NKG2D Signaling Between Human NK Cells Enhances TACE-Mediated TNF- Α Release Neekun Sharma, Camille V

NKG2D Signaling between Human NK Cells Enhances TACE-Mediated TNF- α Release Neekun Sharma, Camille V. Trinidad, Andrew P. Trembath and Mary A. Markiewicz This information is current as of September 26, 2021. J Immunol published online 11 September 2017 http://www.jimmunol.org/content/early/2017/09/09/jimmun ol.1700647 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published September 11, 2017, doi:10.4049/jimmunol.1700647 The Journal of Immunology

NKG2D Signaling between Human NK Cells Enhances TACE-Mediated TNF-a Release

Neekun Sharma, Camille V. Trinidad, Andrew P. Trembath, and Mary A. Markiewicz

NK group 2 member D (NKG2D) is a strong NK cell–activating receptor, with engagement by ligands triggering granule release and cytokine production. The function of NKG2D signaling in NK cells has largely been studied in the context of engagement of the receptor by ligands expressed on the surface of target cells. We report that upon activation with IL-12, IL-15, and IL-18 human NK cells express NKG2D ligands of the UL16 binding protein family on the cell surface. NKG2D-ligand interaction between cytokine-stimulated NK cells increases the activity of the metalloprotease TNF-a–converting enzyme. This enhanced TNF-a–converting enzyme activity signiﬁcantly increases the release of TNF-a and UL16 binding protein from the surface of the NK cells. These results demonstrate that NKG2D signaling is critical for maximal TNF-a release by NK cells. Further, they

demonstrate a role for NKG2D-ligand interaction via homotypic NK cell contact in NK cell effector function. The Journal of Downloaded from Immunology, 2017, 199: 000–000.

atural killer cells are important effector cells that bridge protein ectodomain shedding has been known for years. However, the innate and adaptive immune response. As such, they little is known about how TACE activity is regulated in NK cells. N play a critical role in antitumor and antimicrobial im- We report in this study that upon activation with IL-12, IL-15, munity (1). NK cell activation is controlled by the engagement of and IL-18, human NK cells express ULBP family members on the http://www.jimmunol.org/ activating and inhibitory receptors, as well as by cytokines, in- cell surface, and that NKG2D signaling controls the magnitude of cluding IL-2, IL-12, IL-15, IL-18, and IFN-a (2, 3). One of the this expression. We demonstrate that this is the result of increased best-characterized NK cell–activating receptors is the NK group 2 activity of the metalloprotease TACE. Further, we show NKG2D- member D (NKG2D) C-type lectin-like receptor. NKG2D is induced TACE activity signiﬁcantly increases the release of TNF-a expressed by all human NK cells and recognizes a number of from NK cells. These results demonstrate that NKG2D signaling endogenous ligands that are structurally similar to MHC class I is critical for maximal TNF-a release by NK cells. Further, they molecules, namely class I–related chain A and B (MICA/B) and demonstrate a role for NKG2D-ligand interaction via homotypic UL16 binding proteins (ULBPs) (ULBP1-6) (reviewed in Ref. 4). NK cell contact in human NK cell effector function.

