Supramolecular attack particles are autonomous killing entities released from cytotoxic T cells

Abstract

Cytotoxic T lymphocytes (CTLs) kill infected and cancerous cells. We detected transfer of cytotoxic multiprotein complexes, called supramolecular attack particles (SMAPs), from CTLs to target cells. SMAPs were rapidly released from CTLs and were autonomously cytotoxic. Mass spectrometry, immunochemical analysis, and CRISPR editing identified a carboxyl-terminal fragment of thrombospondin-1 as an unexpected SMAP component that contributed to target killing. Direct stochastic optical reconstruction microscopy resolved a cytotoxic core surrounded by a thrombospondin-1 shell of ~120 nanometer diameter. Cryo-soft x-ray tomography analysis revealed that SMAPs had a carbon-dense shell and were stored in multicore granules. We propose that SMAPs are autonomous extracellular killing entities that deliver cytotoxic cargo targeted by the specificity of shell components.

Fig. 1. SMAPs were released at the IS and displayed autonomous cytotoxicity.

(A) Time-lapse confocal images depicting the transfer of Gzmb-mCherry⁺ (green) and WGA (magenta) labeled SMAPs from an antigen-specific CTL clone into pp65-pulsed JY target cells (Target). Arrows and inset indicate the presence of SMAPs inside the target. Scale bar, 10 μm. Quantification of Gzmb mean fluorescence intensity (MFI) and number of double-positive particles inside the target cell in CTL conjugates with unpulsed or pulsed target cells. Each dot represents one target cell (< 50 cells). Horizontal lines and error bars represent mean ± SD from 2 independent experiments. ****, p < 0.0001 (B) Live cell imaging of SMAPs release by CD8⁺ T-cells transfected with Gzmb-mCherry-SEpHluorin (magenta/green) on activating SLB. IRM, interference reflection microscopy. Scale bar, 5 μm. (C) Schematic of the working model for capturing SMAPs released by activated CD8⁺ T-cells. CD8⁺ T-cells (grey) were incubated on SLB presenting activating ligands for the indicated time. Cells were removed with cold PBS leaving the released SMAPs (purple) on the SLB. Elements are not drawn to scale. (D) TIRFM images of CD8⁺ T-cells incubated on activating SLB in the presence of anti-Prf1 (green) and anti-Gzmb (magenta) antibodies (top panels). After cell removal, Prf1⁺ and Gzmb⁺ SMAPs remained on the SLB (bottom panels). The formation of a mature IS is indicated by an ICAM-1 ring (blue). IRM, interference reflection microscopy. Scale bar, 5 μm. (E) Target cell cytotoxicity induced by density-dependent release of SMAPs captured on SLB measured by LDH release assay. Data points and error bars represent mean ± SEM from 3 independent experiments.

Fig. 2. TSP-1 was a major constituent of SMAPs and contributed to CTL killing of targets.

(A) Two-set Venn diagram showing the number of individual and common proteins identified by MS analysis of material released by CD8⁺ T-cells incubated on non-activating (ICAM-1) or activating (ICAM-1 + anti-CD3ε) SLB. Representative of 3 independent experiments with 8 donors. (B) Normalized abundance of the 285 proteins identified by MS in each condition. Cytotoxic proteins are highlighted in red, chemokine/cytokines in blue and adhesion proteins in green. (C) TIRFM images of SMAPs released from CD8⁺ T-cells transfected with TSP-1-GFPSpark (green; top row) or non-transfected cells (bottom row). Released SMAPs were further stained with anti-Gzmb (yellow) and anti-Prf1 (magenta) antibodies. IRM, interference reflection microscopy. BF, bright field microscopy. Scale bar, 5 μm. (D) Percentage of galectin-1 and TSP-1 knockout in CD8⁺ T-cells by CRISPR/Cas9 genome editing measured from immuno-blotting analysis (left). Each colored dot represents one donor. Bars represent mean ± SEM. Representative immuno-blot for galectin-1 (Lgals1) and TSP-1 in Lgals1 and TSP-1, respectively edited CD8⁺ T-cells (right). CD8⁺ T-cells (Blast) were analyzed in parallel as a control. (E) Target cell cytotoxicity mediated by galectin-1 (Lgals1-CRISPR) or TSP-1 (TSP-1-CRISPR) gene edited CD8⁺ T-cells measured by LDH release assay. T cell blasts were used as a control. Bars represent mean ± SEM. **, p < 0.01. Donors are the same as in (D).

Fig. 3. SMAPs shell was rich in glycoproteins, TSP-1 and organic material.

(A) dSTORM image of SMAPs released on activating SLB by multiple cells (left; scale bar, 2 μm) and two examples of individual SMAPs (top right; scale bar, 200 nm), showing their heterogeneity in size. SMAPs were labeled with WGA. Quantification of SMAPs size and number released per cell (bottom right; n>1800 and n=67, respectively). Horizontal lines and error bars represent mean ± SD from five donors. (B) dSTORM images of SMAPs (labeled with WGA, magenta) positive for TSP-1 (green) released on activating SLB. Scale bar, 1 μm. (C) Multiple CSXT examples of released SMAPs after cell removal. Scale bar, 500 nm. (D) CSXT of CD8⁺ T-cells interacting with carbon coated EM grids (note grid holes in C and D) containing ICAM-1 and anti-CD3ε. Scale bar, 2 μm or 500 nm for zoomed in regions (right). Arrows indicate SMAPs.

Fig. 4. SMAPs had a TSP-1 shell and a core of cytotoxic proteins.

(A and B) dSTORM images of individual SMAPs positive for Prf1 (green), Gzmb (magenta) and TSP-1 (A, orange) or stained with WGA (B, orange). Scale bar, 200 nm. (C) Quantification of the size of cytotoxic particles based on their protein composition (n=64 for Prf1^- and Gzmb^- cytotoxic particles, n=149 and n=83 for Prf1⁺ and Gzmb⁺ cytotoxic particles, respectively). ****, p < 0.0001. n.s, not significant. (D) Quantification of the percentage of particles positive and negative for Prf1 or Gzmb. (C-D) Horizontal lines/bars and error bars represent mean ± SD from five donors.