Selective Defect in Antigen-Induced TCR Internalization at the Immune Synapse of CD8 T Cells Bearing the ZAP-70(Y292F) Mutation

Abstract

Cbl proteins have been implicated in ligand-induced TCR/CD3 down-modulation, but underlying mechanisms are unclear. We analyzed the effect of mutation of a cbl-binding site on ZAP-70 (ZAP-Y292F) on dynamics, internalization, and degradation of the TCR/CD3 complex in response to distinct stimuli. Naive CD8 T cells expressing the P14 transgenic TCR from ZAP-Y292F mice were selectively affected in TCR/CD3 down-modulation in response to antigenic stimulation, whereas neither anti-CD3 Ab-, and PMA-induced TCR down-modulation, nor constitutive receptor endocytosis/cycling were impaired. We further established that the defect in TCR/CD3 down-modulation in response to Ag was paralleled by an impaired TCR/CD3 internalization and CD3ζ degradation. Analysis of T/APC conjugates revealed that delayed redistribution of TCR at the T/APC contact zone was paralleled by a delay in TCR internalization in the synaptic zone in ZAP-Y292F compared with ZAP-wild-type T cells. Cbl recruitment to the synapse was also retarded in ZAP-Y292F T cells, although F-actin and LFA-1 redistribution was similar for both cell types. This study identifies a step involving ZAP-70/cbl interaction that is critical for rapid internalization of the TCR/CD3 complex at the CD8 T cell/APC synapse.

ZAP-70 enhances B-cell–receptor signaling despite absent or inefficient tyrosine kinase activation in chronic lymphocytic leukemia and lymphoma B cells

“…This hypothesis is intriguing in view of the associations between ZAP-70 and the c-Cbl and Cbl-b ubiquitin ligases, which negatively regulate BCR and TCR signaling by promoting ligand-induced down-modulation of the antigen receptors and by targeting for degradation specific BCR and TCR components. 22,29,[37][38][39] Consistent with this hypothesis, we observed that ligandinduced BCR internalization was reduced by approximately one third in ZAP-70 ϩ BJAB B cells. Decreased ligand-mediated BCR internalization has already been shown to increase the magnitude and duration of BCR signaling, including activation of the Akt and ERK kinases.…”

“…28,29 Briefly, 2 ϫ 10 6 cells were incubated for each investigated time point in 200 L complete RPMI medium with 20 L R-PE-coupled goat F(abЈ) 2 anti-human IgM (Caltag Laboratories, Burlingame, CA) in microcentrifuge tubes on ice for 30 minutes. Cells were washed, resuspended in 200 L medium, and split in equal amounts into 3 new tubes.…”

Molecular mimicry and immunomodulation by the HRES-1 endogenous retrovirus in SLE

“…While the plasma membrane-associated Lck is bound to CD4, an intracellular pool of Lck is associated with TFR-positive recycling endosomes [116,117]. Based on its homology to Rab4a, HRES-1/Rab4 is likely to be activated by p85-PI3K [118] and its GTP-bound active form associates with the CD2 adaptor protein [119] and the ubiquitin ligase Cbl [120] which are key components of the IS [108,121]. The existing data on direct binding of Rab4 to p85-PI3K and CD2 adaptor protein and regulation of CD4 and TFR recycling strongly suggest that HRES-1/Rab4 is involved in abnormal assembly and signaling through the IS.…”

Cbl- and Nedd4-family ubiquitin ligases: balancing tolerance and immunity

“…Domain organization of these murine proteins is based on the National Center for Biotechnology Information database (http:// www.ncbi.nlm.nih.gov/) Cbl-family targets in T cell activation. Following T cell activation, Cbl E3 ligases ubiquitinate subunits of the TCR as well as phosphorylated signaling components including ZAP70, the p85 subunit of PI3Kinase, Fyn, Lck and Vav [38][39][40][74][75][76][77][78]. This ubiquitination results in dampening of T cell activation signals.…”