Natural killer cells determine the outcome of B cell mediated autoimmunity

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1 Nturl killer cells determine the outcome of B cell medited utoimmunity Fu-Dong Shi 1, *, Hu-Bing Wng 2, *, Hulun Li 2, *, Seokmnn Hong 3, Msru Tniguchi 4, Hns Link 2, Luc Vn Ker 3 nd Hns-Gustf Ljunggren 1 Nturl killer (NK) cells cn ffect the outcome of dptive immune responses. NK cells, ut not NK1.1 + T cells, were found to prticipte in the development of mystheni grvis ( T cell dependent, B cell nd ntiody-medited utoimmune disese) in C57BL/6 mice.the requirement for NK cells ws reflected y the lck of type 1 helper T cell response nd ntiodies to the cetylcholine receptor in oth NK1.1 + cell depleted nd NK cell deficient IL-18 / mice.these findings estlish previously unrecognized link etween NK cells nd utorective T nd B cells. Autoimmune diseses re inflmmtory disorders, mny of which hve suspected infectious etiology. Nturl killer (NK) cells, s first line of defense in comting infections, my e involved in the initition of utoimmunity nd ccumulte in the trget orgns of certin utoimmune diseses 1 3. It hs een suggested tht NK or NK1.1 + T (NKT) cells serve s regultory cells in some T cell medited experimentl utoimmune disese, including murine models of encephlomyelitis 4,5, colitis 6 nd dietes 7. However, severl issues regrding the role of NK cells in the development of utoimmune diseses remin unresolved. Few studies hve een le to distinguish etween effects of NK cells nd effects of NKT cells, nd the mechnism for the regultory effect of NK cells in murine models remins uncler. Also still unknown is the point t which NK cells impct on the development of utoimmune disese, nd the contriution of NK cells to the development of those utoimmune diseses tht re primrily medited y pthogenic ntiodies. Autontiodies produced y B cells re the primry cuse of disese in vriety of utoimmune conditions, including hemolytic nemi, thyroiditis, stiff mn syndrome, pemphigus vulgris nd systemic lupus erythemtosus 8. Mystheni grvis (MG) is one of the est chrcterized ntiody-medited utoimmune diseses ecuse the trget ntigen, the nicotinic cetylcholine receptor (AChR) of neuromusculr junctions, hs een well defined 9. Experimentl utoimmune mystheni grvis (EAMG) in C57BL/6 (B6) mice, induced y repeted immuniztions with Torpedo AChR emulsified in complete Freund s djuvnt (CFA), is prticulrly useful model for identifiction of the pthogenic mechnisms tht cuse MG in humns 10. In oth MG nd EAMG, utorective CD4 + T cells provide help for B cells to produce ntiodies to AChR 9. Although the T H1 cytokines interferon γ (IFN-γ) nd interleukin 12 (IL- 12) re criticlly importnt for the genertion of EAMG in B6 mice 11,12, the production of oth T helper cell susets 1 nd 2 (T H1 nd T H2) cytokines my e required for the development of full-lown EAMG 13. To otin more comprehensive view of the potentil role of NK nd NKT cells in B cell medited utoimmune diseses, we exmined the development of ntiody-medited EAMG in mice depleted of NK1.1 + cells, in NK cell deficient IL-18 / mice, nd in NKT cell deficient mice. Our results demonstrte tht NK cells determine the outcome of utontiody responses to AChR vi control of utorective T c d Figure 1. Incidence nd severity of EAMG in NK1.1 + cell depleted nd NKT cell deficient mice. Mice were immunized with AChR nd CFA, oosted on dy 30 nd 60 fter immuniztion, nd monitored for development of EAMG. (,) B6 mice (, n=35) treted with isotype control ntiody nd B6 mice depleted of NK1.1 + cells (, n=36). (c,d) Wild-type (, n=15) nd J α281 / mice (, n=17). (e,f) Wild-type (, n=8) nd CD1d1 / mice (, n=8). e f 1 Microiology nd Tumor Biology Center, Krolinsk Institutet, S Stockholm, Sweden. 2 Division of Neurology, Huddinge University Hospitl, Krolinsk Institutet, S Stockholm, Sweden. 3 Howrd Hughes Medicl Institute, Deprtment of Microiology nd Immunology, Vnderilt University School of Medicine, Nshville,TN 37232, USA. 4 Division of Moleculr Immunology, Center for Biomedicl Science, School of Medicine, Chi University, Inon, Chuo-ku, Chi, Jpn 260. *These uthors contriuted eqully to this work. Present ddress: Deprtment of Immunology, IMM-23, The Scripps Reserch Institute, North Torrey Pines Rod, L Joll, CA 92037, USA. Correspondence should e ddressed to H.G.L. (hns-gustf.ljunggren@mtc.ki.se). septemer 2000 volume 1 no 3 nture immunology 245

