Degradation of  T Cell Receptor (TCR)–CD3-ζ Complexes after Antigenic Stimulation

T Cell Clones and APCs.

Two DRBI*1104-restricted T cell clones (KS140 and KS70) specific for the tetanus toxin peptide (TT830– 843; QYIKANSKFIGITE) were used. Autologous Epstein–Barr virus (EBV)-B cells were used as APCs (4).

Intracellular Staining for CD3-ε and ζ Chain.

EBV-B cells were pulsed for 2 h at 37°C with various concentrations of TT830–843 in RPMI, 5% FCS. During the last 10 min, 1 μM BCECF-AM (2′,7-bis-(carboxyethyl)-5(6′)-carboxyfluorescin; Calbiochem, San Diego, CA) was added and the cells were washed four times. T cells were mixed with EBV-B cells at a 1:2 ratio in 200 μl RPMI, 5% FCS in U-bottomed microplates, centrifuged 1 min at 1,500 rpm to allow conjugate formation, and incubated at 37°C. In some experiments, T cells were pretreated for 1 h with 10 μg/ml cycloheximide and the drug was present throughout the assay. At different times, the cells were resuspended, washed in PBS, 0.5 mM EDTA and fixed for 10 min with 3% paraformaldehyde. The cells were permeabilized for 10 min at room temperature with washing buffer (Hepes-buffered PBS containing 0.1% saponin) and stained with anti-CD3 (TR66; reference 4), anti-Vβ2 (Immunotech, Marseille, France), or anti-ζ (Santa Cruz Biotechnology, Santa Cruz, CA) in Hepes-buffered PBS containing 0.1% saponin and 2% BSA (or 5% FCS), followed by a goat anti–mouse PE-labeled Ab (SBA, Birmingham, AL). The TCR, CD3, and ζ fluorescence were analyzed on a FACScan^® (Becton Dickinson, Mountain View, CA). EBV-B cells were gated out using both forward and side scatter (FSC/SSC) parameters and green BCECF fluorescence. In some experiments, T cells were conjugated with unstained peptidepulsed EBV-B cells; at different times of incubation at 37°C, the cells were gently resuspended and laid on poly-l-lysine–coated slides for 10 min at 37°C. The cells were fixed for 10 min with 3% paraformaldehyde, permeabilized for 10 min at room temperature with washing buffer, and stained with an anti-ζ (Santa Cruz) in Hepes-buffered PBS containing 0.1% saponin and 4% BSA, followed by FITC-labeled goat anti–mouse (SBA) and anti– human Lamp-1 (provided by Dr. S. Carlsson, Umea University, St. Louis, MO) followed by Texas Red–labeled goat anti–rabbit antibody (SBA). The samples were mounted in 90% glycerol–PBS containing 2.5% 1-4-diazabicyclo (2.2.2) octane (DABCO; Fluka AG, Buchs, Switzerland). In some experiments, T cells were pretreated with 0.5 μM bafilomycin A1 (Calbiochem) or with the vehicle of the drug only (DMSO 0.5%) for 1 h at 37°C before conjugate formation. The samples were examined using a BioRad MRC 1024 confocal microscope (Bio-Rad, Richmond, CA).

ζ Chain Detection by Western Blot.

EBV-B cells were pulsed with various concentrations of TT830–843. 5 × 10⁵ T cells were pretreated for 1 h with 10 μg/ml cycloheximide mixed with 10⁶ EBV-B cells in 500 μl RPMI 5% FCS in U-bottomed tubes, centrifuged to allow conjugate formation, and incubated at 37°C for 2 h in the presence of cycloheximide. In some experiments, T cells were pretreated with 1 μM bafilomycin A1 or with 1 μM folimycin (Calbiochem) or with the vehicle of the drugs only (DMSO 1%) for 1 h at 37°C before conjugate formation. The drugs were present in the culture throughout the assay. At different timepoints, the cells were mixed with PBS, lysed in either ice-cold RIPA buffer containing 1 mM NaVO₄ or in prewarmed Laemmli buffer, sonicated, and boiled. After separation on 12.5% SDS-PAGE and transfer to nitrocellulose, membranes were blocked 1 h at room temperature with blocking buffer (5% nonfat dry milk, 0.05% Tween-20 in Tris-buffered saline) and incubated for 1 h with 1 μg/ml anti-ζ (Santa Cruz) in blocking buffer. After washing, the membranes were incubated for 1 h with HRP-labeled goat anti–mouse Ig (SBA) in blocking buffer. Filters were developed using an enhanced chemiluminescence detection system (Amersham, Arlington Heights, IL). Densitometric analysis was performed using a Computing Densitometer 300A (Molecular Dynamics, Sunnyvale, CA).

