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Complex Induced by MHC:Peptide Ligation Architectural Changes in the TCR:CD3

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Complex Induced by MHC:Peptide Ligation Architectural Changes in the TCR:CD3 The Journal of Immunology Architectural Changes in the TCR:CD3 Complex Induced by MHC:Peptide Ligation1 Nicole L. La Gruta,2* Haiyan Liu,3†‡* Smaroula Dilioglou,* Michele Rhodes,§ David L. Wiest,§ and Dario A. A. Vignali4‡* A hallmark of T cell activation is the ligation-induced down-modulation of the TCR:CD3 complex. However, little is known about the molecular events that drive this process. The CD3 ␨-chain has been shown to play a unique role in regulating the assembly, transport, and cell surface expression of the TCR:CD3 complex. In this study we have investigated the relationship between CD3␨ and the TCR␣␤CD3⑀␦␥ complex after ligation by MHC:peptide complexes. Our results show that there is a significant increase in free surface CD3␨, which is not associated with the TCR:CD3 complex, after T cell stimulation. This may reflect dissociation of CD3␨ from the TCR␣␤CD3⑀␦␥ complex or transport of intracellular CD3␨ directly to the cell surface. We also show that ␨ MHC:peptide ligation also results in exposure of the TCR-associated CD3 NH2 terminus, which is ordinarily buried in the complex. These observations appears to be dependent on Src family protein tyrosine kinases, which are known to be critical for efficient T cell activation. These data suggest a mechanism by which ligated TCR may be differentiated from unligated TCR and selectively down-modulated. The Journal of Immunology, 2004, 172: 3662–3669. cells, at the cellular level, and the TCR, at the molecular unclear what molecular mechanism facilitates the rapid removal of level, play pivotal roles in the initiation and regulation of ligated TCR, which is required to drive serial triggering, while at T the immune response. It has been known for some time the same time maintaining the signaling necessary to activate the that one of the most predictable events that occurs following liga- T cell. tion with MHC:peptide complexes is down-modulation of surface The TCR is expressed as a large multichain complex composed TCR complexes (1–3). However, elucidation of the molecular of at least eight polypeptides (TCR␣␤, CD3⑀␥, ⑀␦, and ␨␨) (10, events that mediate TCR down-modulation and its role in T cell 11). The cytoplasmic tails of the CD3 molecules contain immune signal transduction remain elusive. receptor tyrosine-based activation motifs, which are phosphory- Renewed interest in the potential importance of TCR down- lated upon TCR ligation (12). These act as key docking sites for modulation was initiated by seminal studies suggesting that a sin- Src homology 2 domain-containing proteins that mediate signal ϳ gle MHC:peptide complex can serially ligate and trigger 200 transduction and T cell activation. Our previous studies have sug- TCR, leading to an amplified and sustained signal (3). The serial gested that TCR down-modulation is mediated by the intracellular triggering model is dependent on two factors: optimal kinetics in retention of ligated complexes rather than an increase in the inter- the interaction between the TCR and MHC:peptide complexes and nalization rate (5). However, it is not clear how the intracellular ligation-induced TCR down-modulation. These factors combine to sorting machinery distinguishes between ligated and unligated ensure the rapid clearance of ligated TCR, which releases the spe- complexes. cific MHC:peptide complexes to bind to additional TCR (3, 4). The CD3 ␨-chain has long been considered to have a unique Indeed, the rapid and constitutive internalization of the TCR is relationship with the TCR complex and is thought to play an im- likely to facilitate serial triggering (5). However, it is well estab- portant role in facilitating stable TCR cell surface expression (13, lished that sustained signaling is required, particularly for naive T 14). Studies in CD3␨ knockout mice and CD3␨-deficient T cell cells, to initiate full T cell activation (6–9). Thus, it is currently variants have clearly demonstrated that the TCR is not stably ex- pressed at the cell surface in the absence of CD3␨ (15–21). In this instance, hexameric TCR␣␤CD3⑀␥⑀␦ complexes are assembled in *Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN the endoplasmic reticulum and subsequently transported via the 38105; †Graduate Program in Pathology and ‡Department of Pathology, University of Tennessee Medical Center, Memphis, TN 38163; and §Fox Chase Cancer Center, Golgi apparatus to the lysosomes for degradation (13). Assembly Philadelphia, PA 19111 with CD3␨ is thought to prevent trafficking to the lysosomes and Received for publication May 2, 2003. Accepted for publication January 5, 2004. stabilize surface expression of the TCR by shielding a CD3␥ The costs of publication of this article were defrayed in part by the payment of page dileucine motif, which is known to mediate targeting to lysosomes charges. This article must therefore be