2 dy 49 nd P<0.01 on dy 59 fter immuniztion) of EAMG were significntly lower in mice depleted of NK1.1 + cells (Fig. 1,). Mice depleted of NK1.1 + cells hd lso higher AChR levels in their muscle tissue thn control mice (35±5% versus 68±12%, P<0.05). To determine whether NKT cells re importnt for the development of EAMG in this model, NKT cell deficient J α281 / (ref. 14) nd CD1d1 / (ref. 15) mice were immunized with AChR nd CFA s ove. Both J α281 / mice nd CD1d1 / mice developed EAMG t similr rte to their corresponding wild-type controls (Fig. 1c f). Thus NK cells, ut not NKT cells, ffect the development of AChRinduced EAMG. Figure 2. Functionl properties of NK cells in EAMG. () NK cell medited cytotoxicity of splenocytes from nïve B6 mice ( ), B6 mice immunized with CFA lone ( ) or B6 mice immunized with AChR in CFA ( ) 7 dys fter immuniztion. All effectors were tested ginst YAC-1 trget cells. () Men numers (± s.d.) of IFN-γ spot forming NK cells (SFC) mesured y solid-phse enzyme-linked immunospot (ELISPOT). NK cells were sorted from spleen cells of nïve B6 mice, B6 mice immunized with CFA lone or B6 mice immunized with AChR in CFA y flow cytometry on dy 7 fter immuniztion nd cultured for 48 h without ntigen stimultion. () nd () represents one of two independent experiments. (n=4 mice per group). cells. Results Onset nd severity of murine EAMG To determine whether NK1.1 + cells (NK cells nd NKT cells) re importnt for the development of EAMG, B6 mice or B6 mice depleted of NK1.1 + cells were immunized with AChR nd CFA, oosted on dys 30 nd 60 fter immuniztion, nd monitored for 100 dys fter immuniztion for signs of MG. In B6 mice, the medin EAMG onset ws dy 37, wheres in NK1.1 + depleted mice, the medin onset ws dy 65. Compred to control mice, the incidence (P<0.001 on dy 49 nd P<0.01 on dy 59 fter immuniztion) nd severity (P<0.001 on NK cell ctivtion during primry immuniztion To exmine the mechnisms y which NK cells influence the development of EAMG in mice, we first exmined the sttus of NK cells in mice ctively immunized with AChR + CFA. NK cell ctivity in mice immunized with AChR + CFA, or CFA lone, ws higher thn in nonimmunized mice (Fig. 2). The frequency of NK cells producing IFN-γ ws lso higher in mice immunized with AChR + CFA, or CFA lone, thn in nonimmunized mice (P<0.01) (Fig. 2). Therefore, NK cells re ctivted fter primry immuniztion with AChR + CFA. NK1.1 + cell depletion: prolifertion nd cytokine responses To determine whether NK1.1 + cells influence utorective T cell responses, lymph node mononucler cells (MNCs) from NK1.1 + cell depleted or control mice were isolted t dys 7 nd 100 fter immuniztion, nd exmined for prolifertive nd cytokine responses. Depletion of NK1.1 + cells did not significntly lter T cell prolifertive responses to AChR or its dominnt epitope the peptide AChRα( ) 13 (Fig. 3,). When lymph node MNCs were exmined for cytokine responses, it ws found tht IFN-γ production y AChR-specific T cells ws reduced on dy 7 nd 100 fter immu- Figure 3. NK1.1 + cell depletion efore immuniztion with AChR + CFA leds to enhnced TGF-β1 production y T cells tht down-regulte T H1 type immune responses. B6 mice were immunized with AChR + CFA on dys 0, 30 nd 60. NK1.1 + cells were depleted s descried in the Methods. Mice were killed t dy 7 or dy 100 fter immuniztion nd MNCs isolted from drining lymph nodes were cultured. Prolifertive responses to AChR nd AChRα( ) on () dy 7 nd () dy 100 fter immuniztion (ckground cpm 1306±110 for dy 7 fter immuniztion, 1130±50 for dy 100 fter immuniztion; no difference ws found etween control nd NK1.1 + depleted mice). (c) AChR-specific IFN-γ production in NK1.1 c d e f + cell depleted mice or in mice treted with isotype control ntiody (spontneous relese: 67±21 pg/ml). Neutrlizing trnsforming growth fctor β1 monoclonl ntiody (TGFβ1 ma) injected, s descried in the Methods, on the dy of immuniztion. (d) AChR-specific TGF-β1 production in mice treted with nti-nk1.1 + or in mice treted with isotype control ntiody (spontneous relese: 273±69 pg/ml). (e) AChR-specific IL-4 production in NK1.1 + cell depleted mice or mice treted with control ntiody. Neutrlizing nti TGF-β ws injected, s descried in the Methods (spontneous relese: undetectle). (f) Men numers of AChRspecific TGF-β SFC per 10 5 CD4 + cells mesured y ELISPOT in control nd NK1.1 + cell depleted mice (ckground spots: 3 5).All results re expressed s men vlues ± s.d. nd represent one of eight independent experiments; c f represent one of three independent experiments. Cytokine relese in response to myelin sic protein (MBP) ws similr to spontneous cytokine relese. Comprtive sttisticl nlysis of control nd experimentl groups ws done: **, P<0.01; #, P< (open rs, control ntiody; filled rs, nti-nk1.1; wvey-lined rs, nti-nk1.1 + nti TGF-β, dotted rs nti-nk1.1 + control ntiody). 246 nture immunology volume 1 no 3 septemer

3 niztion in mice depleted of NK1.1 + cells compred to control mice (Fig. 3c; P<0.01 t oth time points). In contrst, TGF-β1 production y AChRspecific T cells ws mrkedly elevted in NK1.1 + cell depleted mice on dys 7 nd 100 fter immuniztion (Fig. 3d; P<0.001 nd 0.01, respectively). No significnt difference in IL-4 production ws oserved in NK1.1-depleted mice versus control mice (Fig. 3e). The numer of CD4 + TGF-β1 producing cells, mesured y ELISPOT ws lso higher in NK1.1 + cell depleted mice (Fig. 3f). Similr results were otined for mice depleted of NK1.1 + cells 7 dys efore immuniztion nd onwrds, s descried in Methods (dt not shown). Tken together, these results suggest tht NK1.1 + cell depletion differentilly regultes cytokine production y AChRspecific T H susets. Neutrliztion of TGF-β in NK1.1 + cell depleted mice TGF-β is potent inhiitor of T cells 16,17. To exmine whether the suppression of IFN-γ producing T H1 cells oserved in NK1.1 + cell depleted mice ws ssocited with incresed production of TGF-β, we inoculted NK1.1 + cell depleted mice with neutrlizing ntiodies to TGF-β. The suppression of IFN-γ production in NK1.1 + cell depleted mice could e reversed, t lest in prt, y neutrlizing ntiodies to TGF-β. Becuse concentrtions of the T H2 cytokine IL-4 were unltered fter NK1.1 + cell depletion, it is less likely tht the suppression of T H1 cells ws ttriuted to effects medited y T H2 cells (Fig. 3c). In line with the cytokine dt, EAMG development in NK1.1 + cell depleted mice inoculted with neutrlizing ntiodies to TGF-β ws significntly enhnced compred to NK1.1 + Tle 1. EAMG development in NK1.1 + cell depleted B6 mice inoculted with neutrlizing ntiodies to TGF-β1 Numer of B6 mice Tretment Muscle wekness Disese incidence Control mas /35 (86%) 36 Anti-NK1.1 +c /36 (36%) 6 Anti-NK nti TGF-β1 c /6 (83%) NK1.1 ma tretment ws initited 2 dys efore immuniztion.anti TGF-β tretment ws initited t the time of immuniztion. Dt on mice injected with control ma or NK1.1 ma only re from Fig. 1. Mice were immunized with AChR nd CFA, nd monitored for EAMG (muscle wekness) grded 0 3 s descried in Methods. c P<0.01 etween groups with nti-nk1.1 treted mice versus nti-nk1.1 treted mice given neutrlizing nti TGF-β1. Figure 4. NK1.1 + cell depletion efore immuniztion with AChR nd CFA impirs nti-achr IgG nd IgG2 isotype responses. () Men numers of nti-achr IgG SFC in control mice nd mice depleted of NK1.1 + cells (n=8 per group, ckground spots 0 3). () Serum nti- AChR mesured y rdioimmunossy using 125 I α-ungrotoxin (α-bgt). (c) Anti- AChR IgG isotypes mesured y ELISA on dy 100 fter immuniztion, nd effects of neutrlizing TGF-β (see Methods). All results re expressed s men vlues ± s.d. n=20 mice per group for nd c. Comprtive sttisticl nlysis of control nd experimentl groups ws done: *, P<0.05, **, P<0.01, #, P< (Open squres, control ntiody; filled squres, nti-nk1.1; wvey-lined rs, nti-nk1.1 + nti-tgf-β, dotted rs