Recognition and Killing of Human and Murine Pancreatic β Cells by the NK Receptor NKp46

This information is current as Chamutal Gur, Jonatan Enk, Sameer A. Kassem, Yaron of September 27, 2021. Suissa, Judith Magenheim, Miri Stolovich-Rain, Tomer Nir, Hagit Achdout, Benjamin Glaser, James Shapiro, Yaakov Naparstek, Angel Porgador, Yuval Dor and Ofer Mandelboim

J Immunol 2011; 187:3096-3103; Prepublished online 17 Downloaded from August 2011; doi: 10.4049/jimmunol.1101269 http://www.jimmunol.org/content/187/6/3096 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2011/08/18/jimmunol.110126 Material 9.DC1 References This article cites 29 articles, 9 of which you can access for free at: http://www.jimmunol.org/content/187/6/3096.full#ref-list-1

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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 © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Recognition and Killing of Human and Murine Pancreatic b Cells by the NK Receptor NKp46

Chamutal Gur,*,†,1 Jonatan Enk,*,1 Sameer A. Kassem,†,‡ Yaron Suissa,x Judith Magenheim,x Miri Stolovich-Rain,x Tomer Nir,x Hagit Achdout,*,2 Benjamin Glaser,†,‡ James Shapiro,{ Yaakov Naparstek,† Angel Porgador,‖,# Yuval Dor,x and Ofer Mandelboim*

Type 1 diabetes is an incurable disease that is currently treated by insulin injections or in rare cases by islet transplantation. We have recently shown that NKp46, a major killer receptor expressed by NK cells, recognizes an unknown ligand expressed by b cells and that in the absence of NKp46, or when its activity is blocked, diabetes development is inhibited. In this study, we investigate whether NKp46 is involved in the killing of human b cells that are intended to be used for transplantation, and we also thoroughly characterize the interaction between NKp46 and its human and mouse b cell ligands. We show that human b cells express an unknown ligand for NKp46 and are killed in an NKp46-dependent manner. We further demonstrate that the expression of the Downloaded from NKp46 ligand is detected on human b cells already at the embryonic stage and that it appears on murine b cells only following birth. Because the NKp46 ligand is detected on healthy b cells, we wondered why type 1 diabetes does not develop in all individuals and show that NK cells are absent from the vicinity of islets of healthy mice and are detected in situ in proximity with b cells in NOD mice. We also investigate the molecular mechanisms controlling NKp46 interactions with its b cell ligand and demonstrate that the recognition is confined to the membrane proximal domain and stalk region of NKp46 and that two glycosylated residues of NKp46, Thr125 and Asn216, are critical for this recognition. The Journal of Immunology, 2011, 187: http://www.jimmunol.org/ 3096–3103.

