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Signals and Sequences That Control CD28 Localization to the Central Region of the Immunological Synapse¹

Mariano Sanchez-Lockhart^*, Beth Graf^* and Jim Miller^2,*,

^* The David H. Smith Center for Vaccine Biology and Immunology and the Department of Microbiology and Immunology, University of Rochester, Rochester, NY 14642

During T cell interaction with APC, CD28 is recruited to the central region (cSMAC) of the immunological synapse. CD28-mediated signaling through PI3K results in the recruitment of protein kinase C- (PKC) to the cSMAC, activation of NF-B, and up-regulation of IL-2 transcription. However, the mechanism that mediates CD28 localization to the cSMAC and the functional consequences of CD28 localization to the cSMAC are not understood. In this report, we show that CD28 recruitment and persistence at the immunological synapse requires TCR signals and CD80 engagement. Addition of mAb to either MHC class II or CD80 results in the rapid displacement of CD28 from the immunological synapse. Ligand binding is not sufficient for CD28 localization to the immunological synapse, as truncation of the cytosolic tail of CD28 disrupts synapse localization without effecting the ability of CD28 to bind CD80. Furthermore, a single point mutation in the CD28 cytosolic tail (tyrosine 188) interferes with the ability of CD28 to preferentially accumulate at the cSMAC. PKC distribution at the immunological synapse mirrors the distribution of tyrosine 188-mutated CD28, indicating that CD28 drives the localization of PKC even when CD28 is not localized to the cSMAC. Mutation of tyrosine 188 also results in diminished activation of NF-B, suggesting that CD28-mediated localization of PKC to the cSMAC is important for efficient signal transduction. These data reinforce the importance of the interplay of signals between TCR and CD28 and suggest that CD28 signaling through PCK may be mediated through localization to the cSMAC region of the immunological synapse.

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 Grant R01-AI063418 from the National Institutes of Health (to J. M.). M.S.L. was supported by National Institutes of Health Training Grant T32-AI007169.

² Address correspondence and reprint requests to Dr. Jim Miller, Center for Vaccine Biology and Immunology, University of Rochester, Box 609, 601 Elmwood Avenue, Rochester, NY 14642-8609. E-mail address: jim_miller{at}urmc.rochester.edu

³ Abbreviations used in this paper: SH, Src homology domain; SMAC, supramolecular activation cluster; cSMAC, central SMAC; pSMAC, peripheral SMAC; PKC, protein kinase C-; IS, immunological synapse; WT, wild type; IRES, internal ribosomal entry site; CT, cytosolic tail; DIC, differential interference contrast; CFP, cyan fluorescent protein.

⁴ The online version of this article contains supplemental material.