nti-nk1.1 + control ntiody.) c cell depleted mice receiving no ntiodies to TGF-β (Tle 1). Antiody responses to AChR In MG nd EAMG, the pthogenic ntiodies to AChR consist predominntly of ll IgG sutypes 9. These ntiodies re responsile for the functionl loss of AChR in the neuromusculr junctions, resulting in muscle wekness 9. The numer of nti-achr IgG secreting cells ws reduced in mice depleted of NK1.1 + cells (Fig. 4). Accordingly, the concentrtion of AChR-specific IgG in serum ws reduced in these mice (Fig. 4). Compred to control mice, AChR-specific IgG2 nd IgG1 concentrtions were not significntly ltered in mice depleted of NK1.1 + cells, ut IgG2 ws significntly reduced (P<0.05, Fig. 4c). In NK1.1 + cell depleted mice receiving nti TGF-β, the concentrtions of nti AChR IgG nd IgG2 were similr to those in control mice (Fig. 4c). These effects were not specific for AChR, s NK1.1 + cell depleted mice immunized with keyhole limpet hemocynin lso hd reduced concentrtion of specific ntiody (dt not shown). NK1.1 + cells influence EAMG during priming To determine the time point t which NK1.1 + cells ffect development of EAMG, mice were treted with nti-nk1.1 every 5 7 dys strting on dy 7 or dy 37 fter immuniztion. Mice depleted of NK1.1 + cells from dys 7 nd 37 fter immuniztion developed EAMG t similr rte to control mice (MG incidence 83.3%, mximum MG severity 1.9; compre with dt in Fig. 1 nd Tle 1). AChR-specific T cell prolifertion (4,220±604 cpm versus 5,170±567 cpm) nd cytokine production (IFN-γ 267±45 pg/ml versus 300±60 pg/ml; IL-4 77±11 pg/ml versus 70±7 pg/ml; TGF-β 455±123 pg/ml versus 423±109 pg/ml) were similr in control mice nd in mice depleted of NK1.1 + cells t dy 7 fter immuniztion These groups of mice hd similr nti-achr IgG (1.23±0.05 versus 0.89±0.24), detected y enzyme-linked immunosorent ssy (ELISA) t A 405 nd of IgG isotypes (IgG1, 0.14±0.03 versus 0.1±0.04; IgG2, 0.15±0.08 versus 0.12±0.05; IgG2, 0.84±0.60 versus 0.61±0.13 detected y ELISA t A 405). Likewise, depletion of NK1.1 + cells from dy 37 fter immuniztion hd no impct on the development of MG or T nd B cell responses to AChR (dt not shown). AChR responses in mice lcking NKT cells To find whether the effects oserved fter removl of NK1.1 + cells were due to NK cells or NKT cells, we immunized NKT cell deficient J α281 / nd CD1d1 / mice, nd corresponding wild-type controls, with AChR in CFA. Seven dys fter immuniztion lymph node MNCs were prepred nd nlyzed for prolifertive nd cytokine responses. AChR- nd AChRα( ) specific prolifertion did not differ significntly etween J α281 / nd wild-type mice (Fig. 5,). MNC from J α281 / mice relesed slightly more AChR- septemer 2000 volume 1 no 3 nture immunology 247

4 c d e specific IFN-γ nd slightly less IL-4 thn wild-type mice (not sttisticlly significnt). No differences in TGF-β1 production were oserved (Fig. 5c,d). Totl IgG, s well s IgG1, concentrtions were not sttisticlly different in J α281 / mice s compred to wild-type mice (Fig. 5e,f). IgG2 ws undetectle t the sme dilution of the ser. CD1d1 / mice exhiited immune responses to AChR similr to J α281 / mice (Fig. 5 f). J α281 / nd CD1d1 / mice treted with ntiodies ginst NK1.1 on dy 2 efore immuniztion with AChR in CFA ehved in similr wy s corresponding wild-type mice treted with the sme ntiodies (Fig. 5; dt not shown). Immune responses to AChR in IL-18 / mice IL-18, originlly identified s IFN-γ inducing fctor, promotes the production of IFN-γ y NK cells nd T H1 cells nd enhnces NK cell ctivity 18. To further clrify the mechnisms y which NK cells ffect EAMG development, we chrcterized the immune responses to AChR in IL-18 / mice 19 immunized with AChR nd CFA. Compred to wild-type mice, IL-18 / mice were resistnt to the induction of MG (Fig. 6,; incidence P<0.01 on dy 55 fter immuniztion, severity P<0.01 on dy 78 fter immuniztion). This ws ssocited with defective T H1 responses nd lower levels of ntiodies to AChR IgG f Figure 5. Effects of NKT cell deficiency on utorective T nd B cell responses. Mice were immunized with AChR nd CFA, nd lymph node MNCs