ype 1 diabetes (T1D) is currently an incurable disease that within a short period of time (2, 3). The reasons for this rejection is mainly treated by daily injections of insulin. In rare are largely unknown. T situations, when metabolic instability and severe hypo- Early reports suggest that in addition to T cells, NK cells are glycemia persist, human islet transplantation is considered as involved in the pathogenesis of T1D (4–6). The NK cell cyto- possible treatment (1) (2). However, such treatment is still not very toxicity is complex and regulated by both inhibitory receptors, effective, and in almost all cases, .90% of the islets are rejected which recognize mainly MHC class I proteins, and activating re- by guest on September 27, 2021 ceptors (7–10). The most prominent NK inhibitory receptors are those that recognize the HLA-C proteins, as all of the HLA-C proteins can be divided into two groups, C1 and C2, that are *The Lautenberg Center for General and Tumor Immunology, Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem recognized by killer Ig-like receptor (KIR) 2DL2 and KIR2DL1, 91120, Israel; †Department of Medicine, Hadassah-Hebrew University Hospital, Jer- respectively (11). Another prominent inhibitory receptor is leu- usalem 91120, Israel; ‡Department of Endocrinology and Metabolism, Hadassah- x kocyte Ig-like receptor-1 (LIR1), which recognizes a broad range Hebrew University Hospital, Jerusalem 91120, Israel; Department of Developmental Biology and Cancer Research, Institute for Medical Research Israel-Canada, Hebrew of MHC class I proteins (11, 12). The NK activating receptors University-Hadassah Medical School, Jerusalem 91120, Israel; {Healthcare Solutions recognize pathogen-derived, stress-induced, tumor-derived, and (Alberta Innovates-Health Solutions), University of Alberta, Edmonton, Alberta T6G ‖ even self-ligands (13–16). Prominent among the killer receptors is 2C8, Canada; Shraga Segal Department of Microbiology and Immunology, Ben- Gurion University of the Negev, Beer-Sheva 84105, Israel; and #National Institute NKp46 (NCR1 in mice), as it is expressed exclusively by NK and for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva NK-like cells (8, 17, 18). The only ligands identified so far for 84105, Israel NKp46 are viral hemagglutinins (HA) (19, 20), and the recogni- 1 C.G. and J.E. contributed equally to this work. tion of viral HA by NKp46 is mediated mainly via a2,6-linked 2 Current address: Department of Infectious Diseases, Israel Institute for Biological sialic acid residues carried by NKp46 and largely relies on the Research, Ness-Ziona, Israel. highly conserved sugar-carrying residue of NKp46, Thr225 (19). Received for publication May 6, 2011. Accepted for publication July 11, 2011. Recently, we showed that NKp46 recognizes an unknown ligand This work was supported by grants from the Israeli Science Foundation and the expressed by pancreatic b cells (21). We showed in murine models Israeli Science Foundation (Morasha), a Croatia Israel Research grant, a Ministry of Science, Culture and Sport-German Cancer Research Center Research grant, the Ro- that NK cells kill pancreatic b cells in an NKp46-dependent setrees trust, the Israel Cancer Association (20100003), an Israeli Cancer Research manner and demonstrated that T1D development could be pre- Fund professorship grant, and by the Association for International Cancer Research (all to O.M.). O.M. is a Crown professor of Molecular Immunology. vented by blocking NKp46 activity using active immunizations (21). Address correspondence and reprint requests to Prof. Ofer Mandelboim, The Lauten- berg Center for General and Tumor Immunology, Hebrew University-Hadassah Med- In this study, we extensively and thoroughly analyze the inter- ical School, Institute for Medical Research Israel-Canada, Jerusalem 91120, Israel. actions between NKp46 and its unknown ligand expressed by both E-mail address: [email protected] human and murine b cells. We demonstrate that human b cells, The online version of this article contains supplemental material. which are intended to be used for transplantation, express the Abbreviations used in this article: CEA, carcinoembryonic Ag; HA, hemagglutinin; unknown ligand of NKp46 and are killed in an NKp46-dependent KIR, killer Ig-like receptor; LIR1, leukocyte Ig-like receptor-1; T1D, type 1 diabetes. manner. We also describe the molecular mechanisms controlling Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 the NKp46 interaction with its mouse and human b cell ligands www.jimmunol.org/cgi/doi/10.4049/jimmunol.1101269 The Journal of Immunology 3097 and identify the binding site on NKp46 that is involved in these Ig, with its membrane proximal domain, which also contains the interactions. stalk region and is named NKp46D2-Ig, and with its membrane distal domain, named NKp46D1-Ig. A schematic representation of Materials and Methods NKp46 is presented in Supplemental Fig. 1. As can be seen in Fig. Mice, sera, and fusion proteins 1, isolated human b cells that express insulin (detected by staining All experiments were performed in a specific pathogen-free unit of the with polyclonal anti-insulin Abs; Fig. 1A) also express a putative Hadassah Medical School (Ein-Kerem, Jerusalem) in accordance with the ligand for NKp46, and the NKp46 binding is confined to the guidelines of the ethical committee. The double-transgenic Insulin-rtTA; b membrane proximal domain D2 (Fig. 1B). TET-DTA mice and the conditional ablation of pancreatic cells were b generated as previously described (22). All fusion proteins used in this We also investigated whether the isolated human cells are study—the NCR1-Ig, NKp46-Ig, NKp46D2-Ig, NKp46D1-Ig, KIR2DL1- recognized by the KIR2DL1-Ig, KIR2DL2-Ig, and LIR1-Ig in- Ig, KIR2DL2-Ig, LIR1-Ig, NKp46-T225V-Ig, NKp46-T125V-Ig, and hibitory receptor fusion proteins. Surprisingly, whereas LIR1-Ig NKp46-N216V-Ig—were generated in COS-7 cells and purified by affinity binding was detected, little or no staining was observed with either chromatography using a protein G column, as previously described (20). Human islets intended to be used for transplantations were derived from the KIR2DL-1-Ig or the KIR2DL2-Ig (Fig. 1B). Because similar healthy donors. The anti-NKp46 and control anti-carcinoembryonic Ag results were observed with b cells obtained from four separate (CEA) sera were generated as previously described (21). donors