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The Journal of Immunology, 2002, 169: 6269-6278.
Copyright © 2002 by The American Association of Immunologists

Regulation of Constitutive TCR Internalization by the {zeta}-Chain

Ugo D’Oro1,*, Ivana Munitic1,{dagger}, George Chacko{dagger}, Tatiana Karpova{ddagger}, James McNally{ddagger} and Jonathan D. Ashwell2,{dagger}

* Chiron, Siena, Italy; {dagger} Laboratory of Immune Cell Biology and {ddagger} Fluorescence Imaging Group, Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The ability of a T cell to be activated is critically regulated by the number of TCRs expressed on the plasma membrane. Cell surface TCR expression is influenced by dynamic processes such as synthesis and transport of newly assembled receptors, endocytosis of surface TCR, and recycling to the plasma membrane of internalized receptors. In this study, the internalization of fluorescently labeled anti-TCR Abs was used to analyze constitutive endocytosis of TCRs on T cells, and to investigate the role of the {zeta}-chain in this process. We found that cell surface TCRs lacking {zeta} were endocytosed more rapidly than completely assembled receptors, and that reexpression of full-length {zeta} led to a dose-dependent decrease in the rate of TCR internalization. Rapid TCR internalization was also observed with CD4+CD8+ thymocytes from {zeta}-deficient mice, whereas TCR internalization on thymocytes from CD3-{delta} deficient animals was slow, similar to that of wild-type thymocytes. This identifies a specific role for {zeta} in the regulation of constitutive receptor internalization. Furthermore, chimeric {zeta} molecules containing non-native intracellular amino acid sequences also led to high levels of TCR expression and reduced TCR cycling. These effects were dependent solely on the length of the intracellular tail, ruling out a role for intracellular {zeta}-specific interactions with other molecules as a mechanism for regulating TCR internalization. Rather, these findings strongly support a model in which the {zeta}-chain stabilizes TCR residency on the cell surface, and functions to maintain cell surface receptor expression by sterically blocking internalization sequences in other TCR components.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
An indispensable function of the immune system in higher vertebrates is the recognition of Ag by T cells. This requires that peptide Ags bound to the surface of APCs interact with the cell surface TCR. The consequences of TCR engagement include complex changes in surface distribution of TCR and other membrane proteins, activation of intracellular signaling pathways, and changes in the transcriptional profile of genes expressed by the T cell (1). The nature and magnitude of these molecular modifications are integrated by the T cell into functional responses such as clonal expansion and cytokine secretion, anergy, or death by apoptosis.

The TCR exists as a cluster of integral membrane proteins comprising three groups of molecules. A disulfide-linked heterodimer of TCR{alpha} and TCR{beta} binds ligand, while the CD3 complex (composed of {gamma}-, {delta}-, and {epsilon}-chains) and a second disulfide-linked dimer of {zeta} transduce binding signals across the plasma membrane. Although the {alpha}- and {beta}-chains of the TCR are clonotypic, the remaining components are invariant, thereby allowing diversity in Ag recognition while conserving mechanisms of signal transduction. The fully assembled TCR contains at least one TCR{alpha}{beta} dimer, CD3{gamma}{epsilon} and CD3{delta}{epsilon} pairs, and a {zeta}-chain dimer, and thus exists as an octamer composed of six different subunits (2). Variations on this theme occur when a TCR{gamma}{delta} dimer is substituted for TCR{alpha}{beta} or when the {zeta} homodimer is replaced by heterodimers of {zeta} and its {eta} splice variant or the {gamma}-subunit common to CD16, CD64, and the high affinity receptor for IgE. The TCR that comprises TCR{alpha}{beta}, CD3{gamma}{delta}{epsilon}, and {zeta} is expressed on the majority of T cells and is the subject of this study.

Assembly of the TCR begins in the endoplasmic reticulum with the formation of dimers of {zeta} and dimers of {alpha}{beta}, and is completed in the trans-Golgi compartment when a TCR{alpha}{beta}:CD3{gamma}{delta}{epsilon} complex associates with a {zeta} dimer to form the octameric holoreceptor (3, 4). Extensive "editing" in the form of pre-Golgi retention ensures that only correctly folded and assembled TCR complexes are allowed to reach the cell surface (5). Stable assembly and expression of surface TCR is limited by the amount of available {zeta}. The majority (85–95%) of newly synthesized {alpha}{beta} and CD3{gamma},{delta},{epsilon} are degraded within 4 hr of synthesis, whereas 80–100% of {zeta} is long-lived and has a half-life of 10–20 h (3). In the case of isolated subunits or partial complexes lacking any chain other than {zeta}, retention and degradation in the pre-Golgi compartments also occur. Hexameric complexes lacking {zeta} are reported to be transported from the Golgi complex to lysosomes where they are degraded. However, a small proportion of {zeta}-deficient TCR is believed to escape to the surface, accounting for the low expression of TCR in T cell hybridomas and mice lacking the {zeta}-chain (6, 7). In contrast, fully assembled octameric complexes are transported to the surface where they are long-lived.

