ARTICLES. MHC class II dependent basophil CD4 + Tcell interactions promote T H 2 cytokine dependent immunity

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1 MHC clss II dependent sophil CD4 + Tcell interctions promote T H 2 cytokine dependent immunity 29 Nture Americ, Inc. All rights reserved. Jcqueline G Perrigoue 1, Steven A Senz 1, Mrk C Sircus 1, Eric J Allenspch 2, Betsy C Tylor 1, Pul R Gicomin 1, Meer G Nir 1, Yurong Du 1, Coly Zph 3, Nico vn Rooijen 4, Michel R Comeu 5, Edwrd J Perce 1, Terri M Lufer 2,6 & Dvid Artis 1 Dendritic cells cn prime nive CD4 + T cells; however, here we demonstrte tht dendritic cell medited priming ws insufficient for the development of T helper type 2 cell dependent immunity. We identify sophils s dominnt cell popultion tht coexpressed mjor histocomptiility complex clss II nd interleukin 4 messge fter helminth infection. Bsophili ws promoted y thymic stroml lymphopoietin, nd depletion of sophils impired immunity to helminth infection. Bsophils promoted ntigen-specific CD4 + T cell prolifertion nd interleukin 4 production in vitro, nd trnsfer of sophils ugmented the popultion expnsion of helminth-responsive CD4 + T cells in vivo. Collectively, our studies suggest tht mjor histocomptiility complex clss II dependent interctions etween sophils nd CD4 + T cells promote T helper type 2 cytokine responses nd immunity to helminth infection. Since the demonstrtion of specifiction of CD4 + helper T cell ftes 1, sustntil dvnces hve een mde in delineting the regultory mechnisms tht promote distinct modules of CD4 + T cell dependent immunity nd inflmmtion 2. The differentition of T helper type 2 cells (T H 2 cells) is dependent on interleukin 4 receptor (IL-4R) nd the trnscription fctors STAT6 nd GATA-3, nd their signture cytokine profile is chrcterized y expression of IL-4 (A1262), IL-5, IL-9 nd IL-13 (ref. 3). The hllmrks of T H 2 cytokine dependent inflmmtion t rrier surfces such s the skin, irwy nd intestine include the recruitment of CD4 + T H 2 cells, eosinophils, mst cells nd sophils, coupled with golet cell hyperplsi, mucus production nd greter smooth muscle contrctility 4. Type 2 inflmmtory responses re required for immunity nd tissue repir fter exposure to helminth prsites. However, T H 2 cytokine responses cn lso promote the pthologicl chnges found in the context of sthm nd llergic diseses 5. Although the sequele of type 2 immunity nd inflmmtion in peripherl tissues re well chrcterized, the innte responses tht promote T H 2 cell development, including the nture of the ntigenpresenting cell (APC) involved, the host microil receptor lignd interctions nd the APC-derived fctors needed to initite nd sustin T H 2 cell differentition remin less well defined 6.Dendritic cells (DCs) re the only APC thought to prime nive T cells, nd the present prdigm suggests tht recognition of conserved pthogenssocited moleculr ptterns through distinct pttern-recognition receptors expressed on DCs promote the pproprite pthogenspecific CD4 + helper T cell responses 7. Activtion of DCs cn result in higher surfce expression of mjor histocomptiility complex (MHC) clss II nd costimultory molecules such s CD4, CD8 nd CD86, s well s the expression of fctors tht cn shpe the nture of the developing dptive immune response 8. However, the criticl DC-derived signls responsile for driving T H 2 cell responses in vivo remin undefined 9. In vitro studies hve indicted tht the requirements for DC-medited T H 2 differentition include differences in expression of the Notch lignd Jgged 1 nd upregultion of the costimultory molecules CD4 (ref. 11) nd OX4L 12. However, whether these pthwys re sufficient for DCs to promote the differentition of CD4 + T H 2 cells in vivo is uncler. The recruitment nd ctivtion of mst cells, eosinophils nd sophils re hllmrks of T H 2 cytokine dependent inflmmtion in peripherl tissues, nd erlier studies hve suggested tht these grnulocyte popultions my function s ccessory cells in the initition of CD4 + T H 2 cell responses. For exmple, mst cells, eosinophils nd sophils re le to produce nd secrete IL-4 from intrcellulr stores, which indictes these popultions re potentil erly sources of IL-4 tht could promote the differentition of CD4 + T H 2 cells In 1 Deprtment of Pthoiology nd 2 Deprtment of Medicine, University of Pennsylvni, Phildelphi, Pennsylvni, USA. 3 The Biomedicl Reserch Center, Deprtment of Pthology nd Lortory Medicine, University of British Columi, Vncouver, British Columi, Cnd. 4 Deprtment of Moleculr Cell Biology, Vrije Universiteit, Amsterdm, The Netherlnds. 5 Inflmmtion Reserch, Amgen, Settle, Wshington, USA. 6 Phildelphi Veterns Affirs Medicl Center, University of Pennsylvni, Phildelphi, Pennsylvni, USA. Correspondence should e ddressed to D.A. (drtis@vet.upenn.edu). Received 19 Decemer 28; ccepted 15 April 29; pulished online 24 My 29; doi:1.138/ni.174 NATURE IMMUNOLOGY VOLUME 1 NUMBER 7 JULY

2 IL-4 (pg/ml) IL-13 (pg/ml) ARTICLES Figure 1 Restriction of MHC clss II expression to CD11c + DC is insufficient to promote type 2 immunity to intestinl helminth infection. Anlysis of littermte control mice (Ctrl) nd MHC II CD11c mice (CII CD11c ) on dy 21 fter no infection or infection with T. muris eggs. () Enzyme-linked immunosorent ssy (ELISA) of IL-4, IL-5, nd IL-13 in superntnts of mln cells otined from nive (N) nd infected (INF) mice nd cultured in vitro for 48 h with nti-cd3 nd nti-cd28. *P ¼.3 (Student s t-test). () Cecl sections from nive nd infected mice, stined with Alcin lue periodic cid Schiff regent for the detection of mucins. Scle rs, 1 mm; originl mgnifiction 2. (c) Cecl sections from infected mice, stined y immunofluorescence for the golet cell mrker Go-5 (green) nd with the DNA-interclting dye c 1,26 63 IL-5 (pg/ml) 14 Ctrl CII CD11c Ctrl CII CD11c Ctrl 7 CII CD11c Go-5 DAPI DAPI (lue). Scle rs, 5 mm. (d) Immunolot nlysis of protein extrcted from pooled fecl pellets of mice, collected on dys 21 fter infection (ove lnes), to ssess luminl secretion of RELM. (e) Cecl worm urdens in infected mice, ssessed y microscopy. *P ¼.6 (Student s t-test). Dt re representtive of three independent experiments with three to five mice per group (men nd s.e.m. in,e). * 1,2 d 6 RELMβ N INF Ctrl CII CD11c Ctrl Nive Ctrl Infected CII CD11c Time (d) CII CD11c Nive Infected e Worm urden 175 * Ctrl CII CD11c 29 Nture Americ, Inc. All rights reserved. ddition, mst cells nd eosinophils cn express MHC clss II, nd eosinophils hve een suggested to e potentil APCs in oth irwy inflmmtion nd helminth infection Bsophil frequencies re higher fter exposure to llergens nd helminth prsites, nd pulished work hs demonstrted tht sophils re dominnt source of IL-4 nd IL-13 fter helminth infection nd contriute to protective immunity Although sophili is common feture of T H 2 cytokine medited inflmmtion, little is known out how these cells re ctivted nd recruited to peripherl tissues. A conserved mechnism for sophil-medited recognition of prsite products nd llergens through protese-dependent ctivtion hs een proposed 22. In tht study, sophils were recruited to the drining lymph node erly fter llergen exposure nd were essentil for the genertion of T H 2 cytokine responses elicited fter ppin immuniztion. However, the potentil ccessory cell functions of sophils during the development of CD4 + T H 2 cells remin unknown. Collectively, the inility of DCs to express IL-4 nd the