The activating receptor NKp46 is essential for the development of type 1 diabetes

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1 A r t i c l e s The ctivting receptor NKp46 is essentil for the development of type 1 dietes Chmutl Gur 1,2, Angel Porgdor 3,6, Morn Eloim 1, Roi Gzit 1, Sr Mizrhi 1, Nom Stern-Ginossr 1, Hgit Achdout 1, Horms Ghdilly 1, Yuvl Dor 4, Tomer Nir 4, Victori Doviner, Oren Hershkovitz 3, Michl Mendelson 3, Ykov Nprstek 2,6 & Ofer Mndeloim 1,6 21 Nture Americ, Inc. All rights reserved. The mechnism of ction of nturl killer (NK) cells in type 1 dietes is still unknown. Here we show tht the ctivting receptor NKp46 recognizes mouse nd humn lignds on pncretic et cells. NK cells ppered in the pncres when insulitis progressed to type 1 dietes, nd NKp46 enggement y et cells led to degrnultion of NK cells. NKp46-deficient mice hd less development of type 1 dietes induced y injection of low dose of streptozotocin. Injection of solule NKp46 proteins into nonoese dietic mice during the erly phse of insulitis nd the predietic stge prevented the development of type 1 dietes. Our findings demonstrte tht NKp46 is essentil for the development of type 1 dietes nd highlight potentil new therpeutic modlities for this disese. Type 1 dietes mellitus is multifctoril utoimmune disese in which insulin-producing et cells in pncretic islets re destroyed y utorective T cells. The most intensively studied model of utoimmune type 1 dietes is the nonoese dietic (NOD) mouse, which develops dietes spontneously fter vrile period of insulitis, similr to humn type 1 dietes. Inflmmtion of pncretic islets (insulitis) is oserved in 4- to -week-old NOD mice, ut dietes does not develop until 1 2 weeks lter, even with mssive infiltrtion of the pncretic islets y T cells 1,2. An dditionl ccepted model of experimentl utoimmune dietes in mice is the induction of dietes y multiple injections of low dose of streptozotocin (LDST) 3,4. Streptozotocin cuses dietes y direct et cell cytotoxicity s well s y initition of T cell medited utoimmune ttck of et cells 4,. Indeed, doptive trnsfer of ctivted splenocytes from LDST-treted mice induces dietes in untreted helthy mice. Although type 1 dietes is considered T cell medited disese, severl studies hve proposed role for the innte immune system in the pthogenesis of this disese. Some dt indicte n essentil contriution of mcrophges to the onset of type 1 dietes 6, nd other reserchers hve found tht nturl killer (NK) cells infiltrte the islets of NOD mice 7. Islet inflmmtion medited minly y NK cells hs lso een reported in humn type 1 dietes 8, nd ltertions in the NK cell comprtments of ptients with type 1 dietes t the onset of the disese or fter long-term hyperglycemi hve een oserved 9. The proportion of NK cells, their numers nd the timing of their entry into the pncres correlte with the severity of type 1 dietes in trnsgenic NOD mice 1. In ddition, depletion of NK cells in trnsgenic NOD mouse models of ccelerted type 1 dietes sustntilly inhiits dietes development 1,11. However, the moleculr mechnisms of the involvement of NK cells in type 1 dietes re still unknown. NK cells hve een detected in trget orgns of ptients suffering from utoimmune diseses 12 nd re le to ttck utologous cells NK cells recognize trget cells vi diverse rry of ctivting nd inhiitory receptors, nd delicte lnce etween inhiitory nd ctivting signls tightly regultes NK cell ctivtion 16. Activting receptors, including the nturl cytotoxicity receptors (NCRs) nd NKG2D, promote NK cell ctivtion 16. The NCRs, which include NKp3, NKp44 nd NKp46, re expressed lmost exclusively on NK cells, wheres NKG2D is lso expressed on other lymphocytes, such s CD8 + T cells 16,17. NKp46 is considered the most specific NK cell mrker for which n orthologous protein (NCR1) hs een found in mice 18,19. Indeed, even cells tht re not cytotoxic nd secrete interleukin 22 (IL-22) re considered n NK cell suset ecuse of the presence of NKp46 (ref. 2). Whether NK cell ctivting receptors re involved in type 1 dietes is still n open question. Although lockde of NKG2D hs een shown to prevent dietes development in NOD mice 21, this effect ws ttriuted to CD8 + T cells nd the exct mechnism of NKG2D ction is still controversil 17,21,22. NKp46, in contrst, is importnt in the defense ginst pthogens 18,23 ; however, the role of NKp46 nd NCR1 in utoimmune diseses nd the cellulr lignds recognized y this receptor re still unknown. 1 The Lutenerg Center for Generl nd Tumor Immunology, Herew University Hdssh Medicl School, Institute for Medicl Reserch Isrel Cnd, Jeruslem, Isrel. 2 Deprtment of Medicine, Hdssh Herew University Hospitl, Jeruslem, Isrel. 3 The Shrg Segl Deprtment of Microiology nd Immunology nd the Ntionl Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Isrel. 4 Developmentl Biology nd Cncer Reserch, Herew University Hdssh Medicl School, Institute for Medicl Reserch Isrel Cnd, Jeruslem, Isrel. Deprtment of Pthology, Hdssh Herew University Hospitl, Jeruslem, Isrel. 6 These uthors eqully contriuted to this work. Correspondence should e ddressed to O.M. (oferm@ekmd.huji.c.il) or A.P. (ngel@gumil.gu.c.il). Received 8 June; ccepted 19 Novemer; pulished online 2 Decemer 29; doi:1.138/ni.1834 nture immunology volume 11 numer 2 ferury

2 21 Nture Americ, Inc. All rights reserved. Figure 1 NKp46 recognizes lignd (or lignds) on et cells. Identifiction of et cells isolted from BALB/c mice, C7BL/6 mice nd predietic femle NOD mice t 8 or 14 weeks of ge (n = 7 mice), stined with CEA-Ig (fusion protein (FP) control),, or NKG2D-Ig nd identified y stining with nti-glut-2. Numers in qudrnts indicte percent positive cells in ech. Dt re representtive of three independent experiments (summry of ll experiments, Supplementry Fig. 1). Here we demonstrte tht NKp46 nd NCR1 specificlly recognize humn nd mouse pncretic et cells nd slivry glnds nd tht NK cells degrnulte fter interction with mouse et cells in n NKp46-dependent mnner. Dietes development ws impired in the sence of NKp46, nd we oserved the highest percentge of NK cells in the pncres t the time when insulitis develops into dietes (the predietic stge). Injection of solule NKp46 vrints into femle NOD mice oth in the erly stge of insulitis nd in the lte predietic stge