Fratricide of Natural Killer Cells Dressed with Tumor-Derived NKG2D Ligand

Fratricide of natural killer cells dressed with tumor-derived NKG2D ligand

Kyohei Nakamuraa,b, Masafumi Nakayamaa, Mitsuko Kawanoa, Ryo Amagaia, Tomonori Ishiib, Hideo Harigaeb, and Kouetsu Ogasawaraa,1

aDepartment of Immunobiology, Institute of Development, Aging, and Cancer, Tohoku University, Sendai 980-8575, Japan; and bDepartment of Hematology and Rheumatology, Tohoku University Graduate School of Medicine, Sendai 980-8574, Japan

Edited* by Lewis L. Lanier, University of California, San Francisco, CA, and approved April 24, 2013 (received for review January 4, 2013) The natural killer group 2 membrane D (NKG2D) activating re- the retinoic acid early-inducible protein 1 (Rae-1), H60, and ceptor plays crucial roles not only in host defense against tumors MULT1 in mice; as well as UL-16 binding proteins, and MHC and viral infections, but also in autoimmune diseases. After class I (MHCI) chain-related molecules A (MICA) and B (MICB) NKG2D-mediated activation, Natural killer (NK) cells must be reg- in humans (10). Although NK cells had been considered to lyse ulated to avoid potentially harmful reactivity. However, the neg- cells that have lost MHCI expression through “missing-self” ative regulation of these activated NK cells is poorly understood. recognition (12), expression of NKG2DL renders cells sus- Here, we reveal that the engagement of NKG2D by its ligand elicits ceptible to NK cytotoxicity, irrespective of MHCI expression not only target cell lysis, but also NK cell fratricide. Conventional (13). Upon recognition of NKG2DL, NKG2D recruits its adaptor mouse NK cells underwent cell death when cocultured with RMA proteins DNAX-activating protein 10 (DAP10) and DAP12 in cells expressing the NKG2D ligand retinoic acid early-inducible pro- conjunction with adhesion molecules at the cell–cell contact site tein 1 (Rae-1), but not with RMA cells lacking MHC class I. NK cells called NK cell immunological synapse (NK-IS), which provides from mice deﬁcient for DAP10 and DAP12 or perforin did not a platform for intracellular signal transduction leading to perforin undergo death, highlighting the importance of the NKG2D degranulation (14–17). pathway for NK cell death. However, NKG2D does not transmit Recently, unique functions of the NK-IS have been reported. direct death signals in NK cells. Rather, the interaction between We have demonstrated that upon NK-IS formation, NKG2D NKG2D and Rae-1 allowed NK cells to acquire tumor-derived Rae- undergoes ligand-induced down-modulation for negative feedback ” ” 1 by a membrane transfer process known as trogocytosis, which regulation (18). In addition, upon NK-IS formation, human NK was associated with clathrin-dependent NKG2D endocytosis. NK cells rapidly acquire MICA and MICB from tumor cells (19, 20). cells dressed with Rae-1 were lysed by neighboring NK cells Such dynamic intercellular protein relocalization is mediated by through the NKG2D-induced perforin pathway in vitro and in the transfer of plasma membrane fragments from one cell to an- vivo. These results provide the unique NKG2D function in neg- other during cell–cell contact; a process known as “trogocytosis” ative regulation of activated NK cells. (21). The trogocytosed MICB masks NKG2D on NK cells and suppresses NKG2D-mediated NK cell functions (20). Given that immunological synapse | intercellular membrane transfer | the NKG2DL-expressing cells are susceptible to NK cytotoxicity immune regulation by induced-self recognition, NK cells exogenously acquired NKG2DL might be lysed by other activated NK cells, called atural killer (NK) cells are lymphocytes that mediate rapid fratricide (22). However, this possibility has not previously been Nresponse against virally infected cells and tumor cells with- addressed, and the molecular mechanisms underlying NKG2D- out prior sensitization. Upon activation, NK cells proliferate and mediated trogocytosis have not been previously deﬁned. exert effector functions including perforin-mediated cytotoxicity We therefore asked whether the interaction between NKG2D γ against target cells and cytokine production such as IFN (IFN)- and NKG2DL is involved in NK cell death. Furthermore, we (1). In contrast to the pivotal role of NK cells in host defense, investigated the possibility that NKG2D-mediated trogocytosis is persistent expansion of activated NK cells in benign and malig- a trigger of NK cell fratricide. nant conditions is often associated with systemic autoimmune diseases (2–4). Therefore, NK cells themselves must be regulated Results after activation. However, the fate of activated NK cells has not NK Cell Death Occurs in an NKG2D-Dependent Manner. To inves- been fully understood. tigate whether NKG2D and NKG2DL are involved in NK cell – In IL-2 activated human NK cells, cross-linking stimulation of death, we addressed the relationship between NK cell cytotoxic γ activating Fc receptor CD16 induces not only cytolytic function, activity and NK cell death. We used IL-2–activated C57BL/6 but also suicide (5), indicating that activating receptor could mouse NK cells as effector cells and three target cell lines: transmit the death signal in NK cells under certain conditions. mouse T-cell lymphoma RMA cells (RMA), RMA lacking Additionally, a subpopulation of NK cells undergoes rapid ap- MHCI expression on their cell surface (RMA-S), and RMA optosis upon interaction with the NK-sensitive tumor cell line stably expressing an NKG2DL, Rae-1δ (RMA/Rae-1δ). Consis- K562 (6, 7), although the molecular mechanism of NK cell death tent with previous reports (13, 23), NK cells effectively lysed both remains largely unknown. Given that K562 cells express natural RMA-S and RMA/Rae-1δ but did not lyse parental RMA (Fig. S1). killer group 2 membrane D ligand (NKG2DL) and are susceptible to NKG2D-mediated cytotoxicity (8), NKG2D as well as