lck of defined mechnisms y which DCs promote T H 2 cell differentition provoked ressessment of the reltive contriution of DCs in promoting T H 2 cytokine responses in vivo. In this study, we demonstrte tht DC-restricted expression of MHC clss II ws insufficient for the genertion of protective CD4 + T H 2 cytokine dependent immunity to the gstrointestinl helminth Trichuris muris. We identify sophils s dominnt ccessory cell popultion tht expressed Il4 messge nd MHC clss II. In vitro studies show tht sophils could promote MHC clss II dependent ntigen-specific prolifertion of CD4 + T cells nd differentition of T H 2 cells. Depletion of sophils in vivo resulted in impired protective immunity to T. muris, wheres doptive trnsfer of primry wildtype sophils ugmented the prolifertion of CD4 + T cells in response to the injection of Schistosom mnsoni eggs. Our studies collectively suggest previously unrecognized function for sophils in MHC clss II dependent cognte interctions with CD4 + T cells tht promote prsite-specific T H 2 cytokine responses nd host protective immunity. RESULTS CD11c-restricted MHC II is insufficient for type 2 immunity To determine whether ntigen presenttion y CD11c + DCs ws sufficient to promote CD4 + T H 2 cell dependent immunity in vivo, we infected mice in which MHC clss II expression is restricted to CD11c + cells (MHC II CD11c mice; Supplementry Fig. 1 online) with the intestinl helminth prsite T. muris. Expulsion of T. muris nd protective immunity is dependent on CD4 + T H 2cells,wheresprsitespecific production of interferon-g (IFN-g) promotes chronic infection T. muris infection provides well-chrcterized in vivo model of T H 2 cytokine dependent immunity tht offers functionl redout of the mgnitude of the host T H 2 cytokine response. As MHC II CD11c mice lck MHC clss II expression on the thymic epithelium 26 nd therefore re unle to positively select CD4 + T cells in the thymus, we gve mice fetl thymic grfts 8 weeks efore infection to provide n endogenous CD4 + T cell popultion. After infection with T. muris, littermte control mice developed pthogeninduced T H 2 cytokine responses chrcterized y production of IL-4, IL-5 nd IL-13 y mesenteric lymph node (mln) cells (Fig. 1). In contrst, MHC II CD11c mice showed miniml infection-induced production of T H 2 cytokines (Fig. 1). Histologicl nlysis of intestinl tissues in infected control mice showed hllmrks of type 2 inflmmtion, including golet cell hyperplsi nd mucin production (Fig. 1) nd expression of Go5 (lso known s mclca3; Fig. 1c), golet cell specific mrker regulted y T H 2 cytokines nd ssocited with type 2 inflmmtion 27. Consistent with lower production of T H 2 cytokines, infected MHC II CD11c mice hd notle sence of golet cells nd golet cell derived proteins (Fig. 1,c). T H 2 cytokine dependent expression nd luminl secretion of golet cell derived resistinlike molecule- (RELM) in resistnt mice provides noninvsive indictor of the kinetics of T H 2 cytokine responses in the intestinl microenvironment 28. As reported efore 28, luminl RELM protein peked in resistnt control mice etween dys 12 nd 18 fter infection, coincident with worm expulsion (Fig. 1d), wheres luminl secretion of RELM in infected MHC II CD11c mice ws much lower in mgnitude (Fig. 1d). Associted with polrized T H 2 cytokine response, littermte control mice lso hd higher titers of immunogloulin G1 (IgG1) nd IgE (dt not shown). However, s MHC II CD11c mice lck MHC clss II expression on B cells 26, we detected no ntigen-specific clss-switched ntiody in infected MHC II CD11c mice (dt not shown). Criticlly, the defect in T H 2 cytokine responses in MHC II CD11c mice resulted in susceptiility to infection in mice on normlly geneticlly resistnt ckground (Fig. 1e). These dt collectively demonstrte tht restriction of MHC clss II dependent ntigen presenttion to CD11c + cells ws insufficient to promote CD4 + T H 2 cell dependent immunity fter intestinl helminth infection. 698 VOLUME 1 NUMBER 7 JULY 29 NATURE IMMUNOLOGY

3 29 Nture Americ, Inc. All rights reserved. Figure 2 Blockde of IFN-g in MHC II CD11c mice restores T H 2 cytokine dependent immunity to T. muris infection. () Totl numer of mln CD4 + T cells from nive nd T. muris infected littermte control nd MHC II CD11c mice, ssessed y flow cytometry. Dt re representtive of three independent experiments with three to five mice per group (men nd s.e.m. of three mice per group). () Quntittive rel-time PCR nlysis of Ifng mrna expression in mlns from infected mice, presented s fold increse reltive to expression in cells from nive control mice. *P ¼.3 (Student s t-test). Dt re representtive of three experiments (men nd s.e.m.). (c) Flow cytometry of intrcellulr IFN-g stining of mln cells from infected control nd MHC II CD11c mice, gted on CD4 + Tcells. Numers djcent to outlined res indicte frequency of cells tht produced IFN-g (old) or mount of IFN-g per cell (men fluorescence intensity; itlics) for cells in the gted re. Dt re representtive of three independent experiments with three to five mice per group. (d) ELISA of IFN-g secretion y mln cells isolted from nive nd infected mice. *P ¼.3 (Student s t-test). Dt re representtive of three experiments (men nd s.e.m.). (e) ELISA of cytokine production y mln cells isolted from infected MHC II CD11c mice given control tretment ( ) or tretment with nti-ifn-g (-IFN-g). *P ¼.3; **P ¼.2; nd ***P ¼.1 (Student s t-test). Dt re representtive of one experiment (men nd s.e.m. of three to four mice per group). (f) Cecl sections from infected littermte control mice or from infected MHC II CD11c mice fter control tretment (Ctrl Tx) or tretment with nti-ifn-g, stined for mucins with Alcin lue periodic cid Schiff. Scle rs, 1 mm. Results re representtive of one experiment with three to four mice per group. (g) Worm urdens t dy 21 fter infection of MHC II CD11c mice given control tretment ( ) or tretment with nti-ifn-g (+). *P o.1 (Student s t-test). Dt re representtive of one experiment (men nd s.e.m. of three to four mice per group). Intct T H 1 differentition in MHC II CD11c mice The extensive physicl nd iochemicl rriers etween ntigenic mteril in the enteric spce nd lymphocytes in the underlying lymphoid follicles nd lmin propri of the intestine crete unique chllenges in ntigen smpling nd presenttion 29. Therefore, the impired T H 2 cytokine responses in T. muris infected MHC II CD11c mice my indicte tht dditionl APCs re required for either the smpling of T. muris ntigens or the provision of signls necessry for the priming, prolifertion nd differentition of pthogen-specific CD4 + T cells. However, fter T. muris infection, oth littermte control mice nd MHC II CD11c mice showed n infection-induced increse in totl CD4 + T cell numers in the drining mlns (Fig. 2), which suggests tht DC-restricted ntigen presenttion ws sufficient for promoting the prolifertion of CD4 + T cells fter infection. To determine whether CD4 + T cells in infected MHC II CD11c mice were unresponsive or hd received signls for lterntive differentition, we isolted mrna from mlns of nive nd infected control nd MHC II CD11c mice nd nlyzed the expression of Il1, Il17, ndifng to ssess the mgnitude of regultory T cell, IL-17-producing T helper cell nd T H 1 cell responses. Although there ws little to no induction of the expression of Il1 nd Il17 in infected control nd MHC II CD11c mice (Supplementry Fig. 2 online), Ifng