Remodeling of secretory lysosomes during education tunes functional potential in NK cells - PubMed (original) (raw)

doi: 10.1038/s41467-019-08384-x.

Benedikt Jacobs 1 2, Michelle L Saetersmoen 1 2, Dennis Clement 1 2, Quirin Hammer 3, Trevor Clancy 1 2, Ellen Skarpen 4, Andreas Brech 4, Johannes Landskron 1 5, Christian Grimm 6, Aline Pfefferle 3, Leonardo Meza-Zepeda 7 8, Susanne Lorenz 8, Merete Thune Wiiger 1 2, William E Louch 9, Eivind Heggernes Ask 1 2, Lisa L Liu 3, Vincent Yi Sheng Oei 1 2, Una Kjällquist 10, Sten Linnarsson 10, Sandip Patel 11, Kjetil Taskén 1 2 5, Harald Stenmark 4, Karl-Johan Malmberg 12 13 14

Affiliations

Remodeling of secretory lysosomes during education tunes functional potential in NK cells

Jodie P Goodridge et al. Nat Commun. 2019.

Abstract

Inhibitory signaling during natural killer (NK) cell education translates into increased responsiveness to activation; however, the intracellular mechanism for functional tuning by inhibitory receptors remains unclear. Secretory lysosomes are part of the acidic lysosomal compartment that mediates intracellular signalling in several cell types. Here we show that educated NK cells expressing self-MHC specific inhibitory killer cell immunoglobulin-like receptors (KIR) accumulate granzyme B in dense-core secretory lysosomes that converge close to the centrosome. This discrete morphological phenotype is independent of transcriptional programs that regulate effector function, metabolism and lysosomal biogenesis. Meanwhile, interference of signaling from acidic Ca2+ stores in primary NK cells reduces target-specific Ca2+-flux, degranulation and cytokine production. Furthermore, inhibition of PI(3,5)P2 synthesis, or genetic silencing of the PI(3,5)P2-regulated lysosomal Ca2+-channel TRPML1, leads to increased granzyme B and enhanced functional potential, thereby mimicking the educated state. These results indicate an intrinsic role for lysosomal remodeling in NK cell education.

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Conflict of interest statement

K.J. Malmberg is a scientific advisor and consultant at Fate Therapeutics. J.P. Goodridge is currently employed as a Scientist at Fate Therapeutics (started after completion of this work). Malmberg and Goodridge are co-inventors on a patent application concerning the use of lysosomal modulation to tune NK and T cell function. The other authors declare no competing interests.

Figures

Fig. 1

Fig. 1

NK cell education is associated with accumulation of granzyme B. a CD107a expression in response to K562 cells by the indicated KIR subset of resting mature CD56dim NKG2A− NKG2C−CD57− NK cells from C1/C1 (n = 38 donors), C1/C2 (n = 31 donors), and C2/C2 (n = 19 donors). b Expression of granzyme B in 2DL3 and 2DL1 single-positive NKG2A−CD57− NK cells from C1/C1 (n = 21 donors), C1/C2 (n = 26 donors), and C2/C2 (n = 12 donors). Donors with less than 100 events in the final KIR gate were excluded. c Expression of granzyme B in 3DL1+/− NKG2A−CD57− NK cells from Bw4+ (n = 20 donors) and Bw4− (n = 7 donors). d Expression of granzyme B in 2DL2/2DS2 or 2DL3-single-positive NKG2A− NKG2C−CD57− NK cells in C1/C1 (n = 9 donors), C1/C2 (n = 8 donors), and C2/C2 (n = 9 donors). e Expression of granzyme B in the indicated NKG2A− NKG2C−CD57− NK cell subset in C2/C2 (n = 7 donors). f Expression of granzyme B in KIR−CD57−NKG2A+/− CD57− NK cells (n = 63 donors). g Expression of granzyme B in YTS and NKL cells transfected with 2DL3 or 2DL1. The graph shows data from one representative experiment of two. Paired _t_-tests were performed in (a–c, f–g). One-way ANOVA tests followed by Tukey's multiple comparison tests were performed in (e, f). Whiskers show 5th to 95th percentile. Bars show the median. ****p < 0.0001; ***p < 0.001; and *p < 0.05. Red and blue circles and box plots represent NK cells with self or non-self KIR, respectively

