《人体的社交网络——共生微生物》课外文献阅读：Anticancer immunotherapy by CTLA-4blockade relies on the gut microbiota

RESEARCH I REPORTS Interspecific competition is thought to be a Victoria Species Survival Program for facitating access to live SUPPLEMENTARY MATERIALS pervasive force in evolution (29, 30), and we and preserved specimens: t www.sciencemagorg/content/350/6264/1077/suppl/dc1 suggest that the pattern we observe across Lakes port and the Tanzania C Victoria and Tanganyika is Figs. SI to S5 Strong, M. Turell, andG. tition between Nile perch and cichlid predators. was provided by NSF grants I0s-0924489DEB-0717009, and DEE Tables s1 to s8 For nearly half a century, the robust pharyn- 061981 to P.C. W and SNSF grant 310034.144046 to O.S. R.Y. N. References(31-76) geal jaws of cichlids, wrasses, and other phary animal protocols comply with UC Davis Guidelines for Animal Care and 8 March 2015, accepted 15 October 2015 ognathous fishes have been considered a classic Use Data are archived in Dryad. cample of evolutionary innovation that ope up new niches through increased trophic flexi bility(18). Although this is almost certainly cor- rect, our results suggest that the innovation CANCER IMMUNOTHERAPY olves a major trade-off that severely limits the ete ef on an a Anticancer immunotherapy by CTLA-4 innovation,The evolutionary inno blockade relies on the gut microbiota ait, but a specialization that can promote ompetitive exclusion and extinction depending on Marie vetizou, 12, Jonathan M. Pitt, 1 2 Romain Daimere, 11, Patricia Lepage, Bertrand Routy,r, Maria P. Roberti, 1, Sylvie Rusakiewicz, 12, 6 ecological context and community composition. Nadine Waldschmitt, Caroline Flament, 1, 2.6 Connie P. M. duong. 1, 2,6 REFERENCES AND NOTES Animal Species and Evolution(Harvard Univ. Press Vichnou Poirier-Colame, 2,6 Antoine Roux, ,7 Sonia Becharef, 2,6 Silvia Formenti, B o寸 idge, MA, 1963 Encouse Golden, Sascha Cording, Gerard Eberl, Andreas Schlitzer, 0 2. Florent Ginhoux, 0 Sridhar Mani, Takahiro Yamazaki, 2,6 Nicolas jacquelot, 1. 2,3 3. 1. P. Hunter, Trends Ecol. Evol. 13, 31-36( 1998). David P Enot, ,7, 2 Marion Berard, Jerome Nigou, ,ls Paule Opolon, Rubenstein,MDShawkey.Proc.Nat.Acad. Alexander Eggermont 1,2, 16 Paul-Louis Woerther, "Elisabeth Chachaty, 7 sciU.sA.110.10687-10692(20 Nathalie Chaput, ls Caroline Robert, 16, 19 Christina Mateus, , 16 5. D L Rabosky. PLOS ONE 9. 889543(2014) Guido Kroemer, 7, 1220, 2122 Didier Raoult, Ivo Gomperts Boneca, 24, 26* a D. V Nitecki, Eds. (Univ. of Chicago Press, Chicago, 1990). Franck Carbonnel, 26* Mathias Chamaillard, * Laurence Zitvogel 2y .6 7. G.J. Vermeij. Biol. I Linn. Soc. Land. 72, 461-508 Antibodies targeting CTLA-4 have been successfully used as cancer immunotherapy 8. G.I. Vermeij. Paleobiology 33, 469-493(2007). We find that the antitumor effects of CTLA-4 blockade depend on distinct Bacteroides 9. G.I. Vermeil. Evol Eco. 26, 357-373 (2012). species. In mice and patients, T cell responses specific for B thetaiotaomicron or B. fragil 335 were associated with the efficacy of CTLA-4 blockade Tumors in antibiotic-treated or Evot.Bl9.255(2009) germ-free mice did not respond to CTLA blockade. This defect was overcome by gavage 12. S A Hodges. M. L Arnold, Proc. Biol. Sci. 262. 343-3 with B. fragilis, by immunization with B fragilis polysaccharides, or by adoptive transfer of B. fragilis-specific T cells. Fecal microbial transplantation from humans to mice 13.D.Schluter. The Ecology of Adaptive Radiation(Oxford Univ. confirmed that treatment of melanoma patients with antibodies against CTLA-4 favored the outgrowth of B fragilis with anticancer properties. This study reveals a key role for wand et al, Nature 513, 375-381(2014) 5. C. Mitter, B. FarrelL, B. Wiegmann. Am. Nat 132, 107-128 Bacteroidales in the immunostimulatory effects of CTLA-4 blockade 16. D J Futuyma, G. Moreno. Annu. Rev. Ecol. Syst. 19, 207-Z33 mab s a fuly human monodonal anti: tomycin(g11 as well as imipenem目 17. T.I. Givnish, in Evolution on islands, P. Grant, Ed.(Oxford Univ. 1& K. s.s em. sast. Bao: 22: 425-41 negative regulator of T cell activation(), ap- antitumor effects of CTLA-4-specificAb.These 19.P.c. Wainwright, I. Zo.213.283-297(198) roved in 2011 for improving the overall sur- results, which suggest that the gut microbiota is 20. P. C. Wainwright et al, Syst. Biol. 61. 1001-1027 vival of patients with metastatic melanoma required for the anticancer effects of CTLA- block- (MM)(2). However, blockade of CTLA-4 by ipili- ade, were confirmed in the Ret melanoma and the 21. P. H. Greenwood, The Haplochroine Fishes of the mumab often results in immune-related adverse MC38 colon cancer models(fig. Sl, A and B).In akes: Collected Papers on Their Taxonomy events at sites that are exposed to commensal mi- addition, in GF or ACS-treated mice, activation of Biology and Evolution( Kraus International Publications croorganisms, mostly the gut(3) Patients treated splenic effector CD"T cells and tumor-infiltrating 0. Seehausen. Proc. a.273,1987-1998(2006 with ipilimumab develop Abs to components of lymphocytes(TILs)induced by Ab against CTLA-4 23. F. Witte et al, Environ Biol. Fishes 34, 1-28(1992). the enteric flora(4). Therefore, given our previous was significantly decreased(Fig.1,DandE, and 24. G. W. Coulter, I-I Tiercelin, Lake Tanganyika and Its Life indings for other cancer therapies(5), addressing fig Sl, C to E) (Oxford Univ. Press, Oxford, 199 the role of gut microbiota in the immunomodu- We next addressed the impact of the gut micro- latory effects of CTLA-4 blockade is crucial for the biota on the incidence and severity of intestinal 26.M. J. P. Van Oijen, Neth. I. Zool 32, 336-363 (1981). future development of immune checkpoint block-lesions induced by CTLA-4Ab treatmentA"sub- J. I. Van Alphen, F. witte, Science 27 ers in oncology clinical colitis" dependent on the gut microbiota 28. I. C. van Rijssel, F. Witte. EvoL. Ecol. 27, 253-267(2013). We compared the relative therapeutic efficacy was observed at late time points(figs. $2 to S5). 