mrna ws selectively nd significntly induced in infected MHC II CD11c mice reltive to its expression in control mice (Fig. 2). Consistent with higher Ifng mrna expression, the frequency of mln CD4 + T cells tht produced IFN-g s well s the mount of IFN-g mde per cell were greter in infected MHC II CD11c mice thn in control mice (Fig. 2c). Secretion of IFN-g ws lso significntly greter fter in vitro stimultion of mln cellsisoltedfrominfectedmhcii CD11c mice thn in control mice (Fig. 2d). Thus, fter intestinl infection, cognte interctions etween ntigen-specific CD4 + T cells nd CD11c + DCs lone were sufficient to promote the priming nd popultion expnsion of CD4 + T cells s well s to provide signls necessry for T H 1 differentition ut were insufficient for the development of T H 2 cytokine dependent immunity. These dt suggested tht CD11c + cells my not e required for T H 2 cytokine dependent immunity. To determine the reltive CD4 + cells ( 1 6 ) e IFN-γ (ng/ml) mln N 1 INF 25 * Ctrl CII CD11c * α-ifn-γ IL-4 (ng/ml).8.4 Ifng (fold) 125 α-ifn-γ Ctrl CIICD11c α-ifn-γ IFN-γ FSC ** 1 18 *** IL-5 (ng/ml).5 c IL-13 (ng/ml) α-ifn-γ Ctrl f CII CD11c CII CD11c 4, Ctrl Ctrl Tx α-ifn-γ α-ifn-γ contriution of CD11c + cells in immunity to T. muris, weusedthe CD11c diphtheri toxin receptor (CD11c-DTR) mouse model, in which delivery of diphtheri toxin to CD11c-DTR mice results in the selective poptosis of CD11c + cells, wheres similr dministrtion to littermte controls hs no effect. To void the toxicity ssocited with long-term tretment of intct CD11c-DTR mice with diphtheri toxin, we used one mrrow chimers creted y the trnsfer of wildtype or CD11c-DTR donor one mrrow into wild-type recipients. Although trnsient depletion of CD11c + cells (Supplementry Fig. 3 online) throughout the course of T. muris infection resulted in significntly fewer mln CD4 + T cells (P ¼.1; Supplementry Fig. 3), there ws no effect on the production of T H 2 cytokines or worm urden (Supplementry Fig. 3c,d). Together these dt suggest tht CD11c + cells my not e essentil for protective immunity to T. muris nd tht nother APC my e required for the development of T H 2 cytokine dependent immunity in vivo. To determine whether ltertions in the cytokine milieu could restore immunity in MHC II CD11c mice, we treted T. muris infected MHC II CD11c mice with monoclonl locking ntiody to IFN-g (nti-ifn-g) during the course of infection. Consistent with findings reported ove (Figs. 1 nd 2d), stimulted T cells isolted from the mlns of infected MHC II CD11c mice showed roust IFN-g response with low concentrtions of IL-4, IL-5 nd IL-13 (Fig. 2e). Associted with the lck of T H 2 cytokines, infected, control-treted MHC II CD11c mice showed lower golet cell responses nd susceptiility to T. muris infection (Fig. 2f,g). Tretment of MHC II CD11c mice with nti-ifn-g resulted in significntly lower IFN-g production nd the emergence of T H 2 cytokine response chrcterized y significntly greter production of IL-4, IL-5 nd IL-13 y mln cells, golet cell hyperplsi nd recovery of immunity to infection (Fig. 2e g). These dt collectively suggest tht fter lockde of nonprotective T H 1 cytokine response, CD11c + cells lone cn provide the ntigen-specific interctions needed to drive CD4 + T H 2 cell differentition nd protective immunity. However, in the presence of endogenous IFN-g signls, non-dc popultions re required for the development of protective T H 2 cytokine responses in vivo. d IFN-γ (ng/ml) g Worm urden N INF * Ctrl CII CD11c * + NATURE IMMUNOLOGY VOLUME 1 NUMBER 7 JULY

4 29 Nture Americ, Inc. All rights reserved. Cells 5, NBNT IL-4 egfp d MHCII MHCII 1 5 NBNT, IL-4 egfp + IL-4 egfp MHCII-KO sophil WT sophil WT B cell c-kit Eosinophils 4, 58 ± 4 35 ± 1 Bsophils express MHC clss II nd Il4 mrna In ddition to DCs, mcrophges nd B cells re professionl APCs involved in the development of dptive CD4 + T cell dependent immunity. However, depletion of mcrophges in mice y tretment with clodronte-loded liposomes hd no effect on cytokinedependent inflmmtion or worm expulsion (Supplementry Fig. 4 c online). Pulished work hs demonstrted tht doptive trnsfer of CD4 + T cells lone into mice lcking oth B cells nd T cells is sufficient to restore immunity to T. muris 3.MicedeficientinBcells (mmt mice) lso showed intct T H 2 cytokine dependent golet cell responses nd protective immunity (Supplementry Fig. 4d f). Collectively, these dt suggest tht lthough mcrophges nd B cells my contriute to immunity to T. muris in physiologicl setting, they do not hve essentil nonredundnt functions in host protective immunity. We therefore focused on identifying cells of the innte immune system tht could oth express MHC clss II nd provide n innte source of IL-4 fter T. muris infection. IL-4 egfp (4get) reporter mice hve een used to trck emerging CD4 + T H 2 responses fter T. muris infection 31. These mice hve n internl riosoml entry site enhnced green fluorescent protein (egfp) element in the Il4 locus tht llows direct ex vivo nlysis of cells le to express IL-4 (ref. 32). We used 4get mice to identify non B, non T cells tht coexpressed Il4 mrna nd MHC clss II molecules. By gting on non B, non T cells, we identified n IL-4 egfp + cell popultion (Fig. 3) tht expressed MHC clss II (Fig. 3). Pulished studies hve shown tht mst cells nd eosinophils cn express MHC 6.1 FcεRI SSC SSC c IL-4 egfp + MHCII + 4, 1 5 Siglec-F CD49 Nive Mst cells 2.2 ± ± Infected 1 5 Bsophils 25 ± 2 42 ± 3 Figure 3 FceRI + CD49 + sophils coexpress MHC clss II nd IL-4 egfp. Flow cytometry of splenocytes isolted from nive nd T. muris infected 4get mice t dy 14 fter infection. () Identifiction of IL-4 egfp + cells with non B, non T cell gte (CD3 B22 CD19 ;NBNT). () Anlysis of the coexpression of MHC clss II (MHCII) nd IL-4 egfp y the cells identified in. (c) Expression of c-kit, Siglec-F, FceRI nd CD49 y IL-4 egfp +, MHC clss II positive (MHCII + ) non B, non T cells. (d) MHC clss II expression on MHC clss II deficient (MHCII-KO) sophils, wild-type (WT) sophils nd wild-type B cells isolted from nive mice. Numers ove rcketed line () or djcent to outlined res(,c) indicte percent cells in gte. Dt re representtive two experiments with three mice per group (men ± s.e.m. in c). 1 5 clss II (refs ) nd re le to produce IL-4 (refs. 13,14). However, we did not find clssicl mst cells (c-kit + side-sctter high (SSC hi )) fter infection with T. muris, nd frequencies of Siglec-F + SSC hi eosinophils were lower fter infection (Fig. 3c). In contrst, CD49 + FceRI + sophils emerged s dominnt cell type tht expressed oth Il4 mrna nd MHC clss II fter T. muris infection (Fig. 3c), consistently comprising 4% of IL-4 egfp + MHC clss II positive cells. Although their MHC clss II expression ws not s high s tht in professionl APCs such s B cells, sophils hd intermedite expression of MHC clss II reltive to tht of MHC clss II deficient sophils (Fig. 3d). Although there hve een reports of MHC clss II expression on eosinophils 17,18,thisis the first report to our knowledge demonstrting MHC clss II expression on mouse sophils, nd this result suggests potentil ccessory cell function for this cell popultion during helminth infection. Bsophil depletion impirs immunity To determine whether sophils re involved in the development of T H 2 cytokine dependent protective immunity, we infected wild-type C57BL/6 mice with T. muris nd treted them with either