Fig. 2

Fig. 2

Granzyme B accumulation is independent of transcription. a Global RNA-Seq of sorted self-KIR+ and non-self-KIR+ NKG2A− NKG2C−CD57− NK cells. The figure depicts one representative C1/C1 donor out of three independent donors. b Quantitative PCR of granzyme B mRNA in sorted NK cells at different stages of differentiation (left, 4 pooled samples) and in NKG2A−NKG2C−CD57− NK cells expressing a self- or non-self KIR (right, n = 5 paired samples). c Expression of granzyme B in the indicated NK cell subsets following stimulation with IL-15 or IL-21 for the indicated length of time (summary of two independent experiments using two different donors). d Expression of granzyme B after 24 h of stimulation with IL-21 or IL-15 in CD56bright NK cells and NKG2A− NKG2C−CD57− single-positive CD56dim NK cells expressing a self- or non-self KIR NK cell subset in the presence or absence of the STAT-5 inhibitor Pimozide (top) (7 μM) and the mTOR inhibitor Torin-1 (100 nM) (bottom). The data in panel (d) are the summary of at least two independent experiments. Whiskers show 5th to 95th percentile. Bars show the median. Wilcoxon paired, non-parametric tests. ****p < 0.0001 and **p < 0.01. Red and blue circles, connecting lines and box plots represent NK cells with self or non-self KIR, respectively

Fig. 3

Fig. 3

Modulation of the lysosomal compartment in educated NK cells. a Confocal microscopy Z-stack showing Pericentrin (PCNT), LAMP-1, and granzyme B (GZMB) staining in sorted mature CD56dim NKG2A−NKG2C−CD57− NK cells expressing non-self or self KIR. Scale bar is 2 μm. b The pixel sum of granzyme B staining in cells expressing non-self or self KIR. c The number of LAMP-1+ lysosomal structures. Data in panels (b) and (c) are aggregated from sorted 2DL1 and 2DL3 single-positive NK cell subsets from C1C1 (n = 5 donors) and C2C2 (n = 5 donors) donors. d Granzyme B expression levels versus the distance from the centrosome in individual lysosomes in sorted NKG2A−NKG2C−CD57− NK cells expressing non-self or self KIR (n = 804 lysosomes from 3 donors were analyzed). Gates were set based on visual inspection to quantify the percentage of granzyme-B dense secretory lysosomes in self and non-self NK cells. e Representative immuno-EM section showing staining with gold-particle coated anti-granzyme B (top) and Chondroitin Sulphate-4 (CS-4) (bottom) of sorted CD56dim NKG2A−NKG2C−CD57− NK cells expressing non-self or self KIR. Scale bar is 200 nm. f Number of gold particles (granzyme B and CS-4) per cell. (non-self n = 83 cells, self n = 109 cells). g Particle count (granzyme B and CS-4) as a function of the lysosomal area. h Density of gold particles (granzyme B and CS-4) per lysosomal area (μm2). Immuno-EM data are from 5 donors and 5 experiments. Paired _t_-tests were performed in panels (b, c, e, and g). ****p < 0.0001 and *p < 0.05. Red and blue circles and box plots represent NK cells with self or non-self KIR, respectively

Fig. 4

Fig. 4

Educated NK cells mobilize dense-core secretory lysosomes. a Representative example of granzyme B and CD107a expression in self KIR+ and non-self KIR+ CD56dim NKG2A−NKG2C−CD57−NK cells following stimulation with K562 cells. b Aggregated data of percent CD107ahigh NK cells following stimulation with K562 cells (n = 5 donors). c Expression of granzyme B in the indicated NK cell subset after stimulation with K562. Summary of data from 4 C1/C1 donors. d Representative images of immuno-EM sections of resting (left) or sorted degranulated CD107ahigh NK cells (right). Scale bar: 200 nm. e Secretory lysosome size and granzyme B content as determined by immuno-EM in resting and sorted CD107ahigh NK cells after stimulation with K562. A Wilcoxon test was performed in panel (b). A non-parametric Friedman test was performed in panel (c). Whiskers show 5th to 95th percentile. Bars show the median. *p < 0.05. Red and blue box plots represent NK cells with self or non-self KIR, respectively