hereby marked advertisement in accordance for degradation (16, 22–25). Finally, CD3␨ appears to turn over with 18 U.S.C. Section 1734 solely to indicate this fact. independently from the rest of the TCR complex on the cell sur- 1 This work was supported by the National Institutes of Health (Grant AI39480), a Cancer Center Support CORE grant (CA21765), and the American Lebanese Syrian face (26). Associated Charities. Collectively, these studies suggested that CD3␨ has a unique and 2 Current address: Department of Microbiology and Immunology, University of Mel- dynamic relationship with the TCR:CD3 complex and plays a key bourne, Melbourne, Victoria 3010, Australia. role in regulating its assembly, transport, and cell surface expres- 3 Current address: Department of Microbiology and Immunology, University of Ne- sion. We hypothesized that CD3␨ may also play a central role in vada School of Medicine, Reno, NV 89557. mediating TCR down-modulation. To examine this possibility, we 4 Address correspondence and reprint requests to: Dr. Dario Vignali, Department of ␨ Immunology, St. Jude Children’s Research Hospital, 332 North Lauderdale, Mem- explored the relationship between CD3 and the TCR:CD3 com- phis, TN 38105-2794. E-mail address: [email protected] plex after T cell activation by MHC:peptide complexes. Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 The Journal of Immunology 3663 Materials and Methods H-2Ak-PE to gate out B cells, and with propidium iodide for live/dead cell Generation of CD3␨ mutant constructs gating. The percent down-modulation of TCR:CD3 was determined from the median values using the unstimulated controls as reference. All CD3␨ mutants were made by recombinant PCR, using murine CD3␨ cDNA as a template (gift from L. Samelson, National Institutes of Health, Bethesda, MD) and cloned into pCIneo (Promega, Madison, WI) (27), Surface biotinylation and immunoprecipitation (IP) pCIneo-internal ribosome entry sequence (IRES)5-green fluorescent pro- tein (GFP), or murine stem cell virus (MSCV)-IRES-GFP, which is an Assays were performed as previously described with some modifications MSCV-based retroviral vector. The latter two of these vectors contain an (5). Briefly, T cells (5 ϫ 106/IP) were cultured at 37°C with APC encephalomyocarditis virus IRES and GFP. IRES allows for translation of (LK35.2 Ϯ 10 ␮M HEL; 5 ϫ 106/IP) for 5 or 24 h. In some experiments the test protein and GFP from the same mRNA, thus facilitating verifica- LK35.2 cells expressing GFP were used to facilitate enumeration and tion of coexpression while avoiding attachment at the protein level. CD3␨- equalization of T cells (GFPϪ) between samples. In Src family protein FLAG was produced by attaching the FLAG peptide to the CD3␨ N ter- tyrosine kinase (PTK) inhibition studies, T cells and APCs were incubated minus. The first two amino acids were duplicated to ensure correct signal separately for2hat37°C in the presence or the absence of 10 ␮M 4-ami- sequence cleavage. Thus, the sequence of the extracellular domain was no-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) (Cal- QSDYKDDDDKQSFGLLDPK (duplicated residues in italics, FLAG pep- biochem, San Diego, CA) before being cultured together for 24 h at 37°C. tide in bold, native CD3␨ extracellular sequence underlined). CD3␨.K9R Cells were surface-biotinylated with 1 mg/ml sulfo-N-hydroxysuccinimide and K9S mutants were produced using oligonucleotide primers that con- (NHS)-biotin (Pierce, Rockford, IL) in HBSS for 30 min on ice. Excess tained an AAA to AGA mutation (K9R) or an AAA to AGC mutation biotin was quenched with 20 mM L-lysine/HBSS, and the cells were (K9S). Details of the construction strategy and oligos used will be provided washed three times. Dead cells were removed by density gradient centrif- on request ([email protected]). ugation using Lymphocyte Separation Medium (Cappel/ICN Pharmaceu- Transfection of T cell hybridomas ticals, Costa Mesa, CA). Cells were lysed in 1% digitonin lysis buffer (1% digitonin (Wako Biochemicals, Osaka, Japan; Calbiochem, San Diego, After sequence verification, CD3␨-wild type (CD3␨-WT) and CD3␨-FLAG CA), 0.05 M Tris (pH 7.4), 0.15 M NaCl, 2 mM Pefablock (Roche Applied were transfected into CD3␨ loss variants of 3A9 (3A9.␨-7 and 3A9.␨-4, hen Science, Indianapolis, IN), 40 ␮g/ml aprotinin, and 20 ␮g/ml leupeptin) at k egg lysozyme (HEL)48–62-specific, H-2A -restricted) (21) and 2B4 4°C for 30 min, and the lysate was precleared with heat-killed formalin k (MA5.8, pigeon cytochrome c (PCC)88–104-specific, H-2E -restricted) (15) fixed Staphylococcus aureus (Pansorbin) cells (Calbiochem) for1hat4°C. by electroporation (27, 28). Transfectants were selected with G418 and in The lysate was then immunoprecipitated with protein A-Sepharose (Am- some cases cloned or bulk sorted by FACS. ersham Pharmacia Biotech, Piscataway, NJ) precoated with anti-TCR-C␤ (H57-597), twice sequentially (1 h each) at 4°C, followed by protein A- Retroviral transduction of T cell hybridomas Sepharose precoated with anti-CD3␨ (H146-968) for1hat4°C.
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