control imunogloulin or monoclonl ntiody (MAR-1) to the receptor FceRI. Studies hve demonstrted efficient depletion of sophils for up to 1 d fter intrperitonel injection of MAR-1 (ref. 33), nd in mice treted with MAR-1, we found 9% depletion of sophils t dy 21 fter infection (Fig. 4). Depletion of sophils in infected mice resulted in lower expression of Il4 mrna (Fig. 4), much less T H 2 cytokine dependent golet cell hyperplsi (Fig. 4c) nd less luminl secretion of RELM in the intestine (Fig. 4d). Loss of sophils nd impired T H 2 cytokine responses were ssocited with impired expulsion of T. muris (Fig. 4e). These dt collectively support the ide of function for sophils in the development of protective type 2 immunity to intestinl helminth infection. Thymic stroml lymphopoietin selectively elicits sophils Essentil functions hve een identified for the intestinl epithelil cell (IEC)-derived cytokines IL-25 (ref. 34) nd thymic stroml lymphopoietin (TSLP; A2363) 27 in the development of T H 2 cytokine dependent immunity to T. muris. In ddition, IEC-derived IL-33 hs een shown to promote T H 2 cytokine responses nd worm expulsion 35, nd severl studies hve demonstrted tht tretment with IL-33 cn directly stimulte cytokine nd chemokine production from sophils nd mst cells in vitro To determine whether IL-25, IL-33 or TSLP contriuted to sophil responses in vivo, weinjected 4get mice with recominnt IL-25, IL-33 or TSLP nd exmined peripherl sophil responses y flow cytometry. As reported efore 39, IL-25 tretment elicited popultion of IL-4 egfp + SSC hi cells (Fig. 5). Tretment with IL-33 lso resulted in higher frequency of IL-4 egfp + SSC hi cells (Fig. 5). However, phenotypic nlysis of these IL-4 egfp + cells showed tht there were two distinct cell 7 VOLUME 1 NUMBER 7 JULY 29 NATURE IMMUNOLOGY

5 Figure 4 Depletion of FceRI + cells in vivo results in impired immunity to T. muris infection. () Flow cytometry of splenic sophils from wildtype mice treted with control immunogloulin (Hmster Ig) or MAR-1 t dy 21 fter infection, gted on CD3 B22 CD19 non B, non T cells. Numers djcent to outlined res indicte percent cells in ech gte. () Rel-time quntittive PCR nlysis of colon tissue from nive nd infected mice treted with control immunogloulin (Ig) or MAR-1, ssessed t dy 21 fter infection, presented s fold increse reltive to expression in nive mice treted with control immunogloulin. (c) Cecl sections from nive nd infected mice treted with control immunogloulin (Hmster Ig) or MAR-1, ssessed t dy 21 fter infection, stined for mucins with Alcin lue periodic cid Schiff. FcεRI CD49 Hmster Ig 1.88 MAR Originl mgnifiction, 2. (d) Immunolot nlysis of RELM in protein extrcted on dys 9, 12 nd 15 fter infection (ove lnes) from pooled fecl pellets of mice treted with control immunogloulin (Ig) or MAR-1. (e) Cecl worm urdens t dy 21 fter infection in mice treted with control immunogloulin (Ig) or MAR-1. Dt re representtive of two independent experiments with three to four mice per group (error rs (,e), s.e.m.). il4 (fold) ifng (fold) Ig MAR-1 N INF c Nive Infected Hmster Ig MAR-1 d RELMβ Time (d) Ig MAR-1 e Worm urden 125 Ig MAR-1 29 Nture Americ, Inc. All rights reserved. popultions selectively elicited y ech cytokine. Tretment with IL-25 resulted in greter frequencies of non B, non T cell c-kit + mst cell like popultion, wheres tretment with IL-33 led to greter frequency of CCR3 + eosinophils (Supplementry Fig. 5 online). Although dministrtion of TSLP lso resulted in threefold greter numer of IL-4 egfp + cells thn did tretment with PBS (Fig. 5), unlike IL-25 nd IL-33, TSLP selectively elicited CD49 + FceRI + sophils (Fig. 5). These dt suggest tht wheres IEC-derived IL-25, IL-33 nd TSLP promote the expnsion of diverse innte cell popultions le to produce IL-4, only TSLP promotes sophil popultion expnsion. Bsophils promote the differentition of CD4 + T H 2cells The demonstrtion tht depletion of sophils resulted in impired immunity to T. muris (Fig. 4), coupled with the coexpression of MHC clss II nd Il4 mrna (Fig. 3), suggested tht they my lso prticipte in MHC clss II dependent cognte interctions with CD4 + T cells to promote T H 2 differentition. To determine whether sophils could present ntigen, we dopted n in vitro coculture system in which we ctivted ntigen-pulsed purified sophils with recominnt IL-3 to provide survivl signls nd promote IL-4 production nd cultured them together with purified ovlumin (OVA)-specific DO11.1 CD4 + T cells leled with the cytosolic dye CFSE. Sorted sophils showed chrcteristic multiloed nuclei nd expressed oth MHC clss II nd IL-4 egfp (Fig. 6). Although we detected miniml prolifertion in the sence of OVA peptide, pproximtely 75% of CD4 + T cells cultured in the presence of ntigenpulsed sophils hd diluted CFSE, consistent with prolifertion (Fig. 6). Bsophilinduced prolifertion of CD4 + T cells ws dependent on MHC clss II expression, s the ddition of locking ntiody to MHC clss II rogted these responses (Fig. 6). To determine whether sophils could influence the differentition of CD4 + T H 2 cells fter ntigen-specific stimultion of T cells, we did intrcellulr cytokine stining for IL-4 NBNT SSC NBNT, IL-4 egfp + CD49 25K 1 5 IL-4 egfp FcεRI (Fig. 6) nd mesured IL-4 in superntnts of cocultured cells (Fig. 6c). Superntnts of sophils nd T cells cultured together in the sence of ntigen contined sl mounts of IL-4 (Fig. 6c), nd fter the ddition of OVA peptide, there ws two- to threefold more IL-4, n enhncement tht ws rogted in the presence of nti MHC clss II (Fig. 6c). Therefore, MHC clss II dependent cognte interctions etween sophils nd CD4 + T cells cn promote ntigenspecific T H 2differentitionin vitro. S. mnsoni eggs recruit sophils to lymph nodes We sought to determine whether the recruitment of IL-4 egfp + MHC clss II positive sophils ws unique to T. muris infection or whether tht ws common event fter exposure to other helminth prsites. For this, we used footpd injection of S. mnsoni eggs, which results in cute nd synchronous T H 2 cytokine responses in the drining poplitel lymph node (pln). Roust prolifertion of CD4 + T cells hs een demonstrted fter egg injection, with over 4% of pln CD4 + T cells ecoming positive for the thymidine nlog BrdU PBS IL-25 IL-33 TSLP Figure 5 TSLP tretment selectively increses sophil frequencies in vivo. Flow cytometry of sophil frequencies in lood of mice treted dily for 4 d with recominnt IL-25, IL-33 or TSLP. () Expression of IL-4 egfp y non B, non T cells from the peripherl lood. () Bsophil frequencies in IL-4 egfp + non B, non T cell popultions. Numers djcent to outlined res indicte percent mong totl cells of gted popultion. Dt re representtive of t lest two independent experiments with three mice per group. NATURE IMMUNOLOGY VOLUME 1 NUMBER 7 JULY 29 71

