Induction by concanavalin A of specific mRNAs and cytolytic function in a CD8-positive T cell hybridoma.
A previous report from this laboratory described the production of CD8+, class I-specific T cell hybridomas which developed specific cytolytic activity and the ability to secrete IL-2 upon Con A or specific Ag stimulation. Unlike normal lymphocytes or long-term CTL lines for which exposure to Ag triggers both differentiation and proliferation, T cell hybridoma lines can be activated functionally against a background of continuous proliferation. They therefore provide a unique system with which to study the molecular events involved in the induction of cytolytic function. The expression of mRNA from a series of genes was evaluated by Northern hybridization at various times after Con A stimulation of the H-2Ld-specific CD8+ 3D9 hybridoma. Induction of the c-fos proto-oncogene by 45 min poststimulation was followed shortly by c-myc induction. Perforin mRNA was expressed at a low level in the unstimulated hybridomas, but was down-regulated upon Con A stimulation to levels undetectable by PCR. Interestingly, production of granzyme A mRNA was strongly induced by 45 min after Con A stimulation. In the CD8+ RT-1.3G3 hybridoma, which is nonlytic and specific for the HIV-1 envelope glycoprotein, c-fos but not granzyme A mRNA was induced by 45 min poststimulation, and no granzyme A mRNA was detectable at any time. Thus, a significant role for granzyme A in the induction of cytolytic activity is suggested. Cytolysis by the 3D9 hybridoma involved both target cell membrane damage and DNA fragmentation, and both Ca(2+)-dependent and Ca(2+)-independent cytolysis were observed. Although TNF-alpha mRNA was induced by 4 h poststimulation, Ab to TNF-alpha failed to inhibit the Ca(2+)-independent lysis observed, leaving the basis for the observed Ca(2+)-independent lysis unexplained.
Department of Microbiology, University of Texas at Austin 78712.
J J
JJ
Gu
J V
JV
Harriss
K
K
Ozato
P D
PD
Gottlieb
D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D018414:CD8-Positive T-Lymphocytes / Q000187:drug effects / Q000276:immunology*; D003208:Concanavalin A / Q000494:pharmacology*; D003601:Cytotoxicity Tests, Immunologic; D003602:Cytotoxicity, Immunologic / Q000187:drug effects* / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects* / Q000276:immunology; D006825:Hybridomas / Q000187:drug effects / Q000276:immunology*; D008562:Membrane Glycoproteins / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D054353:Perforin; D016133:Polymerase Chain Reaction; D052899:Pore Forming Cytotoxic Proteins; D011519:Proto-Oncogenes / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis*
D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D012333:RNA, Messenger; D003208:Concanavalin A; D054353:Perforin