Signaling Through a CD3γ-Deficient TCR/CD3 Complex in Immortalized Mature CD4+ and CD8+ T Lymphocytes This information is current as Alberto Pacheco-Castro, David Alvarez-Zapata, Pilar of September 25, 2021. Serrano-Torres and José R. Regueiro J Immunol 1998; 161:3152-3160; ; http://www.jimmunol.org/content/161/6/3152 Downloaded from References This article cites 47 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/161/6/3152.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 September 25, 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 © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Signaling Through a CD3g-Deficient TCR/CD3 Complex in Immortalized Mature CD41 and CD81 T Lymphocytes1 Alberto Pacheco-Castro,2 David Alvarez-Zapata,2 Pilar Serrano-Torres, and Jose´R. Regueiro3 The biologic role of each CD3 chain and their relative contribution to the signals transduced through the TCR/CD3 complex and to downstream activation events are still controversial: they may be specialized or redundant. We have immortalized peripheral blood CD41 and CD81 T lymphocytes from a human selective CD3g deficiency using Herpesvirus saimiri. The accessibility of the mutant TCR/CD3 complex to different Abs was consistently lower in immortalized CD81 cells when compared with CD41 cells, relative to their corresponding CD3g-sufficient controls. Several TCR/CD3-induced downstream activation events, immediate (calcium flux), early (cytotoxicity and induction of surface CD69 or CD40L activation markers or intracellular TNF-a) and late (proliferation and secretion of TNF-a), were normal in g-deficient cells, despite the fact that their TCR/CD3 complexes were significantly less accessible than those of controls. In contrast, the accumulation of intracellular IL-2 or its secretion after CD3 Downloaded from triggering was severely impaired in g-deficient cells. The defect was upstream of protein kinase C activation because addition of transmembrane stimuli (PMA plus calcium ionophore) completely restored IL-2 secretion in g-deficient cells. These results suggest that the propagation of signals initiated at the TCR itself can result in a modified downstream signaling cascade with distinct functional consequences when g is absent. They also provide evidence for the specific participation of the CD3g chain in the induction of certain cytokine genes in both CD41 and CD81 human mature T cells. These immortalized mutant cells may prove to be useful in isolating cytosolic signaling pathways emanating from the TCR/CD3 complex. The Journal of Immunology, 1998, http://www.jimmunol.org/ 161: 3152–3160. cells detect the presence of Ags by way of a surface het- ing molecules (for a review, see Ref. 4). Isolated CD3e or -z erodimer termed the TCR. TCR molecules are not ex- ITAMs cannot induce mature T cell proliferation (5) and ablation T pressed alone; they require association with a group of of CD3d blocks ab, but not gd T cell development (6). monomorphic proteins, collectively called CD3. At least four types We have attempted to address this question for the CD3g chain of CD3 proteins, termed g, d, e, and z, have been reported. CD3 by studying the functional behavior of human mature T cells de- proteins are believed to maintain TCR/CD3 expression and to par- rived from a natural selective CD3g deficiency (7). To circumvent ticipate in the delivery of signals that drive T cell maturation or the inherent difficulties of growing primary T cells and our inabil- by guest on September 25, 2021 apoptosis in the thymus and T cell activation or anergy in the ity to obtain CD81 T cell lines (8), we have used Herpesvirus periphery (1). During early T cell development, some CD3 chains saimiri (HVS), a common lymphotropic virus of squirrel monkeys, may act alone or assist immature TCR ensembles, such as those known to immortalize both CD41 and CD81 human T lympho- containing pre-TCRa (2). However, their relative contribution to cytes (9, 10). Immortalized cells remain IL-2-dependent, but be- the signals that are propagated through the cytoplasm and that come Ag- and mitogen-independent for their continued growth result in distal activation events is a matter for discussion. CD3 (11). However, they do display normal downstream functional