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The CD3 Leucine-Based Receptor-Sorting Motif Is Required for Efficient Ligand-Mediated TCR Down-Regulation¹

Institute of Medical Microbiology and Immunology, University of Copenhagen, Copenhagen, Denmark

	Abstract

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TCR down-regulation plays an important role in modulating T cell responses both during T cell development and in mature T cells. At least two distinct pathways exist for down-regulation of the TCR. One pathway is activated following TCR ligation and is dependent on tyrosine phosphorylation. The other pathway is dependent on protein kinase C (PKC)-mediated activation of the CD3 di-leucine-based receptor-sorting motif. Previous studies have failed to demonstrate a connection between ligand- and PKC-induced TCR down-regulation. Thus, although an apparent paradox, the dogma has been that ligand- and PKC-induced TCR down-regulations are not interrelated. By analyses of a newly developed CD3-negative T cell variant, freshly isolated and PHA-activated PBMC, and a mouse T cell line, we challenged this dogma and demonstrate in this work that PKC activation and the CD3 di-leucine-based motif are indeed required for efficient ligand-induced TCR down-regulation.

	Introduction

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At least two distinct pathways exist for down-regulation of the TCR (1). One pathway is activated following TCR ligation and is dependent on tyrosine phosphorylation (2, 3). The other pathway is dependent on protein kinase C (PKC)³-mediated activation of the CD3 di-leucine-based (L-based) receptor-sorting motif (4, 5). The dogma has been that these pathways are not interrelated. Accordingly, several studies have demonstrated that ligand-mediated TCR down-regulation is functional in cell lines with a disrupted CD3 L-based motif and, vice versa, that PKC-mediated TCR down-regulation can take place independently of tyrosine kinase activation (1, 6, 7, 8, 9). It has been a puzzle that ligand- and PKC-mediated TCR down-regulations should be two separate, independent processes, as it is known that TCR ligation induces PKC activation with subsequent CD3 phosphorylation and activation of the CD3 L-based receptor-sorting motif (10, 11, 12, 13). Thus, TCR ligation would, in addition to the tyrosine phosphorylation-dependent pathway, be expected to activate the PKC/CD3-dependent pathway. The aim of this study was to determine whether an interplay between ligand- and PKC-mediated TCR down-regulation exists.

	Materials and Methods

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Cells, derivations of mutants, and reagents

The human T cell line Jurkat, the Burkitt’s lymphoma cell line Raji (American Type Culture Collection, Manassas, VA), and the mouse DO11.10 T cell hybridoma (14) were cultured in complete medium (RPMI 1640 medium supplemented with 0.5 IU/L penicillin, 500 mg/L streptomycin, and 10% FCS) at 37°C in 5% CO₂. PBMC were isolated from healthy donors by density centrifugation using Lymphoprep (Nycomed, Oslo, Norway) as previously described (15). Jurkat cells were mutagenized by incubation in complete medium containing methanesulfonic acid ethyl ester (Sigma-Aldrich, St. Louis, MO) at a final concentration of 100 µg/ml for 24 h. The cells were washed and rested for 72 h, and TCR cell surface-positive cells were subsequently depleted by incubation with the anti-TCR Ab F101.01 (16) and Dynabeads (Dynal Biotech, Oslo, Norway) as previously described (17). This procedure was repeated four times resulting in a TCR-negative subpopulation of 25% of the cell population. The cells were subsequently cloned, and TCR-negative clones were selected for further characterization by metabolic labeling and immunoprecipitation as previously described (18). Purified and fluorochrome-conjugated mAb were obtained from BD PharMingen (San Diego, CA) and DAKO (Glostrup, Denmark). Phorbol 12,13-dibutyrate (PDB) was obtained from Sigma-Aldrich. The broad PKC inhibitor Ro 31-8220 (19) was a gift from Dr. D. Bradshaw (Roche Research Center, Welwyn Garden City, U.K.). The anti-mouse V8.2 mAb F23.1 variant 31 with a K_d of 9.5 µM was produced as previously described (20). Superantigen Staphylococcus enterotoxin E (SEE) was purchased from Toxin Technology (Sarasota, FL), and 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF/AM) was obtained from Molecular Probes (Eugene, OR).