NKG2D ligands are not expressed by most healthy tissue, but by guest on September 26, 2021 rather are induced upon cellular stress, such as microbial infec- Materials and Methods tion, cellular transformation, or DNA damage (4). Despite this NK cell purification generality, it is now clear there are cells that are not considered stressed or damaged that also express NKG2D ligands (reviewed Peripheral blood was harvested from healthy volunteers who donated to the University of Kansas Biospecimen Repository Core Facility (http://www. in Ref. 5). These include subsets of hematopoietic cells, including kumc.edu/school-of-medicine/biospecimen.html). This facility is overseen macrophages, monocytes, dendritic cells, and activated T cells and by an interprogrammatic Internal Advisory Board and the University of NK cells. The role for this expression in the immune function of Kansas Medical Center Institutional Review Board. PBMCs were isolated each of these cell types is not known. by density gradient centrifugation using Histopaque (Sigma-Aldrich). NK a cells were then purified by negative selection using the Dynabeads Un- TNF- –converting enzyme (TACE), also known as A dis- touched Human NK cells kit (Invitrogen) following the manufacturer’s integrin and metalloproteinase 17, is expressed constitutively by protocol. The purity of NK cells was assessed by flow cytometry to be NK cells. TACE plays a broad role in cleaving proteins at the cell .90% CD32CD56+CD16+. surface (6), including NKG2D ligands (7, 8). TACE’s role in Abs AF700 anti-CD3 (UCHT1), PE-Cy7 anti-CD16 (3G8), APC anti-CD56 Department of Microbiology, Molecular Genetics and Immunology, University of (B159), and PE anti-TNF-a (MAb11) were purchased from BD Biosci- Kansas Medical Center, Kansas City, KS 66160 ences. PE anti-NKG2D (1D11), PE-Cy7 anti-CD16 (B73.1), BV650 anti- ORCIDs: 0000-0003-3480-208X (C.V.T.); 0000-0001-5685-8573 (M.A.M.). CD62L (DREG-56), and PE mouse IgG1 isotype control (MOPC-21) were Received for publication May 3, 2017. Accepted for publication August 15, 2017. purchased from BioLegend. PE anti-MICA/B (159207), PE anti-ULBP1 (170818), PE anti-ULBP2/5/6 (165903), PE anti-ULBP3 (166510), PE This work was supported by grants from the American Association of Immunologists anti-ULBP4 (709116), PE anti-TACE (FAB9301P), PE mouse IgG iso- Careers in Immunology Fellowship Program (to N.S. and M.A.M.), University of 2A Kansas Cancer Center’s Cancer Support Grant P30 CA168524 (Biospecimen Repos- type control (20102), PE mouse IgG2B isotype control (133303), purified itory), and National Institutes of Health/National Institute of General Medical Sci- anti-NKG2D (149810), and mouse IgG1 isotype control (11711) were ence Grant P30 GMI103326 (flow cytometry core). purchased from R&D Systems. Anti-TACE [D1(A12)] was purchased from EMD Millipore. Address correspondence and reprint requests to Dr. Mary A. Markiewicz, University of Kansas Medical Center, 3901 Rainbow Boulevard, Mailstop 3029, Kansas City, KS 66160. E-mail address: [email protected] NK cell culture and activation The online version of this article contains supplemental material. Purified NK cells were plated at a concentration of 2 3 105 cells per well in Abbreviations used in this article: NKG2D, NK group 2 member D; siRNA, small RPMI 1640 medium supplemented with penicillin-streptomycin-glutamine interfering RNA; TACE, TNF-a–converting enzyme; ULBP, UL16 binding protein. and 10% FCS. The NK cells were cultured either alone or stimulated with 10 ng/ml of recombinant human IL-12 (PeproTech), IL-15 (PeproTech), Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 IL-18 (MBL International), or a combination of IL-12, IL-15, and IL-18. In