prevented the development of dietes lmost entirely. Thus, our results demonstrte tht NKp46 hs criticl role in dietes progression. RESULTS Pncretic et cells express NKp46 lignd(s) As NCR1 is the only NCR expressed in mice, we investigted its involvement in mouse models of type 1 dietes. Becuse the cellulr lignds for NCR1 nd NKp46 re still unknown, we tested whether NKp46 nd NCR1 would recognize lignd(s) on pncretic et cells y using fusion proteins of NKp46 nd NCR1 with immunogloulin ( nd, respectively) 18,24. As negtive controls, we used truncted extrcellulr portion of NKp46 lcking the ligndinding domin (NKp46D1-Ig) 2 nd nother irrelevnt fusion protein contining the protein crcinoemryonic ntigen (CEA-Ig). As positive control, we used the fusion protein NKG2D-Ig. We isolted et cells from femle BALB/c, C7BL/6 nd NOD mice nd doulestined the cells with mouse ntiody to GLUT-2 (nti GLUT-2; this specificlly mrks et cells) nd the vrious immunogloulin fusion proteins. Bet cells derived from ll mouse strins tested were recognized y the mouse nd fusion proteins nd, s expected ecuse of species specificity, the most efficient inding ws otined with mouse (Fig. 1 nd Supplementry Fig. 1). The stining of et cells with nd ws specific, s we found no stining with the control fusion protein CEA-Ig (Fig. 1) or NKp46D1-Ig (fusion protein lcking the NKp46 inding domin; dt not shown). In greement with pulished results demonstrting tht NKG2D on CD8 + T cells is involved in et-cell recognition 17,21, et cells were recognized y NKG2D-Ig. However, not ll et cells expressed NKG2D lignds nd the expression of the NKG2D lignds decresed sustntilly during the progression of dietes (Fig. 1 nd Supplementry Fig. 1). Notly, most of the et cells derived from ll mice strins were recognized y, nd expression of the NCR1 lignd remined constnt during dietes development (Fig. 1). Indeed, we found four supopultions of et cells expressing different mounts of GLUT-2 nd NCR1 lignds (Supplementry Fig. 1). To further confirm tht NKp46 recognizes specific et-cell lignds, we used pulished reporter ssy of BW mouse thymom cells 24. In this system, the extrcellulr portion of NKp46 is fused to the trnsmemrne nd til domins of mouse CD3ζ (NKp46- CD3ζ; Supplementry Fig. 2) nd thus lignd recognition leds to secretion of mouse IL-2. We found sustntil secretion of IL-2 in NKp46-CD3ζ trnsfected BW cells incuted with et cells derived from ll mouse strins (Supplementry Fig. 2). Smll mounts of Anti-GLUT-2 Anti-GLUT-2 Anti-GLUT-2 BALB/c NOD 14 weeks Anti-GLUT C7BL/6 NOD 8 weeks NKp46-lg NCR1-lg NKG2D-lg 27 IL-2 were produced y prentl BW cells incuted with et cells nd y NKp46-CD3ζ trnsfected BW cells incuted with HeL humn cervix crcinom cells (Supplementry Fig. 2), pncretic exocrine tissue derived cells or peripherl lood lymphocytes (dt not shown). To demonstrte tht lignds for NKp46 lso exist on humn et cells nd tht specific stining cn e oserved in the endocrine tissue of the whole pncres, we used immunohistochemicl stining. We found intense stining of et cells in islets of pncretic tissues derived from humns, femle NOD mice nd BALB/c mice (Fig. 2). In femle NOD mice, we found insulitis mnifested y mononucler cell infiltrtion, nd the residul pncretic islet cells were stined y. To investigte the importnce of the NKp46 lignd in the pthogenesis of type 1 dietes, we did immunohistochemicl stining of NOD islets efore nd fter the development of insulitis, including the emryonic period. We did not detect NKp46 lignds in islets t emryonic dy 2 (E2; Fig. 2). However, NKp46 lignds grdully ppered during the postntl stge nd were present efore the development of insulitis (week 2) nd throughout the progression of dietes (weeks 6 nd 14; Fig. 2). Insulitis in the type 1 dietes of NOD mice is ccompnied y utoimmune silitis, nd NKp46 lignds were lso expressed in slivry glnds of femle NOD nd BALB/c mice (Fig. 2c). We found mononucler cell infiltrtion of slivry glnds in NOD mice (Fig. 2c). No other tissues exmined in femle NOD nd BALB/c mice were recognized y (Fig. 2d). To support the oservtion tht the lignd for NKp46 in the pncretic tissue is specificlly expressed on insulin-producing et cells nd not on other islet cells (for exmple, glucgon- or somtosttinproducing cells), we did doule- nd triple-immunofluorescence stining of pncretic tissue derived from BALB/c mice (Fig. 2e), NOD mice (Fig. 2f) nd humns (Fig. 2g). We detected sustntil overlp of stining with nd nti-insulin, which indicted tht NKp46 uniquely stins et cells. We found no stining with the control fusion protein NKp46D1-Ig (Fig. 2e g) or CEA-Ig (dt not shown), nd there ws no overlp for stining with nti-somtosttin volume 11 numer 2 ferury 21 nture immunology

3 21 Nture Americ, Inc. All rights reserved. or nti-glucgon nd (Fig. 2h). Other norml tissues exmined were not recognized y (Fig. 2i). Together, the four different methods indicte tht specific lignd for NKp46 is expressed on et cells in humn nd mice. Bet cells induce NKp46-dependent degrnultion The two min functions of NK cells re cytotoxicity nd the secretion of cytokines, including interferon-γ (IFN-γ) nd tumor necrosis fctor. Our next im, therefore, ws to determine whether NKp46 signling ws ctivted y et cells. To mesure the induction of cytokine secretion, we used NK cells from Ncr1 gfp/gfp nd Ncr1 +/gfp mice 18. In these mice, reporter gene encoding green fluorescent protein (GFP) is inserted into the Ncr1 locus nd thus Ncr1 is knocked out nd ll NK cells re green; Ncr1 +/gfp mice hve NK cell function similr to tht of wild-type mice 18. To ssy cytokine secretion from NK cells, we isolted GFP-expressing NK cells from the splenocytes of Ncr1 +/gfp nd Ncr1 gfp/gfp mice nd incuted them together with et cells derived from NOD, BALB/c nd C7BL/6 mice. Neither IFN-γ (Fig. 3) nor tumor necrosis fctor (dt not shown) ws secreted regrdless of whether NK cells expressed NCR1 or not. However, e Humn NOD BALB/c NOD BALB/c BALB/c pncres Anti-insulin Merge Emryonic Preinsulitis (week 1) Preinsulitis (2 weeks) Erly insulitis (6 weeks) Lte insulitis (14 weeks) f NOD pncres Anti-insulin Ncr1 gfp/gfp NK cells secreted less IFN-γ thn did Ncr1 +/gfp NK cells when incuted together with mouse rhdomyosrcom cell line. Thus, we concluded tht the interction