and because all fusion proteins stained their corresponding Immunohistochemical and immunofluorescence staining MHC ligands on various 721.221 transfectants (12) (Supplemental Fig. 2), we concluded that the HLA-C is either not expressed on Paraffin-embedded sections of pancreatic tissues were prepared from female these human b cells or that its expression levels are too low to be NOD mice at various weeks of age (including the embryonic period) from 8– Downloaded from 12-wk-old BALB/C mice and from double-transgenic Insulin-rtTA;TET- detected by the appropriate NK inhibitory receptors. Currently, DTA mice. Archival human pancreatic tissue sections were obtained from there is no specific mAb that recognizes HLA-C, and we are autopsies (male and females) ranging in age from 17 wk of gestation to therefore unable to differentiate between these two options. We adulthood. All fetuses, infants, and adults died as a result of diseases not also stained the b cells derived from various donors for the ex- related to the pancreas, and all autopsies were performed due to medical pression of the NKG2D ligands MICA, MICB, ULBP1, ULBP2, reasons according to accepted procedures. The immunohistochemical and immunofluorescence stainings were performed as previously described (21). and ULBP3 and observed no staining (data not shown). http://www.jimmunol.org/ Isolation of b cells, FACS staining with fusion proteins, The killing of primary human b cells is NKp46 dependent isolation of human NK cells, in vitro cytotoxicity assay, and Because we observed that human b cells express a ligand for CD107 assays NKp46 and because they cannot be recognized by the HLA-C– Isolation of murine b cells was performed as previously described using binding NK inhibitory receptors, we wondered whether they will apoolofb cells derived from 8–10-wk-old BALB/C mice (n . 8/ be killed by NK cells in an NKp46-dependent manner. We b experiment) (21). For FACS staining, human and murine cells therefore initially used the same primary human b cells, described (75,000/well) were incubated on ice with the appropriate fusion protein (5 mg/well) and the biotinylated anti-mouse GLUT-2 mAb (BAM1440, clone above, in CD107a degranulation assays. As can be seen in Fig. 1, 205115; R&D Systems), or with the guinea pig anti-insulin Ab for 2 h. incubation of NK cells with human b cells leads to NK cell de- Cells were then incubated with fluorochrome-conjugated secondary Abs granulation (Fig. 1C–E). Importantly, as can be seen in Fig. 1E, by guest on September 27, 2021 for 30 min and analyzed by FACS using CellQuest software. the NK cell degranulation was NKp46 dependent, as it was re- Human NK cells were isolated from peripheral blood using the Human NK Cell Isolation Kit and the autoMACS instrument (Miltenyi Biotec) duced upon preincubation of bulk NK cells with anti-NKp46 sera according to the manufacturer’s instructions. Purified NK cells from pe- (produced by injecting mice with NKp46-Ig) (21) (Supplemental ripheral blood were plated at 1 cell/well in 96 U-well plates in RPMI 1640 Fig. 3) as compared with preincubation of the NK cells with medium supplemented with 10% human serum, 50 U/ml recombinant control sera (produced by injecting mice with CEA-Ig) (21) human IL-2 (Boehringer Mannheim, Indianapolis, IN), and 1 ng/ml PHA 3 4 (Supplemental Fig. 3). Similar results were obtained with human for 2 wk. Irradiated feeder cells (2.5 10 allogeneic PBMCs from two b donors and 5 3 103 RPMI 8866 cell line in each well) were added. Pro- cells derived from two donors. liferating clones were verified to be positive for CD56 and negative for However, NK cell degranulation does not always correlate with CD3 markers and were subsequently combined and grown for an addi- actual killing of the target cells. Therefore, to make sure that hu- tional week to generate bulk polyclonal NK cell lines. Purity of NK cell man b cells are indeed killed in an NKp46-dependent manner, we lines was routinely verified to be .99%. For the in vitro cytotoxicity assay, 35 took advantage of the fact that human b cells are metabolically human islets were labeled with [ S]methionine, for 24 h at 37˚C, sepa- 35 rated with Cell Dissociation Solution Non-enzymatic 13 (Sigma, C5914), active and labeled them with [ S]methionine for 24 h. We then and incubated for 5 h at 37˚C with effector NK cells at various E:T ratios isolated human NK cells from the PBLs of various donors, grew as previously described (20). The CD107a assays were performed as them in culture, preincubated them with anti-NKp46 sera and previously described (21). control sera (as described above), and then incubated them with Detection of NK cells in situ the labeled b cells to determine whether human b cells are killed For detection of NK cells in situ, pancreases were dissected out from eight in an NK-46–dependent manner. As can be seen in Fig. 1F,effi- 9- to 10-wk-old female nondiabetic NOD and BALB/C mice, immersed in cient killing of human b cells was noted, and such killing was fixative solution for 3 h, and rinsed for 24 h in PBS containing 30% sucrose. NKp46-dependent, as it could be blocked by using the anti- The tissue was frozen, cryostat sectioned into 14-mm thickness, and thaw- NKp46 sera mentioned above (Fig. 1F). Thus, primary b cells, b mounted onto glass slides. The pancreatic cells were detected by poly- which are used for transplantation, express an unknown NKp46 clonal guinea pig anti-mouse insulin (Abcam). The tyramide signal am- plification kit (PerkinElmer) was used to visualize NKp46 by polyclonal ligand and are killed in an NKp46-dependent manner. goat Abs against mouse NKp46 (catalog number AF2225; R&D Systems) b as previously described (23). Expression of NKp46 ligand on human cells before and following birth Results The human b cell distribution in the pancreas undergoes pro- b Primary human cells express a ligand for NKp46 gressive changes during development and in the uterus; around the To test whether primary human islets that are used for human age of 6 mo, the b cells are organized into islets (24). Therefore, transplantation express an unknown ligand for NKp46, we isolated we next studied whether the expression of the NKp46 ligand is b cells from such islets and stained them with the intact NKp46- associated with the organization of the b cells into islet structures. 3098 b CELL RECOGNITION BY NKp46 Downloaded from http://www.jimmunol.org/

