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 expressed 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.

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 protein (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. Sepharose CD3␨.WT, K9R and K9S DNA constructs were cloned into beads were washed with 0.2% digitonin buffer (three times) and immuno- MSCV.IRES.GFP. Ecotropic retrovirus was produced in 293T cells by precipitated proteins eluted in an equal volume of 2ϫ SDS sample buffer FuGENE-mediated transfection. Briefly, 293T cells (provided by D. Bal- at 75°C for 5 min. timore; 2 ϫ 106 cells in a 10-cm tissue culture petri dish, cultured over- In some experiments proteins bound to anti-TCR-C␤-coated protein A- night) were transfected with the CD3␨ constructs (4 ␮g) and the helper Sepharose beads were eluted, and the protein complexes were disrupted by plasmids pEQ.PAM-E (4 ␮g) and pVSVg (2 ␮g) using FuGENE transfec- boiling for 5 min in 1% SDS. Supernatants were collected, and 900 ␮lof tion reagent (Roche, Basel, Switzerland) according to the manufacturer’s 1% Nonidet P-40 lysis buffer was added. CD3␨-FLAG proteins were then instructions and were incubated overnight at 37°C. Medium was changed immunoprecipitated from the lysate with anti-CD3␨-coated protein after 24 h, and after 72 h supernatant was collected and filtered (0.45 ␮m A-Sepharose beads for1hat4°C. Unbound proteins remaining in the pore size filter). Viral titer was determined by 3T3 transduction, and the lysate were precipitated with 12% TCA by vortexing and placing on ice for percentage of GFP cells was determined by flow cytometry. T cell hybrid- 45 min. The precipitated proteins were pelleted by centrifugation at 4 omas (10 in 1 ml medium plus 6 ␮g/ml polybrene) were transduced at a 12,000 ϫ g for 10 min at 4°C. The pellet was then washed with acetone multiplicity of infection of 5:1 and were cultured at 37°C for 12 h. To (Ϫ20°C), vortexed, centrifuged again, allowed to air dry, and resuspended overcome the low level of ecotropic receptor on the surface of hybridomas, in 1ϫ SDS running buffer. Samples were subsequently treated as described this transduction procedure was repeated five times. Expression was as- above. sessed by flow cytometry. Eluted proteins were resolved by 10% SDS-PAGE or 12% NuPage bis- Ag presentation assay Tris precast gels (Invitrogen, San Diego, CA) and transferred onto a poly- vinylidene difluoride membrane. Blots were blocked with 5% milk powder Assays were performed as previously described (29). Briefly, hybridomas in PBS-Tween 20 (1 h at room temperature), followed by streptavidin-HRP (5 ϫ 104/well) were stimulated with LK35.2 cells (2.5 ϫ 104/well; murine or streptavidin-alkaline phosphatase (streptavidin-AP; 60 min at room tem- B cell lymphoma; H-2Akd) as APC and peptides or HEL protein (Sigma- perature). Blots were developed using ECL Plus (Amersham Pharmacia Aldrich, St. Louis, MO) at the concentration indicated. Supernatants were Biotech, Little Chalfont, U.K.) or Vistra ECF substrate (Amersham Phar- removed after 24 h for estimation of IL-2 secretion by culture with the macia Biotech). Occasionally, membranes were stripped in 100 mM 2-ME, IL-2-dependent T cell line, CTLL-2, as described. For some assays, IL-2 2% (w/v) SDS, and 0.1 M Tris (pH 6.7) in PBS at 50°C for 30 min. was determined using a particle-based flow cytometric assay as previously Membranes were then washed in PBS/0.2% Tween 20 and incubated with described (30). a 1/4 dilution of anti-CD3␨ (H146-968) and anti-CD3⑀ (HMT3.1) hybrid- Downmodulation and flow cytometry oma supernatants. Ab binding was detected using protein A/G-AP (Am- ersham Pharmacia Biotech). Densitometric analysis was performed with The assay for down-modulation of TCR:CD3 was performed as described QuantityOne (Bio-Rad, Hercules, CA) or ImageQuant (Amersham Phar- previously (5, 27). Briefly, T cell hybridomas (5 ϫ 104; 100 ␮l) were macia Biotech). cultured in flat-bottom, 96-well microtiter plates with LK35.2 cells that had ␮ been prepulsed overnight with or without 10 M peptide (HEL48–62 for 3A9 or PCC88–104 for 2B4). At the time points indicated, cells were