6 29 Nture Americ, Inc. All rights reserved. FcεRI Cells IL Sorted sophils CD49 Bso + T cell Medi Cytospin Bso + T cell OVA peptide MHCII Bso + T cell OVA peptide + α-mhcii CFSE CD4 nd 2% expressing IL-4 egfp 4,whichprovidespowerfulin vivo model for exmining helminth-induced innte nd dptive responses. We delivered S. mnsoni eggs into the footpds of 4get mice nd collected plns t vrious times fter injection. Trnsient recruitment of sophils to the drining pln occurred y dy 2 fter injection, with n over 2-fold higher frequency (Fig. 7) nd numer (Fig. 7) tht ws sent y dy 5 (dt not shown). Sorted IL-4 egfp + sophils from mice injected with S. mnsoni eggs showed chrcteristic multiloulr nuclei y cytospin nlysis (Fig. 7c), were Events (% of mximum) , IL-4 egfp c IL-4 (U/ml) ,5 Medi OVA peptide OVA peptide α-mhcii Figure 6 Bsophils promote MHC clss II dependent ntigen-specific CD4 + Tcell prolifertion nd T H 2 cytokine production in vitro. () Flow cytometry (left) nd DiffQuick stining (middle) of sorted TSLP-elicited sophils; numer djcent to outlined re (left) indictes percent FceRI + CD49 + cells. Right, expression of MHC clss II nd IL-4 egfp (solid lines) on sorted TSLP-elicited sophils; shded histogrms, fluorescence minus one (FMO) controls. Numers in plots indicte men fluorescence intensity. () CFSE dilution (top) y DO11.1 CD4 + T cells fter 4 d of culture together with sophils (Bso) in medi lone or with OVA peptide or OVA peptide plus MHC clss II locking ntiody M5114 (-MHCII). Below, frequency of IL-4 + CD4 + T cells (numers djcent to outlined res), ssessed y intrcellulr cytokine stining. Numers in plots indicte percent CFSE lo cells (divided cells, left of ornge verticl line, top) or percent IL-4 + CD4 + cells (elow). (c) ELISA of IL-4 in superntnts of sophil CD4 + T cell cocultures treted s descried in. Dt re representtive of four independent experiments with results of five to ten mice pooled per experiment () orre representtive of two independent experiments (,c). FceRI + nd expressed MHC clss II, s ssessed y oth flow cytometry (Fig. 7d) nd immunofluorescence (Fig. 7e). We next investigted whether helminth-elicited sophils could influence the prolifertion of CD4 + T cells in vivo. To ddress this, we doptively trnsferred CFSE-leled CD4 + T cells into MHC II CD11c mice. We then used the S. mnsoni egg injection model to ssess whether doptive trnsfer of sophils influenced helminth-induced prolifertion of CD4 + T cells in the drining pln. We sorted wild-type sophils from mice injected with S. mnsoni eggs nd doptively trnsferred the cells into nive MHC II CD11c recipients. In the sence of dditionl ntigen stimultion in recipient mice, trnsfer of CD Nive IL-4 egfp.4 S. mnsoni eggs.9 Bsophils ( 1 4 ) 2 1 c d N INJ FcεRI MHCII Figure 7 IL-4 egfp + MHC clss II positive sophils re recruited to the drining lymph node fter exposure to S. mnsoni eggs, nd they ugment CD4 + Tcell prolifertion in vivo. () Flow cytometry of sophil frequencies in plns from nive 4get mice or 4get mice injected with S. mnsoni eggs, ssessed t dy 2 fter injection. Numers djcent to outlined res indicte percent CD49 + IL-4 egfp + cells. () Totl numers of sophils in the lymph nodes of nive mice (N) or mice injected with S. mnsoni eggs (INJ). (c) Cytospin of sorted S. mnsoni elicited sophils stined with Diffquick. (d) Flow cytometry of sorted S. mnsoni elicited sophils stined for FceRI or MHC clss II. Shded histogrms indicte CD4 T cells ( 1 3 ) Eggs Eggs + so expression on CD3 + cells; lck lines re sorted sophils. Numers in plots indicte men fluorescence intensity. (e) Confocl microscopy of sorted S. mnsoni elicited sophils. Green, GFP; red, MHC clss II; lue, DAPI. Originl mgnifiction, 1. (f) Totl CD4 + cells in the nondrining pln (ndpln) nd drining pln (d-pln) of MHC CII CD11c mice tht received S. mnsoni eggs lone (Eggs) or in comintion with sophils (Eggs + so). (g) Flow cytometry of CFSE dilution y CD4 + T cells from MHC CII CD11c mice tht received S. mnsoni eggs lone (shded histogrm) or with sophils (lck line). Numers in plots indicte percent CFSE lo cells. Dt re representtive of five independent experiments with three to five mice per group ( e) orone experiment with two mice per group (f,g; error rs (,f), s.e.m.). Events (% of mximum) f nd-pln d-pln g Events (% of mximum) 1 e 5 67 CFSE µm VOLUME 1 NUMBER 7 JULY 29 NATURE IMMUNOLOGY

7 29 Nture Americ, Inc. All rights reserved. sophils lone did not induce the recruitment of ntigen-specific T cells to the pln (dt not shown). We therefore chllenged MHC II CD11c mice tht hd received oth T cells nd sophils with S. mnsoni eggs in the footpd. After egg injection, MHC II CD11c mice tht received eggs lone hd four- to fivefold more CD4 + Tcells in the drining pln thn in the nondrining pln (Fig. 7f), nd 5% of pln CD4 + T cells were CFSE lo (Fig. 7g). In contrst, prolifertion of CD4 + T cells ws sustntilly ugmented in MHC II CD11c mice tht hd received ctivted sophils. At dy 4 fter delivery of S. mnsoni eggs, there were over 14-fold more pln CD4 + Tcells(Fig. 7f), nd nerly 7% of the CD4 + TcellswereCFSE lo (Fig. 7g). This prolifertion wsconsistentwiththemgnitudeofcd4 + T cell responses reported efore in egg-injected wild-type mice 4. In ddition, pulished studies hve shown tht unlike T cells doptively trnsferred into recipient mice deficient in recomintion-ctivting gene 1 or 2, donor CD4 + Tcells delivered into MHC II CD11c mice do not undergo homeosttic prolifertion,prolyecusemhcii CD11c mice hve norml CD8 + Tcellcomprtment 26,41. Collectively, these dt demonstrte tht MHC clss II positive sophils re rpidly recruited to the lymph node fter exposure to helminth ntigens nd suggest potentil coopertion etween sophils nd DCs in the efficient popultion expnsion of helminth-responsive CD4 + T cells in vivo. DISCUSSION Bsophils re rre circulting cells tht mke up less thn.5% of totl lood leukocytes yet re evolutionrily conserved cross ll verterte species nd cn ccumulte in peripherl tissues in mny settings ssocited with type 2 inflmmtion. Although sophils were first descried 13 yers go 42, their scrcity, coupled with pucity of regents, hs mde it difficult to study their function in vivo. The vilility of new regents hs llowed the identifiction of distinct nonredundnt functions for sophils in ugmenting CD4 + T H 2 cytokine responses 22,43, in providing B cell help for IgE clss-switch recomintion nd enhnced humorl immune responses 33,44,ndin the initition nd mintennce of chronic llergic inflmmtion 45.To those functions, the results of our study here dd previously unrecognized function for sophils s ccessory cells tht cn promote the differentition of CD4 + T H 2 cells in prt through MHC clss II dependent cognte interctions, s indicted y sophil CD4 + Tcell coculture experiments. A criticl question tht emerges from these findings is where the functionl sophil T cell cognte interctions occur in vivo. Bsophils re redily found in the lood nd spleen ut hve een reported to e excluded from lymph nodes, where CD4 + Tcellprimingprolytkesplce 46. However, sophils hve een shown to e trnsiently recruited to drining lymph nodes fter llergen exposure 22. In this study, we hve demonstrted tht sophils were rpidly recruited to the lymph node fter exposure to S. mnsoni eggs. Bsophils tht ccumulted in lymph nodes expressed oth MHC clss II nd Il4 mrna, which suggests tht they re le to interct directly with nive T cells in peripherl lymphoid tissues. Consistent with involvement of sophils in the development of T H 2 cell responses, in vivo depletion of sophils resulted in impired expression of T H 2 cytokines nd host protective immunity, wheres doptive trnsfer of sophils ugmented helminth-induced prolifertion of CD4 + T cells. In ddition to their potentil involvement in the initil priming of nive CD4 + T cells in the lymph node, sophils my ct s ccessory cells t the site of inflmmtion, where ctivted T cells my require dditionl cognte interctions to promote or mintin T H 2 cell differentition nd effector function. In