WIP is essential for lytic granule polarization and NK cell cytotoxicity - PubMed (original) (raw)

WIP is essential for lytic granule polarization and NK cell cytotoxicity

Konrad Krzewski et al. Proc Natl Acad Sci U S A. 2008.

Abstract

Natural killer (NK) cells play important roles in host immunity by killing virus-infected and tumor cells. Killing of the target cell is achieved by formation of an immune synapse and localized secretion of lytic granules containing perforin and granzymes. Here, we demonstrate that Wiskott-Aldrich syndrome protein (WASp)-interacting protein (WIP), important in generation of a large complex of proteins involved in actin cytoskeleton rearrangements, is indispensable for NK cell cytotoxicity. WIP knockdown completely inhibited cytotoxicity, whereas overexpression of WIP enhanced NK cell cytolytic ability. WIP was found to colocalize with lytic granules. WIP segregated to the lysosomal fraction, where granzyme B activity was also found, and the interaction between WIP and granules was independent of WASp. Importantly, WIP knockdown inhibited polarization of lytic granules to the immune synapse, but not conjugate formation. These results indicate that WIP is involved in lytic granule transport and is essential for regulation of NK cell cytotoxic function.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

WIP is associated with lytic granules in NK cells. (A) Colocalization of FLAG-WIP and perforin in resting (Left) and activated (Center) YTS cells. YTS cells expressing FLAG-WIP, unconjugated or conjugated by mixing for 10 min at 37°C with 721.221 target cells, were stained with Cy3-conjugated anti-FLAG mAb (red) and AlexaFluor 647-conjugated anti-perforin (green) antibodies. (Scale bar: 5 μm.) The graph (Right) displays the percentage of FLAG-WIP colocalizing with perforin (black bars) and perforin that colocalized with FLAG-WIP (gray bars) in either resting or activated NK cells (n ≥ 10). Values above each bar represent Manders' colocalization coefficients. Error bars represent SD. (B) WIP and WASp are found in the lysosomal fraction of NK cells. YTS cells expressing FLAG-WIP were homogenized and fractionated by centrifugation, and crude lysosomal fraction was resolved on an 8–27% Iodoxanol density gradient. Proteins from gradient fractions were resolved on a NuPage gel, transferred to a PVDF membrane, and subsequently immunobloted with anti-FLAG, anti-WASp, and anti-granzyme B antibodies. The serine esterase activity (a marker for granzyme activity) was determined in each density gradient fraction and displayed graphically below. (C) WIP association with lytic granules is independent of WASp and requires intact granules. YTS cells, transfected with either FLAG-WIP or FLAG-WIPΔ460–503 or FLAG expression vector alone, were lysed by homogenization. Crude lysosomal fractions either nontreated or treated with 1% Nonidet P-40 (to solubilize cell membranes) were immunoprecipitated with anti-FLAG mAb. Immunoprecipitated proteins were resolved on a NuPage gel, transferred to a PVDF membrane, and immunobloted with anti-FLAG, anti-WASp, and anti-granzyme B antibodies. The molecular masses of the proteins, relative to masses of molecular markers, are shown in parentheses.

Fig. 2.

Fig. 2.

WIP knockdown inhibits NK cell cytotoxicity despite normal conjugate formation. (A) Cytolytic activity of YTS (Upper) and pNK (Lower) cells, untransfected or transfected with either FLAG-WIP (YTS only) or WIP RNAi. Cells transfected with FLAG expression vector (FLAG; YTS only) or empty RNAi vector (RNAi vector) were used as additional controls. (Insets) Western blots show decrease of WIP expression due to WIP knockdown, with anti-actin immunostaining as a loading control. The percentage of 721.221 target cell lysis for different effector-to-target ratio is shown. Error bars represent SD. The values are based on five and three separate experiments for YTS and pNK cells, respectively. The asterisks indicate statistical significance (*, P < 0.05; **, P < 0.01 by Student's t test) when compared with control-transfected cells. (B) YTS cells, untransfected or transfected with either WIP RNAi, empty RNAi vector (RNAi vector), FLAG expression vector (FLAG), or with FLAG-WIP, were stained with CD56-APC antibodies and mixed with RFP-expressing 721.221 target cells. Cells were allowed to form conjugates for 30 min at 37°C, fixed, and analyzed by flow cytometry. The graphs represent the population of YTS cells conjugated with target cells, as determined by the measurement of the percentage of CD56+RFP+ double-positive cells from the total pool of live CD56+ cells.

Fig. 3.

Fig. 3.

Knockdown of WIP prevents granule but not F-actin polarization to the immune synapse in NK cells. YTS (A) and pNK (B) cells, untransfected (Top) or transfected with empty RNAi vector (Middle) or WIP RNAi (Bottom), were activated by mixing with 721.221 target cells for 15 min at 37°C. Cells were next stained with AlexaFluor 568-conjugated phalloidin (red) and AlexaFluor 647-conjugated anti-perforin antibodies (green) to visualize F-actin and lytic granules, respectively. (Scale bar: 5 μm.) (C) The percentages of F-actin and perforin polarization toward the cell–cell contact site (immune synapse) in YTS (Left) and pNK (Right) cells conjugated with 721.221 target cells. Error bars represent SD. The values were determined by evaluation of 100–200 conjugates in two to four separate experiments. The asterisks indicate statistical significance (*, P < 0.05; **, P < 0.01 by Student's t test) when compared with control.

Fig. 4.

Fig. 4.

WIP knockdown does not prevent F-actin polarization, but affects the area of F-actin accumulation at the cell–cell interface. (A) F-actin accumulation at the cell–bead interface. YTS cells either untransfected or transfected with empty RNAi vector, WIP RNAi, or FLAG-WIP were mixed with anti-LFA1 mAb-coated polystyrene beads for 60 min at 37°C. The cells were then stained with AlexaFluor 568-conjugated phalloidin to visualize F-actin. (Scale bar: 5 μm.) (B) The area of F-actin accumulated at the cell–cell contact site. Untransfected YTS cells or YTS cells transfected with empty RNAi vector, WIP RNAi, or FLAG-WIP were mixed with 721.221 target cells 10 min at 37°C, followed by staining with AlexaFluor 568-conjugated phalloidin and AlexaFluor 647-conjugated anti-perforin antibodies (to distinguish NK cells from target cells). F-actin at the immune synapse was visualized by taking a series of images of conjugated cells in the z plane, from which the area of F-actin accumulation was measured. n ≥ 14. Error bars represent SD. The asterisk indicates statistical significance (P < 0.05 by Student's t test) when compared with control.

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