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Specific Target Cell Lysis Cells and Facilitates Exocytosis-Independent Antigen-Dependent Release of IFN-γ by Cytotoxic T Cells Up-Regulates Fas on Target Cells and Facilitates Exocytosis-Independent Specific Target Cell Lysis This information is current as of September 27, 2021. Arno Müllbacher, Mario Lobigs, Ron Tha Hla, Thao Tran, Thomas Stehle and Markus M. Simon J Immunol 2002; 169:145-150; ; doi: 10.4049/jimmunol.169.1.145 http://www.jimmunol.org/content/169/1/145 Downloaded from References This article cites 38 articles, 22 of which you can access for free at: http://www.jimmunol.org/content/169/1/145.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 27, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Antigen-Dependent Release of IFN-␥ by Cytotoxic T Cells Up-Regulates Fas on Target Cells and Facilitates Exocytosis-Independent Specific Target Cell Lysis Arno Mu¨llbacher,1* Mario Lobigs,* Ron Tha Hla,* Thao Tran,† Thomas Stehle,† and Markus M. Simon† Effector cytolytic T (Tc) lymphocytes, deficient in the exocytosis-mediated pathway of target cell lysis, induce Fas on target cells and, in turn, delayed cell death and apoptosis via the Fas ligand-Fas interaction. The induction of Fas can be blocked by anti- IFN-␥ Abs. This Fas up-regulation on initially Fas-negative target cells is not mediated by TCR-MHC/peptide signaling per se, but by secreted IFN-␥ from Tc cells after Ag engagement. The Fas up-regulation by Tc cells can be mimicked by treatment of target cells with rIFN-␥. Tc cells from IFN-␥ knockout mice do not induce Fas expression on target cells. Tc cell-mediated Fas expression Downloaded from on third party, bystander, target cells does not enhance their susceptibility to lysis by these nominal effector cells. The results are discussed as to the possible relevance of the phenomenon in efficiency and regulation of the Tc cell response to infections by viruses. The Journal of Immunology, 2002, 169: 145–150. D8ϩ effector T cells exert their biological activity via We have shown previously (17) that delayed target cell lysis and two quite distinct means upon specific encounter with apoptosis by alloreactive cytolytic T cells from perf-deficient http://www.jimmunol.org/ C target cells. One is the synthesis and release of cytokines (perfϪ/Ϫ) mice proceed via the Fas pathway. This conclusion was such as IFN-␥ (1) and TNF-␣ (2), which may act proximal and reached for the following reasons: 1) Fas expression, on originally distal to the effector cell. The other is direct cytolysis and apoptosis Fas-negative (Fasneg) targets, after incubation with Tc cells from Ϫ Ϫ of the bound target cell. These two distinct effector mechanisms perf / mice, was increased in a time-dependent manner; 2) target are not necessarily executed by the same CD8ϩ T cell. Evidence cells had to be biosynthetically active, i.e., RNA and protein syn- for such is the vastly different numbers of Ag-specific CD8ϩ T thesis was required as well as protein transport for lysis to occur; cells identified as IFN-␥ producers vs estimates of cytolytic T 3) Abs to Fas or soluble Fas-Fc inhibited cytolysis and nucleolysis; (Tc)2 cells using limiting dilution techniques (3), and our recent 4) poxvirus-encoded serpins, in particular SPI-2, a strong inhibitor finding that in response to flavivirus infection, Tc cells with lytic of the Fas pathway (18–20), completely inhibited lysis of target by guest on September 27, 2021 Ϫ Ϫ activity are not always producers of IFN-␥ (4). cells by perf / Tc cells; 5) brefeldin A treatment of Tc cells Apoptosis and lysis of target cells by cytolytic lymphocytes (NK severely reduced lysis, which interferes with polypeptide transport, and Tc cells) can be executed via at least two distinct pathways: but only marginally with concanamycin A, an inhibitor of exocy- one, the exocytosis pathway mediated by perforin (perf) and gran- tosis (21); 6) target cells derived from the Fas-defective mutant zymes (gzm); the other, by the Fas pathway involving the Fas mouse (lpr) were refractory to lysis; and finally 7) Tc effector cells Ϫ/Ϫ ligand (FasL) on the effector cell engaging the Fas receptor (Fas or from perf ϫ gld mice, defective in both cytolytic effector path- neg CD95) on the target cell (5–12). It is generally believed that the ways, did not exhibit this lytic phenotype on Fas target cells. exocytosis pathway is primarily involved in the elimination and/or In this study, we present evidence as to the mechanism by which neg control of intracellular pathogens such as viruses. The Fas pathway Tc cells induce Fas expression