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  • Recombinant anti-CD4 antibody 13B8.2 blocks membrane-proximal events by excluding the Zap70 molecule and downstream targets SLP-76, PLC gamma 1, and Vav-1 from the CD4-segregated Brij 98 detergent-resistant raft domains.

Recombinant anti-CD4 antibody 13B8.2 blocks membrane-proximal events by excluding the Zap70 molecule and downstream targets SLP-76, PLC gamma 1, and Vav-1 from the CD4-segregated Brij 98 detergent-resistant raft domains.

Journal of immunology (Baltimore, Md. : 1950) (2007-06-21)
Myriam Chentouf, Soufiane Ghannam, Cédric Bès, Samuel Troadec, Martine Cérutti, Thierry Chardès
ABSTRACT

The biological effects of rIgG(1) 13B8.2, directed against the CDR3-like loop on the D1 domain of CD4, are partly due to signals that prevent NF-kappaB nuclear translocation, but the precise mechanisms of action, particularly at the level of membrane proximal signaling, remain obscure. We support the hypothesis that rIgG(1) 13B8.2 acts by interfering with the spatiotemporal distribution of signaling or receptor molecules inside membrane rafts. Upon cross-linking of Jurkat T lymphocytes, rIgG(1) 13B8.2 was found to induce an accumulation/retention of the CD4 molecule inside polyoxyethylene-20 ether Brij 98 detergent-resistant membranes at 37 degrees C, together with recruitment of TCR, CD3zeta, p56 Lck, Lyn, and Syk p70 kinases, linker for activation of T cells, and Csk-binding protein/phosphoprotein associated with glycosphingolipid adaptor proteins, and protein kinase Ctheta, but excluded Zap70 and its downstream targets Src homology 2-domain-containing leukocyte protein of 76 kDa, phospholipase Cgamma1, and p95(vav). Analysis of key upstream events such as Zap70 phosphorylation showed that modulation of Tyr(292) and Tyr(319) phosphorylation occurred concomitantly with 13B8.2-induced Zap70 exclusion from the membrane rafts. 13B8.2-induced differential raft partitioning was epitope, cholesterol, and actin dependent but did not require Ab hyper-cross-linking. Fluorescence confocal imaging confirmed the spatiotemporal segregation of the CD4 complex inside rafts and concomitant Zap70 exclusion, which occurred within 10-30 s following rIgG(1) 13B8.2 ligation, reached a plateau at 1 min, and persisted until the end of the 1-h experiment. The differential spatiotemporal partitioning between the CD4 receptor and the Zap70-signaling kinase inside membrane rafts interrupts the proximal signal cross-talk leading to subsequent NF-kappaB nuclear translocation and explains how baculovirus-expressed CD4-CDR3-like-specific rIgG(1) 13B8.2 acts to induce its biological effects.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Brij® 93, average Mn ~357
Sigma-Aldrich
Brij® O10
Sigma-Aldrich
ECO BRIJ® O10
Sigma-Aldrich
ECO BRIJ® O20, average Mn ~1,150
Sigma-Aldrich
BRIJ® O20, average Mn ~1,150