support of tht ide, pulished report of cytokine reporter mice hs demonstrted tht cytokine mrna expression nd cytokine protein expression re uncoupled fter the priming nd popultion expnsion of nive T cells, which suggests tht dditionl ctivtion t the site of infection my e needed to license effector function 47. Depletion of sophils lso suggests tht these cells my provide chemotctic fctors, either directly or indirectly, tht re required for the recruitment of eosinophils to peripherl tissues 48. Microrry nlyses of sophils sorted from the lung during infection with Nippostrongylus rsiliensis hs lso shown high expression of the chemokines CCL3 (MIP1), CCL4, (MIP1), CCL6 (C1) nd CCL17 (TARC), which supports the ide tht sophils re involved in the recruitment of ctivted CD4 + T cells to the site of infection 21. Therefore, identifying the fctors tht regulte sophil prolifertion nd recruitment could e n importnt trget for modulting erly events in the genertion of CD4 + T H 2 cell dependent immunity nd inflmmtion. Pulished reports hve identified T cell derived IL-3 s criticl cytokine for sophili during intestinl helminth infection 49.However, whether other cytokines derived from cells of the innte immune system contriute to erly sophil responses is uncler. A criticl function hs een identified for IEC ctivtion in the genertion of protective T H 2 cytokine dependent immunity to T. muris, ndtslp hs een shown to e n importnt prt of the IEC response required for immunity to infection 27,5. Here we hve shown tht delivery of recominnt TSLP resulted in the selective ccumultion of sophils in the periphery, which identifies previously unpprecited function for TSLP in promoting sophili. TSLP hs een linked efore to the promotion of type 2 inflmmtion in the skin nd lung through effects on cells of oth the innte nd dptive immune systems 51,52. Although there ws no nlysis of sophil responses in the TSLPtrnsgenic mice used in those erlier studies, it is plusile tht component of the enhnced type 2 inflmmtion oserved could hve een consequence of enhnced sophil responses. In ddition to TSLP, the IEC-derived cytokines IL-25 nd IL-33 hve lso een linked to the promotion of type 2 inflmmtion, nd IL-33 cn directly ctivte sophils 34,36 38,53 56.However,wehve shown tht these cytokines, lthough they were le to promote the ccumultion of IL-4 egfp + innte cells, did not promote sophili in vivo. Insted, IL-25 promoted the prolifertion nd/or ccumultion of c-kit + cells, wheres IL-33 promoted peripherl eosinophili. Although TSLP seems to hve selective effect on sophil responses, the influence of TSLP, IL-25 nd IL-33 in comintion with other stimuli, such s IL-3, IL-18, Toll-like receptor ligtion nd FceRI crosslinking, on sophil cytokine production, recruitment to the lymph nodes nd APC function remins to e determined. In ddition to IEC-derived cytokines, there is evidence to suggest tht sophils cn e directly ctivted y either helminth-derived products or llergens tht my ct s superllergens to stimulte FceRI crosslinking in non ntigen-specific wy 57. For exmple, IPSE1, glycoprotein derived from S. mnsoni eggs, hs een shown to directly stimulte the production of IL-4 y sophils vi n IgE-dependent ut non ntigen-specific mechnism 58. Thus, comintion of cytokines derived from nonhemtopoietic cells nd cells of the innte immune system, coupled with direct stimultion y helminth products or llergens, my ct together to elicit sophil prolifertion nd ctivtion in vivo. In ddition to function for sophils in the MHC clss II dependent promotion of T H 2 differentition nd immunity to T. muris infection, criticl function for sophils hs een found in the development of llergen-specific T H 2 cytokine responses. In those studies, llergen-stimulted sophils expressed MHC clss II, the trnscriptionl coctivtor CIITA, the invrint chin nd costimultory molecules nd promoted llergen-specific CD4 + T H 2 cell NATURE IMMUNOLOGY VOLUME 1 NUMBER 7 JULY 29 73

8 29 Nture Americ, Inc. All rights reserved. differentition (R. Medzhitov, personl communiction). These findings collectively indicte tht sophil-medited recognition of llergens nd helminth-derived products, coupled with their MHC clss II dependent promotion of T H 2 cell responses, my e n evolutionrily conserved pthwy tht serves crdinl function in the development of type 2 inflmmtion t mucosl sites. METHODS Methods nd ny ssocited references re ville in the online version of the pper t Accession codes. UCSD-Nture Signling Gtewy ( signling-gtewy.org): A1262 nd A2363. Note: Supplementry informtion is ville on the Nture Immunology wesite. ACKNOWLEDGMENTS We thnk M. Mohrs (Trudeu Institute) for 4get mice; A. Troy for criticl reding of this mnuscript; E. Tit for generting the one mrrow chimers; nd the University of Pennsylvni Flow Cytometry Core nd Center for Moleculr Studies in Digestive nd Liver Diseses Morphology Core for ssistnce with sorting nd immunofluorescence stining. Supported y the Ntionl Institutes of Helth (Artis l: AI6157 nd AI74878 to D.A., T32 trining grnt AI to J.G.P., F31 trining grnt GM82187 to S.A.S. nd T32 CA914-3 to B.C.T.; Perce l: AI32573 nd AI53825; nd Lufer l), the Burroughs Wellcome Fund (Artis l: D.A.), the Ntionl Institute of Dietes nd Digestive Kidney Diseses (Artis l: DK536), the Crohn s nd Colitis Foundtion of Americ (Artis l: D.A. nd M.G.N.) nd the University of Pennsylvni (Artis l: D.A. nd T.M.L.). COMPETING INTERESTS STATEMENT The uthors declre competing finncil interests: detils ccompny the full-text HTML version of the pper t Pulished online t Reprints nd permissions informtion is ville online t reprintsndpermissions/ 1. Mosmnn, T.R., Cherwinski, H., Bond, M.W., Giedlin, M.A. & Coffmn, R.L. Two types of murine helper T cell clone. I. Definition ccording to profiles of lymphokine ctivities nd secreted proteins. J. Immunol. 136, (1986). 2. Reiner, S.L. Development in motion: helper T cells t work. Cell 129, (27). 3. Mowen, K.A. & Glimcher, L.H. Signling pthwys in Th2 development. Immunol. Rev. 22, (24). 4. Anthony, R.M., Rutitzky, L.I., Urn, J.F., Jr., Stdecker, M.J. & Guse, W.C. Protective immune mechnisms in helminth infection. Nt. Rev. Immunol. 7, (27). 5. Cohn, L., Elis, J.A. & Chupp, G.L. Asthm: mechnisms of disese persistence nd progression. Annu. Rev. Immunol. 22, (24). 6. Perrigoue, J.G., Mrshll, F.A. & Artis, D. On the hunt for helminths: innte immune cells in the recognition nd response to helminth prsites. Cell. Microiol. 1, (28). 7. Medzhitov, R. Recognition of microorgnisms nd ctivtion of the immune response. Nture 449, (27). 8. Kpsenerg, M.L. Dendritic-cell control of pthogen-driven T-cell polriztion. Nt. Rev. Immunol. 3, (23). 9. McDonld, A.S. & Mizels, R.M. Alrming dendritic cells for Th2 induction. J. Exp. Med. 25, (28). 1. Amsen, D. et l. InstructionofdistinctCD4Thelpercellftesydifferentnotch lignds on ntigen-presenting cells. Cell 117, (24). 11. McDonld, A.S., Strw, A.D., Dlton, N.M. & Perce, E.J. Cutting edge: Th2 response induction y dendritic cells: role for CD4. J. Immunol. 168, (22). 12. Ekkens, M.J. et l. The role of OX4 lignd interctions in the development of the Th2 response to the gstrointestinl nemtode prsite Heligmosomoides polygyrus. J. Immunol. 17, (23). 13. Gessner, A., Mohrs, K. & Mohrs, M. Mst cells, sophils, nd eosinophils cquire constitutive IL-4 nd IL-13 trnscripts during linege differentition tht re sufficient for rpid cytokine production. J. Immunol. 