RT-PCR and sequencing

RNA was isolated using the RNeasy kit from Qiagen (Hilden, Germany), and RT-PCR was performed using the Reverse-iT kit from Abgene (Surrey, U.K.) as described by the manufacturers. The PCR products were sequenced using the BigDye terminator cycle sequencing kit and analyzed on an ABI 310 genetic analyzer from Applied Biosystems (Warrington, U.K.).

TCR down-regulation and phosphotyrosine blots

Flat-bottomed Nuclon MicroWell Plates (Life Technologies, Paisley, U.K.) were coated with the indicated concentrations of anti-TCR mAb F101.01 for 16 h at 4°C and subsequently washed three times with PBS. The cells were adjusted to 4 x 10⁵/ml complete medium and incubated in the mAb-precoated wells for the time indicated. The cells were subsequently transferred to 4°C and analyzed for TCR expression by incubation with saturating amounts of F101.01 for 30 min at 4°C, followed by FITC- or PE-conjugated goat anti-mouse Ig Ab from Jackson ImmunoResearch Laboratories (West Grove, PA). For experiments with DO11.10 cells, the F23.1 variant 31 was coated on protein A-precoated plates as previously described (20), and TCR expression was analyzed using FITC-conjugated anti-mouse CD3 mAb 145-2C11. For stimulation with SEE, Raji cells were pulsed for 2 h with the indicated concentrations of SEE, stained with BCECF/AM for 10 min, washed four times, and cocultured with Jurkat cells at a ratio of 1:1 for the time indicated. The cells were subsequently transferred to 4°C and analyzed for TCR expression by incubation with PE-conjugated anti-CD3 mAb and gating out cells with green fluorescence (BCECF/AM). The mean fluorescence intensity (MFI) was determined by flow cytometry and used calculating the percentage of anti-TCR binding: (MFI of treated cells/MFI of untreated cells) x 100%. Phosphotyrosine blots were performed as previously described (21)

	Results

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Generation of a CD3-negative Jurkat variant

The fact that TCR ligation leads to activation of PKC with subsequent CD3 serine 126 phosphorylation and activation of the CD3 L-based motif combined with the observation that CD3 does not play a role in ligand-induced TCR down-regulation has been an apparent paradox for several years. However, most of the studies on CD3 have been performed using the CD3-negative Jurkat variant JGN (1, 4, 6, 22, 23). This variant was produced by exposing Jurkat cells to 250 rad of gamma radiation (18), and the possibility existed that other genes beside CD3 were affected by this treatment. Therefore, we decided to produce new, independently derived CD3-negative Jurkat variants to further study the role of CD3 in ligand-induced TCR down-regulation.

To produce new CD3-negative variants Jurkat cells were mutagenized using methanesulfonic acid ethyl ester. TCR cell surface-negative clones were isolated and screened by metabolic labeling, immunoprecipitation, and SDS-PAGE. One clone (E3) that failed to express the CD3 protein was isolated (Fig. 1A). Full-length CD3 cDNA could be isolated from E3 cells by RT-PCR (data not shown), and this was subsequently sequenced. The E3 cells carried the same frameshift mutation as the JGN variant, namely a deletion of one of the nine adenines (bases 242–250) in exon 3 encoding most of the extracellular domain of CD3 (18). The position of the mutation in E3 supported the proposal that the human CD3 gene has a mutational hot spot in the nucleotide sequence of nine adenosines in exon 3 (24).

