- Email: [email protected]
LAK cells specific mRNAs identified by differential display
NK cells
25 June 1997 - Poster presentations
1P.1.09.34 1 Establishment and characterixation of porcine cytoiytic ceil lines and clones M. de Bruin, J. Vcermans, A. Bianchi, T. Kimman. ID-DLO Dept. Mammalian Vimbgy AB Lelystad. The Netherlands Introduction: Non-MHC restricted cytolytic cells appear to significantly influence antiviral immunity in pigs, but the phenotype and functional characteristics of these cells are not well defined. To allow an analysis of these subsets, we established and characterized cell lines and clones of IL-2 activated cytolytic cells. Materlds and Methods: Cell lines and clones were obtained from PBMC of minipigs of the MHC d/d haplotype. Cells were cultured in the presence of human rll-2 and cloned by double limiting dilution in the presence of gamma-irradiated L14 feeder cells (MHC d/d). Cytolytic cell lines and clones were characterized for their ability to kill diierent target cells and for their cell surface phenotype. All clones were CD2+CD4-CD6+. Reauttr: The following three subsets of cytolytic cells were identified: 1) CD3- CD5- cells that killed K562 cells (natural killer cell target), as well as the pseudorabies (PRV) Virus-infected or uninfected porcine kidney cells. These cells were considered to be typical natural killer cells. 2) CD3y/&+ CD5- T-cells that killed K562 cells and PRV virus-infected or uninfected porcine kidney cells, and infected-, gB- and gC-transfected or uninfected L14 cells. These cells were considered to be y/6 T-cells with natural killer activity. 3) CD3@+ CD5+ T-cells that killed L14 cells, PRV-infected L14 cells, and PRV gB- and gC-transfected L14 cells. These cells were possibly induced by the 114 feeder cells, used in the in vitro culture system. Conclusion: We describe a method to isolate, clone, and culture cytolytic cells from pigs. The clones could be cultured for 5 months, tiich allowed phenotypic and functional characterization of the clones. Two of the subsets, CD3y/6 T- and the natural killer cell subset may be involved in antiviral immunity in this species.
1P.1.09.35 1 HLA-G: Public ilgand for natural killer ceil inhibitory receptors Nathalie Rouas-Freiss, Rachel Marchal, Marek Kirszenbaum, Jean Dausset ‘, Edgardo D. Camsella ‘. ServicedeRecherches en Hdmato-lmmundogie, Commissadat & I’Energie Atomique-DRM-DSV Hdpitai Saint-Louis, 1, avenue Claude-Vellefaux, Paris CBdex, France, ’ Fondation Jean Dausset, 27, rue Juliet&Dodu, Paris C&LX, France HLA-G is a non-classical major histocompatibility complex class I antigen selectively expressed on extra-vlllous trophoblast cells at the fetal-maternal interface. The lack of classical HLA class I expression on trophoblast cells is known to not protect them from natural killer (NK) cytotoxicity. It was thus proposed that HLA-G mediates protection from NK cells, thereby playing an important role in maintaining maternal immune tolerance of the semi-allogenic fetus. In this context, we have investigated the protectiie role of the membrane-bound HLA-Gl and HLA-G2 isofonns against NK cytotoxicity. For this purpose, HLA-Gl and HLA-GP cDNAs were transfected into the HLA class l-negative human K562 cell line, a known reference target for NK lysis. The HLA-Gl protein, encoded by a full-length mRNA, presents a structure similar to that of classical HLA class I antigens. The HIA-GL protein, deduced from an alternatively spliced transcript, consists of the crl domain linked to the ~3 domain. We used either PBMC or poiyclonal NK cells (CD3- CD16+ CD56’) obtained from twenty healthy adult donors as effector cells. In this study we demonstrate that: (i) HLA-GP is present at the cell surface as a truncated class I molecule associated with 82 m; (ii) NK cytolysis, observed in PBMC and in polyclonal NK cells obtained from twenty donors, is inhibited by both HLA-Gl and HLA-GP. This HIA-G-mediated inhibition is reversed by blocking H&G with a specific mAb. This led us to the conjecture that HlA-G is the public ligand for natural killer inhibitory receptors present in all individuals; and (iii) The al domain common to HLA-Gl and HLA-GP could mediate this protection from NK lysis.