CD16 may also elicit activation-induced NK cell death. Author contributions: K.N., M.N., and K.O. designed reseach; K.N. and M.N. performed NKG2D is an extensively characterized activating NK receptor research; M.N., M.K., R.A., T.I., and H.H. analyzed data; and K.N., M.N., and K.O. wrote the (9). NKG2D is expressed on essentially all NK cells, and plays paper. a crucial role in NK cell-mediated effector functions (10). Nev- The authors declare no conﬂict of interest. ertheless, the involvement of NKG2D in activation-induced *This Direct Submission article had a prearranged editor. NK cell death remains unclear. NKG2D recognizes stress- 1To whom correspondence should be addressed. E-mail: [email protected]. induced self ligands on abnormal cells through a process known This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. IMMUNOLOGY as “induced-self” recognition (11). These NKG2DLs include 1073/pnas.1300140110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1300140110 PNAS | June 4, 2013 | vol. 110 | no. 23 | 9421–9426 Downloaded by guest on September 24, 2021 apoptosis-inducing ligand (TRAIL) (24) nor another NK acti- A Gated on R1 BCR1 Gate R2 Gate 104 80 R1 30 40 103 vating receptor DNAX accessory molecule 1 (DNAM-1) (25) is

2 NK alone CFSE 10 6.7% ** E 1 involved in this process (Fig. 1 ). RMA-S are well known to be 10 Cell number ** **

100 0 * 100 101 102 103 104 100 101 102 103 104 104 80 ** ** recognized as missing-self cells to be killed through perforin by R1 20 NK 103 ** * ** * A B 2 ** NK cells (23). However, NK cells did not die (Fig. 1 and ) + 10 12.4% * 1 20 RMA 10 Cell number R2 when lysed RMA-S (Fig. S1 and Fig. 1C), excluding the possi- 100 0 ** 100 101 102 3 104 0 1 2 3 4 4 10 10 10 10 10 10 ** 10 80 10 ** 3 R1 % NK cell death bility that NK cells simply lyse themselves via self-released per- NK 10 + 102 11.6% % Target cell death * 101 forin. We next addressed the possibility that NKG2D directly

RMA-S Cell number R2 100 0 100 101 102 103 104 100 101 102 103 104 transmits death signals in NK cells; however, cross-linking of 104 80 R1 0 0 NK 103 0.3 1 3 10 0.3 1 3 10 NKG2D induced IFN-γ production (Fig. S2A), but not NK cell 102 22.4% + E/T ratio E/T ratio 101 RMA/Rae-1δ Cell number B R2 death (Fig. S2 ), indicating that NKG2D per se does not 100 0 NK+RMA NK+RMA-S NK+RMA/Rae-1δ 100 101 102 103 104 100 101 102 103 104 NK1.1 PI transmit death signals. Collectively, these results indicate that

