Fas Ligand Costimulates the In Vivo Proliferation of CD8 + T Cells Ivy Suzuki, Stefan Martin, Tamar E. Boursalian, Courtney Beers and Pamela J. Fink This information is current as of October 2, 2021. J Immunol 2000; 165:5537-5543; ; doi: 10.4049/jimmunol.165.10.5537 http://www.jimmunol.org/content/165/10/5537 Downloaded from References This article cites 29 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/165/10/5537.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • 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 *average by guest on October 2, 2021 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 © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Fas Ligand Costimulates the In Vivo Proliferation of CD8؉ T Cells1 Ivy Suzuki, Stefan Martin,2 Tamar E. Boursalian, Courtney Beers, and Pamela J. Fink3 Fas ligand (FasL/CD95L/APO-1L) is one of a growing number of TNF family members whose triggering costimulates maximal proliferation of activated T cells. In this study we show that maximal Ag-dependent accumulation of transferred TCR-transgenic CD8؉ T cells requires Fas (CD95/APO-1) expression by the adoptive hosts. Additionally, adoptively transferred FasL؉ CD8؉ T cells demonstrate a 2-fold advantage in Ag-driven expansion over their FasL؊counterparts. This study illustrates the in vivo role of TCR-dependent FasL costimulation in the Ag-specific proliferation of both heterogeneous and homogeneous populations of primary CD8؉ T cells and long-term CTL lines. Thus, cross-linking FasL on naive and Ag-experienced CD8؉ T cells whose Ag-specific TCRs are engaged is required to drive maximal cellular proliferation in vivo. The Journal of Immunology, 2000, 165: 5537–5543. Downloaded from embers of the TNF and TNF receptor (TNFR)4 fami- by direct cell-cell contact, little is known about the mechanism of lies comprise the majority of activation-induced co- reverse signaling. It is likely that the cytoplasmic domains of these M stimulatory receptor-ligand pairs, which serve both to molecules are involved in signal transduction, as the cytoplasmic enhance T cell expansion and differentiation and to mediate T tails of TNF family members are distinct from each other yet con- cell-B cell interactions (reviewed in Refs. 1 and 2). The TNFR served across species (1). family includes CD30, CD40, Fas, TNFR1, and TNFR2, all type I Our work has focused on bipolar signaling by FasL. Previous in http://www.jimmunol.org/ membrane proteins whose extracellular ligand-binding region is vitro studies from our laboratory provide compelling evidence that characterized by repeating cysteine-rich domains. The TNF family FasL acts as a costimulatory receptor in CD8ϩ T cells. From our includes CD30 ligand (CD30L), CD40L, Fas ligand (FasL), lym- initial studies (7), three lines of evidence support a role for FasL in photoxin ␣ (LT␣), LT␤, TNF-␣, and TNF-␤. With the exception positive reverse signaling. First, multiple CD8ϩ CTL cell lines, of LT␣, these molecules are type II membrane proteins character- independently derived from FasLϪ gld mice on a C57BL/6 (B6) ized by a homologous sequence in the extracellular C terminus. background, exhibit depressed proliferation upon alloantigenic The crystallization of TNFR1, LT␣, and TNF illustrates that the stimulation compared with CTL lines derived from wild-type B6 receptor-ligand interactions between these families involve one mice or FasϪ lpr mice. This was not due to differences in kinetics homotrimeric ligand binding to three cross-linked receptors orga- of proliferation among the cell lines, overexpression of Fas on the by guest on October 2, 2021 nized in a predictable cluster (3, 4). gld cell lines (which could result in increased apoptosis of these The TNF family members 4-1BBL, CD27L, CD30L, CD40L, cells), or a general defect in signaling in the gld cells. Our findings FasL, OX40L, and TNF-␣ all exhibit the capacity to reverse signal, suggest that the presence of FasL is important for inducing max- to transduce a signal inward upon binding specific receptors (2, imal proliferation of CTLs. The second major line of evidence 5–8). Bipolar signaling may turn out to be the norm for this family implicating FasL in positive signaling used soluble FasIgG fusion of molecules, thereby blurring the distinction between receptor and protein (a chimeric molecule of human IgG and the ligand-binding ligand. Although it is known that these ligands are mainly mem- portion of murine Fas) to block cell surface FasL-Fas interactions. brane-bound molecules that