DNA released from dying host cells mediates aluminum adjuvant activity

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1 DNA relesed from dying host cells medites luminum djuvnt ctivity Thoms Mrichl 1, Keiichi Oht 2, Denis Bedoret 1, Clire Mesnil 1, Ctherine Stel 1, Kouji Koiym 2,3, Pierre Lekeux 1, Cevyir Con 2, Shizuo Akir 2, Ken J. Ishii 2,3,4, Frice Bureu 1,4, Christophe J. Desmet 1,4. 1 Lortory of Cellulr nd Moleculr Physiology, GIGA-Reserch nd Fculty of Veterinry Medicine, B34, University of Liege, 1 Avenue de l'hopitl, B4 Liège, Belgium 2 WPI Immunology Frontier Reserch Center, Osk University, 3-1 Ymdok, Suit, Osk, Jpn 3 Lortory of Adjuvnt Innovtion, Ntionl Institute of Biomedicl Innovtion, Asgi Sito Irki-City Osk, Jpn 4 These uthors contriuted eqully to this work. Correspondence should e ddressed to C.J.D. (christophe.desmet@ulg.c.e), F.B. (frice.ureu@ulg.c.e) or K.J.I. (kenishii@iken.osk-u.c.jp). Supplementry figures nd legends 1-17 Supplementry methods Nture Medicine doi:1.138/nm.243

Supplementry figure 1 c Unstined Free doule-strnded DNA (g per lvge) d 7-AAD + cells per lvge (%) 4 3 2 1.9 i.p..64 3 6 12 24 Time (h) 1. 7.5 5. 2.5. OVA + lum mg OVA + lum.

() Concentrtion of free doule-strnded (ds)dna in the cellulr frction of the muscle lvge fluid of mice treted i.m. with incresing doses of lum, mesured through time using quntittive fluorescent doule-strnded DNA stin.

2 Supplementry figure 1 c Unstined Free doule-strnded DNA (g per lvge) d 7-AAD + cells per lvge (%) i.p Time (h) OVA + lum mg OVA + lum.3 mg OVA + lum 1 mg i.m..64 Free doule-strnded DNA (g per lvge) e DAPI i.p AAD + cells per lvge (%) Alum (mg) i.m..68 Supplementry Figure 1 Alum nd AlHydrogel induce cell deth nd relese of host DNA t sites of injection. () Concentrtion of free doule-strnded (ds)dna in the cellulr frction of the muscle lvge fluid of mice treted i.m. with incresing doses of lum, mesured through time using quntittive fluorescent doule-strnded DNA stin. () Extrcellulr DNA deposition in lum mcroscopic i.m. depots stined with 4',6-dimidino-2- phenylindole (DAPI) (scle r: 25 m). (c) Cell deth rte in the peritonel lvge fluid of mice treted i.m. with incresing doses of lum, ssessed y stining with 7-minoctinomycin D (7-AAD) nd flow cytometry. (d,e) Comprison of cell deth rte (d) nd dsdna relese (e) t i.p. nd i.m. injection sites etween lum- nd AlHydrogel-treted mice. n=5 (,c-e). Dt re representtive of one of three independent experiments. Nture Medicine doi:1.138/nm.243

3 Supplementry figure 2 OVA-IgM (AU ml 1 ) 5 OVA OVA + lum.3 mg OVA + lum 2 mg OVA + DNA 1.5 g OVA + DNA 6g 25 ns ns Time (d) OVA-IgG1 (AU ml 1 x 1 3 ) c Time (d) ns ns OVA-IgE (AU ml 1 ) 1 ns ns Time (d) Supplementry Figure 2 Alum nd host DNA injected i.m. potentites type 2 humorl responses.() Serum titers of OVA-specific IgM, () IgG1 nd (c) IgE mesured on indicted dys in mice immunized i.m. with OVA lone, OVA nd lum, or OVA nd DNA on dys nd 14, nd oosted with OVA on dy 21. n=5. Dt re representtive of one of two experiments. (AU, ritrry unit). Nture Medicine doi:1.138/nm.243