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700647 2 NKG2D SIGNALING BETWEEN HUMAN NK CELLS ENHANCES TNFa RELEASE blocking experiments, the cells were incubated with Human BD Fc block ACG GTG CCA TGG AAT TT-39. Relative expression was calculated by (2.5 mg/ml) and 20 mg/ml anti-NKG2D (149810) or mouse IgG1 isotype the cycling threshold method as 2–DDCt. control (11711) throughout the culture period. When indicated, the TACE inhibitor TAPI-0 (Peptides International, Louisville, KY) or anti-TACE Ab TACE activity assay were added at a concentration of 1 mMor6mg/ml, respectively. The cells 3 TACE (A disintegrin and metalloproteinase 17) activity was determined were analyzed after 18 h of culture. For the cell count experiments, 4 using the SensoLyte 520 TACE (a-Secretase) Activity Assay Kit (AnaSpec) 105 cells per well supplemented with IL-12, IL-15, and IL-18 were plated m according to the manufacturer’s protocol. Cell lysates were generated from by adding 20 g/ml of anti-NKG2D or IgG1 isotype control Abs, and the 5 3 105 cells using CytoBuster protein Extraction Reagent (EMD Millipore). live cells were counted 18 h later. Fluorescence was measured in a fluorescence microplate reader (Synergy RT-PCR H1; BioTek) at excitation/emission = 490 nm/520 nm. RNA was extracted from NK cells using Trizol reagent (Invitrogen) and Measurement of TNF-a release reverse transcription performed with the QuantiTect Reverse Transcription a Kit (Qiagen). Real time quantitative PCR was then performed using the TNF- release was measured in the supernatant by cytometric bead array Power SYBR Green PCR Master Mix (Applied Biosystems) and a real time (BD Biosciences) and an LSR II (BD Biosciences) according to the PCR machine (Roche) using primers to specific genes. Transcript levels manufacturer’s recommended procedure. Data were analyzed using FCAP array software (BD Biosciences). were normalized to the endogenous housekeeping gene GAPDH. The primers for ULBP2 (PPH15076B), ULBP5 (PPH57805B), and ULBP6 Intracellular TNF-a measurement (PPH17958A) were purchased from Qiagen. The sequence of the primers for TNF-a and GAPDH were as follows: TNF-a;F–59-CCC AGG GAC BD GolgiPlug (BD Biosciences) was added (1 ml/ml) during the final 4 h of CTC TCT CTA ATC A-39;R–59-AGC TGC CCC TCA GCT TGA G-39 NK cell culture. The cells were washed, stained with anti-CD3, anti-CD56, and GAPDH; F – 59-GCC ATC AAT GAC CCC TTC ATT-39;R–59-TTG anti-CD16, and anti-NKG2D mAbs, fixed, permeabilized, and then stained Downloaded from http://www.jimmunol.org/

FIGURE 1. Activation with the combination of IL-12, IL-15, and IL-18 induces ULBP expression on human NK cells. (A and B)Repre- sentative ﬂow cytometric (A)histo- by guest on September 26, 2021 gram and (B) dot plots showing cell surface expression of NKG2D ligands compared with isotype control Abs on primary human NK cells culturedinmediumalonefor18h. The numbers shown indicate the percentage of cells with staining above that of the isotype control. (C and D) Representative ﬂow cytometric (C) histogram and (D) dot plots showing cell surface expression of NKG2D ligands compared with isotype control Abs on primary human NK cells stimulated with IL-12, IL-15, and IL-18 for 18 h. The numbers shown indicate the percentage of cells with staining above that of the isotype control. (E and F) Combined expression data of (E) ULBP2/5/6 and (F) ULBP4 on NK cells from six independent donors. **p , 0.01 in two-tailed Mann– Whitney U test. The Journal of Immunology 3 Downloaded from

FIGURE 2. The combination of IL-12, IL-15, and IL-18 is required to induce ULBP expression on human NK cells. (A–C) Percentage of human NK http://www.jimmunol.org/ cells expressing ULBP2/5/6 cultured without cytokine or with IL-12, IL-15, and IL-18 alone or in combination for (A)2,(B) 6, and (C)18h.(D–F) Percentage of human NK cells expressing ULBP4 cultured without cytokines or with IL-12, IL-15, and IL-18 either alone or in combination for (D)2,(E)6, and (F) 18 h. Combined data (mean 6 SEM) from three independent donors are shown in each panel. with anti-human TNF-a or with isotype control Ab. Cells were subse- killing of M21 cells by NK cells was calculated according the following quently washed, resuspended in PBS, and analyzed using a BD LSR II (BD equation: [(number of target cells at beginning of assay 2 number of live Biosciences). The data were analyzed using FlowJo (Tree Star, Ashland, target cells at end of assay)/number of target cells at beginning of assay] 3 100. OR). Knockdown of NKG2D and ULBP4 by RNA interference Tumor-killing assay by guest on September 26, 2021 NKG2D and ULBP4 were knocked down by RNA interference. For IL-12, IL-15, and IL-18–stimulated NK cells (effector cells) were cultured hNKG2D (AM16708A), the following silencer small interfering RNAs with 1 mM CFSE- (Invitrogen) labeled M21 target cells in triplicate at (siRNAs) (Thermo Fisher Scientiﬁc) were used: 108247 (siRNA#1), varying E:T ratios and incubated for 4 h. The number of live (7AAD2) 108248 (siRNA#2), 108249 (siRNA#3). Additionally, a fourth siRNA of CFSE+ cells was then determined using ﬂow cytometry. The percentage the following sequence was used: 59-CGGGGUCAGGGAGGUGGUGUU-39