D E 60 F 30 NK cell death is mediated by the NKG2D-induced perforin ﬁ DIC * * pathway, but NKG2D ligation alone is insuf cient to cause NK ** cell death. 40 20 ** ** ** * ** Cell Surface Expression of Rae-1 on NK Cells Is Induced After Coculture TAMRA ** with RMA/Rae-1δ. We hypothesized that NK cells themselves could 20 10 turn to be target cells after interaction with RMA/Rae-1δ and % NK cell death Annexin V to be lysed by perforin. Thus, we evaluated alterations of the % inhibition of NK cell death cell surface expression of NKG2DL on NK cells. Although 0 0 0.3 1 3 10 Rae-1 was not expressed on NK cells, cell surface expression E/T ratio Merged WT NK was dramatically induced 2 h after coculture with RMA/Rae-1δ

control Ig -/- -/- anti-FasL DAP10 /DAP12 NK anti-TRAIL

anti-NKG2D -/- (Fig. 2). MULT1, another NKG2DL, was not expressed on these

anti-DNAM-1 Prf NK tumor cells, and MULT1 was not induced on NK cells (Fig. 2). Fig. 1. NK cell death is mediated through the NKG2D-induced perforin Thus, upon coculture with RMA/Rae-1δ, NK cells turn to be – pathway. (A C) NK cell death and target cell death in the same culture. NK Rae-1δ–positive, which may be recognized by other NK cells, cells were labeled with CFSE and then cocultured with RMA, RMA-S, or RMA/ Rae-1δ at a 1:1 ratio (A) or the indicated ratios (B and C) for 2 h. Cells were and undergo fratricide. stained with anti-NK1.1 mAb and propidium iodide (PI), and then percent + + + + + cell death of R1-gated NK (PI CFSE NK1.1 cells/CFSE NK1.1 cells) and of + − − − − R2-gated tumor (PI CFSE NK1.1 cells/CFSE NK1.1 cells) was calculated. 104 104 Representative PI staining of NK cells (gray histograms) are shown in A. Percent NK cell death (B) and percent tumor cell death (C) are shown as 103 103 CFSE means plus SDs of triplicates. *P < 0.05, **P < 0.01. (D) TAMRA-labeled NK CFSE 102 102 cells cocultured with RMA/Rae-1δ were stained with FITC-conjugated NK only annexin V and analyzed by confocal microscopy. (Scale bars, 5 μm.) (E) CFSE- 101 101 labeled NK cells were treated with the indicated blocking mAb. Percent 100 100 inhibition of cell death by mAbs was calculated by comparing the percent NK < 100 101 102 103 104 100 101 102 103 104 cell death in the presence of mAb with that in the absence of mAb. *P 0.05; 4 4 − − − − − − 10 10 compared with control rat IgG. (F) NK cells from DAP10 / /DAP12 / ,Prf / ,or WT mice were cocultured with RMA/Rae-1δ, and percent NK cell death was NK 103 103 analyzed. Data represent means plus SDs of triplicates. *P < 0.05, **P < + 102 102 0.01; compared with WT NK. Similar results were obtained in three (A–C) or two (D–F) independent experiments. RMA 101 101

100 100 100 101 102 103 104 100 101 102 103 104 To address NK cell death, we labeled NK cells with 5-(and-6)- 104 104 carboxyfluorescein diacetate succinimidyl ester (CFSE) and 3 measured the percentage of propidium iodide (PI)-positive cells NK 10 103 + + in CFSE NK1.1 NK cells cocultured with tumor cells. Sur- + 102 102 prisingly, within 2 h, more than 20% of NK cells died only when − − RMA-S 101 101 cocultured with RMA/Rae-1δ (Fig. 1 A and B). CFSE NK1.1 RMA/Rae-1δ were lysed by NK cells in an effector/target (E/T) 100 100 0 2 3 4 ratio-dependent manner (Fig. 1C), whereas effector NK cell 10 101 10 10 10 100 101 102 103 104 104 104 death was independent of the E/T ratio (Fig. 1B). Confocal microscopy showed that 5-(and-6)-carboxyfluorescein diacetate NK 103 103 succinimidyl ester (TAMRA)-labeled NK cells turned to be + 102 102 annexin V-positive after cocultured with RMA/Rae-1δ (Fig. 1D). RMA/Rae-1δ 1 101 This NK cell death was significantly inhibited by anti-NKG2D 10 blocking mAb (Fig. 1E). These results suggest that NKG2D and 100 100 Rae-1 interaction induces not only tumor cell death but also NK 100 101 102 103 104 100 101 102 103 104 cell death. Furthermore, NK cells deficient for NKG2D adaptor Rae-1 MULT1 − − − − molecules DAP10 and DAP12 (DAP10 / /DAP12 / ) did not −/− Fig. 2. Cell surface expression of NKG2DL on NK cells before and after undergo cell death (Fig. 1F). Perforin-deficient (Prf ) NK cells δ F coculture with RMA/Rae-1 . CFSE-labeled NK cells were cocultured with tu- did not undergo cell death (Fig. 1 ), suggesting that NK cell mor cells at a 1:1 ratio for 2 h. Then cells were stained with anti–Rae-1 mAb death occurs through the NKG2D-induced perforin pathway. Nei- (Left) or anti-MULT1 mAb (Right). Representative data from three in- ther death-inducing ligand such as Fas ligand and TNF-related dependent experiments are shown.