trans- Purification of biotinylated proteins ferred to V-well microtiter plates for flow cytometry, which was performed as detailed previously (27, 29). Briefly, cells were stained with anti-CD3⑀- Biotinylated proteins were purified from cell lysates using SoftLink Soft FITC or -PE (all Abs from BD PharMingen (San Diego, CA) unless stated Release Avidin Resin (Promega). Briefly, resin was washed with 0.1 M otherwise) and anti-FLAG-biotin (cation-independent M2 mAb; Sigma- NaPO4 (pH 7.0) and preadsorbed in 5 mM biotin/0.1 M NaPO4 for 20 min Aldrich) plus streptavidin-allophycocyanin. Cells were stained with anti- at 4°C with rocking. Beads were regenerated by washing three times with

1 ml of 10% acetic acid, followed by three times with 1 ml of 0.1 M NaPO4 (pH 7.0). For the ﬁnal wash, the beads were incubated for 30 min at 4°C 5 Abbreviations used in this paper; IRES, internal ribosome entry sequence; AP, al- with rocking to allow avidin to refold. Lysate containing biotinylated pro- kaline phosphatase; GFP, green ﬂuorescent protein; HEL, hen egg lysozyme; IP, teins was then added to the resin and incubated at 4°C for 30 min with immunoprecipitation; LAT, linker for activation of T cells; MSCV, murine stem cell virus; NHS, N-hydroxysuccinimide; PCC, pigeon cytochrome c; PTK, protein tyrosine rocking. The resin was washed with three times with 1 ml of 0.2% digi- kinase; WT, wild type; PP2, 4-amino-5-(4-chlorophenyl)-8-(t-butyl)pyrazolo[3,4-d] tonin wash buffer, and biotinylated proteins were eluted with 10 mM biotin pyrimidine. in 1% digitonin lysis buffer (three times, 500 ␮l). 3664 ARCHITECTURAL CHANGES IN THE TCR COMPLEX

Results function (data not shown). As TCR is not stably expressed on the CD3␨ extracellular domain becomes exposed upon TCR ligation cell surface in the absence of CD3␨ (13, 15, 16, 21), it is reason- able to assume that all surface TCR:CD3 complexes on the CD3␨- CD3␨ loss variants of the 3A9 and 2B4 T cell hybridomas FLAG transfectants contain FLAG-tagged CD3␨. Indeed, both (3A9.␨Ϫ and 2B4.␨Ϫ) were transfected with either CD3␨-WT or CD3␨-WT and CD3␨-FLAG restored TCR expression comparably an N-terminal FLAG-tagged CD3␨ (CD3␨-FLAG) to follow the (Fig. 1A). However, minimal FLAG staining was observed, sug- fate of CD3␨ after TCR ligation with MHC:peptide complexes. gesting that the epitope is concealed within the complex (Fig. 1A). Both CD3␨ constructs restored surface expression of the TCR:CD3 As expected, both the 3A9 and 2B4 transfectants down-modulated complex as well as function, as determined by IL-2 production, to TCR following stimulation with Ag-pulsed B cells (Fig. 1C). Sur- levels comparable to those in the parental 3A9 and 2B4 T cell prisingly, there was a substantial increase in FLAG staining, which hybridomas (Fig. 1, A and B, and data not shown). Mock trans- rose ϳ25-fold in the 2B4 transfectants and ϳ400-fold in the 3A9 fections with empty vector failed to restore TCR expression or transfectants 10–12 h poststimulation. These data imply that after TCR ligation, the TCR:CD3 complex undergoes structural alterations, such that the extracellular portion of CD3␨-FLAG becomes exposed for Ab binding. The high level of CD3␨-FLAG staining compared with TCR staining at 12 h poststimulation suggests that although TCR ligation induces down-modulation of the TCR:CD3 complex, CD3␨ may remain on the cell surface. Interestingly, the increase in FLAG staining was delayed relative to maximal TCR down-modulation. Although 3A9 TCR down-modulation was maximal after 3 h, CD3␨-FLAG increase was not evident until 5 h. The reason for this delay is unclear, but it is possible that some time is required before a sufficient quantity of free CD3␨ has ac- cumulated to a detectable level by flow cytometry. Alternatively, CD3␨ may be down-modulated with the ligated TCR complex, separated in an intracellular compartment, and thus not be seen as free CD3␨ until it was recycled back to the cell surface. This would be synonymous with our previous suggestions regarding the transport of nonligated TCR (5). Of course, such a lag may also be a reflection of the appearance of newly synthesized CD3␨ after TCR ligation.

