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Recombinant Anti-CD4 Antibody 13B8.2 Blocks Membrane-Proximal Events by Excluding the Zap70 Molecule and Downstream Targets SLP-76, PLC1, and Vav-1 from the CD4-Segregated Brij 98 Detergent-Resistant Raft Domains¹

Myriam Chentouf^*, Soufiane Ghannam^*, Cédric Bès^2,*, Samuel Troadec^*, Martine Cérutti and Thierry Chardès^3,*

^* Centre National de la Recherche Scientifique (CNRS) Unité Mixte de Recherche 5236, Centre d’études d’agents Pathogènes et Biotechnologies pour la Santé, Faculté de Pharmacie, Montpellier, France; and CNRS Unité Propre de Service 3040, Saint Christol-Les-Alès, France

The biological effects of rIgG₁ 13B8.2, directed against the CDR3-like loop on the D1 domain of CD4, are partly due to signals that prevent NF-B nuclear translocation, but the precise mechanisms of action, particularly at the level of membrane proximal signaling, remain obscure. We support the hypothesis that rIgG₁ 13B8.2 acts by interfering with the spatiotemporal distribution of signaling or receptor molecules inside membrane rafts. Upon cross-linking of Jurkat T lymphocytes, rIgG₁ 13B8.2 was found to induce an accumulation/retention of the CD4 molecule inside polyoxyethylene-20 ether Brij 98 detergent-resistant membranes at 37°C, together with recruitment of TCR, CD3, 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 C, but excluded Zap70 and its downstream targets Src homology 2-domain-containing leukocyte protein of 76 kDa, phospholipase C1, and p95^vav. Analysis of key upstream events such as Zap70 phosphorylation showed that modulation of Tyr²⁹² and Tyr³¹⁹ 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 rIgG₁ 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-B nuclear translocation and explains how baculovirus-expressed CD4-CDR3-like-specific rIgG₁ 13B8.2 acts to induce its biological effects.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ T.C. was supported by a grant from the Ligue Nationale Contre le Cancer, Comité de l’Hérault. S.T. held, successively, doctoral fellowships from the Ligue Nationale contre le Cancer, Comité de l’Hérault, and the Association de Recherche contre le Cancer. M.Ch. was a recipient of a doctoral fellowship from the Ligue Nationale contre le Cancer, Comité de l’Hérault. C.B. was a recipient of a postdoctoral fellowship from the Fondation de France.

² Current address: Institut de Recherche Pierre Fabre, 5 Avenue Napoléon III, BP 60497, 74164 Saint-Julien en Genevois, France.

³ Address correspondence and reprint requests to Dr. Thierry Chardès, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5236, Centre d’études d’agents Pathogènes et Biotechnologies pour la Santé, Faculté de Pharmacie, 15 Avenue Charles Flahault, BP 14491, 34093 Montpellier Cedex 5, France. E-mail address: thierry.chardes{at}univ-montp1.fr

⁴ Abbreviations used in this paper: lo, liquid ordered; DRM, detergent-resistant membrane; PKC, protein kinase C; SLP-76, Src homology 2-domain-containing leukocyte protein of 76 kDa; PLC1, phospholipase C1; Vav-1, p95^vav; LAT, linker for activation of T cells; GM1, ganglioside M1; Cbp/PAG, Csk-binding protein/ phosphoprotein associated with glycosphingolipid; MCD, methyl--cyclodextrin; PBS-T, PBS containing 0.1% Tween 20.

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