of NKp46 with its lignd on et cells does not led to cytokine secretion. Next, we mesured NK degrnultion 26,27 (which indictes moiliztion of CD17 to the cell surfce), rther thn direct cytotoxicity, ecuse mouse et cells do not proliferte much t ll nd thus it is lmost impossile to lel them with rdioctive isotopes. We incuted NK cells from the spleens of Ncr1 +/gfp nd Ncr1 gfp/gfp mice (Supplementry Fig. 3) with et cells derived from BALB/c nd NOD mice. We found significntly less degrnultion in the Ncr1 gfp/gfp cells incuted with et cells derived from ech mouse strin (Fig. 3 nd Supplementry Fig. 3). In contrst, Ncr1 +/gfp nd Ncr1 gfp/gfp NK cells showed similr degrnultion in response to mouse YAC-1 lymphom cells (which re killed in n NCR1- independent mnner 18 ; Fig. 3 nd Supplementry Fig. 3). We found miniml NK cell degrnultion in response to the negtive control humn HeL cells. Finlly, we exmined the in vivo degrnultion stte of pthogenic pncretic NK cells, which re present in the islets of NOD mice c Merge g Humn pncres Anti-insulin d Lung Muscle Liver Spleen Thyroid NOD BALB/c Merge h Anti-insulin Anti-somtosttin Merge Figure 2 recognizes mouse nd humn et cells in situ. () Prffin-emedded sections of pncretic tissues otined from nondietic humn utopsy (top) nd from femle NOD nd BALB/c mice, stined with or Anti-insulin Anti-glucgon Merge NKp46D1-Ig (). Originl mgnifiction, 4 (humn), 2 (mouse) or 4 (insets). () Prffin-emedded sections of pncretic tissues otined t E2 (top; rrows Humn thyroid Humn spleen indicte islets) nd from femle NOD mice during the course of dietes development. Originl mgnifiction, 1 (min imges) or 4 (insets). (c) Slivry glnds derived from femle NOD nd BALB/c mice nd stined with or NKp46D1-Ig (). Originl mgnifiction, 4 (top) or 4 (middle nd ottom). (d) Tissues derived from femle NOD nd BALB/c mice nd stined with. Originl mgnifiction, 1 (lung, liver nd thyroid) or 4 (muscle nd spleen). (e g) Prffin-emedded sections of pncretic tissues derived from BALB/c mice (e), femle NOD mice (f) nd nondietic humn utopsy (g) nd incuted with nti-insulin (green) nd or NKp46D1-Ig (; red). Scle rs, 1 µm. (h) Prffin-emedded sections of pncretic tissue derived from BALB/c mice nd incuted with nti-insulin (green), (red) nd nti-somtosttin or nti-glucgon (lue). (i) Thyroid nd spleen smples from nondietic humn utopsy, stined with. Blue, nucler stining with the DNA-interclting dye DAPI (4,6-dimidino-2-phenylindole). Results re representtive of six (), four (,e g), three (c,d,i) or two (h) independent experiments. i nture immunology volume 11 numer 2 ferury

4 Figure 3 NKp46-medited killing of et cells. () Enzyme-linked immunosorent ssy of IFN-γ production y purified NK cells from Ncr1 gfp/gfp mice (KO) or Ncr1 +/gfp mice (HET) ctivted with polyinosinic-polycytidylic cid nd incuted for 48 h with et cells derived from C7BL/6, BALB/c or predietic femle NOD mice. RMS, methylcholnthrene-induced rhdomyosrcom cell line (positive control). P <. (Student s t-test). Dt re representtive of two independent experiments. () Flow cytometry nlysis of splenic NK cells cultured together with et cells from NOD or BALB/c mice or with YAC-1 or HeL cells (effector/trget rtio, 1:1), then stined with llophycocynin-conjugted nti-cd17, gted on NK cells (GFP + CD3 ). CD17 + cells re presented s percent of totl NK cells. P <. (Student s t-test). Dt re representtive of three independent experiments. (c) NK cells otined from pncretic islets (PNK) or the pncretic lymph nodes (PLN NK) of 12-week-old predietic femle.18 c HET HET KO KO NOD mice (n = 6 7) nd stined with llophycocynin-conjugted nti-cd17; plots re gted on NCR1 + cells. CD17 + cells re presented s percent of totl NK cells. P <.1 (Student s t-test). Dt re representtive of two independent experiments. IFN-γ (ng/ml) NOD BALB/c C7BL/6 RMS CD17 + cells (%) YAC-1 HeL NOD BALB/c CD17 + cells (%) PLN NK PNK 21 Nture Americ, Inc. All rights reserved. during dietes development (discussed elow). We isolted NK cells from the pncretic lymph nodes nd et cell islets of predietic femle NOD mice (12 weeks old) nd stined them for CD17 expression. We detected sustntil dergnultion of the pthogenic pncretic islets NK cells ut little or no degrnultion of pncretic lymph node NK cells (Fig. 3c). In greement with the results reported ove, we found no IFN-γ secretion from the pthogenic pncretic islet NK cells (dt not shown). Thus, the pthogenic NK cells present in vivo in the pncretic islets hd degrnulted. Impired dietes development in the sence of NCR1 Our next gol ws to test the function of NKp46 in the development of type 1 dietes in vivo. We injected sex- nd ge-mtched Ncr1 gfp/gfp nd wild-type mice intrperitonelly for consecutive dys with streptozotocin nd mesured lood glucose concentrtions from dy 7 up to 4 d fter injection. In the sence of NCR1, dietes development ws significntly impired (Fig. 4). Hyperglycemi, defined s nonfsting lood glucose concentrtion of >2 mg/dl in two sequentil mesurements, ws less severe in the Ncr1 gfp/gfp dietic mice (Fig. 4). NCR1-deficient mice lso showed less-severe insulitis, s determined y pulished pthologicl insulitis scle 28 (Supplementry Fig. 4). These results indicte tht NKp46 is importnt for dietes development nd islet destruction in the LDST model. Appernce of NK cells in the pncres during type 1 dietes As the unknown lignd for NKp46 is expressed on norml et cells, why does dietes not develop in every individul? Our hypothesis ws tht NK cells nd T cells tht re normlly not found in the pncres pper in this orgn fter dietes development. To test our hypothesis, we monitored the ppernce of NK cells in the pncretic tissues in two mouse models of type 1 dietes. We trcked NK cells in the pncres of NOD mice during the emryonic period (E2), preinsulitis (3 4 weeks of ge), erly insulitis (6 8 weeks of ge), predietes (12 14 weeks of ge; mice with norml fsting glucose concentrtions ut pthologicl intrperitonel Dietes-free mice (%) 1..8 KO WT Blood glucose (mg/dl) WT 1 KO Time fter LDST injection (d) Time fter LDST injection (d) glucose tolernce test) nd lte overt dietes (2 3 weeks fter the dignosis of dietes). The highest percentge (round 4%) of NK cells expressing NCR1 in the pncres ws in the predietic stge (Fig. ). We lso monitored