␨ TCR ligation induces an increase in CD3 NH2-terminal biotinylation The relationship between CD3␨ and the TCR␣␤CD3⑀␥␦ complex was studied further using surface biotinylation and sequential IP. The CD3␨-WT and CD3␨-FLAG transfectants of the 3A9␨- hybridoma were surface-biotinylated 5 and 24 h after incubation with B cells precultured with or without Ag. Dead cells were removed to ensure that only surface-biotinylated proteins from viable cells were included. Cell lysates were immunoprecipitated sequentially with anti-TCR-C␤ three times to remove TCR complex-associated CD3␨, followed by anti-CD3␨ to determine the amount of free surface-biotinylated CD3␨. In unstimulated CD3␨-WT transfectants, there was some label- ing of TCR-associated CD3␨; however, very little free CD3␨ was observed. In the CD3␨-FLAG transfectants, some biotinylated free CD3␨ was seen (Fig. 2). This increased biotin label on free CD3␨ may be due to increased biotinylation efficiency, as the FLAG tag contains two additional lysine residues. The relatively small amount of free CD3␨-FLAG is consistent with the minimal surface FLAG staining observed on resting T cells (Fig. 1A). Interestingly, no TCR-associated CD3␨-FLAG biotinylation was detected in un- ␨ FIGURE 1. Increased accessibility to the CD3 NH2 terminus after stimulated cells, suggesting that the tag is buried in the complex. TCR ligation. CD3␨ loss mutants of 2B4 and 3A9 (2B4.␨ and 3A9.␨) were Efficient biotinylation may also have been precluded if the extra- transfected with CD3␨-WT and CD3␨-FLAG. A, Unstimulated cells were cellular lysine residues had formed stable interactions with adja- ⑀ analyzed by flow cytometry with anti-CD3 and anti-FLAG (cation-inde- cent residues in the complex. It is important to note that the lack pendent M2 mAb). B, The functional capacity of the CD3␨-FLAG trans- of CD3␨-FLAG biotinylation on resting cells is not due to an ab- fectants was evaluated by IL-2 production after stimulation with LK35.2 B ␨ cells plus the peptides indicated. C, Modulation of TCR and CD3␨-FLAG sence of associated protein, as the presence of CD3 -FLAG was ␨ after stimulation with Ag-pulsed B cells was determined by flow cytom- confirmed by subsequent immunoblot analysis using an anti-CD3 etry. In all cases, homogeneous down-modulation was observed. Data are Ab (data not shown and Figs. 3 and 4). After antigenic stimulation, either representative of two experiments (A) or are the mean of three ex- two changes in the pattern of TCR:CD3 biotinylation were ob- periments (B and C). served. First, there was an increase in the TCR-associated CD3␨ The Journal of Immunology 3665

FIGURE 3. The increased intensity of the ϳ16-kDa band represents CD3␨ and not recruitment of other proteins. CD3␨-FLAG transfectants of a 3A9 CD3␨ loss mutant were incubated with LK35.2 B cells pulsed, or not, with 10 ␮M HEL. After 24 h, cells were surface-biotinylated and lysed in 1% digitonin buffer. After IP of cell lysates with anti-TCR-C␤, half the sample was removed for immunoblot (lanes 1 and 2). Bound proteins were eluted from the remainder of the sample by boiling for 5 min in 1% SDS and were added to 900 ␮l 1% Nonidet P-40 lysis buffer. CD3␨-FLAG proteins were immunoprecipitated from the lysate with anti-CD3␨ Ab (lanes 3 and 4). Unbound proteins were precipitated with TCA (lanes 5 and 6). Biotinylated proteins were analyzed by probing the Western blot with streptavidin-AP (A). Membranes were stripped and reprobed with anti- CD3␨ Ab (B). Images were produced using a fluorescence-based detection FIGURE 2. Altered relationship between CD3␨ and the system on a phosphorimager. TCR␣␤CD3⑀␥␦ complex after ligation by MHC:peptide complexes. CD3␨-WT (A) and CD3␨-FLAG (B) transfectants of the 3A9 CD3␨ loss mutant were incubated with LK35.2 B cells or LK35.2 prepulsed with 10 ␮M HEL. After 5 and 24 h, cells were surface-biotinylated with NHS- tated with TCA before electrophoresis (Fig. 3A, lanes 5 and 6). biotin and lysed in 1% digitonin. Cell lysates were immunoprecipitated After detection of biotinylated proteins using streptavidin-AP, the three times sequentially with anti-TCR-C␤ (only the first and third are blot was stripped and reprobed with anti-CD3␨ Ab to verify that all shown), followed by anti-CD3␨. Nonspecific binding of streptavidin-HRP the CD3␨ protein had been immunoprecipitated, and none