174, (25). 14. Mohrs, K., Wkil, A.E., Killeen, N., Locksley, R.M. & Mohrs, M. A two-step process for cytokine production reveled y IL-4 dul-reporter mice. Immunity 23, (25). 15. Min, B. et l. Bsophils produce IL-4 nd ccumulte in tissues fter infection with Th2-inducing prsite. J. Exp. Med. 2, (24). 16. Skokos, D. et l. Mst cell-derived exosomes induce phenotypic nd functionl mturtion of dendritic cells nd elicit specific immune responses in vivo. J. Immunol. 17, (23). 17. McKenzie, J.R., Mttes, J., Dent, L.A. & Foster, P.S. Eosinophils promote llergic disese of the lung y regulting CD4 + Th2 lymphocyte function. J. Immunol. 167, (21). 18. Pdigel, U.M. et l. Eosinophils ct s ntigen-presenting cells to induce immunity to Strongyloides stercorlis in mice. J. Infect. Dis. 196, (27). 19. Voehringer, D., Reese, T.A., Hung, X., Shinki, K. & Locksley, R.M. Type 2 immunity is controlled y IL-4/IL-13 expression in hemtopoietic non-eosinophil cells of the innte immune system. J. Exp. Med. 23, (26). 2. Ohnmcht, C. & Voehringer, D. Bsophil effector function nd homeostsis during helminth infection. Blood 113, (28). 21. Voehringer, D., Shinki, K. & Locksley, R.M. Type 2 immunity reflects orchestrted recruitment of cells committed to IL-4 production. Immunity 2, (24). 22. Sokol, C.L., Brton, G.M., Frr, A.G. & Medzhitov, R. A mechnism for the initition of llergen-induced T helper type 2 responses. Nt. Immunol. 9, (28). 23. Else, K.J. & Finkelmn, F.D. Intestinl nemtode prsites, cytokines nd effector mechnisms. Int. J. Prsitol. 28, (1998). 24. Cliffe, L.J. & Grencis, R.K. The Trichuris muris system: prdigm of resistnce nd susceptiility to intestinl nemtode infection. Adv. Prsitol. 57, (24). 25. Else, K.J., Finkelmn, F.D., Mliszewski, C.R. & Grencis, R.K. Cytokine-medited regultion of chronic intestinl helminth infection. J. Exp. Med. 179, (1994). 26. Lemos, M.P., Fn, L., Lo, D. & Lufer, T.M. CD8 + nd CD11 + dendritic cell-restricted MHC clss II controls Th1 CD4 + Tcellimmunity.J. Immunol. 171, (23). 27. Zph, C. et l. Epithelil-cell-intrinsic IKK- expression regultes intestinl immune homeostsis. Nture 446, (27). 28. Artis, D. et l. RELM/FIZZ2 is golet cell-specific immune-effector molecule in the gstrointestinl trct. Proc. Ntl. Acd. Sci. USA 11, (24). 29. Artis, D. Epithelil-cell recognition of commensl cteri nd mintennce of immune homeostsis in the gut. Nt. Rev. Immunol. 8, (28). 3. Else, K.J. & Grencis, R.K. Antiody-independent effector mechnisms in resistnce to the intestinl nemtode prsite Trichuris muris. Infect. Immun. 64, (1996). 31. Zph, C. et l. Persistence nd function of centrl nd effector memory CD4 + Tcells following infection with gstrointestinl helminth. J. Immunol. 177, (26). 32. Mohrs, M., Shinki, K., Mohrs, K. & Locksley, R.M. Anlysis of type 2 immunity in vivo with icistronic IL-4 reporter. Immunity 15, (21). 33. Denzel, A. et l. Bsophils enhnce immunologicl memory responses. Nt. Immunol. 9, (28). 34. Owyng, A.M. et l. Interleukin 25 regultes type 2 cytokine-dependent immunity nd limits chronic inflmmtion in the gstrointestinl trct. J. Exp. Med. 23, (26). 35. Humphreys, N.E., Xu, D., Hepworth, M.R., Liew, F.Y. & Grencis, R.K. IL-33, potent inducer of dptive immunity to intestinl nemtodes. J. Immunol. 18, (28). 36. Suzukw, M. et l. An IL-1 cytokine memer, IL-33, induces humn sophil ctivtion vi its ST2 receptor. J. Immunol. 181, (28). 37. Pecric-Petkovic, T., Didichenko, S.A., Kempfer, S., Spiegl, N. & Dhinden, C.A. Humn sophils nd eosinophils re the direct trget leukocytes of the novel IL-1 fmily memer IL-33. Blood 113, (29). 38. Smithgll, M.D. et l. IL-33 mplifies oth Th1- nd Th2-type responses through its ctivity on humn sophils, llergen-rective Th2 cells, inkt nd NK cells. Int. Immunol. 2, (28). 39. Fllon, P.G. et l. Identifiction of n interleukin (IL)-25-dependent cell popultion tht provides IL-4, IL-5, nd IL-13 t the onset of helminth expulsion. J. Exp. Med. 23, (26). 4. Tylor, J.J., Mohrs, M. & Perce, E.J. Regultory T cell responses develop in prllel to Th responses nd control the mgnitude nd phenotype of the Th effector popultion. J. Immunol. 176, (26). 41. Allenspch, E.J., Lemos, M.P., Porrett, P.M., Turk, L.A. & Lufer, T.M. Migrtory nd lymphoid-resident dendritic cells cooperte to efficiently prime nive CD4 T cells. Immunity 29, (28). 42. Ehrlich, P. Beitrge zur Kenntins der grnulierten Bindegewes zellen und der eosinophilen Leukocythen. Arch Ant Physiol Lpz 3, (1879). 43. Oh, K., Shen, T., Le Gros, G. & Min, B. Induction of Th2 type immunity in mouse system revels novel immunoregultory role of sophils. Blood 19, (27). 44. Gucht, J.F. et l. Induction of humn IgE synthesis in B cells y mst cells nd sophils. Nture 365, (1993). 45. Ot, K. et l. Bsophils re essentil inititors of novel type of chronic llergic inflmmtion. Blood 11, (27). 46. Min, B., Le Gros, G. & Pul, W.E. Bsophils: potentil liison etween innte nd dptive immunity. Allergol. Int. 55, (26). 47. Scheu, S. et l. Activtion of the integrted stress response during T helper cell differentition. Nt. Immunol. 7, (26). 48. Brown, S.J., Glli, S.J., Gleich, G.J. & Askense, P.W. Altion of immunity to Amlyomm mericnum y nti-sophil serum: coopertion etween sophils nd eosinophils in expression of immunity to ectoprsites (ticks) in guine pigs. J. Immunol. 129, (1982). 74 VOLUME 1 NUMBER 7 JULY 29 NATURE IMMUNOLOGY

9 49. Shen, T. et l. T cell-derived IL-3 plys key role in prsite infection-induced sophil production ut is dispensle for in vivo sophil survivl. Int. Immunol. 2, (28). 5. Tylor, B.C. et l. TSLP regultes intestinl immunity nd inflmmtion in mouse models of helminth infection nd colitis. J. Exp. Med. 26, (29). 51. Yoo, J. et l. Spontneous topic dermtitis in mice expressing n inducile thymic stroml lymphopoietin trnsgene specificlly in the skin. J. Exp. Med. 22, (25). 52. Zhou, B. et l. Thymic stroml lymphopoietin s key inititor of llergic irwy inflmmtion in mice. Nt. Immunol. 6, (25). 53. Schmitz, J. et l. IL-33, n interleukin-1-like cytokine tht signls vi the IL-1 receptorrelted protein ST2 nd induces T helper type 2-ssocited cytokines. Immunity 23, (25). 54. Angksekwini, P. et l. Interleukin 25 promotes the initition of prollergic type 2 responses. J. Exp. Med. 24, (27). 55. Allkhverdi, Z., Smith, D.E., Comeu, M.R. & Delespesse, G. Cutting edge: the ST2 lignd IL-33 potently ctivtes nd drives mturtion of humn mst cells. J. Immunol. 179, (27). 56. Senz, S.A., Tylor, B.C. & Artis, D. Welcome to the neighorhood: epithelil cellderived cytokines license innte nd dptive immune responses t mucosl sites. Immunol. Rev. 226, (28). 57. Flcone, F.H., Pritchrd, D.I. & Gis, B.F. Do sophils ply role in immunity ginst prsites? Trends Prsitol. 17, (21). 58. Schrmm, G. et l. Cutting edge: IPSE/-1, glycoprotein from Schistosom mnsoni eggs, induces IgE-dependent, ntigen-independent IL-4 production y murine sophils in vivo. J. Immunol. 178, (27). 29 Nture Americ, Inc. All rights reserved. NATURE IMMUNOLOGY VOLUME 1 NUMBER 7 JULY 29 75