NK cell ppernce (y GFP expression) in the pncretic tissues of LDST-injected Ncr1 gfp/gfp mice during the emryonic period (E2), preinsulitis (dy ; the dy of LDST injection), erly insulitis (dy 7 fter LDST injection), predietes (dy 9 fter LDST injection) nd lte overt dietes (dy 4 fter LDST injection). We found the most NK cells in the pncres in the predietic stge, on dy 9 fter the injection, the sme dy t which the trnsition from insulitis to dietes usully strts in this model (Figs. 4 nd ). The few NK cells oserved in the pncres during the emryonic period nd the preinsulitis stge in oth models were proly contminting lymphocytes. By monitoring CD3 expression, we found tht like NK cells, T cells were normlly not found in the pncres nd tht they ppered in the pncres concomitntly with NK cells (dt not shown). Thus, in norml conditions, despite the fct tht pncretic et cells of oth humn nd mice express lignds for NKp46, dietes does not develop, proly ecuse NK cells nd T cells re sent from the pncres. NKp46 proteins prevent type 1 dietes when injected erly Our next im ws to demonstrte tht NKp46 is indeed involved in dietes development in the NOD mouse model nd, in prllel, to develop new therpeutic tool for the tretment of type 1 dietes. Tretment of NOD mice with nondepleting NKG2D-specific monoclonl ntiodies ttenutes the development of dietes y impiring the function nd migrtion of utorective CD8 + T cells 21. Unfortuntely, however, no locking, nondepleting ntiody directed ginst mouse NKp46 is ville. However, injection of the fusion protein results in the production of NKp46-specific ntiodies tht lock the killing of virus-infected cells 24. Therefore, we decided to induce locking NKp46-specific ntiodies in NOD mice y repeted injection of the fusion protein. We used nd nd, to prevent nonspecific inding, we lso used n dditionl version of the mouse NCR1 receptor tht lcks the complementnd Fc-inding sites (). Figure 4 Impired dietes development in the sence of NKp46. () Kpln-Meier nlysis of the development of dietes in Ncr1 gfp/gfp mice (KO) nd Ncr1 +/+ mice (WT) fter streptozotocin injection (LDST). P <.1 (log-rnk test). Dt re representtive of three independent experiments. () Blood glucose concentrtions up to 4 d fter the first streptozotocin injection. Dt re representtive of three independent experiments (error rs, s.e.m.). 124 volume 11 numer 2 ferury 21 nture immunology

5 LDST model NOD model GFP Anti-mNCR1 1 4 Emryo Preinsulitis Erly insulitis Predietes Lte dietes NK cells (%) 6 NOD model Emryo Pre-insulitis Erly insulitis Pre-dietes Lte dietes NK cells (%) LDST model Emryo Pre-insulitis Erly insulitis Pre-dietes Lte dietes Figure Appernce of NK cells in the pncres during the development of dietes. NK cells in the pncres of femle NOD mice (NOD model) or Ncr1 gfp/gfp mice injected with streptozotocin (LDST model), identified y stining with nti mouse NCR1 (NOD model) or s GFP + cells (LDST model). Cells were stined during severl stges of insulitis nd dietes development; for ech stge, lymphocytes were purified from two to three pncretic tissues derived from femle NOD mice nd Ncr1 gfp/gfp mice, except t the emryonic nd the preinsulitis stges, for which eight to ten pncretic tissues were used. P <. (Student s t-test). Dt re from three independent experiments (men nd s.d.). 21 Nture Americ, Inc. All rights reserved. We injected femle NOD mice intrperitonelly with the vrious fusion proteins (t dose of. g per kg ody weight) or twice week, strting from 6 weeks of ge. Mice treted with lone egn to develop dietes t 1 weeks of ge, nd 67% were dietic y 24 weeks, s expected in this model (Fig. 6). At this point, we stopped the immunogloulin tretment. We did not find dietes in ny NOD mice treted with the fusion proteins during the first 19 weeks of tretment (Fig. 6). Most of the fusion protein treted NOD mice remined vitl (Supplementry Movies 1 nd 2) nd disese-free up to 36 weeks of ge. In contrst, 89% of the -treted mice ecme dietic, nd most of them died efore 33 weeks of ge (Fig. 6). Hemtoxylin nd eosin stining of pncretic tissue derived from ll fusion protein treted mice showed mny residul islets with usully only mild insulitis, sitution similr to tht of pncretic tissue derived from helthy, 3-week-old, untreted nondietic femle NOD mice (Fig. 6). In contrst, we detected no pncretic islets in mice injected with (Fig. 6). We noted no side effects in the treted mice either y gross exmintion or y histologicl nlysis (Fig. 6c). To investigte the mechnism responsile for the protective effect medited y the NKp46 fusion proteins, we collected from the vrious mouse groups during the course of the experiment. As predicted, injection of the vrious vrints resulted in the genertion of specific ntiodies directed ginst NKp46 nd NCR1, ut injection of did not (Fig. 7). The NKp46- nd NCR1-specific ntiodies were mostly of the IgM isotype, were present in the strting t 2 nd 4 weeks, respectively, fter injection of the solule fusion protein, nd remined in the for up to 36 weeks of ge (end of experiment; dt not shown). To gin insight into the mechnism y which the NKp46 fusion proteins inhiited dietes development, we first excluded the possiility tht these fusion proteins cted y depleting NK cells or y suppressing the ppernce of NK cells in the pncres (dt not shown). Tht finding is in contrst to the oservtion of lower CD8 + T cell percentges in the helthy pncres of mice treted with n NKG2D-specific ntiody 21. In ddition, et cells derived from NOD mice treted with or NKp46 fusion proteins expressed similr mounts of the unknown NKp46 lignd (dt not shown). We lso ruled out the possiility of potentil involvement of the Fc portion ecuse (the truncted version of the fusion protein lcking the complement- nd Fc-inding domin) ws lmost s effective s the other fusion proteins in suppressing dietes (Fig. 6). Dietic mice (%) 1 NKp46-lg 9 8 NCR1-lg c NKp46-lg NCR1-lg Helthy NOD Adrenl Hert Intestine Liver Bone mrrow Lung Brin Kidney Begin Tx Age (weeks) End Tx Figure 6 Tretment with fusion proteins prevents the development of dietes in NOD mice. () Development of dietes (lood glucose concentrtion >2 mg/dl in two consecutive mesurements) in femle NOD mice treted with, or (. g per kg ody weight, injected intrperitonelly twice weekly) or strting from 6 weeks of ge nd continuing up to 24 weeks of ge (tretment (Tx), upwrd rrows). P <.2, fusion protein versus (Kpln-Meyer nlysis, log-rnk test). Dt re representtive of two independent experiments with eight to nine mice per group. () Hemtoxylin nd eosin stining of pncretic tissues derived from the treted groups in (36 weeks of ge) nd from n untreted 3-week-old nondietic femle NOD mouse (fr right). Originl mgnifiction, 4. Dt re representtive of two experiments with eight to nine mice. (c) Hemtoxylin nd eosin stining of vrious tissues derived from -treted mice. Originl mgnifiction, 4. Dt re representtive of two experiments with eight to nine mice. nture immunology volume 11 numer 2 ferury 21 12

