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+ APCs. Monoclonal antibodies used for flow cytometry and blocking experiments were as follows (4): OKT6, which blocks CD1a; WM-25, which blocks CD1b; 10C3, which blocks CD1c; OKT4, which recognizes CD4; and OKT8, which recognizes CD8a; antibodies to αβ TCR (Becton-Dickinson, San Jose, CA) and to γδ TCR, LNK16, and CD16 (Serotec, Washington, DC).
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2+ was chosen as a selective inhibitor of granule-dependent lysis, because the calcium chelator EGTA, which blocks the perforin pathway, also inhibits up-regulation of FasL, as required in our studies with primary human T cells.
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19
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1842274077
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Tumor necrosis factor-α (TNF-α) induces lysis of a murine fibrosarcoma cell line independently of perforin or Fas-FasL (19). This cytokine did not contribute to lysis of the CTLs used in this study because addition of blocking antibody to TNF-α did not inhibit the cytotoxicity.
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21
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1842276052
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51Cr release by macrophages in the absence of T cells was <15%.
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1842382776
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note
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51Cr release was determined after a 4-hour incubation. Expression of Fas on the target cells was not affected by infection with M. tuberculosis, as determined by flow cytometry.
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40
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1842350920
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L-lysine thiobenzyl (BLT)-esterase in the supernatant was determined by the BLT-esterase assay (23). The supernatants (20 μl) were coincubated with 35 μl of 1 mM BLT (Sigma), 35 μl of 1 mM 5,5′-dithio-bis-(2-nitrobenzoic acid) (Sigma), and 10 μl of a 0.1% Triton X-100 (Sigma) solution. After a 30-min incubation at 37°C, the absorbance at 405 nm was determined.
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32P and polynucleotide kinase (Boehringer Mannheim, Germany) and visualized by autoradiography.
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1842303014
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note
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We are indebted to M. Horwitz for use of his P3 laboratory and P. Sieling for continuous support and helpful discussions. Supported by the AIDS Stipendium; Deutsches Krebsforschungszentrum, Heidelberg (S.S.); NIH (R.L.M., S.A.P., B.R.B., M.B.B.); the Arthritis Foundation (S.A.P.); the Howard Hughes Medical Institute (B.R.B.); the Swiss Foundation for Grants in Medicine and Biology (J.-P.R); and the United Nations Development Programme-World Bank-World Health Organization Special Program for Research and Training in Tropical Diseases (IMMLEP) and the Dermatologic Research Foundation of California.
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