1P.1.09.36 I A novel putative inhibitory NK receptor belonging to the immunogiobuiin-superfamiiy C. Cantoni ‘, S. Verdiani ‘, M.Falco ‘, R.Conte ‘, L. Moretta I,*, R. Biassoni ‘.
’ ISTKBA Genova, I&I&, 2lstifuto di Patolcgia Genetale Univ di Geneva, Geneva, Italy NK cells display specific receptors (NKR) for different alleles of major histocompatibility complex (MHC) class I molecules expressed on normal cells. So far, different inhibitory receptors specific for distinct groups of HLA-C (p56/CD156), HIA-B (p70/NKBl) or HLA-A (~140) alleles have been identified that belong to the lmmunoglobulin superfamily (Ig-SF); they are type I transmembmne proteins, characterized by two (~56) or three (p7O/NKBl and ~140) extracellular Ig-like domains and containing two Immuno-receptor Tymsine-based lnhibitoly Motifs (ITIMs) in their cytoplasmic tails.
389
Molecular cloning of full length cDNAs was performed by RT-PCR utilizing selected NK and T/NKR+ cell clones. Here we describe three different cDNAs sharing a high degree of sequence homology with the genes encoding for the ~56, ~70, ~140 HLA-specific NK receptors and with the recently characterized KIRl03AS cDNA. The prototype of these cDNAs is ~1.15.212that encodes for a typs I transmembrane protein belonging to the Ig superfamily, characterized by 2 extracellular lg-like domains and a 115 amino acid cytoplasmic tail containing only one ITIM-motif. Amino acid sequence of cl.15.21P-encoded protein was compared to amino acid sequences of different members of the NKR belonging to the Ig-SF giving about 50% sequence homology. Other homologous cDNAs have been amplified that display only one lg-iike domain. Differently from the other HlAclass I specific NK receptors (~56, p70 and ~140) and from KIRl03AS cDNA ~1.15.212transcdpt is not clonallv distributed. since it aooears to be expressed bv all NK cell clones as well as 7 cell clones. expressing NKR. Differ&t human. haemopoietic cell lines, such as Jurkat, Rajl, HLBO, K562 and 721.221 B-EBV cell lines lack cl. 15.212/cl. 12.11 specific transcripts More interestingly, isoforms that lack sequences encoding for the transmembrane portion have been isolated that may possibly encode for molecules produced in soluble form. The ~1.15.212and related cDNAs may encode for inhibitory receptors with a broad NK cell distribution.
I P.1.09.37 I ~sM&is
specific mFiNAs identified by differential
E. Capelli. S. Comincini, G. Damiani, S. Pa&Ii, M. Cuccia. Dip. di Genefica e Microbiologia, Universita di Pavia, /ta/y Introduction: LAK activity represents a function of lymphoid subsets with cytotoxic activity against tumoral cells. At present it is not clear if lAK cells belong to Natural Killer (NK) subsets or represent different subpopulations with a particularly high specific antitumoral activity. The aim of the study was the identification of Lymphokine Activated Killers (LAK) cells specific mRNAs. Matirfals and Methods:cDNAs were obtained from mRNAs of Peripheral Blood Mononucleated Cells (PBMC) from healthy donors and stimulated in vitro with recombinant IL-2 (rlL-2, 50 p@ml) for eight days. Freshly isolated PBMC, PHA-stimulated PBMC and K562 eritroleukemic cells were used as controls. Molecular markers for LAK cells were searched by means of Differential Display method. Differentially expressed cDNAs were cloned in bacterial host cells and analysed in their sequence by means of BLAST Network service. Results:Cells with LAK activity showing CD4, CD45RO positive phenotype were obtained. Survival tests evidenced the ability of these cells to kill target transformed cells (Chang tumorigenic line). Differential Display analyses evidenced: a) for a first clone, a significant homology for a Zinc finger domain preferentially located In the C-terminal region of many proteins; b) for a second clone, a significant homology with a cytokine domain; c) for a third clone, a significant homology for tralgene (human homologue of murfne tumor rejection antigen gp96). Concluslons:Our study revealed possible molecular markers useful for elucidating the genetic bases of IAK activity and for a rapid screening of differences between the various cell subsets stimulated with IL-2.