TCR expression is dynamic. A cycling pool of TCR is constitutively internalized and re-expressed (8, 9, 10). The functional consequence of rapid cycling is that surface receptor levels, which regulate the ability of the T cell to be activated (11, 12, 13), respond rapidly to small changes in the rate constants for internalization and surface transport. Changes in surface TCR also occur physiologically. TCR engagement induces down-regulation of surface TCR levels, activation of protein kinases, and targeting of activated receptors to lysosomes for degradation (14, 15). Down-regulation of unengaged receptors is also reported to occur during activation (16). In addition, TCR levels are dynamically regulated during thymic development, with immature CD4+CD8+ cells having ~10-fold lower cell surface TCR than more mature T cells that have undergone positive selection (17). Experimentally, reductions in surface TCR can be achieved with reagents that induce protein kinase C (PKC)3 activity, while elevations can be obtained with glycolipids such as ceramide, which can activate serine/threonine phosphatases (8, 9, 13). There is evidence for additional complexity in the assembly and dynamic expression of the TCR. It has been reported that {zeta} can be expressed at the surface independently of the other subunits and cycles at a different rate from the other components (18, 19), and that functional dissociation of TCR{alpha}{beta} from CD3{gamma}{delta}{epsilon} occurs after activation (20). Furthermore, recent data indicate the presence of two TCR{alpha}{beta} dimers in a single TCR complex (21). The factors that regulate transport to the cell surface, internalization, and cycling of the TCR are important modulators of T cell function because they determine the ability of the T cell to recognize ligand and be activated.

In this study, we examine constitutive TCR internalization in living cells expressing either octameric or hexameric (lacking {zeta}) TCRs. We find that cell surface TCRs lacking {zeta} are endocytosed more rapidly than wild-type TCRs, suggesting that {zeta} plays an important role in regulating constitutive internalization. Furthermore, using forms of {zeta} that express heterologous, non-native intracellular sequences, we observed that the ability of {zeta} to support cell surface TCR expression and regulate internalization is independent of the primary structure of the intracellular domain but is dependent upon the length of this region, strongly suggesting that the function of {zeta} in maintaining cell surface TCR levels is to mask internalization sequences inherent in other TCR components.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

C57BL/6 mice are maintained in our breeding colony. {zeta}-deficient mice (22) were provided by Dr. E. Shores (Food and Drug Administration, Bethesda, MD) and CD3{delta}-deficient mice were provided by Drs. D. Kappes (Fox Chase Cancer Center, Philadelphia, PA) and A. Singer (National Cancer Institute, Bethesda, MD).

Reagents

R-PE and Alexa Fluor 488 were purchased from Molecular Probes (Eugene, OR). Succinimidyl 4-(p-maleidophenyl) butyrate (SMPB) and 2-iminothiolane (Traut’s Reagent) were purchased from Pierce (Rockford, IL). Dithioerythritol, aprotinin, leupeptin, pepstatin, PMSF, and iodoacetamide were purchased from Sigma-Aldrich (St. Louis, MO). Ceramic hydroxyapatite (type II) was purchased from Bio-Rad (Hercules, CA). Protein-A I125 was purchased from ICN Pharmaceuticals (Costa Mesa, CA). Protein A and protein G-Sepharose were purchased from Zymed Laboratories (San Francisco, CA). mAbs H57-597 (H57, anti-TCR{beta}), H146-968 (anti-TCR{zeta}), and 145-2C11 (2C11, anti-CD3{epsilon}) were purified from culture supernatants by protein A affinity chromatography (18, 23, 24, 25). mAb H57-PE, 2C11-PE, anti-mouse CD4-FITC, anti-mouse CD8{alpha}-CyChrome, anti-mouse CD8{alpha}-Cy5, hamster IgG-PE, and purified control mouse IgG were obtained from BD PharMingen (San Diego, CA). Monoclonal anti-hemagglutinin (HA) Ab 12CA5 was purchased from Roche (Milan, Italy). Goat anti-mouse IgG conjugated to FITC (GAM-FITC) was from Jackson ImmunoResearch Laboratories (West Grove, PA). Fab of H57 were generated by digestion of intact Ab with immobilized papain according to the manufacturer’s instructions (Pierce). Fc fragments and intact Ab were removed by passing the digest over protein A-Sepharose beads. The Fab preparation was free of intact Ab when visualized by Coomassie Blue staining after electrophoresis on SDS-PAGE gels. Coupling of Fab to PE was performed according to a protocol kindly provided by Dr. H. Petrie (Memorial Sloan-Kettering Cancer Center, New York, NY). Briefly, Fab were dialyzed in 100 mM sodium phosphate, pH 6.8, then treated with SMPB to introduce reactive maleimide groups while PE was treated with Traut’s Reagent to introduce sulfhydryl groups. After desalting on PD-10 columns to remove excess SMPB and Traut’s Reagent, respectively, modified PE and Fab were allowed to react with each other for 16 h at room temperature. Free sulfhydryl groups were quenched by sequential treatment with dithioerythriotol and iodoacetamide. Fab-PE conjugates were dialyzed in 2 mM phosphate, pH 7.0, and were purified by hydroxyapatite chromatography. Coupling of Fab to Alexa Fluor 488 (H57-Fab Alexa) was performed according to the manufacturer’s instructions.

Cell lines

The T cell hybridomas 2B4.11 and MA5.8 were maintained in RPMI 1640 (Biofluids, Gaithersburg, MD) supplemented with 10% heat-inactivated FCS, 250 µg/ml gentamicin, 100 U/ml penicillin, 4 mM glutamine, and 5 µM 2-ME (complete medium). For imaging experiments, the RPMI used was free of phenol red. The cell lines MA{zeta}.1, MA {zeta}.2, and MA{zeta}.3 (originally named 2A7-7, 2A7-37, and 2B4.99) were previously cloned from G-418-resistant cells generated by stable transfection of the cDNA for murine TCR{zeta} in MA5.8 cells (26) and unpublished cell lines were generously provided by A. Weissman (National Cancer Institute).