FIGURE 3. NKG2D blockade increases IL-12, IL-15, and IL-18– induced ULBP expression on human NK cells. (A and C) Representative ﬂow cytometric plots of (A) ULBP2/ 5/6 and (C) ULBP4 staining on primary human NK cells stimulated with IL-12, IL-15, and IL-18 for 18 h with or without NKG2D blockade. (B and D) Combined data from eight independent experiments showing percentage (mean 6 SEM) of NK cells expressing ULBP2/5/6 and ULBP4 upon NKG2D blockade. *p , 0.05 in two-tailed Mann–Whitney U test. ns, not signiﬁcant. 4 NKG2D SIGNALING BETWEEN HUMAN NK CELLS ENHANCES TNFa RELEASE

(9) (siRNA#4). The siRNAs used for ULBP4 (4392420) were s43926 We next asked whether activation of NK cells with cytokines (siRNA#1) and s43928 (siRNA#2) (Thermo Fisher Scientific). The silencer could increase this expression. To do this, we stimulated the cells negative control siRNA (AM4611) (Thermo Fisher Scientific) was used for with IL-12, IL-15, IL-18, or a combination of IL-12, IL-15, and comparison. The siRNAs (5 nM) were transfected into the NK cells using a Nucleofector II (Lonza) following the manufacturer’s instructions. Then IL-18. ULBP4 expression was significantly increased on the cells 24 h following transfection, the cells were analyzed for NKG2D and stimulated with the combination of IL-12, IL-15, and IL-18 ULBP4 surface expression and TNF-a release using flow cytometry and (Fig. 1C, 1D, 1F). These cells also exhibited staining with an cytometric bead array, respectively. Ab that detects ULBP2, 5, and 6 (ULBP2/5/6) (Fig. 1C–E). This Statistical analysis expression could be observed by 6 h post-cytokine treatment, but was highest following overnight culture (Fig. 2). In contrast to the All statistical analysis was performed with GraphPad Prism Software (GraphPad Software). combined cytokine treatment, single treatment with IL-12, IL-15, or IL-18 alone did not induce ULBP expression (Fig. 2). These Results results demonstrate that activation with the combination of IL-12, IL-15, and IL-18 induces high ULBP family member expression Human NK cells express ULBP family members upon on human NK cells. activation with IL-12, IL-15, and IL-18 We hypothesized that NKG2D-ligand interaction between NK cells NKG2D expression on human NK cells is unaffected by could play a role in NK cell effector responses. To test this, we first activation with IL-12, IL-15, and IL-18 analyzed expression of all eight ligands on NK cells purified from Sustained NKG2D engagement can induce internalization of

PBMCs of healthy donors. We found no expression of MICA, NKG2D from the cell surface, resulting in an inability of cells Downloaded from MICB, ULBP1, 2, 3, 5, or 6, but did find low expression of ULBP4 to respond to NKG2D ligands (10–13). Therefore, we asked on NK cells purified from all individuals tested (Fig. 1A, 1B, 1F). whether the induction of NKG2D ligands on NK cells by IL-12, http://www.jimmunol.org/ by guest on September 26, 2021 FIGURE 4. Inhibition of TACE activity increases ULBP expression on IL-12, IL-15, and IL-18–stimulated human NK cells. (A–C) Relative expression of (A) ULBP2, (B) ULBP5, and (C) ULBP6 mRNA in IL-12, IL-15, and IL-18–stimulated human NK cells with or without NKG2D blockade. (D and F) Repre- sentative flow cytometric dot plots showing percentage of IL-12, IL-15 and IL-18–stimulated NK cells expressing (D) ULBP2/5/6 and (F) ULBP4 in the presence or absence of the TACE inhibitor TAPI-0. (E and G) Combined data from five independent experiments showing percentage of NK cells expressing ULBP2/5/6 and ULBP4 upon TACE inhibition. **p , 0.01 in two-tailed Mann–Whitney U test. ns, not significant. The Journal of Immunology 5

