Syntaxin 11 marks a distinct intracellular compartment recruited to the immunological synapse of NK cells to colocalize with cytotoxic granules

Abstract

The syntaxin 11 (STX11) gene is mutated in a proportion of patients with familial haemophagocytic lymphohistiocytosis (FHL) and exocytosis of cytotoxic granules is impaired in STX11-deficient NK cells. However, the subcellular localization, regulation of expression and molecular function of STX11 in NK cells and other cytotoxic lymphocytes remain unknown. Here we demonstrate that STX11 expression is strictly controlled by several mechanisms in a cell-type-specific manner and that the enzymatic activity of the proteasome is required for STX11 expression in NK cells. In resting NKL cells, STX11 was localized in the cation-dependent mannose-6-phosphate receptor (CD-M6PR)-containing compartment, which was clearly distinct from cytotoxic granules or Rab27a-expressing vesicles. These subcellular structures appeared to fuse at the contact area with NK-sensitive target cells as demonstrated by partial colocalization of STX11 with perforin and Rab27a. Although STX11-deficent allo-specific cytotoxic T-lymphocytes efficiently lysed target cells and released cytotoxic granules, they exhibited a significantly lower extent of spontaneous association of perforin with Rab27a as compared with STX11-expressing T cells. Thus, our results suggest that STX11 promotes the fusion of Rab27a-expressing vesicles with cytotoxic granules and reveal an additional level of complexity in the spatial/temporal segregation of subcellular structures participating in the process of granule-mediated cytotoxicity.

Fig 1

Endogenous and ectopic expression of STX11 in human cell lines of different origin. (A) AA-B1, HeLa, NKL, NK-92 and SK-N-BE cells were transfected with the indicated control vectors or plasmids encoding the wild-type or 3FLAG-tagged STX11. Transfected cells were grown in relevant selection media for at least 2 months before analysis by immunoblot-ting with the indicated specific antibodies as described in Materials and Methods. Samples were analysed in overlapping sets loaded on the same gel to allow proper comparison of relative expression levels of the wild-type or FLAG-tagged STX11. Total cell lysate of MON-B1 LCL was used as a positive control for each blot. (B) Real-time PCR-based quantification of STX11 and STXBP2 mRNA expression in the indicated cell lines was performed as described in Materials and Methods. The levels of expression are shown relative to GAPDH expression determined and expressed as 1 for each individual sample. The mean ± S.D. of triplicates.

Fig 2

Expression of endogenous and ectopic STX11 in NK cells requires proteasome-dependent protein degradation. Total cell lysates of untreated (control) and inhibitor treated cells were analysed by immunoblotting with antibodies specific to the indicated proteins. (A) Control NKL cells, along with NKL cells transfected with either empty or STX11-encoding pCMV-3FLAG plasmid, were cultured overnight in the presence of lactacystin (LAC, 10 μM) or chloroquine (CLQ, 25 μM). Lysates of AA-B1 and MON-B1 cells were used, respectively, as a negative and positive control of STX11 expression. (B) NKL cells were cultured overnight in the presence of lactacystin, chloroquine, the pan-caspase inhibitor Z-VAD (50 μM) or TPPII inhibitor AAF-cmk (5 μM). Inhibitors were added either alone or in combinations as indicated in the figure. CTLs from a healthy donor were included as a positive and AA-B1 cells as a negative control. (C) Proteasome but not lysosome inhibitors suppress STX11 expression in the indicated human NK lines. (D) Human primary NK cells and T cells were purified from the peripheral blood of healthy donors by negative selection with immunomagnetic beads and then incubated in the absence or presence of lactacystin along with NK92 cells and in vitro cultured human CTLs. (E) Human primary NK cells and CD8⁺ T cells were purified as described in (D). NK cells were activated by coculturing with irradiated MHC class I negative K562 cells. CD8⁺ cells were activated by culturing with beads conjugated with CD3- and CD8-specific antibodies. Following activation, NK and CD8⁺ T cells were cultured in the presence of recombinant IL-2 for 7 days and treated with the indicated protease inhibitors or solvent alone (DMSO) as described in (A).