Buffers

FACS buffer was PBS containing 0.1% sodium azide and 1% BSA. Stripping buffers were either 0.5 M NaCl, 0.5 M acetic acid, pH 2.5 (acetate stripping buffer) or 100 mM glycine, 100 mM NaCl, pH 2.5 (glycine stripping buffer). Triton X-100 lysis buffer was 1% Triton X-100, 100 mM NaCl, 20 mM Tris-HCl, pH 8.0, to which the protease inhibitors leupeptin, pepstatin, aprotinin, at a final concentration of 1 µg/ml and PMSF at 17 µM, were added immediately before use.

Transfection

2B4.11 or MA5.8 cells were resuspended at 108/ml in RPMI without serum. 200 µl of cell suspension was gently mixed with 10 µg of plasmid DNA in an electroporation cuvette. Electroporation was performed using a Bio-Rad Gene Pulser at 960 µF and 250 V. Cells were then either transferred to 5 ml of complete medium and analyzed by flow cytometry 18 later, or were selected with G-418 (Life Technologies, Grand Island, NY) 48 h after electroporation and stable-expressing clones were isolated by limiting dilution.

Plasmids

Murine {zeta} devoid of the leader sequence and containing native amino acids 21–164 (wt) or amino acid residues 21–65 ({zeta}t65) were obtained by PCR amplification using the following oligonucleotides: 5'-AAAAAAAGCTTGGCCCAGCCGGCCCAGAGCTTTGGTCTGCTG-3' and 5'-AAAAACCCGGGATTAGCGAGGGGCCAGGGTCTG-3' (for {zeta} wt) or 5'-AAAAACCCGGGACTACAGGTTGGCAGCAGTCTCTGC-3' (for {zeta}t65). The PCR products were digested with SfiI and XbaI and ligated in pDisplay (Invitrogen, Carlsbad, CA) inframe with the Ig leader and the HA sequence present in this plasmid. All three immunoreceptor tyrosine-based activation motifs are missing in {zeta}t65, effectively limiting the length of the intracellular domain to 14 amino acids. Two additional mutants that read through the stop codon of {zeta}t65 and add irrelevant sequences of 29 ({zeta}t65 + 29) and 67 ({zeta}t65 + 67) amino acids at the C terminus were obtained by PCR amplification. A plasmid expressing {zeta} truncated at amino acid 65 in frame with a green fluorescent protein (GFP) sequence at the C-terminal was obtained by PCR amplification using the following oligonucleotides: 5'-AAAGGTACCATCCCAGGGAAGCAGAAGATGAAGTGGAAAGTGTCT-3' and 5'-TGGATCCCGGGCCTGCAGGTTGGCAGCAGTCTCTGC-3'. The PCR product was digested with KpnI and XbaI before ligation in pEGFP-N1 (Clontech Laboratories, Palo Alto, CA). The sequence of constructs obtained by PCR was confirmed by DNA automated sequencing.

TCR internalization assay

2B4.11 and MA5.8 cells were incubated in 48-well plates at 200,000/ml in complete medium at 37°C for described intervals in the presence of H57-PE at a concentration of 1 µg/ml. Each well contained 750 µl of cell suspension. Internalization was terminated by the addition of an equal volume of ice-cold FACS buffer. The contents of each well were then removed, pelleted at 3000 rpm for 1 min (room temperature) in an Eppendorf 5415 microcentrifuge and resuspended in 100 µl of ice-cold FACS buffer. To remove surface-bound Ab, 300 µl of stripping buffer was added to the resuspended cells at room temperature. Twenty seconds later for acetate stripping buffer or 2 min later for glycine stripping buffer, 1 ml of ice-cold FACS buffer was added and cells were centrifuged as before. Supernatants were removed; the cells were resuspended in 1 ml of ice-cold FACS buffer. The centrifugation procedure was repeated once more and cells were resuspended in 0.4 ml of FACS buffer and stored on ice. For some of the experiments, H57-PE was allowed to bind to the cells for 45 min on ice, and the unbound Abs were removed by washing with cold complete medium. The cells were warmed to 37°C and at varying periods of time the cells were acid-stripped and internalized fluorescence was measured. To measure intracellular fluorescence, cells were analyzed on a FACScan flow cytometer (BD Biosciences, Mountain View, CA). Live cells were identified by forward and side scatter profiles and the median fluorescence of 10,000 gated events was measured for each experimental condition. Internalization of TCR was expressed as the percentage of a surface equivalent of TCR and was calculated by the following formula: % SRt = 100 x (ARFt-AF)/(SF-AF) where SRt is the percent of surface receptor internalized at time "t", ARFt is the acid-resistant fluorescence at time "t", AF indicates cellular autofluorescence of cells (median fluorescence of unstained cells or cells that were stained and then acid-stripped), and SF refers to the median fluorescence of cells that were stained with H57-PE for 20 min at 4°C. The actual data points are plotted, as well as linear regression line-calculated with CA-Cricket Graph (Computer Associates International, Islandia, NY) software.

Thymocytes

Thymocytes were collected from C57BL/6 and {zeta}-deficient mice on the same background (kindly provided by Dr. E. Shores, Food and Drug Administration). The preparation of thymocytes was performed in ice-cold complete medium until they were warmed to 37°C at the beginning of the experiment. Thymocytes were plated in 96-well plates at a density of 106 cells per 200 µl of complete medium at 37°C. TCR internalization of H57-PE was measured as described above, with the exception that exposure to glycine stripping buffer was limited to 80 s and terminated by the addition of 1 ml of FACS buffer adjusted to pH 10. The cells were stained with anti-CD4-FITC and anti-CD8-CyChrome (in the case of cells cultured with Ab, after acid-stripping). Propidium iodide was included so that dead cells could be eliminated from analysis. A minimum of 10,000 live CD4+CD8+ cells were analyzed with a FACSVantage flow cytometer (BD Biosciences).