6 21 Nture Americ, Inc. All rights reserved. Figure 7 Impired NKp46 function. () Flow cytometry of BW trnsfectnts stined with specific ntiodies (A; top row) or with derived from 3-week-old treted mice. Blck lines, specific stining; gry histogrms, secondry ntiody stining. Inset (top row), stining of BWNKp46 cells with nti-ncr1. Dt re representtive of three independent experiments. () CD17 expression on NK cells isolted from NOD splenocytes preincuted with (horizontl xis) nd then incuted with et cells t n effector/trget rtio of 1:1. NK cells were identified y NCR1 expression. CD17 + cells re presented s percent of totl NK cells. P <. (Student s t-test). Dt re representtive of two independent experiments (error rs, s.d.). (c) Flow cytometry of NK cells derived from splenocytes of fusion protein treted mice (lck lines) or -treted mice (gry filled histogrms) t 36 weeks of ge nd stined with nti-ncr1. Dt re representtive of two independent experiments. (d,e) CD17 + cells mong NK cells derived from the splenocytes of 36-week-old - or fusion protein treted treted mice, incuted with et cells (d) or other trget cells (e) t n effector/trget rtio of 1:1 nd stined with llophycocynin-conjugted nti-cd17; plots re gted on NCR1 + cells. CD17 + cells re presented s percent of totl NK cells. P <.1 (Student s t-test). Dt re representtive of two independent experiments (error rs, s.d.). We lso determined whether the NKp46-specific ntiodies generted fter injection of fusion proteins could lock the degrnultion of NK cells. For this, we cultured NK cells derived from the spleens of 8-week-old nondietic femle NOD mice with et cells otined from predietic femle NOD mice nd did the CD17-moiliztion ssy. Degrnultion of NK cells ws significntly lower fter preincution with derived from mice treted with the NKp46 fusion proteins, ut norml or derived from -treted mice did not lter NK cell degrnultion (Fig. 7). As injection of nti-nkg2d, or solule NKG2D lignd, induces internliztion of NKG2D 17,29, we next determined whether NCR1 ws lso downregulted ecuse of the genertion of NKp46-specific ntiodies in the fusion protein treted mice. We noted lower expression of NCR1 on splenic NK cells derived from mice treted with the fusion proteins thn on those from the -treted group (Fig. 7c). The NCR1 reduction ws systemic, s we found similrly lower expression on NK cells derived from the lood (discussed elow) or the pncres (dt not shown). Stining with nti mouse IgG ws similr on NK cells from - nd fusion protein treted mice (dt not shown), which indicted tht the NK cells were not simply coted with the mouse NKp46 specific ntiodies. The NCR1 downregultion ffected the degrnultion of NK cells, s shown y CD17 stining of NK cells isolted from splenocytes of 36-week-old mice (12 weeks fter the end of tretment with or fusion proteins) nd incuted with et cells in vitro. -treted mice were lredy dietic nd hd low ut still significnt percentge of CD17 + NK cells (Fig. 7d). In contrst, NK cells derived from helthy mice treted with NKp46 fusion proteins filed to express CD17 fter interction with et cells (Fig. 7d). The reltively low function of NK cells derived from the -treted group compred with tht of cells from the helthy control 8-week-old mice (Fig. 7) could hve een due to the effect of hyperglycemi. Finlly, to demonstrte tht the impirment in NK cell ctivity ws specific to NKp46, we mesured the degrnultion ility of NK cells derived from - or fusion protein treted mice fter incution with NKp46-dependent trget cells (such s PD1.6 nd RMAS cells 18,3 ) or NKp46-independent, NKG2D-dependent trget cells (such s YAC-1 cells 18 ). All trget cells whose killing ws NKp46 dependent induced miniml CD17 expression on NK cells derived from mice treted with the vrious fusion proteins ut induced Specific A CD17 + cells (%) 1 BW No + BWNKp46 BWNCR1 BWNKp3 BWNKp44 d CD17 + cells (%) sustntil CD17 expression on NK cells derived from -treted mice (Fig. 7e). In contrst, NK cells derived from ll groups expressed CD17 fter incution with YAC-1 cells (Fig. 7e). Lte injection of NKp46 fusion proteins prevents type 1 dietes To investigte whether our tretment modlity ws le to prevent or dely the onset of dietes even in the lte predietic stge nd to demonstrte tht injection of the immunogloulin fusion proteins did not result in nonspecific effect, we injected 11- to 12-week-old nondietic femle NOD mice with,, or the irrelevnt fusion protein CEA-Ig. As expected, ll mice developed ntiodies directed ginst the injected fusion protein, including the control CEA-Ig (Fig. 8). We detected NCR1-specific ntiodies in the t round 4 weeks fter the first injection (proly ecuse tolernce hd to e roken), nd in the other groups, we detected fusion protein specific ntiodies s erly s 2 weeks fter the injection. Most ntiodies were of the IgM isotype (dt not shown). We injected NOD mice with the vrious fusion proteins or from weeks of ge until 2 weeks of ge. The dietes dignosis nd schedule nd dose of fusion protein injection were the sme s in the erly-injection experiment (Fig. 6). CEA-Ig did not prevent dietes development nd even cused slight ccelertion in the disese reltive to tht of the -treted group (Fig. 8). However, 67% of the treted mice nd ll of the treted mice remined dietes-free until 2 weeks of ge nd during the first 6 weeks fter therpy ws hlted (Fig. 8). Antiody genertion resulted in the specific downregultion of NCR1 t 2 or 4 weeks fter injection of or, respectively (Fig. 8c). In ddition, lthough round 3% of the pthogenic pncretic NK cells derived from groups treted with CEA-Ig or expressed CD17 fter incution with et cells, less thn 1% of the NK cells from mice treted with or hd sustntil expression of CD17 (Fig. 8d). Together, these findings indicte tht NKp46 is criticl for the development of dietes nd tht NKp46 therpy might e used to prevent the development of dietes t the lte predietic stge, when et cells fce immedite destruction y NK cells. e CD17 + cells (%) 6 c 4 YAC-1 PD1.6 3 RMAS 2 1 Anti-mNCR-1 Anti-mNCR-1 Anti-mNCR volume 11 numer 2 ferury 21 nture immunology