isolted 7 dys fter immuniztion (,) Prolifertive responses to AChR nd AChRα( ) (ckground cpm 1637±122). (c,d) AChR-specific cytokine relese (spontneous relese: IFN-γ,85±15 pg/ml; IL-4, undetectle;tgf-β1, 159±23 pg/ml). (e,f) Anti-AChR IgG nd IgG isotypes mesured y ELISA on dy 21 fter immuniztion. All results re expressed s men vlues ± s.d. (n=8 per group). Comprtive sttisticl nlysis of control nd experimentl groups ws done: *, P<0.05; **, P<0.01; #, P< (In,c,e, open rs, J α281 +/+ ; wvey-lined rs, J α281 / ; filled rs, J α281 / nti-nk1.1. In,d,f, open rs, CD1d1 +/+ ; wvey-lined rs CD1d1 / ; filled rs, CD1d1 / nti-nk1.1.) nd IgG2, nd incresed TGF-β1 production (Fig. 6c,d), wheres AChR-rective T cell prolifertive responses were not impired in the IL-18 / mice (dt not shown). Therefore, the clinicl nd immunologicl prmeters in the induction of MG pper similr in B6 mice receiving nti-nk1.1 nd in IL-18 / mice. c d Figure 6. Resistnce to EAMG induction in IL-18 / mice is ssocited with defective NK cell function. Mice were immunized with AChR nd CFA, oosted on dy 30 nd 60 fter immuniztion, nd monitored for development of EAMG. () EAMG incidence nd () severity in wild-type (n=19) nd IL-18 / mice (n=20). (c) Effects of reconstitution of NK cells in IL-18 / mice on AChR-specific cytokine relese. IL-18 / mice were reconstituted with 10 7 NK cells from either RAG-1 / or RAG-1 / IFN-γ / mice one dy efore immuniztion (n=4, see Methods). Drining lymph node MNC culture superntnts were collected on dy 7 fter immuniztion nd nlyzed for IFN-γ nd TGF-β1 production y ELISA (spontneous relese: IFN-γ, 58±23 pg/ml;tgf-β1, 122±15 pg/ml). (d) AChR-specific ntiody responses in IL-18 / mice. Serum smples were collected on dy 7 fter immuniztion from mice used in c.antiodies were detected y ELISA (n=4). (e) Numers of IFN-γ SFC mesured y ELISPOT. NK cells were sorted y flow cytometry from spleen cells of wild-type nd IL-18 / mice immunized with AChR in CFA on dy 7 fter immuniztion nd cultured for 48 h without ntigen stimultion (n=4). All results re expressed s men vlues ± s.d. *, P<0.05; **, P<0.01 in experiments where wild-type mice were compred with IL-18 / mice. #, P<0.05 when IL-18 / mice were compred with IL-18 / mice which hd een reconstituted with NK cells (RAG-1 / derived). (Open rs, IL-18 +/+ ; light htched rs, IL-18 +/+, NK cells (RAG-1 / ); wveylined rs, IL-18 +/+, NK cells (RAG-1 / IFN-γ / ); filled rs, IL-18 / ; shded rs, IL-18 /, NK cells (RAG-1 / ); drk htched rs, IL-18 /, NK cells (RAG-1 / IFN-γ / )). e 248 nture immunology volume 1 no 3 septemer

5 Tle 2. EAMG development in IL-18 / mice reconstituted with NK cells Numer of IL-18 / mice Tretment Muscle wekness Disese incidence PBS uffer c /7 (29%) 5 NK cells (RAG-1 / ) c,d /5 (80%) 5 NK cells (RAG-1 / IFNγ / ) d /5 (20%) Mice were intrvenously inoculted with 10 7 NK cells t the time of immuniztion. Mice were immunized with AChR nd CFA, nd monitored for EAMG (muscle wekness) grded 0 3 s descried in Methods. c P < 0.01 for PBS uffer inoculted mice versus NK cell (RAG-1 / derived) inoculted mice. d P < 0.05 for NK cell (RAG-1 / IFN-γ / derived) inoculted mice versus NK cell (RAG-1 / derived) inoculted mice. Reconstitution of NK cells in IL-18 / mice Becuse erly production of IFN-γ y NK cells my promote susequent T H1 responses 20,21, the defective AChR rective T H1 responses could e due to either the sence of direct IL-18 ction on T H1 cells, reduced IFN-γ production y NK cells, or oth. Despite norml development of NK cells, NK cell ctivity with respect to cytotoxicity nd production of IFN-γ is severely impired in IL-18 / mice 19 (Fig. 6e). RAG-1 / mice 22 hve norml NK cells ut no NKT cells. To ddress directly whether IFN-γ derived from NK cells cn influence utorective T H1 cell responses nd disese development in the EAMG model, we sorted NK cells from RAG-1 / or RAG-1 / IFNγ / (doule mutnt) 23 mice. Although NK cells from RAG-1 / mice redily produced IFN-γ s determined y ELISPOT nlysis, only ckground levels of IFN-γ production were detected in NK cells from RAG-1 / IFNγ / mice (dt