Modulation of Secretory Lysosomes During NK Cell Education Leads to Accumulation of Granzyme B and Enhanced Functional Potential

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. We found that educated NK cells expressing self-MHC specific inhibitory killer cell immunoglobulin-like receptors (KIR) show accumulation of granzyme B, localized in dense-core secretory lysosomes, converged close to the centrosome. This discrete morphological phenotype persists in self-KIR+ NK cells independently of transcriptional programs that regulate effector function, metabolism and lysosomal biogenesis. The granzymeB dense, large secretory lysosomes in self-KIR+ NK cells were efficiently released upon target cell recognition, contributing to their enhanced cytotoxic capacity. Secretory lysosomes are part of the acidic lysosomal compartment, which has been shown to channel calcium and mediate intracellular signalling in several cell types. Interference of signaling from acidic Ca2+ stores in primary NK cells reduced both 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 led to increased levels of granzyme B and enhanced functional potential. These results indicate an intrinsic role for lysosomal homeostasis in NK cell education.

both target-specific Ca 2+ -flux, degranulation and cytokine production. Furthermore, 1 5 inhibition of PI(3,5)P 2 synthesis or genetic silencing of the PI(3,5)P 2 -regulated 1 6 lysosomal Ca 2+ -channel TRPML1 led to increased levels of granzyme B and that defines the educated NK-cell state. Functional readouts used to distinguish self-1 specific NK cells from hyporesponsive NK cells do not provide information about the 2 prior events that culminate in the development of effector potential. Apart from 3 differences in the relative levels and distribution of NK cell receptors at the cell 4 membrane, 14, 15 transcriptional and phenotypic readouts at steady state provide scant 5 differences between self and non-self specific NK cells. 16,17 Whether inhibitory 6 signaling is converted into a paradoxical gain of function through an as yet unknown 7 mechanism (e.g. arming/stimulatory licensing), or whether expression of self-specific 8 inhibitory receptors protect the cell from tonic activation that would otherwise lead to 9 erosion of function over time (e.g. disarming/inhibitory licensing) remains to be 1 0 determined. 3,18,19 1 1 Here we show that expression of self-specific inhibitory receptors influences ligand, HLA-C2/C2 (Fig. 1c)., In order to control for the stage of differentiation, 1 which is known to influence expression of effector molecules, 22 these analyses were 2 performed in NK cells that were NKG2A negative and CD57 negative. Corroborating 3 the link between inhibitory input through self-KIR and granzyme B expression, 4 donors that were heterozygous for HLA-C1/C2 had similarly high levels of granzyme 5 B in both 2DL1 and 2DL3 single-positive NK cells. Granzyme B expression was also 6 higher in 3DL1 + NK cells from donors positive for its cognate ligand HLA-Bw4 ( Fig.   7 1d). NK cells with higher levels of 3DL1 surface expression, also known to have a 8 higher functional capacity, 23 exhibited greater expression of granzyme B 9 ( Supplementary Fig. 1a). It is well established that NKG2A/HLA-E interactions 1 0 contribute to education of NK cells. 24,25,26 In line with the results of single KIR + NK 1 1 cell subsets, NKG2A + KIR -CD57 -NK cells expressed slightly higher levels granzyme 1 2 B (Supplementary Fig. 1b Fig. 2). 27 These results were confirmed in a panel of 8 selected 1 6 qPCR targets comprising transcription factors and canonical cell surface markers 1 7 linked to NK cell differentiation (Fig 2b and Supplementary Fig. 3). Together, these 1 8 data demonstrated that the increased levels of granzyme B detected by flow cytometry 1 9 in self-KIR + NK cells occur independently of transcriptionally regulated programs, 2 0 including differences in tonic metabolic input to the cell.