re- to the anti-CD3␨ L chain is shown (IgL) and was also seen in lanes con- mained in the unbound fraction (Fig. 3B). It was clear from this taining the IP Ab alone (data not shown). The results of two representative experiment that essentially all the ϳ16-kDa biotinylated protein ␨ experiments with CD3 -FLAG transfectants and one experiment with detected after anti-TCR-C␤ IP was CD3␨, as evidenced by its com- ␨ CD3 -WT are shown. Comparable results were obtained using a fluores- plete removal from the lysate after IP with anti-CD3␨. cence-based detection system (ECF, Amersham Pharmacia Biotech) and Secondly, as visualization of proteins in this system is depen- direct quantitation on a phosphorimager (N. La Gruta and D. A. A. Vignali, unpublished observations). dent on their ability to be biotinylated, it was unclear whether the increased CD3␨ signal intensity represented an increase in the amount of CD3␨ protein or an increase in the biotinylation state of biotin signal (Fig. 2). Second, the free CD3␨ biotin signal in- CD3␨. Differentiation between these possibilities required selec- creased significantly, which was particularly evident after 24 h. In tive quantitation of cell surface CD3␨ protein. Cells were surface- contrast, there was little change in the level of CD3⑀␥␦ biotinylated and lysed as described; however, before IP with anti- biotinylation. TCR-C␤, biotinylated proteins were purified from the lysate using Based on these observations, three key issues arose. Firstly, it a SoftLink monomeric avidin system. This allowed for the selec- was possible that the increased signal intensity at ϳ16 kDa was tive IP of cell surface proteins. The eluted biotinylated proteins due to recruitment of another protein(s) to the TCR:CD3 complex were then sequentially immunoprecipitated with anti-TCR-C␤ and after TCR ligation. To assess this possibility, CD3␨-FLAG trans- anti-CD3␨ as described above. Upon probing with streptavidin- fectants of the 3A9.␨- hybridoma were surface-biotinylated 24 h AP, the characteristic pattern of increased TCR-associated and free after incubation with B cells precultured with or without Ag. We CD3␨ signal intensity was observed after Ag stimulation (Fig. 4A, preferentially chose to use the CD3␨-FLAG transfectants for these upper panel; compare lanes 1 and 4, and lanes 3 and 6), with experiments because it is more readily biotinylated and thus easier densitometric analyses revealing 2.4- and 8.5-fold increases in to detect. Cell lysates were then immunoprecipitated with anti- TCR-associated and free CD3␨, respectively (Fig. 4B, upper pan- TCR C␤ Ab (Fig. 3A, lanes 1 and 2). As shown in Fig. 2, increased els). The blots were then stripped and reprobed with a mixture of signal intensity at ϳ16 kDa was again evident after Ag stimulation anti-CD3␨ and anti-CD3⑀ Abs (Fig. 4A, lower panel). The anti- without significant alteration in the biotinylation of CD3⑀␥␦. Pro- CD3⑀ Ab was included to enable normalization of the CD3␨ signal teins bound to the anti-TCR C␤ Sepharose beads were eluted, and intensity relative to the rest of the TCR:CD3 complex. Densito- the TCR complex was disrupted by boiling in the presence of 1% metric analyses of both TCR-associated and free CD3␨ protein SDS. CD3␨-FLAG proteins were subsequently immunoprecipi- revealed no increase in TCR-associated CD3␨ protein. However, a tated with anti-CD3␨ Ab (Fig. 3A, lanes 3 and 4). The remaining 3-fold increase in free CD3␨ protein was detected after Ag stim- proteins that were not bound by the anti-CD3␨ Ab were precipi- ulation. The reduced CD3␥␦⑀-biotin and CD3⑀ protein signal after 3666 ARCHITECTURAL CHANGES IN THE TCR COMPLEX

FIGURE 4. The amount of CD3␨ protein associated with the TCR remains unchanged despite the increased biotin signal after Ag stimulation. CD3␨-FLAG transfectants of a 3A9 CD3␨ loss mutant were incubated with LK35.2 B cells pulsed, or not, with 10 ␮M HEL. After 24 h, cells were surface-biotinylated and lysed in 1% digitonin buffer. Biotinylated proteins were purified from the cell lysates using SoftLink soft release avidin resin (Promega). Purified biotinylated proteins were immunoprecipitated twice sequentially with anti-TCR-C␤, followed by anti-CD3␨. Biotinylated proteins were analyzed by probing the Western blot with streptavidin-AP (A, upper panel). Membranes were stripped and reprobed with anti-CD3␨ and anti-CD3⑀ Abs, washed, and probed with protein A/G-AP (Amersham; A, lower panel). A fluorescence-based detection system was used (ECF, Am- ersham Pharmacia Biotech), and bands were visualized on a phosphorimager for direct quantitation of biotin and Ab signals. Graphs are expressed as the ratio of CD3⑀ and CD3␨ arbitrary densitometric units (B: upper graph, biotin signal; lower graph, Ab signal). FIGURE 5. Biotinylation of the CD3␨ extracellular domain does not involve the K9 residue. 