Imaging of live cells

2B4.11 cells were incubated with H57-Fab Alexa (10 µg/ml) for 30–45 min on ice, after which time one group was washed and maintained on ice and another was warmed to 37°C, cultured for the indicated times, washed, and maintained on ice until analyzed. Cells were resuspended at three million cells/ml in complete medium without phenol red. The cell suspension (300 µl) was pipetted into a chambered coverglass system and imaged without further manipulation. Imaging was performed with a Zeiss LSM 510 confocal microscope (Oberkochen, Germany), with the stage maintained at room temperature, and a 100x oil immersion objective lens. Excitation was with the 488-nm line of an argon laser and emissions were monitored with a 505-nm long pass filter.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Internalization of cell surface TCR

In initial experiments, we used confocal microscopy to analyze the distribution and fate of internalized Ab in single cells. H57 (anti-TCR{beta}) Fab was coupled to the green fluorescent dye Alexa, a fluorophore that is optimized for use with an argon laser in confocal imaging systems. 2B4.11 T hybridoma cells were incubated on ice or at 37°C in the presence of H57 Fab-Alexa. For cells maintained on ice, the TCR was readily detected on the plasma membrane, with only a small amount of intracellular fluorescence detectable (Fig. 1Go, A and C). Cell surface TCR was also detected on cells cultured at 37°C, but in addition there was the appearance of highly fluorescent large intracellular vesicles (Fig. 1Go, B and D). Cell surface staining and accumulation of fluorescent label was inhibited by an excess of unlabeled Ab (data not shown). This accumulation of fluorescence in discrete vesicles is indicative of a regulated process that transports internalized TCR inside the cytoplasm.



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  		FIGURE 1. Abs bound to constitutively internalized TCR are transported to discrete intracellular vesicles. 2B4.11 hybridoma cells incubated with H57 Fab-Alexa on ice (A) or after incubation on ice were incubated at 37°C for 30 min (B). C and D, Enlarged images of 2B4.11 cells incubated as above except incubation at 37°C was for 60 min. Imaging at x100 was performed by confocal microscopy. The scale bars indicate 10 µm. Arrowheads indicate a few of the many fluorescent vesicles.

 
Although it appeared that internalized TCR-bound fluorescent Ab continued to accumulate in cells over time, this is difficult to quantitate using single cell imaging. Another approach that has been used to follow receptor internalization is to bind radiolabeled receptor-specific Abs to intact cells and, over time, ask what fraction is protected from acid-stripping (9). This has been extended to the use of fluorescently labeled anti-TCR Abs to follow receptor internalization (27, 28). We chose to use this method to study TCR internalization because it is sensitive and quantitative, and allows one to follow receptor fate in individual cells. Quantitation of fluorescence at the individual cell level was done by flow cytometry. Cells were acid-stripped to remove cell surface TCR-bound Abs (which typically removed >95% of the surface bound Ab; data not shown) (9) and the amount of internalized Ab relative to the length of incubation was determined. As shown in Fig. 2GoA, a time-dependent increase in intracellular fluorescence was observed when the 2B4.11 T cell hybridoma was cultured in the presence of H57-PE. This increase was linear over time, up to at least 4 h (Fig. 2Go and data not shown). Control experiments with PE-labeled IgG with an irrelevant specificity showed no appreciable accumulation over time (Fig. 2GoB). By comparing the amount of internalized fluorescence to the amount of surface fluorescence, it is possible to calculate the fraction of surface TCR internalized over time (Fig. 2GoC). For example, in 2B4.11 cells, ~70% of the steady-state cell surface complement of TCRs was internalized per hour. Stated differently, it takes ~90 min to internalize the entire pool of cell surface receptors, or 1.1% of surface TCR internalized per minute. Similar results were obtained with PE-coupled H57 Fab, although the absolute measured rates of turnover with Fab were approximately half that of intact Ab, perhaps because of the lesser avidity (data not shown; see Fig. 3Go). The rapid accumulation of intracellular fluorescence to levels greater than that seen with cell surface labeling is most likely a reflection of TCR recycling (10), although newly synthesized receptors may contribute as well. These observations are consistent with previous reports using radiolabeled anti-TCR Ab and 2B4.11 cells, in which ~50% of surface TCRs were internalized per hour (9).



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  		FIGURE 2. Measurement of TCR endocytosis. A, Time-dependent accumulation of acid-resistant H57-PE fluorescence. 2B4.11 cells were incubated with H57-PE (1 µg/ml) for 30, 60, 90, and 120 min. Cells were washed once to remove free Ab, then surface-bound Ab was stripped at low pH, and internalized Ab was quantified by flow cytometry. Background fluorescence is indicated by the dotted histogram. Inset, Surface TCR levels in 2B4.11 cells were measured by staining with H57-PE at 4°C for 30 min then washing to remove free Ab. B, Comparison of internalized anti-TCR Ab with irrelevant IgG at 2 h. C, TCR internalization in 2B4.11 cells as a fraction of surface TCR level.

