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,
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* Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN 38105;
Graduate Program in Pathology and
Department of Pathology, University of Tennessee Medical Center, Memphis, TN 38163; and
Fox Chase Cancer Center, Philadelphia, PA 19111
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Abstract |
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-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 TCRCD3 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. |
Introduction |
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Renewed interest in the potential importance of TCR down-modulation was initiated by seminal studies suggesting that a single MHC:peptide complex can serially ligate and trigger 
200 TCR, leading to an amplified and sustained signal (3). The serial triggering model is dependent on two factors: optimal kinetics in the interaction between the TCR and MHC:peptide complexes and ligation-induced TCR down-modulation. These factors combine to ensure the rapid clearance of ligated TCR, which releases the specific MHC:peptide complexes to bind to additional TCR (3, 4). Indeed, the rapid and constitutive internalization of the TCR is likely to facilitate serial triggering (5). However, it is well established that sustained signaling is required, particularly for naive T cells, to initiate full T cell activation (6, 7, 8, 9). Thus, it is currently unclear what molecular mechanism facilitates the rapid removal of ligated TCR, which is required to drive serial triggering, while at the same time maintaining the signaling necessary to activate the T cell.
The TCR is expressed as a large multichain complex composed of at least eight polypeptides (TCR, CD3, , and ) (10, 11). The cytoplasmic tails of the CD3 molecules contain immune receptor tyrosine-based activation motifs, which are phosphorylated upon TCR ligation (12). These act as key docking sites for Src homology 2 domain-containing proteins that mediate signal transduction and T cell activation. Our previous studies have suggested that TCR down-modulation is mediated by the intracellular retention of ligated complexes rather than an increase in the internalization rate (5). However, it is not clear how the intracellular sorting machinery distinguishes between ligated and unligated complexes.
The CD3 -chain has long been considered to have a unique relationship with the TCR complex and is thought to play an important role in facilitating stable TCR cell surface expression (13, 14). Studies in CD3 knockout mice and CD3-deficient T cell variants have clearly demonstrated that the TCR is not stably expressed at the cell surface in the absence of CD3 (15, 16, 17, 18, 19, 20, 21). In this instance, hexameric TCRCD3 complexes are assembled in the endoplasmic reticulum and subsequently transported via the Golgi apparatus to the lysosomes for degradation (13). Assembly with CD3 is thought to prevent trafficking to the lysosomes and stabilize surface expression of the TCR by shielding a CD3 dileucine motif, which is known to mediate targeting to lysosomes for degradation (16, 22, 23, 24, 25). Finally, CD3 appears to turn over independently from the rest of the TCR complex on the cell surface (26).
Collectively, these studies suggested that CD3 has a unique and dynamic relationship with the TCR:CD3 complex and plays a key role in regulating its assembly, transport, and cell surface expression. We hypothesized that CD3 may also play a central role in mediating TCR down-modulation. To examine this possibility, we explored the relationship between CD3 and the TCR:CD3 complex after T cell activation by MHC:peptide complexes.
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Materials and Methods |
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mutant constructs
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), pCIneo-internal ribosome entry sequence (IRES)5-green fluorescent protein (GFP), or murine stem cell virus (MSCV)-IRES-GFP, which is an MSCV-based retroviral vector. The latter two of these vectors contain an encephalomyocarditis virus IRES and GFP. IRES allows for translation of the test protein and GFP from the same mRNA, thus facilitating verification of coexpression while avoiding attachment at the protein level. CD3-FLAG was produced by attaching the FLAG peptide to the CD3 N terminus. The first two amino acids were duplicated to ensure correct signal sequence cleavage. Thus, the sequence of the extracellular domain was QSDYKDDDDKQSFGLLDPK (duplicated residues in italics, FLAG peptide in bold, native CD3 extracellular sequence underlined). CD3.K9R and K9S mutants were produced using oligonucleotide primers that contained an AAA to AGA mutation (K9R) or an AAA to AGC mutation (K9S). Details of the construction strategy and oligos used will be provided on request (dario.vignali{at}stjude.org).
Transfection of T cell hybridomas
After sequence verification, CD3-wild type (CD3-WT) and CD3-FLAG were transfected into CD3 loss variants of 3A9 (3A9.-7 and 3A9.-4, hen egg lysozyme (HEL)48–62-specific, H-2Ak-restricted) (21) and 2B4 (MA5.8, pigeon cytochrome c (PCC)88–104-specific, H-2Ek-restricted) (15) by electroporation (27, 28). Transfectants were selected with G418 and in some cases cloned or bulk sorted by FACS.
