; n=3) and chemotactic responsiveness to C5a and fMLP (132% of controls; n=6), whereas the oxidative burst response to fMLP stimulation was unaffected (98% of controls; n=3). Further studies are in progress with tetranectin, which may show to be an important new cytokine.
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Further Characterization of Human Natural Killer Cvtotoxic Factors fNKCF). Lawrence T. Nakamura and Benjamin Bonavida. UCLA School of Medicine, Los Angeles, CA 90024. Several techniaues have been used to ourifv natural killer cytotoxic faciors (NKCF) from culture' supirnates of human peripheral blood lymphocytes (PBL). We have achieved enrichment of cytotoxic material by fractionation of concentrated culture supernates on an FPLC chromatofocusing column (Mono P). The observed cytotoxicity does not appear to be tumor necrosis factor (TNF) and is specific for NK sensitive target cells, but not for NK resistant target cells. Recently, we generated a rabbit antiserum to the NKCF enriched Mono P fractions to facilitate concentration NKCF The salt-fractionated antiserum from culture supernates. inhibited the cytotoxic activity of Mono P fractions but did not inhibitrecombinant human TNF or lymphotoxin (LT). We have observed a significant inhibition of NK-cell mediated cytotoxicity (CMC), when effector cells were preincubated with specific antiserum, although conjugate frequency was not affected. Further, when effector cells were ppreincubated with a combination of specific antiserum and anti-TNF antiserum, an almost complete inhibition of NK-CMC was observed. Efforts are currently in progress to complete characterization of the specific antiserum and to identify a single protein in the Mono P fractions which may be responsible for the observed lysis of NK sensitive target cells. (Supported by NIH CA 35791 from the NIH.)
INTERLEUKIN 6 (IL6) AND IL6-LIKE ACTIVITIES PRODUCED BY RAT W. Northemann, T.A. MACROPHAGES AND HEPATOMA CELL LINES. and G.H. Fey. Dept. Immunology, Scripps Braciak, J. Gauldie*, Clinic, La Jolla, USA; *McMaster Univ., Hamilton, Canada. Interleukin 6 is a multifunctional hormone with both differentiation and growth promoting activities. It is produced by activated macrophages, monocytes, fibroblasts, and endothelial cells in response to inflammation, and by a number of tumors and tumor-derived cell lines, including rat hepatoma cells. During the inflarmnatory response circulating IL6 stimulates the production of the acute phase proteins in the liver. The rat hepatoma lines HTC, H5, and FtoZB produce IL6 activities. In addition, these lines secreted high amounts of a novel, IL6-like activity (IL6-LA). IL6-LA shares with IL6 its hepatocyte stimulating function, being capable of inducing of rat acute phase genes. However, IL6-LA is structurally distinct from IL6, because it migrates with a chromatographic mobility of 60 kDa1 and cannot be neutralized with anti-IL6 sera. We believe IL6-LA to be a novel cytokine. Since IL6-LA was only observed in hepatoma cells, but not in normal rat liver, we anticipate this cytokine to be involved in the growth regulation of hepatocytes. To relate this novel cytokine to IL6 and to investigate the structural variants of IL6 and their respective functions we have isolated and sequenced a cloned 10.1 kb chromosomal DNA fragment containing the rat IL6 gene (5.8 kb) and mapped its 5' an 3' ends. The rat IL6 gene is a single copy gene with 5 exons coding for two IL6 mRNA species, a major 1.2 kb and a minor 2.4 kb species.
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ACT-2: A GENE THAT ENCODES A SECRETED PROTEIN ACTIVATION. MonicaNapolitanoandWarrenJ.Leonard,NIH,Bethesda, MD 20892.
UPON
We recently identified, cloned and sequenced a cDNA, ACT-2 (M. Lipes etal., PNAS 85, 9704-9708, 1988)thatis expressed in PHA/PMA or anti-T3 activated T cells, SAC I activated B cells and LPS activated monocytes. The ACT-2 cDNA was isolated using a differential hybridization technique from a PHA/PMA activated PBMC library. The time course of activation of the ACT-2 gene shows a very rapid induction of mRNA with the peak of expression being approximately 4 hours. The deduced amino acid sequence shows a very hydrophobic Nterminus, possibly representing a signal peptide. We then translated the ACT-2 mRNA in reticulocyte or wheat germ lysate translation systems with canine microsomal membranes but were unsuccessful in demonstrating the cleavage. We therefore expressed the ACT-2 cDNA in a baculovirus expression system and we demonstrated that ACT-2 encodes a secreted protein. In the 3’ UTR of the ACT-2 cDNA are present “AT rich motifs” that are also present in many protooncogenes and lymphokines. We have also isolated genomic phage clones corresponding to the ACT-2 gene; sequencing of the region 5’ to the mRNA cap site revealed a classical TATA box. These 5’ flanking sequences serve as functional promoter for driving CAT gene expression in Jurkat cells activated by mitogens and by the transactivator of HTLV-I tax product.