4 Supplementry figure 3 Peritonel lvge superntnt t12h Donor OVA + lum i.p. th DNAse I In vitro Serum d17 i.p. d OVA i.p. d1 Recipient 12 c 1 OVA-IgE (AU ml "1 ) 8 4 OVA-IgG1 (AU ml "1 x 1 3 ) Alum (donor) DNse I " " #" #" " #" Alum (donor) DNse I " " #" #" " #" Supplementry Figure 3 DNA relesed upon lum tretment is necessry nd sufficient to oost humorl responses. () Experimentl outline. In vitro, we mock-treted or sumitted to DNse I tretment the cellulr frction of peritonel lvge fluid from OVA- or OVA nd lum-treted mice, efore trnsferring it to nïve recipient mice with 1g OVA. We oosted recipient mice i.p. with OVA 1 d lter. (,) ELISA mesurement of OVA-specific IgE () nd IgG1 (c) serum titers 7 d lter. n=5. Dt re representtive of one of four independent experiments. (AU, ritrry unit). Nture Medicine doi:1.138/nm.243

5 Supplementry figure 4 OVA - shm OVA + DNA - shm 2.86% 26.9% OVA + lum - shm OVA + lum - DNse 32.7% 1.94% CD4 + CFSE low cells (%) CD4 CFSE Supplementry Figure 4 Host DNA relesed y lum cytotoxicity medites lum djuvnt ctivity on endogenous T cell responses. We treted mice i.p. with OVA, OVA nd DNA, OVA nd lum or OVA nd lum followed y DNse I tretment. Five dys lter, we isolted ronchil lymph node (BLN) cells, leled them with CFSE nd restimulted them in vitro with OVA for 5 dys. Cell viility remined high following croxyfluorescein succinimidyl ester (CFSE) leling (dt not shown). We estimted the prolifertion of OVAspecific CD4+ T cells y mesuring the percentge of CFSE low CD4 + T cells y flow cytometry (inserts indicte the percentge of CFSE low CD4 + T cells). n=5. Dt re representtive of one of two independent experiments. Nture Medicine doi:1.138/nm.243

6 Supplementry figure OVA-IgE (AU ml 1 ) 1 5 Alum WT Nlrp3 / Csp-1 / OVA-IgG1 (AU ml 1 x 1 3 ) 4 2 Alum WT Nlrp3 / Csp-1 / Supplementry Figure 5 Nlrp3 or Csp1 deficiency does not significntly impct on the djuvnt ctivity of lum on humorl responses. Serum OVA-specific IgE () nd IgG1 () serum ntiody titers mesured on dy 28 in WT, Nlrp3 / nd Csp1 / mice immunized i.m. with OVA or OVA nd.3 mg lum on dys nd 14 nd oosted with OVA on dy 21. n=5. Dt re representtive of one of two independent experiments. (AU, ritrry unit). Nture Medicine doi:1.138/nm.243

7 Supplementry figure 6 HSA-IgE (AU ml 1 ) AlHydrogel (mg) WT Irf3 / HSA-IgG1 (AU ml 1 x 1 3 ) 1 5 AlHydrogel (mg) WT Irf3 / Supplementry Figure 6 The djuvnt ctivity of lum on ntigen-specific IgE responses requires Irf3 independently of lum type nd ntigen. Serum HSA-specific IgE () nd IgG1 () ntiody titers mesured on dy 28 in Irf3 / mice immunized i.p. with HSA or HSA comined with the indicted doses of AlHydrogel on dys nd 14 nd oosted with HSA i.p. on dy 21. n=5. Dt re representtive of one of two independent experiments. (AU, ritrry unit). Nture Medicine doi:1.138/nm.243

8 Supplementry figure 7 15 c 2 OVA-IgE (AU ml "1 ) 1 5 OVA-IgE (AU ml "1 ) Alum (mg) DNA (g) WT Irf3 / WT Irf3 / d OVA-IgG1 (AU ml "1 x 1 3 ) 1 5 OVA-IgG1 (AU ml "1 x 1 3 ) 1 5 Alum (mg) DNA (g) WT Irf3 / WT Irf3 / Supplementry Figure 7 The djuvnt ctivity of lum nd host DNA on ntigen-specific IgE responses requires Irf3 independently of the site of injection. Serum OVA-specific IgE (, c) nd IgG1 (, d) ntiody titers mesured on dy 28 in WT nd Irf3 / mice immunized i.m. with OVA or OVA comined with the indicted doses of lum or DNA on dys nd 14 nd oosted with OVA on dy 21. n=5. Dt re representtive of one of two independent experiments. (AU, ritrry unit). Nture Medicine doi:1.138/nm.243

