Cytolytic Mechanisms and Expression of Activation-Regulating Receptors on Effector-Type CD8+CD45RA+CD27− Human T Cells This information is current as of September 29, 2021. Paul A. Baars, Laura M. Ribeiro do Couto, Jeanette H. W. Leusen, Berend Hooibrink, Taco W. Kuijpers, Susanne M. A. Lens and René A. W. van Lier J Immunol 2000; 165:1910-1917; ; doi: 10.4049/jimmunol.165.4.1910 Downloaded from http://www.jimmunol.org/content/165/4/1910 References This article cites 45 articles, 19 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/165/4/1910.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 29, 2021 • Fast Publication! 4 weeks from acceptance to publication *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 © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Cytolytic Mechanisms and Expression of Activation-Regulating Receptors on Effector-Type CD8؉CD45RA؉CD27؊ Human T Cells Paul A. Baars,1* Laura M. Ribeiro do Couto,*1,2,3 Jeanette H. W. Leusen,† Berend Hooibrink,* Taco W. Kuijpers,* Susanne M. A. Lens,4* and Rene´A. W. van Lier5* Circulating CD8؉ T cells with a CD45RA؉CD27؊ phenotype resemble cytolytic effector cells because they express various cytolytic mediators and are able to execute cytotoxicity without prior stimulation in vitro. We here demonstrate that .CD8؉CD45RA؉CD27؊ T cells can use both granule exocytosis and Fas/Fas ligand pathways to induce apoptosis in target cells The availability of these cytolytic mechanisms in circulating T cells suggests that the activity of these cells must be carefully controlled to prevent unwanted tissue damage. For this reason, we analyzed the expression of surface receptors that either enhance Downloaded from or inhibit T cell function. Compared with memory-type cells, effector cells were found to express normal levels of CD3⑀ and TCR␨ and relatively high levels of CD8. CTLA-4 was absent from freshly isolated effector cells, whereas a limited number of unstimu- lated memory cells expressed this molecule. In line with recent findings on CD8؉CD28؊ T cells, CD45RA؉CD27؊ T cells were unique in the abundant expression of NK cell-inhibitory receptors, both of Ig superfamily and C-type lectin classes. Binding of NK cell-inhibitory receptors to classical and nonclassical MHC class I molecules may inhibit the activation of the cytolytic machinery ؉ ؉ ؊ induced by either Ag receptor-specific or nonspecific signals in CD8 CD45RA CD27 T cells. The Journal of Immunology, 2000, http://www.jimmunol.org/ 165: 1910–1917. ffective immune responses against viruses depend for a assumption, cytokine secretion profile, activation requirements, large part on the induction of CTL that lyse virally in- and the expression of adhesion and homing receptors on fected cells. Studies initially performed in mice have CD45RAϪCD45R0ϩ T cells resemble those of Ag-experienced E Ϫ ϩ shown that after infection, viral peptide-specific T cells undergo T cells (8, 9). Next to the CD45RA CD45R0 subset, a second massive expansion and rapidly acquire effector function (1–3). Af- subpopulation with features of in vivo priming is found in hu- ter elimination of the pathogen, most of the CD8ϩ Ag-specific man peripheral blood. This population is characterized by the lymphocytes die as a result of apoptosis. However, a portion of absence of the costimulatory molecules CD28 (10) and CD27 by guest on September 29, 2021 specific T cells persists (1) and constitutes a pool of memory cells, (7), the presence of both CD45RA and CD57 Ags (11), and which on challenge with the same or a similar pathogen is able to abundant expression of CD11a (12). Because CD8ϩ T cells mount a more forceful immune response (4). Recent studies on within this subset express cytolytic mediators such as perforin, EBV infection have shown that similar mechanisms are opera- granzyme A and B, and Fas ligand (FasL) 6 mRNA and are able tional in the human system (5). to execute cytotoxicity without prior in vitro stimulation, it is In humans, cell surface marker analysis has been thoroughly suggested that they represent effector cells in vivo (13). Inter- ϩ used to discern functional distinct subsets of CD8 T cells. estingly, although these effector type cells have poor prolifer- Proliferative responses to viral Ags are predominantly confined ative potential in vitro, it has been documented that these cells Ϫ ϩ to the CD45RA CD45R0 subset, which suggests that this increase with age (14). The recent observation (15) that these population contains memory-type cells (6, 7). In line with this cells lack the chemokine receptor CCR7 indicates that they do not recirculate through the secondary lymphoid organs but *Department of