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* Cardiovascular Biology Research Program;
Arthritis and Immunology Program, Oklahoma Medical Research Foundation,
Department of Microbiology and Immunology;
Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104
Polarization of membrane rafts and signaling proteins to form an immunological synapse is a hallmark of T cell stimulation. However, the kinetics of raft polarization and associated proteins in relation to the initial contact of the T cell with the APC are poorly defined. We addressed this question by measuring the distribution of membrane-targeted fluorescent protein markers during initial T cell interactions with B cell APCs. Experiments with unpulsed B cells lacking cognate Ag demonstrated an MHC class II-independent capping that was specific to membrane raft markers and required actin rearrangements and signals from Src kinases and PI3K. By live cell imaging experiments, we identified a similar specific polarization of membrane raft markers before TCR-dependent stop signals, and which occurred independently of cognate peptide-MHC class II. T cells conjugated to unpulsed B cells exhibited capping of CD4 and microclusters of the TCR 
-chain, but only the CD4 enrichment was cholesterol dependent. Furthermore, raft association of CD4 was necessary for its efficient targeting to the Ag-independent caps. Interestingly, anergic Vβ8+ T cells isolated from staphylococcal enterotoxin B-injected mice did not exhibit Ag-independent capping of membrane rafts, showing that inhibition of these early, Ag-independent events is a property associated with tolerance. Altogether, these data show that membrane raft capping is one of the earliest events in T cell activation and represents one avenue for promoting and regulating downstream peptide-MHC-dependent signaling within the T cell.
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 This work was supported by grants from the National Institutes of Health (P50 RR015577), R01 GM070001 (to W.R.), and R01 AI 48097 (to A.D.F.) and the Oklahoma Center for the Advancement of Science and Technology Grants HR02-009 (to W.R.) and HR02-48 (to A.D.F.).
2 Address correspondence and reprint requests to Dr. William Rodgers, Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, 825 NE 13th Street, MS 45, Oklahoma City, OK 73104. E-mail address: william-rodgers{at}omrf.ouhsc.edu
3 Abbreviations used in this paper: pMHC, peptide-MHC; CFP, cyan flourescent protein; Ctx, cholera toxin B subunit; DIC, differential interference contrast; IS, immunological synapse; LAT, linker for activation of T cells; Lat B, latrunculin B; PIP3, phosphatidylinositol 3,4,5-triphosphate; MβCD, methyl-β-cyclodextrin; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; TX-100, Triton X-100; PH, pleckstrin homology.
4 The online version of this article has supplemental material.
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