《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

RESEARCH I REPORTS Nationale de la Recherche. AS was supported by BMSI YIG 2014. E.G. study have been submitted to the NCBI under theBicproject Figs. SI to s22 Reed Archive ables s1 to s5 sociation pour la Recherche contre le References(19-35) 200851) F.C. was supported by INCA-DGOS (GOLD sRR28006.SRR2758031,SRR275878.sRR275879.sRR2758180 2012-1-RT-14-GR-01). L'Oreal awarded a prize to M V. We are grateful SUPPLEMENTARY MATERIALS 21 October 2015 gelique, N. Chanthapathet, www.sciencemag.org/content/350/6264/1079/suppl/dcl ublished online 5 November 2015 and S. Zuberagoitia for technical help. DNA sequence reads from this Materials and Methods 10.1126/science aad1329 CANCER IMMUNOTHERAPY and JAX mice appeared to acquire the jAX pheno- type, which suggested that JAX mice may be col- Commensal bifidobacterium onized by commensal microbes that dominantly facilitate antitumor immunity To directly test the role of commensal bacteria promotes antitumor immunity and in regulating antitumor immunity, we transferred JAX or TAC fecal suspensions into TAC and JAX facilitates anti-PD-Ll efficacy recipients by oral gavage before tumor implan- tation (fig. SIA). We found that prophylactic trans- fer of JAX fecal material, but not saline or TAc Ayelet Sivan, Leticia Corrales, Nathaniel Hubert, Jason B. williams, fecal material, into TAC recipients was sufficient Keston Aquino-Michaels, " Zachary M. Earley, Franco w Benyamin, 'Yuk Man Lei, to delay tumor growth(Fig 2A)and to enhance Bana Jabri, Maria-Luisa Alegre, Eugene B Chang, Thomas F Gajewski,t T cells(Fig. 2, B and C, and fig SIB), which sup- s T cell infiltration of solid tumors is associated with favorable patient outcomes, yet the ported a microbe-derived effect. Reciprocal trans. mechanisms underlying variable immune responses between individuals are not well fer of TAC fecal material into JAX recipients had understood. One possible modulator could be the intestinal microbiota. We compared a minimal effect on tumor growth rate and anti- sD melanoma growth in mice harboring distinct commensal microbiota and observed differences in spontaneous antitumor immunity, which were eliminated upon cohousing SIB), consistent with the JAX-dominant effects g observed upon cohousing or after fecal transfer Sequencing of the 16S ribosomal RNA identified Bifidobacterium asTo test whether manipulation of the microbial associated with the antitumor effects Oral administration of Bifidobacterium alone improved tumor control to the same degree as programmed cell death protein l ligand community could be effective as a therapy, we ad- 1(PD-L1)-specific antibody therapy(checkpoint blockade), and combination treatment ministered jAX fecal material alone or in combi nearly abolished tumor outgrowth. Augmented dendritic cell function leading to enhanced nation with antibodies targeting PD-Ll(aPD-Ll) 35 CD8" T cell priming and accumulation in the tumor microenvironment mediated the effect. to TAc mice bearing established tumors.Trans- Our data suggest that manipulating the microbiota may modulate cancer immunotherapy. icantly slower tumor growth (Fig 2D, ccompanied a by increased tumor-specific T cell responses E armessing the host immune system