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In mouse NK cells, expression of granzyme B is regulated by cytokine-induced 2 3 translation from a preexisting pool of mRNA transcript. 28 Therefore, we explored the 2 4 possibility that self and non-self specific NK cells may respond differentially to 2 5 cytokine priming in vivo, resulting in divergent steady-state levels of expressed 1 granzyme B. To address this possibility, NK cells exposed to IL-15 or IL-21 for 2 various lengths of time were monitored for granzyme B content using flow cytometry 3 (Fig. 2c). Both self and non-self KIR + CD56 dim NK cells displayed increased levels of 4 granzyme B in response to IL-15 and IL-21 stimulation. Notably, the relative 5 differences in granzyme B between self and non-self specific NKG2A -CD57 -NK 6 cells were similar after stimulation with IL-15 or IL-21 (Fig. 2c)  activation may occur in NK cells that lack self-specific inhibitory KIR, which in turn 1 9 would affect lysosome stability and/or retention through the mechanism described 2 0 here.

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The dose dependent induction of granzyme B expression in response to 2 2 cytokine, or viral infection, is connected to activation of the metabolic check-point 2 3 kinase mTOR. 83 Notably, however, we did not observe any transcriptional imprint in 2 4 the mTOR pathway when we examined circulating NK cells at rest, arguing against a  MicroImaging GmbH, Jena, Germany) and Imaris 7.7.2 (Bitplane AG, Zürich, 1 7 Switzerland). Confocal z-stacks were deconvolved using Huygens Essential 14.06 1 8 (Scientific Volume Imaging b.v., VB Hilversum, The Netherlands). ImarisCell was 1 9 used to identify secretory lysosomes and centrosomes in confocal Z-stacks of single 2 0 cells, while Imaris Venture was used to find the correlation between the intensity of 2 1 the individual secretory lysosome and their distance to the centrosome center.  Target cell killing was determined using a combination of viability stains.

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Cytotoxicity assays were performed using an NK cell to target cell ratio of 5:1 at 37C

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Read alignment was carried out using Bowtie (version 2.0.5.0) and Tophat (version    l  l  m  o  l  e  c  u  l  e  r  e  s  t  o  r  e  s  f  u  n  c  t  i  o  n  t  o  T  R  P  M  L  1  m  u  t  a  n  t  i  s  o  f  o  r  m  s  r  e  s  p  o  n  s  i  b  l  e  f  o  r  m  u  c  o  l  i  p  i  d  o  s  i  s  t  y  p  e  I  V A . e t a l .  D  u  a  l  e  n  h  a  n  c  e  m  e  n  t  o  f  T  a  n  d  N  K  c  e  l  l  f  u  n  c  t  i  o  n  b  y  p  u  l  s  a  t  i  l  e  i  n  h  i  b  i  t  i  o  n  o  f  S  H  I  P  1  i  m  p  r  o  v  e  s  a  n  t  i  t  u  m  o  r  i  m  m  u  n  i  t  y  a  n  d  s  u  r  v  i  v  a  l  .   S  c  i  e  n  c  e  s  i  g  n  a  l  i  n  g   1  0   (  2  0  1  7  )  .   6  7  .  G  u  m  b  l  e  t  o  n  ,  M  .  ,  V  i  v  i  e  r  ,  E  .  &  K  e  r  r  ,  W  .  G  .  S  H  I  P  1  i  n  t  r  i  n  s  i  c  a  l  l  y  r  e  g  u  l  a  t  e  s  N  K  c  e  l  l  s  i  g  n  a  l  i  n  g  a  n  d  e  d  u  c  a  t  i         Model describing the putative pathway downstream of activating receptors leading to activation of PIKfyve and TRPML1, lysosomal fission and a hyporesponsive state.

Figure Legends
Self-specific inhibitory KIRs interfere with activation signals at a proximal level and allow accumulation of dense-core secretory lysosomes.    Distance from centrosome (μm) Non-Self Self Non-Self Self Non-Self Self Non-Self Self Non-Self Self Secretory lysosome area (μm 2 ) Non-Self Self Non-