9 Supplementry figure 8 WT Irf3 / Isotype CD4.59% 1.59% CFSE OVA 23.1% 5.% OVA + DNA 37.3% 8.31% OVA + lum WT - DNA Irf3 / - DNA WT - lum Irf3 / - lum.66%.57%.92%.84% 2.27% 1.8% 3.26% 1.67% CD4 + CFSE low cells (%) 75 OVA OVA + DNA OVA + lum 5 25 IL-4 + in CD4 + CFSE low cells (%) 8 Isotype Anti-IL WT Irf3 / IL-4 CD4 WT Irf3 / WT Irf3 / DNA lum Supplementry Figure 8 Irf3 is essentil for the oosting of type 2 T cell responses y lum nd genomic DNA. We treted WT nd Irf3 / mice i.p. with OVA, OVA nd DNA or OVA nd lum. Five dys lter, we isolted BLN cells, leled them with CFSE nd restimulted them in vitro with OVA for 5 dys. Cell viility remined high following croxyfluorescein succinimidyl ester (CFSE) leling nd ws not different etween WT nd Irf3 / cells (dt not shown). () Prolifertion of OVA-specific CD4+ T cells estimted y mesuring the percentge of CFSE low CD4 + T cells y flow cytometry (inserts indicte the percentge of CFSE low CD4 + T cells). () Percentges of IL4 + cells mong CD4 + CFSE low cells ssessed y intrcellulr stining nd flow cytometry (inserts indicte the percentge of IL4 + CFSE low CD4 + T cells). n=5. Dt re representtive of one of three independent experiments. Nture Medicine doi:1.138/nm.243

10 Supplementry figure 9 3 H-thymidine uptke (CPM x 1 3 ) OVA ("g ml -1 ) WT OVA WT OVA + lum Irf3 / OVA Irf3 / OVA + lum 5 Cytokine concentrtion (pg ml 1 ) 1, IL-4 IL-5 IL-13 c OVA-IgE (AU ml 1 ) Alum + + WT Irf3 / d OVA-IgG1 (AU ml 1 x 1 4 ) Alum + + WT Irf3 / Supplementry Figure 9 Irf3 is essentil for the oosting of cnonicl Th2 cell differentition nd IgE responses in n lum-immuniztion-sed sthm model. We chllenged OVA- nd OVA nd lum-sensitized WT nd Irf3 / mice with erosolized OVA nd nlyzed for type 2 T cell nd humorl responses. () BLN cell prolifertion in response to 3 dys in vitro OVA stimultion ssessed y the mesurement of 3 H-thymidine uptke. () ELISA mesurement of IL-4, IL-5 nd IL-13 concentrtions in culture superntnts of OVA-stimulted BLN cells. Serum OVA-specific IgE (c) nd IgG1 (d) titers. n=5. Dt re representtive of one of three independent experiments. (CPM, counts per minute). Nture Medicine doi:1.138/nm.243

11 Supplementry figure 1 3 H-thymidine uptke (CPM x 1 3 ) Cytokine concentrtion (pg ml 1 ) CD3 - CD28.49 WT WT Irf3 /.53 Irf3 /.41 IL-4 IL-5 IL-13 Supplementry Figure 1 WT nd Irf3 / T lymphocytes hve similr potentil for prolifertion nd Th2 cytokine secretion. () Prolifertion ssessed y mesuring 3 H-thymidine incorportion during the lst 16 hours of 2-dy culture of T cells (2 1 5 cells, >95% purity) purified from the BLNs of nïve WT nd Irf3 / mice nd cultured with CD28-specific ntiodies into pltes coted with CD3-specific ntiodies. We cultured controls in uncoted wells without CD28-specific ntiodies. () ELISA mesurement of IL-4, IL-5, IL-13 in the superntnt of the cells in. n=5. Dt re representtive of one of three independent experiments. (CPM, counts per minute) Nture Medicine doi:1.138/nm.243