View larger version (37K): [in this window] [in a new window]   FIGURE 1. Characterization of the new CD3-negative Jurkat variant E3. A, WT Jurkat cells (lane 1), E3 cells (lane 2), and E3-WT cells (lane 3) were metabolically labeled for 30 min at 37°C before solubilization in digitonin lysis buffer and immunoprecipitation with anti-CD3 mAb (UCHT1). Immunoprecipitates were analyzed by SDS-PAGE under nonreducing conditions on 12% acrylamide gels. The positions of the TCR chains are indicated (fg, fully glycosylated; pg, partially glycosylated). B, WT Jurkat cells, E3 cells, and E3-WT cells were incubated with anti-CD3 mAb UCHT1, anti-TCR mAb F101.01, or anti-V8 mAb, followed by staining with PE-conjugated goat anti-mouse Ig Ab (filled histograms). Nonspecific fluorescence was assessed by staining only with PE-conjugated goat anti-mouse Ig Ab (open histograms). The abscissa gives the fluorescence intensity in a logarithmic scale in arbitrary units. The ordinate gives the relative cell number.

The CD3 L-based motif is required for PKC-mediated TCR down-regulation

Detailed studies using the JGN variant have pinpointed the essential amino acids in the CD3 L-based receptor-sorting motif in PKC-mediated TCR down-regulation. Thus, CD3 serine 126 is the phosphoacceptor, and leucine 131 and 132 are essential docking sites for the adaptor proteins of clathrin-coated vesicles (4, 22, 23, 25). Selected CD3 constructs were transfected into E3 cells (Fig. 2), and TCR-positive transfectants expressing comparable amounts of TCR were subsequently analyzed for their ability to down-regulate their TCR following PDB-induced PKC activation. As shown in Fig. 2B, PKC-mediated TCR down-regulation was almost abolished in the E3-LLAA and E3-S126V cells and was preserved in E3-WT and E3-S123V cells exactly as previously found for JGN cells (1). Thus, these analyses confirmed the general composition and role of the CD3 L-based motif in PKC-mediated TCR down-regulation.

View larger version (27K): [in this window] [in a new window]   FIGURE 2. The CD3 L-based motif is required for PKC-mediated TCR down-regulation. A, Schematic presentation of the amino acid sequences in the cytoplasmic tails of the different CD3 molecules used in this study. B, Cells were incubated with various concentrations of the PKC activator PDB for 1 h, and TCR down-regulation was determined as described in Materials and Methods. C, FACS histograms of untreated cells (open histograms) and cells treated with PDB (111 nM) for 1 h. The cell line and the percentage of anti-TCR binding following PDB treatment are given in the upper left corner of each histogram.

An intact CD3 L-based motif is required for efficient ligand-induced TCR down-regulation

To determine whether the CD3 L-based motif was involved in ligand-mediated TCR down-regulation, cells were stimulated with plate-bound anti-TCR mAb for 30 min. Cells with an intact CD3 L-based motif (E3-WT and E3-S123V) clearly down-regulated TCR more efficiently than cells with a disrupted motif (E3-LLAA and E3-S126V). Thus, 3- to 10-fold less ligand was required to induce the same degree of TCR down-regulation in cells with an intact CD3 L-based motif compared with cells with a disrupted motif (Fig. 3A). Time-course experiments using low concentrations of ligand further demonstrated that an intact CD3 L-based motif was absolutely required for fast and efficient ligand-induced TCR down-regulation (Fig. 3, B and C).

View larger version (15K): [in this window] [in a new window]   FIGURE 3. An intact CD3 L-based motif is required for efficient ligand-induced TCR down-regulation. A and D, Cells were incubated with various concentrations of plate-bound anti-TCR mAb for 30 min, and TCR down-regulation was determined as described in Materials and Methods. B and C, Cells were incubated with plate-bound anti-TCR mAb corresponding to 100 (B) and 400 (C) ng/ml for different time intervals, and TCR down-regulation was determined as described in Materials and Methods. One representative experiment of at least three is shown.