29. D. W. Pfennig, K. S. Pfennig, Evolution 's Wedge: Competition of the CTLA-4-specific D9 Ab against established However, shortly (by 24 hours)after the first ad- and the Origins of Diversity(Univ of California Press, Berkeley. MCA205 sarcomas in mice housed in specific ministration of CTLA-4 Ab, we observed increased pathogen-free(SPF)versus germ-free(GF)condi- cell death and proliferation of intestinal epithelial 30. D.L. Rabosky. Annu. Rex. Ecol Evol. Syst 44. 481-502(2013) tions. Tumor progression was controlled by Ab cells(IECs)residing in the ileum and colon, as ACKNOWLEDGMENTS against CTLA-4 in SPF but not in GF mice(Fig. 1, shown by immunohistochemistry using Ab-cleaved We thank R Bireley. I Clton L DeMason R Robbins. D. Schumacher, A and B). Moreover, a combination of broad pase-3 and Ki67 Ab, respectively(Fig 2A and W. Wong, 0. Selz. A. Taverna, M. Kayeha, M. Haluna and the Lake spectrum antibiotics [ampicillin +colistin +strep- fig S6A) The CTLA-4 Ab-induced IEC proliferation SCIENCE sciencemag. org 27 NOVEMBER 2015. VOL 350 ISSUE 6264 1079

Interspecific competition is thought to be a pervasive force in evolution (29, 30), and we suggest that the pattern we observe across Lakes Victoria and Tanganyika is likely due to compe￾tition between Nile perch and cichlid predators. For nearly half a century, the robust pharyn￾geal jaws of cichlids, wrasses, and other pharyn￾gognathous fishes have been considered a classic example of evolutionary innovation that opened up new niches through increased trophic flexi￾bility (18). Although this is almost certainly cor￾rect, our results suggest that the innovation involves a major trade-off that severely limits the size of prey that can be eaten, facilitating com￾petitive inferiority in predatory niches and ex￾tinction in the presence of a predatory invader lacking the innovation. The evolutionary inno￾vation of pharyngognathy is not a uniformly ben￾eficial trait, but a specialization that can promote competitive exclusion and extinction depending on ecological context and community composition. REFERENCES AND NOTES 1. E. Mayr, Animal Species and Evolution (Harvard Univ. Press Cambridge, MA, 1963). 2. S. B. Heard, D. L. Hauser, Hist. Biol. 10, 151–173 (1995). 3. J. P. Hunter, Trends Ecol. Evol. 13, 31–36 (1998). 4. R. Maia, D. R. Rubenstein, M. D. Shawkey, Proc. Natl. Acad. Sci. U.S.A. 110, 10687–10692 (2013). 5. D. L. Rabosky, PLOS ONE 9, e89543 (2014). 6. J. Cracraft, in Evolutionary Innovations, M. H. Nitecki, D. V. Nitecki, Eds. (Univ. of Chicago Press, Chicago, 1990), pp. 21–44. 7. G. J. Vermeij, Biol. J. Linn. Soc. Lond. 72, 461–508 (2001). 8. G. J. Vermeij, Paleobiology 33, 469–493 (2007). 9. G. J. Vermeij, Evol. Ecol. 26, 357–373 (2012). 10. M. E. Alfaro, C. D. Brock, B. L. Banbury, P. C. Wainwright, BMC Evol. Biol. 9, 255 (2009). 11. T. J. Givnish et al., Evolution 54, 1915–1937 (2000). 12. S. A. Hodges, M. L. Arnold, Proc. Biol. Sci. 262, 343–348 (1995). 13. D. Schluter, The Ecology of Adaptive Radiation (Oxford Univ. Press, Oxford, 2000). 14. D. Brawand et al., Nature 513, 375–381 (2014). 15. C. Mitter, B. Farrell, B. Wiegmann, Am. Nat. 132, 107–128 (1988). 16. D. J. Futuyma, G. Moreno, Annu. Rev. Ecol. Syst. 19, 207–233 (1988). 17. T. J. Givnish, in Evolution on Islands, P. Grant, Ed. (Oxford Univ. Press, Oxford, 1998), pp. 281–304. 18. K. F. Liem, Syst. Biol. 22, 425–441 (1973). 19. P. C. Wainwright, J. Zool. 213, 283–297 (1987). 20. P. C. Wainwright et al., Syst. Biol. 61, 1001–1027 (2012). 21. P. H. Greenwood, The Haplochromine Fishes of the East African Lakes: Collected Papers on Their Taxonomy, Biology and Evolution (Kraus International Publications, Munich, 1981). 22. O. Seehausen, Proc. Biol. Sci. 273, 1987–1998 (2006). 23. F. Witte et al., Environ. Biol. Fishes 34, 1–28 (1992). 24. G. W. Coulter, J.-J. Tiercelin, Lake Tanganyika and Its Life (Oxford Univ. Press, Oxford, 1991). 25. Materials and methods are available as supplementary materials on Science Online. 26. M. J. P. Van Oijen, Neth. J. Zool. 32, 336–363 (1981). 27. O. Seehausen, J. J. Van Alphen, F. Witte, Science 277, 1808–1811 (1997). 28. J. C. van Rijssel, F. Witte, Evol. Ecol. 27, 253–267 (2013). 29. D. W. Pfennig, K. S. Pfennig, Evolution's Wedge: Competition and the Origins of Diversity (Univ. of California Press, Berkeley, CA, 2012). 