10 29 Nture Americ, Inc. All rights reserved. ONLINE METHODS Mice nd prsites. C57BL/6 mice nd timed pregnnt femle B6.SJL mice 6 8 weeks of ge were from The Jckson Lortories. MHC II CD11c mice (lso known s CD11c-A mice; generted s descried 26 ), MHC clss II deficient (H2-A1 / ) mice, 4get (IL-4eGFP; C.129-IL4 tm1lky /J) mice (from M. Mohrs), B cell deficient (mmt) mice, CD11c-DTR mice nd DO11.1 mice were red nd housed in specific pthogen free conditions t the University of Pennsylvni. Littermte control mice were shm grfted nd MHC II CD11c mice were given sucutneous thymic grfts from neontl ( 2 d of ge) B6.SJL mice t 4 6 weeks of ge nd then were llowed 8 weeks to reconstitute CD4 + T cells efore experimentl use. Bone mrrow chimers were generted y intrvenous injection of one mrrow cells from wild-type or CD11c-DTR mice into irrdited (5 rds, twice) wild-type recipients. Recipient mice were given ntiiotics for 2 weeks nd were llowed 8 weeks to reconstitute. All experiments were ccordnce with the guidelines of the Institutionl Animl Cre nd Use Committee of the University of Pennsylvni. T. muris ws mintined in geneticlly susceptile mouse strins nd eggs were collected s descried 31 ; mice were infected y orl gvge with 2 3 emryonted T. muris eggs. S. mnsoni eggs were prepred s descried 4 ; mice were injected in the footpd with 2,5 eggs in 5 ml PBS. Polyclonl T cell stimultion. Single-cell suspensions of mlns were prepred in complete medium (DMEM supplemented with 1% (vol/vol) hetinctivted FBS, 2 mm glutmine, 1 U/ml of penicillin, 1 mg/ml of streptomycin, 25 mm HEPES, ph 7.4, nd 5 mm -mercptoethnol). The mln cells were seeded in 48-well pltes t density of per well nd were incuted for 48 h with medi or 1 mg/ml of solule nti-cd3 (145-2C11) nd nti-cd28 (37-51) ebioscience). Cell-free superntnts were collected nd cytokine production ws determined y sndwich ELISA (ll ntiody pirs from ebioscience: AN-18 nd R4-6A2 (nti-ifn-g); 11B11 nd BVD6-24G2 (nti-il-4); TRFK5 nd TRFK4 (nti-il-5); nd ebio13a nd ebio 1316H (nti-il-13)). Immunolot. Fecl protein ws isolted s descried 28 ; 3mg protein per smple ws seprted y SDS-PAGE nd nlyzed y immunolot for RELM with polyclonl rit nti mouse RELM (Peprotech). Rel-time PCR. RNA ws isolted from intestinl tissues of mice y TRizol extrction (Invitrogen) nd from mln cells with RNEsy Spin Columns (Qigen). Tissues were disrupted with tissue homogenizer (Tissue- Lyzer; Qigen) nd cdna ws synthesized with SuperScript Reverse Trnscriptse (Invitrogen). Quntittive rel-time PCR nlysis of cdna smples used commercil primer sets (Il4, QT16678; Ifng, QT138821; Il1, QT16169; nd Il17, QT13278; Qigen) nd SYBR Green. All rections were run on n ABI 75 Fst Rel-Time PCR System (Applied Biosystems). Smples were normlized to nive controls unless otherwise stted. Histology nd immunofluorescence. Cecl tips were fixed in 4% (vol/vol) prformldehyde nd were emedded in prffin. Sections 5 mm in thickness were cut nd were stined with hemtoxylin nd eosin or Alcin lue periodic cid Schiff. Unstined sections prepred on immunoslides were stined for Go5 y immunofluorescence s descried 27. Smples were deprffinized y consecutive rinses in methnol nd ethnol, then were oiled in citric cid uffer nd stined overnight t 4 1C with nti-go5, followed y stining with crocynine-conjugted nti-got ( ; Jckson Immunoreserch). Sorted sophils were sujected to cytospin nd were fixed overnight t 4 1C in 2% (vol/vol) prformldehyde. Slides were wshed in PBS, then cells were mde permele in Triton-X, locked with streptvidin nd iotin nd stined overnight t 4 1C with iotin nti-mhc clss II (M5114) nd nti-gfp ( ; ebioscience). Slides were then wshed with PBS nd stined for 2 h t 25 1C with streptvidin-indocrocynine nd crocynine-conjugted donkey nti-rit ( ; Jckson Immunoreserch), then were wshed gin with PBS nd nuclei were stined with DAPI (4,6-dimidino- 2-phenylindole). Neutrlizing nd depleting ntiodies nd recominnt cytokines. Neutrlizing monoclonl nti-ifn-g (XMG-6) ws purified from scites fluid (grown y Hrln Bioscience) y mmonium sulfte precipittion nd ws dilyzed ginst PBS. Mice were given 1 mg ntiody intrperitonelly every 3 5 d during the course of infection, strting t dy. Mice were depleted of sophils y intrperitonel injection of 1 mg nti-fceri (MAR-1; ebioscience) on dys, 1, 2 nd dys 1, 11, 12 fter infection. Recominnt mouse IL-25 (4 mg/ml), IL-33 (2 mg/ml) nd TSLP (.1 mg/ml; ll from R&D Systems) nd 1 ml PBS were injected intrperitonelly once dily for 4 d. Bsophil isoltion nd CD4 + T cell coculture. CD4 + T cells were isolted from spleens y negtive selection y incution with hyridom superntnts (nti-b22 (RAE), nti-fcr (24G2), nti-cd8 (2.43) nd nti MHC clss II (M5114); prepred in-house ) followed y mgnetic ed purifiction (Qigen). To otin purified sophils, lood, spleen nd mln cells were isolted from 4get mice injected intrperitonelly with 1 mg recominnt TSLP (R&D Systems) once dily for 4 d for enrichment of sophils, were positively selected for CD49 expression y purifiction on MACS column (Milltenyi) nd were stined with fluorochromeconjugted monoclonl nti-b22 (RA3-6B2), nti-cd3e (145-2C11), nti-c-kit (2B8), nti-cd49 (HM2) nd nti-fceri (MAR-1; ll from BD Biosciences nd ebioscience). Bsophils were sorted with FACSAri (BD Bioscience) on the sis of negtive stining for B22, CD3 nd c-kit, positive stining for CD49 nd FceRI, nd expression of IL-4 egfp. After purifiction, sorted sophils were resuspended t density of cells per ml in complete medium; 1 ml of the sophils were used for cytospin nd were stined y Diffquick for confirmtion of cellulr morphology. Between nd sophils were cultured together with purified, CFSE (croxyfluorescein dicette succinimidyl ester) leled DO11.1 CD4 + Tcells with recominnt IL-3 (1 ng/ml R&D Systems) in the presence or sence of OVA peptide (1 mg/ml) nd locking ntiody to MHC clss II (5 mg/ml; M5/114; ebioscience). After 4 d of culture, cells were stimulted for 4 h with phorol 12-myristte 13-cette, ionomycin nd refeldin A. Cells were pelleted for 5 min t 485g. Superntnts were collected for ELISA nd cells were wshed in flow cytometry uffer, incuted for 1 min t 4 1C with Fc Block (2.4G2 nd rt IgG), stined with fluorochrome-conjugted monoclonl nti-cd4 (RM4-5; ebioscience) nd fixed with 2% (vol/vol) prformldehyde. Cells were mde permele with.4% (wt/vol) sponin in flow cytometry uffer nd were stined for intrcellulr cytokines with fluorochrome-conjugted monoclonl nti-il-4 (11B11; ebioscience) nd nti-il-13 (ebio13a; ebioscience). Adoptive trnsfer of sophils. C57BL/6 mice were injected with S. mnsoni eggs in ech footpd, then plns, spleen nd lood were pooled 2 d lter nd sophils were purified y sequentil CD49 enrichment nd cell sorting s descried ove. Recipient MHC II CD11c mice were given purified CFSE-leled CD4 + T cells from nive C57BL/6 mice 1 d efore egg injection. Sorted sophils were resuspended in suspension of PBS plus S. mnsoni eggs, nd ech recipient MHC II CD11c mouse ws given either sophils nd S. mnsoni eggs or S. mnsoni eggs lone in the right footpd in volume of 5 ml. Cells from drining nd nondrining plns were isolted 4 d fter egg injection, then were stined with fluorochromeconjugted monoclonl ntiodies nd nlyzed y flow cytometry on FACSCnto II (BD Biosciences). Mcrophge nd DC depletion. Liposomes loded with PBS or clodronte (Roche Dignostics) were prepred s descried 59 nd 15 ml of liposomes were injected intrvenously every 2 d during the course of infection. CD11c-DTR mice were depleted of DCs y intrperitonel injection of diphtheri toxin (1 ng per mouse) every 3 d during the course of infection. Sttistics. Sttisticl significnce ws determined y the Student s t-test. 59. Vn Rooijen, N. The liposome-medited mcrophge suicide technique. J. Immunol. Methods 124, 1 6 (1989). NATURE IMMUNOLOGY doi:1.138/ni.174