3A9 CD3␨- hybrids were retrovirally transduced with virus encoding WT CD3␨, CD3␨.K9R, or CD3␨.K9S. A, The func- ligation is due to TCR down-modulation. Thus, the increased tional capacity of the CD3␨ transductants was evaluated by IL-2 production CD3␨ biotin signal observed after TCR ligation corresponds to an after stimulation with either plate-bound anti-TCR␤ (H57; left graph)or increased biotinylation state of TCR-associated CD3␨ rather than a HEL plus LK35.2 B cells (right graph). The IL-2 concentration was de- change in the amount of CD3␨ protein associated with the TCR termined using a particle-based flow cytometric assay (30). BÐD, The 3A9 complex. For free CD3␨, the increased biotin signal appears to be cells were incubated for 24 h with LK cells precultured with or without Ag. the consequence of an increased amount of protein. Cells were then surface-biotinylated, lysed, and immunoprecipitated with ␤ Thirdly, what residue(s) in CD3␨ is biotinylated? The CD3 anti-TCR-C . Bound proteins were separated by SDS-PAGE, immuno- blotted, and probed with streptavidin-AP (A). Membranes were stripped ␨-chain has an extremely short, nine-amino acid extracellular do- and reprobed with anti-CD3␨ and anti-CD3⑀ Abs, followed by protein main (NH2-QSFGLLDPK). The most likely candidate for biotiny- A/G-AP (B). Quantitative analysis of the CD3␨ band intensity was per- lation is the lysine residue at position 9 (K9). To determine formed as described in Fig. 4 (C). whether enhanced biotinylation at K9 is responsible for the increased CD3␨ biotin signal after TCR ligation, CD3␨ constructs were generated in which the lysine residue was mutated to either cell systems used. Transductants were then stimulated, surface- arginine (K9R) or serine (K9S). 3A9 CD3␨- hybridomas were biotinylated, and immunoprecipitated with anti-TCR-C␤ as de- transduced with retrovirus encoding the mutant CD3 ␨-chains. scribed. The data clearly show that mutation of K9 had no effect on TCR expression and T cell function were comparable to those in T CD3␨ biotinylation (Fig. 5B). After detection of biotinylated pro- cells expressing wild-type CD3␨ (Fig. 5A and data not shown). teins, the blot was stripped and probed with a mixture of anti-CD3␨ Previous studies have suggested an important role for the K9 res- and anti-CD3⑀ Abs (Fig. 5C). This blot clearly demonstrates that idue in the extracellular domain of CD3␨ in signal transduction as despite a weak biotin signal, CD3␨ is associated with TCR com- well as in stabilizing the association of CD3␨ with the TCR:CD3 plexes in unstimulated cells. It also supports our previous finding complex (31). Thus, we were surprised to find that these mutations that the increased biotin signal of TCR-associated CD3␨ is not due had no effect on the function of 3A9. Although the reason for this to an increased amount of protein. The relative increase in the discrepancy is unclear, it may simply represent differences in the T CD3␨:CD3⑀ biotin ratio between unstimulated and stimulated cells The Journal of Immunology 3667

was essentially identical (densitometric analysis: CD3␨-WT, 2.09- fold; CD3␨-K9R, 2.2-fold; CD3␨-K9S, 1.8-fold; Fig. 5D). Thus, it appears that the increase in CD3␨ biotinylation is not mediated via the K9 residue. Taken together, our data strongly suggest that TCR ligation by MHC:peptide causes alterations in the TCR:CD3 complex, such that the short extracellular domain of CD3␨ becomes more acces- sible for biotinylation. Furthermore, ligation appears to cause an increase in the amount of free CD3␨ on the cell surface. There are two possible explanations for the latter. First, de novo synthesis of CD3␨ induced by TCR ligation may escape intracellular retention and be expressed on the cell surface. Second, free CD3␨ may result from dissociation between CD3␨ and the rest of the TCR:CD3 complex.