30. D. L. Rabosky, Annu. Rev. Ecol. Evol. Syst. 44, 481–502 (2013). ACKNOWLEDGMENTS We thank R. Bireley, J. Clifton, L. DeMason, R. Robbins, D. Schumacher, W. Wong, O. Selz, A. Taverna, M. Kayeba, M. Haluna, and the Lake Victoria Species Survival Program for facilitating access to live and preserved specimens; the Tanzania Fisheries Research Institute for support and the Tanzania Commission for Science and Technology for research permits to O.S.; and R. Grosberg, D. Schluter, T. Schoener, D. Strong, M. Turelli, and G. Vermeij for manuscript comments. Funding was provided by NSF grants IOS-0924489, DEB-0717009, and DEB- 061981 to P.C.W. and SNSF grant 31003A_144046 to O.S. R.Y.N. was supported by a Sloan Foundation grant to J. Eisen. All live animal protocols comply with UC Davis Guidelines for Animal Care and Use. Data are archived in Dryad. SUPPLEMENTARY MATERIALS www.sciencemag.org/content/350/6264/1077/suppl/DC1 Materials and Methods Figs. S1 to S5 Tables S1 to S8 References (31–76) 8 March 2015; accepted 15 October 2015 10.1126/science.aab0800 CANCER IMMUNOTHERAPY Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota Marie Vétizou,1,2,3 Jonathan M. Pitt,1,2,3 Romain Daillère,1,2,3 Patricia Lepage,4 Nadine Waldschmitt,5 Caroline Flament,1,2,6 Sylvie Rusakiewicz,1,2,6 Bertrand Routy,1,2,3,6 Maria P. Roberti,1,2,6 Connie P. M. Duong,1,2,6 Vichnou Poirier-Colame,1,2,6 Antoine Roux,1,2,7 Sonia Becharef,1,2,6 Silvia Formenti,8 Encouse Golden,8 Sascha Cording,9 Gerard Eberl,9 Andreas Schlitzer,10 Florent Ginhoux,10 Sridhar Mani,11 Takahiro Yamazaki,1,2,6 Nicolas Jacquelot,1,2,3 David P. Enot,1,7,12 Marion Bérard,13 Jérôme Nigou,14,15 Paule Opolon,1 Alexander Eggermont,1,2,16 Paul-Louis Woerther,17 Elisabeth Chachaty,17 Nathalie Chaput,1,18 Caroline Robert,1,16,19 Christina Mateus,1,16 Guido Kroemer,7,12,20,21,22 Didier Raoult,23 Ivo Gomperts Boneca,24,25* Franck Carbonnel,3,26* Mathias Chamaillard,5 * Laurence Zitvogel1,2,3,6† Antibodies targeting CTLA-4 have been successfully used as cancer immunotherapy. We find that the antitumor effects of CTLA-4 blockade depend on distinct Bacteroides species. In mice and patients, T cell responses specific for B. thetaiotaomicron or B. fragilis were associated with the efficacy of CTLA-4 blockade. Tumors in antibiotic-treated or germ-free mice did not respond to CTLA blockade. This defect was overcome by gavage with B. fragilis, by immunization with B. fragilis polysaccharides, or by adoptive transfer of B. fragilis–specific T cells. Fecal microbial transplantation from humans to mice confirmed that treatment of melanoma patients with antibodies against CTLA-4 favored the outgrowth of B. fragilis with anticancer properties. This study reveals a key role for Bacteroidales in the immunostimulatory effects of CTLA-4 blockade. I pilimumab is a fully human monoclonal anti￾body (Ab) directed against CTLA-4, a major negative regulator of T cell activation (1), ap￾proved in 2011 for improving the overall sur￾vival of patients with metastatic melanoma (MM) (2). However, blockade of CTLA-4 by ipili￾mumab often results in immune-related adverse events at sites that are exposed to commensal mi￾croorganisms, mostly the gut (3). Patients treated with ipilimumab develop Abs to components of the enteric flora (4). Therefore, given our previous findings for other cancer therapies (5), addressing the role of gut microbiota in the immunomodu￾latory effects of CTLA-4 blockade is crucial for the future development of immune checkpoint block￾ers in oncology. We compared the relative therapeutic efficacy of the CTLA-4–specific 9D9 Ab against established MCA205 sarcomas in mice housed in specific pathogen–free (SPF) versus germ-free (GF) condi￾tions. Tumor progression was controlled by Ab against CTLA-4 in SPF but not in GF mice (Fig. 1, A and B). Moreover, a combination of broad￾spectrum antibiotics [ampicillin + colistin + strep￾tomycin (ACS)] (Fig. 1C), as well as imipenem alone (but not colistin) (Fig. 1C), compromised the antitumor effects of CTLA-4–specific Ab. These results, which suggest that the gut microbiota is required for the anticancer effects of CTLA-4 block￾ade, were confirmed in the Ret melanoma and the MC38 colon cancer models (fig. S1, A and B). In addition, in GF or ACS-treated mice, activation of splenic effector CD4+ T cells and tumor-infiltrating lymphocytes (TILs) induced by Ab against CTLA-4 was significantly decreased (Fig. 1, D and E, and fig. S1, C to E). We next addressed the impact of the gut micro￾biota on the incidence and severity of intestinal lesions induced by CTLA-4 Ab treatment. A “sub￾clinical colitis” dependent on the gut microbiota was observed at late time points (figs. S2 to S5). However, shortly (by 24 hours) after the first ad￾ministration of CTLA-4 Ab, we observed increased cell death and proliferation of intestinal epithelial cells (IECs) residing in the ileum and colon, as shown by immunohistochemistry using Ab-cleaved caspase-3 and Ki67 Ab, respectively (Fig. 2A and fig. S6A). The CTLA-4 Ab–induced IEC proliferation SCIENCE sciencemag.org 27 NOVEMBER 2015 • VOL 350 ISSUE 6264 1079 RESEARCH | REPORTS on June 24, 2016 http://science.sciencemag.org/ Downloaded from

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 SCIENCE

was 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