Fig. 5

Fig. 5

Compromising lysosomal activity decreases NK cell function. a Global Ca2+-flux in resting bulk NK cells measured by Fluo-4 _F_1/_F_0 ratio following stimulation with biotinylated anti-DNAM-1/anti-2B4 (top) or biotinylated anti-CD16 (bottom) crosslinked at the indicated time-point with streptavidin in the presence (added at the onset of stimulation and maintained throughout the assay) or absence of 50 μM GPN. b Representative example of CD107a expression following stimulation of NK cells with K562 cells in the presence or absence of GPN. c Frequency of CD107high+ and d IFNγ+ self-KIR+ and non-self-KIR+ NK cells following stimulation with K562 cells in the presence or absence of 50 μM GPN or 10 μM Mefloquine (MEF). Friedman’s tests were performed followed by Dunn’s multicomparison tests. Whiskers show 5th to 95th percentile. Bars show the median. ****p < 0.0001; ***p < 0.001; **p < 0.01; and *p < 0.05. Red and blue box plots represent NK cells with self or non-self KIR, respectively

Fig. 6

Fig. 6

Enlarging the secretory lysosomes leads to enhanced NK cell function. a Confocal Z-stack showing MHC-I, LAMP-1, and granzyme B (GZMB) staining in primary NK cells following PIKfyve inhibition using overnight incubation with 1 μM vacuolin-1, 1 μM apilimod, or 1 μM YM201636. Scale bar is 5 μm. b Intracellular granzyme B expression in self-KIR+ and non-self-KIR+ NK cells following overnight incubation with the indicated PIKfyve inhibitor assessed by flow cytometry (n = 5 independent donors). c Representative example of a confocal image of primary NK cells treated overnight with 1 μM vacuolin-1 or DMSO. Scale bar is 2 μm. d Compiled confocal data on the volume of LAMP-1+ structures from cells treated overnight with DMSO or vacuolin-1 (n = 149 LAMP-1+ structures). e Cytosolic Ca2+-flux in NK cells in response to stimulation with biotinylated anti-DNAM-/2B4 (top) or anti-CD16 (bottom) crosslinked with streptavidin at the indicated timepoint. Cells were treated with 10 μM vacuolin-1 added directly before the assay and then maintained throughout the incubation time. f Representative FACS histogram of granzyme B versus CD107a expression following stimulation with K562 cells in the presence of DMSO or 10 μM vacuolin-1. g Frequency of CD107ahigh+ and h IFNγ+ self-KIR+ and non-self-KIR+ NK cells after stimulation with K562 in the presence of DMSO or 10 μM vacuolin-1. i FACS-based killing assay showing NK cell killing of K562 cells after treatment with DMSO or 10 μM vacuolin-1 (n = 6 donors). j Relative phosphorylation of the indicated signaling molecules following stimulation with biotinylated anti-CD16 (10 μg/mL) crosslinked with streptavidin in the presence of 50 μM GPN or 10 μM vacuolin-1. Friedman’s test was used in panel (b). A non-paired _t_-test was used in panel (d). Paired _t_-test was used in panels (g–i). Whiskers show 5th to 95th percentile. Bars show the median. ****p < 0.0001; **p < 0.01; and *p < 0.05. Red and blue circles and box plots represent NK cells with self or non-self KIR, respectively. In panels (i) and (j), red and blue colors indicate cells treated with the indicated compounds