consti- mediating immune activation in response to chemo(Fig. 2E)and infiltration of antigen-specific Tcells tutes a promising cancer therapeutic be- therapeutic agents has been demonstrated (10, Ir). into the tumor(Fig. 2F), to the same degree as auseofitspotentialtospecificallytargetHowever,itisnotknownwhethercommensaltreatmentwithsystemicapd-llmab.combina- tumor cells although limiting harm to nor. microbiota influence spontaneous immune re- tion treatment with both JAX fecal transfer and 3 mal tissue. Enthusiasm has been fueled sponses against tumors and thereby affect the aPD-LImAb improved tumor control (Fig 2D)and a specifically CTLA-4 and the axis between pro- antibodies(mAbs). on accumulation of activated t cells within the grammed cell death protein 1(PD-1) and its To address this question, we compared sub- tumor microenvironment (Fig. 2F). Consistent with igand1(PD-LD(1, 2).Clinical responses to these cutaneous B16 STY melanoma growthin genetically these results, aPD-LI therapy alone was signifi- are more frequent in patients similar C57BL/6 mice derived from two different cantly more efficacious in JAX mice compared who show evidence of an endogenous T cell re- mouse facilities, Jackson Laboratory (AX) and with TAC mice(Fig. 2G), which paralleled improved sponse ongoing in the tumor microenvironment Taconic Farms(TAC), which have been shown to antitumor T cell responses(fig. SIC). These data before therapy(3-6). However, the mechanisms differ in their commensal microbes(12). We found indicate that the commensal microbial compo- hat govern the presence or absence of this phe- that JAX and TAC mice exhibited significant sition can influence spontaneous antitumor im- notype are not well understood. Theoretical sources differences in B16 STY melanoma growth rate, munity, as well as a response to immunotherapy of interpatient heterogeneity include host germ- with tumors growing more aggressively in TAc with aPD-LI mAb ine genetic differences, variability in patterns of mice(Fig. 1A). This difference was immune- To identify specific bacteria associated with im- somatic alterations in tumor cells, and environ- mediated: Tumor-specific T cell responses(Fig. 1, proved antitumor immune responses, we moni mental differences B and C) and intratumoral CDS*T cell accumu- tored the fecal bacterial content over time of mice he gut microbiota plays an important role in lation(Fig. ID)were significantly higher in JAX that were subjected to administration of fecal shaping systemicimmune responses(7-9). In the than in TAC mice. To begin to address whether permutations, using the 16S ribosomal RNA(rRNA) cancer context, a role for intestinal microbiota in this difference could be mediated by commensal miSeq Illumina platform. Principal coordinate microbiota, we choused JAX and TAC mice be nalysis revealed that fecal samples analyzed from fore tumor implantation. We found that cohous- TAC mice that received JAX fecal material grad ent of Medicine, University of Chicago, ing ablated the differences in tumor growth(Fig. ually separated from samples obtained from sham- Chicago, IL 60637, USA 3Section of Genetic Medicine 1E) and immune responses(Fig. 1, F to h)be- d TAC feces-inoculated TAC "These authors contributed equally to this work. cOrresponding tween the two lations,which sug.(P=0.001 and P=0.003, respect NOSIM author. E-mail: tgajewsk@medicine bsd uchicago. ed gested an enviro nce Choused TAc I multivariate data analysis )and 1084 27 NOVEMBER 2015. VOL 350 ISSUE 6264 sciencemag. org SCIENCE