RESEARCH I REPORTS to do so(table S2 and Fig 3A). Similarly, oral of CTLA-4 Ab was further demonstrated by the ipilimumab in 25 individuals with MM(table feeding with Bf, which colonized the mucosal adoptive transfer of memory Bf-specific(but not S4). A clustering algorithm based on genus ayer of GF mice(fig. S8)(n), induced T helper B. distasonis-specific) THI cells into GF or ACS- composition of the stools(12,13)distinguished 1(THI) immune responses in the tumor-draining treated tumor bearers(Fig. 3F and fig S12), which three clusters(Fig 4A and table S5)with Al- odes and promoted the maturation of partially restored the efficacy of the immune lopreuotella or Prevotella driving cluster Aand ntratumoral dendritic cells (DCs), which culmi- ckpoint blocker distinct Bacteroides spp driving clusters B and nated in the restoration of the therapeutic respons The microbiota-dependent immunostimula- C(Fig. 4B). During ipilimumab therapy, the of GF tumor bearers to CTLA-4 Ab(Fig. 3B and tory effects induced by CTLA-4 blockade de- proportions of MM patients falling into cluster ig S9, A and B) pended on the mobilization of lamina propria C increased, at the expense of those belonging We analyzed the dynamics of memory T cell CDllb" DC that can process zwitterionic poly- to cluster B(Fig. 4B and fig.$20A).We next responses directed against distinct bacterial spe- saccharides(9)and then mount interleukin-12 performed fecal microbial t Station o es in mice and humans during CTLA-4 block-(IL-12-dependent cognate THl immune responses feces harvested from different MM patients ade. CD4 T cells harvested from spleens of arides(figs. S13 from each cluster, 2 weeks before tumor inocula- CTLA-4 Ab-treated mice(Fig 3C)or from blood and S14) However, they did not appear to result tion into GF mice that were subsequently treated taken from individuals with MM or non-small from TLR2/TLRA-mediated innate signaling(7, 8) with anti-CTLA4 Ab. Tumors growing in mice cell lung carcinoma (NSCLC) patients after two in the context of a compromised gut tolerance that had been transplanted with feces from administrations of ipilimumab(Fig 3, D and E,(figs. S15 to S19). cluster C patients markedly responded to CTLA+ and table S3)tended to recover a THI phenotyp To address the clinical relevance of these find- blockade, contrasting with absent anticance (figs. S10 and S1l). The functional relevance of ings, we analyzed the composition of the gut effects in mice transplanted with cluster B- such T cell responses for the anticancer activity I microbiome before and after treatment with I related feces(Fig. 4C). QPCR analyses revealed Iso Ctrl CCTLA4 lleum nd intestinal dysbiosis after CTLA-4 Ab injec- 5 tion (A)(left) Representative micrograph pic- tures of distal ileum after staining with Ab-cleaved 9D9(or lso Ctri) Ab in naive mice with or without 玉 prior depletion of CD4 and CD8 T cells. Inset periments. (B)(left)Representative micrographs g 3D enteroid cocultures stimulated (or not)wit TLR agonists and incubated with IELs harvested B CCasp3 from 9D9(or iso CtrD) Ab-treated mice in hema 35 oxylin and eosin(H&E), then(middle)stained with 题! Medium cCasp3-specific Ab (Right) Data concatenated from ages of apoptotic cells to organoid in 20 organoids. +IELs Iso Ctrl (C) Sequencing of 16s rRNA gene i SI feces from tumor bearers before and 48 hours Principal component analysis( PCA)on a relative 2 abundance matrix of genus repartition highlighting a +ELS OCTLA4 No lELs Iso ctrl aCTLA4 the clustering between baseline, Iso Ctrl Ab-, and 9D9 Ab-treated animals after one injection( five to Bacteroidales Burkholderiales Clostridiales six mice per group). Ellipses are presented around the centroids of the resulting three clusters. The first two components explain 34. 41% of total 导 ariance(Component 1: 20.04% Component 2: 14.35%)(Monte-Carlo test with 1000 replicate P=0.0049).(C)(right) Means t SEM of relative abundance for each three orders for five mice per group are shown.(D)QPCR analyses targeting three distinct Bacteroides spp in ileal mucosae performed 24 to 48 hours after Ab introduction. Results are represented as 2-ACt x 103, nor- D malized to 16s rDNA and to the basal time poir 15 8. thetaiotaomicron B. uniforn (before treatment). Each dot represents one mouse in two gathered experiments. *P 0.05 3 **P<0.01: ++*P<0.001: ns, not significant. 10 ● 0 .lO SCIENCE sciencemag. org 27 NOVEMBER 2015. VOL 350 ISSUE 6264 1081

to do so (table S2 and Fig. 3A). Similarly, oral feeding with Bf, which colonized the mucosal layer of GF mice (fig. S8) (11), induced T helper 1 (TH1) immune responses in the tumor-draining lymph nodes and promoted the maturation of intratumoral dendritic cells (DCs), which culmi￾nated in the restoration of the therapeutic response of GF tumor bearers to CTLA-4 Ab (Fig. 3B and fig. S9, A and B). We analyzed the dynamics of memory T cell responses directed against distinct bacterial spe￾cies in mice and humans during CTLA-4 block￾ade. CD4+ T cells harvested from spleens of CTLA-4 Ab–treated mice (Fig. 3C) or from blood taken from individuals with MM or non–small cell lung carcinoma (NSCLC) patients after two administrations of ipilimumab (Fig. 3, D and E, and table S3) tended to recover a TH1 phenotype (figs. S10 and S11). The functional relevance of such T cell responses for the anticancer activity of CTLA-4 Ab was further demonstrated by the adoptive transfer of memory Bf-specific (but not B. distasonis-specific) TH1 cells into GF or ACS￾treated tumor bearers (Fig. 3F and fig. S12), which partially restored the efficacy of the immune checkpoint blocker. The microbiota-dependent immunostimula￾tory effects induced by CTLA-4 blockade de￾pended on the mobilization of lamina propria CD11b+ DC that can process zwitterionic poly￾saccharides (9) and