 


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  		FIGURE 3. Enhanced TCR endocytosis in {zeta}-deficient cells. A, Surface TCR levels. 2B4.11 (solid line) and MA5.8 (dashed line) cells were stained on ice with H57-PE. One aliquot of cells was acid-stripped (for clarity, only 2B4.11 is shown, dotted line). B, TCR internalization after 60 min of incubation. 2B4.11 and MA 5.8 cells were acid-stripped of cell surface H57-PE after 60 min of incubation at 37°C and analyzed for internalized fluorescence. The dotted line represents 2B4.11 cells that were incubated with H57-PE on ice and then acid-stripped. C and D, TCR internalization as a function of cell surface levels. 2B4.11 and MA5.8 cells were incubated with PE-conjugates of intact (C) or Fab (D) of H57 for the indicated times. E, 2B4.11 and MA5.8 cells were incubated with PE-2C11 and % CD3 internalization was determined at the indicated times. F, 2B4.11 and MA5.8 cells were stained with H57-PE on ice, washed, warmed to 37°C, and acid-resistant intracellular fluorescence was measured over time. The fraction of the initial fluorescence remaining after acid stripping is shown in duplicate cultures, ± SD. G, 2B4.11 and MA5.8 cells were incubated with anti-CD69-PE and percent internalization was determined at the indicated times. One representative experiment of seven (C), two (D), three (E), or three (G) is shown.

 
Rapid endocytosis of TCR in {zeta}-deficient cells

TCRs lacking {zeta} are expressed on the cell surface at much reduced levels, which has been attributed to bypass of transport to the surface by direct shunting of hexameric TCR (TCR{alpha},{beta} plus CD3{gamma}{epsilon} and {delta}{epsilon}) from the Golgi to lysosomes (5, 6). However, an alternative possibility is that these partial receptor complexes take the normal route of transport to the cell surface, but are then rapidly internalized and subsequently degraded in lysosomes. To determine whether the latter was possible, TCR internalization was studied in the MA5.8 cell, a 2B4.11 variant that lacks expression of {zeta} but synthesizes all other subunits at the same rate as 2B4.11 cells (6). Despite the relatively low levels of TCR expressed on MA5.8 cells, turnover of surface TCRs occurred at a substantially greater rate than in the parental cell line (Fig. 3GoC). This difference was also observed when PE-labeled H57 Fab (Fig. 3GoD) or PE-labeled anti-CD3{epsilon} (Fig. 3GoE) was used. In contrast to 2B4.11 cells, {zeta}-deficient MA5.8 cells replaced one surface equivalent every 27 min, or 3.7% of surface receptor levels per minute. We also examined the internalization rate of the cohort of receptors present on the cell surface at a given time. 2B4.11 and MA5.8 cells were stained with anti-TCR Abs on ice, washed to remove unbound Ab, warmed to 37°C, and at different periods of time were acid-stripped and analyzed for intracellular fluorescence (Fig. 3GoF). Even as early as 7 min after warming, 25% of TCRs were internalized in MA5.8 cells, with the amount reaching ~60% by 21 min and plateauing at ~90% by 42 min. In contrast, 2B4.11 cells internalized only 5% of its receptors at 7 min, and 16% by 21 min. Internalization on 2B4.11 cells continued at this slow rate up to 3 h, at which time ~70% were internalized (data not shown). Thus, the majority of surface receptors internalize in both cells, and this occurs much more rapidly when TCRs lack {zeta}.

It is possible that the rapid internalization of TCRs on MA5.8 cells could be due to cell-specific factors other than lack of {zeta} expression. One argument against this is that similar results were obtained with an independently derived {zeta}-deficient variant of 2B4.11, 2M.2 (Ref. 29 and data not shown). Furthermore, internalization of other cell surface molecules, such as CD69 (Fig. 3GoG) and Thy-1 (data not shown), was similar between 2B4.11 and MA5.8 cells. Nonetheless, to directly address this issue, TCR turnover was analyzed with MA5.8 cells in which the {zeta}-chain had been reintroduced and is stably expressed (26). Stable expression of {zeta} in MA5.8 complemented the deficiency, inducing both up-regulation of surface TCR and a reduction in the rate of receptor turnover (Fig. 4Go). Importantly, the effect of {zeta} on TCR cycling was {zeta} gene dose-dependent, and stable transfectants that express levels of {zeta} higher than that of the parental 2B4.11 cells actually had lower rates of TCR cycling than 2B4.11. For example, MA{zeta}.2 cells, which express the highest level of {zeta} (Fig. 4Go, inset), replace one surface equivalent every 271 min (0.37% of surface TCR is replaced every minute), a 10-fold difference compared with the {zeta}-deficient cell line. Therefore, the half-life of the TCR at the cell surface correlates directly with the level of {zeta}.



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  		FIGURE 4. TCR internalization rate is inversely related to {zeta} expression. Rates of TCR internalization were measured for a cohort of T cell hybridomas with varying surface TCR levels. 2B4.11, MA5.8, and three independent stable wild-type transfectants derived from MA5.8 were incubated with H57-PE for the indicated times. Receptor internalization as a fraction of cell surface levels is plotted as a function of time. Numbers in parentheses indicate cell surface TCR levels defined as median fluorescence in arbitrary units of cells stained with H57-PE at 4°C. Inset, {zeta} expression in each cell line was determined by immunoprecipitation and then immunoblotting with an anti-{zeta} mAb.