Retroviral transduction of T cell hybridomas
CD3.WT, K9R and K9S DNA constructs were cloned into MSCV.IRES.GFP. Ecotropic retrovirus was produced in 293T cells by FuGENE-mediated transfection. Briefly, 293T cells (provided by D. Baltimore; 2 x 106 cells in a 10-cm tissue culture petri dish, cultured overnight) were transfected with the CD3 constructs (4 µg) and the helper plasmids pEQ.PAM-E (4 µg) and pVSVg (2 µg) using FuGENE transfection reagent (Roche, Basel, Switzerland) according to the manufacturer’s instructions and were incubated overnight at 37°C. Medium was changed after 24 h, and after 72 h supernatant was collected and filtered (0.45 µm pore size filter). Viral titer was determined by 3T3 transduction, and the percentage of GFP cells was determined by flow cytometry. T cell hybridomas (104 in 1 ml medium plus 6 µg/ml polybrene) were transduced at a multiplicity of infection of 5:1 and were cultured at 37°C for 12 h. To overcome the low level of ecotropic receptor on the surface of hybridomas, this transduction procedure was repeated five times. Expression was assessed by flow cytometry.
Ag presentation assay
Assays were performed as previously described (29). Briefly, hybridomas (5 x 104/well) were stimulated with LK35.2 cells (2.5 x 104/well; murine B cell lymphoma; H-2Akd) as APC and peptides or HEL protein (Sigma-Aldrich, St. Louis, MO) at the concentration indicated. Supernatants were removed after 24 h for estimation of IL-2 secretion by culture with the IL-2-dependent T cell line, CTLL-2, as described. For some assays, IL-2 was determined using a particle-based flow cytometric assay as previously described (30).
Downmodulation and flow cytometry
The assay for down-modulation of TCR:CD3 was performed as described previously (5, 27). Briefly, T cell hybridomas (5 x 104; 100 µl) were cultured in flat-bottom, 96-well microtiter plates with LK35.2 cells that had been prepulsed overnight with or without 10 µM peptide (HEL48–62 for 3A9 or PCC88–104 for 2B4). At the time points indicated, cells were transferred to V-well microtiter plates for flow cytometry, which was performed as detailed previously (27, 29). Briefly, cells were stained with anti-CD3-FITC or -PE (all Abs from BD PharMingen (San Diego, CA) unless stated otherwise) and anti-FLAG-biotin (cation-independent M2 mAb; Sigma-Aldrich) plus streptavidin-allophycocyanin. Cells were stained with anti-H-2Ak-PE to gate out B cells, and with propidium iodide for live/dead cell gating. The percent down-modulation of TCR:CD3 was determined from the median values using the unstimulated controls as reference.
Surface biotinylation and immunoprecipitation (IP)
Assays were performed as previously described with some modifications (5). Briefly, T cells (5 x 106/IP) were cultured at 37°C with APC (LK35.2 ± 10 µM HEL; 5 x 106/IP) for 5 or 24 h. In some experiments LK35.2 cells expressing GFP were used to facilitate enumeration and equalization of T cells (GFP-) between samples. In Src family protein tyrosine kinase (PTK) inhibition studies, T cells and APCs were incubated separately for 2 h at 37°C in the presence or the absence of 10 µM 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) (Calbiochem, San Diego, CA) before being cultured together for 24 h at 37°C. Cells were surface-biotinylated with 1 mg/ml sulfo-N-hydroxysuccinimide (NHS)-biotin (Pierce, Rockford, IL) in HBSS for 30 min on ice. Excess biotin was quenched with 20 mM L-lysine/HBSS, and the cells were washed three times. Dead cells were removed by density gradient centrifugation using Lymphocyte Separation Medium (Cappel/ICN Pharmaceuticals, Costa Mesa, CA). Cells were lysed in 1% digitonin lysis buffer (1% digitonin (Wako Biochemicals, Osaka, Japan; Calbiochem, San Diego, CA), 0.05 M Tris (pH 7.4), 0.15 M NaCl, 2 mM Pefablock (Roche Applied Science, Indianapolis, IN), 40 µg/ml aprotinin, and 20 µg/ml leupeptin) at 4°C for 30 min, and the lysate was precleared with heat-killed formalin fixed Staphylococcus aureus (Pansorbin) cells (Calbiochem) for 1 h at 4°C. The lysate was then immunoprecipitated with protein A-Sepharose (Amersham Pharmacia Biotech, Piscataway, NJ) precoated with anti-TCR-C (H57-597), twice sequentially (1 h each) at 4°C, followed by protein A-Sepharose precoated with anti-CD3 (H146-968) for 1 h at 4°C. Sepharose beads were washed with 0.2% digitonin buffer (three times) and immunoprecipitated proteins eluted in an equal volume of 2x SDS sample buffer at 75°C for 5 min.