IL-15, and IL-18 affected NKG2D surface expression by the NK ULBP2/5/6-specific Ab (Fig. 4D, 4E). Confirming this was due to cells. Despite the striking increase in ULBP expression (Fig. 1), inhibition of TACE, treatment with a TACE-specific blocking Ab we did not observe any change in NKG2D surface expression fol- similarly increased ULBP2/5/6 staining (Supplemental Fig. 3A). lowing cytokine activation (Supplemental Fig. 1A, 1B). Additionally, By contrast, ULBP-4 surface expression was unchanged by TACE no effect on NK target-cell killing was observed (Supplemental inhibition (Fig. 4F, 4G). Fig. 2). NKG2D signaling regulates TACE activity in human NK cells NKG2D signaling decreases NKG2D ligand expression on The result that both NKG2D blockade and TACE inhibition in- human NK cells creased surface staining with the ULBP2/5/6-specific Ab suggested We next asked whether NKG2D signaling affected NK cell survival that NKG2D signaling enhanced TACE activity. Therefore, we next or ULBP expression induced by IL-12, IL-15, and IL-18. To do this, directly compared TACE activity between the cells when NKG2D we tested the effect of NKG2D blockade during incubation with the was blocked. We did not observe any difference in TACE activity cytokines. We observed no effect of NKG2D blockade on NK cell between unstimulated and IL-12, IL-15, and IL-18–stimulated NK survival (Supplemental Fig. 1C) or ULBP4 expression (Fig. 3C, cells (Fig. 5A), but did observe increased TACE expression on the 3D). By contrast, inclusion of an NKG2D inhibitory Ab resulted cell surface of the cytokine-stimulated cells (Supplemental Fig. in increased staining with the Ab that detects ULBP2/5/6 (Fig. 3A, 3B). There was no effect of NKG2D blockade on TACE activity in 3B). the unstimulated NK cells (Fig. 5B). By contrast, NKG2D blockade resulted in a significant decrease in TACE activity in the TACE enhances cleavage of ULBP2/5/6 on human NK cells

cytokine-treated NK cells (Fig. 5C) without a change in TACE Downloaded from The change in ULBP expression observed with NKG2D blockade surface expression (Supplemental Fig. 3B). These results dem- was not the result of increased gene transcription, as similar levels onstrate that NKG2D-ligand interaction between human NK cells of ULBP2, ULBP5 and ULBP6 transcripts were present with or enhances TACE activity following activation with IL-12, IL-15, without NKG2D blockade (Fig. 4A–C). Therefore, we next asked and IL-18. whether the increase could be due to decreased release of one or a more of the ligands from the cell surface. All ULBP family NKG2D signaling enhances TNF- release by human NK cells http://www.jimmunol.org/ members can be released as soluble proteins. However, the A critical effector function of NK cells is the release of proin- mechanism of release varies. Soluble ULBP1, 2, 3, and 6 are flammatory cytokines (17). One of these cytokines, TNF-a,is generated by proteolytic cleavage from the plasma membrane (7, first produced and expressed as a transmembrane protein on the 8, 14). By contrast, soluble ULBP4 and 5 are generated by al- cell surface. The production of soluble TNF-a requires the ternative splicing (15, 16). Given that NKG2D inhibition altered cleavage of membrane TNF-a by TACE (18). Therefore, we staining with the ULBP2/5/6-specific, but not the ULBP4-specific, hypothesized that the decreased TACE activity we observed Ab, we hypothesized NKG2D signaling was involved in increas- with inhibition of NKG2D signaling would result in decreased ing cleavage of ligands from the cell surface. TNF-a release by the cytokine-treated NK cells. We first con-

Multiple studies demonstrate that A disintegrin and metal- firmedwithchemicalinhibitionthatTACEwasrequiredfor by guest on September 26, 2021 loproteinase family members can cleave NKG2D ligands from the TNF-a release from the cytokine-stimulated NK cells (Fig. 6A). cell surface (8). One of these metalloproteases, TACE, is consti- We next determined whether TNF-a release by these cells was tutively expressed in NK cells where it plays a critical role in affected by NKG2D signaling. Inhibition of NKG2D signaling shedding protein ectodomains at the cell surface (6). Therefore, by Ab blockade (Fig. 6B), siRNA knockdown of NKG2D we wondered whether the increase in surface staining with the (Fig. 6C, 6D), or siRNA knockdown of ULBP4 (Fig. 6E, 6F) ULBP2/5/6-specific Ab on NK cells with NKG2D blockade could significantly reduced the amount of TNF-a present in the culture be due to altered enzymatic activity of TACE. To test this, we first supernatant. measured the effect of NKG2D blockade on ULBP expression in To confirm NKG2D signaling enhanced TNF-a release, rather the presence of a TACE inhibitor (TAPI-0). Similar to NKG2D than production, we compared the amount of TNF-a protein and blockade, TAPI-0 treatment increased surface staining with the transcript inside the cells. As expected, the unstimulated NK cells

