RESEARCH I REPORTS was absent in ReglllB-deficient mice(fig. S6A). feces(Fig. 2C and table SI). Quantitative p ut was not significantly increased with CTLA-4 Concomitantly, the transcription levels of 117a, merase chain reaction(QPCR) analyses Ab(fig. S7). Ifng, Idol, type I Ifn-related gene products and geting the Bacteroides genus and species(spp Next, to establish a cause-and-effect rela a out not 176), which indicate ongoing in- in small intestine mucosa and feces contents tionship between the dominance of distinct tory processes, significantly increased by showed a trend toward a decreased relative Bacteroides spp in the small intestine and the in the distal ileum of CTLA-4 Ab-treated abundance of such bacteria in the feces, which anticancer efficacy of CTLA-4 blockade, we re- nice(fig. S6, B to D). Depletion of T cells, includ- contrasted with a relative enrichment in pa colonized Acs-treated and gF mice wit ng intraepithelial lymphocytes (Els)(by ular species [such as B. thetaiotaomicron(Bt) bacterial species associated with CTI tion of Abs specific for CD4 and CD8), abolished and B uniformis] in the small intestine mu- treated intestinal mucosae as well as the induction of IEC apoptosis by CTLA-4 e cosa 24 to 48 hours after one CTLA-4 Ab injec- treated mice orally fed with Bt, Bf, Burkholderia Ab(Fig 2A). When crypt -derived three-dimensional tion(Fig 2D and fig S7). One of the most cepacia(Bc), or the combination of Bf and Bc mall intestinal enteroids(6)were exposed to Toll- regulatory Bacteroides isolates, B. fragilis(B) recovered the anticancer response to CTLA-4Ab like receptor(TLR)agonists(which act as mi-(7-10), was detectable by PCR in colon mucosae I contrasting with all the other isolates that failed crobial ligands in this assay) and subsequent admixed with iels harvested from mice treated with Ab against CTLA-4 (but not isotype Cu),I A SPF B IECs within the enteroids underwent apoptosis 2501。koct oo Ctrl (Fig 2B). Hence, CTLA-4 Ab compromises the -.-oCTLA4 200— aCTAS4 homeostatic IEC-IEL equilibrium, favoring the apoptotic demise of IEC in the presence of mi-oN1 crobial product 100 ieC death could induce perturbations of the25 50 microbiota composition, we performed high throughput pyrosequencing of 16S ribosomal 101520 RNA (rRNA) gene amplicons of feces. The prin- cipal component analysis indicated that a single Days after tumor inoculation Days after tumor inoculation injection of CTLA-4 Ab sufficed to significantly affect the microbiome at the genus level (Fig. 20). C a CTLA blockade induced a rapid underrepre- sentation of both Bacteroidales and burkholder- les, with a relative increase of clos. ales, in E200 -+aCTLA E300° Institut de Cancerologie Gustave (GRcC), I4 rue Edouard Vaillant, 94805 Villejuif, France. 50 emlin-Bicetre. france. Institut National de la 15 CCTLA4 Centre Hospitalier Regional Universitaire de Lille, Institu Days after tumor inoculation Water Antibiotics Imipenem Colistin d'lmmunite de Lille( CIL). F-59000 Lille France. Center of pcc1.104 pe2i10-3 FgMicrobota-dependent Chinical Investigations in Biotherapies of Cancer 1428. llejuif, France. 'Universite Paris Descartes, Sorbonne Paris of CTLA-4 Ab Tumor growth 8 Cite. Paris france of MCA205 in SPF(A) GF(B) mice treated with five injections(compare the ngapore, Singapore. Department of Genetics and Department of Medicine A arrows)of 9D9 or isotype Metabolomics Platform, GRCC, Villejuif. oo8c A A control(lso Ctrl) Ab (C)Tumor 81∞ growth as in(A)and(B)in ence (left)of ACS or(right) of single-antibiotic regimen in France. uNiversite de Toulouse. Universite Paul Sabatier. Water Antibiotics later Antibiotics >20 mice per group. Flow ps1.10-4 and ICOS expression and (E)TH cytokines on splenic 2DINSERM U848, villejuif, franc TILs(E)2 days after the third Cancer centre administration of 9Dg or lso ctrl Recherche des Cordeliers INSERM Ul138, Paris, france Ab Each dot represents on mouse in two to three indepen ique-Hopitaux de Paris, Paris, dent experiments of five mice Mediterranee, Marseille. France. 24lnstitut Pasteur, Unit of Aaa per group. Pvalues corrected Biology and Genetics of the bacterial Cell Wall, Paris, France. NSERM, Equipe Aveni, Paris, France. Gastroenterology variation between three indi- Bicetre, Assistance Publique-Hopitaux Water Antibiotics Water Antibiotics idual experiments in(D). *P< These authors contributed equally to this work. cOrresponding 005P<001*P<0001; author. E-mail: laurence. zitvogelegustaveroussy fr ns, not significant 1080 27 NOVEMBER 2015. VOL 350 ISSUE 6264 sciencemag. org SCIENCEwas absent in RegIIIb-deficient mice (fig. S6A). Concomitantly, the transcription levels of Il17a, Ifng, Ido1, type 1 Ifn-related gene products and Ctla4 (but not Il6), which indicate ongoing in￾flammatory processes, significantly increased by 24 hours in the distal ileum of CTLA-4 Ab–treated mice (fig. S6, B to D). Depletion of T cells, includ￾ing intraepithelial lymphocytes (IELs) (by injec￾tion of Abs specific for CD4 and CD8), abolished the induction of IEC apoptosis by CTLA-4–specific Ab (Fig. 2A).When crypt-derived three-dimensional small intestinal enteroids (6) were exposed to Toll￾like receptor (TLR) agonists (which act as mi￾crobial ligands in this assay) and subsequently admixed with IELs harvested from mice treated with Ab against CTLA-4 (but not isotype Ctl), IECs within the enteroids underwent apoptosis (Fig. 2B). Hence, CTLA-4 Ab compromises the homeostatic IEC-IEL equilibrium, favoring the apoptotic demise of IEC in the presence of mi￾crobial products. To explore whether this T cell–dependent IEC death could induce perturbations of the microbiota composition, we performed high￾throughput pyrosequencing of 16S ribosomal RNA (rRNA) gene amplicons of feces. The prin￾cipal component analysis indicated that a single injection of CTLA-4 Ab sufficed to significantly affect the microbiome at the genus level (Fig. 2C). CTLA-4 blockade induced a rapid underrepre￾sentation of both Bacteroidales and Burkholder￾iales, with a relative increase of Clostridiales, in feces (Fig. 2C and table S1). Quantitative poly￾merase chain reaction (QPCR) analyses tar￾geting the Bacteroides genus and species (spp.) in small intestine mucosa and feces contents showed a trend toward a decreased relative abundance of such bacteria in the feces, which contrasted with a relative enrichment in partic￾ular species [such as B. thetaiotaomicron (Bt) and B. uniformis] in the small intestine mu￾cosa 24 to 48 hours after one CTLA-4 Ab injec￾tion (Fig. 2D and fig. S7). One of the most regulatory Bacteroides isolates, B. fragilis (Bf) (7–10), was detectable by PCR in colon mucosae but was not significantly increased with CTLA-4 Ab (fig. S7). Next, to establish a cause-and-effect rela￾tionship between the dominance of distinct Bacteroides spp. in the small intestine and the anticancer efficacy of CTLA-4 blockade, we re￾colonized ACS-treated and GF mice with several bacterial species associated with CTLA-4 Ab– treated intestinal mucosae as well as Bf. ACS￾treated mice orally fed with Bt, Bf, Burkholderia cepacia (Bc), or the combination of Bf and Bc, recovered the anticancer response to CTLA-4 Ab, contrasting with all the other isolates that failed 1080 27 NOVEMBER 2015 • VOL 350 ISSUE 6264 sciencemag.org SCIENCE 1 Institut de Cancérologie Gustave Roussy Cancer Campus (GRCC), 114 rue Edouard Vaillant, 94805 Villejuif, France. 2 INSERM U1015, GRCC, Villejuif, France. 3 University of Paris Sud XI, Kremlin-Bicêtre, France. 4 Institut National de la Recherche Agronomique (INRA), Micalis-UMR1319, 78360 Jouy-en-Josas, France. 5 University of Lille, CNRS, INSERM, Centre Hospitalier Régional Universitaire de Lille, Institut Pasteur de Lille, U1019, UMR 8204, Centre d'Infection et d'Immunité de Lille (CIIL), F-59000 Lille, France. 6 Center of Clinical Investigations in Biotherapies of Cancer 1428, Villejuif, France. 7 Université Paris Descartes, Sorbonne Paris Cité, Paris, France. 8 Department of Radiation Oncology, New York University, New York, NY, USA. 9 Microenvironment and Immunity Unit, Institut Pasteur, Paris, France. 10Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore. 11Department of Genetics and Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA. 12Metabolomics Platform, GRCC, Villejuif, France. 13Animalerie Centrale, Institut Pasteur, Paris, France. 14Centre National de la Recherche Scientifique, Institut de Pharmacologie et de Biologie Structurale (IPBS), Toulouse, France. 15Université de Toulouse, Université Paul Sabatier, IPBS, F-31077 Toulouse, France. 16Department of Medical Oncology, Institut Gustave Roussy, Villejuif, France. 17Service de microbiologie, GRCC, Villejuif, France. 18Laboratory of Immunomonitoring in Oncology, UMS 3655 CNRS/US 23 INSERM, GRCC, Villejuif, France. 19INSERM U981, GRCC, Villejuif, France. 20INSERM U848, Villejuif, France. 21Equipe 11 Labellisée—Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, INSERM U1138, Paris, France. 22Pôle de Biologie, Hôpital Européen Georges Pompidou, Assistance Publique—Hôpitaux de Paris, Paris, France. 23Unité des Rickettsies, Faculté de Médecine, Université de la Méditerranée, Marseille, France. 24Institut Pasteur, Unit of Biology and Genetics of the Bacterial Cell Wall, Paris, France. 25INSERM, Equipe Avenir, Paris, France. 26Gastroenterology Department, Hôpital Bicêtre, Assistance Publique—Hôpitaux de Paris, Paris, France. *These authors contributed equally to this work. †Corresponding author. E-mail: laurence.zitvogel@gustaveroussy.fr Fig. 1. Microbiota-dependent immunomodulatory effects of CTLA-4 Ab.Tumor growth of MCA205 in SPF (A) or GF (B) mice treated with five injections (compare the arrows) of 9D9 or isotype control (Iso Ctrl) Ab. (C) Tumor growth as in (A) and (B) in the presence (left) of ACS or (right) of single-antibiotic regimen in >20 mice per group. Flow cytometric analyses of (D) Ki67 and ICOS expression and (E) TH1 cytokines on splenic CD4+Foxp3– Tcells (D) and TILs (E) 2 days after the third administration of 9D9 or Iso Ctrl Ab. Each dot represents one mouse in two to three indepen￾dent experiments of five mice per group. P values corrected for interexperimental baseline variation between three indi￾vidual experiments in (D). *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant. RESEARCH | REPORTS on June 24, 2016 http://science.sciencemag.org/ Downloaded from