not shown). We then reconstituted IL-18 / mice with these two types of NK cells 1 dy efore immuniztion with AChR nd CFA. Trnsfer of or NK cells from RAG-1 / IFNγ / mice to IL-18 / mice hd no detectle effects on cytokine production y T cells (dt not shown). However, trnsfer of or NK cells from RAG-1 / mice led to incresed levels of IFN-γ production y MNCs from IL- 18 / mice nd the production of nti-achr IgG2 (Fig. 6c,d; dt not shown). TGF-β1 production ws suppressed, wheres IL-4 production ws unltered y reconstitution of NK cells (Fig. 6c; dt not shown). IFN-γ producing CD4 + T cells from IL-18 / mice, s mesured y ELISPOT, were lso rescued (dt not shown). AChRimmunized IL-18 / mice reconstituted with NK cells derived from RAG-1 / mice exhiited clinicl MG similr to tht of wild-type mice (Tle 2). Tken together, these results suggest tht IFN-γ production y ctivted NK cells cn promote T H1 responses nd enhnce B cell medited utoimmunity. Discussion In this study we provide evidence tht NK cells, mjor rm of innte immunity, prticipte in the development of MG, primry B cell nd ntiody medited utoimmune disese, in mice. The requirement of NK cells ws reflected y the filure to mount T H1 type nd nti-achr responses fter NK1.1 + cell depletion in wild-type mice nd in NK cell deficient IL-18 / mice. NK cells were involved in the initition of disese, nd sence of NK cells resulted in enhnced TGF-β production y AChR-specific T cells. These effects were not oserved in mice lcking NKT cells. Collectively, these dt revel criticl link etween NK cells nd utorective T nd B cells. The mechnisms y which NK cells modulte dptive immune responses re not fully understood. Fctors tht determine the differentition of nïve T cells into either T H1 (IL-2, IFN-γ, TNF-α), or T H2 (IL-4, IL-10) phenotypes would e expected to hve n impct on the development of utoimmune responses. It hs een proposed tht intrcellulr cteri nd viruses stimulte NK cells to produce IFN-γ nd y this mens promote the differentition of ntigen-specific T cells into T H1 phenotype 20,21. Our findings support this notion. However, the production of IFN-γ y NK cells, lthough importnt, my not e sufficient for the induction of fulllown T H1 response 20 (nd s this study shows). TGF-β is potent inhiitor of T cell medited responses 16,17. Antigen-specific triggering of T H3 cells to produce TGF-β confers protection ginst EAMG induction 24. TGF-β ppers to contriute to the suppression of disese development in the current model. Our dt suggest n interply etween NK cells nd ntigen-specific T nd B cells: NK cells promote T H1 responses in prt y erly production of IFN-γ, nd further y suppression of TGF-β producing T cells. These effects of NK cells influence the ntigen-specific ntiody response. These oservtions do not exclude the possiility tht other signls from NK cells, or other inhiitory molecules involved in NK cell T cell interctions, my contriute to down-regultion of T H1 responses in this model. It hs een proposed tht n inhiitory circuit in which TGF-β produced y NK cells serves s criticl costimultory signl to induce CD8 + T suppressor cells 25,26. It is lso possile tht cytokines produced y NK cells collorte with cytokines produced y T cells to regulte utorective B cell responses. NK cells my lso directly interct with B cells 27. We oserved profound effects of NK1.1 + cell depletion on ntiody responses. However, in nother study it ws shown tht depletion of NK cells y nti-nk1.1 reduced host defense ginst mlignncy without loss of cellulr or humorl immunity 28. These different results re most likely due to the different sttus of the NK cells in the experimentl systems used. In n erlier study, NK cells did not receive n infectious signl nd thus my not hve een ctivted. The frequent ssocition etween infections nd utoimmune diseses in humns nd the use of cteril djuvnt to induce experimentl utoimmune diseses indictes tht initition of utoimmunity often involves NK cell ctivtion. The findings presented here further demonstrte tht NK cell ctivtion cn result in ltered ntiody responses, including