FIGURE 1. Primary human b cells express a ligand for NKp46 and are killed in an NKp46-dependent manner. A, Staining of human b cells derived from healthy human donor for insulin. B, The human b cells that are shown in A were stained with various domains of NKp46 (red histograms) and with inhibitory receptors (blue histograms). The filled gray histogram is the background staining performed with secondary mAb only. Representative of four independent experiments. C, Expression of CD107a on human NK cells incubated with primary human b cells (characterized in A) at different E:T ratios. Summary of three stainings is shown in D. E, Bulk NK cell cultures were preincubated with anti-NKp46 sera (Blocking NKp46 sera, produced by injecting mice with NKp46-Ig) or with control sera (Control sera, produced by injecting mice with CEA-Ig) directed against CEA and then incubated with human b cells at an E:T ratio of 1:3. CD107a on the NK cells was monitored by FACS. Representative of two independent experiments. *p = 0.013. F, Bulk NK cell cultures were preincubated with anti-NKp46 sera (gray filled triangle) or with control sera (produced against CEA-Ig; black filled square) and were then 35 incubated with [ S]methionin-labeled human b cells for 5 h. Shown are mean values of primary human b cell killing (represented as percentages) 6 SD. by guest on September 27, 2021 Error bars (SD) are derived from triplicates. Data from one of two independent assays are shown. *p = 0.038, **p = 0.009, ***p = 0.0049.

