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Human JEG-3 cells transfected with murine MHC class II Aq genes can present antigen to Aq-restricted T-cell hybridomas
12
Abstracts
Lack of placental MHC class II mRNA expression in IFNy- and 5-azacytidinetreated mice Erika Gustafsson, Marie Arvola, Anita Mattsson, Ragnar Mattsson. Department of Animal Development and Genetics, Uppsala University, Norbyvtigen 18A, S- 75236 Uppsala, Sweden
Trophoblast cells do not normally express major histocompatibility complex (MHC) class II antigens during placental development in either mice or rats. There exist, however, different opinions on whether interferon-y (IFNy) or the drug 5-azacytidine are capable of inducing MHC class II expression in murine placental cells. The present study was performed to clarify whether treatment of pregnant mice with recombinant IFNy or 5-azacytidine can induce MHC class II mRNA expression in murine placentas. A strain of transgenic mice carrying a cytomegalovirus (CMV)-regulated MHC class II Abq transgene, which was expressed in the placenta, was used as positive control in all in situ hybridizations and ribonuclease protection analyses (RPA). Treatment of pregnant mice with IFNy did not induce detectable class II expression in the placental cells, whereas the maternal decidua showed expression both at the mRNA and protein level. Similarly, treatment with 5-azacytidine did not induce class II expression in the placenta, while a slight increase in mRNA expression was detected in the maternal decidual and uterine tissues. These results strongly indicate that MHC class II mRNA cannot be induced in murine placental cells following IFNy or 5-azacytidine treatments. Keywords: 5-azacytidine; Interferon-y; MHC class II expression; pregnancy; Placenta; Trophoblast; Transgenic mice
Mouse
Human JEG3 cells transfected with murine MHC class II Aq genes can present antigen to Aq-restricted T-cell hybridomas
Marie Arvola”, Erika Gustafsson”, Ulrica Brunsbergb, Anita Mattsson”, Ragnar Mattsson a. “Department of Animal Development and Genetics, Uppsala University, Norbyviigen 18A, S- 7.5234 Uppsala, Sweden; bDepartment of Medical Injlammation Research, Lund University, P.O.Box 94, S-22200 Lund, Sweden
Surface MHC class II molecules are normally not expressed on placental cells or cell lines of placental origin. This could contribute to the relative low incidence of harmful anti-placental immune reactions in mammals. We have studied whether the human choriocarcinoma-derived cell line JEG-3 is capable of expressing a functional murine class II molecule. The JEG-3 cells
Abstracts
73
were transiently co-transfected with SV40- and CMV-regulated class II Aq alpha and beta genes in vitro. More than 30% of the transfected JEG-3 cells showed surface expression of murine class II Aq molecule within 2 days of transfection. Positive cells were recognized by staining viable cells with FITC-conjugated class II antibodies. In order to test whether the detected class II molecules were functional, the cells were analysed by an antigen-presentation assay in vitro. The transfected JEG-3 cells were incubated with collagen peptides (processed antigen) and cocultured with a T-cell hybridoma capable of recognizing collagen peptides if presented in association with class II Aq. The T-cell hybridoma released IL-2 when collagen peptides were added to transfected JEG-3 cells, indicating that the class II was expressed in a functional way. If non-processed collagen was added to the cultures no IL-2 response could be detected. The results indicate that murine class II molecules can be expressed in a functional way by JEG-3 cells, but that JEG-3 cells are unable to process antigen in a proper way. Similar studies of murine placenta-derived cell lines are in progress. Keywords: MHC class II; JEG-3 cells; Antigen presentation
061
HLA-G present peptides to murine CDS T cells in transgenic mice A. Horuzsko, V. Portik-Dobos, A.L. Mellor. Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912, USA’
HLA-G, a human MHC class I molecule expressed on the cytotrophoblast during pregnancy, was expressed in transgenic mice by recombining the HLA-G gene with a transcriptional promoter from a murine H-2 MHC class I gene. HLA-G transgenic mice were immunized with a peptide Gl (RHPKYKTEL) that binds to HLA-G molecules. Gl-peptide specific CTL responses were detected when T cells were isolated from that HLA-G immunized transgenic mice. These results demonstrate molecules bind and present peptide to murine T cells and induce them to differentiate into effector T cells (CTL) that recognize the HLA-G/peptide Gl complex on target cells. We conclude that HLA-G molecules behave as classical restriction elements presenting peptides to T cells that recognize HLA-G/peptide complex in vivo. This implies that HLA-G molecules affect thymocyte selection in mice by influencing the development of thymocytes that have low affinity for HLA-G molecules. Keywords:
HLA-G; Peptide presentation;
CTL responses; Transgenic mice
Abstracts
Lack of placental MHC class II mRNA expression in IFNy- and 5-azacytidinetreated mice Erika Gustafsson, Marie Arvola, Anita Mattsson, Ragnar Mattsson. Department of Animal Development and Genetics, Uppsala University, Norbyvtigen 18A, S- 75236 Uppsala, Sweden
Trophoblast cells do not normally express major histocompatibility complex (MHC) class II antigens during placental development in either mice or rats. There exist, however, different opinions on whether interferon-y (IFNy) or the drug 5-azacytidine are capable of inducing MHC class II expression in murine placental cells. The present study was performed to clarify whether treatment of pregnant mice with recombinant IFNy or 5-azacytidine can induce MHC class II mRNA expression in murine placentas. A strain of transgenic mice carrying a cytomegalovirus (CMV)-regulated MHC class II Abq transgene, which was expressed in the placenta, was used as positive control in all in situ hybridizations and ribonuclease protection analyses (RPA). Treatment of pregnant mice with IFNy did not induce detectable class II expression in the placental cells, whereas the maternal decidua showed expression both at the mRNA and protein level. Similarly, treatment with 5-azacytidine did not induce class II expression in the placenta, while a slight increase in mRNA expression was detected in the maternal decidual and uterine tissues. These results strongly indicate that MHC class II mRNA cannot be induced in murine placental cells following IFNy or 5-azacytidine treatments. Keywords: 5-azacytidine; Interferon-y; MHC class II expression; pregnancy; Placenta; Trophoblast; Transgenic mice
Mouse
Human JEG3 cells transfected with murine MHC class II Aq genes can present antigen to Aq-restricted T-cell hybridomas
Marie Arvola”, Erika Gustafsson”, Ulrica Brunsbergb, Anita Mattsson”, Ragnar Mattsson a. “Department of Animal Development and Genetics, Uppsala University, Norbyviigen 18A, S- 7.5234 Uppsala, Sweden; bDepartment of Medical Injlammation Research, Lund University, P.O.Box 94, S-22200 Lund, Sweden
Surface MHC class II molecules are normally not expressed on placental cells or cell lines of placental origin. This could contribute to the relative low incidence of harmful anti-placental immune reactions in mammals. We have studied whether the human choriocarcinoma-derived cell line JEG-3 is capable of expressing a functional murine class II molecule. The JEG-3 cells
Abstracts
73
were transiently co-transfected with SV40- and CMV-regulated class II Aq alpha and beta genes in vitro. More than 30% of the transfected JEG-3 cells showed surface expression of murine class II Aq molecule within 2 days of transfection. Positive cells were recognized by staining viable cells with FITC-conjugated class II antibodies. In order to test whether the detected class II molecules were functional, the cells were analysed by an antigen-presentation assay in vitro. The transfected JEG-3 cells were incubated with collagen peptides (processed antigen) and cocultured with a T-cell hybridoma capable of recognizing collagen peptides if presented in association with class II Aq. The T-cell hybridoma released IL-2 when collagen peptides were added to transfected JEG-3 cells, indicating that the class II was expressed in a functional way. If non-processed collagen was added to the cultures no IL-2 response could be detected. The results indicate that murine class II molecules can be expressed in a functional way by JEG-3 cells, but that JEG-3 cells are unable to process antigen in a proper way. Similar studies of murine placenta-derived cell lines are in progress. Keywords: MHC class II; JEG-3 cells; Antigen presentation
061
HLA-G present peptides to murine CDS T cells in transgenic mice A. Horuzsko, V. Portik-Dobos, A.L. Mellor. Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912, USA’
HLA-G, a human MHC class I molecule expressed on the cytotrophoblast during pregnancy, was expressed in transgenic mice by recombining the HLA-G gene with a transcriptional promoter from a murine H-2 MHC class I gene. HLA-G transgenic mice were immunized with a peptide Gl (RHPKYKTEL) that binds to HLA-G molecules. Gl-peptide specific CTL responses were detected when T cells were isolated from that HLA-G immunized transgenic mice. These results demonstrate molecules bind and present peptide to murine T cells and induce them to differentiate into effector T cells (CTL) that recognize the HLA-G/peptide Gl complex on target cells. We conclude that HLA-G molecules behave as classical restriction elements presenting peptides to T cells that recognize HLA-G/peptide complex in vivo. This implies that HLA-G molecules affect thymocyte selection in mice by influencing the development of thymocytes that have low affinity for HLA-G molecules. Keywords:
HLA-G; Peptide presentation;
CTL responses; Transgenic mice