1P.1.09.38 I Effect of ehydroxynonenal, a product of ilpid peroxldatlon, on NK susceptibility of human K5S2 target cells M. Tdcadco ‘, S.A. Ciafre’ 1,2,P. Spinsanti ‘, M.G. Farace 1,2,E. Bonmassar 1,2, V.M Fazio’,3, M. Rinaldi ‘. l/nstitute of Experimental Medicine, CNR, Rome, ha/i 2Depattment of Experimental Medicine and Biochemical Sciences, Univetsifyof Rome %r Vergata”, /tak 31nstiiu~eof General Pathdogy; Catholic univefs* Rome, fiWy
Introduction: 4-hydroxynonenal (HNE) is one of the major breakdown products of cellular lipid peroxidation. The level of lipid peroxidation and the concentration of its products are inversely related to the rate of cell proliferation and directly related to the level of cell differentiation. It has been demonstrated that HNE can load to inhibition of proliferation and induction of differentiation in the HL60 cell line. In the present study the effect of HNE, at concentration close to those found in the normal tissue, on the NK susceptibility of the human K562 target cells was analvzed. Materialsand Methods:Human K562 cells in RPM1 1640 medium plus 10% FCS were diluted at the concentration of 1OO,OOO/ml. Addiion of HNE (final concentration 1 FM) was performed at regular intervals of time (45 minut&) up to 16 treatments. Hemin was added at a final concentration of 30 PM. Growth rate and cell viability were monitored daily. Mononuclear cells (MNC) from peripheral blood of healthy donors were separated on Ficoll Hypaque gradient, and used as effector cells (E) in cytotoxicity assays. Cytotoxic activity of MNC was performed in a 4-hr 51Cr-release assay using K562 as target cells (T) at the selected E:T ratio.
25 June 1997 - Poster presentations
1P.1.09.34 1 Establishment and characterixation of porcine cytoiytic ceil lines and clones M. de Bruin, J. Vcermans, A. Bianchi, T. Kimman. ID-DLO Dept. Mammalian Vimbgy AB Lelystad. The Netherlands Introduction: Non-MHC restricted cytolytic cells appear to significantly influence antiviral immunity in pigs, but the phenotype and functional characteristics of these cells are not well defined. To allow an analysis of these subsets, we established and characterized cell lines and clones of IL-2 activated cytolytic cells. Materlds and Methods: Cell lines and clones were obtained from PBMC of minipigs of the MHC d/d haplotype. Cells were cultured in the presence of human rll-2 and cloned by double limiting dilution in the presence of gamma-irradiated L14 feeder cells (MHC d/d). Cytolytic cell lines and clones were characterized for their ability to kill diierent target cells and for their cell surface phenotype. All clones were CD2+CD4-CD6+. Reauttr: The following three subsets of cytolytic cells were identified: 1) CD3- CD5- cells that killed K562 cells (natural killer cell target), as well as the pseudorabies (PRV) Virus-infected or uninfected porcine kidney cells. These cells were considered to be typical natural killer cells. 2) CD3y/&+ CD5- T-cells that killed K562 cells and PRV virus-infected or uninfected porcine kidney cells, and infected-, gB- and gC-transfected or uninfected L14 cells. These cells were considered to be y/6 T-cells with natural killer activity. 3) CD3@+ CD5+ T-cells that killed L14 cells, PRV-infected L14 cells, and PRV gB- and gC-transfected L14 cells. These cells were possibly induced by the 114 feeder cells, used in the in vitro culture system. Conclusion: We describe a method to isolate, clone, and culture cytolytic cells from pigs. The clones could be cultured for 5 months, tiich allowed phenotypic and functional characterization of the clones. Two of the subsets, CD3y/6 T- and the natural killer cell subset may be involved in antiviral immunity in this species.