primarily interact with their receptors In a dose-dependent manner, soluble FasIgG, but not isotype- matched control human IgG, attenuated the proliferation of wild- type B6 CTLs down to the level achieved by gld responders. A Department of Immunology, University of Washington, Seattle, WA 98195 third line of evidence showed that cross-linking FasL with plate- Received for publication February 28, 2000. Accepted for publication August 21, 2000. bound FasIgG, in conjunction with suboptimal amounts of anti- CD3, delivered a costimulatory signal for proliferation to FasLϩ The costs of publication of this article were defrayed in part by the payment of page Ϫ charges. This article must therefore be hereby marked advertisement in accordance but not FasL CTL. We extended these initial studies to examine with 18 U.S.C. Section 1734 solely to indicate this fact. the role of FasL costimulation in CD8ϩ vs CD4ϩ peripheral T 1 This work was supported by National Institutes of Health Research Grants AG13078 cells (9). We found that naive CD4ϩ T cells can also respond to and AI 44130, CFS900 from the Cystic Fibrosis Foundation, and Basic Immunology Training Grant CA09537A. FasL costimulation when Fas-mediated death is prevented, and ϩ ϩ 2 Current address: Clinical Research Group, Department of Dermatology, University that the observed differences between CD4 and CD8 T cells of Freiburg, Hauptstrasse 7, D-79104 Freiburg, Germany. stem partly from the differential control of FasL expression in 3 Address correspondence and reprint requests to Dr. Pamela J. Fink, Department of these two cell types. FasL costimulation occurs very early during Immunology, University of Washington, Box 357650, Room H574A, Seattle, WA the course of an MLC, at a time when FasL expression is induced 98195. E-mail address: pfi[email protected] on both CD4ϩ and CD8ϩ T cells meeting Ag for the first time. In 4 Abbreviations used in this paper: TNFR, TNF receptor; B6, C57BL/6; B6.gld, gld lpr a b contrast, Fas-mediated death occurs late in an immune response B6Smn.C3H-Fasl ; B6.lpr, B6.MRL-Fas ; B6.Ly5.1, B6.SJL-Ptprc Pep3 /BoyJ ϩ (Ly5.1); B6.Thy-1.1, B6.PL-Thy-1a/Cy; B6.wt, wild-type C57BL/6; C3H, C3HeB/ when high levels of FasL expression are maintained on CD4 T FeJ; L, ligand; LN, lymph node; LT, lymphotoxin; OT-1, line of OVA-specific TCR- cells, rendering them sensitive to apoptosis, whereas CD8ϩ T transgenic mice; OT-1.gld, OT-1 on a B6.gld background; OT-1.Thy-1.1, OT-1 on a B6.Thy-1.1 background; OVAp, OVA peptide; VSV, vesicular stomatitis virus; cells are relatively insensitive to this signal. Taken together, VSVp, VSV peptide. these in vitro data provide evidence that FasL plays dual Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 5538 FAS LIGAND COSTIMULATION IN VIVO functions in the regulation of CD8ϩ and CD4ϩ T cells, having Flow cytometry the capacity to both positively and negatively regulate the PBLs, splenocytes, and lymph node (LN) cells were stained as described peripheral T cell compartment. previously (19) and analyzed on a FACScan using CellQuest software The in vivo consequence of FasL costimulation is the focus of (Becton Dickinson, Mountain View, CA). Dead cells were excluded on the the current study. In general, costimulation has multiple functions. basis of forward and side scatter profiles, and at least 104 live-gated events First, costimulation may regulate the production of cytokines and were collected. their receptors that together help regulate cell proliferation. Sec- ond, costimulation may strengthen the TCR signal in both ampli- Generation and maintenance of Ag-specific CTLs tude and duration, possibly by enhancing the redistribution and Alloreactive H-2k-specific CTLs were generated by incubating naive clustering of raft microdomains at the TCR engagement site (10, spleen and LN cells from age-matched B6, B6.lpr, and B6.gld mice with an 11). Third, costimulation may enhance the long-term survival of equal number of irradiated (3000 rad) C3H splenocytes, as previously de- scribed (7). Lines were maintained by stimulation every 8–10 days. After Ag-activated T lymphocytes (12). Fourth, costimulation may play the third stimulation, the medium was supplemented with 50 mM ␣-methyl a role in the differentiation of T cells into mature effector subsets mannoside and 5% supernatant from rat cells stimulated with 3 ␮g/ml Con by altering the Th1/Th2 balance (13, 14) and regulating CTL ef- A for 2 days. All CTL lines were routinely monitored by flow cytometry fector development (15). and CTL assay.