For this purpose, we stained human pancreases derived from Interaction of NKp46 with the mouse NKp46 ligand fetuses, infants, and adults with anti-insulin p-Ab and NKp46D2-Ig Working with primary human b cells is very difficult, not only (we used the D2 domain of NKp46 for this purpose because it gives because they are hard to manipulate in vitro, but also because of a better staining as compared with the intact NKp46-Ig). As can the limited supply. Thus, it is quite essential to determine the be seen in Fig. 2, the expression of the NKp46 ligand is already murine b cell’s ligand properties. In this regard, we have recently detected during the embryonic period, almost concomitantly with shown that murine b cells express the unknown ligand for NKp46 the detection of insulin (Fig. 2A). From that point on, the ex- and that this ligand can be recognized by both the human and pression of the NKp46 ligand is consistent, and its expression is murine NKp46 (21). Therefore, we next investigated whether the b increased with age concomitantly with the increase in cell mass D2 domain (including the stalk) of the human NKp46 is also in- (Fig. 2A,2B). As presented in Fig. 2A and 2B, the NKp46 staining volved in the recognition of mouse b cells by using two modali- is very similar to the insulin staining. However, a closer exami- ties: FACS and immunohistochemistry. For immunohistochem nation of the staining with higher magnification revealed that, istry, pancreatic BALB/C paraffin sections were stained with the indeed, the insulin staining merged with the NKp46D2-Ig staining NKp46D1-Ig and NKp46D2-Ig fusion proteins, and for the FACS (Fig. 2C, left panels, red staining alone could hardly be detected). staining, b cells derived from BALB/C mice were stained with However, it is also shown that the NKp46D2-Ig staining is not a combination of fusion proteins and anti-mouse GLUT-2 mAb. identical to the insulin staining, and areas inside the cells can As can be seen in Fig. 3A and 3B, in both assays, b cell staining be observed that are free from insulin and are stained with was obtained only with the usage of the NKp46D2-Ig, indicating NKp46D2-Ig (observe the green D2 speckles, presumably corre- that similar to human b cells, this portion of the NKp46 receptor sponding to distinct cellular vesicles that do not contain insulin contains the binding site for the murine b cell ligand. Similar [red], in Fig. 2C). Nevertheless, to make sure that the costaining results were obtained with b cells derived from C57BL/6 and observed with NKp46D2-Ig and insulin is not an artifact that nondiabetic NOD mice (n . 8/experiment). occurred due to Ab cross-reactivity, we repeated the staining with the same Abs and the control NKp46D1-Ig, as can be seen in Fig. NK cells are not found in the endocrine tissue of normal 2C, right panels; in this case, only insulin and DAPI staining is pancreases detected (no green or yellow colors). In addition, we verified that In our recent publication (21) and in this study, we have demon- the secondary Abs used in this assay recognize only their appro- strated that the unknown NKp46 ligand is expressed by all b cells priate primary Abs (data not shown). derived from healthy humans, healthy mice, and prediabetic NOD The Journal of Immunology 3099