Fig 3

STX11 resides in a subset of CD-M6PR-expressing vesicles in NKL cells. The indicated cell lines were stably transfected with the pCMV-3FLAG-STX11 plasmid and subcellular localization of STX11 was identified by staining with FLAG-specific antibodies alone (A) or in combination with CD-M6PR-specific antibodies (B) and confocal microscopy. The scale bars correspond to 5 μm. Analysis of 30 randomly selected individual cells generated an overlap coefficient of 0.74 ± 0.1 for CD-M6PR- and STX11-specific signals.

Fig 4

STX11, perforin and Rab27a localize to different vesicular compartments in resting NKL cells. Confocal microscopy of resting NKL cells stably transfected with pCMV-3FLAG-STX11 was performed after immunostaining with FLAG-, perforin- or Rab27a-specific antibodies. The panels show data representative of three to five independent experiments. The scale bars correspond to 5 μm. Analysis of 30 individual cells generated the following values of overlap coefficient for the indicated pairs of signals: FLAG/perforin-10 ± 5; FLAG/Rab27a-7 ± 2.5; perforin/Rab27a-6 ± 5.

Fig 5

Activation of NKL cells induces partial colocalization of STX11, perforin and Rab27a at the NK cell/target cell contact area. NKL cells were activated by coculturing with K562 cells for 20 min, fixed and costained with antibodies specific to the indicated proteins and analysed by confocal microscopy. Scale bars, 5 μm. Images are representative of three to five independent experiments. Statistical analysis of 30 individual images produced the following values of Pearson's correlation coefficient: FLAG/perforin, 0.35 > r > 0.67; FLAG/Rab27a, 0.48 > r > 0.68; perforin/Rab27a, 0.47 > r > 0.74.

Fig 6

Composition and cytotoxic activity of allo-specific CTL cultures generated from STX11-deficient or STX11-proficient individuals. (A) Cultures of allo-specific T cells were generated from PBLs of a healthy donor, patients 1 and 3 with STX11 deficiency, the mother of patient 3 as well as patient 2 with deficiency in perforin expression. PBLs were subjected to consecutive re-stimulations with the allogeneic MON-B1 LCL and expansion in IL-2 containing medium. IFN-γ production by CD8⁺ T cells was measured by intracellular immunostaining and FACS analysis before and after stimulation with MON-B1 cells at a responder/stimulator ratio of 5:1. Numbers in the plots represent percentages of CD8⁺ cells in the cultures and percentages of IFN-γ-producing cells in the CD8⁺ cell populations. One representative of three experiments. (B) Cytotoxic activity of the indicated CTL cultures was tested in ⁵¹Cr-release assays. Effectors were added at ratios generating comparable numbers of CD8⁺ T cells producing IFN-γ in response to stimulation with MON-B1 cells, which served as targets in the assay. The kinetics of cytotoxicity was analysed as described in Materials and Methods. Filled symbols represent cytotoxicity exhibited by the indicated CTL cultures after CMA-induced disruption of cytotoxic granules and inhibition of perforin activity achieved by pre-incubation with CMA for 2 hrs. The error bars show standard deviations of experimental triplicates. One representative of three experiments.

Fig 7

Spontaneous and activation-induced colo-calization of perforin and Rab27a in STX11-deficient and STX11-proficient CTLs. CTLs from a healthy donor (A) and FHL patient 1 (B) were analysed by immunostaining with perforin- or Rab27a-specific antibodies and confocal microscopy either directly from culture (i, ii) or after 20 min of activation by coincubation with MON-B1 LCLs (iii). Images representative for non-activated CTLs which did not exhibit detectable overlapping of perforin- and Rab27a-specific signal (i), images of cells in which colocalization of the signals was observed without specific CTL activation (ii), and re-localization of perforin and Rab27a into the area of immunological synapse following activation with MON-B1 cells (iii) are shown. (C) The extent of colocalization of the two signals was calculated as overlap coefficient assessed for 30 individual cells observed in 10–15 randomly selected fields of view. The statistical analysis of data was performed using Student's two-tailed unequal variance t-test.