Immunobiology and Laboratory for Experimental and Clinical Im- rather migrate to sites of inflammation. Finally, compatible with munology, Academic Medical Center, CLB, University of Amsterdam, Amsterdam, selection by specific Ag in vivo is the observation that their † The Netherlands; and Department of Cell Biology, University Medical Center Utre- ␤ cht, Utrecht, The Netherlands TCR V repertoire is strongly skewed when compared with ϩ Received for publication January 18, 2000. Accepted for publication June 6, 2000. either naive or memory CD8 T cells (16). Indeed, recent stud- ies using HLA/viral-peptide tetrameric complexes have demon- The costs of publication of this article were defrayed in part by the payment of page ϩ Ϫ charges. This article must therefore be hereby marked advertisement in accordance strated that specific CD8 T cells with CD28 and with 18 U.S.C. Section 1734 solely to indicate this fact. CD45RAϩCD27Ϫ phenotypes can be found in EBV, hepatitis C 1 P.A.B. and L.M.R. contributed equally to this study. virus, and CMV carriers (17, 18). 2 L.R. was supported by Fundac¸a˜o para a Cieˆncia e a Tecnologia, Portugal (Grant Studies with in vitro expanded T cell clones and experiments in PRAXIS XXI/BD/9156/96). mutant mice have provided evidence that CTL can exert their ef- 3 Current address: Department of Molecular Immunology, National Institute of Public fector functions by at least two independent pathways: granule Health and Environmental Protection, P.O. Box 1, 3720 BA Bilthoven, The Netherlands. exocytosis and the Fas/FasL pathway (19). The granule exocytosis 4 Current address: Institut Biochimi Universite´de Lausanne, Chemin de Boveresses 155, 1066 Epalinges, Switzerland. 5 Address correspondence and reprint requests to Dr. Rene´A. W. van Lier, Depart- ment of Immunobiology, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands. 6 Abbreviations used in this paper: FasL, Fas ligand; RT, room temperature; CMA, E-mail address: [email protected] concanamycin A; NKRs, NK cell-inhibitory receptors. Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 The Journal of Immunology 1911 pathway involves secretion of granules containing cytotoxic effec- cells with anti-human granzyme A Ab (45 min at RT). Cells were then tor molecules onto the surface of target cells. Perforin plays a washed three times and incubated with goat anti-mouse IgG FITC (30 min critical role in this pathway, because it can polymerize to form at RT). Moviol containing 2.5% diazabicyclo octane (Fluka, Buchs, Swit- zerland) was used to mount on the cells. For immediate analysis, we made channel-like structures in target cell membrane, through which use of a Leitz DMIRB fluorescence microscope (Leica, Voorburg, The granzymes can enter and subsequently activate the death machin- Netherlands) interfaced with a Leica TCS4D confocal laser microscope ery (20). Alternatively, CTLs can use the Fas pathway to kill their (Leica, Heidelberg, Germany). Images were imported in Adobe Photoshop target cells. This cytolytic pathway is mainly based on cell-cell 4.0 (Adobe Systems, San Jose, CA). interaction between Fas, expressed on the target cell, and FasL, Induction and measurement of granzyme release expressed on the CTL (21). Engagement of the Fas receptor results ϩ in the aggregation of its intracellular death domains, followed by CD8 subpopulations were isolated as described above. Cells were cul- tured in triplicate in 96-well plates (0.3 ϫ 106 cells/well) in either the the activation of several caspases with the ultimate death of the presence or the absence of 1 ng/ml PMA and 1 ␮M ionomycin (both from target cell (22). We here show that both granule exocytosis and Sigma, St. Louis, MO). Supernatants were harvested after 1 h culture, and FasL pathways are readily operational in circulating effector-type the levels of soluble granzyme A and B were measured by ELISA. Stim- CD8ϩ T cells. Furthermore, in search for potential mechanisms ulated cells were used for intracellular evaluation of these molecules ac- that could control the cytotoxic machinery of these cells in vivo, cording to the protocol described below. ⑀ ␨ Granzyme A and B concentrations in culture supernatants were mea- we found normal expression of CD3 and CD3 chains and ab- sured by specific solid phase sandwich ELISA as previously described (27) sence of CTLA-4 expression. However, in agreement with recent with some modifications. The granzyme A ELISA was performed using the finding on CD28Ϫ T cells (23, 24), a strong increase in the ex- GA29 mAb (coating Ab; CLB) and the biotinylated GA28 mAb (detection pression of various classes of killer-inhibitory receptors was ob- Ab; CLB).