FIGURE 5. NKG2D-ligand interaction between IL-12, IL-15, and IL-18–activated human NK cells increases TACE activity. (A) Combined data from six individuals showing TACE activity in total cell lysates from NK cells cultured in medium alone or with IL-12, IL-15 and IL-18 for 18 h measured by relative ﬂuorescence intensity (RFI). (B and C) TACE activity in total cell lysates from (B) unstimulated NK cells and (C) IL-12, IL-15, and IL-18–treated NK cells cultured with isotype control Ab or blocking NKG2D Ab measured by RFI. These data are representative of two and seven independent experiments, respectively. **p , 0.01 in paired two-tailed Student t test. ns, not signiﬁcant. 6 NKG2D SIGNALING BETWEEN HUMAN NK CELLS ENHANCES TNFa RELEASE

together, these results demonstrate that NKG2D signaling between NK cells does not increase TNF-a production by the cells, but rather increases TACE-mediated TNF-a release. By contrast, the residual TACE activity in NKG2D-blocked cells was sufﬁcient to cleave two other targets, CD16 and CD62L (6) (Supplemental Fig. 4A, 4B).

Discussion In this study we demonstrate that ULBP family members are induced on human NK cells following activation with the combination of IL-12, IL-15, and IL-18. These three cytokines act synergistically to activate cytokine production from NK cells (6). Our data demonstrate that this cytokine combination is similarly required to induce high expression of ULBPs on human NK cells. Further, we show that NKG2D signaling induced by these ULBPs is critical for maximum TACE-mediated cleavage of TNF-a from the surface of NK cells. To our knowledge, this is the ﬁrst report of

a role for NKG2D ligand expression by NK cells in NK cell Downloaded from function. Despite a signiﬁcant increase in ULBP expression, we did not observe a change in NKG2D expression following activation of human NK cells with IL-12, IL-15, and IL-18. This was somewhat surprising given that sustained NKG2D engagement often leads to