7 Specific A CEA-Ig BWNKp BWNCR CEA BWNKp3 Dietic mice (%) c Anti-mNCR Age (weeks) Begin Tx 1 4 End Tx d CD17 + cells (%) NCR-1 (MFI) CEA-Ig Nkp46-Ig CEA-Ig Nkp46-Ig 21 Nture Americ, Inc. All rights reserved. Figure 8 Tretment with NKp46 t lte predietic stge prevents the development of dietes. () Flow cytometry of BW trnsfectnts nd trnsfectnts (Epstein-Brr virus trnsfected B cell line) stined with specific ntiodies (top row) or with otined from 16-week-old treted mice. Blck lines, specific stining; gry histogrms, secondry ntiody stining. Dt re representtive of two independent experiments. () Development of dietes in NOD mice treted with,, CEA-Ig () or strting t weeks of ge nd continuing to 2 weeks of ge (n = 8 9 mice per group). P <.1, nd versus nd (Kpln-Meyer nlysis, log-rnk test). Dt re representtive of two independent experiments. (c) NK cells isolted from peripherl lood lymphocytes of 16-week-old treted mice (tretment, ove plots) nd stined with got nti mouse NCR1. Gry filled histogrms, CEA-Ig (); lck lines, or. Right, men fluorescent intensity (MFI) of NCR1 stining. P <. (Student s t-test). Dt re representtive of two independent experiments (error rs, s.d.). (d) CD17 + cells mong pthogenic NK cells derived from the islets of treted mice nd stined with CD17; plots re gted on NCR1 + cells. CD17 + cells re presented s percent of totl NK cells. P <.3, CEA-Ig versus, or P <.7, CEA-Ig versus (Student s t-test). Dt re representtive of two independent experiments (error rs, s.d.). DISCUSSION Although it is known tht NK cells hve crucil role in dietes development 1,11, the mechnisms tht control NK cell function in type 1 dietes remin unknown. Here we hve demonstrted tht NKp46 is criticlly involved in dietes development. In the sence of NKp46, or when the function of NKp46 ws locked, dietes ws prevented in most cses. NK cells derived from ll mouse strins tested degrnulted fter enggement with et cells in n NKp46-dependent mnner. Furthermore, pthogenic pncretic NK cells derived from the islet cells of NOD mice degrnulted in vivo, ut those from other orgns did not. In norml conditions, NK cells do not infiltrte the helthy pncres, so NK cell medited killing of et cells is prevented. NK cells pper in the pncres concomitntly with T cells when dietes strts to develop. We suggest tht NKp46 is crucil t this point for islet cell destruction nd, consequently, for dietes progression. In support of those oservtions, severl studies hve shown tht ctivted NK cells cn kill utologous et cells 14,31,32. Notly, oth norml nd pthologicl pncretic tissues nd slivry glnds, ut no other norml humn or mouse tissues exmined, expressed lignd for NKp46. It is still unknown why NKp46 lignds re expressed on helthy et cells. Islets derived from pncretic tissues of emryonic NOD mice do not express lignds for NCR1 nd contin no infiltrting NK cells. Thus, it seems tht NK cells do not hve criticl role during the emryonic period of norml pncretic tissue development. Lignds for other stress receptors such s NKG2D re lso present in the pncres (s presented here nd in refs. 17,21). However, the expression of NKG2D lignds ws restricted to supopultion of et cells nd diminished s dietes progressed, which suggests tht some of the NKG2D-Ig positive et cells were killed during dietes development or tht the NKG2D lignds were downregulted. In contrst, lmost ll et cells expressed the NCR1 lignd nd its expression remined constnt during dietes progression. The NKp46 lignd ppered t the postntl stge, nd we still do not know its identity or why norml et cells express lignd with the potentil to cuse hrm. The function of the NKp46 lignd in et cells is proly not relted to NK cell ctivity, ecuse NK cells re sent from the helthy pncres. It is possile tht the NKp46 lignds in the pncres resemle foreign lignds recognized y NKp46 nd tht NK cells ttck et cells unintentionlly ecuse of this moleculr mimicry. Indeed, infectious diseses hve een suggested s potentil triggers for mny utoimmune diseses, nd link etween viruses nd type 1 dietes hs een demonstrted 33,34. Expression of the NCR1 lignd is lso pprent in the slivry glnds. The slivry glnds of NOD mice lso serve s trget orgn in the utoimmune process, nd insulitis is usully ccompnied y silitis 3. Notly, progenitor cells in the slivry glnds of severl niml strins cn differentite into functionl et cells fter injury 36. Although NK cells degrnulted fter interction with et cells in n NKp46- dependent mnner nd NK cells present in the et cells of the pncres express CD17, it is possile tht NK cells lso ffect dietes development indirectly, for exmple, through cytokine secretion. However, we did not detect cytokine secretion y NK cells cultured together with et cells. Notly, injection of NKp46 vrints into NOD mice, either t n erly stge of insulitis or in the lte predietic stge, locked dietes development lmost entirely. Our model to explin these oservtions is tht NKp46-specific ntiodies (which contin locking ntiodies) re generted nd tht these ntiodies cuse systemic downregultion of NKp46. Such downregultion is the min cuse of the NK cell dysfunction. nture immunology volume 11 numer 2 ferury