9422 | www.pnas.org/cgi/doi/10.1073/pnas.1300140110 Nakamura et al. Downloaded by guest on September 24, 2021 Lymphocyte gated Gated on DX5+ A 50 20 D G 5.5 102 B16 s.c. R1 5.8 NK alone

0 0 100 101 102 103 104 100 101 102 103 104 50 40

Cell number 4.9 101 B16/Rae-1ε s.c. R1 29.8

0 0 100 101 102 103 104 100 101 102 103 104 DX5 Rae-1 B Lymphocyte gated Gated on DX5+ 100 100 40 0 2 4 6 24 8.9 Rae-1 expression level (MFI) Time (hour) RMA-S s.c. R1 9.0 0 0 1 2 3 4 0 10 10 10 10 10 100 101 102 103 104 E 100 100 40 6.0 47.2 R1 NK + RMA/Rae-1δ RMA/Rae-1δ s.c. Cell number 42.9 Cell-cell contact 0 0 0 1 2 3 104 0 100 101 102 103 104 10 10 10 10 100 101 102 103 104 DX5 Rae-1 100 5.5 C Cell number Intrasplenic injection Lymphocyte gated Gated on NK1.1+ Transwell plate 200 50 0 RMA 4.1 100 101 102 103 104 ↓ R1 Rae-1 on NK Rag-1-/- mouse 0 0 50 100 101 102 103 104 100 101 102 103 104 F 200 50 RMA/Rae-1δ 8.1 NK + RMA/Rae-1γ

↓ Cell number R1 -/- 0 Rag-1 mouse 0 0 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 NK1.1 Rae-1 Rae-1γ on NK

Fig. 3. Rae-1 is transferred from RMA/Rae-1 to NK cells via trogocytosis. (A and B) B16 (5 × 105), B16/Rae-1e (2 × 106)(A), RMA-S (5 × 105), or RMA/Rae-1δ (1 × + 106)(B) were s.c. inoculated into C57BL/6 mice. Two weeks later, tumor-infiltrating lymphocytes were prepared, and Rae-1 expression on DX5 NK cells was analyzed. Gray and dotted line histograms indicate anti–Rae-1 mAb and isotype control mAb staining, respectively. Numbers indicate the mean fluorescence − − + intensity (MFI). (C) RMA or RMA/Rae-1δ (1 × 107) was injected into spleen of Rag-1 / mice. After 2 h, Rae-1 expression on NK1.1 splenocytes was analyzed as described in B.(D) CFSE-labeled NK cells were cocultured with RMA/Rae-1δ at a 1:1 ratio for different periods of time (10 min, 30 min, 3 h, 6 h, and 24 h). The MFI of anti–Rae-1 mAb staining (closed circles) or isotype control mAb staining (open circles) of CFSE+ NK cells is shown. (E) CFSE-labeled NK cells were cocultured with RMA/Rae-1δ for 2 h together (cell–cell contact) or separated by a semipermeable membrane (Transwell) on a 12-well plate. Then NK cells were stained with isotype control mAb (dotted line histograms), or anti–Rae-1 mAb (gray histograms). Solid line white histograms indicate anti–Rae-1 mAb staining of NK cells without coculture. (F) Cell surface expression of Rae-1γ on NK cells after coculture with RMA/Rae-1γ was analyzed by anti–Rae-1γ specific mAb (gray histogram) or isotype control mAb (dotted line histogram). Solid line white histogram indicates anti–Rae-1γ specific mAb staining of NK cells without coculture. (G) NK cells were stained with biotinylated anti-NK1.1 mAb, followed by DyLight 594-conjugated streptavidin. After 30 min of coculture with RMA/ Rae-1δ, cells were stained with FITC-conjugated anti–Rae-1 mAb. Confocal microscopy imaging of NK cells alone (Left) and NK cells with RMA/Rae-1δ (Right) are shown. (Scale bars, 10 μm.) Representative data from three independent experiments are shown.