Nationale de la Recherche. A.S. was supported by BMSI YIG 2014. F.G. is supported by SIgN core funding. L.Z., M.C., and I.B.G. are all sponsored by Association pour la Recherche contre le Cancer (PGA120140200851). F.C. was supported by INCA-DGOS (GOLD H78008). N.C. was supported by INCA-DGOS (GOLD study; 2012-1-RT-14-IGR-01). L’Oreal awarded a prize to M.V. We are grateful to the staff of the animal facility of Gustave Roussy and Institut Pasteur. We thank P. Gonin, B. Ryffel, T. Angelique, N. Chanthapathet, H. Li, and S. Zuberogoitia for technical help. DNA sequence reads from this study have been submitted to the NCBI under the Bioproject IDPRJNA299112 and are available from the Sequence Read Archive (SRP Study accession SRP065109; run accession numbers SRR2758006, SRR2758031, SRR2758178, SRR2758179, SRR2758180, SRR2758181, SRR2768454, and SRR2768457. SUPPLEMENTARY MATERIALS www.sciencemag.org/content/350/6264/1079/suppl/DC1 Materials and Methods Figs. S1 to S22 Tables S1 to S5 References (19–35) 3 April 2015; accepted 21 October 2015 Published online 5 November 2015; 10.1126/science.aad1329 CANCER IMMUNOTHERAPY Commensal Bifidobacterium promotes antitumor immunity and facilitates anti–PD-L1 efficacy Ayelet Sivan,1 * Leticia Corrales,1 * Nathaniel Hubert,2 Jason B. Williams,1 Keston Aquino-Michaels,3 Zachary M. Earley,2 Franco W. Benyamin,1 Yuk Man Lei,2 Bana Jabri,2 Maria-Luisa Alegre,2 Eugene B. Chang,2 Thomas F. Gajewski1,2† T cell infiltration of solid tumors is associated with favorable patient outcomes, yet the mechanisms underlying variable immune responses between individuals are not well understood. One possible modulator could be the intestinal microbiota. We compared melanoma growth in mice harboring distinct commensal microbiota and observed differences in spontaneous antitumor immunity, which were eliminated upon cohousing or after fecal transfer. Sequencing of the 16S ribosomal RNA identified Bifidobacterium as associated with the antitumor effects. Oral administration of Bifidobacterium alone improved tumor control to the same degree as programmed cell death protein 1 ligand 1 (PD-L1)–specific antibody therapy (checkpoint blockade), and combination treatment nearly abolished tumor outgrowth. Augmented dendritic cell function leading to enhanced CD8+ T cell priming and accumulation in the tumor microenvironment mediated the effect. Our data suggest that manipulating the microbiota may modulate cancer immunotherapy. H arnessing the host immune system consti￾tutes a promising cancer therapeutic be￾cause of its potential to specifically target tumor cells although limiting harm to nor￾mal tissue. Enthusiasm has been fueled by recent clinical success, particularly with anti￾bodies that block immune inhibitory pathways, specifically CTLA-4 and the axis between pro￾grammed cell death protein 1 (PD-1) and its ligand 1 (PD-L1) (1, 2). Clinical responses to these immunotherapies are more frequent in patients who show evidence of an endogenous T cell re￾sponse