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: 19.75). 10/2009; 31(4):621-31. DOI: 10.1016/j.immuni.2009.08.024
Source: PubMed

ABSTRACT 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.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Cytotoxic lymphocytes kill target cells through the polarized release of the contents of intracellular perforin-containing granules. In natural killer (NK) cells, the binding of β2 integrin to members of the intercellular adhesion molecule family is sufficient to promote not only the adhesion of NK cells to target cells but also the polarization of intracellular lytic granules toward the target. We used NK cells in an experimental system designed to enable us to study the polarization of lytic granules in the absence of their release through degranulation, as well as β2 integrin signaling independently of inside-out signals from other receptors. Through a proteomics approach, we identified a signaling network centered on an integrin-linked kinase (ILK)-Pyk2-paxillin core that was required for granule and microtubule-organizing center (MTOC) polarization. The conserved Cdc42-Par6 signaling pathway, which controls cell polarity, was also activated by ILK and was required for granule polarization toward the target cell. A subset of the signaling components required for polarization contributed also to the convergence of granules on the MTOC. These results delineate two connected signaling networks that are stimulated upon β2 integrin engagement and control the polarization of the MTOC and associated lytic granules toward the site of contact with target cells to mediate cellular cytotoxicity.
    Science Signaling 10/2014; 7(346):ra96. DOI:10.1126/scisignal.2005629 · 7.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Cytotoxic lymphocytes destroy pathogen-infected and transformed cells through the cytotoxic granule exocytosis death pathway, which is dependent on the delivery of proapoptotic granzymes into the target cell cytosol by the pore-forming protein, perforin. Despite the importance of mouse models in understanding the role of cytotoxic lymphocytes in immune-mediated disease and their role in cancer immune surveillance, no reliable intracellular detection method exists for mouse perforin. Consequently, rapid, flow-based assessment of cytotoxic potential has been problematic, and complex assays of function are generally required. In this study, we have developed a novel method for detecting perforin in primary mouse cytotoxic T lymphocytes by immunofluorescence and flow cytometry. We used this new technique to validate perforin colocalization with granzyme B in cytotoxic granules polarized to the immunological synapse, and to assess the expression of perforin in cytotoxic T lymphocytes at various stages of activation. The sensitivity of this technique also allowed us to distinguish perforin levels in Prf1(+/+) and Prf1(+/-) mice. This new methodology will have broad applications and contribute to advances within the fields of lymphocyte biology, infectious disease, and cancer.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Failure of cytotoxic T lymphocytes (CTLs) or natural killer (NK) cells to kill target cells by perforin (Prf)/granzyme (Gzm)-induced apoptosis causes severe immune dysregulation. In familial hemophagocytic lymphohistiocytosis, Prf-deficient infants suffer a fatal "cytokine storm" resulting from macrophage overactivation, but the link to failed target cell death is not understood. We show that prolonged target cell survival greatly amplifies the quanta of inflammatory cytokines secreted by CTLs/NK cells and that interferon-γ (IFN-γ) directly invokes the activation and secondary overproduction of proinflammatory IL-6 from naive macrophages. Furthermore, using live cell microscopy to visualize hundreds of synapses formed between wild-type, Prf-null, or GzmA/B-null CTLs/NK cells and their targets in real time, we show that hypersecretion of IL-2, TNF, IFN-γ, and various chemokines is linked to failed disengagement of Prf- or Gzm-deficient lymphocytes from their targets, with mean synapse time increased fivefold, from ∼8 to >40 min. Surprisingly, the signal for detachment arose from the dying target cell and was caspase dependent, as delaying target cell death with various forms of caspase blockade also prevented their disengagement from fully competent CTLs/NK cells and caused cytokine hypersecretion. Our findings provide the cellular mechanism through which failed killing by lymphocytes causes systemic inflammation involving recruitment and activation of myeloid cells. © 2015 Jenkins et al.
    Journal of Experimental Medicine 03/2015; 212(3). DOI:10.1084/jem.20140964 · 13.91 Impact Factor

Full-text (2 Sources)

Available from
May 22, 2014