 
Rapid TCR turnover in {zeta}-deficient, but not CD3{delta}-deficient, thymocytes

To determine whether TCRs lacking {zeta} also internalize rapidly in normal cells, TCR internalization was quantitated using thymocytes from mice deficient in {zeta} expression ({zeta}-KO mice) (22). The loss of {zeta} in these animals results in arrested T cell development, with very few cells being able to progress beyond the CD4+CD8+ (double-positive (DP)) stage of development. As previously reported, surface staining with H57-PE of unmanipulated {zeta}-deficient DP thymocytes demonstrated that TCR levels were much lower than on wild-type DP thymocytes (Fig. 5GoA). Thymocytes from wild-type or {zeta}-deficient animals were cultured with H57-PE for varying periods of time and, after acid-stripping, were analyzed for internalized fluorescence. One representative experiment of four is shown in Fig. 5GoB. To allow comparison between wild-type and {zeta}-deficient cells, analysis was restricted to DP cells. Both wild-type and {zeta}-KO DP thymocytes showed a time-dependent accumulation of fluorescence. Although there was heterogeneity with regard to the amount of increase, there was no obvious difference between the two thymocyte sources. Strikingly, despite the very different levels of cell surface TCR, over the course of the incubation the amount of fluorescence accumulated by the {zeta}-KO thymocytes approached the amount accumulated by wild-type cells. When normalized to the amount of TCR expressed, it is apparent that intracellular fluorescence accumulated much faster in {zeta}-deficient than in wild-type thymocytes (Fig. 5GoC). Therefore, just as with T cell hybridomas, the lack of {zeta} results in an increase in the rate of internalization of cell surface TCR.



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  		FIGURE 5. TCR internalization is more rapid in {zeta}-deficient than in wild-type thymocytes. A, Steady-state cell surface TCR expression. Wild-type and {zeta}-deficient thymocytes were stained with H57-PE on ice and then analyzed by flow cytometry before (solid line) or after (dashed line) acid stripping. Profiles of DP cells are shown. B, Time course of TCR internalization. Wild-type or {zeta}-deficient thymocytes were incubated with H57-PE at 37°C for the indicated times, acid-stripped, and analyzed by flow cytometry. Profiles of DP cells are shown. One representative experiment of five is shown. C, The data in B are plotted as percent surface TCR internalized over time.

 
It is conceivable that the apparent rate of internalization of receptors expressed at low levels might be influenced by factors other than {zeta}-chain expression. For example, if a component of the endocytic machinery is limiting and thus the absolute number of receptor internalized was fixed, it would then appear that receptors expressed at low levels were being internalized more rapidly then ones expressed at higher levels. One way to address this issue is to quantitate receptor turnover in T cells expressing normal levels of {zeta}-chain but low levels of cell surface TCR due to deficiency of a different subunit. DP thymocytes from CD3{delta}-deficient mice have low levels of cell surface TCR, comparable to the levels found in the absence of {zeta}, both having ~5% of wild-type levels (Ref. 30 and Fig. 6GoA). TCR internalization of CD3{delta}-deficient DP thymocytes was compared with wild type (Fig. 6GoB). Unlike {zeta}-deficient thymocytes, the rate of TCR internalization on CD3{delta}-deficient thymocytes was indistinguishable from that of wild-type cells. Therefore, simply having low steady-state levels of TCR on the surface does not result in an increase in the determined rate of internalization, but rather the {zeta}-chain has a specific role in maintaining receptors on the plasma membrane.



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  		FIGURE 6. TCR internalization is similar between CD3{delta}-deficient and wild-type thymocytes. A, Steady-state cell surface TCR expression on wild-type and CD3{delta}-deficient thymocytes was determined as in Fig. 5GoA. B, Time course of TCR internalization with wild-type or CD3{delta}-deficient thymocytes was determined as in Fig. 5GoB. One representative experiment of four is shown. C, Internalized fluorescence is plotted as percent surface TCR internalized over time. The first four time points for each group were performed in duplicate cultures, and the arithmetic mean of duplicate samples is shown.

 
Sequence-independent regulation of surface TCR levels by {zeta}

There are a number of different mechanisms by which {zeta} could stabilize expression of the TCR on the cell surface. For example, {zeta} associates with the cytoskeleton (31, 32) and a T cell-specific transmembrane disulfide-linked homodimer termed TRIM (33), which may allow stable expression on the plasma membrane. Other models propose that {zeta} sterically obscures internalization sequences present in the intracellular tails of other TCR components (28). Analysis of chimeric receptors containing internalization signals from the cytoplasmic domains of CD3{gamma} and CD3{delta} have shed light on a possible role for {zeta} whereby it masks internalization signals present in the TCR complex from the cellular endocytic machinery (28, 34). Consistent with this masking role, reports on the ability of C-terminal deletion or internal deletion mutants of {zeta} in cell lines and transgenic mice to support TCR expression have indicated that the extent of deletions of the native sequence correlates with reduced ability to support surface expression (7, 35, 36, 37).

