Distinct structural and catalytic roles for Zap70 in formation of the immunological synapse in CTL
- University of Cambridge, United Kingdom
- University of California, San Francisco, United States
- Howard Hughes Medical Institue, University of California, San Francisco, United States
- National Institutes of Health, United States
Figures
Figure 1
T cell killing and cytokine production is dependent on the catalytic activity of Zap70.
(A) Target cell lysis of P815 targets by Zap70(AS) CTL in the presence (squares) or absence (circles) of 10 µM 3-MB-PP1. Graphs show the mean percentage cytotoxicity of triplicates ± SD for effector to target (E:T) ratios shown; representative of three independent experiments. (B) FACS analysis of intracellular staining for IFN-γ (y-axes) TNF-α and IL2 (x-axes) production by Zap70(AS) CTL stimulated with anti-CD3 ±10 µM 3-MB-PP1.
Figure 2
Inhibition of Zap70 activity impairs formation of both the cSMAC and pSMAC.
(A–C) Confocal projections of Zap70(AS) CTL conjugated to P815 targets ±10 µM 3-MB-PP1. Cells are labelled with Hoechst (nuclei, blue) and antibodies against γ-tubulin (AlexaFluor 546; red) and either talin (A), Lck (B) or PKC-θ (C) (AlexaFluor-488; green) in the xy plane (scale bar, 5 µm) or as 1 µm reconstructions en face across the synapse in the xz plane (insets, scale bar, 3 µm). Graphs show the quantitation of conjugates with the percentages of conjugates displaying talin rings (A), Lck cSMACs (B) and PKC-θ cSMACs (C) at the synapse.
Figure 3
Actin clearance from synapses formed by CTL requires Zap70 activity.
(A) Confocal projections of Zap70(AS) CTL conjugated to P815 targets showing actin organisation at the synapse ±10 µM 3-MB-PP1, in the xy plane (scale bar, 5 µm) or as 1 µm reconstructions en face across the synapse in the xz plane (insets, scale bar, 3 µm). Labelling with Hoechst (blue) and antibodies against actin (AlexaFluor-546; red) and Lck (AlexaFluor-488; green). (B) Quantitation of actin organisation at the synapse in conjugates in the presence (n = 83) or absence (n = 64) of 10 µM 3-MB-PP1 and (C) for conjugates in the presence of 10 µM 3-MB-PP1 (n = 83), in which a cSMAC, identified by Lck clustering, is present (cSMAC) or absent (no cSMAC).
Figure 4 with 1 supplement
TCR signalling downstream of Zap70 is impaired in the absence of Zap70 catalytic activity.
(A) In vitro generated Zap70+/− and Zap70(AS) CTLs were left unstimulated or were stimulated for 2 min by soluble anti-CD3 (10 μg/ml) and cross-linking secondary antibodies, in the presence of vehicle alone (DMSO) or 5 or 10 μM 3-MB-PP1. The phosphorylation status of the indicated TCR signalling molecules was determined by Western blot analysis. Molecular weights: ZAP-70, 70kD; LAT, 38kD; PLCγ 150kD; ERK 42-44kD; Akt, 60kD; Vav-1, 100kD. (B) Vav-1 immunoprecipitated from Zap70+/− OT-I and Zap70(AS) OT-I CTL treated with vehicle or 3-MB-PP1 as shown. Immunoprecipitates were probed for phosphorylation on tyrosine 160 or total tyrosine phosphorylation of Vav-1.
Figure 4—figure supplement 1
ICAM adhesion assays.
Zap70+/− and Zap-70(AS) CTL were stimulated with soluble anti-CD3 in the presence of 10 μM 3-MB-PP1 or DMSO alone. The graphs show the percentage of cells adherent to ICAM-1 coated plates after 10 min of stimulation. Data shown are the mean ± SEM from triplicate samples from one of three independent experiments.
Figure 5
Centrosome and granule polarisation to the synapse is impaired in Zap70 inactive CTL.
(A and C) Confocal projections of Zap70(AS) CTL conjugated to P815 targets, labelled with Hoechst (blue) and antibodies against Lck (AlexaFluor-488; green), γ-tubulin (AlexaFluor-546; red) and (C) LAMP-1 (AlexaFluor-633; white) (scale bars, 5 µm) illustrating centrosome (A) and granule (C) polarisation in CTL. Quantitation of conjugates in the presence (n = 325) or absence (n = 163) of 10 µM 3-MB-PP1, showing distance of centrosome from the synapse (B) or granule polarisation phenotypes (D), illustrated in (A) and (C), as a percentage of total conjugates formed. (NB LAMP-1 stains both CTL and target lysosomes.)
