Systems Imaging of the Immune Synapse

Ambler, Rachel; Ruan, Xiangtao; Murphy, Robert F.; Wülfing, Christoph

doi:10.1007/978-1-4939-6881-7_25

Cited by 10 publications

(23 citation statements)

References 21 publications

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“…We characterized T cell signaling organization as extensively described before (Ambler et al, 2017;Roybal et al, 2015;Singleton et al, 2009). Briefly, in vitro primed 5C.C7 T cells are retrovirally transduced to express fluorescent signaling intermediates or sensors, FACS sorted to low expression as close as possible to endogenous signaling intermediate concentrations and imaged in three dimensions over time during restimulation with APC and 10µM MCC peptide ('full stimulus').…”

Section: Results µM Scale Lat Accumulation At the Center Of The T Celmentioning

confidence: 99%

“…To ensure that overall interface accumulation of the targeted LAT constructs was comparable, we measured (Ambler et al, 2017;Roybal et al, 2016) their interface recruitment upon costimulation blocked conditions. All constructs showed substantial interface recruitment with moderately less LAT Vav recruitment at the last four time points ( Fig.…”

Section: Fusion Of Lat With Protein Domains With Pronounced Interfacementioning

confidence: 99%

“…The cells were adjusted to 4 x 10 6 cells/ml and MCC peptide was added to a final concentration of 3 µM. T cells were transduced with MMLV-derived retroviruses for the expression of signaling sensors, commonly signaling intermediates fused with GFP as described in detail (Ambler et al, 2017;Roybal et al, 2015;Singleton et al, 2009). All animals were maintained in pathogen-free animal facilities at the University of Bristol under a University mouse breeding Home Office License.…”

Section: Mice and Cellsmentioning

confidence: 99%

“…Our imaging and image analysis protocols have recently been described in great detail in a dedicated publication (Ambler et al, 2017). Briefly, time-lapse fluorescence microscopy was performed with retrovirally transduced T cells, FACS-sorted to the lowest detectable sensor expression of 2 µM, and CH27 cells loaded with 10µM MCC.…”

Section: Time-lapsed Imaging Of T Cell:apc Interactionsmentioning

confidence: 99%

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Transient protein accumulation at the center of the T cell antigen presenting cell interface drives efficient IL-2 secretion

Clark

McMillan

Massoué

et al. 2018

Preprint

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Supramolecular signaling assemblies are of interest for their unique signaling properties. A µm scale signaling assembly, the central supramolecular signaling cluster (cSMAC), forms at the center of the interface of T cells activated by antigen presenting cells. We have determined that it is composed of multiple complexes of a supramolecular volume of up to 0.5µm 3 and associated with extensive membrane undulations. To determine cSMAC function, we have systematically manipulated the localization of three adaptor proteins, LAT, SLP-76, and Grb2. cSMAC localization varied between the adaptors and was diminished upon blockade of the costimulatory receptor CD28 and deficiency of the signal amplifying kinase Itk. Reconstitution of cSMAC localization restored IL-2 secretion which is a key T cell effector function as dependent on reconstitution dynamics. Our data suggest that the cSMAC enhances early signaling by facilitating signaling interactions and attenuates signaling thereafter through sequestration of a more limited set of signaling intermediates.

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Section: Results µM Scale Lat Accumulation At the Center Of The T Celmentioning

confidence: 99%

Section: Fusion Of Lat With Protein Domains With Pronounced Interfacementioning

confidence: 99%

Section: Mice and Cellsmentioning

confidence: 99%

Section: Time-lapsed Imaging Of T Cell:apc Interactionsmentioning

confidence: 99%

See 2 more Smart Citations

Transient protein accumulation at the center of the T cell antigen presenting cell interface drives efficient IL-2 secretion

Clark

McMillan

Massoué

et al. 2018

Preprint

Self Cite

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show abstract

“…Procedures used for the isolation and stimulation of TCR transgenic T cells have been extensively detailed elsewhere (22, 29, 57). Briefly, red blood cells were removed from dissociated spleens using ACK lysis buffer (Gibco) and splenocytes were plated at a density of 5×10 6 cells per well, in 1 mL of complete medium, in a 24-well plate.…”

Section: Methodsmentioning

confidence: 99%

PD-1 suppresses the maintenance of cell couples between cytotoxic T cells and tumor target cells within the tumor

Ambler

Edmunds

Toti

et al. 2018

Preprint

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CD8+ T cell killing of tumor cells is suppressed by the tumor microenvironment. Inhibitory receptors, prominently PD-1, are key mediators of this suppression. To discover cellular defects triggered by tumor exposure and associated PD-1 signaling, we have established an ex vivo imaging approach to investigate CD8+ tumor infiltrating lymphocytes (TILs) interacting with tumor targets. Whilst TIL:tumor cell couples formed effectively, couple stability deteriorated within 1-2 minutes. This was associated with excessive cofilin recruitment to the cellular interface, coincident deterioration of f-actin structures, increased TIL locomotion, and impaired tumor cell killing. Diminished engagement of PD-1 within the tumor, but not acute ex vivo blockade, partially restored cell couple maintenance and killing. PD-1 thus suppresses TIL function by inducing a polarization-impaired state.

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F-Actin-Driven CD28-CD80 Localization in the Immune Synapse

et al. 2018

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During immunological synapse (IS) formation, T cell receptor (TCR) signaling complexes, integrins, and costimulatory molecules exhibit a particular spatial localization. Here, we develop an agent-based model for the IS formation based on TCR peptide-bound major histocompatibility complex (pMHC) and leukocyte-function-associated antigen 1 (LFA-1) intracellular activation molecule 1 (ICAM-1) dynamics, including CD28 binding to a costimulatory ligand, coupling of molecules to the centripetal actin flow, and size-based segregation (SBS). A radial gradient of LFA-1 in the peripheral supramolecular activation cluster (pSMAC) toward the central supramolecular activation cluster (cSMAC) emerged as a combined consequence of actin binding and diffusion and modified the positioning of other molecules. The simulations predict a mechanism of CD28 movement, according to which CD28-CD80 complexes passively follow TCR-pMHC microclusters. However, the characteristic CD28-CD80 localization in a ring pattern around the cSMAC only emerges with a particular CD28-actin coupling strength that induces a centripetal motion. These results have implications for the understanding of T cell activation and fate decisions.

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Systems Imaging of the Immune Synapse

Cited by 10 publications

References 21 publications

Transient protein accumulation at the center of the T cell antigen presenting cell interface drives efficient IL-2 secretion

Transient protein accumulation at the center of the T cell antigen presenting cell interface drives efficient IL-2 secretion

PD-1 suppresses the maintenance of cell couples between cytotoxic T cells and tumor target cells within the tumor

F-Actin-Driven CD28-CD80 Localization in the Immune Synapse

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