those directed ginst utontigens. In reltion to the effects oserved on IgG2 concentrtions in NK cell depleted mice, it hs een proposed tht oth IFN-γ nd TGF-β regulte production of IgG2 to T cell dependent ntigens 29. However, TGF-β does not ffect IgG2 production in ll experimentl systems 29,30. In situtions when levels of oth IFN-γ nd TGF-β re ltered, it is uncler which one of these cytokines is most criticl for control of IgG2 production. In EAMG, results from severl lortories indicte tht nti-achr IgG2 is preferentilly suject to regultion y IFN-γ, in prticulr in situtions where multiple cytokines re ltered This pttern is in line with the oservtions presented here. Recent studies suggest tht susceptiility to T H1-medited utoimmune diseses in mice cn e ttriuted, t lest in prt, to the ville numers of NKT cells 7,31. It hs een suggested tht NKT cells confer protection due to their ility to promote T H2 development septemer 2000 volume 1 no 3 nture immunology 249

6 We lso oserved tht the NKT cell deficient mice exhiited tendency towrd reduced IL-4 production nd concomitnt increse in IFN-γ production. However, the two mouse strins deficient in NKT cells developed MG to similr extent s wild-type mice, suggesting tht this defect is not sufficient to enhnce disese. Nevertheless, these findings do not exclude possile role for NKT cells in the current model. The current dt provide insights into the possile mechnisms of utoimmune pthology. In tenttive scenrio, one my envisge n intrcellulr cteril or virus infection tht ctivtes NK cells. Next, NK cells my promote utoggresssion vi control of utorective T nd B cells during the initil ctivtion phse of these cells. Decresed NK cell function hs een oserved in fulminnt MG 32, s well s in some other utoimmune diseses 33. These studies of humn sujects my represent only prt of the history of utoimmune diseses, essentilly events in the estlished phse. Our study lso suggests tht n orchestr of NK cells nd utorective T nd B cells contriute to the genertion of destructive utoimmunity. Thus therpy tking into ccount ll these cellulr components, nd trgeting the criticl link etween them, could e successful in preventing utoimmune diseses. Methods Mice. B6 mice were otined from The Jckson Lortory (Br Hror, ME). The NKT cell deficient strins Jα281 / (ref. 14), CD1d1 / (ref. 15), IL-18 / (ref. 19), RAG-1 / (ref. 22) nd IFN-γ / (ref. 23) were ll ckcrossed to B6 ckground. All mice were housed in specific pthogen-free condition t the niml fcilities of the Microiology nd Tumor Biology Center, Krolinsk Institutet. Femle mice, ged 8 10 weeks t the initition of the experiments, were used. Antigens. AChR ws purified from the electric orgns of Torpedo clifornic (Pcific Biomrine, Venice, CA) y ffinity chromtogrphy on α-corotoxin grose resin (Sigm) 34. The isolted product ws pure s judged y SDS-PAGE. The purified Torpedo AChR ws used to induce EAMG nd for stimultion in vitro. Muscle AChR extrct from B6 mice ws prepred 34 nd used s ntigen for detection of ntiodies to mouse AChR. MBP used s control ntigen ws purified from norml mouse rins 35. AChRα( ): LGIWTYDGTKVSISPES 13 ws synthesized t the Swedish Institute for Infectious Disese Control, Stockholm, Sweden. Antiodies. The clone PK136 (nti-nk1.1) 36 ws otined from Americn Type Culture Collection (Mnsss, VA). Mouse IgG2 (Sigm) ws used s isotype control ntiody. For in vivo depletion of NK1.1 + cells, 300 µg monoclonl ntiody to NK1.1 ws dministered y intrperitonel (i.p.) injection to ech mouse 2 dys efore immuniztion unless otherwise stted. Every 5 7 dys therefter, 150 µg nti-nk1.1, or control ntiodies were injected vi the i.p. route until the termintion of experiments. Depletion efficcy ws checked y flow cytometry with nti-nk1.1 or nti-dx5 (oth from PhrMingen, L Joll, CA). In some experiments, the nti TGF-β 1D11.16 (mouse IgG1) specific for TGF-β1, TGF-β2, nd TGF-β3 nd 1410KG7 (mouse IgG1) isotype control ma 37 were injected vi the i.p route to neutrlize TGF-β. In these experiments, mice were treted with 1 mg nti TGF-β or control ma t the time of immuniztion, followed