ongoing in the tumor microenvironment before therapy (3–6). However, the mechanisms that govern the presence or absence of this phe￾notype are not well understood. Theoretical sources of interpatient heterogeneity include host germ￾line genetic differences, variability in patterns of somatic alterations in tumor cells, and environ￾mental differences. The gut microbiota plays an important role in shaping systemic immune responses (7–9). In the cancer context, a role for intestinal microbiota in mediatingimmune activationin response to chemo￾therapeutic agents has been demonstrated (10,11). However, it is not known whether commensal microbiota influence spontaneous immune re￾sponses against tumors and thereby affect the therapeutic activity of immunotherapeutic inter￾ventions, such as anti–PD-1/PD-L1 monoclonal antibodies (mAbs). To address this question, we compared sub￾cutaneous B16.SIYmelanoma growthin genetically similar C57BL/6 mice derived from two different mouse facilities, Jackson Laboratory (JAX) and Taconic Farms (TAC), which have been shown to differ in their commensal microbes (12). We found that JAX and TAC mice exhibited significant differences in B16.SIY melanoma growth rate, with tumors growing more aggressively in TAC mice (Fig. 1A). This difference was immune￾mediated: Tumor-specific T cell responses (Fig. 1, B and C) and intratumoral CD8+ T cell accumu￾lation (Fig. 1D) were significantly higher in JAX than in TAC mice. To begin to address whether this difference could be mediated by commensal microbiota, we cohoused JAX and TAC mice be￾fore tumor implantation. We found that cohous￾ing ablated the differences in tumor growth (Fig. 1E) and immune responses (Fig. 1, F to H) be￾tween the two mouse populations, which sug￾gested an environmental influence. Cohoused TAC and JAX mice appeared to acquire the JAX pheno￾type, which suggested that JAX mice may be col￾onized by commensal microbes that dominantly facilitate antitumor immunity. To directly test the role of commensal bacteria in regulating antitumor immunity, we transferred JAX or TAC fecal suspensions into TAC and JAX recipients by oral gavage before tumor implan￾tation (fig. S1A). We found that prophylactic trans￾fer of JAX fecal material, but not saline or TAC fecal material, into TAC recipients was sufficient to delay tumor growth (Fig. 2A) and to enhance induction and infiltration of tumor-specific CD8+ T cells (Fig. 2, B and C, and fig. S1B), which sup￾ported a microbe-derived effect. Reciprocal trans￾fer of TAC fecal material into JAX recipients had a minimal effect on tumor growth rate and anti￾tumor T cell responses (Fig. 2, A to C, and fig. S1B), consistent with the JAX-dominant effects observed upon cohousing. To test whether manipulation of the microbial community could be effective as a therapy, we ad￾ministered JAX fecal material alone or in combi￾nation with antibodies targeting PD-L1 (aPD-L1) to TAC mice bearing established tumors. Trans￾fer of JAX fecal material alone resulted in signif￾icantly slower tumor growth (Fig. 2D), accompanied by increased tumor-specific T cell responses (Fig. 2E) and infiltration of antigen-specific T cells into the tumor (Fig. 2F), to the same degree as treatment with systemic aPD-L1 mAb. Combina￾tion treatment