If {zeta} simply sterically blocked internalization sequences present in other TCR chains, we reasoned that the size of the intracellular tail, rather than amino acid sequence specific to {zeta}, might be all that is needed to support expression of the fully assembled receptor. Therefore, we generated a series of {zeta} variants (Fig. 7GoA) containing irrelevant sequences in the intracellular portion of the molecule, and asked how well they support TCR expression when transiently expressed in MA 5.8 cells. The constructs contained an amino-terminal HA tag, so that transfected cells could be simultaneously stained for expression of the transfected gene and the TCR. In transient expression experiments under conditions where expression of full-length {zeta} reconstituted surface expression in the MA5.8 cell line, a truncation mutant that is almost devoid of a cytoplasmic domain ({zeta}t65) was essentially unable to do so (Fig. 7GoB). Chimeric {zeta} molecules were generated in which irrelevant sequences of 29 ({zeta}t65 + 29) and 67 ({zeta}t65 + 67) were added to the cytoplasmic tail. Expression of these constructs reconstituted cell surface TCR expression, with the longer form being almost as good as wild-type {zeta}. Furthermore, expression of {zeta}t65 plus GFP (248 amino acids) was as efficient as wild-type {zeta} in restoring cell surface TCR levels (Fig. 8Go). Stable expression of {zeta}WT (Fig. 9GoA) or {zeta}t65 + 67 (Fig. 9GoB) in MA5.8 cells also restored full TCR expression. Furthermore, the rates of TCR internalization for these cells were similar to 2B4.11 cells (Fig. 9GoC). These observations strongly support a model in which steric hindrance of internalization by the cytoplasmic domain of {zeta} regulates the half-life of TCR at the cell surface.



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  		FIGURE 7. The function of {zeta} in maintaining surface TCR maps to a sequence-independent region in its intracytoplasmic domain. A, Schematic representation of TCR {zeta} constructs used for transfection. The length of each schematic molecule shown is not proportional to the real length. HA, hemagglutinin tag epitope; EC, extracellular region; TM, transmembrane region. B, MA5.8 cells were transiently transfected with a control vector or a vector expressing the indicated form of chimeric TCR {zeta} protein. After 18 h, cells were stained with anti-HA Ab (12CA5), washed in FACS buffer, and incubated with GAM-FITC. After washing in FACS buffer, cells were incubated with an excess of a control mouse IgG to saturate residual binding sites of the GAM-FITC, washed in FACS buffer again, stained with H57-PE, and analyzed by flow cytometry. Dead cells were excluded by staining with propidium iodide.

 


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  		FIGURE 8. GFP can substitute for the intracytoplasmic domain of {zeta} in maintaining surface TCR. MA5.8 cells were transiently transfected with a control vector or a vector expressing the indicated form of chimeric TCR {zeta} protein or GFP. After 18 h, the cells were analyzed as in Fig. 7Go except that cells transfected with GFP or the {zeta}/GFP chimeric molecule were gated on GFP fluorescence.

 


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  		FIGURE 9. Stable expression of wild-type {zeta} and {zeta}t65 + 67 in {zeta}-deficient cells restores TCR expression and slows TCR internalization. A, MA5.8 cells and two independent stable transfectants expressing {zeta}WT (clones 39 and 77) were stained with H57-PE and analyzed by flow cytometry; staining of MA5.8 cells with control hamster IgG conjugated to PE is also shown. B, Two independent stable transfectants expressing {zeta}t65 + 67 (clones 20 and 75) were stained with H57-PE and analyzed by flow cytometry. MA5.8 H57-PE and control Ab staining are reproduced from A. C, TCR internalization was measured in 2B4.11, MA5.8, and two {zeta}-reconstituted MA5.8 clones and plotted as a function of time. One representative experiment of three is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Cell surface expression of the TCR is a dynamic process, and a number of previous studies have assessed its constitutive turnover. One early approach was to radioiodinate intact T cells on ice, culture them thereafter for varying periods of time at 37°C, remove sialic acid with neuraminidase, and detect labeled TCR by immunoprecipitation and gel electrophoresis (8). This allowed one to quantitate the loss of cell surface TCRs over time, because internalized receptors would be protected from desialylation. Using a human T leukemic cell line, it was shown that upon warming there was a rapid internalization of receptor, with equilibration between surface and intracellular pools by 60 min. Neuraminidase treatment of labeled cells in which equilibration of receptors had been achieved revealed that TCRs, perhaps those with phosphorylated CD3{gamma}, did recycle from the intracellular compartment to the cell surface. Another study measured receptor turnover on 2B4.11 cells with a radiolabeled intact Ab or Fab that recognized the TCR{alpha} chain (9). After culturing cells in the presence of Ab for defined periods of time, the cells were washed at low pH to remove cell surface-bound material and the fraction of TCR that had internalized was determined. Approximately 30% of cell surface receptors internalized within 30 min. A more recent analysis followed the fate of TCRs derivatized with a cleavable biotin group on the surface of T cell hybridomas and normal resting T cells, and it was estimated that TCRs internalize at a rate of ~0.6–1.5% per min (10). We have used the intracellular accumulation of fluorescent anti-TCR Abs over time to measure TCR internalization. This technique yielded results that are very similar to the earlier reports, namely that octameric TCRs constitutively internalize at a rate of ~1% per minute.

TCRs lacking the {zeta} chain are expressed at much lower levels on the cell surface than wild-type receptors, a finding that has been attributed to shunting of these partial complexes from the Golgi to lysosomes (6). Our analyses reveal that the turnover rate of these complexes on the cell surface is much greater than for the normally assembled receptor. This was true for {zeta}-deficient thymocytes as well as {zeta}-deficient T cell hybridomas. Reconstitution with wild-type {zeta} in the latter demonstrated that the rate of turnover is proportional to the amount of {zeta} expressed. How might {zeta} regulate the cell surface residency time of the TCR? It is possible that the intracellular portion of the {zeta} chain contains specific sequences that stabilize the receptor at the cell surface. Our results argue strongly that this is not the case, because chimeric molecules containing the transmembrane portion of {zeta} and non-native intracellular amino acid sequences were comparable to wild-type {zeta} in reducing the rate of TCR internalization. These results indicate another mechanism, that the intracellular portion of {zeta} sterically blocks internalization sequences present in the other TCR subunits.

