Modulation of TCR expression levels is a central event during T cell development and activation, and it probably plays an important role in adjusting T cell responsiveness. Conflicting data have been published on down-regulation and degradation rates of the individual TCR subunits, and several divergent models for TCR down-regulation and degradation have been suggested. The aims of this study were to determine the rate constants for constitutive and ligand-induced TCR degradation and to determine whether the TCR subunits segregate or are processed as an intact unit during TCR down-regulation and degradation. We found that the TCR subunits in nonstimulated Jurkat cells were degraded with rate constants of ∼0.0011 min−1, resulting in a half-life of ∼10.5 h. Triggering of the TCR by anti-TCR Abs resulted in a 3-fold increase in the degradation rate constants to ∼0.0033 min−1, resulting in a half-life of ∼3.5 h. The subunits of the TCR complex were down-regulated from the cell surface and degraded with identical kinetics, and most likely remained associated during the passage throughout the endocytic pathway from the cell surface to the lysosomes. Similar results were obtained in studies of primary human Vβ8+ T cells stimulated with superantigen. Based on these results, the simplest model for TCR internalization, sorting, and degradation is proposed.

TCR internalization takes place both in resting T cells as part of constitutive TCR cycling, after PKC activation, and during TCR triggering. It is still a matter of debate whether these pathways represent distinct pathways. Thus, some studies have indicated that ligand-induced TCR internalization is regulated by mechanisms distinct from those involved in constitutive internalization, whereas other studies have suggested that the ligand-induced TCR internalization pathway is identical with the constitutive pathway. To resolve this question, we first identified requirements for constitutive TCR cycling. We found that in contrast to PKC-induced TCR internalization where both CD3γ-S126 and the CD3γ leucine-based internalization motif are required, constitutive TCR cycling required neither PKC nor CD3γ-S126 but only the CD3γ leucine-based motif. Having identified these requirements, we next studied ligand-induced internalization in cells with abolished constitutive TCR cycling. We found that ligand-induced TCR internalization was not dependent on constitutive TCR internalization. Likewise, constitutive internalization and recycling of the TCR were independent of an intact ligand-induced internalization of the TCR. In conclusion, ligand-induced TCR internalization and constitutive cycling of the TCR represents two independent pathways regulated by different mechanisms.

One of the earliest events following TCR triggering is TCR down-regulation. However, the mechanisms behind TCR down-regulation are still not fully known. Some studies have suggested that only directly triggered TCR are internalized, whereas others studies have indicated that, in addition to triggered receptors, nonengaged TCR are also internalized (comodulated). In this study, we used transfected T cells expressing two different TCR to analyze whether comodulation took place. We show that TCR triggering by anti-TCR mAb and peptide-MHC complexes clearly induced internalization of nonengaged TCR. By using a panel of mAb against the Tiβ chain, we demonstrate that the comodulation kinetics depended on the affinity of the ligand. Thus, high-affinity mAb (KD = 2.3 nM) induced a rapid but reversible comodulation, whereas low-affinity mAb (KD = 6200 nM) induced a slower but more permanent type of comodulation. Like internalization of engaged TCR, comodulation was dependent on protein tyrosine kinase activity. Finally, we found that in contrast to internalization of engaged TCR, comodulation was highly dependent on protein kinase C activity and the CD3γ di-leucine-based motif. Based on these observations, a physiological role of comodulation is proposed and the plausibility of the TCR serial triggering model is discussed.

The T-cell receptor (TCR) is a multimeric receptor composed of the Ti alpha beta heterodimer and the noncovalently associated CD3 gamma delta epsilon and zeta(2) chains. All of the TCR chains are required for efficient cell surface expression of the TCR. Previous studies on chimeric molecules containing the di-leucine-based endocytosis motif of the TCR subunit CD3 gamma have indicated that the zeta chain can mask this motif. In this study, we show that successive truncations of the cytoplasmic tail of zeta led to reduced surface expression levels of completely assembled TCR complexes. The reduced TCR expression levels were caused by an increase in the TCR endocytic rate constant in combination with an unaffected exocytic rate constant. Furthermore, the TCR degradation rate constant was increased in cells with truncated zeta. Introduction of a CD3 gamma chain with a disrupted di-leucine-based endocytosis motif partially restored TCR expression in cells with truncated zeta chains, indicating that the zeta chain masks the endocytosis motif in CD3 gamma and thereby stabilizes TCR cell surface expression.

Interferon (IFN)-␣/␤ is produced by virally infected cells and is believed to play an important role in earlyIn conclusion, we provide evidence that IFN-␣ both upand down-regulates IL-4-mediated STAT6 signaling and thereby regulates the sensitivity to IL-4 in human T lymphocytes. Thus, our findings suggest that IFN-␣ has a complex regulatory role in adaptive immunity, which is different from the "classical" Th1 profile of IFN-␥.