12 Supplementry figure OVA-IgG1 (AU ml 1 x 1 3 ) OVA-IgE (AU ml 1 ) CFA + IFA + + WT Irf3 / CFA + IFA + + WT Irf3 / c OVA-IgG2c (AU ml 1 x 1 3 ) CFA + IFA + + WT Irf3 / Supplementry Figure 11 Irf3 / mice hve norml immuniztion potentil in response to Irf3-independent djuvnts. Serum OVA-specific IgG1 (), IgE (), nd IgG2c (c) ntiody titers mesured on dy 28 in WT nd Irf3 / mice immunized s.c. with OVA nd CFA on dy nd OVA nd IFA on dy 14, nd oosted with OVA i.p. on dy 21. n=5. Dt re representtive of one of two independent experiments. (AU, ritrry unit). Nture Medicine doi:1.138/nm.243

13 Supplementry figure 12 imonos cdcs "#$ Numer of cells per lvge x 1 3 5, 4, 3, 2, 1,.56 WT OVA WT OVA + lum Irf3 / OVA Irf3 / OVA + lum Time (h) Time (h) % & '( () Time (h) Mcrophges Neutrophils Eosinophils Numer of cells per lvge x 1 3 1,5 1, 5 WT OVA WT OVA + lum Irf3 / OVA Irf3 / OVA + lum 2, 1,5 1, , Time (h) Time (h) Time (h) Supplementry Figure 12 Alum induces similr recruitment of innte immune cells in WT nd Irf3 / mice. Recruitment of innte immune cells through time in the peritonel lvge fluid of WT nd Irf3 / mice treted i.p. with OVA or OVA nd lum, ssessed y flow cytometry. () We defined inflmmtory monocytes (imonos) s F4/8 int CD11 + Ly6C + Ly6G cells, conventionl DCs (cdcs) s MHCII + CD11c + F4/8 low Ly6C cells, nd plsmcytoid DCs (pdcs) s B22 + Ly6G + CD11c int F4/8 low cells. () We defined peritonel mcrophges s F4/8 high CD11 + SSC high cells, neutrophils s CD11 + Ly6C + Ly6G + F4/8 cells, nd eosinophils s CD11 + Ly6C int Ly6G int F4/8 int cells. n=5. Dt re representtive of one of four independent experiments. Nture Medicine doi:1.138/nm.243

Supplementry figure 13 P1 P2 idcs (P1 x P2) CD11c in P1 Ly6C CD11 Ly6G cdcs (P3

B22 CD11c idc numer per node (x 1 3 ) 75 5 25 WT OVA WT OVA + DNA WT OVA + lum.

Supplementry Figure 13 Gting strtegy nd lum- nd DNA-induced migrtion of idcs.

conventionl DCs (cdcs) nd plsmcytoid DCs (pdcs) in the BLNs of mice y flow

We defined idcs s CD11c int/+ CD11 + Ly6C + Ly6G - cells, cdcs s MHCII + CD11c +

() Comprison y flow cytometric nlysis of the recruitment of idcs to the BLNs of

14 Supplementry figure 13 P1 P2 idcs (P1 x P2) CD11c in P1 Ly6C CD11 Ly6G cdcs (P3 x P4) P3 in P3 P4 MHC-II CD11c F4/8 Ly6C P5 pdcs (P5 x P6) in P5 P6 Ly6G F4/8 B22 CD11c idc numer per node (x 1 3 ) WT OVA WT OVA + DNA WT OVA + lum Irf3 / OVA Irf3 / OVA + DNA Irf3 / OVA + lum Time (h) Supplementry Figure 13 Gting strtegy nd lum- nd DNA-induced migrtion of idcs. () Gting strtegy for the identifiction of inflmmtory dendritic cells (idcs), conventionl DCs (cdcs) nd plsmcytoid DCs (pdcs) in the BLNs of mice y flow cytometry. We defined idcs s CD11c int/+ CD11 + Ly6C + Ly6G - cells, cdcs s MHCII + CD11c + F4/8 low Ly6C - cells nd pdcs s B22 + Ly6G + CD11c int F4/8 low cells. () Comprison y flow cytometric nlysis of the recruitment of idcs to the BLNs of WT nd Irf3 -/- mice treted i.m. with OVA, OVA nd DNA or OVA nd lum. n=5. Dt re representtive of one of more thn 4 () nd one of two () independent experiments. Nture Medicine doi:1.138/nm.243