In some experiments proteins bound to anti-TCR-C-coated protein A-Sepharose beads were eluted, and the protein complexes were disrupted by boiling for 5 min in 1% SDS. Supernatants were collected, and 900 µl of 1% Nonidet P-40 lysis buffer was added. CD3-FLAG proteins were then immunoprecipitated from the lysate with anti-CD3-coated protein A-Sepharose beads for 1 h at 4°C. Unbound proteins remaining in the lysate were precipitated with 12% TCA by vortexing and placing on ice for 45 min. The precipitated proteins were pelleted by centrifugation at 12,000 x g for 10 min at 4°C. The pellet was then washed with acetone (-20°C), vortexed, centrifuged again, allowed to air dry, and resuspended in 1x SDS running buffer. Samples were subsequently treated as described above.
Eluted proteins were resolved by 10% SDS-PAGE or 12% NuPage bis-Tris precast gels (Invitrogen, San Diego, CA) and transferred onto a polyvinylidene difluoride membrane. Blots were blocked with 5% milk powder in PBS-Tween 20 (1 h at room temperature), followed by streptavidin-HRP or streptavidin-alkaline phosphatase (streptavidin-AP; 60 min at room temperature). Blots were developed using ECL Plus (Amersham Pharmacia Biotech, Little Chalfont, U.K.) or Vistra ECF substrate (Amersham Pharmacia Biotech). Occasionally, membranes were stripped in 100 mM 2-ME, 2% (w/v) SDS, and 0.1 M Tris (pH 6.7) in PBS at 50°C for 30 min. Membranes were then washed in PBS/0.2% Tween 20 and incubated with a 1/4 dilution of anti-CD3 (H146-968) and anti-CD3 (HMT3.1) hybridoma supernatants. Ab binding was detected using protein A/G-AP (Amersham Pharmacia Biotech). Densitometric analysis was performed with QuantityOne (Bio-Rad, Hercules, CA) or ImageQuant (Amersham Pharmacia Biotech).
Purification of biotinylated proteins
Biotinylated proteins were purified from cell lysates using SoftLink Soft Release Avidin Resin (Promega). Briefly, resin was washed with 0.1 M NaPO4 (pH 7.0) and preadsorbed in 5 mM biotin/0.1 M NaPO4 for 20 min 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 with rocking to allow avidin to refold. Lysate containing biotinylated proteins was then added to the resin and incubated at 4°C for 30 min with rocking. The resin was washed with three times with 1 ml of 0.2% digitonin wash buffer, and biotinylated proteins were eluted with 10 mM biotin in 1% digitonin lysis buffer (three times, 500 µl).
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Results |
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extracellular domain becomes exposed upon TCR ligation
CD3 loss variants of the 3A9 and 2B4 T cell hybridomas (3A9.- and 2B4.-) were transfected with either CD3-WT or an N-terminal FLAG-tagged CD3 (CD3-FLAG) to follow the fate of CD3 after TCR ligation with MHC:peptide complexes. Both CD3 constructs restored surface expression of the TCR:CD3 complex as well as function, as determined by IL-2 production, to levels comparable to those in the parental 3A9 and 2B4 T cell hybridomas (Fig. 1, A and B, and data not shown). Mock transfections with empty vector failed to restore TCR expression or function (data not shown). As TCR is not stably expressed on the cell surface in the absence of CD3 (13, 15, 16, 21), it is reasonable to assume that all surface TCR:CD3 complexes on the CD3-FLAG transfectants contain FLAG-tagged CD3. Indeed, both CD3-WT and CD3-FLAG restored TCR expression comparably (Fig. 1A). However, minimal FLAG staining was observed, suggesting that the epitope is concealed within the complex (Fig. 1A). As expected, both the 3A9 and 2B4 transfectants down-modulated TCR following stimulation with Ag-pulsed B cells (Fig. 1C). Surprisingly, there was a substantial increase in FLAG staining, which rose 25-fold in the 2B4 transfectants and 400-fold in the 3A9 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 accumulated 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.