on Fas target cells, investigate its of cytotoxicity, in contrast, was thought to be in essence immu- consequence on bystander killing, and speculate on the signifi- noregulatory (11, 13, 14). However, some recent studies (15), in- cance of this process in recovery from viral infections. cluding our own on flavivirus-induced cytotoxicity (16), suggest that in certain virus infections both pathways are operative. Materials and Methods Animals C57BL/6 (KbDb) (B6), AKR/N (KkDk) (AKR), B10.HTG (KdDb) (HTG), the perfϪ/Ϫ mutant (8), the triple knockout mouse, deficient in perf plus Ϫ Ϫ *Division of Immunology and Cell Biology, John Curtin School of Medical Research, gzm A and B (perfϫgzmAϫB / ) (17), and the IFN-␥-deficient mouse Australian National University, Canberra, Australia; and †Max-Planck-Institut fu¨r Im- (IFN-␥Ϫ/Ϫ) (22) were bred under pathogen-free conditions at the Animal munbiologie, Stu¨beweg, Freiburg, Germany Breeding Facility of the John Curtin School of Medical Research or the Received for publication February 28, 2002. Accepted for publication May 1, 2002. animal facilities of the Max-Planck-Institute for Immunbiology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance Cell lines with 18 U.S.C. Section 1734 solely to indicate this fact. The mouse cell lines L929 (H-2k), L1210 (H-2d), and L1210.Fas (trans- 1 Address correspondence and reprint requests to Dr. Arno Mu¨llbacher, Division of fected with mouse Fas; kindly provided by P. Golstein, Marseilles, France) Immunology and Cell Biology, John Curtin School of Medical Research, Australian were grown in Eagle’s MEM (EMEM) supplemented with 10% FCS. National University, P.O. Box 334, Canberra, ACT 2601, Australia. E-mail address: [email protected] FACS analysis 2 Abbreviations used in this paper: Tc, cytolytic T; DC, dendritic cell; Ect, Ectrome- lia; EMEM, Eagle’s MEM; FasL, Fas ligand; gzm, granzyme; neg, negative; perf, Cells were stained for Fas expression using the FITC-conjugated mAb perforin. specific for mouse Fas (Jo-2; PharMingen, Hamburg, Germany). Cells Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00 146 Tc CELLS UP-REGULATE Fas ON TARGETS BY SECRETED IFN-␥ were examined with a FACScan flow cytometer (BD Biosciences, San lation. Initially, we explored whether signaling by the Tc cell via Jose, CA). MHC class I on the target cell was responsible for inducing Fas neg Treatment of L929 cells with rIFN-␥ expression on Fas targets. We failed to mimic Fas up-regulation on Fasneg L929 (H-2k) targets by a panel of anti-Kk or anti-Dk Abs. L929 were plated in 24-well Costar (Cambridge, MA) plates and treated neg ␥ ␮ In addition, Fas L929 target cells transfected with MHC class I with 300 U rIFN- (Genzyme, Cambridge, MA; MG-IFN; 200 U/ l) for 4, d 6, and 22 h. Cells were trypsinized and aliquots were stained for cell sur- K cDNA, lacking the cytoplasmic domain, still induced Fas after d face Fas expression and Fas transcript quantitation by PCR. recognition by K -reactive Tc cells (data not shown). Thus, it was unlikely that Fas expression was induced by signaling via MHC Quantitation of Fas transcripts class I directly. Therefore, we considered the second option, i.e., mRNA was isolated from cell lines using TriReagent, following the in- cytokine-induced up-regulation of Fas on target cells. struction of the manufacturer (T9424; Sigma-Aldrich, St. Louis, MO). Af- ter treatment with 2 ␮l(2␮g) DNase (Roche Molecular Biochemicals, Ag-mediated release of soluble mediators induces Fas Mannheim, Germany), mRNA was incubated with 1 ␮l oligo(dT) 12–18 expression primer (500 ng; Pharmacia, Freiburg, Germany) and Omniscript RT (4 U; Qiagen, Hilden, Germany). RT-PCR was done as described by Qiagen. Double-chamber experiments were performed to investigate Aliquots of 50–100 ng cDNA were used as template for PCR amplification whether soluble mediators were responsible for the up-regulation of mouse Fas using the primer pairs, as described (17, 23). PCR products Ϫ Ϫ of Fas on Fasneg L929 targets. Splenocytes from perfϫgzmAϫB / were amplified with 35 cycles, separated by 1.5% agarose gel electrophore- b sis, and visualized by ethidium bromide staining. Quantitation of PCR mice (H-2 )deficient in exocytosis-mediated cytotoxicity were cocul- products was done using the NIH Image software (version 1.62; freeware tured in vitro with B10.HTG (KdDb) stimulator cells and tested for from National Institutes of Health download page) by analyzing the density their ability to lyse Fasneg L1210 targets after 6- and 20-h assay time Downloaded from of the scanned bands.
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