then mount interleukin-12 (IL-12)–dependent cognate TH1 immune responses against Bf capsular polysaccharides (figs. S13 and S14). However, they did not appear to result from TLR2/TLR4-mediated innate signaling (7, 8) in the context of a compromised gut tolerance (figs. S15 to S19). To address the clinical relevance of these find￾ings, we analyzed the composition of the gut microbiome before and after treatment with ipilimumab in 25 individuals with MM (table S4). A clustering algorithm based on genus composition of the stools (12, 13) distinguished three clusters (Fig. 4A and table S5) with Al￾loprevotella or Prevotella driving cluster A and distinct Bacteroides spp. driving clusters B and C (Fig. 4B). During ipilimumab therapy, the proportions of MM patients falling into cluster C increased, at the expense of those belonging to cluster B (Fig. 4B and fig. S20A). We next performed fecal microbial transplantation of feces harvested from different MM patients from each cluster, 2 weeks before tumor inocula￾tion into GF mice that were subsequently treated with anti–CTLA-4 Ab. Tumors growing in mice that had been transplanted with feces from cluster C patients markedly responded to CTLA-4 blockade, contrasting with absent anticancer effects in mice transplanted with cluster B– related feces (Fig. 4C). QPCR analyses revealed SCIENCE sciencemag.org 27 NOVEMBER 2015 • VOL 350 ISSUE 6264 1081 Fig. 2. IEC-IEL dialogue causes IEC apoptosis and intestinal dysbiosis after CTLA-4 Ab injec￾tion. (A) (left) Representative micrograph pic￾tures of distal ileum after staining with Ab-cleaved caspase 3 (cCasp3) Ab 24 hours after one injection of 9D9 (or Iso Ctrl) Ab in naïve mice with or without prior depletion of CD4+ and CD8+ T cells. Inset enlarged 18-fold. (Right) Concatanated data of two experiments. (B) (left) Representative micrographs of 3D enteroid cocultures stimulated (or not) with TLR agonists and incubated with IELs harvested from 9D9 (or Iso Ctrl) Ab–treated mice in hema￾toxylin and eosin (H&E), then (middle) stained with cCasp3-specific Ab. (Right) Data concatenated from two experiments counting themeans ± SEM percent￾ages of apoptotic cells to organoid in 20 organoids. (C) Sequencing of 16S rRNA gene amplicons of feces from tumor bearers before and 48 hours after one administration of 9D9 or Iso Ctrl Ab. (Left) Principal component analysis (PCA) on a relative abundance matrix of genus repartition highlighting the clustering between baseline, Iso Ctrl Ab–, and 9D9 Ab–treated animals after one injection (five to six mice per group). Ellipses are presented around the centroids of the resulting three clusters. The first two components explain 34.41% of total variance (Component 1: 20.04%; Component 2: 14.35%) (Monte-Carlo test with 1000 replicates, P = 0.0049). (C) (right) Means ± SEM of relative abundance for each three orders for five mice per group are shown. (D) QPCR analyses targeting three distinct Bacteroides spp. in ileal mucosae performed 24 to 48 hours after Ab introduction. Results are represented as 2–DDCt × 103 , nor￾malized to 16S rDNA and to the basal time point (before treatment). Each dot represents one mouse in two gathered experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant. RESEARCH | REPORTS on June 24, 2016 http://science.sciencemag.org/ Downloaded from

RESEARCH I REPORTS A Bf and anticancer efficacy of CTLA-4 block ade.(A and B) Tumoricidal effects of Bf, Bt, and/or B. cepacia( Bc)administered by oral feeding of ACS- treated or GF mice(also refer to fig S8A).(a) g 8=8 tumor, and graphs depict two to three experiments of five mice per group. (Middle)Histopathological aCTLA bearers receiving 9D9 Ab after oral gavage with ° ■3- severe 2-moderate colons monitoring microscopic lesions as described 5::1-mild in materials and methods at day 20 after treat Antibiotics Antibiotics ment in five animals per group on at least six B independent areas.(Right) Representative micro- graphs are shown, scale bar, 100 um.(B)Tumor- icidal effects of Bf in GF mice as indicated. (c to E fragilis Recall responses of CD4 T cells in mice and patients to various bacterial strains after CTLA-4 blockade. DCs loaded with bacteria of the indi ated strain were incubated with cD4- T cells 20 lays after three intraperitoneal (p) CTLA-4 Ab in mice, and after at least two injections of Days after tumor inoculation iimumab(ipi)in patients. The graphs represe 8. dista nterferon-y(IFN-y)concentrations from coculture D olena ematants at 24 hours in mice(C)and 48 hour B fragilis B. thetaiotaomicron MM patients(D).