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NH2-terminal biotinylation
The relationship between CD3 and the TCRCD3 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 labeling 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 unstimulated cells, suggesting that the tag is buried in the complex. Efficient biotinylation may also have been precluded if the extracellular lysine residues had formed stable interactions with adjacent residues in the complex. It is important to note that the lack of CD3-FLAG biotinylation on resting cells is not due to an absence of associated protein, as the presence of CD3-FLAG was confirmed by subsequent immunoblot analysis using an anti-CD3 Ab (data not shown and Figs. 3 and 4). After antigenic stimulation, two changes in the pattern of TCR:CD3 biotinylation were observed. First, there was an increase in the TCR-associated CD3 biotin signal (Fig. 2). Second, the free CD3 biotin signal increased significantly, which was particularly evident after 24 h. In contrast, there was little change in the level of CD3 biotinylation.
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16 kDa was due to recruitment of another protein(s) to the TCR:CD3 complex after TCR ligation. To assess this possibility, CD3-FLAG transfectants of the 3A9.-hybridoma were surface-biotinylated 24 h after incubation with B cells precultured with or without Ag. We preferentially chose to use the CD3-FLAG transfectants for these experiments because it is more readily biotinylated and thus easier to detect. Cell lysates were then immunoprecipitated with anti-TCR C Ab (Fig. 3A, lanes 1 and 2). As shown in Fig. 2, increased signal intensity at 16 kDa was again evident after Ag stimulation without significant alteration in the biotinylation of CD3. Proteins bound to the anti-TCR C Sepharose beads were eluted, and the TCR complex was disrupted by boiling in the presence of 1% SDS. CD3-FLAG proteins were subsequently immunoprecipitated with anti-CD3 Ab (Fig. 3A, lanes 3 and 4). The remaining proteins that were not bound by the anti-CD3 Ab were precipitated with TCA before electrophoresis (Fig. 3A, lanes 5 and 6). After detection of biotinylated proteins using streptavidin-AP, the blot was stripped and reprobed with anti-CD3 Ab to verify that all the CD3 protein had been immunoprecipitated, and none remained in the unbound fraction (Fig. 3B). It was clear from this experiment that essentially all the 16-kDa biotinylated protein detected after anti-TCR-C IP was CD3, as evidenced by its complete removal from the lysate after IP with anti-CD3.
Secondly, as visualization of proteins in this system is dependent 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 CD3. Differentiation between these possibilities required selective quantitation of cell surface CD3 protein. Cells were surface-biotinylated and lysed as described; however, before IP with anti-TCR-C, biotinylated proteins were purified from the lysate using a SoftLink monomeric avidin system. This allowed for the selective IP of cell surface proteins. The eluted biotinylated proteins were then sequentially immunoprecipitated with anti-TCR-C and anti-CD3 as described above. Upon probing with streptavidin-AP, the characteristic pattern of increased TCR-associated and free CD3 signal intensity was observed after Ag stimulation (Fig. 4A, upper panel; compare lanes 1 and 4, and lanes 3 and 6), with densitometric analyses revealing 2.4- and 8.5-fold increases in TCR-associated and free CD3, respectively (Fig. 4B, upper panels). The blots were then stripped and reprobed with a mixture of anti-CD3 and anti-CD3 Abs (Fig. 4A, lower panel). The anti-CD3 Ab was included to enable normalization of the CD3 signal intensity relative to the rest of the TCR:CD3 complex. Densitometric analyses of both TCR-associated and free CD3 protein revealed no increase in TCR-associated CD3 protein. However, a 3-fold increase in free CD3 protein was detected after Ag stimulation. The reduced CD3-biotin and CD3 protein signal after ligation is due to TCR down-modulation. Thus, the increased CD3 biotin signal observed after TCR ligation corresponds to an increased biotinylation state of TCR-associated CD3 rather than a change in the amount of CD3 protein associated with the TCR complex. For free CD3, the increased biotin signal appears to be the consequence of an increased amount of protein.