CD3{gamma} and CD3{delta} contain a leucine-based (di-leucine) receptor sorting motif (SDKQTLL for {gamma}), and when this sequence is fused with Tac (human IL-2R {alpha}-chain) or CD4, the resulting protein is targeted to lysosomes for degradation (34, 38). Furthermore, chimeric molecules containing the di-leucine motif that reached the cell surface are rapidly internalized as measured by cointernalization of fluorescently labeled Ab, an approach similar to that used in the present report. In the context of a complete TCR, PKC-mediated phosphorylation of the serine residue preceding the CD3{gamma} di-leucine motif induces rapid internalization, presumably by causing a conformational change that allows this sequence to bind adaptor proteins (AP-1 and AP-2), components of clathrin-coated pits and vesicles (38, 39). CD3{gamma} and CD3{delta} also have a tyrosine-based motif, YXXØ (where Y is tyrosine, X is any amino acid, and Ø is an amino acid with a bulky hydrophobic group), that can cause internalization and lysosomal targeting when fused to Tac (34). The relevance of these sequences to the regulation of TCR expression is unclear. A previous study addressed the issue of the role of {zeta} in TCR turnover indirectly, using chimeric molecules containing the intracellular domain of CD3{gamma} and the transmembrane and extracellular portion of CD16 (which associates with {zeta}) or CD4 (which does not associate with {zeta}) (28). The former was highly expressed on CD3{gamma}-deficient Jurkat T cells and had a low rate of spontaneous internalization, whereas the latter was rapidly internalized from the cell surface, a phenomenon that required the di-leucine motif in the CD3{gamma} tail. This was interpreted as a demonstration that {zeta} shielded di-leucine motif from adaptor proteins. The findings in the present report extend these observations in several ways. First, although valuable tools for investigating individual intermolecular interactions, as mentioned above, chimeric molecules of single TCR subunits do not faithfully mimic the turnover and trafficking of the fully assembled octameric TCR. We analyzed expression and turnover of the octameric TCR containing either wild-type or chimeric {zeta} molecules, thus addressing the behavior of an intact receptor complex. Second, we introduced chimeric {zeta} molecules in which the bulk of the intracellular portion is composed of non-native and irrelevant amino acid sequences. The finding that even these unrelated sequences restored TCR expression (and rates of internalization) to normal argues strongly that the role of {zeta} in stabilizing the TCR is not dependent upon {zeta}-specific signaling sequences, but rather that the intracellular portion of {zeta} sterically masks targeting sequences present in other TCR components. The precise identity of these internalization signals remains to be determined. In preliminary studies we have been unable to affect spontaneous internalization in wild-type or {zeta}-deficient cells with inhibitors of PKC or Src family kinases. Studies with CD3 subunits in which the di-leucine motif has been mutated are ongoing.

The present observations raise the possibility that {zeta}-deficient TCRs do not sort directly from the Golgi to lysosomes as previously thought, but may first be expressed on the cell surface before internalization. However, it is important to note that the rapid internalization is not itself the cause of the low TCR expression on {zeta}-deficient cells: there is no increase in the amount of intracellular TCR in MA5.8 cells at steady state, and inhibitors of lysosomal degradation increase the amount and half-life of CD3 components (Ref. 6 and our unpublished observation). The increase in internalization and lysosomal degradation could be related if the fate of an internalized receptor-to cycle back to the cell surface or to sort to a lysosome-is stochastic. In that case, the more often a receptor internalizes the more likely it would be to sort to a lysosome and be degraded. Furthermore, if routing to lysosomes depends on sequences that are exposed only in the {zeta}-less internalized TCR, this receptor would be preferentially degraded in MA5.8 cells. Whether this is in fact the case, or whether the small amount of {zeta}-less receptor expressed on the cell surface represents a small pool that has escaped targeting to lysosomes, remains to be determined. Another interesting possibility is that internalization of octameric receptors is in fact secondary to dissociation of {zeta} from the rest of the complex. {zeta} can exist on the cell surface unassociated with {alpha}{beta}/CD3 (18). If {zeta} can dissociate from the rest of the receptor in the plasma membrane, one would predict that the other components would rapidly internalize. In this fashion, the dynamic equilibrium of TCR subunit associations would actually determine the residency time of the receptor on the cell surface. This issue might possibly be addressed by characterization of the TCR composition in the cycling endocytic pool.


   Acknowledgments
 
We thank Elizabeth Shores for {zeta}-KO mice, Dietmar Kappes and Alfred Singer for CD3{delta}-deficient mice, Howard Petrie for the PE-coupling protocol, Allan Weissman for cell lines, Ralph Kubo for anti-TCR-{zeta} Ab, Susan Sharrow and Tony Adams for help with flow cytometric analysis, and Remy Bosselut for helpful suggestions and critical review of this manuscript.


   Footnotes
 
1 U.D. and I.M. contributed equally to this work. Back

2 Address correspondence and reprint requests to Dr. Jonathan D. Ashwell, National Institutes of Health, Room 1B-40, Building 10, 9000 Rockville Pike, Bethesda, MD 20892. E-mail address: jda{at}pop.nci.nih.gov Back

3 Abbreviations used in this paper: PKC, protein kinase C; HA, hemagglutinin; GFP, green fluorescent protein; DP, double positive; SMPB, succinimidyl 4-(p-maleidophenyl) butyrate. Back

Received for publication June 3, 2002. Accepted for publication September 23, 2002.


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