15 Supplementry figure 14 WT 2,63% 15,7% + WT imonos Irf3 / 1,24% 3,3% 1,32% CD4 CFSE CD4 + CFSE low cells (%) 4 OVA OVA + lum OVA + lum - WT imonos OVA.8 WT Irf3 / OVA + lum IL-4 + in CD4 + CFSE low cells (%) imonos 8 Isotype Anti-IL-4 in WT WT.2 Irf3 / Supplementry Figure 14 Deficient inflmmtory monocyte function is responsile for impired type 2 responses in the lymph nodes drining lum injection sites in Irf3 / mice. We treted Irf3 / mice i.p. with OVA nd lum nd, 6 h lter, injected them i.p imonos isolted from the peritonel cvity of OVA nd lumtreted WT mice. Five dys lter, we leled the BLN cells of recipient mice with CFSE nd restimulted them in vitro with OVA for 5 dys. () Prolifertion of OVA-specific CD4 + T cells estimted y mesuring the percentge of CFSE low CD4 + T cells y flow cytometry (Inserts indicting the percentge of CFSE low CD4 + T cells). () Percentges of IL4 + cells mong CD4 + CFSE low cells ssessed y intrcellulr stining nd flow cytometry. We used BLN cells from WT nd Irf3 / mice tht received PBS with OVA lone or OVA nd lum s controls. n=4. Dt re representtive of one of three independent experiments. Nture Medicine doi:1.138/nm.243

16 Supplementry figure 15 CD4 + CFSE low cells (%) 3 OVA ( g ml -1 ) 25 OVA (5 g ml -1 ) OVA OVA + imonos OVA (g ml -1 ) OVA (5g ml -1 ) CD4 1,85% 2,55% CFSE OVA,95% 14,6% OVA + imonos c d IL-4 + in CD4 + CFSE low cells (%) 3 Isotype Anti-IL Cytokine concentrtion (pg ml "1 ) 1 OVA OVA + imonos OVA OVA + imonos IL-4 IL-5 ÌL-13 IFN- Supplementry Figure 15 Inflmmtory monocytes re sufficient to induce type 2 responses in the lymph nodes drining lum injection sites. We gve WT mice i.p. 1 g OVA lone or OVA with imonos isolted from the peritonel cvity of OVA nd lum-treted WT mice (OVA + imonos). Five dys lter, we leled the BLN cells of recipient mice with CFSE nd restimulted them in vitro with or without OVA for 5 dys. () Prolifertion of OVAspecific CD4 + T cells estimted y mesuring the percentge of CFSE low CD4 + T cells y flow cytometry. () Representtive histogrms of smples compred in (), with inserts indicting the percentge of CFSE low CD4 + T cells. (c) Percentges of IL4 + cells mong CD4 + CFSE low cells ssessed y intrcellulr stining nd flow cytometry. (d) ELISA mesurement of IL-4, IL-5, IL-13 nd IFN- concentrtions in the superntnt of the OVAstimulted BLN cells. n=5. Dt re representtive of one of two independent experiments. (imonos, inflmmtory monocytes). Nture Medicine doi:1.138/nm.243