Thirdly, what residue(s) in CD3 is biotinylated? The CD3 -chain has an extremely short, nine-amino acid extracellular domain (NH2-QSFGLLDPK). The most likely candidate for biotinylation is the lysine residue at position 9 (K9). To determine 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 arginine (K9R) or serine (K9S). 3A9 CD3-hybridomas were transduced with retrovirus encoding the mutant CD3 -chains. TCR expression and T cell function were comparable to those in T cells expressing wild-type CD3 (Fig. 5A and data not shown). Previous studies have suggested an important role for the K9 residue in the extracellular domain of CD3 in signal transduction as well as in stabilizing the association of CD3 with the TCR:CD3 complex (31). Thus, we were surprised to find that these mutations had no effect on the function of 3A9. Although the reason for this discrepancy is unclear, it may simply represent differences in the T cell systems used. Transductants were then stimulated, surface-biotinylated, and immunoprecipitated with anti-TCR-C as described. The data clearly show that mutation of K9 had no effect on CD3 biotinylation (Fig. 5B). After detection of biotinylated proteins, the blot was stripped and probed with a mixture of anti-CD3 and anti-CD3 Abs (Fig. 5C). This blot clearly demonstrates that despite a weak biotin signal, CD3 is associated with TCR complexes in unstimulated cells. It also supports our previous finding that the increased biotin signal of TCR-associated CD3 is not due to an increased amount of protein. The relative increase in the CD3:CD3 biotin ratio between unstimulated and stimulated cells 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.
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becomes more accessible 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-based activation motif residues and activation of ZAP-70 (32, 33). The question arose of whether the architectural changes observed after TCR ligation were dependent on Src family PTKs. The role of these kinases in the induction of TCR:CD3 architectural changes could provide information regarding the order of events following TCR ligation. 3A9 T cell hybridomas or 3A9 CD3 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 surface-biotinylated, and cell lysates were immunoprecipitated twice sequentially with anti-TCR-C, followed by anti-CD3. Immunoprecipitated proteins were resolved by SDS-PAGE, blotted, and probed with streptavidin-AP for detection of biotinylated proteins, as described above. Parallel experiments showed that TCR down-modulation was significantly decreased, and IL-2 production was abrogated after PP2 treatment of 3A9 T cells (data not shown). After Ag stimulation, there was a substantial increase in the TCR-associated CD3 signal as well as an increase in free CD3, as described previously (Fig. 6). Pretreatment of the cells with PP2 almost completely abrogated these changes (Fig. 6). Taken together, these data suggest that the structural changes induced in the TCR:CD3 complex after TCR ligation are highly dependent on Src family PTKs and thus presumably occur after or concurrently with kinase activation.
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Discussion |
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NH2 terminus, which is ordinarily buried in the complex. For discussion purposes, we have generally interpreted this event as reflecting architectural changes in the TCR:CD3 complex. We would also propose, based on increased free CD3 levels following stimulation, that such architectural changes may ultimately lead to dissociation of the CD3 dimer from the TCR:CD3 complex. However, the origin of the free cell surface CD3 has not yet been formally demonstrated, and thus we cannot rule out the possibility that this free CD3 derives from de novo synthesis induced by TCR ligation. This would be surprising, because it is generally thought that the free CD3 seen after stimulation are products of de novo synthesis and cannot escape intracellular retention (13, 15, 16, 21). Further studies are required to resolve this issue. Regardless, we propose that the architectural changes occurring within the TCR:CD3 complex after TCR ligation may be a mechanism by which T cells recognize ligated TCR and direct them to lysosomal compartments for degradation. Recently, D’Oro and colleagues (42) proposed that the CD3 -chain maintains cell surface receptor expression by sterically blocking internalization sequences in other TCR components, using chimeric CD3 -chains expressing intracellular domains of varying lengths to show that the size of the intracellular tail, rather than the amino acid sequence, was all that was required to support the expression of the fully assembled receptor. We have made similar observations (A. Szymczak, H. Liu, and D. A. A. Vignali, unpublished observations). Other studies with CD16/CD3 chimeras have suggested that CD3 may shield the CD3 dileucine motif (24). As this motif is known to mediate targeting to lysosomes for degradation (22, 23), structural changes in the TCR:CD3 complex, particularly those involving CD3, may result in targeting of ligated TCR:CD3 complexes to lysosomes for degradation, rather than recycling back to the cell surface. It is also possible that other motifs are involved in this process, as T cells lacking the cytoplasmic tail of CD3 and CD3 still down-modulate TCR after Ag stimulation (43). Indeed, an endocytosis signal in CD3 has recently been described (44). Thus, TCR down-modulation may be determined by the exposure of intracellular retention motifs that are normally concealed by CD3.
