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

The David H Smith Center for Vaccine Biology and Immunology, University of Rochester, Rochester, NY 14642, USA.
The Journal of Immunology (Impact Factor: 4.92). 12/2009; 181(11):7639-48. DOI: 10.4049/jimmunol.181.11.7639
Source: PubMed

ABSTRACT 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-theta (PKCtheta) to the cSMAC, activation of NF-kappaB, 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. PKCtheta distribution at the immunological synapse mirrors the distribution of tyrosine 188-mutated CD28, indicating that CD28 drives the localization of PKCtheta even when CD28 is not localized to the cSMAC. Mutation of tyrosine 188 also results in diminished activation of NF-kappaB, suggesting that CD28-mediated localization of PKCtheta 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 PCKtheta may be mediated through localization to the cSMAC region of the immunological synapse.

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Available from: Mariano Sanchez-Lockhart, Jun 17, 2015
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    • "TCR signaling can induce a rapid reorientation of the cytosolic tail domains within the CD28 homodimer as detected by a change in fluorescence resonance energy transfer (FRET) [14]. TCR signaling is also necessary for sustained localization of CD28 to the immunological synapse [11], [13] and TCR signaling can induce CD28 polarization toward CD80-positive cells [14]. These results raise the possibility that TCR signaling may be able to enhance CD28 localization at the immunological synapse by regulating the ability of CD28 to interact with ligand. "
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    ABSTRACT: T cell activation takes place in the context of a spatial and kinetic reorganization of cell surface proteins and signaling molecules at the contact site with an antigen presenting cell, termed the immunological synapse. Coordination of the activation, recruitment, and signaling from T cell receptor (TCR) in conjunction with adhesion and costimulatory receptors regulates both the initiation and duration of signaling that is required for T cell activation. The costimulatory receptor, CD28, is an essential signaling molecule that determines the quality and quantity of T cell immune responses. Although the functional consequences of CD28 engagement are well described, the molecular mechanisms that regulate CD28 function are largely unknown. Using a micropipet adhesion frequency assay, we show that TCR signaling enhances the direct binding between CD28 and its ligand, CD80. Although CD28 is expressed as a homodimer, soluble recombinant CD28 can only bind ligand monovalently. Our data suggest that the increase in CD28-CD28 binding is mediated through a change in CD28 valency. Molecular dynamic simulations and in vitro mutagenesis indicate that mutations at the base of the CD28 homodimer interface, distal to the ligand-binding site, can induce a change in the orientation of the dimer that allows for bivalent ligand binding. When expressed in T cells, this mutation allows for high avidity CD28-CD80 interactions without TCR signaling. Molecular dynamic simulations also suggest that wild type CD28 can stably adopt a bivalent conformation. These results support a model whereby inside-out signaling from the TCR can enhance CD28 ligand interactions by inducing a change in the CD28 dimer interface to allow for bivalent ligand binding and ultimately the transduction of CD28 costimulatory signals that are required for T cell activation.
    PLoS ONE 02/2014; 9(2):e89263. DOI:10.1371/journal.pone.0089263 · 3.23 Impact Factor
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    • "During conjugate formation, many proteins depend on motifs found in the cytoplasmic tail for proper localization. For instance, CD28 requires Y188 in its cytoplasmic tail for localization towards the IS 44. Likewise, CD43, which moves opposite the immunological synapse and to the distal pole complex, requires its cytoplasmic tail for this localization 6. "
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    ABSTRACT: The interaction between T cells and APCs bearing cognate antigen results in the formation of an immunological synapse (IS). During this process, many receptors and signaling proteins segregate to regions proximal to the synapse. This protein movement is thought to influence T cell function. However, some proteins are transported away from the IS, which is controlled in part by ERM family proteins. Tim-1 is a transmembrane protein with co-stimulatory functions that is found on many immune cells, including T cells. However, the expression pattern of Tim-1 on T cells upon activation by APCs has not been explored. Interestingly, in this study we demonstrate that the majority of Tim-1 on activated T cells is excluded from the IS. Tim-1 predominantly resides outside of the IS, and structure/function studies indicate that the cytoplasmic tail influences Tim-1 polarization. Specifically, a putative ERM binding motif (KRK 244-246) in the Tim-1 cytoplasmic tail appears necessary for proper Tim-1 localization. Furthermore, mutation of the KRK motif results in enhanced Tim1-mediated early tyrosine phosphorylation downstream of TCR/CD28 stimulation. Paradoxically however, the KRK motif is necessary for Tim-1 induced NFAT/AP-1 activation and co-stimulation of cytokine production. This work reveals unexpected complexity underlying Tim-1 localization and suggests potentially novel mechanisms by which Tim-1 modulates T cell activity.
    F1000 Research 10/2012; 1:10. DOI:10.12688/f1000research.1-10.v1
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    • "T-cell activation is initiated by the immunological synapse (IS) formed at the intercellular contacts between APCs and antigen-specific T-cells. The IS has been demonstrated to be a dynamic complex that consists not only the TCRs, but also sets of co-stimulatory receptors and ligands [42], [43], [44], [45], [46], [47], [48], [49], [50], [51]. CD28, the most powerful T-cell co-stimulatory receptors for CD80 or CD86, has been shown to localize coordinately with TCRs to form microclusters that are able to recruit protein kinase C θ (PKCθ) to initiate T-cell activation [45], [52], [53]. "
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    ABSTRACT: Functional T-cell responses are initiated by physical interactions between T-cells and antigen-presenting cells (APCs), including dendritic cells (DCs) and B-cells. T-cells are activated more effectively by DCs than by B-cells, but little is known about the key molecular mechanisms that underpin the particular potency of DC in triggering T-cell responses. To better understand the influence of physical intercellular interactions on APC efficacy in activating T-cells, we used single cell force spectroscopy to characterize and compare the mechanical forces of interactions between DC:T-cells and B:T-cells. Following antigen stimulation, intercellular interactions of DC:T-cell conjugates were stronger than B:T-cell interactions. DCs induced higher levels of T-cell calcium mobilization and production of IL-2 and IFNγ than were elicited by B-cells, thus suggesting that tight intercellular contacts are important in providing mechanically stable environment to initiate T-cell activation. Blocking antibodies targeting surface co-stimulatory molecules CD80 or CD86 weakened intercellular interactions and dampen T-cell activation, highlighting the amplificatory roles of CD80/86 in regulating APC:T-cell interactions and T-cell functional activation. The variable strength of mechanical forces between DC:T-cells and B:T-cell interactions were not solely dependent on differential APC expression of CD80/86, since DCs were superior to B-cells in promoting strong interactions with T-cells even when CD80 and CD86 were inhibited. These data provide mechanical insights into the effects of co-stimulatory molecules in regulating APC:T-cell interactions.
    PLoS ONE 09/2012; 7(9):e45185. DOI:10.1371/journal.pone.0045185 · 3.23 Impact Factor
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