NK Cells Acquire Rae-1 from Target Cells via Trogocytosis. To address did not occur on NK cells cultured with RMA/Rae-1δ in whether NK cells become Rae-1–positive after Rae-1–expressing Transwell plates (Fig. 3E). Notably, C57BL/6 mouse NK cells tumor cells in vivo, we inoculated wild-type (WT) C57BL/6 mice turned to be BALB/c allele Rae-1γ–positive when cocultured s.c. with B16/Rae-1e (Fig. 3A) or RMA/Rae-1δ (Fig. 3B). Two with RMA/Rae-1γ, reinforcing the fact that NK cells acquire + weeks after the inoculation, we observed that DX5 NK cells tumor-derived Rae-1 (Fig. 3F). Confocal microscopy revealed infiltrated into tumors, and that these NK cells become Rae-1– that NK cells acquired Rae-1 from RMA/Rae-1δ in a cell–cell positive (Fig. 3 A and B). Because NK cells turn to be Rae-1– contact-dependent manner (Fig. 3G). We next evaluated positive at 2 h coculture with RMA/Rae-1δ in vitro (Fig. 2), we whether NK cells acquire the naturally processed soluble form of next performed the short-term in vivo study. Specifically, we NKG2DL, which has been reported in human, but not in mouse injected RMA/Rae-1δ into spleen of recombination activating (10). Human NK cells also acquire MICA in a cell–cell contact- − − gene-1–deficient (Rag-1 / ) mice that lack mature T and B dependent manner, but not soluble MICA (Fig. S5), suggesting lymphocytes, and found that splenic NK cells turned to be Rae- that NK cells acquire only the membrane-bound form of 1–positive at 2 h after injection (Fig. 3C). We further aimed to NKG2DL. Collectively, these results indicate that NK cells ac- elucidate the mechanism underlying Rae-1 induction on the NK quire tumor-derived Rae-1 via trogocytosis. cell surface in vitro. First, we investigated the kinetics of cell surface expression of Rae-1 on NK cells and found it to occur on Trogocytosis of Rae-1 Is Coupled with Clathrin-Dependent NKG2D both freshly isolated NK cells (Fig. S3) and IL-2–activated NK Endocytosis. Upon ligation, NKG2D accumulates at the NK-IS, cells (Fig. 3D) within 10 min after coculture. Although Rae-1 which provides a platform not only for signal transduction, but transcript was very low but detectable in NK cells (Fig. S4)as also for trogocytosis (20). Therefore, we examined the in- well as a wide variety of normal cells (26, 27), the rapid ap- volvement of NK-IS components in trogocytosis. DAP10 and pearance of Rae-1 (Fig. 3D) lead us to address the possibility DAP12, which associate with NKG2D at the NK-IS (17), were that NK cells acquire Rae-1 from target cells, rather than involved in the trogocytosis of Rae-1 (Fig. 4A). Intercellular through de novo synthesis. We and others have recently reported adhesion molecule-1 (ICAM-1), which is recruited to NK-IS that NK cells have the ability to acquire cell surface proteins (30), was not involved in the trogocytosis (Fig. 4B). At NK-IS, from target cells in a cell–cell contact-dependent manner, re- a coactivation receptor, 2B4 is engaged by CD48 on target cells

ferred to as trogocytosis (21, 28, 29). Likewise, acquisition of (31). Both ligand and receptor is involved in the trogocytosis IMMUNOLOGY Rae-1 expression occurred only with direct cell–cell contact and (Fig. 4B). We next investigated NKG2D signaling leading to

Nakamura et al. PNAS | June 4, 2013 | vol. 110 | no. 23 | 9423 Downloaded by guest on September 24, 2021 + cells in spleen, suggesting that Rae-1acq NK cells are eliminated A 50 C + B100 100 A B acq 2.7 in vivo (Fig. 5 and ). We next adoptively transferred Rae-1 WT NK 92.1 − * ** ** NK1.1 BALB/c NK cells into C57BL/6 mice or NK-depleted 0 100 101 102 103 104 C57BL/6 mice that had been injected with anti-NK1.1 mAb. 50 ** ** ** ** acq+ 3.6 50 ** ** ** 5050 These Rae-1 BALB/c NK cells were eliminated in C57BL/6 DAP10-/-/12-/- NK 16.2 **

Cell number mice, but not in NK-depleted C57BL/6 mice (Fig. S7), suggesting 0 + 3 4 acq 100 101 102 10 10 % Trogocytosis % Trogocytosis that Rae-1 NK cells are eliminated by NK cells in vivo. To 50 + 0 acq -/- 3.4 0 further address whether Rae-1 NK cells directly lysed by Prf NK 100.3 00 12.512.5 2525 5050 100100 other activated NK cells, called fratricide, we next performed in 0 + 100 101 102 103 104 acq anti-2B4 LY294002 vitro cytotoxicity assay using sort-puriﬁed Rae-1 NK cells Rae-1 control Ig anti-CD48 anti-ICAM-1 anti-NKG2D Piceatannol (Fig. S8A) as target cells on which Rae-1 remains until 24 h after + LY294002+Piceatannol trogocytosis (Fig. S8B). As we expected, Rae-1acq NK cells were − −

