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Short-lived TCR microclusters and a longer-lived protein kinase Ctheta-focusing central supramolecular activation cluster (cSMAC) have been defined in model immunological synapses (IS). In different model systems, CD28-mediated costimulatory interactions have been detected in microclusters, the cSMAC, or segregated from the TCR forming multiple distinct foci. The relationship between TCR and costimulatory molecules in the physiological IS of T cell-dendritic cell (DC) is obscure. To study the dynamic relationship of CD28-CD80 and TCR interactions in the T cell-DC IS during Ag-specific T cell activation, we generated CD80-eCFP mice using bacterial artificial chromosome transgenic technology. In splenic DCs, endogenous CD80 and CD80-eCFP localized to plasma membrane and Golgi apparatus, and CD80-eCFP was functional in vivo. In the OT-II T cell-DC IS, multiple segregated TCR, CD80, and LFA-1 clusters were detected. In the T cell-DC synapse CD80 clusters were colocalized with CD28 and PKCtheta, a characteristic of the cSMAC. Acute blockade of TCR signaling with anti-MHC Ab resulted in a rapid reduction in Ca(2+) signaling and the number and size of the CD80 clusters, a characteristic of TCR microclusters. Thus, the T cell-DC interface contains dynamic costimulatory foci that share characteristics of microclusters and cSMACs.

Type

Journal article

Journal

J Immunol

Publication Date

01/10/2008

Volume

181

Pages

4852 - 4863

Keywords

Amino Acid Sequence, Animals, B7-1 Antigen, CD28 Antigens, CHO Cells, Chromosomes, Artificial, Bacterial, Cricetinae, Cricetulus, Dendritic Cells, Green Fluorescent Proteins, Immunological Synapses, Isoenzymes, Lymphocyte Activation, Mice, Mice, Inbred C57BL, Mice, Knockout, Mice, Transgenic, Molecular Sequence Data, Protein Kinase C, Protein Kinase C-theta, Protein Transport, Receptors, Antigen, T-Cell, T-Lymphocyte Subsets