the internalization of NKG2D from the cell surface (10–13, 19). http://www.jimmunol.org/ This may be because both IL-12 and IL-15 signaling increase transcription of the gene encoding NKG2D (20, 21). Since their initial description, NKG2D ligands have been labeled stress ligands due to their induction upon conditions of cellular stress, such as DNA damage, viral infection, or cellular transformation (5). However, more recently it has become clear that there are cells that are generally not considered stressed that also express NKG2D ligands, including multiple hematopoietic cells. One previous study demonstrated NKG2D ligand expression was by guest on September 26, 2021 induced on primary human NK cells activated with IL-2 (22). However, expression of only five of the eight ligands was assessed FIGURE 6. NKG2D-induced TACE activity enhances TNF-a release and no function for this expression was elucidated. from IL-12, IL-15, IL-18–activated human NK cells. (A) TNF-a release in One proposed hypothesis for NKG2D ligand expression by NK cells stimulated with IL-12, IL-15, and IL-18 for 18 h in the presence immune cells is that it is a mechanism to downregulate the immune or absence of the TACE inhibitor TAPI-0. These data are representative of response. This is because NKG2D ligand expression by immune three individual experiments. (B) TNF-a release from NK cells stimulated cells can make the cells sensitive to lysis by NK cells (5, 23–25). with IL-12, IL-15, and IL-18 for 18 h with or without NKG2D blockade (mean 6 SD). These data are representative of at least three individual Supporting this idea, NK cells were shown to gain surface ex- experiments. (C) Representative overlay histogram showing NKG2D expression of NKG2D ligands by trogocytosis upon interacting with pression in IL-12, IL-15, IL-18–activated human NK cells that had been NKG2D ligand-expressing target cells, leading to fratricide of the transfected with four different NKG2D-specific siRNAs or negative con- NK cells (26). However, we did not observe decreased NK cell trol siRNA. The siRNAs shown are as follows: NKG2D siRNA#1 (solid survival with endogenously expressed ULBPs. Similarly, Brennan line), NKG2D siRNA#2 (dotted line), NKG2D siRNA#3 (dashed line), et al. (22) did not observe NK cell fratricide upon ligand ex- NKG2D siRNA#4 (long dashes), and negative control siRNA (solid filled pression following IL-2 stimulation. Taken together, these data D gray histograms). The cells were analyzed 24 h after transfection. ( ) suggest there is a differential effect of endogenously induced a 6 TNF- release (mean SD) from human NK cells transfected with NKG2D ligands on NK cells compared with those gained by NKG2D-specific siRNAs or negative control siRNA. These data are rep- E trogocytosis. resentative of two individual experiments. ( ) Representative overlay a histogram showing ULBP4 expression in IL-12, IL-15, and IL-18– The biological role of TACE in cleavage of TNF- from NK activated human NK cells that had been transfected with ULBP4-specific cells is well known (27). In this study we identified a novel role for siRNA#1 (long dashes), ULBP4 siRNA#2 (solid line) or negative control this metalloprotease in controlling surface ULBP expression on siRNA (solid filled gray histogram). (F) TNF-a release from human NK activated human NK cells. The signals involved in regulating cells transfected with ULBP4-specific siRNAs or negative control siRNA. TACE activity in NK cells have not been clearly defined. Our These data are representative of three individual experiments. *p , 0.05, studies demonstrate a role for NKG2D-ligand engagement in this **p , 0.01, ***p , 0.001 in paired two-tailed Student t test. NC, negative regulation. This is likely due to activation of ERK and p38 MAPK control. signaling. These MAPK signaling pathways are required to release inhibition of TACE by tissue inhibitor metalloproteases 3 (28). contained little TNF-a protein or transcript (Fig. 7A, 7E). Upon NKG2D, IL-12, IL-15, and IL-18 all induce these MAPK sig- stimulation with IL-12, IL-15, and IL-18, there was both increased naling pathways in human NK cells (20, 29–31). We found that TNF-a transcript and protein produced. However, this production inducing NKG2D signaling by Ab cross-linking was insufficient was unaffected by blocking NKG2D signaling (Fig. 7B–E). Taken to increase TACE activity in unstimulated NK cells (data not The Journal of Immunology 7

FIGURE 7. NKG2D-ULBP interaction does not alter TNF-a transcription or protein production in IL-12, IL-15, IL-18–activated human NK cells. (A and B) Representative flow cytometry plots showing intracellular staining of TNF-a in human NK cells cultured in (A) medium alone or (B) with IL-12, IL-15, and IL-18 for 18 h with or without NKG2D blockade. The numbers shown indicate the percentage of cells with staining above that of the isotype control. (C and D) Combined data from six independent experiments showing the (C) percentage (mean 6 SEM) and (D) geometric mean fluorescence intensity (MFI) Downloaded from (mean 6 SEM) of TNF-a–positive cells. (E) Relative expression of TNF-a mRNA in nonstimulated or IL-12, IL-15, and IL-18–stimulated human NK cells with or without NKG2D blockade. ns, not significant. http://www.jimmunol.org/