(E)IFN-7/IL-10 ratios were mon- itored in DC-T cell cocultures of NSCLC patients at 48 hours. No cytokine release was observed in 8 the absence of bacteria or Tcells(fig. Sll with H) Each dot represents one patient or mouse. Paired are represented by linking dots pre- and T cells harvested from spleens of mice exposed to CTLA-4 Ab and restimulated with Bf Melanoma 0.05 versus b. distasonis or bone marrow dcs alone Pre Post Pre Post F (CD4 NT) were infused intravenously in day 6 CD4*B. distasonis CD4*Bfragilis MCA205 tumor-bearing GF mice. A representative experiment containing five to six mice per group shown. *P< 0.05 * ot significant. that, although bacteria from the Bacteroidales achieved with vancomycin, which could boost the I probiotics. " The geodistribution of Bf in the order equally colonized the recipient murine antitumor effects of CTLA-4 blockade(p mucosal layer of the intestine (fig. SS)and its ntestine,stools from cluster C(but not A or B) ably by inducing the overrepresentation of Bac- association with Burkholderiales-recognize ndividuals specifically facilitated the coloni- teroidales at the expense of Clostridiales)but ugh the pyrin-caspase-l inflammasome zation of the immunogenic bacteria Bf and worsened the histopathological score (fig. S21). d synergizing with TLR2/TLR4 signaling path (7-10, 14, 15)(Fig. 4D). Moreover, after CTLA-4 Ab In support of this notion, Bf maintained its reg- ways(fi S15)-may account for the immunomod- herapy,only cluster C (not A or B)recipient mice ulatory properties in the context of CTLA 4 block- ulatory effects of CTLA-4 Ab. Future investigations had outgrowth of Bf(fig. S20B) Note that the ade(fig S22)(7. will determine whether a potential molecular fecal abundance of Bf (but not B. distasonis ol Hence, the efficacy of CTLA-4 blockade is in- mimicry between distinct commensals and/or B uniformis)negatively correlated with tumor fluenced by the microbiota composition(B fragilis pathobionts and tumor neoantigens could ac- size after CTLA-4 blockade in cluster C-recipient and/or B thetaiotaomicron and Burkholderiales ) count for the toxicity andyor efficacy of immune mice(Fig. 4E and fig. S20C) Hence, ipilimumab The microbiota composition affects interleukin checkpoint blockers. Prospective studies in MM an modify the abundance of immunogenic Bac- 12(IL-12-dependent THl immune responses d or NSClC may validate the relevance of the teroides spp in the gut, which in turn affects its which facilitate tumor control in mice and pa- enterotypes described herein in the long-term anticancer efficacy. tients while sparing intestinal integrity. In ac- efficacy of immune checkpoint blockers, with Finally, intestinal reconstitution of ACS-treated cord with previous findings(16), colitis(observed the aim of compensating cluster B-driven pa nimals with the combination of Bf and Be did in the context of IL-10 deficiency and CTLA-4 tients with live and immunogenicorrecombinant not increase but rather reduced histopathological blockade)(fig. S17)could even antagonize anti- Bacteroides spp(18)or fecal microbial transplanta- signs of colitis induced by CTLA-4 blockade(Fig 3A). cancer efficacy. Several factors may dictate why tion from cluster C-associated stools to improve This efficacy-toxicity uncoupling effect was not I such commensals could be suitable "anticancer their antitumor immune responses. 1082 27 NOVEMBER 2015. VOL 350 ISSUE 6264 sciencemag. org SCIENCE

that, although bacteria from the Bacteroidales order equally colonized the recipient murine intestine, stools from cluster C (but not A or B) individuals specifically facilitated the coloni￾zation of the immunogenic bacteria Bf and Bt (7–10,14,15) (Fig. 4D). Moreover, after CTLA-4 Ab therapy, only cluster C (not A or B) recipient mice had outgrowth of Bf (fig. S20B). Note that the fecal abundance of Bf (but not B. distasonis or B. uniformis) negatively correlated with tumor size after CTLA-4 blockade in cluster C–recipient mice (Fig. 4E and fig. S20C). Hence, ipilimumab can modify the abundance of immunogenic Bac￾teroides spp. in the gut, which in turn affects its anticancer efficacy. Finally, intestinal reconstitution of ACS-treated animals with the combination of Bf and Bc did not increase but rather reduced histopathological signs of colitis induced by CTLA-4 blockade (Fig. 3A). This efficacy-toxicity uncoupling effect was not achieved with vancomycin, which could boost the antitumor effects of CTLA-4 blockade (presum￾ably by inducing the overrepresentation of Bac￾teroidales at the expense of Clostridiales) but worsened the histopathological score (fig. S21). In support of this notion, Bf maintained its reg￾ulatory properties in the context of CTLA-4 block￾ade (fig. S22) (7). Hence, the efficacy of CTLA-4 blockade is in￾fluenced by the microbiota composition (B. fragilis and/or B. thetaiotaomicron and Burkholderiales). The microbiota composition affects interleukin 12 (IL-12)–dependent TH1 immune responses, which facilitate tumor control in mice and pa￾tients while sparing intestinal integrity. In ac￾cord with previous findings (16), colitis (observed in the context of IL-10 deficiency and CTLA-4 blockade) (fig. S17) could even antagonize anti￾cancer efficacy. Several factors may dictate why such commensals could be suitable “anticancer probiotics.” The geodistribution of Bf in the mucosal layer of the intestine (fig. S8) and its association with Burkholderiales—recognized through the pyrin–caspase-1 inflammasome (17) and synergizing with TLR2/TLR4 signaling path￾ways (fig. S15)—may account for the immunomod￾ulatory effects of CTLA-4 Ab. Future investigations will determine whether a potential molecular mimicry between distinct commensals and/or pathobionts and tumor neoantigens could ac￾count for the toxicity and/or efficacy of immune checkpoint blockers. Prospective studies in MM and/or NSCLC may validate the relevance of the enterotypes described herein in the long-term efficacy of immune checkpoint blockers, with the aim of compensating cluster B–driven pa￾tients with live and immunogenic or recombinant Bacteroides spp. (18) or fecal microbial transplanta￾tion from cluster C–associated stools to improve their antitumor immune responses. 