The Strength of T Cell Receptor Signal Controls the Polarization of Cytotoxic Machinery to the Immunological Synapse

Cambridge Institute for Medical Research, Addenbrooke's Hospital, Cambridge CB2 0XY, UK.
Immunity (Impact Factor: 21.56). 10/2009; 31(4):621-31. DOI: 10.1016/j.immuni.2009.08.024
Source: PubMed


Killing by cytotoxic T lymphocytes (CTLs) is mediated by the secretion of lytic granules. The centrosome plays a key role in granule delivery, polarizing to the central supramolecular activation complex (cSMAC) within the immunological synapse upon T cell receptor (TCR) activation. Although stronger TCR signals lead to increased target cell death than do weaker signals, it is not known how the strength of TCR signal controls polarization of the centrosome and lytic granules. By using TCR transgenic OT-I CTLs, we showed that both high- and low-avidity interactions led to centrosome polarization to the cSMAC. However, only high-avidity interactions, which induced a higher threshold of intracellular signaling, gave rise to granule recruitment to the polarized centrosome at the synapse. By controlling centrosome and granule polarization independently, the centrosome is able to respond rapidly to weak signals so that CTLs are poised and ready for the trigger for granule delivery.

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    • "Previous studies have shown that phosphorylated ERK may play a role in actin reorganisation as ERK co-localises with actin at the immune synapse, is implicated in granule and MTOC polarisation, and is required for CTL degranulation (Robertson et al., 2005; Jenkins et al., 2009). "
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    ABSTRACT: T cell receptor (TCR) activation leads to a dramatic reorganisation of both membranes and receptors as the immunological synapse forms. Using a genetic model to rapidly inhibit Zap70 catalytic activity we examined synapse formation between cytotoxic T lymphocytes and their targets. In the absence of Zap70 catalytic activity Vav-1 activation occurs and synapse formation is arrested at a stage with actin and integrin rich interdigitations forming the interface between the two cells. The membranes at the synapse are unable to flatten to provide extended contact, and Lck does not cluster to form the central supramolecular activation cluster (cSMAC). Centrosome polarisation is initiated but aborts before reaching the synapse and the granules do not polarise. Our findings reveal distinct roles for Zap70 as a structural protein regulating integrin-mediated control of actin vs its catalytic activity that regulates TCR-mediated control of actin and membrane remodelling during formation of the immunological synapse. DOI:
    eLife Sciences 03/2014; 3(3):e01310. DOI:10.7554/eLife.01310 · 9.32 Impact Factor
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    • "Although formation of a stable immune synapse between CTL and target cell may not be required for CTL cytolytic activity [55], high avidity CTL/target cell interactions such as those involving the CD8+ OT1 CTL/OVA peptide-loaded target cell system used in our study have been shown to lead to granule polarization to the immune synapse [56]. Accordingly, in our live video experiments, we observed the movement of the red GZMB-Tom granules towards the CTL/target cell tight interaction zone corresponding to the immune synapse. "
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    ABSTRACT: To evaluate acquisition and activation of cytolytic functions during immune responses we generated knock in (KI) mice expressing Granzyme B (GZMB) as a fusion protein with red fluorescent tdTomato (GZMB-Tom). As for GZMB in wild type (WT) lymphocytes, GZMB-Tom was absent from naïve CD8 and CD4 T cells in GZMB-Tom-KI mice. It was rapidly induced in most CD8 T cells and in a subpopulation of CD4 T cells in response to stimulation with antibodies to CD3/CD28. A fraction of splenic NK cells expressed GZMB-Tom ex vivo with most becoming positive upon culture in IL-2. GZMB-Tom was present in CTL granules and active as a protease when these degranulated into cognate target cells, as shown with target cells expressing a specific FRET reporter construct. Using T cells from mice expressing GZMB-Tom but lacking perforin, we show that the transfer of fluorescent GZMB-Tom into target cells was dependent on perforin, favoring a role for perforin in delivery of GZMB at the target cells' plasma membranes. Time-lapse video microscopy showed Ca++ signaling in CTL upon interaction with cognate targets, followed by relocalization of GZMB-Tom-containing granules to the synaptic contact zone. A perforin-dependent step was next visualized by the fluorescence signal from the non-permeant dye TO-PRO-3 at the synaptic cleft, minutes before the labeling of the target cell nucleus, characterizing a previously undescribed synaptic event in CTL cytolysis. Transferred OVA-specific GZMB-Tom-expressing CD8 T cells acquired GZMB-Tom expression in Listeria monocytogenes-OVA infected mice as soon as 48h after infection. These GZMB-Tom positive CD8 T cells localized in the splenic T-zone where they interacted with CD11c positive dendritic cells (DC), as shown by GZMB-Tom granule redistribution to the T/DC contact zone. GZMB-Tom-KI mice thus also provide tools to visualize acquisition and activation of cytolytic function in vivo.
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    • "Shortly after, this molecular pattern is inverted forming a central cluster of TCR/pMHC molecules known as central supramolecular activation cluster (cSMAC), which is surrounded by a peripheral ring of LFA- 1/ICAM-1 molecules and is named the peripheral supramolecular activation cluster (pSMAC) [20, 78–80], defining a mature IS. Importantly, this process depends on significant cytoskeleton rearrangements involving the microtubule organizing center (MTOC) and cytotoxic granules polarizing to the IS [81] [82] [83]. In fact, disruption of actin polymerization by cytochalasin D has been shown to disrupt IS formation [84] [85]. "
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