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PPD-specific IL-2 expanded CTL are restricted by antigens encoded from the HLA-D region
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Annual AACHT Meeting, 1984 duction and expression of antibiotic resistance and SV40 large T antigen in a human T cell leukemia line. The transfecting vector was the Berg plasmid pSV3neo which carries the genes for SV40 large T antigen, resistance to G418 (an antibiotic which is cytotoxic for eukaryotic cells), and other regulatory genetic elements. Transfection was achieved by protoplast fusion of the Jurkat T cell leukemia line with E. coil K-12 strain HB I 01 bearing the plasmid. Protoplasts were prepared using a modification of the procedure described by Oi et al. (1983). Presence of the transfected genes was shown by Southern hybridization using plasmid pSV3neo labeled with 32-p, as a hybridization probe. Expression of transfected genes was shown by growth in the antibiotic G418, as opposed to death of untransfected cells in 5-12 days. Nuclear localization of SV40 large T antigen was shown by immunofluorescence using hamster anti-SV40 large T, followed by FITC labeled goat anti-hamster Ig. Human lymphoid cells of T cell origin are shown to be acceptable recipients of transfected DNA, and thus are possible recipients of cloned T cell specific sequences.
A B S T R A C T SESSION 3: HLA SPECIFICITY OF T CELLS PPD-SPECIFIC IL-2 EXPANDED CTL ARE RESTRICTED BY ANTIGENS ENCODED FROM THE HLA-D REGION. Peter Wendelboe Hansen and Tom Kristensen; Tissue Typing Laboratory, University Hospital. Aarhus. Denmark Unexpectedly, we found human CTL directed against PPD pulsed monocyte targets to be guided by HLA class II but not class I antigens. In order to further dissect the nature and degree of specificity of the CTL restricting HLA-D region product(s), 20 bulk-expanded CTL were tested against (PPD-) antigen pulsed to unpulsed monocyte targets from a panel of unrelated donors as well as in selected families using a standard 5~Cr-release assay. Analysis of the results revealed a general strong correlation between PPD-specific lysis and sharing between CTL and target cell donors of HLA-DR antigens indicating DR rather than DQ or DP restricted clones to be predominant. Analysis of the role of individual antigens within the HLA-DR series showed a particularly strong correlation concerning HLA-DR1, -2, -3, -4, and -7, whereas the correlation was less pronounced for HLA-DR5, w6 and -w8. A limited number of consistent positive and negative exceptions was found, however. These have been investigated further using extended serology and family studies. ISOLATION AND CHARACTERIZATION OF LYMPHOCYTES FROM PULMONARY LAVAGE FLUIDS OF HEART/LUNG TRANSPLANT RECIPIENTS. A Zeevi, I. Paradis, J. H. Dauber, R. L. Hardesty, B. P Griffith, and R. J. Duquesnoy; University of Pittsburgh, Pittsburgh, PA Testing of pulmonary lavage fluids (PLF) has provided unique opportunities to study in situ transplant immunity in patients with heart-lung transplants. In five patients studied so far, the cell number in PLF ranged from 30-120 x 10° (normal values 5-15 x 10~') and three major cell types were observed: macrophages: 35-75%; lymphocytes: 10-60%; PMNs: 2-12% (normal values are 80-90%, 5-10%, and 2-3%, respectively). The lymphocytes in PLF from transplant recipients were primarily T cells and always showed an inverted helper
Annual AACHT Meeting, 1984 duction and expression of antibiotic resistance and SV40 large T antigen in a human T cell leukemia line. The transfecting vector was the Berg plasmid pSV3neo which carries the genes for SV40 large T antigen, resistance to G418 (an antibiotic which is cytotoxic for eukaryotic cells), and other regulatory genetic elements. Transfection was achieved by protoplast fusion of the Jurkat T cell leukemia line with E. coil K-12 strain HB I 01 bearing the plasmid. Protoplasts were prepared using a modification of the procedure described by Oi et al. (1983). Presence of the transfected genes was shown by Southern hybridization using plasmid pSV3neo labeled with 32-p, as a hybridization probe. Expression of transfected genes was shown by growth in the antibiotic G418, as opposed to death of untransfected cells in 5-12 days. Nuclear localization of SV40 large T antigen was shown by immunofluorescence using hamster anti-SV40 large T, followed by FITC labeled goat anti-hamster Ig. Human lymphoid cells of T cell origin are shown to be acceptable recipients of transfected DNA, and thus are possible recipients of cloned T cell specific sequences.
A B S T R A C T SESSION 3: HLA SPECIFICITY OF T CELLS PPD-SPECIFIC IL-2 EXPANDED CTL ARE RESTRICTED BY ANTIGENS ENCODED FROM THE HLA-D REGION. Peter Wendelboe Hansen and Tom Kristensen; Tissue Typing Laboratory, University Hospital. Aarhus. Denmark Unexpectedly, we found human CTL directed against PPD pulsed monocyte targets to be guided by HLA class II but not class I antigens. In order to further dissect the nature and degree of specificity of the CTL restricting HLA-D region product(s), 20 bulk-expanded CTL were tested against (PPD-) antigen pulsed to unpulsed monocyte targets from a panel of unrelated donors as well as in selected families using a standard 5~Cr-release assay. Analysis of the results revealed a general strong correlation between PPD-specific lysis and sharing between CTL and target cell donors of HLA-DR antigens indicating DR rather than DQ or DP restricted clones to be predominant. Analysis of the role of individual antigens within the HLA-DR series showed a particularly strong correlation concerning HLA-DR1, -2, -3, -4, and -7, whereas the correlation was less pronounced for HLA-DR5, w6 and -w8. A limited number of consistent positive and negative exceptions was found, however. These have been investigated further using extended serology and family studies. ISOLATION AND CHARACTERIZATION OF LYMPHOCYTES FROM PULMONARY LAVAGE FLUIDS OF HEART/LUNG TRANSPLANT RECIPIENTS. A Zeevi, I. Paradis, J. H. Dauber, R. L. Hardesty, B. P Griffith, and R. J. Duquesnoy; University of Pittsburgh, Pittsburgh, PA Testing of pulmonary lavage fluids (PLF) has provided unique opportunities to study in situ transplant immunity in patients with heart-lung transplants. In five patients studied so far, the cell number in PLF ranged from 30-120 x 10° (normal values 5-15 x 10~') and three major cell types were observed: macrophages: 35-75%; lymphocytes: 10-60%; PMNs: 2-12% (normal values are 80-90%, 5-10%, and 2-3%, respectively). The lymphocytes in PLF from transplant recipients were primarily T cells and always showed an inverted helper