D E anti-CD48+anti-2B4 lysed by WT NK cells, but not by NK cells from DAP10 / / 60 − − − − 100100 DAP12 / or Prf / mice, in an E/T ratio-dependent manner, Control C D Filipin complex (Fig. 5 and ). We next addressed whether cell surface ex- 40 Chlorpromazine pression of Rae-1 is sufficient for NK cell fratricide by using 5050 Hypertonic media NK cells from Rae-1 transgenic mice (tg) (Fig. S9A)astarget 20 cells. Rae-1 tg NK cells are lysed by NK cells in an E/T ratio- − − dependent manner, but not by NK cells from DAP10 / / −/− −/− B 0 0 DAP12 or Prf (Fig. S9 ). Overall, these results suggest 00:00:2 1:0 4:0 00 0: 00 102: : 00 0:4 0 0 24 0 2 4 that NK cell fratricide is mediated via the NKG2D-induced perforin pathway (Fig. 5E). Fig. 4. Trogocytosis of Rae-1 is coupled with clathrin-dependent NKG2D − − − − − − down-modulation. (A) WT, DAP10 / /DAP12 / ,orPrf/ NK cells were Discussion δ cocultured with RMA/Rae-1 for 1 h, and then Rae-1 acquisition by NK cells In this study, we revealed a unique pathway for NK cell frat- was analyzed as described in Fig. 3E.(B and C) WT NK cells were treated with the indicated blocking mAbs (B) or indicated inhibitors (C) for 30 min, and ricide: NK cells acquire NKG2DL from target tumor cells via then cocultured with RMA/Rae-1δ as described in A. Percent trogocytosis was trogocytosis and are subsequently lysed by other NK cells calculated as (MFI of anti–Rae-1 mAb staining in the presence of inhibitor)/ through the NKG2D-induced perforin pathway (Fig. 5E). Our (MFI of anti–Rae-1 mAb staining in the absence of inhibitor). Data represent findings may explain the previously observed phenomena that means plus SDs of triplicates. *P < 0.05, **P < 0.01, compared with the ab- NK cells undergo death when they interact with tumor cells (6, 7). sence of the inhibitor. (D and E) NK cells were cocultured with RMA/Rae-1δ We further showed that trogocytosis of Rae-1 is coupled with in the presence or absence of the indicated inhibitors or in the medium clathrin-dependent NKG2D endocytosis. containing 0.45 M sucrose for the indicated periods of time. Then cell surface Trogocytosis was first observed 40 y ago on mouse T cells, expression of NKG2D and Rae-1 on NK cells was analyzed. Delta MFI was which acquire MHCII from B cells (37). However, the molecular calculated by subtracting MFI of isotype control mAb staining from MFI of specific mAb staining. Similar results were obtained in three independent mechanism and physiological relevance of trogocytosis remained experiments. unknown for a long period. Recently, it was revealed that the trogocytosis of MHCII by T-cell receptor (TCR) requires the driving force of clathrin-independent TCR internalization, which trogocytosis by using chemical inhibitors listed in Fig. S6A. is dependent on small GTPases TC21 and Rho G-mediated DAP10 and DAP12 recruit PI3-kinase (PI3K) and spleen tyro- phagocytic machinery (38, 39). Unlike TCR trogocytosis, NKG2D sine kinase (Syk), respectively (10). Combined inhibition of trogocytosis may require the clathrin-dependent NKG2D in- both pathways by LY294002 (PI3K inhibitor) with piceatannol ternalization. In contrast to rapid internalization of NKG2D, the (Syk inhibitor) markedly inhibited the trogocytosis, although trogocytosed ligand Rae-1 remains on the NK cell surface at LY294002 alone also inhibited at high dose (Fig. 4C). U0126 a substantial level for at least 24 h (Fig. S8B). Therefore, tumor- [mitogen-activated protein kinase kinase/extracellular signal- experienced NK cells probably lose NKG2D-mediated effector regulated kinase (MEK/ERK) inhibitor] or PP2 (Src inhibitor) function and are subsequently lysed by other NK cells. Indeed, sort- significantly inhibit the trogocytosis only at high dose (Fig. S6B). purified Rae-1–dressed NK cells alone do not die (Fig. 5C;E/T Because we have previously demonstrated that ligand-induced ratio of 0), and these NK cells were lysed by newly added NK cells down-modulation of NKG2D is mediated by clathrin-dependent in an E/T ratio-dependent manner (Fig. 5C), suggesting that Rae- endocytosis (18), we next examined the effect of various en- 1–dressed NK cells do not attack each other. NK cells that com- docytosis inhibitors (32–34) on trogocytosis (Fig. 4 C and D). mitted fratricide may sequentially acquire Rae-1 and turn to be Both cytochalasin D (actin polymeization inhibitor) and methyl- target cells, generating a negative feedback loop. When NK cells β-cyclodextrin (broad endocytosis inhibitor) inhibited Rae-1 trogo- were cocultured with RMA/Rae-1δ, NK cells died most at E/T cytosis (Fig. 4 D and E and Fig. S6D). Interestingly, chlorpromazine ratio of 1 (Fig. 1B). The trogocytosis and fratricide may occur (an inhibitor of clathrin-dependent endocytosis), but not filipin most efficiently at E/T ratio of 1. A previous study showed that (an inhibitor of caveolae-dependent endocytosis), inhibited both human NK cell death occurred in an E/T ratio-independent NKG2D down-modulation and trogocytosis of Rae-1 (Fig. 4 D manner when cocultured with K562 (6). It is possible that trogo- and E, and Fig. S6D). In addition, hypertonic media that block cytosis-mediated NK cell fratricide may be one of the mechanisms clathrin-coated pit formation (35, 36) showed both inhibitions of the NK cell death. (Fig. 4 D and E and Fig. S6D). These results suggest that NKG2D- NK cells and cytotoxic T lymphocytes (CTLs) were considered mediated trogocytosis is coupled with clathrin-dependent NKG2D to be perforin resistant (40, 41), whereas perforin-mediated down-modulation. fratricide of CTLs was frequently observed (42–44). Therefore, besides Fas ligand, perforin may also play an important role for NK Cells Eliminate Rae-1–Dressed NK Cells in Vitro and in Vivo. To activation-induced cell death, in particular fratricide. Fratricide + address the fate of Rae-1–dressed (Rae-1acq ) NK cells in vivo, is proposed to contribute to memory T-cell homeostasis (45). + − we injected CFSE-labeled Rae-1acq NK cells or Rae-1acq NK For instance, CTLs acquire MHCI from their targets (42), and − − cells into spleen of Rag-1 / mice. After 4 h, we found a signifi- this trogocytosis is considered to promote self-recognition by + cant decrease in the percentage of transferred Rae-1acq NK CTLs, which may be involved in down-regulating the immune

