Limits...
The immune synapse clears and excludes molecules above a size threshold.

Cartwright AN, Griggs J, Davis DM - Nat Commun (2014)

Bottom Line: Dextran sized ≤4 nm move in and out of the IS, but access is significantly reduced (by >50%) for dextran sized 10-13 nm, and dextran ≥32 nm is almost entirely excluded.Depolymerization of F-actin abrogated exclusion.Therefore, the IS can clear and exclude molecules above a size threshold, and drugs designed to target synaptic cytokines or cytotoxic proteins must fit these dimensions.

View Article: PubMed Central - PubMed

Affiliation: 1] Manchester Collaborative Centre for Inflammation Research, University of Manchester, 46 Grafton Street, Manchester M13 9NT, UK [2] Division of Cell and Molecular Biology, Imperial College London, London SW7 2AZ, UK.

ABSTRACT
Natural killer cells assess target cell health via interactions at the immune synapse (IS) that facilitates signal integration and directed secretion. Here we test whether the IS also functions as a gasket. Quantitative fluorescence microscopy of nanometer-scale dextrans within synapses formed by various effector-target cell conjugates reveal that molecules are excluded in a size-dependent manner at activating synapses. Dextran sized ≤4 nm move in and out of the IS, but access is significantly reduced (by >50%) for dextran sized 10-13 nm, and dextran ≥32 nm is almost entirely excluded. Depolymerization of F-actin abrogated exclusion. Unexpectedly, larger-sized dextrans are cleared as the IS assembles in a zipper-like manner. Monoclonal antibodies are also excluded from the IS but smaller single-domain antibodies are able to penetrate. Therefore, the IS can clear and exclude molecules above a size threshold, and drugs designed to target synaptic cytokines or cytotoxic proteins must fit these dimensions.

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Dextran is excluded in a size-dependent manner by activating but not inhibitory synapses.(a,b) Graphs show the mean relative fluorescence intensity of dextran of the sizes indicated for synapses formed by pNK cells co-incubated with (a) Daudi cells or (b) K562 cells. Error bars show mean±s.d. of all data points. n=40, 39, 35 and 35 conjugates for Daudi cells and 40, 38, 38 and 42 conjugates for K562 cells from three independent experiments. Data were analysed using a one-way analysis of variance (ANOVA) with Tukey corrections. ****P<0.0001. (c) Panels show bright-field (BF) images of YTS/KIR2DL1 cells in conjugate with 221/Cw6 target cells overlaid with nuclear stained target cell (blue;upper row) and the corresponding fluorescence image of dextrans (lower row), sizes as indicated. Scale bars, 10 μm. (d) Graphs show mean raw fluorescence intensities of dextran (at the size indicated) along a line drawn perpendicular to the synapse. (e) Graph shows the mean relative fluorescence intensities of dextran within synapses formed by YTS/KIR2DL1 and 221/Cw6 cells. Bars show the mean for all data points. n=42, 45, 41, 45, 35 and 35 from three independent experiments. Data were analysed using a one-way ANOVA with Tukey corrections. (f) Panels show BF images overlaid with nuclear stained target cell (upper row) and the corresponding fluorescence image of dextran (lower row), size as indicated, of Jurkat cells in conjugate with superantigen-loaded Raji target cells. Scale bars, 10 μm. (g) Graph shows the mean relative fluorescence intensities of each dextran within synapses formed by T cells and Raji target cells. Bars show mean from all data points. n=34, 36, 33 and 37 from three independent experiments. Data were analysed using a one-way ANOVA with Tukey corrections. *P<0.1, ****P<0.0001.
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f2: Dextran is excluded in a size-dependent manner by activating but not inhibitory synapses.(a,b) Graphs show the mean relative fluorescence intensity of dextran of the sizes indicated for synapses formed by pNK cells co-incubated with (a) Daudi cells or (b) K562 cells. Error bars show mean±s.d. of all data points. n=40, 39, 35 and 35 conjugates for Daudi cells and 40, 38, 38 and 42 conjugates for K562 cells from three independent experiments. Data were analysed using a one-way analysis of variance (ANOVA) with Tukey corrections. ****P<0.0001. (c) Panels show bright-field (BF) images of YTS/KIR2DL1 cells in conjugate with 221/Cw6 target cells overlaid with nuclear stained target cell (blue;upper row) and the corresponding fluorescence image of dextrans (lower row), sizes as indicated. Scale bars, 10 μm. (d) Graphs show mean raw fluorescence intensities of dextran (at the size indicated) along a line drawn perpendicular to the synapse. (e) Graph shows the mean relative fluorescence intensities of dextran within synapses formed by YTS/KIR2DL1 and 221/Cw6 cells. Bars show the mean for all data points. n=42, 45, 41, 45, 35 and 35 from three independent experiments. Data were analysed using a one-way ANOVA with Tukey corrections. (f) Panels show BF images overlaid with nuclear stained target cell (upper row) and the corresponding fluorescence image of dextran (lower row), size as indicated, of Jurkat cells in conjugate with superantigen-loaded Raji target cells. Scale bars, 10 μm. (g) Graph shows the mean relative fluorescence intensities of each dextran within synapses formed by T cells and Raji target cells. Bars show mean from all data points. n=34, 36, 33 and 37 from three independent experiments. Data were analysed using a one-way ANOVA with Tukey corrections. *P<0.1, ****P<0.0001.