with both JAX fecal transfer and aPD-L1 mAb improved tumor control (Fig. 2D) and circulating tumor antigen–specific T cell responses (Fig. 2E), although there was little additive effect on accumulation of activated T cells within the tumor microenvironment (Fig. 2F). Consistent with these results, aPD-L1 therapy alone was signifi￾cantly more efficacious in JAX mice compared with TACmice (Fig. 2G), which paralleled improved antitumor T cell responses (fig. S1C). These data indicate that the commensal microbial compo￾sition can influence spontaneous antitumor im￾munity, as well as a response to immunotherapy with aPD-L1 mAb. To identify specific bacteria associated with im￾proved antitumor immune responses, we moni￾tored the fecal bacterial content over time of mice that were subjected to administration of fecal permutations, using the 16S ribosomal RNA (rRNA) miSeq Illumina platform. Principal coordinate analysis revealed that fecal samples analyzed from TAC mice that received JAX fecal material grad￾ually separated from samples obtained from sham￾and TAC feces–inoculated TAC mice over time (P = 0.001 and P = 0.003, respectively, ANOSIM multivariate data analysis) and became similar 1084 27 NOVEMBER 2015 • VOL 350 ISSUE 6264 sciencemag.org SCIENCE 1 Department of Pathology, University of Chicago, Chicago, IL 60637, USA. 2 Department of Medicine, University of Chicago, Chicago, IL 60637, USA. 3 Section of Genetic Medicine, University of Chicago, Chicago, IL 60637, USA. *These authors contributed equally to this work. †Corresponding author. E-mail: tgajewsk@medicine.bsd.uchicago.edu RESEARCH | REPORTS on June 24, 2016 http://science.sciencemag.org/ Downloaded from

Anticancer immunotherapy by CtLA-4 blockade relies on the Science gut microbiota Marie vetizou, Jonathan M. Pitt, Romain Daillere, Patricia Lepage Nadine Waldschmitt, Caroline Flament, Sylvie Rusakiewicz, NAAAS Bertrand Routy, Maria P Roberti, Connie P. M. Duong, Vichnou Poirier-Colame. Antoine Roux. Sonia Becharef. Silvia Formenti Encouse Golden, Sascha Cording, Gerard Eberl, Andreas Schlitzer Florent Ginhoux. Sridhar Mani. Takahiro Y amazaki. Nicolas Jacquelot, David P Enot, Marion Berard, Jerome Nigou, Paule Opolon, Alexander Eggermont, Paul-Louis Woerther, Elisabeth hachaty, Nathalie Chaput, Caroline Robert, Christina Mateus undo Kroemer, Didier Raoult, Ivo Gomperts Boneca, Franck Carbonnel, Mathias Chamaillard and laurence Zitvogel(November Science350(6264),1079-1084.[doi:10.1126/ science aad l329 originally published online November 5, 2015 Editor's Summary Gut microbes affect immunotherapy The unleashing of antitumor T cell responses has d in a new era of cancer treatment Although these therapies can cause dramatic tumor regre In some patients, many patients lexplicably see no benefit. Mice have been used to investigate what might be happening Perspective by Snyder et al. ) vetizou et al. found that optimal responses to anticytotoxic T lymphocyte ntigen blockade required specific Bacteroides spp. Similarly, Sivan et al. discovered that Bifidobacterium spp. enhanced the efficacy of antiprogrammed cell death ligand I therapy Science, this issue, p. 1079 and p. 1084; see also p. 1031 09月55885 This copy is for your personal, non-commercial use only =88 Article