9424 | www.pnas.org/cgi/doi/10.1073/pnas.1300140110 Nakamura et al. Downloaded by guest on September 24, 2021 A C Gated on CFSE+ NK E Lymphocyte gated acq- acq+ NK cell Target cell 104 Rae-1 NK Rae-1 NK 1.42% 80 80 NKG2D Rae-1 3 E/T ratio Rae-1acq- NK 10 11.2% 17.9% NK1.1 0 102 (Target only) -/- 0 0 Rag-1 mouse 0 1 2 3 4 0 1 2 3 4 101 10 10 10 10 10 10 10 10 10 10 Target killing Target cell death 80 80 100 0 1 2 3 4 27.1% 10 10 10 10 10 3 11.5% 104 0 0 acq+ 0.14% 1 2 3 4 0 1 2 3 4 Rae-1 NK 103 100 10 10 10 10 10 10 10 10 10 80 80 Trogocytosis of Rae-1 102 NKG2D down-modulation Rag-1-/- mouse 10 13.2% 32.1% 101 Adjacent NK cell Rae-1-dressed NK cell 0 0 0 1 2 3 4 0 1 2 3 4 0 10 10 10 10 10 10 10 10 10 10 10 40 60 100 101 102 103 104 CFSE 30 21.3% 51.1%

0 0 NK cell fratricide NK cell death 100 101 102 103 104 100 101 102 103 104 B D PI 3.0 Rae-1acq- NK 60 ** * Rag-1-/- mouse cells + 2.0 ** Rae-1acq+ NK

NK1.1 ** + 30 1.0 Rag-1-/- mouse %CFSE % Target NK cell death 0 0 E/T ratio : 3 10 30 3 10 30 3 10 30 Effector : WT NK DAP10-/-/12-/- NK Prf-/- NK Target : Rae-1acq- NK Rae-1acq+ NK