FIGURE 2. Expression of the NKp46 ligands during human pan- creatic development. Paraffin-embed- ded sections derived from various human embryonic stages (indicated in weeks [W] following inception, A; indicated in months [M] and years [Y], B) and in the adult pan- creas (B). The left panels represent staining with anti-insulin Ab (red). The middle panels represent stain- ing with the NKp46D2-Ig (green), and the right panels represent merge sig- nal (yellow). C, Representative stain- ing of human paraffin-embedded sections derived from the human embryonic pancreatic tissue (27 wk)

with the anti-insulin p-Ab (red), Downloaded from NKp46D2-Ig (green, left panels), NKp46D1-Ig (green, right panels), and DNA staining (blue). Original magnification 3400 (A, B), 3600 (C, upper panels), and 31800 (C, lower panels). Scale bars, 50 mm.

Representative of three independent http://www.jimmunol.org/ experiments.

mice. We therefore wondered why diabetes does not develop in used the anti-NKp46 mAb in immunofluorescence staining of every individual and hypothesized that NK cells are not found in cryosections derived from female BALB/C or NOD mice pan- the vicinity of healthy pancreatic islets. To investigate this, we creases at the prediabetic stage. Confirming our hypothesis, NK by guest on September 27, 2021

FIGURE 3. Detection of the NKp46 ligand and NK cells. A, b cells were isolated from BALB/C mice and stained with the various fusion proteins (indicated at the top of the figure) together with anti-mouse GLUT-2 Ab. Representative of five independent experiments (n . 8/experiment). B, Paraffin- embedded sections of pancreatic tissues were obtained from BALB/C mice and stained with NKp46 D2-Ig or NKp46 D1-Ig. Original magnification 3200. Representative of five independent experiments. C and D, Cryosections derived from 9- to 10-wk-old female BALB/C mice (original magnification 3400) and prediabetic NOD mice (D, upper panels, original magnification 3400; lower panels, original magnification 31200). NK cells (green) were detected by using anti-mouse NKp46 mAb, insulin in red, and nuclear staining in blue. Figures show two representative images of ∼50 sections performed for each group of mice. Scale bars, 50 mm. The large green spot observed in the upper panels of D is probably artifactual staining of the content of a pancreatic duct. 3100 b CELL RECOGNITION BY NKp46 cells were detected in situ, in close proximity with b cells only in specific and observed no staining with NKp46D1-Ig (Fig. 4D, the pathological NOD mice pancreases and only rarely observed right panel). In addition, we verified that the secondary Abs used in the exocrine pancreatic tissue of the normal mice (examples are in this assay recognize only their appropriate primary Abs (data shown in Fig. 3C and 3D). not shown). Thus, because the human and mouse NKp46 ligands seem to be similar (see above) and because the NKp46 staining Expression of NKp46 ligand on murine b cells before and does not always correlate with the insulin staining, we concluded following birth that insulin is not the NKp46 ligand. Indeed, in separate experi- The results obtained so far suggest that human and mice b cells ments, purified insulin was not recognized by NKp46 (not shown). express a similar ligand for NKp46. We therefore tested whether b the expression pattern of the mouse NKp46 ligand is similar to Expression of the NKp46 ligand following cell ablation and that of the human b cells, hoping that such an investigation would regeneration provide us with essential clues concerning the identity of the To gain further information concerning the identity of the NKp46 NKp46 ligand. For this purpose, we performed immunofluores- ligand, we investigated whether its expression is stable (i.e., cence staining for the expression of insulin and the unknown whether under certain conditions the expression of the ligand is NKp46 ligand in pancreases obtained from NOD mice during the altered). Because we observed that ligand expression persists on embryonic phase, immediately after birth, and up to the late stage b cells that survived the immune cell attack in NOD mice (21), of insulitis. As can be seen in Fig. 4A, a few insulin-producing we tested whether its expression would be maintained following cells are present in the embryonic pancreas as early as day 14.5 of cytotoxic destruction of the islets and subsequent b cell re- embryonic development, but the organization into islets is only generation. For this purpose, we used a combined transgenic Downloaded from seen at around day 18.5. Interestingly, and in contrast to the hu- mouse in which, upon the administration of doxycycline, rtTA man (Fig. 2), in the mouse, little or no expression of the NKp46 induces the expression of diphtheria toxin A, causing b cell ap- ligand was observed during the embryonic period (Fig. 4A). optosis and diabetes (22). As can be seen in Fig. 5, following However, following birth, the expression of the NKp46 ligand treatment with doxycycline for 1 wk, a severe loss of b cells was merged with that of insulin (Fig. 4B,4C). Similar to the human noted (Fig. 5, upper left panel, detected by anti-insulin staining). b staining, a closer examination of the staining with higher magni- Interestingly, following such ablation, the remaining few cells http://www.jimmunol.org/ fication revealed that the NKp46D2-Ig staining of the mouse b that still express insulin also express the NKp46 ligand, and the cells is not identical to the insulin staining, and areas inside the expression of the NKp46 ligand merged almost completely, but cells can be observed that are either free from insulin or free from not entirely, with that of insulin (Fig. 5, upper center and right the NKp46-ligand (Fig. 4D). Again, we verified that the staining is panels). Furthermore, when the mice were left to recuperate for by guest on September 27, 2021