re- proteins may have partially overlapping functions, as all CD3 com- sponses (proliferation, cytokine synthesis, induction of activation 4 ponents display a shared amino acid motif called ITAM (immu- markers, cytotoxicity, etc.) when their TCR/CD3 activation path- noreceptor tyrosine-based activation motif) in their cytoplasmic way is triggered (12, 13). domains, which can by itself transduce several T cell differentia- tion and activation signals (for a review, see Ref. 3). Alternatively, they may have specialized functions, as ITAMs belonging to dif- Materials and Methods ferent CD3 chains show different affinities for downstream signal- Cell lines HVS-transformed T cell lines were derived from PBL of a healthy con- Inmunologı´a, Facultad de Medicina, Universidad Complutense, Madrid, Spain genital CD3g-deficient individual (DSF or III-2) (7, 14) and normal do- Received for publication December 4, 1997. Accepted for publication May 6, 1998. nors, as previously described (10). Briefly, PBLs were resuspended (2 3 6 The costs of publication of this article were defrayed in part by the payment of page 10 cells/ml) in a mixture (1:1 proportions) of two culture media [RPMI charges. This article must therefore be hereby marked advertisement in accordance 1640 medium from Biochrom (Berlin, Germany) and cell growth (CG) with 18 U.S.C. Section 1734 solely to indicate this fact. medium from Vitromex (Vilshofen, Germany)] supplemented with 10% 1 This work was supported by PR112/96 (Ministerio de Educacio´n y Cultura), 13/97 FCS (Flow Laboratories, Rockville, MD), 1% L-glutamine (BioWhitaker, (Comunidad Auto´noma de Madrid) and SAF96/119 (Comisio´n Interministerial de Berkshire, U.K.), 50 IU/ml human rIL-2 (Hoffmann-La Roche, Nutley, Ciencia y Tecnologı´a) grants to Jose´R. Regueiro. Alberto Pacheco-Castro and David NJ), and 1 mg/ml phytohemagglutinin (PHA; Difco Laboratories, Detroit, Alvarez-Zapata were supported by the Ministerio de Educacio´n y Cultura. MI). The same day of isolation for CD81 cells, or at day 3 for CD41 cells, 2 A.P.-C. and D.A.-Z. are joint first authors. they were resuspended at 2 3 106 cells/ml in CG/RPMI medium contain- 3 Address correspondence and reprint requests to Dr. Jose´R. Regueiro, Inmunologı´a, ing 50 IU/ml human rIL-2 and exposed once to 1 ml of HVS supernatant Facultad de Medicina, Universidad Complutense, 28040 Madrid, Spain. E-mail ad- in 24-well plates (Costar, Cambridge, MA). Thereafter, medium was re- dress: [email protected] placed every 3 to 4 days (no PHA, only rIL-2). An immortalized phenotype 4 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-based activation was indicated by the death of control cultures (i.e., non-HVS-exposed) vs motif; HVS, Herpesvirus saimiri C-488; CG, cell growth medium; PE, phycoerythrin; the sustained growth, presence of HVS genomes, and T lymphoblast cell MFI, mean fluorescence intensity. morphology of test cultures, as described (9, 15). HVS-exposed T cells had Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 The Journal of Immunology 3153 Table I. Surface markers of CD41 and CD81 CD3g-deficient (DSF4 and DSF8, respectively) and -sufficient (CTC4 and CTO8, respectively) HVS T cells Cell Type CD41 CD81 Percentagea Percentagea Molecule mAb CTC4 DSF4 MFI ratiob CTO8 DSF8 MFI ratiob CD3c Leu4 98 96 3.3 6 0.9 100 96 6.0 6 1.1 CD3 IOT3b 99 82 3.5 6 0.2 100 89 4.3 6 1.4 CD3 OKT3 97 74 3.7 6 0.7 99 73 4.7 6 0.2 CD3 X35 99 78 4.0 6 0.5 98 75 4.3 6 1.4 TCRabc BMA031 98 27 5.6 6 0.7 99 9 12.1 6 1.1 TCRab OKT3a 98 17 5.7 6 0.3 98 18 7.7 6 0.3 CD4 Leu3a 99 99 0.9 6 0.2 0 0 — CD8 Leu2a 0 0 — 99 100 0.6 6 0.2 CD2 T11 100 100 0.8 6 0.1 100 100 1.4 6 0.4 CD18 IOT18 100 96 0.9 6 0.3 99 99 1.0 6 0.3 CD5 CD5 25 40 1.1 6 0.5 27 90 1.2 6 0.7 CD45RO Leu45RO 100 100 0.6 6 0.1 100 100 0.3 6 0.1 Downloaded from HLA-DR HLA-DR 100 100 0.7 6 0.3 100 100 1.2 6 0.5 a Expressed as representative percentages of gated lymphoid cells with fluorescence intensities above the upper limit of the negative control for the indicated molecules using the corresponding Abs. All cells were negative for CD28, CD45RA, CD16, CD20, and TCRgd molecules. Four other unrelated CD3-g-sufficient HVS T cell lines (two CD41, two CD81) showed comparable phenotypes to CTC4 and CTO8, respectively.