TCR ligation-induced architectural changes in the TCR:CD3 complex are Src family PTK-dependent Two members of the Src family of PTKs, p56lck and p59fyn, have been shown to play a critical role in T cell development and activation by mediating two of the earliest events following TCR ligation, phosphorylation of CD3 chain immune receptor tyrosine- FIGURE 6. Architectural changes within the TCR:CD3 complex after based activation motif residues and activation of ZAP-70 (32, 33). Ag stimulation are Src family PTK-dependent. 3A9 hybridomas (A)orthe CD3␨-FLAG transfectants (B) were incubated with LK35.2 B cells pulsed, The question arose of whether the architectural changes observed or not, with 10 ␮M HEL in the presence or the absence of the Src family after TCR ligation were dependent on Src family PTKs. The role PTK inhibitor PP2 (10 ␮M). After 24 h, cells were surface-biotinylated and of these kinases in the induction of TCR:CD3 architectural lysed in 1% digitonin buffer. Cell lysates were immunoprecipitated twice changes could provide information regarding the order of events sequentially with anti-TCR-C␤, followed by anti-CD3␨. Biotinylated pro- following TCR ligation. 3A9 T cell hybridomas or 3A9 CD3␨ teins were analyzed by probing the Western blot with streptavidin-AP. mutant hybridomas stably transfected with FLAG-tagged CD3␨ were pretreated and stimulated in the presence or the absence of PP2, a selective inhibitor of Src family PTKs (34). Cells were tion. How does the T cell recognize ligated TCR and subsequently surface-biotinylated, and cell lysates were immunoprecipitated direct these molecules to lysosomes for degradation? Our results twice sequentially with anti-TCR-C␤, followed by anti-CD3␨. Im- suggest that MHC:peptide ligation of the TCR:CD3 complex re- ␨ munoprecipitated proteins were resolved by SDS-PAGE, blotted, sults in exposure of the CD3 NH2 terminus, which is ordinarily and probed with streptavidin-AP for detection of biotinylated pro- buried in the complex. For discussion purposes, we have generally teins, as described above. Parallel experiments showed that TCR interpreted this event as reflecting architectural changes in the down-modulation was significantly decreased, and IL-2 production TCR:CD3 complex. We would also propose, based on increased was abrogated after PP2 treatment of 3A9 T cells (data not shown). free CD3␨ levels following stimulation, that such architectural After Ag stimulation, there was a substantial increase in the TCR- changes may ultimately lead to dissociation of the CD3␨ dimer associated CD3␨ signal as well as an increase in free CD3␨,as from the TCR:CD3 complex. However, the origin of the free cell described previously (Fig. 6). Pretreatment of the cells with PP2 surface CD3␨ has not yet been formally demonstrated, and thus we almost completely abrogated these changes (Fig. 6). Taken to- cannot rule out the possibility that this free CD3␨ derives from de gether, these data suggest that the structural changes induced in the novo synthesis induced by TCR ligation. This would be surprising, TCR:CD3 complex after TCR ligation are highly dependent on Src because it is generally thought that the free CD3␨ seen after stim- family PTKs and thus presumably occur after or concurrently with ulation are products of de novo synthesis and cannot escape intra- kinase activation. cellular retention (13, 15, 16, 21). Further studies are required to resolve this issue. Regardless, we propose that the architectural Discussion changes occurring within the TCR:CD3 complex after TCR liga- The expression of TCR on the surface of T cells is a dynamic tion may be a mechanism by which T cells recognize ligated TCR process. In resting T cells, for example, a cycling reservoir of TCR and direct them to lysosomal compartments for degradation. Re- is constitutively internalized and recycled back to the cell surface cently, D’Oro and colleagues (42) proposed that the CD3 ␨-chain (5, 13, 35). T cell stimulation also results in internalization of the maintains