Fig. 5. Rae-1–dressed NK cells are efﬁciently lysed by other NK cells both in vitro and in vivo. (A and B) CFSE-labeled NK cells (5 × 106) were cocultured with − − RMA or RMA/Rae-1δ for 15 min. Then, these cells were injected into spleen of Rag-1 / mice (n = 4, each group). After 4 h, splenocytes were stained with anti- + + NK1.1 mAb, and the percentage of CFSE NK1.1 cells in the lymphocyte gate was calculated. (C and D) CFSE-labeled NK cells were cocultured with RMA or RMA/Rae-1δ for 15 min, and then CFSE+ NK cells were sort puriﬁed as shown in Fig. S8A. These NK cells were used as target cells and cocultured with effector WT NK cells for 2 h. Percent cell death of target NK (PI+CFSE+ cells/CFSE+ cells) was calculated. Data are presentedasthemeansplusSDsoftriplicates. − *P < 0.05, **P < 0.01, compared with Rae-1acq NK cells. Representative data from two independent experiments are shown. (E) Model for NK cell fratricide via NKG2D-mediated trogocytosis. Interaction between NKG2D on NK cells and Rae-1 on target cells results in target cell lysis and trogocytosis of Rae-1 by NK cells. NK cells dressed with tumor-derived Rae-1 are lysed by other NK cells via the NKG2D-induced perforin pathway.

response (46). Likewise, we propose that NKG2D trogocytosis- mAb (clone GK1.5) and anti-CD8 mAb (clone 53-6.7), followed by magnetic mediated NK cell fratricide could contribute to maintenance of beads coated with goat anti-mouse Ig and goat anti-rat Ig Abs (Qiagen). NK cell homeostasis. If tumor cells actively give their NKG2DL Then CD4-positive cells, CD8-positive cells, and surface Ig-positive cells were to NK cells for immune escape, the perturbation of NKG2D removed by magnetic cell sorting. Subsequently, NK cells were positively se- lected by using phycoerythrin (PE)-labeled DX5 (anti-CD49b) mAb (BioLegend) trogocytosis-mediated NK cell fratricide may enhance NK cell- and anti–PE-conjugated MACS beads (Miltenyi Biotec). Purified NK cells mediated antitumor immunity. However, it may also cause NK were cultured in growth medium supplemented with recombinant human cell-mediated host injury. Given that trogocytosis of Rae-1 IL-2 (rhIL-2; 1,000 units/mL; Wako) for 5 d. requires NKG2D signal leading to generation of the pulling force, NK cells may intentionally acquire tumor-derived Rae-1 Cell Lines. James C. Ryan (University of California, San Francisco) kindly for the purpose of activation-induced regulation. Although the provided the RMA (H-2b-positive) lymphoma cells and the RMA-S (H-2b- specific inhibition of trogocytosis is impossible at this moment, deficient) cells. RMA cells stably expressing Rae-1γ (RMA/Rae-1γ) or Rae-1δ δ e e identification of a specific molecule that regulates trogocytosis (RMA/Rae-1 ), and B16 melanoma stably expressing Rae-1 (B16/Rae-1 ), were established as previously described (13). would reveal the physiological relevance of trogocytosis. – × Materials and Methods Trogocytosis and NK Fratricide Assay. CFSE-labeled IL-2 activated NK cells (1 105 per well) and Rae-1–expressing target cells (1 × 105 per well) were Mice. C57BL/6 and BALB/c female mice (6 wk old) were obtained from cocultured in a 96-well flat-bottom plates for indicated periods at 37 °C. Cells CLEA Japan. Toshiyuki Takai (Tohoku University, Sendai, Japan) kindly were stained with anti–Rae-1 mAb (186107; R&D Systems), and acquisition of fi fi + provided DAP10 and DAP12 double-de cient mice. Perforin-de cient Rae-1 by CFSE NK cells was analyzed by FACSCanto II (BD Biosciences). As – fi mice and Rag-1 de cient mice were obtained from The Jackson Laboratory. for the NK fratricide assay, the cytotoxic activity against sort-purified Rae-1– Rae-1e transgenic mice were generated as previously described (47). These dressed NK cells was analyzed. fi mice were maintained under speci c pathogen-free conditions and used Further details for materials and experimental procedures are described in according to the guidelines of the Tohoku University Institutional Animal SI Materials and Methods. Care and Use Committee. ACKNOWLEDGMENTS. We thank Dr. Lewis Lanier for helpful suggestions, Isolation of Mouse NK Cells. NK cells were prepared from mouse splenocytes as Chika Takahashi and Madoka Itabashi for technical assistance, and Hiromi IMMUNOLOGY described previously (28). In brief, splenocytes were incubated with anti-CD4 Yoshida for cell sorting. This work was supported by Grants-in-Aid for

Nakamura et al. PNAS | June 4, 2013 | vol. 110 | no. 23 | 9425 Downloaded by guest on September 24, 2021 Scientiﬁc Research from the Japanese Ministry of Education, Culture, for Scientiﬁc Research from the Ministry of Health, Labor, and Welfare Sports, Science, and Technology (to K.O., M.K., and M.N.); and Grants-in-Aid of Japan [H22-meneki-ippan-004 (to K.O.) and 005 (to M.N.)].

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