1P.1.09.35 1 HLA-G: Public ilgand for natural killer ceil inhibitory receptors Nathalie Rouas-Freiss, Rachel Marchal, Marek Kirszenbaum, Jean Dausset ‘, Edgardo D. Camsella ‘. ServicedeRecherches en Hdmato-lmmundogie, Commissadat & I’Energie Atomique-DRM-DSV Hdpitai Saint-Louis, 1, avenue Claude-Vellefaux, Paris CBdex, France, ’ Fondation Jean Dausset, 27, rue Juliet&Dodu, Paris C&LX, France HLA-G is a non-classical major histocompatibility complex class I antigen selectively expressed on extra-vlllous trophoblast cells at the fetal-maternal interface. The lack of classical HLA class I expression on trophoblast cells is known to not protect them from natural killer (NK) cytotoxicity. It was thus proposed that HLA-G mediates protection from NK cells, thereby playing an important role in maintaining maternal immune tolerance of the semi-allogenic fetus. In this context, we have investigated the protectiie role of the membrane-bound HLA-Gl and HLA-G2 isofonns against NK cytotoxicity. For this purpose, HLA-Gl and HLA-GP cDNAs were transfected into the HLA class l-negative human K562 cell line, a known reference target for NK lysis. The HLA-Gl protein, encoded by a full-length mRNA, presents a structure similar to that of classical HLA class I antigens. The HIA-GL protein, deduced from an alternatively spliced transcript, consists of the crl domain linked to the ~3 domain. We used either PBMC or poiyclonal NK cells (CD3- CD16+ CD56’) obtained from twenty healthy adult donors as effector cells. In this study we demonstrate that: (i) HLA-GP is present at the cell surface as a truncated class I molecule associated with 82 m; (ii) NK cytolysis, observed in PBMC and in polyclonal NK cells obtained from twenty donors, is inhibited by both HLA-Gl and HLA-GP. This HIA-G-mediated inhibition is reversed by blocking H&G with a specific mAb. This led us to the conjecture that HlA-G is the public ligand for natural killer inhibitory receptors present in all individuals; and (iii) The al domain common to HLA-Gl and HLA-GP could mediate this protection from NK lysis.
1P.1.09.36 I A novel putative inhibitory NK receptor belonging to the immunogiobuiin-superfamiiy C. Cantoni ‘, S. Verdiani ‘, M.Falco ‘, R.Conte ‘, L. Moretta I,*, R. Biassoni ‘.
’ ISTKBA Genova, I&I&, 2lstifuto di Patolcgia Genetale Univ di Geneva, Geneva, Italy NK cells display specific receptors (NKR) for different alleles of major histocompatibility complex (MHC) class I molecules expressed on normal cells. So far, different inhibitory receptors specific for distinct groups of HLA-C (p56/CD156), HIA-B (p70/NKBl) or HLA-A (~140) alleles have been identified that belong to the lmmunoglobulin superfamily (Ig-SF); they are type I transmembmne proteins, characterized by two (~56) or three (p7O/NKBl and ~140) extracellular Ig-like domains and containing two Immuno-receptor Tymsine-based lnhibitoly Motifs (ITIMs) in their cytoplasmic tails.
389
Molecular cloning of full length cDNAs was performed by RT-PCR utilizing selected NK and T/NKR+ cell clones. Here we describe three different cDNAs sharing a high degree of sequence homology with the genes encoding for the ~56, ~70, ~140 HLA-specific NK receptors and with the recently characterized KIRl03AS cDNA. The prototype of these cDNAs is ~1.15.212that encodes for a typs I transmembrane protein belonging to the Ig superfamily, characterized by 2 extracellular lg-like domains and a 115 amino acid cytoplasmic tail containing only one ITIM-motif. Amino acid sequence of cl.15.21P-encoded protein was compared to amino acid sequences of different members of the NKR belonging to the Ig-SF giving about 50% sequence homology. Other homologous cDNAs have been amplified that display only one lg-iike domain. Differently from the other HlAclass I specific NK receptors (~56, p70 and ~140) and from KIRl03AS cDNA ~1.15.212transcdpt is not clonallv distributed. since it aooears to be expressed bv all NK cell clones as well as 7 cell clones. expressing NKR. Differ&t human. haemopoietic cell lines, such as Jurkat, Rajl, HLBO, K562 and 721.221 B-EBV cell lines lack cl. 15.212/cl. 12.11 specific transcripts More interestingly, isoforms that lack sequences encoding for the transmembrane portion have been isolated that may possibly encode for molecules produced in soluble form. The ~1.15.212and related cDNAs may encode for inhibitory receptors with a broad NK cell distribution.