Tools Visit the online version of this article to access the personalization and article tools http://science.sciencemag.org/content/350/6264/1079 Permissions Obtain information about reproducing this article http://www.sciencemag.org/about/permissions.dtl Science(print IssN 0036-8075 online Issn 1095-9203)is published weekly, except the last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue Nw, Washington, DC 20005. Copyright 2016 by the American Association for the Advancement of Science; all rights reserved. The title Science is a registered trademark of AAAs

originally published online November 5, 2015 Science 350 (6264), 1079-1084. [doi: 10.1126/science.aad1329] 5, 2015) Carbonnel, Mathias Chamaillard and Laurence Zitvogel (November Guido Kroemer, Didier Raoult, Ivo Gomperts Boneca, Franck Chachaty, Nathalie Chaput, Caroline Robert, Christina Mateus, Opolon, Alexander Eggermont, Paul-Louis Woerther, Elisabeth Jacquelot, David P. Enot, Marion Bérard, Jérôme Nigou, Paule Florent Ginhoux, Sridhar Mani, Takahiro Yamazaki, Nicolas Encouse Golden, Sascha Cording, Gerard Eberl, Andreas Schlitzer, Poirier-Colame, Antoine Roux, Sonia Becharef, Silvia Formenti, Bertrand Routy, Maria P. Roberti, Connie P. M. Duong, Vichnou Nadine Waldschmitt, Caroline Flament, Sylvie Rusakiewicz, Marie Vétizou, Jonathan M. Pitt, Romain Daillère, Patricia Lepage, gut microbiota Anticancer immunotherapy by CTLA-4 blockade relies on the Editor's Summary Science, this issue, p. 1079 and p. 1084; see also p. 1031 Bifidobacterium spp. enhanced the efficacy of antiprogrammed cell death ligand 1 therapy. antigen blockade required specific Bacteroides spp. Similarly, Sivan et al. discovered that Perspective by Snyder et al.). Vétizou et al. found that optimal responses to anticytotoxic T lymphocyte Specific members of the gut microbiota influence the efficacy of this type of immunotherapy (see the inexplicably see no benefit. Mice have been used in two studies to investigate what might be happening. Although these therapies can cause dramatic tumor regressions in some patients, many patients The unleashing of antitumor T cell responses has ushered in a new era of cancer treatment. Gut microbes affect immunotherapy This copy is for your personal, non-commercial use only. Article Tools http://science.sciencemag.org/content/350/6264/1079 article tools: Visit the online version of this article to access the personalization and Permissions http://www.sciencemag.org/about/permissions.dtl Obtain information about reproducing this article: Advancement of Science; all rights reserved. The title Science is a registered trademark of AAAS. Avenue NW, Washington, DC 20005. Copyright 2016 by the American Association for the in December, by the American Association for the Advancement of Science, 1200 New York Science (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week on June 24, 2016 http://science.sciencemag.org/ Downloaded from