cell surface receptor expression by sterically blocking TCR:CD3 complex. However, this is dependent on ligation of the internalization sequences in other TCR components, using chi- TCR by Ab or MHC:peptide complexes and, unlike internalization meric CD3 ␨-chains expressing intracellular domains of varying in resting cells, results in a rapid and net reduction of TCR on the lengths to show that the size of the intracellular tail, rather than the cell surface (36, 37). TCR ligation-mediated down-modulation is a amino acid sequence, was all that was required to support the consequence of TCR degradation within lysosomal compartments expression of the fully assembled receptor. We have made similar (5, 38, 39). Some studies have proposed that TCR ligation induces observations (A. Szymczak, H. Liu, and D. A. A. Vignali, unpub- the down-modulation of nonengaged receptors (40), although oth- lished observations). Other studies with CD16/CD3 chimeras have ers argue that only specific, ligated receptors are down-modulated suggested that CD3␨ may shield the CD3␥ dileucine motif (24). As after ligation (4). Support for the latter theory comes from a recent this motif is known to mediate targeting to lysosomes for degra- study involving mathematical modeling of MHC:peptide-induced dation (22, 23), structural changes in the TCR:CD3 complex, par- TCR down-modulation (41). In it, the researchers propose that an ticularly those involving CD3␨, may result in targeting of ligated activated TCR remains marked for internalization after dissociat- TCR:CD3 complexes to lysosomes for degradation, rather than ing from the MHC:peptide complex. This raises an important ques- recycling back to the cell surface. It is also possible that other 3668 ARCHITECTURAL CHANGES IN THE TCR COMPLEX motifs are involved in this process, as T cells lacking the cyto- implications for our understanding of the molecular events that plasmic tail of CD3␥ and CD3␦ still down-modulate TCR after Ag initiate or perpetuate signal transduction via multichain receptor stimulation (43). Indeed, an endocytosis signal in CD3⑀ has re- complexes. cently been described (44). Thus, TCR down-modulation may be determined by the exposure of intracellular retention motifs that Acknowledgments are normally concealed by CD3␨. We are very grateful to Ralph Kubo for the Abs, Mark Davis for the 3A9 Until recently, the idea that the TCR:CD3 complex undergoes TCR transgenic mice, Elio Vanin and David Baltimore for the 293T cells, conformational change after TCR ligation was speculative. How- Emil Unanue for M12.C3.F6, and Larry Samelson for the CD3␨ cDNA. ever, a study by Gil and colleagues (45) demonstrated that ligation We also thank Richard Cross and Mahnaz Paktinat for assistance with the of TCR resulted in the exposure of a proline-rich sequence in flow cytometry, Janet Gatewood for performing the particle-based flow CD3⑀, which, in turn, leads to recruitment of the adaptor protein, cytometric cytokine assay, Elio Vanin for assistance and reagents for es- Nck. Interestingly, these events occurred before and independently tablishing retroviral transduction in our laboratory, and members of the of tyrosine kinase activation and appeared to be involved in early Vignali laboratory for constructive criticisms and comments. signaling processes. In our study the observed architectural changes initiated by TCR ligation were heavily dependent on Src References ␨ 1. Zanders, E. D., J. R. Lamb, M. Feldmann, N. Green, and P. C. Beverley. 1983. family PTK activity, suggesting that the exposure of CD3 and its Tolerance of T-cell clones is associated with membrane antigen changes. Nature subsequent dissociation from the remainder of the TCR:CD3 com- 303:625. plex are relatively late events and may occur downstream of CD3⑀ 2. Schonrich, G., U. Kalinke, F. Momburg, M. Malissen, A. M. Schmitt-Verhulst, B. Malissen, G. J. Hammerling, and B. Arnold. 1991. Down-regulation of T cell exposure and Nck recruitment. 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