8 21 Nture Americ, Inc. All rights reserved. These results highlight res for potentil specific new therpeutic modlities for type 1 dietes. An NKp46-dependent phrmcologicl intervention during the honeymoon period of dietes development might slow down or rrest the ongoing destruction of the remining et cells. We nticipte two possile scenrios for such tretment, ech with its own dvntges nd disdvntges. The first is pssive vccintion pproch in which ptients would e treted with nti-nkp46 to lock NKp46 function; this would ypss the need to induce ntiodies, which, s shown here, might e time consuming. The min disdvntge of such tretment is the reltively rpid genertion of neutrlizing ntiodies to the therpeuticlly injected monoclonl ntiodies, s noted with the nti tumor necrosis fctor therpies used in utoimmune diseses 37. Alterntively, it might e possile to ctively vccinte ptients with NKp46 protein to generte long lsting NKp46-specific ntiodies. The min disdvntges of this pproch re tht ntiody genertion tkes time nd tht the NKp46 dysfunction might ffect not only dietes development ut lso NKp46-medited ctivity ginst tumors nd viruses. Nevertheless, our study hs provided new insight into the reltionship etween NK cells nd dietes nd my id the development of therpeutic nd imging pproches sed on the unique interction of the NKp46 lignd nd the ctivting receptor NKp46. Methods Methods nd ny ssocited references re ville in the online version of the pper t Note: Supplementry informtion is ville on the Nture Immunology wesite. Acknowledgments We thnk E. Pikrsky for help nd dvice; E. Horowitz for ssistnce; memers of the Mndeloim lortory for discussions nd the Physicin Reserch Progrm of Hdssh Hospitl for ssistnce. Supported y the Juvenile Dietes Reserch Foundtion (O.M.), the Isrel Science Foundtion (Morsh grnt to C.G.) nd the Leifermnn Foundtion (Y.N.). AUTHOR CONTRIBUTIONS C.G. designed ll experiments, did ll experiments, nlyzed the dt nd wrote the mnuscript; A.P. mde the initil oservtion tht NKp46 recognizes et cells nd supervised the project; M.E., R.G., S.M., N.S.-G., H.A., H.G., T.N., O.H. nd M.M. contriuted regents; Y.D. provided guidnce nd regents; V.D. helped in the immunohistochemicl experiments nd in determining the insulitis scoring; Y.N. supervised the project nd contriuted regents; nd O.M. supervised the entire project nd nlyzed the dt, nd ll experiments were done in the O.M. lortory under the guidnce of O.M. Pulished online t Reprints nd permissions informtion is ville online t reprintsndpermissions/. 1. Delovitch, T.L. & Singh, B. The nonoese dietic mouse s model of utoimmune dietes: immune dysregultion gets the NOD. Immunity 7, (1997). 2. Kikutni, H. & Mkino, S. The murine utoimmune dietes model: NOD nd relted strins. Adv. Immunol. 1, (1992). 3. Like, A.A. & Rossini, A.A. Streptozotocin-induced pncretic insulitis: new model of dietes mellitus. Science 193, (1976). 4. O Brien, B.A., Hrmon, B.V., Cmeron, D.P. & Alln, D.J. Bet-cell poptosis is responsile for the development of IDDM in the multiple low-dose streptozotocin model. J. Pthol. 178, (1996).. Pik, S.G., Fleischer, N. & Shin, S.I. Insulin-dependent dietes mellitus induced y sudietogenic doses of streptozotocin: oligtory role of cell-medited utoimmune processes. Proc. Ntl. Acd. Sci. USA 77, (198). 6. Hutchings, P. et l. Trnsfer of dietes in mice prevented y lockde of dhesionpromoting receptor on mcrophges. Nture 348, (199). 7. Miyzki, A. et l. Predominnce of T lymphocytes in pncretic islets nd spleen of pre-dietic non-oese dietic (NOD) mice: longitudinl study. Clin. Exp. Immunol. 6, (198). 8. Dott, F. et l. Coxsckie B4 virus infection of et cells nd nturl killer cell insulitis in recent-onset type 1 dietic ptients. Proc. Ntl. Acd. Sci. USA 14, (27). 9. Rodcki, M. et l. Altered nturl killer cells in type 1 dietic ptients. Dietes 6, (27). 1. Poirot, L., Benoist, C. & Mthis, D. Nturl killer cells distinguish innocuous nd destructive forms of pncretic islet utoimmunity. Proc. Ntl. Acd. Sci. USA 11, (24). 11. Al, A. et l. Nturl killer cells re required for ccelerted type 1 dietes driven y interferon-β. Clin. Exp. Immunol. 11, (28). 12. Flodstrom, M., Shi, F.D., Srvetnick, N. & Ljunggren, H.G. The nturl killer cell friend or foe in utoimmune disese? Scnd. J. Immunol., (22). 13. Hnsson, M., Kiessling, R. & Andersson, B. Humn fetl thymus nd one mrrow contin trget cells for nturl killer cells. Eur. J. Immunol. 11, 8 12 (1981). 14. Nkmur, N. et l. Intrinsic cytotoxicity of nturl killer cells to pncretic islets in vitro. Dietes 39, (199). 1. Morse, R.H., Seguin, R., McCre, E.L. & Antel, J.P. NK cell-medited lysis of utologous humn oligodendrocytes. J. Neuroimmunol. 116, (21). 16. Krre, K. NK cells, MHC clss I molecules nd the missing self. Scnd. J. Immunol., (22). 17. Ogswr, K. et l. Impirment of NK cell function y NKG2D modultion in NOD mice. Immunity 18, 41 1 (23). 18. Gzit, R. et l. Lethl influenz infection in the sence of the nturl killer cell receptor gene Ncr1. Nt. Immunol. 7, (26). 19. Morett, L. Lymphocyte effector mechnisms in innte nd dptive immunity. Curr. Opin. Immunol. 17, 33 3 (2). 2. Stoh-Tkym, N. et l. The nturl cytotoxicity receptor NKp46 is dispensle for IL-22-medited innte intestinl immune defense ginst Citrocter rodentium. J. Immunol. 183, (29). 21. Ogswr, K. et l. NKG2D lockde prevents utoimmune dietes in NOD mice. Immunity 2, (24). 22. Mier, L.M. et l. NKG2D-RAE-1 receptor-lignd vrition does not ccount for the NK cell defect in nonoese dietic mice. J. Immunol. 181, (28). 23. Morett, A., Bottino, C., Mingri, M.C., Bissoni, R. & Morett, L. Wht is nturl killer cell? Nt. Immunol. 3, 6 8 (22). 24. Mndeloim, O. et l. Recognition of hemgglutinins on virus-infected cells y NKp46 ctivtes lysis y humn NK cells. Nture 49, 1 16 (21). 2. Arnon, T.I. et l. The mechnisms controlling the recognition of tumor- nd virusinfected cells y NKp46. Blood 13, (24). 26. Akts, E., Kucuksezer, U.C., Bilgic, S., Erten, G. & Deniz, G. Reltionship etween CD17 expression nd cytotoxic ctivity. Cell. Immunol. 24, (29). 27. Alter, G., Mlenfnt, J.M. & Altfeld, M. CD17 s functionl mrker for the identifiction of nturl killer cell ctivity. J. Immunol. Methods 294, 1 22 (24). 28. Flodstrom, M., Tyrerg, B., Eizirik, D.L. & Sndler, S. Reduced sensitivity of inducile nitric oxide synthse-deficient mice to multiple low-dose streptozotocininduced dietes. Dietes 48, (1999). 29. Lodoen, M. et l. NKG2D-medited nturl killer cell protection ginst cytomeglovirus is impired y virl gp4 modultion of retinoic cid erly inducile 1 gene molecules. J. Exp. Med. 197, (23). 3. Hlfteck, G.G. et l. Enhnced in vivo growth of lymphom tumors in the sence of the NK-ctivting receptor NKp46/NCR1. J. Immunol. 182, (29). 31. Kitgw, Y. et l. Islet cells ut not thyrocytes re susceptile to lysis y NK cells. J. Autoimmun. 4, (1991). 32. McKy, P., Jcoson, J. & Rinovitch, A. Spontneous dietes mellitus in the Bio-Breeding/Worcester rt. Evidence in vitro for nturl killer cell lysis of islet cells. J. Clin. Invest. 77, (1986). 33. Foulis, A.K., McGill, M., Frquhrson, M.A. & Hilton, D.A. A serch for evidence of virl infection in pncreses of newly dignosed ptients with IDDM. Dietologi 4, 3 61 (1997). 34. Horwitz, M.S. et l. Dietes induced y Coxsckie virus: initition y ystnder dmge nd not moleculr mimicry. Nt. Med. 4, (1998). 3. Lodde, B.M. et l. NOD mouse model for Sjogren s syndrome: lck of longitudinl stility. Orl Dis. 12, (26). 36. Mtsumoto, S. et l. Isoltion of tissue progenitor cells from duct-ligted slivry glnds of swine. Cloning Stem Cells 9, (27). 37. Bert, F. et l. Influence of immunogenicity on the long-term efficcy of inflixim in Crohn s disese. N. Engl. J. Med. 348, (23). 128 volume 11 numer 2 ferury 21 nture immunology