FIGURE 4. Expression of the NKp46 ligands dur- ing murine pancreatic development. A–C, Paraffin- embedded sections derived from various embryonic stages (indicated as embryonic days [E] in A), from the neonatal stage (indicated as days [D] in B), and from adult female NOD mice (indicated in days [D] or weeks [W] in C) were coincubated with anti-insulin Ab and NKp46-Ig. The left panels represent staining with anti-insulin Ab (red). The middle panels repre- sent staining with the NKp46-Ig (green), and the right panels represent merge signal (yellow). D, Repre- sentative staining of murine paraffin-embedded sec- tions derived from prediabetic female NOD mice with anti-insulin pAb (red), NKp46D2-Ig (green, left panel), NKp46D1-Ig (green, right panel), and DNA staining (blue). Representative of three independent experiments. Original magnification 31200. Scale bars, 50 mm. The Journal of Immunology 3101

on Asn216 (Supplemental Fig. 1) (19, 25). Because we have pre- viously demonstrated that Thr225 plays a critical role in the rec- ognition of the influenza virus HA (19), we wondered whether the glycosylated residues of NKp46 are involved in its binding to b cells. We initially treated the NKp46-Ig fusion protein with neuraminidase, which cleaves sialic acid residues, and observed that it did not affect the binding to b cells (data not shown). As previously reported (19), it severely abrogated the binding of NKp46-Ig to influenza-infected cells (data not shown). Never- theless, we continued our research and investigated whether the glycosylated residues of NKp46 are involved in its binding to the b cell ligand in a sialic acid-independent manner. To test this, we mutated each of the three glycosylated residues of the NKp46 FIGURE 5. The expression of the NKp46 ligand is maintained follow- and replaced them with Valin, cloned them in frame with Ig, ing b cell ablation and regeneration. Paraffin sections were derived from 4- and produced the corresponding fusion proteins NKp46T225V, wk-old double-transgenic Insulin-rtTA;TET-DTA mice after doxycycline- NKp46T125V, and NKp46N216V. The various fusion proteins induced ablation (upper panels, ablation) and 2 mo after doxycycline were then used to stain murine and human b cells. As can be seen withdrawal (lower panels, recovery). The left panels represent staining in Fig. 6A, whereas the mutation in Thr225 did not significantly with anti-insulin Ab (red). The middle panels represent staining with the influence the NKp46 recognition of murine b cells, the two other Downloaded from NKp46-Ig (green), and the right panel represents merge signal (yellow). mutations, at Thr125 and at Asn216, completely abolished the Representative of two independent experiments. Original magnification NKp46-Ig binding to murine b cells, using two modalities: im- 3400. Scale bar, 50 mm. munohistochemistry (Fig. 6A, upper panels) and FACS staining several months, the expression of the NKp46 ligand was detected (Fig. 6A, lower panels). The residual staining observed with the almost entirely on all insulin-producing cells (Fig. 5, bottom NKp46N216V-Ig using FACS is probably nonspecific, as no panels). These experiments led us to conclude that once the staining was detected with this fusion protein in the immunohis- http://www.jimmunol.org/ NKp46 ligand is expressed, its expression is stable. Thus, although tochemistry experiments. Importantly, a similar staining pattern these experiments did not provide us with further information was observed with human b cells, and mutating the 216 or the 125 concerning the identity of the ligand, they suggest that almost at residues completely abolished NKp46 binding (Fig. 6B). To verify 125 216 any time point, b cells are at risk for being attacked by NK cells that the Thr and the Asn mutations did not somehow disrupt through NKp46-mediated activation. the NKp46 structure, thus interfering with binding to its ligand, we stained influenza-infected cells with the same fusion proteins. Two glycosylated residues are crucial for the binding of NKp46 Importantly, mutating the Thr125 or the Asn216 residues did not b to its human and murine cell ligand affect binding to the infected 721.221 cells, whereas as previously The NKp46 protein displays two putative O-linked glycosyla- reported (19), the mutation in the Thr225 residue severely reduced by guest on September 27, 2021 tions, on Thr125 and on Thr225, and one N-linked glycosylation the binding (Fig. 6C). Thus, whereas the sialic acid residues of