Figure 6 with 1 supplement
CTL lacking Zap70 catalytic activity show abortive centrosome polarisation.
Single frames from Videos 3 and 4 showing 3 min intervals of Zap70(AS) OT-I CTL ±10 µM 3-MB-PP1. CTL were transfected with Lifeact-EGFP (green) and mPACT-RFP (centrosome marker, red), and target cells expressing farnesylated mTagBFP2 (blue). Scale bar, 5 µm; n = 50 for inhibitor and 34 for control treatments.
Figure 6—figure supplement 1
Lysis of targets by Zap70(AS) OT-I CTL is specifically inhibited by 3-MB-PP1.
Lysis of EL4 target cells by Zap70(AS) OT-I CTL ±10 µM 3-MB-PP1 with percentage target cell death (y-axis) and E:T ratio (x-axis).
Figure 7 with 1 supplement
The structure of the immunological synapse is severely impaired in CTL upon Zap70 inhibition.
Electron micrographs of single (A–F, left panel G) or non-sequential serial (right panels, G) thin (50–70 nm, A–E, G) or semi-thin (70–100 nm, F) lead-stained sections through the contact site formed between Zap70(AS) CTL (CTL) and P815 target cells (target), conjugated for 25 (A and B), 40 (C and D) or 60 (E–G) min at 37C in the absence (A, C, E, G) or presence (B, D, F) of 10 µM 3-MB-PP1. Secretory cleft (SC); interdigitations, between CTL and targets (black arrowheads); centrioles (white arrowheads), lytic granules (asterisks); Golgi elements (G) and nuclei (N) in CTL, and endoplasmic reticular (ER) and mitochondria (m) in target cells, are indicated in lower power images for (A–F) and all images for (G). Scale bars: low power images, 2 μm; high power images bar, 1 μm. Only the ends of the mother centriole appendages are visible in (B).
Figure 7—figure supplement 1
EM quantitation.
Quantitation of images of Zap70(AS) CTL-target conjugates formed ±3-MB-PP1 at 25, 40, or 60 min after conjugation in EM images in which CTL showed both contact with a target and at least one centriole within the section; (n = 24 @ 25 min, 51@ 40 min and 22 @ 60min +10 µM 3-MB-PP1 ; n = 26 @ 25 min, 30 @ 40 min and 60 @ 60 min −10 µM 3-MB-PP1). Graphs show percentage conjugates with (A) a secretory cleft at the contact site in the absence (black bars) or presence (grey bars) of 10 µM 3-MB-PP1; (B) centriole distances from the synapse; (C) 0, 1–2, 3–4 or 5–6 membrane projections >1000 nm/cell across the contact site (y-axis).
Videos
Video 1
Live cell imaging of Zap70(AS) OT-I CTL transfected with Lifeact-EGFP (green) moving on a glass coverslip coated with 0.5 µg/ml ICAM−1 + 0.1% DMSO.
https://doi.org/10.7554/eLife.01310.011
Video 2
Live cell imaging of Zap70(AS) OT-I CTL transfected with Lifeact-EGFP (green) moving on a glass coverslip coated with 0.5 µg/ml ICAM−1 +10 µM 3-MB-PP1.
https://doi.org/10.7554/eLife.01310.012
Video 3
Live cell imaging of Zap70(AS) OT-I CTL transfected with Lifeact-EGFP (green) and mPACT-RFP (centrosome marker, red), with EL4 target cells expressing farnesylated mTagBFP2 (blue) +0.1% DMSO.
https://doi.org/10.7554/eLife.01310.013
Video 4
Live cell imaging of Zap70(AS) OT-I CTL transfected with Lifeact-EGFP (green) and mPACT-RFP (centrosome marker, red), with target cells labelled with EL4 target cells expressing farnesylated mTagBFP2 (blue) +10 µM 3-MB-PP1.
https://doi.org/10.7554/eLife.01310.014Download links
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Distinct structural and catalytic roles for Zap70 in formation of the immunological synapse in CTL
eLife 3:e01310.
https://doi.org/10.7554/eLife.01310