1082 27 NOVEMBER 2015 • VOL 350 ISSUE 6264 sciencemag.org SCIENCE Fig. 3. Memory T cell responses against Bt and Bf and anticancer efficacy of CTLA-4 block￾ade. (A and B) Tumoricidal effects of Bf, Bt, and/or B. cepacia (Bc) administered by oral feeding of ACS￾treated or GF mice (also refer to fig. S8A). (A) (left) Tumor sizes at day 15 after 9D9 or Iso Ctrl Ab treatment are depicted. Each dot represents one tumor, and graphs depict two to three experiments of five mice per group. (Middle) Histopathological score of colonic mucosae in ACS-treated tumor bearers receiving 9D9 Ab after oral gavage with various bacterial strains, assessed on H&E-stained colons monitoring microscopic lesions as described in materials and methods at day 20 after treat￾ment in five animals per group on at least six independent areas. (Right) Representative micro￾graphs are shown; scale bar, 100 mm. (B) Tumor￾icidal effects of Bf in GF mice as indicated. (C to E) Recall responses of CD4+ T cells in mice and patients to various bacterial strains after CTLA-4 blockade. DCs loaded with bacteria of the indi￾cated strain were incubated with CD4+ T cells, 2 days after three intraperitoneal (ip) CTLA-4 Ab in mice, and after at least two injections of ipilimumab (ipi) in patients. The graphs represent interferon-g (IFN-g) concentrations from coculture supernatants at 24 hours in mice (C) and 48 hours in MM patients (D). (E) IFN-g/IL-10 ratios were mon￾itored in DC–T cell cocultures of NSCLC patients at 48 hours. No cytokine release was observed in the absence of bacteria or Tcells (fig. S11 with HV). Each dot represents one patient or mouse. Paired analyses are represented by linking dots pre- and post-ipi. (F) T cells harvested from spleens of mice exposed to CTLA-4 Ab and restimulated with Bf versus B. distasonis or bone marrow DCs alone (CD4+ NT) were infused intravenously in day 6 MCA205 tumor-bearing GF mice. A representative experiment containing five to six mice per group is shown. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant. RESEARCH | REPORTS on June 24, 2016 http://science.sciencemag.org/ Downloaded from

RESEARCH I REPORTS ed ruminococcaceae hascolarctobadterun Faecalibacterium Scoresland dasses Mean Decrease Accuracy B n=18 B。 bacterium mpnwsolate groi of Bamesiella inp, CCUG 39913 意人8盒A家 Stools Before After ipilimumab cluster B Cluster C 2001 Clister A Cluster B Cluster C Days after treatment Bacteroides Prevotella B. fragilis B. thetaiotaomicron Cluster C 寸69月85885 Fg.4. cal significance of ipilimumab-induced dysbiosis in patients. ipilimumab from eight patents falling into each of the three clusters(stool selec- 3 k means clustering algorithm was applied on the basis of genus composition tion for fecal microbial transplantation marked with an asterisk*in fig. S20A) into Calinski-Harabasz index(14) and showed good performance in recovering three SEM of tumor sizes depicted for each cluster over time. (D)QPCR analyses of feces clusters before and after therapy(interclass PCA): (A)(left)(Monte-Carlo test, DNA of the recipient before(2 weeks postcolonization) and 2 weeks after ipi, P=0.000199)(A)(right) Random Forest analysis was applied to decipher the targeting Bacteroidales and Bacteroides spp. Results are represented as 2-aCtx nain genera responsible for this significant clustering( B)(right) The relative 10, normalized to 16S rDNA No significant difference in the relative abundance of abundance of main Bacteroides spp significantly differed between clusters b Bf was detectable in the donors of cluster B versus c before colonization(not d C(B)(left) The proportions of patients falling into each cluster were ana- shown ).(E) Spearman correlations between the amount of Bf in stools 15 days lyzed in a nonpaired manner before versus after ipi injections regardless of the after treatment with 9D9 Ab and tumor sizes across cluster B-and C-recipient time point(fig. S20A).(C) Fecal microbial transplantation after introduction of mice. *P <0.05: **P<0.01; ***P<0.001 REFERENCES AND NOTES 13. 1. Qin et al, Nature 464, 59-65(2010 supported by La Ligue contre le cancer and ARC, respectively. L2 1. K.S. Peggs, S. A. Quezada, A.J. Korman, J. P. Allison 14. A. Cebula et al, Nature 497. 258-262(2013) received a special prize from the Swiss Bridge Foundation and ISREC. Curr. Opin. Immunol 18, 206-213(2006). 15.IL Sonnenburg.C. T.Chen, 1.L.Gordon. PLOS Biol. 4, e413 GK and L.Z. were supported by the Ligue Nationale contre le Cancer 2. F.S. Hodi et al, N. Engl. I. Med. 363. 711-723(2010). (2006) 16. W. Lam et al., Sci. Trans. Med. 2, 45ra59(2010) 5. S Viaud et a, Science 342, 971-976(2013) 11Hx知时乱,N加m51.237-2402041.casm1/ uropean Research Council Advanced Investigator Grant(oGK 18. M Mimee, A. C. Tucker, C. A. Voigt, T.K. Fondation pour la Recherche Medicale(FRM). Institut National du 62-71(2015) Methods421.8-95(2015) ridge Foundation. the LabEx 7. S Dasgupta, D.Erturk-Hlasderni, 1. O0choa-Reparaz,HC. Reinecke.ACKNOWLEDGMENTS D L Kasper, Cel Host Microbe 15. 413-423(2014). We are grateful to the staff of the 8. S K Mazmanian, C H. Liu, A 0. Tzianabos, D L Kasper, Cell and Institut Pasteur. The data pre 122107-118(2005) aper and in 9.F. Stingele et at.mno.172.1483-1490(2004) LZ, MV, and PL have filed patent 二 asy Oncology Cell DNA Repair and Tumor the SIRIC Cancer Research and Personal zed Medicine(CARPEM), an supported by NIH(RO1 CA161879, as Principal Investigator).MC was aA.0. Tzianabos et af. 1. Biol. Chem. 267. 18230-18235(1992) that relates to the following: Methods and products for modulating Fondation l1.1YHargSM.Lee,SK. Mazmarian, Anaerobe 17, 137-141(201). microbiota composition for improving the efficacy of a car ARC pour la recherche sur le cancer, and Institut Nationale du Cancer. 12. M. Arumugam ef al. Nature 473, 174-180(2011) treatment with an immune checkpoint blocker M.V. and JMP.were N.W. is a recipient of a postdoctoral Fellowship from the Agence SCIENCE sciencemag. org 27 NOVEMBER 2015. VOL 350 ISSUE 6264 1083

