Regulation of Constitutive TCR Internalization by the ζ-Chain

Mice

C57BL/6 mice are maintained in our breeding colony. ζ-deficient mice (22) were provided by Dr. E. Shores (Food and Drug Administration, Bethesda, MD) and CD3δ-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-TCRβ), H146-968 (anti-TCRζ), and 145-2C11 (2C11, anti-CD3ε) 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 CD8α-CyChrome, anti-mouse CD8α-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 MAζ.1, MA ζ.2, and MAζ.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 TCRζ 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 ζ devoid of the leader sequence and containing native amino acids 21–164 (wt) or amino acid residues 21–65 (ζt65) were obtained by PCR amplification using the following oligonucleotides: 5′-AAAAAAAGCTTGGCCCAGCCGGCCCAGAGCTTTGGTCTGCTG-3′ and 5′-AAAAACCCGGGATTAGCGAGGGGCCAGGGTCTG-3′ (for ζ wt) or 5′-AAAAACCCGGGACTACAGGTTGGCAGCAGTCTCTGC-3′ (for ζt65). 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 ζt65, effectively limiting the length of the intracellular domain to 14 amino acids. Two additional mutants that read through the stop codon of ζt65 and add irrelevant sequences of 29 (ζt65 + 29) and 67 (ζt65 + 67) amino acids at the C terminus were obtained by PCR amplification. A plasmid expressing ζ 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: % SR_t = 100 × (ARF_t−AF)/(SF−AF) where SR_t is the percent of surface receptor internalized at time “t”, ARF_t 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 ζ-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 100× 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.

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-TCRβ) 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. 1⇓, 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. 1⇓, 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 ×100 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. 2⇓A, 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. 2⇓ and data not shown). Control experiments with PE-labeled IgG with an irrelevant specificity showed no appreciable accumulation over time (Fig. 2⇓B). 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. 2⇓C). 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. 3⇓). 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 ζ-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 ζ-deficient cells

TCRs lacking ζ 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 (TCRα,β plus CD3γε and δε) 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 ζ 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. 3⇑C). This difference was also observed when PE-labeled H57 Fab (Fig. 3⇑D) or PE-labeled anti-CD3ε (Fig. 3⇑E) was used. In contrast to 2B4.11 cells, ζ-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. 3⇑F). 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 ζ.

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

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FIGURE 4.

TCR internalization rate is inversely related to ζ 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, ζ expression in each cell line was determined by immunoprecipitation and then immunoblotting with an anti-ζ mAb.

Rapid TCR turnover in ζ-deficient, but not CD3δ-deficient, thymocytes

To determine whether TCRs lacking ζ also internalize rapidly in normal cells, TCR internalization was quantitated using thymocytes from mice deficient in ζ expression (ζ-KO mice) (22). The loss of ζ 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 ζ-deficient DP thymocytes demonstrated that TCR levels were much lower than on wild-type DP thymocytes (Fig. 5⇓A). Thymocytes from wild-type or ζ-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. 5⇓B. To allow comparison between wild-type and ζ-deficient cells, analysis was restricted to DP cells. Both wild-type and ζ-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 ζ-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 ζ-deficient than in wild-type thymocytes (Fig. 5⇓C). Therefore, just as with T cell hybridomas, the lack of ζ 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 ζ-deficient than in wild-type thymocytes. A, Steady-state cell surface TCR expression. Wild-type and ζ-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 ζ-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 ζ-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 ζ-chain but low levels of cell surface TCR due to deficiency of a different subunit. DP thymocytes from CD3δ-deficient mice have low levels of cell surface TCR, comparable to the levels found in the absence of ζ, both having ∼5% of wild-type levels (Ref. 30 and Fig. 6⇓A). TCR internalization of CD3δ-deficient DP thymocytes was compared with wild type (Fig. 6⇓B). Unlike ζ-deficient thymocytes, the rate of TCR internalization on CD3δ-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 ζ-chain has a specific role in maintaining receptors on the plasma membrane.

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FIGURE 6.

TCR internalization is similar between CD3δ-deficient and wild-type thymocytes. A, Steady-state cell surface TCR expression on wild-type and CD3δ-deficient thymocytes was determined as in Fig. 5⇑A. B, Time course of TCR internalization with wild-type or CD3δ-deficient thymocytes was determined as in Fig. 5⇑B. 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 ζ

There are a number of different mechanisms by which ζ could stabilize expression of the TCR on the cell surface. For example, ζ 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 ζ 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 CD3γ and CD3δ have shed light on a possible role for ζ 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 ζ 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 ζ 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 ζ, might be all that is needed to support expression of the fully assembled receptor. Therefore, we generated a series of ζ variants (Fig. 7⇓A) 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 ζ reconstituted surface expression in the MA5.8 cell line, a truncation mutant that is almost devoid of a cytoplasmic domain (ζt65) was essentially unable to do so (Fig. 7⇓B). Chimeric ζ molecules were generated in which irrelevant sequences of 29 (ζt65 + 29) and 67 (ζt65 + 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 ζ. Furthermore, expression of ζt65 plus GFP (248 amino acids) was as efficient as wild-type ζ in restoring cell surface TCR levels (Fig. 8⇓). Stable expression of ζWT (Fig. 9⇓A) or ζt65 + 67 (Fig. 9⇓B) 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. 9⇓C). These observations strongly support a model in which steric hindrance of internalization by the cytoplasmic domain of ζ regulates the half-life of TCR at the cell surface.

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FIGURE 7.

The function of ζ in maintaining surface TCR maps to a sequence-independent region in its intracytoplasmic domain. A, Schematic representation of TCR ζ 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 ζ 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 ζ in maintaining surface TCR. MA5.8 cells were transiently transfected with a control vector or a vector expressing the indicated form of chimeric TCR ζ protein or GFP. After 18 h, the cells were analyzed as in Fig. 7⇑ except that cells transfected with GFP or the ζ/GFP chimeric molecule were gated on GFP fluorescence.

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FIGURE 9.

Stable expression of wild-type ζ and ζt65 + 67 in ζ-deficient cells restores TCR expression and slows TCR internalization. A, MA5.8 cells and two independent stable transfectants expressing ζWT (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 ζt65 + 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 ζ-reconstituted MA5.8 clones and plotted as a function of time. One representative experiment of three is shown.