shown). This suggests a synergism in the signaling induced by cells has almost exclusively been studied in the context of en- NKG2D and these cytokines that is required for TACE activation. gagement of the receptor by ligands expressed on the surface of This synergy could be at the level of MAPK signaling. Alterna- target cells, such as tumor cells. To our knowledge, this is the first tively, it could be due to increased phosphorylation of DAP10, the report of a role for NKG2D-ligand interaction during homotypic adaptor protein required for NKG2D signaling, by IL-15 (32). NK cell contact. Increasing this signaling could potentially be A previous study demonstrated increased TACE activity at the used to enhance NK cell–based immunotherapies. by guest on September 26, 2021 plasma membrane with cytokine stimulation, which resulted in cleavage of CD16 and CD62L from the surface of human NK cells Acknowledgments (6). Our results demonstrate that this increased TACE activity at We thank Dr. Jeff Bose (University of Kansas Medical Center) for advice in the cell membrane is a result of increased TACE surface expres- writing this manuscript. We acknowledge support from the University of sion, rather than an increase in total TACE activity within the Kansas Cancer Center’s Biospecimen Repository Core Facility staff for cells. Despite reduced TACE activity and TNF-a release with helping obtain healthy human blood samples. NKG2D blockade, we did not observe enhanced accumulation of CD16 and CD62L on the plasma membrane in our study. This Disclosures suggests that a higher level of TACE activity is required for full The authors have no financial conflicts of interest. release of TNF-a compared with CD16 and CD62L. Somewhat surprisingly, IL-12, IL-15, and IL-18–treated cells did not contain greater total TACE activity compared with un- References 1. Raulet, D. H., and R. E. Vance. 2006. Self-tolerance of natural killer cells. Nat. treated cells. However, NKG2D-ligand interaction was required to Rev. Immunol. 6: 520–531. maintain TACE activity following cytokine treatment. This means 2. Biron, C. A., K. B. Nguyen, G. C. Pien, L. P. Cousens, and T. P. Salazar-Mather. that if NKG2D ligands were not expressed, TACE activity would 1999. Natural killer cells in antiviral defense: function and regulation by innate cytokines. Annu. Rev. Immunol. 17: 189–220. decrease with IL-12, IL-15, and IL-18 treatment. These data 3. Vivier, E., E. Tomasello, M. Baratin, T. Walzer, and S. Ugolini. 2008. Functions would seem inconsistent with the finding that IL-12, IL-15, and of natural killer cells. Nat. Immunol. 9: 503–510. IL-18 treatment increases TACE-mediated cleavage of proteins at 4. Lanier, L. L. 2015. NKG2D receptor and its ligands in host defense. Cancer Immunol. Res. 3: 575–582. the cell surface (6). However, in these previous studies, total 5. Eagle, R. A., I. Jafferji, and A. D. Barrow. 2009. Beyond stressed self: evidence TACE activity was not directly measured; rather, functional TACE for NKG2D ligand expression on healthy cells. Curr. Immunol. Rev. 5: 22–34. 6. Romee, R., B. Foley, T. Lenvik, Y. Wang, B. Zhang, D. Ankarlo, X. Luo, activity was measured by cleavage of membrane proteins at the S. Cooley, M. Verneris, B. Walcheck, and J. Miller. 2013. NK cell CD16 surface cell surface. Consistent with this, we demonstrate that IL-12, expression and function is regulated by a disintegrin and metalloprotease-17 IL-15, and IL-18 treatment increases TACE expression at the cell (ADAM17). Blood 121: 3599–3608. 7. Waldhauer, I., and A. Steinle. 2006. Proteolytic release of soluble UL16-binding surface. Therefore, although total TACE is not increased with the protein 2 from tumor cells. Cancer Res. 66: 2520–2526. cytokine treatment, surface expression of TACE is, allowing for 8. Chitadze, G., J. Bhat, M. Lettau, O. Janssen, and D. Kabelitz. 2013. Generation increased TACE-mediated cleavage at the cell surface. of soluble NKG2D ligands: proteolytic cleavage, exosome secretion and functional implications. Scand. J. Immunol. 78: 120–129. In conclusion, our results demonstrate that NKG2D engagement 9. Verneris, M. R., M. Karimi, J. Baker, A. Jayaswal, and R. S. Negrin. 2004. Role by ULBPs during homotypic NK cell–NK cell contact enhances of NKG2D signaling in the cytotoxicity of activated and expanded CD8+ T cells. a [Published erratum appears in 2015 Blood 125: 2583.] Blood 103: 3065–3072. the production of soluble TNF- in response to the combination of 10. Groh, V., J. Wu, C. Yee, and T. Spies. 2002. Tumour-derived soluble MIC li- IL-12, IL-15, and IL-18. The function of NKG2D signaling in NK gands impair expression of NKG2D and T-cell activation. Nature 419: 734–738. 8 NKG2D SIGNALING BETWEEN HUMAN NK CELLS ENHANCES TNFa RELEASE

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