TCR Downregulation, IFN-γ Production, and [Ca²⁺] Measurement.

TCR downregulation, IFN-γ production, and [Ca²⁺]; were measured as previously described (4, 5). In some experiments, T cells were pretreated with 1 μM bafilomycin A1 or with 1 μM folimycin for 1 h at 37°C before conjugate formation and the drugs were kept in the culture through all the assay. Control cultures were done in the presence of vehicle only (1% DMSO).

Reduced Content of TCR-β, CD3-ε, and ζ Chains in T Cells after Antigen Stimulation.

T cells conjugated with peptide-pulsed APCs undergo a rapid downregulation of triggered TCR–CD3 complexes that reaches a plateau in 1–2 h (5). To investigate whether downregulated receptors accumulate inside of T cells or are degraded, we measured the surface levels and the total cellular content of different TCR–CD3 components by FACS^® analysis. As shown in Fig. 1, A–B, in T cells conjugated with peptide-pulsed APCs the surface level of CD3-ε and the total content of CD3-ε and ζ chains decreased in a parallel fashion with increasing doses of antigen. Blocking of protein synthesis did not affect the extent of CD3-ε and ζ chain decrease, demonstrating that disappearance of these components was not due to a decreased synthesis (Fig. 1 C ). Similar results were observed when levels of CD3-ε and TCR-β were measured in a different T cell clone (Fig. 1 D).

To investigate whether the reduced staining for CD3-ε and ζ chain could be due to the localization of triggered receptors in cellular compartments not accessible to antibodies, we measured ζ chain expression by Western blot. As shown in Fig. 2 A, the lysates of T–APC conjugates showed a dose-dependent decrease in the content of ζ chain. These results are comparable to those observed by FACS^® analysis (Fig. 1). In addition, the time kinetics of ζ chain decrease parallels that of surface TCR downregulation (5, 6) reaching a plateau in about 2 h (Fig. 2 B).

The above results demonstrate that TCR triggering by specific antigen results in a net decrease in the cellular content of TCR and CD3-ε and ζ molecules. This indicates that downregulated TCR–CD3-ζ complexes are promptly degraded.

Lysosomal Degradation of CD3-ζ in Activated T Cells.

A wellknown mechanism for the inactivation of hormone and growth factor receptors is their internalization upon ligand binding followed by their degradation in lysosomes (7). To find whether triggered TCRs are degraded via a similar mechanism we stimulated T cells with peptide-pulsed APCs in the presence of bafilomycin A1 or folimycin, drugs that affect lysosomal function by increasing lysosomal pH (8, 9). These drugs do not interfere with TCR downregulation, sustained [Ca²⁺]_i increase, and IFN-γ production (Fig. 3), indicating that they are not toxic for the T cell biological response. However, bafilomycin A1 and folimycin dramatically inhibited ζ chain degradation as detected by Western blot analysis on total cell lysates (Fig. 4). The inhibition of ζ chain degradation by bafilomycin A1 and folimycin was also observed when protein synthesis was blocked by pretreating T cells with cycloheximide (Fig. 4). This result indicates that the effect of bafilomycin A1 and folimycin is not due to the accumulation of newly synthesized unassembled components, but actually results from a block of the degradation of triggered receptors.

Morphological Evidence for Targeting of Triggered CD3-ζ to the Lysosomes.

To identify better the site of TCR–CD3 complex degradation, we investigated by confocal microscopy the fate of triggered TCRs. T cells were conjugated with peptide-pulsed or unpulsed APCs and after 2 h the conjugates were fixed, permeabilized, and stained with anti-ζ chain antibodies.

In unstimulated T cells most of the ζ chain is associated with the plasma membrane and this staining pattern is not affected by preincubation with bafilomycin A1 (Fig. 5, A, B, E ). In T cells that had been conjugated for 2 h with peptide-pulsed APCs, the staining was much weaker and did not show the ring-shaped pattern of ζ chain surface expression (Fig. 5 C ). Strikingly, when conjugates were formed in the presence of bafilomycin A1 a dramatic accumulation of ζ chain was observed in intracellular vesicles (Fig. 5 D). In these vesicles, most of the internalized ζ chain colocalized with the lysosomal marker LAMP-1 (Fig. 5 F). These results demonstrate that upon TCR/peptide– MHC interaction ζ chains are promptly removed from the cell surface and degraded in the lysosomes.