y 500 µg weekly dministrtion until the termintion of the experiments. Induction of EAMG nd mesurement of muscle AChR content. Mice were immunized sucutneously with 20 µg AChR in CFA in totl volume of 100 µl, nd t dys 30 nd 60 fter immuniztion oosted twice with 20 µg of AChR in CFA with sucutneous injection. The mice were scored every other dy fter the second immuniztion for signs of muscle wekness chrcteristic of EAMG. The disese symptoms were grded etween 0 nd 3 10 : 0, no definite muscle wekness; 1, norml strength t rest, ut wek with chin on the floor nd inility to rise the hed fter exercise consisting of 20 consecutive pw grips; 2, s grde 1, nd wekness t rest; 3, moriund, dehydrted nd prlyzed. Clinicl EAMG ws confirmed y injection of neostigmine romide nd tropine sulphte 10. For mesurement of muscle AChR content, two pm liquots of 125 I α-ungrotoxin (Amershm) leled Triton X-100 soluilized mouse muscle extrct ws mixed with stndrd pooled mouse AChR ntiserum in triplicte. After incution, rit nti-mouse immunogloulin (Dkoptts, Copenhgen, Denmrk) ws dded. The precipittes were counted in γ-counter (Pckrd Instrument Co., Meriden, CT) 34. Culture medium. Cells were suspended in Dulecco s modifiction of Egle medium (Gico, Pisley, UK) supplemented with 1% (v/v) minimum essentil medium (Gico), 2 mm glutmine (Flow L., Irvine, UK), 50 IU/ml penicillin nd 50 mg/ml streptomycin nd 10% (v/v) fetl clf serum (oth from Gico). Superntnts to e ssyed for TGF-β1 content were generted in Aim V serum-free medium (Life Technologies, Grnd Islnd, NY). Cytotoxicity ssy. NK cell medited cytotoxicity ws mesured using stndrd 51 Crrelese ssy 38. Spleen cells were incuted with 51 Cr-leled YAC-1 trget cells t the indicted effector/trget rtios. After 4 h of culture, superntnts were counted for 51 Cr relese in γ-counter (Pckrd Instrument Co.). Cell isoltion, sorting nd trnsfer. MNC were otined y mincing the poplitel nd inguinl lymph nodes through wire mesh. Spleen NK cells were sorted using FACStr plus (Becton Dickinson, Mountin View, CA). The sorted cells were >99% pure upon renlysis y flow cytometry. After overnight culture, the sorted NK cells were injected vi the intrvenous route into recipient mice. T cell prolifertion nd cytokine induction MNC were incuted in 200 µl culture medium in 96-well round-ottom microtiter pltes (Nunc, Copenhgen, Denmrk). 10 µl liquots of either AChR, AChRα( ), MBP or Con A (oth from Sigm) were dded into wells t finl concentrtions of 10 µg/ml (AChR, peptide or MBP) or 5 µg/ml (LPS nd Con A). After 4 dys of incution, the cells were pulsed for 18 h with 10 µl liquots contining 1 µci of [ 3 H]methylthymidine (specific ctivity 42 Ci/mmol; Amershm, Arlington Heights, IL). Cells were collected onto glss fier filters nd thymidine incorportion ws mesured. For cytokine induction, superntnts were collected t 48 h fter in vitro oosting. IFN-γ nd IL-4 were mesured y opteia kits (PhrMingen). TGF-β1 ws mesured with n ELISA kit (Promeg). The sensitivities of these ELISA ssys were 31.3 pg/ml for IFN-γ, 7.8 pg/ml for IL-4 nd 30 pg/ml for TGF-β1. Cytokine ELISPOT. An ELISPOT ssy ws used to detect cytokine secretion t the single cell level 33. Plstic pltes (Dyntech, Chntilly, VA) were coted with 100 µl IFN-γ cpture ntiody (Innogenetics, Genth, Belgium) t 15 µg/ml sorted NK1.1 + cells were cultured for 48 h. Secreted nd ound IFN-γ were visulized y ppliction of iotinylted detector ntiody (Innogenetics), nd ABC (Dkoptts). Similrly, IFN-γ nd TGF-β1 cytokine producing CD4 + lymph node cells were detected s descried previously 39. Assys of ntiodies to AChR IgG. An ELISPOT ssy ws used for enumerting nti- AChR IgG secreting cells 13,24. Anti-AChR were mesured y rdioimmunossy 34. Isotypes of nti-achr IgG were detected y ELISA using rit nti-mouse IgG1, IgG2, or IgG2 (Dkoptts) s descried 24. Sttisticl nlysis. Differences etween groups were evluted y ANOVA. Disese incidence nd severity were nlyzed y Fisher s exct test nd Mnn-Whitney s U-test, respectively. Acknowledgements We thnk N. 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