A NEW MITOGEN FOR TUMOR INFILTRATING LYMPHOCYTES (TILS) DERIVED FROM A TUMOR CELL LINE. Beverly S. Packard. Division of Cytokine Biology,CBER, FDA,Bethesda,MD. 20892. The idea that the tumor environment is intrinsically immunogenic and therefore contains immunocytes that can specifically recognize and, if expanded, lead to the induction of regression of said tumor has led to clinical trials in which patients are reinfused with their own lymphocytes (TILs) that have been isolated directly from their tumors and expanded ex vivo in the presence of interleukin-2 (IL-2). In the present study it is shown that the presence of irradiated cultured human tumor cells can stimulate the proliferation of human TlLs in the absence of IL-2. Tumor cell-mediated stimulation can be effected by supernatants from at least two tumor cell lines. A biologic activity profile from reverse phase hplc fractions of a tumor cell-derived serum-free supernatant indicates the presence of at least three domains of mitogenic activity. The fraction containing the major biologic activity has been further characterized by additional hplc and SDS-PAGE analyses; they indicate nonidentity with known factors. Further biochemical characterization of this new factor will be presented.
WORKSHOP
ON CYTOKINES
439
442
A NOVEL HUMAN MONOCYTE-DERIVED CHEMOTACTIC PEPTIDE WITH APPARENT MONOCYTE SPECIFICITY: PURIFICATION AND PARTIAL BIOLOGIC CHARACTERIZATION. v, Mrowietz, J.-M. SchrBder, E. ChristoDhers, Dept. of Dermatology, University of Kiel, Kiel, FRG. Accumulation of monocytes (MO) and MO-derived macrophages during some inflammatory processes indicate the existence of MO-specific chemotaxins. In this study MO-chemotactic activity was detected in the culture supernatants of lipopolysaccharide stimulated purified human MO. purification of MOchemotactic factors by different reversed phase HPLC-procedures revealed three peaks of MOwhich were distinct from chemotactic activity, other cytokines like IL-1 or TNF. One of these MOchemotaxins could be purified to apparent homogeneity giving upon SDS-PAGE a single band corresponding to a ML. of 10 kD. Dose response studies revealed a chemotaxis maximum at about 100 rig/ml. Significant MO chemotactic activity could still be seen at concentrations below 1 rig/ml. At concentrations higher than 30 rig/ml weak chemotactic activity was detectable using human neutrophils. From our results we conclude that human MO secrete at least three MO-specific chemotaxins, one being a 10 kD polypeptide, which appears to be different from known MO-chemotaxins.
TETRANECTIN: A NOVEL SECRETORY PROTEIN FROM HUMAN MONOCYTES. and I. Clemmensen. Statens Seruminstitut, Dept Clin --H. Nielsen Microbiolmigshospitaret, Copenhagen, OK-2200 Denmark. Tetranectin (TN) is a recently characterized protein in human plasma composed of four 181 amino acid polypeptide chains. TN has been demonstrated in many types of human cells including neutrophils (Christensen & Clemm?lisen. Histochemistry, in press). We now extend these findings and report that human monocytes are diffusely stained for TN by immunocytochemical methods. By immunoblotting the serum-free culture supernatant of human monocyte-derived macrophages #as confirmed to contain a single 20 kD band identical to plasma TN. Stimulation of macrophages with PMLP, C5a or PMA induced6release of TN in small but reproducible quantities (20 ng/h/lO cells). The biological role of TN is at present unknown, but preliminary experiments suggest that preincubation with highly purified TN may modulate monocyte function; i.e. increase spontaneous migration (150% of controls; n=3) and chemotactic responsiveness to C5a and fMLP (132% of controls; n=6), whereas the oxidative burst response to fMLP stimulation was unaffected (98% of controls; n=3). Further studies are in progress with tetranectin, which may show to be an important new cytokine.
440
443
Further Characterization of Human Natural Killer Cvtotoxic Factors fNKCF). Lawrence T. Nakamura and Benjamin Bonavida. UCLA School of Medicine, Los Angeles, CA 90024. Several techniaues have been used to ourifv natural killer cytotoxic faciors (NKCF) from culture' supirnates of human peripheral blood lymphocytes (PBL). We have achieved enrichment of cytotoxic material by fractionation of concentrated culture supernates on an FPLC chromatofocusing column (Mono P). The observed cytotoxicity does not appear to be tumor necrosis factor (TNF) and is specific for NK sensitive target cells, but not for NK resistant target cells. Recently, we generated a rabbit antiserum to the NKCF enriched Mono P fractions to facilitate concentration NKCF The salt-fractionated antiserum from culture supernates. inhibited the cytotoxic activity of Mono P fractions but did not inhibitrecombinant human TNF or lymphotoxin (LT). We have observed a significant inhibition of NK-cell mediated cytotoxicity (CMC), when effector cells were preincubated with specific antiserum, although conjugate frequency was not affected. Further, when effector cells were ppreincubated with a combination of specific antiserum and anti-TNF antiserum, an almost complete inhibition of NK-CMC was observed. Efforts are currently in progress to complete characterization of the specific antiserum and to identify a single protein in the Mono P fractions which may be responsible for the observed lysis of NK sensitive target cells. (Supported by NIH CA 35791 from the NIH.)
