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. 1997 May 19;185(10):1859-64.
doi: 10.1084/jem.185.10.1859.

Degradation of T cell receptor (TCR)-CD3-zeta complexes after antigenic stimulation

S Valitutti  1 , S Müller, M Salio, A Lanzavecchia
Affiliations

Degradation of T cell receptor (TCR)-CD3-zeta complexes after antigenic stimulation

S Valitutti et al. J Exp Med. .

Abstract

T cell activation by specific antigen results in a rapid and long-lasting downregulation of triggered T cell receptors (TCRs). In this work, we investigated the fate of downregulated TCR- CD3-zeta complexes. T cells stimulated by peptide-pulsed antigen-presenting cells (APCs) undergo an antigen dose-dependent decrease of the total cellular content of TCR-beta, CD3-epsilon, and zeta chains, as detected by FACS(R) analysis on fixed and permeabilized T-APC conjugates and by Western blot analysis on cell lysates. The time course of CD3-zeta chain consumption overlaps with that of TCR downregulation, indicating that internalized TCR-CD3 complexes are promptly degraded. Inhibitors of lysosomal function (bafilomycin A1, folimycin) markedly reduced zeta chain degradation, leading to the accumulation of zeta chain in large Lamp1(+) vesicles. These results indicate that in T cell-APC conjugates, triggered TCRs are rapidly removed from the cell surface and are degraded in the lysosomal compartment.

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Figures

Figure 1
Figure 1
Parallel decrease in surface expression and total cellular content of TCR-β, CD3-ε and ζ chains in T cells as a function of antigenic stimulation. T cells (clone KS140) were conjugated at 37°C with APCs pulsed with various doses of peptide. After 2 h the cells were either fixed and stained with anti-CD3-ε or anti-Vβ2, or fixed, permeabilized, and stained with anti-CD3-ε, anti-Vβ2, or anti ζ. (A) Staining for total CD3-ε (left) and ζ (right) in T cells conjugated with unpulsed or peptide-pulsed APCs. (B and C ) Levels of surface CD3-ε (○), total CD3-ε (•) and total ζ (▴) chains as a function of antigen concentration. In C, T cells were pretreated for 1 h with cycloheximide and the drug was present throughout the assay. (D) Levels of surface and total Vβ2 and CD3-ε in T cell clone KS70 stimulated with 100 nM (diagonal stripes) or 10 μM (empty) peptide expressed as percent of the staining of unstimulated cells (closed).
Figure 2
Figure 2
Dose response and kinetics of antigen-induced ζ chain loss. T cells were conjugated in the presence of cycloheximide with APCs pulsed with various peptide concentrations. At the indicated time points the cells were lysed and the amount of ζ chain was measured by Western blot. (A) ζ chain loss as a function of antigen concentration. (B) Time course of ζ chain loss in T cells conjugated with APC pulsed with 10 μM peptide.
Figure 3
Figure 3
Bafilomycin A1 and folimycin do not interfere with T cell activation induced by specific antigen. T cells pretreated for 1 h with bafilomycin A1 (▪), folimycin (▴), or vehicle only (•) were conjugated with APCs pulsed with various peptide concentrations and the drugs were left in culture throughout the assay. (A) CD3 downregulation after 4 h; (B) IFN-γ production after 4 h; (C ) [Ca2+]i increase in T cells conjugated with unpulsed APC (a and d ) with APC pulsed with 25 nM (b and e), or 10 μM peptide (c and f  ). T cells were either pretreated with bafilomycin A1 (d, e, f  ) or with the vehicle only (a, b, c).
Figure 4
Figure 4
Antigen-induced degradation of ζ chain is prevented by bafilomycin A1 and folimycin. T cells were pretreated for 1 h with bafilomycin A1 or folimycin in the presence or absence of cycloheximide. The cells were conjugated with APCs pulsed with 10 μM peptide and total content of ζ chain was determined after 2 h by Western blot. The drugs were present throughout the assay. (A) Western blot. (B) Densitometric analysis of the bands obtained in the absence (closed ) or in the presence (empty) of cycloheximide.
Figure 5
Figure 5
Triggered CD3-ζ is targeted to the lysosomes for degradation. T cells were conjugated with either unpulsed APCs (a, b, e) or peptide-pulsed (10 μM) APCs (c, d, f  ). T cells were treated with bafilomycin A1 1 h before conjugate formation (b, d, e, f  ) or with vehicle only (a and c) the drug was left in culture throughout the assay. After 2 h the conjugates were fixed, permeabilized, and stained with anti-ζ (green) and anti-Lamp-1 (red ).
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