Until recently, the idea that the TCR:CD3 complex undergoes conformational change after TCR ligation was speculative. However, a study by Gil and colleagues (45) demonstrated that ligation of TCR resulted in the exposure of a proline-rich sequence in CD3, which, in turn, leads to recruitment of the adaptor protein, Nck. Interestingly, these events occurred before and independently of tyrosine kinase activation and appeared to be involved in early signaling processes. In our study the observed architectural changes initiated by TCR ligation were heavily dependent on Src family PTK activity, suggesting that the exposure of CD3 and its subsequent dissociation from the remainder of the TCR:CD3 complex are relatively late events and may occur downstream of CD3 exposure and Nck recruitment. It is therefore possible that the architectural changes observed in this study may not be directly involved in the signaling process, but, rather, are required for the downstream effects of TCR ligation, such as TCR down-modulation. Importantly, it has been shown that the expression of constitutively active p56lck induces TCR down-modulation (39).
An interesting question remains of which residue in the short extracellular domain of CD3 is biotinylated upon TCR ligation? Previous studies have suggested an important role for the K9 residue in the extracellular domain of CD3 in signal transduction as well as in stabilizing the association of CD3 with the TCR:CD3 complex (31). The NHS-ester used to label proteins with biotin preferentially labels lysine residues, but can also label the protein NH2 terminus. Although other residues, such as arginine, histidine, and cysteine, can be labeled, the frequency and efficiently of this reaction at physiological pH are very low. Thus, the K9 residue appeared to be the most likely candidate. We hypothesized that K9 may, in unligated TCR:CD3 complexes, be involved in interactions with amino acids in other TCR or CD3 chains, preventing its biotinylation. Upon TCR ligation, it is possible that this association is broken, exposing the K9 residue for biotinylation. However, our studies clearly showed that the K9R and K9S mutations had no effect on CD3 biotinylation. Although it remains to be determined which residue/moiety in the extracellular domain of CD3 is biotinylated, the most likely candidate is the NH2 terminus, although these are usually capped in eukaryotic cells.
An interesting question raised by our data is whether there are other multichain transmembrane receptors that undergo architectural changes and/or dissociation upon ligand binding. CD3 is part of a family of molecules that form an integral part of many different receptors (11). For instance, CD3
(a splice variant of CD3) and the FcR can substitute for CD3 in the TCR:CD3 complex (46, 47, 48, 49). In addition, CD3 and FcR form part of the FcRIII (CD16) Ig FcR expressed on T and NK cells. FcR is also incorporated into several other FcRs, such as FcRI, FcRII, and FcRI, on macrophages, monocytes, neutrophils, mast cells, and basophils (49). Lastly, the CD3-like membrane adapter proteins, DAP12 and DAP10 (KAP10), form NK cell-activating receptors with CD94/NKG2C and NKG2D, respectively (50, 51, 52, 53). Thus, it will be interesting to determine whether the ligand-induced alteration and/or dissociation of such transmembrane adapter molecules from multichain receptor complexes are common events. Given the large number of receptors that incorporate CD3-like transmembrane adapter molecules, which are represented in all leukocytes and parts of the CNS (49, 50, 51, 52, 53, 54), our demonstration of ligand-induced architectural changes in the TCR may have broad implications for our understanding of the molecular events that initiate or perpetuate signal transduction via multichain receptor complexes.
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Acknowledgments |
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cDNA. We also thank Richard Cross and Mahnaz Paktinat for assistance with the flow cytometry, Janet Gatewood for performing the particle-based flow cytometric cytokine assay, Elio Vanin for assistance and reagents for establishing retroviral transduction in our laboratory, and members of the Vignali laboratory for constructive criticisms and comments. |
Footnotes |
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2 Current address: Department of Microbiology and Immunology, University of Melbourne, Melbourne, Victoria 3010, Australia.
3 Current address: Department of Microbiology and Immunology, University of Nevada School of Medicine, Reno, NV 89557.
4 Address correspondence and reprint requests to: Dr. Dario Vignali, Department of Immunology, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105-2794. E-mail address: dario.vignali{at}stjude.org
5 Abbreviations used in this paper; IRES, internal ribosome entry sequence; AP, alkaline phosphatase; GFP, green fluorescent protein; HEL, hen egg lysozyme; IP, immunoprecipitation; LAT, linker for activation of T cells; MSCV, murine stem cell virus; NHS, N-hydroxysuccinimide; PCC, pigeon cytochrome c; PTK, protein tyrosine kinase; WT, wild type; PP2, 4-amino-5-(4-chlorophenyl)-8-(t-butyl)pyrazolo[3,4-d]pyrimidine.
Received for publication May 2, 2003. Accepted for publication January 5, 2004.
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receptor subunit (FcRI) facilitates T cell receptor expression and antigen/major histocompatibility complex-driven signaling in the absence of CD3 and CD3. J. Biol. Chem. 266:15974.Related articles in The JI:
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