INTERLEUKIN 6 (IL6) AND IL6-LIKE ACTIVITIES PRODUCED BY RAT W. Northemann, T.A. MACROPHAGES AND HEPATOMA CELL LINES. and G.H. Fey. Dept. Immunology, Scripps Braciak, J. Gauldie*, Clinic, La Jolla, USA; *McMaster Univ., Hamilton, Canada. Interleukin 6 is a multifunctional hormone with both differentiation and growth promoting activities. It is produced by activated macrophages, monocytes, fibroblasts, and endothelial cells in response to inflammation, and by a number of tumors and tumor-derived cell lines, including rat hepatoma cells. During the inflarmnatory response circulating IL6 stimulates the production of the acute phase proteins in the liver. The rat hepatoma lines HTC, H5, and FtoZB produce IL6 activities. In addition, these lines secreted high amounts of a novel, IL6-like activity (IL6-LA). IL6-LA shares with IL6 its hepatocyte stimulating function, being capable of inducing of rat acute phase genes. However, IL6-LA is structurally distinct from IL6, because it migrates with a chromatographic mobility of 60 kDa1 and cannot be neutralized with anti-IL6 sera. We believe IL6-LA to be a novel cytokine. Since IL6-LA was only observed in hepatoma cells, but not in normal rat liver, we anticipate this cytokine to be involved in the growth regulation of hepatocytes. To relate this novel cytokine to IL6 and to investigate the structural variants of IL6 and their respective functions we have isolated and sequenced a cloned 10.1 kb chromosomal DNA fragment containing the rat IL6 gene (5.8 kb) and mapped its 5' an 3' ends. The rat IL6 gene is a single copy gene with 5 exons coding for two IL6 mRNA species, a major 1.2 kb and a minor 2.4 kb species.
441
444
ACT-2: A GENE THAT ENCODES A SECRETED PROTEIN ACTIVATION. MonicaNapolitanoandWarrenJ.Leonard,NIH,Bethesda, MD 20892.
UPON
We recently identified, cloned and sequenced a cDNA, ACT-2 (M. Lipes etal., PNAS 85, 9704-9708, 1988)thatis expressed in PHA/PMA or anti-T3 activated T cells, SAC I activated B cells and LPS activated monocytes. The ACT-2 cDNA was isolated using a differential hybridization technique from a PHA/PMA activated PBMC library. The time course of activation of the ACT-2 gene shows a very rapid induction of mRNA with the peak of expression being approximately 4 hours. The deduced amino acid sequence shows a very hydrophobic Nterminus, possibly representing a signal peptide. We then translated the ACT-2 mRNA in reticulocyte or wheat germ lysate translation systems with canine microsomal membranes but were unsuccessful in demonstrating the cleavage. We therefore expressed the ACT-2 cDNA in a baculovirus expression system and we demonstrated that ACT-2 encodes a secreted protein. In the 3’ UTR of the ACT-2 cDNA are present “AT rich motifs” that are also present in many protooncogenes and lymphokines. We have also isolated genomic phage clones corresponding to the ACT-2 gene; sequencing of the region 5’ to the mRNA cap site revealed a classical TATA box. These 5’ flanking sequences serve as functional promoter for driving CAT gene expression in Jurkat cells activated by mitogens and by the transactivator of HTLV-I tax product.
A NEW MITOGEN FOR TUMOR INFILTRATING LYMPHOCYTES (TILS) DERIVED FROM A TUMOR CELL LINE. Beverly S. Packard. Division of Cytokine Biology,CBER, FDA,Bethesda,MD. 20892. The idea that the tumor environment is intrinsically immunogenic and therefore contains immunocytes that can specifically recognize and, if expanded, lead to the induction of regression of said tumor has led to clinical trials in which patients are reinfused with their own lymphocytes (TILs) that have been isolated directly from their tumors and expanded ex vivo in the presence of interleukin-2 (IL-2). In the present study it is shown that the presence of irradiated cultured human tumor cells can stimulate the proliferation of human TlLs in the absence of IL-2. Tumor cell-mediated stimulation can be effected by supernatants from at least two tumor cell lines. A biologic activity profile from reverse phase hplc fractions of a tumor cell-derived serum-free supernatant indicates the presence of at least three domains of mitogenic activity. The fraction containing the major biologic activity has been further characterized by additional hplc and SDS-PAGE analyses; they indicate nonidentity with known factors. Further biochemical characterization of this new factor will be presented.