Characterization of the Fas ligand/Fas-dependent apoptosis of antiretroviral, class I MHC tetramer-defined, CD8+ CTL by in vivo retrovirus-infected cells.
C57BL/6 (B6; H-2(b)) mice mount strong AKR/Gross murine leukemia virus (MuLV)-specific CD8(+) CTL responses to the immunodominant K(b)-restricted epitope, KSPWFTTL, of endogenous AKR/Gross MuLV. In sharp contrast, spontaneous virus-expressing AKR.H-2(b) congenic mice are low/nonresponders for the generation of AKR/Gross MuLV-specific CTL. Furthermore, when viable AKR.H-2(b) spleen cells are cocultured with primed responder B6 antiviral precursor CTL, the AKR.H-2(b) cells function as "veto" cells that actively mediate the inhibition of antiviral CTL generation. AKR.H-2(b) veto cell inhibition is virus specific, MHC restricted, contact dependent, and mediated through veto cell Fas ligand/responder T cell Fas interactions. In this study, following specific priming and secondary in vitro restimulation, antiretroviral CD8(+) CTL were identified by a labeled K(b)/KSPWFTTL tetramer and flow cytometry, enabling direct visualization of AKR.H-2(b) veto cell-mediated depletion of these CTL. A 65-93% reduction in the number of B6 K(b)/KSPWFTTL tetramer(+) CTL correlated with a similar reduction in antiviral CTL cytotoxicity. Addition on sequential days to the antiviral CTL restimulation cultures of either 1) AKR.H-2(b) veto cells or 2) a blocking Fas-Ig fusion protein (to cultures also containing AKR.H-2(b) veto cells) to block inhibition demonstrated that AKR.H-2(b) veto cells begin to inhibit B6 precursor CTL/CTL expansion during days 2 and 3 of the 6-day culture. Shortly thereafter, a high percentage of B6 tetramer(+) CTL cocultured with AKR.H-2(b) veto cells was annexin V positive and Fas(high), indicating apoptosis as the mechanism of veto cell inhibition. Experiments using the irreversible inhibitor emetine demonstrated that AKR.H-2(b) cells had to be metabolically active and capable of protein synthesis to function as veto cells. Of the tetramer-positive CTL that survived veto cell-mediated apoptosis, there was no marked skewing from the preferential usage of Vbeta4, 8.1/8.2, and 11 TCR normally observed. These findings provide further insight into the complexity of host/virus interactions and suggest a fail-safe escape mechanism by virus-infected cells for epitopes residing in critical areas of viral proteins that cannot accommodate variations of amino acid sequence.
Rich
Robert F
RF
Department of Microbiology and Immunology and The Norris Cotton Cancer Center, Dartmouth Medical School, Lebanon, NH 03756, USA.
Green
William R
WR
D000406:AKR murine leukemia virus / Q000254:growth & development / Q000276:immunology*; D000818:Animals; D017209:Apoptosis / Q000276:immunology*; D002455:Cell Division / Q000276:immunology; D017637:Clonal Deletion; D003602:Cytotoxicity, Immunologic; D018984:Epitopes, T-Lymphocyte / Q000032:analysis / Q000276:immunology; D053222:Fas Ligand Protein; D006131:Growth Inhibitors / Q000096:biosynthesis / Q000502:physiology; D006183:H-2 Antigens / Q000032:analysis*; D018960:Hyaluronan Receptors / Q000096:biosynthesis; D007700:Kinetics; D008024:Ligands; D008213:Lymphocyte Activation; D018655:Lymphocyte Count; D008562:Membrane Glycoproteins / Q000502:physiology*; D051379:Mice; D008806:Mice, Inbred AKR; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D010446:Peptide Fragments / Q000096:biosynthesis / Q000276:immunology; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000096:biosynthesis; D016176:T-Lymphocyte Subsets / Q000166:cytology / Q000276:immunology / Q000378:metabolism / Q000821:virology*; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology / Q000276:immunology / Q000378:metabolism / Q000821:virology*; D014407:Tumor Cells, Cultured; D019014:fas Receptor / Q000096:biosynthesis / Q000378:metabolism / Q000502:physiology
D018984:Epitopes, T-Lymphocyte; D053222:Fas Ligand Protein; C505658:Fasl protein, mouse; D006131:Growth Inhibitors; D006183:H-2 Antigens; C032876:H-2Kb protein, mouse; D018960:Hyaluronan Receptors; D008024:Ligands; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D016693:Receptors, Antigen, T-Cell, alpha-beta; D019014:fas Receptor