17 Supplementry figure 16 IFN-1 concentrtion (pg ml -1 ) imonos Non-iMonos WT Irf3 "/" Supplementry Figure 16. Imonos re mjor source of type I IFN production in lum-treted mice. IFN-1 immunotrpping nd ELISA detection in the superntnts of imonos nd negtive frction cells (non-imonos) FACS-sorted from the peritonel cvity of WT nd Irf3 "/" mice 18h fter OVA nd lum tretment. n=5. Dt re representtive of one of two independent experiments. Nture Medicine doi:1.138/nm.243

cnonicl Th2 response IgE production Supplementry Figure 17 Proposed model for

18 Supplementry figure 17 Alum Cell deth Irf3-independent Free host cell DNA Irf3-dependent T FH2 -relted T cell response idc ctivtion IgG1 production cnonicl Th2 response IgE production Supplementry Figure 17 Proposed model for the djuvnt effect of host cell DNA upon lum immuniztion. Nture Medicine doi:1.138/nm.243

19 Supplementry methods Antiodies. Allophycocynin- nd phycoerythrin-conjugted F4/8 (BM8)-, llophycocynin-conjugted V2 TCR (B2.1)-, llophycocynin-efluor78-conjugted CD11c (N418)-, efluor45-conjugted CD11 (M1/7)-, fluorescein isothiocynteconjugted B22 (RA3-6B2)-, iotinylted CCR7 (4B12)-, CD86 (GL1)-, efluor45- conjugted CD4 (RM4-5)-, CD3e (17A2)-specific ntiodies nd phycoerythrin-cynin7- conjugted streptvidin were from ebioscience. Biotinylted nti MHC clss II (I-A; AF6-12.1)-, fluorescein isothiocynte-conjugted-ly6c (AL-21)-, llophycocynin-conjugted- Ly6G (1A8)-, peridinin chlorphyll protein-cynin5.5-conjugted-ly6c (AL-21)-, llophycocynin-conjugted IL-4- (11.B.11)-specific ntiodies nd phycoerythrinconjugted streptvidin were from BD Biosciences. Fluorescein isothiocynte conjugted nti-cd4 (3/23) ws from Serotec. Pcific-Blue-conjugted streptvidin ws from Moleculr Proes Invitrogen. Lvge of injection sites nd mesurement of free doule-strnded DNA concentrtions nd cell deth rte. We performed lvges with 1 ml ice-cold Mg- nd C-free PBS contining.6 mm EDTA. We removed cells nd lum crystls from the lvge fluid of mice y 2 successive centrifugtions t 1, g for 4 min t 4 C. We mesured doule-strnded DNA in the cellulr frction of the lvge fluid using Qunt-iT PicoGreen dsdna regent (Invitrogen) ccording to the mnufcturer's protocol. We ssessed cell deth rte following lum tretment y stining with 5% (vol/vol) 7-AAD (e-bioscience), followed y flow cytometric nlysis. Fluorescence microscopy. We isolted lum depots from injection sites 12 hours fter tretment nd incuted them for 1 minutes with 4',6-dimidino-2-phenylindole (DAPI). We then plced the depots in RMPI without phenol red, in 35-mm glss ottom dish. We recorded imges with n Olympus FV1 confocl microscope equipped with 6x oil ojective nd n incution chmer to mintin the cells t 37 C in 5% CO 2 humidified tmosphere. We visulized DAPI fluorescence with 45 nm excittion nd nm emission window. Peritonel lvge trnsfer experiments. We performed peritonel lvges 12 h fter tretment with OVA nd lum or OVA lone nd removed cells nd deris. We sumitted peritonel lvge fluids to DNse I digestion (Roche) for 5 h following the mnufcturer's protocol. We mock-treted control peritonel lvge fluids. We then gve recipient mice 4 µl of donor peritonel lvge fluid or PBS mixed with 1 µg OVA i.p. We gve mice n i.p. oost of 2 µg OVA 1 d lter. Mice were scrificed for serum nlysis one week lter. Immuniztions with Freund s djuvnt. We injected mice on d sucutneously with 1 µg OVA lone or in conjunction with 4 "g CFA (Pierce Biochemicls). We collected serum on d 1. We injected mice sucutneously on d 14 with 1 "g OVA lone or in conjunction with 4 "g IFA (Pierce Biochemicls). On d 21, we oosted mice i.p. with 2 "g lone nd collected serum for nlysis on d 28. Restimultion of BLN cells. We cultured BLN cells (2 x 1 5 cells in 96-well plte) in Click s medium (2.1 5 cells in 2 µl, in 96-well pltes) supplemented with.5% (vol/vol) het-inctivted C57 Bl/6 mouse serum (Hrln Netherlnd), 8 mm L-glutmin, 5 UI ml -1 G-penicillin nd 5 µg ml -1 streptomycin, with or without OVA (OVA grde V, Sigm) (5 µg ml -1 ). We collected culture superntnts for cytokine detection y ELISA. We mesured cell prolifertion s 3 H-thymidine incorportion during the lst 16 h of 4-d culture. Nture Medicine doi:1.138/nm.243