Science www.sciencemag.org/cgi/content/full/science.aad1329/dci NAAAS Supplementary Materials for Anticancer immunotherapy by Ctla-4 blockade relies on the gut microbiota Marie Vetizou, Jonathan M. Pitt, Romain Daillere, Patricia Lepage, Nadine Waldschmitt Caroline Flament, Sylvie Rusakiewicz, Bertrand Routy, Maria P. Roberti, Connie P. M Duong, Vichnou Poirier-Colame, Antoine Roux, Sonia Becharef, Silvia Formenti, Encouse Golden, Sascha Cording, Gerard Eberl, Andreas Schlitzer, Florent Ginhoux Sridhar Mani, Takahiro Yamazaki, Nicolas Jacquelot, David P. Enot, Marion Berard Jerome Nigou, Paule Opolon, Alexander Eggermont, Paul-Louis Woerther, Elisabeth Chachaty, Nathalie Chaput, Caroline Robert, Christina Mateus, Guido Kroemer, Didier Raoult, Ivo Gomperts Boneca, Franck Carbonnel, Mathias Chamaillard Laurence zitvogel *Corresponding author. E-mail: laurence. zitvogel@gustaveroussyfr Published 5 November 2015 on Science Express DOI: 10.1 126/science. aad 1329 This pdf file includes Materials and method nces

www.sciencemag.org/cgi/content/full/science.aad1329/DC1 Supplementary Materials for Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota Marie Vétizou, Jonathan M. Pitt, Romain Daillère, Patricia Lepage, Nadine Waldschmitt, Caroline Flament, Sylvie Rusakiewicz, Bertrand Routy, Maria P. Roberti, Connie P. M. Duong, Vichnou Poirier-Colame, Antoine Roux, Sonia Becharef, Silvia Formenti, Encouse Golden, Sascha Cording, Gerard Eberl, Andreas Schlitzer, Florent Ginhoux, Sridhar Mani, Takahiro Yamazaki, Nicolas Jacquelot, David P. Enot, Marion Bérard, Jérôme Nigou, Paule Opolon, Alexander Eggermont, Paul-Louis Woerther, Elisabeth Chachaty, Nathalie Chaput, Caroline Robert, Christina Mateus, Guido Kroemer, Didier Raoult, Ivo Gomperts Boneca, Franck Carbonnel, Mathias Chamaillard, Laurence Zitvogel* *Corresponding author. E-mail: laurence.zitvogel@gustaveroussy.fr Published 5 November 2015 on Science Express DOI: 10.1126/science.aad1329 This PDF file includes Materials and Methods Figs. S1 to S22 Tables S1 to S5 References

Supplemental materials vetizou et al One Sentence Summary: Bacteroides involved in anti-CTLA4 Ab-mediated cancer Abbreviations list: ACS: antibiotic treatment with ampicillin, colistin and streptomycin, Bc: Burkholderia cepacia, Bf Bacteroides fragilis, BM-DC: Bone marrow-derived dendritic cells, CTLA4: Cytotoxic T-Lymphocyte Antigen-4, DC: Dendritic cells, EMA: European Medicine Agency, FDA: Food and drug administration, FItC: fluorescein isothiocyanate, FMT: fecal microbial transplantation, GF: Germ-free, GM-CSF: Granulocyte-macrophage colony-stimulating factor, HV: Healthy volunteers, IBD: Inflammatory bowel diseases, ICB Immune checkpoint blocker, ICOS: Inducible T-cell costimulatory, IEC: intestinal epithelial cells, IEL: intraepithelial lymphocytes, IL-12: Interleukin-12, LP: Lamina propria, mAb Monoclonal antibody, MHC II: class II molecules, mLN: Mesenteric lymph node, MM Metastatic melanoma, MOI: Multiplicity of infection, NOD2: Nucleotide-binding oligomerization domain-containing protein 2, PCA: Principle component analysis, PD1 Programmed cell death protein 1, PS: Polysaccharide, PBMC: peripheral blood mononuclear cells, SPF: Specific pathogen free, Tcl: Type 1 cytotoxic T-cells, Th1: T helper type 1, TLR: Toll like receptor, Trl: Type 1 regulatory T-cells, Tregs: Regulatory T cells, WT: Wild type Key words: CTLA4, ipilimumab, cancer, immunity, Bacteroides fragilis, Bacteroides thetaiotaomicron, Burkholderia cepacia, microbiome, IL-12 Acknowledgments: We are grateful to the staff of the animal facility of Gustave