REFERENCES AND NOTES 1. K. S. Peggs, S. A. Quezada, A. J. Korman, J. P. Allison, Curr. Opin. Immunol. 18, 206–213 (2006). 2. F. S. Hodi et al., N. Engl. J. Med. 363, 711–723 (2010). 3. K. E. Beck et al., J. Clin. Oncol. 24, 2283–2289 (2006). 4. D. Berman et al., Cancer Immun. 10, 11 (2010). 5. S. Viaud et al., Science 342, 971–976 (2013). 6. A. Rogoz, B. S. Reis, R. A. Karssemeijer, D. Mucida, J. Immunol. Methods 421, 89–95 (2015). 7. S. Dasgupta, D. Erturk-Hasdemir, J. Ochoa-Reparaz, H. C. Reinecker, D. L. Kasper, Cell Host Microbe 15, 413–423 (2014). 8. S. K. Mazmanian, C. H. Liu, A. O. Tzianabos, D. L. Kasper, Cell 122, 107–118 (2005). 9. F. Stingele et al., J. Immunol. 172, 1483–1490 (2004). 10. A. O. Tzianabos et al., J. Biol. Chem. 267, 18230–18235 (1992). 11. J. Y. Huang, S. M. Lee, S. K. Mazmanian, Anaerobe 17, 137–141 (2011). 12. M. Arumugam et al., Nature 473, 174–180 (2011). 13. J. Qin et al., Nature 464, 59–65 (2010). 14. A. Cebula et al., Nature 497, 258–262 (2013). 15. J. L. Sonnenburg, C. T. Chen, J. I. Gordon, PLOS Biol. 4, e413 (2006). 16. W. Lam et al., Sci. Transl. Med. 2, 45ra59 (2010). 17. H. Xu et al., Nature 513, 237–241 (2014). 18. M. Mimee, A. C. Tucker, C. A. Voigt, T. K. Lu, Cell Systems 1, 62–71 (2015). ACKNOWLEDGMENTS We are grateful to the staff of the animal facility of Gustave Roussy and Institut Pasteur. The data presented in this manuscript are tabulated in the main paper and in the supplementary materials. L.Z., M.V., and P.L. have filed patent application no. EP 14190167 that relates to the following: Methods and products for modulating microbiota composition for improving the efficacy of a cancer treatment with an immune checkpoint blocker. M.V. and J.M.P. were supported by La Ligue contre le cancer and ARC, respectively. L.Z. received a special prize from the Swiss Bridge Foundation and ISREC. G.K. and L.Z. were supported by the Ligue Nationale contre le Cancer (Equipes labelisées), Agence Nationale pour la Recherche (ANR AUTOPH, ANR Emergence), European Commission (ArtForce), European Research Council Advanced Investigator Grant (to G.K.), Fondation pour la Recherche Médicale (FRM), Institut National du Cancer (INCa), Fondation de France, Cancéropôle Ile-de-France, Fondation Bettencourt-Schueller, Swiss Bridge Foundation, the LabEx Immuno-Oncology, the Institut national du cancer (SIRIC) Stratified Oncology Cell DNA Repair and Tumor Immune Elimination (SOCRATE); the SIRIC Cancer Research and Personalized Medicine (CARPEM), and the Paris Alliance of Cancer Research Institutes (PACRI). S.M. was supported by NIH (R01 CA161879, as Principal Investigator). M.C. was supported by the Fondation pour la Recherche Médicale, the Fondation ARC pour la recherche sur le cancer, and Institut Nationale du Cancer. N.W. is a recipient of a Postdoctoral Fellowship from the Agence SCIENCE sciencemag.org 27 NOVEMBER 2015 • VOL 350 ISSUE 6264 1083 Fig. 4. Biological significance of ipilimumab-induced dysbiosis in patients. The k means clustering algorithm was applied on the basis of genus composition before and during ipilimumab treatment in 25 MM patients, validated using the Calinski-Harabasz index (14), and showed good performance in recovering three clusters before and after therapy (interclass PCA); (A) (left) (Monte-Carlo test, P = 0.000199). (A) (right) Random Forest analysis was applied to decipher the main genera responsible for this significant clustering. (B) (right) The relative abundance of main Bacteroides spp. significantly differed between clusters B and C. (B) (left) The proportions of patients falling into each cluster were ana￾lyzed in a nonpaired manner before versus after ipi injections regardless of the time point (fig. S20A). (C) Fecal microbial transplantation after introduction of ipilimumab from eight patients falling into each of the three clusters (stool selec￾tion for fecal microbial transplantation marked with an asterisk * in fig. S20A) into GF animals. One representative experiment out of three is shown with means ± SEM of tumor sizes depicted for each cluster over time. (D) QPCR analyses of feces DNA of the recipient before (2 weeks postcolonization) and 2 weeks after ipi, targeting Bacteroidales and Bacteroides spp. Results are represented as 2–DCt x 103 , normalized to 16S rDNA. No significant difference in the relative abundance of Bf was detectable in the donors of cluster B versus C before colonization (not shown). (E) Spearman correlations between the amount of Bf in stools 15 days after treatment with 9D9 Ab and tumor sizes across cluster B- and C-recipient mice. *P < 0.05; **P < 0.01; ***P < 0.001. RESEARCH | REPORTS on June 24, 2016 http://science.sciencemag.org/ Downloaded from