FIGURE 6. Glycosylated residues of NKp46 are crucial for its binding to b cells. For the immunohistochemical staining, paraffin-embedded sections of pancreatic tissues obtained from BALB/C mice (A) and from human autopsies (B) were stained with the mutated proteins indicated above the figure. Representative of three independent experiments. Original magnification 3600. For the FACS experiments, the various NKp46-mutated proteins (indicated above the histograms) were incubated either with purified mouse b cells (A, bottom panels) or influenza PR8-infected 721.221 cells (C). The gray histogram represents the background secondary Ab staining only. The mean fluorescence intensity and Xmean values are indicated in the corresponding histograms. Representative of three independent experiments. 3102 b CELL RECOGNITION BY NKp46

Thr225 are critical for the binding of NKp46 to HA (this study and expression of the NKp46 ligand, b cells are in risk of being Ref. 19), the glycosylated residues Asn216 and Thr125 are critically attacked by NK cells via the activation of NKp46. involved in the binding of NKp46 to its human and mouse b cell It is still unclear why b cells of both humans and mice express ligand in a sialic acid-independent manner. a ligand that can potentially harm them. It is possible that the NKp46 ligands in the pancreas resemble foreign ligands recog- nized by NKp46 and that NK cells attack b cells unintentionally Discussion because of this molecular mimicry. Indeed, infectious diseases Unfortunately, despite decades of research, T1D, which affects have been suggested as potential triggers for many autoimmune millions of people around the world, is still an incurable disease. To diseases, and a link between viruses and type 1 diabetes has been date, the only treatment that releases T1D patients from their in- demonstrated (26, 27). sulin dependency is allogeneic transplantation of islets obtained Finally, we show that two glycosylated residues of NKp46, from deceased donors. This treatment, however, still suffers from Thr125 and Asn216, are critical for its b cell recognition. These many difficulties, and, in addition to scarcity of pancreatic islets, observations, together with our previous ones (19), suggest that one of the major obstacles is the loss of a large portion of the NKp46 interacts with at least three different ligands by using transplanted islets within the first weeks of transplantation, even different means of recognition. Although Thr225 is critical for the while using immunosuppressive agents (2, 3). The reasons ac- NKp46 interaction with HA and some tumor cells (19), the two counting for the transplanted islets’ rejection are largely unknown. other residues, Thr125 and Asn216, but not Thr225, are important for In this study, we show that human b cells that are used for the NKp46 recognition of its human and murine b cell ligand. transplantation express an unknown ligand for NKp46, do not The identification of the NKp46 ligands is a very difficult task. Downloaded from express the NKG2D ligands, and are not recognized by the NK One of the problems that has so far prevented identification the inhibitory receptors KIR2DL1 and KIR2DL2, which together NKp46 ligands is, as shown in this study and previously (19, 21, 28, recognize the entire spectrum of all HLA-C proteins. This makes 29), ligand diversity. Still, the identification of the b cell ligand for the human b cells vulnerable to NK cell attack, and, indeed, hu- NKp46 is of particular importance, as it can possibly lead to the man NK cells degranulate, and b cells are killed in an NKp46- development of novel therapy modalities for T1D.

dependent manner. These results are in agreement with our recent http://www.jimmunol.org/ publication, in which we showed that NKp46 is involved in the Disclosures killing of murine b cells and that pathogenic murine NK cells The authors have no financial conflicts of interest. extracted from the pancreases of NOD mice are degranulated (21). The important implication of this research is that blocking of NKp46 function during diabetes development and following hu- References man b cell transplantation might help prevent T1D development 1. Shapiro, A. M., J. R. Lakey, E. A. Ryan, G. S. Korbutt, E. Toth, G. L. Warnock, N. M. Kneteman, and R. V. Rajotte. 2000. Islet transplantation in seven patients and islet graft rejection. with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive We have demonstrated, both in this study and previously (21), regimen. N. Engl. J. Med. 343: 230–238. that b cells of healthy mice and humans express an unknown li- 2. Ricordi, C., and T. B. 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