Mentions: We next tested whether synapses formed between NK cells and different target cells similarly excluded extracellular molecules in a size-dependent manner. To test this, pNK cells were co-incubated with a B-cell lymphoblast cell line, Daudi, or a myeloid leukaemia cell line, K562, and differently sized fluorescein-labelled dextrans. As observed with 221 target cells, the IS formed between pNK cells and Daudi (Fig. 2a) or K562 (Fig. 2b) cells excluded the 32-nm (relative intensity 0.02±0.01 with both targets) and the 54-nm (0.02±0.01 and 0.01±0.01, respectively) dextrans, while the 4-nm (0.11±0.05 with both targets) and the 13-nm (0.06±0.04 with both targets) dextrans were able to access the synapse. The intensities of dextran within synapses were comparable to those measured when pNK cells were co-incubated with 221 cells (Fig. 1). These data establish that, regardless of the target cell, NK cells exclude extracellular molecules above a size threshold from the IS.


The immune synapse clears and excludes molecules above a size threshold.

Cartwright AN, Griggs J, Davis DM - Nat Commun (2014)

Dextran is excluded in a size-dependent manner by activating but not inhibitory synapses.(a,b) Graphs show the mean relative fluorescence intensity of dextran of the sizes indicated for synapses formed by pNK cells co-incubated with (a) Daudi cells or (b) K562 cells. Error bars show mean±s.d. of all data points. n=40, 39, 35 and 35 conjugates for Daudi cells and 40, 38, 38 and 42 conjugates for K562 cells from three independent experiments. Data were analysed using a one-way analysis of variance (ANOVA) with Tukey corrections. ****P<0.0001. (c) Panels show bright-field (BF) images of YTS/KIR2DL1 cells in conjugate with 221/Cw6 target cells overlaid with nuclear stained target cell (blue;upper row) and the corresponding fluorescence image of dextrans (lower row), sizes as indicated. Scale bars, 10 μm. (d) Graphs show mean raw fluorescence intensities of dextran (at the size indicated) along a line drawn perpendicular to the synapse. (e) Graph shows the mean relative fluorescence intensities of dextran within synapses formed by YTS/KIR2DL1 and 221/Cw6 cells. Bars show the mean for all data points. n=42, 45, 41, 45, 35 and 35 from three independent experiments. Data were analysed using a one-way ANOVA with Tukey corrections. (f) Panels show BF images overlaid with nuclear stained target cell (upper row) and the corresponding fluorescence image of dextran (lower row), size as indicated, of Jurkat cells in conjugate with superantigen-loaded Raji target cells. Scale bars, 10 μm. (g) Graph shows the mean relative fluorescence intensities of each dextran within synapses formed by T cells and Raji target cells. Bars show mean from all data points. n=34, 36, 33 and 37 from three independent experiments. Data were analysed using a one-way ANOVA with Tukey corrections. *P<0.1, ****P<0.0001.
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f2: Dextran is excluded in a size-dependent manner by activating but not inhibitory synapses.(a,b) Graphs show the mean relative fluorescence intensity of dextran of the sizes indicated for synapses formed by pNK cells co-incubated with (a) Daudi cells or (b) K562 cells. Error bars show mean±s.d. of all data points. n=40, 39, 35 and 35 conjugates for Daudi cells and 40, 38, 38 and 42 conjugates for K562 cells from three independent experiments. Data were analysed using a one-way analysis of variance (ANOVA) with Tukey corrections. ****P<0.0001. (c) Panels show bright-field (BF) images of YTS/KIR2DL1 cells in conjugate with 221/Cw6 target cells overlaid with nuclear stained target cell (blue;upper row) and the corresponding fluorescence image of dextrans (lower row), sizes as indicated. Scale bars, 10 μm. (d) Graphs show mean raw fluorescence intensities of dextran (at the size indicated) along a line drawn perpendicular to the synapse. (e) Graph shows the mean relative fluorescence intensities of dextran within synapses formed by YTS/KIR2DL1 and 221/Cw6 cells. Bars show the mean for all data points. n=42, 45, 41, 45, 35 and 35 from three independent experiments. Data were analysed using a one-way ANOVA with Tukey corrections. (f) Panels show BF images overlaid with nuclear stained target cell (upper row) and the corresponding fluorescence image of dextran (lower row), size as indicated, of Jurkat cells in conjugate with superantigen-loaded Raji target cells. Scale bars, 10 μm. (g) Graph shows the mean relative fluorescence intensities of each dextran within synapses formed by T cells and Raji target cells. Bars show mean from all data points. n=34, 36, 33 and 37 from three independent experiments. Data were analysed using a one-way ANOVA with Tukey corrections. *P<0.1, ****P<0.0001.
Mentions: We next tested whether synapses formed between NK cells and different target cells similarly excluded extracellular molecules in a size-dependent manner. To test this, pNK cells were co-incubated with a B-cell lymphoblast cell line, Daudi, or a myeloid leukaemia cell line, K562, and differently sized fluorescein-labelled dextrans. As observed with 221 target cells, the IS formed between pNK cells and Daudi (Fig. 2a) or K562 (Fig. 2b) cells excluded the 32-nm (relative intensity 0.02±0.01 with both targets) and the 54-nm (0.02±0.01 and 0.01±0.01, respectively) dextrans, while the 4-nm (0.11±0.05 with both targets) and the 13-nm (0.06±0.04 with both targets) dextrans were able to access the synapse. The intensities of dextran within synapses were comparable to those measured when pNK cells were co-incubated with 221 cells (Fig. 1). These data establish that, regardless of the target cell, NK cells exclude extracellular molecules above a size threshold from the IS.

Bottom Line: Dextran sized ≤4 nm move in and out of the IS, but access is significantly reduced (by >50%) for dextran sized 10-13 nm, and dextran ≥32 nm is almost entirely excluded.Depolymerization of F-actin abrogated exclusion.Therefore, the IS can clear and exclude molecules above a size threshold, and drugs designed to target synaptic cytokines or cytotoxic proteins must fit these dimensions.

View Article: PubMed Central - PubMed

Affiliation: 1] Manchester Collaborative Centre for Inflammation Research, University of Manchester, 46 Grafton Street, Manchester M13 9NT, UK [2] Division of Cell and Molecular Biology, Imperial College London, London SW7 2AZ, UK.

ABSTRACT
Natural killer cells assess target cell health via interactions at the immune synapse (IS) that facilitates signal integration and directed secretion. Here we test whether the IS also functions as a gasket. Quantitative fluorescence microscopy of nanometer-scale dextrans within synapses formed by various effector-target cell conjugates reveal that molecules are excluded in a size-dependent manner at activating synapses. Dextran sized ≤4 nm move in and out of the IS, but access is significantly reduced (by >50%) for dextran sized 10-13 nm, and dextran ≥32 nm is almost entirely excluded. Depolymerization of F-actin abrogated exclusion. Unexpectedly, larger-sized dextrans are cleared as the IS assembles in a zipper-like manner. Monoclonal antibodies are also excluded from the IS but smaller single-domain antibodies are able to penetrate. Therefore, the IS can clear and exclude molecules above a size threshold, and drugs designed to target synaptic cytokines or cytotoxic proteins must fit these dimensions.

Show MeSH