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T Cell-Dendritic Cell Immunological Synapses Contain TCR-dependent CD28-CD80 Clusters That Recruit Protein Kinase C¹

Su-Yi Tseng^2,*, Janelle C. Waite^*, Mengling Liu, Santosha Vardhana^* and Michael L. Dustin^3,*

^* Program in Molecular Pathogenesis, Helen L. and Martin S. Kimmel Center for Biology and Medicine of the Skirball Institute of Biomolecular Medicine and Department of Pathology, New York University School of Medicine, New York, NY 10016; and Division of Biostatistics, New York University Cancer Institute, New York, NY 10016

Short-lived TCR microclusters and a longer-lived protein kinase C-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 PKC, a characteristic of the cSMAC. Acute blockade of TCR signaling with anti-MHC Ab resulted in a rapid reduction in Ca²⁺ 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.

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 National Institutes of Health Grants AI55037, AI43542, and AI44931 (to M.L.D.). S.-Y.T. was a recipient of Leukemia and Lymphoma Society Fellowship Grant 5456-04. M.L.D. was supported by National Cancer Institute Cancer Center Support Grant P30 CA016087.

² Current address: Geron Corporation, 230 Constitution Drive, Menlo Park, CA 94025.

³ Address correspondence and reprint requests to Dr. Michael L. Dustin, Helen L. and Martin S. Kimmel Center for Biology and Medicine of the Skirball Institute of Biomolecular Medicine, New York University School of Medicine, 540 First Avenue, New York, NY 10016. Email address: dustin{at}saturn.med.nyu.edu

⁴ Abbreviations used in this paper: MHCp, MHC class II-antigenic peptide complex; eCFP, enhanced cyan fluorescence protein; IS, immunological synapse; DC, dendritic cell; SMAC, supramolecular activation cluster; cSMAC, central SMAC; PKC, protein kinase C; pSMAC, peripheral SMAC; BAC, bacterial artificial chromosome; Tg, transgenic; CHO, Chinese hamster ovary; 3D, three dimensional; qvtr, quick-time virtual reality; WT, wild type; MTOC, microtubule-organizing center; Treg, regulatory T cell.

⁵ The online version of this article contains supplemental material.