9 21 Nture Americ, Inc. All rights reserved. ONLINE METHODS Mice. The genertion of Ncr1 gfp/gfp mice hs een descried 18. All experiments were done in specific, pthogen-free unit of the Hdssh Medicl School (Ein-Krem, Jeruslem) in ccordnce with the guidelines of the locl ethicl committee. Cells nd fusion proteins. Cell lines used in this study were s follows: HeL, BW (nd BW trnsfectnts), RMA-s, PD1.6, YAC-1, RMS nd (nd the CEA trnsfectnt).,,, NKG2D-Ig, CEA-Ig nd NKp46D1-Ig fusion proteins were generted in COS-7 monkey kidney cells nd were purified on protein G column s descried 2. Immunohistochemicl nd immunofluorescence stining. Prffin-emedded sections of pncretic tissues were prepred from nondietic humn utopsy, from NOD mice emryos (E2) nd from 1- to 14-week-old femle NOD mice nd 8- to 12-week-old femle BALB/c mice. After ntigen retrievl, sections were incuted for 2 h with fusion proteins. Sections were then incuted with polyclonl iotin-leled rit ntiodies directed ginst the humn Fcγ prt of the fusion proteins ( ; Jckson ImmunoReserch). For immunohistochemicl stining, the EnVision+ system (K42; Dko) ws used; this is sed on horserdish peroxidse leled polymer conjugted to nti-rit. After 3 min of incution with nti-rit, slide stining ws completed y 1 3 min of incution with DAB+ Chromogen (3,3 -diminoenzidine) nd then counterstining with hemtoxylin. For immunofluorescence stining, in ddition to eing incuted with fusion proteins, tissues were lso incuted with polyclonl nti mouse insulin (A64; DkoCytomtion,), nti-somtosttin (som-18; Bet Cell Biology Consortium) nd nti-glucgon (glu-1; Bet Cell Biology Consortium), followed y incution with mixture of the following three fluorochrome-conjugted secondry polyclonl ntiodies (ll from Jckson ImmunoReserch): indocrocynine-conjugted nti guine pig ( ), crocynine-conjugted nti-mouse ( ) nd indodicrocynine-conjugted nti-rit ( ). As control, smples were stined with ech fusion protein nd regent (primry nd secondry ntiodies) individully. For oth immunohistochemicl nd the immunofluorescence stining, n fusion protein contining the inding D2 domin nd the stlk region only ws used 2, which gve etter stining thn intct. Isoltion of et cells. Pncretic islets from norml nd NOD mice were prepred with solution of collgense XI (Sigm) diluted in Hnk s lnced-slt solution (Biologicl Industries Kiutz Beit Hemek) t concentrtion of 1 mg/ml. The solution ws first injected into the pncretic duct efore removl of the pncres, followed y digestion for 1 23 min t 37 C. Individul islets were selected y hnd with microscope nd then were seprted into single cells. CD17 moiliztion nd BW reporter ssy. Anlysis of cell surfce moilized CD17 hs een descried 27. In some experiments, NK cells were preincuted for 1 h with derived from pool from ech group of treted mice, diluted to titer of 1:1,. For mesurement of CD17 in vivo, NK cells derived from the islets were stined for CD17 expression. For flow cytometry stining, et cells were stined with 1 µg iotin-conjugted nti mouse GLUT-2 (211; R&D Systems) nd µg fusion protein. The genertion nd use of BW cells expressing NKp46 fused to the trnsmemrne nd til domins of mouse CD3ζ hve een descried 24. Streptozotocin-induced dietes. For the multiple-ldst model, ten to twelve sex- nd ge-mtched mice 8 1 weeks of ge were injected intrperitonelly for consecutive dys with streptozotocin (Sigm) dissolved in citrte uffer, ph 4., t concentrtion of mg per kg ody weight. Dy ws defined s the first dy of injection of streptozotocin. Blood glucose concentrtions were mesured with glucometer (Accu-Check; Roche Dignostics) t dy 7 nd up to 4 d fter first injection. Sttisticl nlysis of multiple LDST experiments ws done y Kpln-Meier nlysis with the log-rnk test for comprison of survivl curves of the two groups nd y nlysis of vrince with repeted mesures model for ssessment the time effect, the group effect nd the interction etween time nd group during the development of dietes. Flow cytometry ntiodies nd enzyme-linked immunosorent ssy. Before pncretic lymphocytes were isolted, pncretic lymph nodes were removed to void lymphocyte contmintion. Next, pncretic tissues were cut into pieces 1 mm 3 nd were digested with 1. mg type I DNAse nd 1 mg type IV collgense (Sigm). Superntnts were pssed through 4-µm cell striner nd then were loded on Ficoll density grdient for purifiction of the lymphocyte popultion. Peripherl lood ws otined from the til vein. A monoclonl ntiody specific for CD16 nd CD32 (93; Biolegend) ws used for lockde of Fc receptors efore stining. NK cells derived from NOD mice were detected y stining with phycoerythrin-conjugted polyclonl got ntiody to mouse NCR1 nd NKp46 (FAB222P; R&D systems). For stining of BW cells trnsfected with NKp46, NKp3 or NKp44, cells, nd cells trnsfected with CEA, specific ntiodies to NKp46 (9E2; BioLegend), NKp3 (2184; BioLegend), NKp44 (2341; R&D Systems) nd CEA (ASL-32; BioLegend) were used. For stining of BW cells trnsfected with NCR1, phycoerythrin-conjugted got polyclonl ntiody to mouse NCR1 nd NKp46 (FAB222P; R&D Systems) ws used. For mesurement of the secretion of mouse IL-2 from the BW trnsfectnts or IFN-γ from mouse NK cells, stndrd enzyme-linked immunosorent ssy ws used with pirs of ntiodies to mouse IL-2 (ges6h4 nd ges6112; BioLegend) or IFN-γ (R4-6A2 nd XMG1.2; BD Phrmingen). doi:1.138/ni.1834 nture immunology