Synaptic secretion from human natural killer cells is diverse and includes supramolecular attack particles

Abstract

Natural killer (NK) cells form immune synapses to ascertain the state of health of cells they encounter. If a target cell triggers NK cell cytotoxicity, lytic granules containing proteins including perforin and granzyme B, are secreted into the synaptic cleft inducing target cell death. Secretion of these proteins also occurs from activated cytotoxic T lymphocytes (CTLs) where they have recently been reported to complex with thrombospondin-1 (TSP-1) in specialized structures termed supramolecular attack particles (SMAPs). Here, using an imaging method to define the position of each NK cell after removal, secretions from individual cells were assessed. NK cell synaptic secretion, triggered by ligation of NKp30 or NKG2D, included vesicles and SMAPs which contained TSP-1, perforin, and granzyme B. Individual NK cells secreted SMAPs, CD63+ vesicles, or both. A similar number of SMAPs were secreted per cell for both NK cells and CTLs, but NK cell SMAPs were larger. These data establish an unexpected diversity in NK cell synaptic secretions.

Keywords: immune synapse; natural killer cells; supramolecular attack particles.

Fig. 1.

NK secretions at the immune synapse are heterogeneous. (A–E) NK cells incubated on slides coated with ICAM-1, ICAM-1 + MICA, or ICAM-1 + αNKp30. (A) IFNγ secreted from NK cells [n = 6; Kruskal–Wallis test]. (B) F-actin ring formation on coated slides [n = 5; one-way ANOVA]. (C) TIRF imaging of perforin and NK cell shadows following cell detachment. (D) Percentage of cells secreting perforin [n = 5; one-way ANOVA]. (E) Intensity of secreted perforin/cell [n = 3; Kruskal–Wallis test]. (F) Intensity of secreted perforin against IFNγ secretion from NK cells incubated on ICAM-1 plus indicated ligands [n = 2–5; mean ± SEM]. (G–L) NK cells activated with αNKp30. (G) TIRF imaging of perforin and CD63 secretions following NK cell detachment. Bottom shows expanded views of boxed regions in the composite image. (H) Percentage of cells secreting perforin, CD63, or both. (I) Intensity of secreted perforin and CD63 with and without 100 nM cambinol [n = 2; ≥20 cells/donor; Mann–Whitney U test]. (J) TIRF images of secretions stained for perforin, WGA, and DiD as indicated. (K) Intensity of secreted perforin, WGA, and DiD (n = 2; ≥20 cells/donor; Mann–Whitney U test). (L) Pearson’s correlation coefficient of WGA compared to perforin in secretions (n = 3). Cells from individual donors are color coded. Mean ± SD unless stated. * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001, and **** = P ≤ 0.0001.

Fig. 2.

NK cells secrete SMAPs upon activation. (A) TIRF microscopy of secretions from αNKp30-activated and detached NK cells. (B) Intensity of secreted TSP-1, serglycin, and galectin-1. (C) Pearson’s correlation coefficient for TSP-1, serglycin, and galectin-1 compared to perforin (B and C; n = 3; one-way ANOVA). (D–H) STORM imaging and quantification of perforin and TSP-1 secretions from MICA-activated NK cells. (D) Representative STORM images. (E) Number of SMAPs secreted/cell. (F) Diameter of SMAPs. (G) Percentage of SMAPs that contain a detectable level of perforin. (H) Coordinate-based colocalization comparing perforin to TSP-1. (E–H; n = 3; ≥5 cells/donor; Kruskal–Wallis test). (I) STORM imaging of perforin and granzyme B in secretions from MICA-activated and detached NK cells. (J) STORM images of SMAPs secreted from MICA-activated NK cells and αCD3-activated CTLs. (K) Number of SMAPs secreted/cell. (L) Diameter of SMAPs. Cells from individual donors are color coded. Mean ± SD ** = P ≤ 0.01 and **** = P ≤ 0.0001.