20 Supplementry methods T-cell stimultion. We purified cells T cells from BLNs using the Pn T cell Isoltion Kit (Miltenyi Biotec) nd ssessed for purity y stining for CD3e followed y flow cytometry. We cultured cells with CD28-specific ntiodies (5 mg ml -1 ; 37.51, ebioscience) in RPMI supplemented with 1% (vol/vol) het-inctivted FCS nd dditives into 96 well pltes coted with CD3-specific ntiodies (1 mg ml -1 ; 145-2C11, ebioscience). We cultured controls in uncoted wells without CD28-specific ntiodies. We mesured cell prolifertion ws mesured s 3 H-thymidine incorportion during the lst 16 h of 2-d culture. CFSE leling. We incuted splenic nd lymph node cells from OT-II trnsgenic mice (5 # 1 7 cells/ml) with CFSE (5 µm in PBS) for 1 minutes t 37 C. We wshed cells in PBS contining 1% FCS nd then twice in PBS nd injected them in the cudl vein of mice. Alum-induced sthm model. We chllenged OVA- nd OVA nd lum-sensitized mice from d 21 to 25 with erosolized OVA 1% (wt/vol) in PBS for 1 h per dy. We performed roncho-lveolr lvges nd cytology. Briefly, we ctheterized the trche nd wshed the lungs with 1 ml ice-cold Mg- nd C-free PBS contining.6 mm EDTA. We ssessed cell density in roncholveolr lvge fluid using hemocytometer. We performed differentil cell counts on cytospin preprtions stined with Diff-Quick (Dde Behring). We fixed lungs in 1% formlin, prffin-emedded them, nd cut them in 5-mm sections. We estimted the extent of perironchil inflmmtion y score clculted y mens of quntifiction of perironchil inflmmtory cell lyers in lung sections stined with hemtoxylin nd eosin. We quntified mucus production s the percentge of periodic cid-schiff-stined golet cells per totl epithelil cells in rndomly selected ronchi. We rndomly selected nd nlyzed seven sections per lung. ELISA. We ssyed culture superntnts for mouse IL-4, IL-5, IL-13 nd IFN-$ y ELISA (Biosource/Invitrogen) ccording to the mnufcturer s protocol. We ssyed peritonel lvge superntnts for mouse IFN-1 nd IL-1 y ELISA (PBL Interferon Source nd Imtec Dignostics NV, respectively) ccording to the mnufcturer s protocol. We performed IFN-1 immunotrpping y culturing FACS-sorted imonos nd negtive frction (4.1 6 cells/ml) overnight in cpture ntiody-coted 96 wells pltes, followed y clssicl ELISA. Cell viility. We ssessed the viility of CFSE-leled imonos prior to doptive trnsfer y mens of DAPI stining nd susequent flow cytometric nlysis. The survivl of imonos following leling ws high nd comprle etween WT nd Irf3 -/- cells (dt not shown). Cell trnsfer experiments. For the study of migrtion, we purified imonos from the peritonel lvge fluid of OVA nd lum-treted mice y FACS 18 h post-tretment (>95% purity). We stined imonos with CFSE t concentrtion of 2 µm for 1 min t 37 C, wshed nd tested their viility. We then injected cells i.p. into recipient mice treted with OVA nd lum 12 h efore trnsfer, or into nïve WT mice. We gve control mice vehicle PBS. For the ssessment of their effects on type 2 nd humorl responses, we purified imonos s ove 6 h post-tretment, nd injected cells i.p. into recipient mice treted with OVA nd lum 6 h efore trnsfer. Nture Medicine doi:1.138/nm.243

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