PKC activation is required for efficient ligand-induced TCR down-regulation

The CD3 L-based motif is activated by PKC-mediated phosphorylation of CD3 serine 126 (4, 23). If the CD3 L-based motif is required for fast and efficient ligand-induced TCR down-regulation, it follows that activation of the relevant PKC must also be required. To determine whether PKC activation was required for efficient ligand-induced TCR down-regulation, cells were pretreated for 30 min with the broad PKC inhibitor Ro 31-8220 at a final concentration of 4 µM or were left untreated. The cells were next stimulated with plate-bound anti-TCR mAb as described above and tested for TCR down-regulation. PKC inhibitor treatment of E3 cells with an intact CD3 L-based motif reduced their sensitivity to ligand stimulation to the same level as E3 cells with a disrupted CD3 L-based motif (Fig. 4, A and B). In contrast, E3 cells with a disrupted CD3 L-based motif were unaffected by PKC inhibitor treatment (Fig. 4B). As previously found, JGN-WT cells were unaffected by PKC inhibitor treatment (data not shown) (1). These results indicated that the PKC inhibitor only affected the PKC/CD3 L-based part of ligand-induced TCR down-regulation and left the phosphotyrosine-dependent part unaffected. To further analyze whether the PKC inhibitor affected tyrosine phosphorylation, Jurkat cells were stimulated with anti-TCR mAb in the absence or the presence of 4 µM Ro 31-8220. The cells were subsequently lysed, and the lysates were analyzed by Western blotting using the anti-phosphotyrosine mAb 4G10. As judged from the blots, Ro 31-8220 in the concentrations used in this study did not affect ligand-induced tyrosine phosphorylation (Fig. 4C). Likewise, phosphotyrosine blots of anti-CD3 immunoprecipitates of cells stimulated with anti-TCR mAb did not indicate any effect of Ro 31-8220 on tyrosine phosphorylation (data not shown).

View larger version (39K): [in this window] [in a new window]   FIGURE 4. PKC activation is required for efficient ligand-induced TCR down-regulation. E3-WT (A) and E3-S123V and E3-S126V (B) cells were pretreated for 30 min with the broad PKC inhibitor Ro 31-8220 (4 µM) or were left untreated before stimulation with various concentrations of plate-bound anti-TCR mAb for 30 min. TCR down-regulation was subsequently determined as described in Materials and Methods. One representative experiment of three is shown. The experiment shown in A was performed in triplicate, and the results are given as the mean ± SD. C, Jurkat cells were pretreated for 30 min with Ro 31-8220 (4 µM; lanes 2 and 4) or were left untreated (lanes 1 and 3) before stimulation with 10 µg/ml anti-TCR mAb (lanes 3 and 4) or PBS (lanes 1 and 2) for 5 min. Cell lysates were subsequently analyzed by Western blot using the phosphotyrosine-specific mAb 4G10.

View larger version (15K): [in this window] [in a new window]   FIGURE 5. PKC activation is also required for efficient ligand-induced TCR down-regulation in PBMC and mouse T cell lines. Freshly isolated human PBMC (A), PHA-stimulated human PBMC (B), and the mouse T cell hybridoma DO11.10 (C) were pretreated for 30 min with the broad PKC inhibitor Ro 31-8220 (4 µM) or were left untreated before stimulation with various concentrations of plate-bound anti-TCR mAb for 30 min. TCR down-regulation was subsequently determined as described in Materials and Methods. Different scales have been applied for the ordinates, as freshly isolated PBMC do not down-regulate the TCR to the same extent as PHA-stimulated cells or cell lines. The scale for the abscissa is the same in all histograms to clearly demonstrate the equal dependency of PKC activation for efficient TCR down-regulation in all examined cell types. One representative experiment of at least three is shown.