Fig. 7

Fig. 7

TRPML1-mediated modulation of secretory lysosomes. a mRNA expression (RNA-Seq) of TRPML1 in the indicated NK cell subsets sorted from PBMC and analyzed directly. b Granzyme B expression in NK cells treated for 2 h with 10 μM of the TRPML1 agonist MK6-83. (Summary of n = 23 donors.) c Degranulation (left) and IFNγ responses (right) by resting primary NK cells following stimulation with K562 cells for 4 h in the presence or absence of 10 μM MK6-83. Data are the summary from two independent experiments with 7 donors. d Relative mRNA expression (qPCR) of TRPML1 72 h after siRNA silencing in NK cells cultured for 3 days in 1 ng/mL IL-15. e Granzyme B expression in NK cells 72 h after siRNA silencing of TRPML1. f Confocal microscopy image showing LAMP-1 and granzyme B (GZMB) staining in siRNA TRPML1 silenced NK cells. Scale bar is 2 μm. g Summary of integrated granzyme B intensity per cell as quantified with ImageJ (n = 3 experiments). AU arbitrary units. h Representative example of FACS plot showing granzyme B expression versus CD107a in siRNA TRPML1 silenced NK cells after 4-h stimulation with K562 cells. Compiled data on i CD107a and j IFNγ production in TRPML1-silenced primary NK cells. Data are from nine donors with confirmed siRNA silencing. Paired _t_-tests were performed in panels (b, c, e, i, and j). Non-paired _t_-test was performed in panel (g). Whiskers show 5th to 95th percentile. Bars show the median. ****p < 0.0001; **p < 0.01; and *p < 0.05. Red and blue box plots represent NK cells treated with the indicated compounds or siRNA

Fig. 8

Fig. 8

Model describing the distinct fates of NK cells during NK cell education. NK cells lacking self-specific receptors receive tonic stimulatory input through activating receptors and show poor functional responses, a process referred to as disarming. We found that such cells exhibit lower levels of the granule matrix protein serglycin and effector molecules granzyme B and perforin and lack dense-core secretory lysosomes. One putative pathway downstream of activation receptor signaling is PI3K/AKT that stimulate the enzyme PIKfyve, which converts PI3P to PI(3,5)P2 and thereby positively regulate the lysosome-specific Ca2+ channel TRPML1. PIKfyve and TRPML1 are critically involved in lysosomal modulation in several cell types, . Inhibitory KIRs interfere with activation signals at a proximal level and thereby shut down any signals that could drive such lysosomal modulation. In support of this notion, we found that pharmacological interference with PIKfyve or silencing of TRPML1 replicated the educated state with enlarged lysosomes, increased granzyme B loads and more potent effector function. The secretory lysosome is part of the acidic Ca2+ stores and may thus potentiate receptor-mediated Ca2+ release from the ER–. Interference with signaling from the acidic Ca2+ stores resulted in the loss of NK cell function. Thus, the accumulation of dense-core secretory lysosomes during NK cell education may contribute to the increased function, not only through the increased cytotoxic payload, but also through enhanced signaling from acidic Ca2+ stores

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References

    1. Karre K, Ljunggren HG, Piontek G, Kiessling R. Selective rejection of H-2-deficient lymphoma variants suggests alternative immune defence strategy. Nature. 1986;319:675–678. doi: 10.1038/319675a0. - DOI - PubMed
    1. Anfossi N, et al. Human NK cell education by inhibitory receptors for MHC class I. Immunity. 2006;25:331–342. doi: 10.1016/j.immuni.2006.06.013. - DOI - PubMed
    1. Elliott JM, Yokoyama WM. Unifying concepts of MHC-dependent natural killer cell education. Trends Immunol. 2011;32:364–372. doi: 10.1016/j.it.2011.06.001. - DOI - PMC - PubMed
    1. Kim S, et al. Licensing of natural killer cells by host major histocompatibility complex class I molecules. Nature. 2005;436:709–713. doi: 10.1038/nature03847. - DOI - PubMed
    1. Joncker NT, Shifrin N, Delebecque F, Raulet DH. Mature natural killer cells reset their responsiveness when exposed to an altered MHC environment. J. Exp. Med. 2010;207:2065–2072. doi: 10.1084/jem.20100570. - DOI - PMC - PubMed

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