I P.1.09.37 I ~sM&is
specific mFiNAs identified by differential
E. Capelli. S. Comincini, G. Damiani, S. Pa&Ii, M. Cuccia. Dip. di Genefica e Microbiologia, Universita di Pavia, /ta/y Introduction: LAK activity represents a function of lymphoid subsets with cytotoxic activity against tumoral cells. At present it is not clear if lAK cells belong to Natural Killer (NK) subsets or represent different subpopulations with a particularly high specific antitumoral activity. The aim of the study was the identification of Lymphokine Activated Killers (LAK) cells specific mRNAs. Matirfals and Methods:cDNAs were obtained from mRNAs of Peripheral Blood Mononucleated Cells (PBMC) from healthy donors and stimulated in vitro with recombinant IL-2 (rlL-2, 50 p@ml) for eight days. Freshly isolated PBMC, PHA-stimulated PBMC and K562 eritroleukemic cells were used as controls. Molecular markers for LAK cells were searched by means of Differential Display method. Differentially expressed cDNAs were cloned in bacterial host cells and analysed in their sequence by means of BLAST Network service. Results:Cells with LAK activity showing CD4, CD45RO positive phenotype were obtained. Survival tests evidenced the ability of these cells to kill target transformed cells (Chang tumorigenic line). Differential Display analyses evidenced: a) for a first clone, a significant homology for a Zinc finger domain preferentially located In the C-terminal region of many proteins; b) for a second clone, a significant homology with a cytokine domain; c) for a third clone, a significant homology for tralgene (human homologue of murfne tumor rejection antigen gp96). Concluslons:Our study revealed possible molecular markers useful for elucidating the genetic bases of IAK activity and for a rapid screening of differences between the various cell subsets stimulated with IL-2.
1P.1.09.38 I Effect of ehydroxynonenal, a product of ilpid peroxldatlon, on NK susceptibility of human K5S2 target cells M. Tdcadco ‘, S.A. Ciafre’ 1,2,P. Spinsanti ‘, M.G. Farace 1,2,E. Bonmassar 1,2, V.M Fazio’,3, M. Rinaldi ‘. l/nstitute of Experimental Medicine, CNR, Rome, ha/i 2Depattment of Experimental Medicine and Biochemical Sciences, Univetsifyof Rome %r Vergata”, /tak 31nstiiu~eof General Pathdogy; Catholic univefs* Rome, fiWy
Introduction: 4-hydroxynonenal (HNE) is one of the major breakdown products of cellular lipid peroxidation. The level of lipid peroxidation and the concentration of its products are inversely related to the rate of cell proliferation and directly related to the level of cell differentiation. It has been demonstrated that HNE can load to inhibition of proliferation and induction of differentiation in the HL60 cell line. In the present study the effect of HNE, at concentration close to those found in the normal tissue, on the NK susceptibility of the human K562 target cells was analvzed. Materialsand Methods:Human K562 cells in RPM1 1640 medium plus 10% FCS were diluted at the concentration of 1OO,OOO/ml. Addiion of HNE (final concentration 1 FM) was performed at regular intervals of time (45 minut&) up to 16 treatments. Hemin was added at a final concentration of 30 PM. Growth rate and cell viability were monitored daily. Mononuclear cells (MNC) from peripheral blood of healthy donors were separated on Ficoll Hypaque gradient, and used as effector cells (E) in cytotoxicity assays. Cytotoxic activity of MNC was performed in a 4-hr 51Cr-release assay using K562 as target cells (T) at the selected E:T ratio.