An intact CD3 L-based motif and PKC are required for efficient superantigen-induced TCR down-regulation

To further extend our analyses we next examined whether the CD3 L-based motif was involved in superantigen/APC-mediated TCR down-regulation. Jurkat cells were incubated for 60 min with Raji cells prepulsed with SEE. Cells with an intact CD3 L-based motif (E3-WT) clearly down-regulated the TCR more efficiently than cells with a disrupted motif (E3-LLAA and E3-S126V), especially during exposure to low concentrations of SEE (Fig. 6A). Time-course experiments using SEE at 50 ng/ml further demonstrated that an intact CD3 L-based motif was absolutely required for fast and efficient ligand-induced TCR down-regulation (Fig. 6B).

View larger version (18K): [in this window] [in a new window]   FIGURE 6. An intact CD3 L-based motif is required for efficient superantigen-induced TCR down-regulation. A, E3 transfectants were incubated for 60 min with Raji cells prepulsed with various concentrations of SEE, and TCR down-regulation was subsequently determined as described in Materials and Methods. B, Cells were incubated for different time intervals with Raji cells prepulsed with 50 ng/ml SEE, and TCR down-regulation was subsequently determined as described in Materials and Methods. One representative experiment of three is shown.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Treatment of E3 cells with an intact CD3 L-based motif (E3-WT and E3-S123V) with a PKC inhibitor reduced their sensitivity to ligand to the same level as E3 cells with a disrupted CD3 L-based motif showing that, in addition to an intact CD3 L-based motif, PKC activation was required for efficient ligand-induced TCR down-regulation. In contrast, PKC inhibitor treatment of E3 cells with a disrupted CD3 L-based motif and JGN cells did not affect ligand-induced TCR down-regulation, demonstrating that the PKC inhibitor did not affect PKC/CD3-independent TCR down-regulation. In agreement with this, we found that the PKC inhibitor did not affect ligand-induced tyrosine phosphorylation at the concentrations used in this study.

Previous studies have probably not been able to demonstrate the dependency of PKC and the CD3 L-based motif for efficient ligand-induced TCR down-regulation, as these studies either have examined the JGN variant or have applied too high concentrations of ligand for long time intervals (1, 6, 7, 8, 9). We believe that identifying the defect in JGN could significantly contribute to our understanding of the connection between ligand- and PKC-induced TCR down-regulation, and we are presently in the process of identifying this defect.

Several important roles can be envisioned for the CD3 L-based motif in ligand-induced TCR down-regulation, e.g., it could be required for optimal T cell activation by allowing serial triggering (29), or it could be required as a negative feedback mechanism to prevent detrimental over-stimulation of T cells, etc. Further clarification of this issue awaits analyses of the CD3 di-leucine-mutated knockin mice (C. Menné, M. C. Haks, A. M. Kruisbeek, and C. Geisler, manuscript in preparation).

	Footnotes

 
¹ This work was supported by the Danish Cancer Society, the Danish Medical Research Council, the Carlsberg Foundation, the Novo Nordisk Foundation, and the Director Leo Nielsen Foundation. J.D. is the recipient of a postdoctoral scholarship from the Carlsberg Foundation. P.S.A. is the recipient of a postdoctoral scholarship from the Danish Medical Research Council. C.M. and J.P.H.L. are recipients of doctoral scholarships from University of Copenhagen. M.v.E. is the recipient of a scholarship from the Danish Cancer Society.

² Address correspondence and reprint requests to Dr. Carsten Geisler, Institute of Medical Microbiology and Immunology, The Panum Institute, Blegdamsvej 3, DK-2200 Copenhagen, Denmark. E-mail address: cgtcr{at}biobase.dk

³ Abbreviations used in this paper: PKC, protein kinase C; BCECF/AM, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester; L-based, di-leucine based; MFI, mean fluorescence intensity; PDB, phorbol 12,13-dibutyrate; SEE, Staphylococcus enterotoxin E; WT, wild type.

Received for publication January 7, 2002. Accepted for publication March 6, 2002.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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