roussy and Institut Pasteur. We thank P Gonin, B. Ryffel, T. Angelique, N. Chanthapathet, H. Li and S. Zuberogoitia for technical help. The data presented in this manuscript are tabulated in the main paper and in the supplementary materials. DNA sequence reads from this study have been submitted to the NCBI under the Bioproject ID PRJNA299112. LZ, MV and PL have filed patent applications n EP 14190167 that relates to specific topic: Methods and products for modulating microbiota composition for improving the efficacy of a cancer treatment with an immune checkpoint blocker. MV and JMP were supported by La Ligue contre le cancer and ARC respectively. Lz received a special prize from the Swiss bridge Foundation and ISREC. GK and Lz were supported by the ligue Nationale contre le cancer (Equipes labellisees), Agence Nationale pour la Recherche (ANR AUTOPH, ANR Emergence), European Commission (ArtForce), European Research Council Advanced Investigator Grant(to GK), Fondation pour la Recherche Medicale(FRM), Institut National du Cancer (INCa), Fondation de france, Canceropole Ile-de-France, Fondation Bettencourt- Schueller, Swiss Bridge Foundation, the LabEx Immuno-Oncology, the 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. SM was supported by nih (ro1 CA161879 PI: SM). MC was supported by the Fondation la Recherche Medical the Fondation ARC pour la recherche sur le cancer and Institut Nationale du

’’Ž‡‡ƒŽƒ‡‹ƒŽ•±‹œ‘‡ƒŽǤ One Sentence Summary: Bacteroides involved in anti-CTLA4 Ab-mediated cancer immunosurveillance. „„‡‹ƒ‹‘• Ž‹•ǣǣ ƒ‹„‹‘‹… ‡ƒ‡ ™‹Š ƒ’‹…‹ŽŽ‹ǡ …‘Ž‹•‹ ƒ† •‡’‘›…‹ǡ …ǣ Š‘Ž†‡‹ƒ…‡’ƒ…‹ƒǡ ˆǣƒ…‡‘‹†‡•ˆƒ‰‹Ž‹•ǡ Ǧǣ ‘‡ ƒ‘™Ǧ†‡‹‡† †‡†‹‹… …‡ŽŽ•ǡ Ͷǣ ›‘‘š‹… Ǧ›’Š‘…›‡ ‹‰‡ǦͶǡ ǣ ‡†‹‹… …‡ŽŽ•ǡ ǣ ‘’‡ƒ ‡†‹…‹‡ ‰‡…›ǡ ǣ ‘‘† ƒ† †‰ ƒ†‹‹•ƒ‹‘ǡ ǣ ˆŽ‘‡•…‡‹ ‹•‘Š‹‘…›ƒƒ‡ǡ ǣ ˆ‡…ƒŽ ‹…‘„‹ƒŽ ƒ•’Žƒƒ‹‘ǡ ǣ ‡Ǧˆ‡‡ǡ Ǧ ǣ ƒŽ‘…›‡Ǧƒ…‘’Šƒ‰‡ …‘Ž‘›Ǧ•‹Žƒ‹‰ ˆƒ…‘ǡǣ‡ƒŽŠ›‘Ž‡‡•ǡ ǣˆŽƒƒ‘›„‘™‡Ž†‹•‡ƒ•‡•ǡǣ ‡…Š‡…’‘‹„Ž‘…‡ǡǣ†…‹„Ž‡Ǧ…‡ŽŽ…‘•‹Žƒ‘›ǡ ǣ‹‡•‹ƒŽ‡’‹Š‡Ž‹ƒŽ …‡ŽŽ•ǡ ǣ ‹ƒ‡’‹Š‡Ž‹ƒŽ Ž›’Š‘…›‡•ǡ Ǧͳʹǣ ‡Ž‡‹Ǧͳʹǡ ǣ ƒ‹ƒ ’‘’‹ƒǡ „ǣ ‘‘…Ž‘ƒŽ ƒ‹„‘†›ǡ  ǣ …Žƒ••  ‘Ž‡…Ž‡•ǡ ǣ ‡•‡‡‹… Ž›’Š ‘†‡ǡ ǣ ‡ƒ•ƒ‹… ‡Žƒ‘ƒǡ ǣ Ž‹’Ž‹…‹› ‘ˆ ‹ˆ‡…‹‘ǡ ʹǣ …Ž‡‘‹†‡Ǧ„‹†‹‰ ‘Ž‹‰‘‡‹œƒ‹‘ †‘ƒ‹Ǧ…‘ƒ‹‹‰ ’‘‡‹ ʹǡ ǣ ‹…‹’Ž‡ …‘’‘‡ ƒƒŽ›•‹•ǡ ͳǣ ‘‰ƒ‡† …‡ŽŽ †‡ƒŠ ’‘‡‹ ͳǡ ǣ ‘Ž›•ƒ……Šƒ‹†‡ǡ ǣ ’‡‹’Š‡ƒŽ „Ž‘‘† ‘‘…Ž‡ƒ…‡ŽŽ•ǡ ǣ’‡…‹ˆ‹…’ƒŠ‘‰‡ˆ‡‡ǡ…ͳǣ›’‡ͳ…›‘‘š‹…Ǧ…‡ŽŽ•ǡŠͳǣŠ‡Ž’‡ ›’‡ ͳ ǡ ǣ ‘ŽŽŽ‹‡ ‡…‡’‘ǡ ͳǣ ›’‡ ͳ ‡‰Žƒ‘› Ǧ…‡ŽŽ•ǡ ‡‰•ǣ‡‰Žƒ‘›  …‡ŽŽ•ǡ ǣ‹Ž†›’‡Ǥ Key words: CTLA4, ipilimumab, cancer, immunity, Bacteroides fragilis, Bacteroides thetaiotaomicron, Burkholderia cepacia, microbiome, IL-12. …‘™Ž‡†‰‡•ǣ‡ ƒ‡ ‰ƒ‡ˆŽ ‘ Š‡ •ƒˆˆ ‘ˆ Š‡ ƒ‹ƒŽ ˆƒ…‹Ž‹› ‘ˆ •ƒ‡ ‘••› ƒ†•‹ƒ•‡Ǥ‡ŠƒǤ ‘‹ǡǤ›ˆˆ‡ŽǡǤ‰‡Ž‹‡ǡǤŠƒŠƒ’ƒŠ‡ǡǤ‹ƒ† Ǥ„‡‘‰‘‹‹ƒ ˆ‘ ‡…Š‹…ƒŽŠ‡Ž’ǤŠ‡†ƒƒ’‡•‡‡†‹ Š‹•ƒ•…‹’ƒ‡ ƒ„Žƒ‡†‹ Š‡ƒ‹’ƒ’‡ƒ†‹ Š‡•’’Ž‡‡ƒ›ƒ‡‹ƒŽ•Ǥ•‡‡…‡‡ƒ†• ˆ‘ Š‹••†› Šƒ‡ „‡‡ •„‹‡† ‘ Š‡  †‡ Š‡ ‹‘’‘Œ‡…   ʹͻͻͳͳʹǤǡ  ƒ†  Šƒ‡ ˆ‹Ž‡† ’ƒ‡ ƒ’’Ž‹…ƒ‹‘• ιͳͶͳͻͲͳ͸͹Šƒ ‡Žƒ‡• ‘ •’‡…‹ˆ‹… ‘’‹…ǣ‡Š‘†•ƒ† ’‘†…• ˆ‘ ‘†Žƒ‹‰ ‹…‘„‹‘ƒ …‘’‘•‹‹‘ ˆ‘ ‹’‘‹‰ Š‡ ‡ˆˆ‹…ƒ…› ‘ˆ ƒ …ƒ…‡ ‡ƒ‡™‹Šƒ‹‡…Š‡…’‘‹ „Ž‘…‡Ǥƒ† ™‡‡ •’’‘‡† „› ƒ ‹‰‡ …‘‡Ž‡ …ƒ…‡ ƒ†  ‡•’‡…‹‡Ž›Ǥ  ‡…‡‹‡† ƒ •’‡…‹ƒŽ ’‹œ‡ ˆ‘ Š‡ ™‹••‹†‰‡ ‘†ƒ‹‘ƒ†Ǥ ƒ†™‡‡•’’‘‡†„›Š‡‹‰‡ƒ‹‘ƒŽ‡…‘‡Ž‡ƒ…‡ ȋ‹’‡• Žƒ„‡ŽŽ‹•±‡•Ȍǡ ‰‡…‡ ƒ‹‘ƒŽ‡ ’‘ Žƒ ‡…Š‡…Š‡ ȋ ǡ  ‡‰‡…‡Ȍǡ ‘’‡ƒ ‘‹••‹‘ ȋ ‘…‡Ȍǡ ‘’‡ƒ ‡•‡ƒ…Š ‘…‹Ž †ƒ…‡† ‡•‹‰ƒ‘ ƒȋ‘ Ȍǡ ‘†ƒ‹‘’‘Žƒ‡…Š‡…Š‡±†‹…ƒŽ‡ȋ Ȍǡ•‹ƒ‹‘ƒŽ †ƒ…‡ȋƒȌǡ ‘†ƒ‹‘†‡ ƒ…‡ǡƒ…±‘’ØŽ‡Ž‡Ǧ†‡Ǧ ƒ…‡ǡ ‘†ƒ‹‘‡‡…‘Ǧ …Š‡ŽŽ‡ǡ ™‹•• ‹†‰‡ ‘†ƒ‹‘ǡ Š‡ ƒ„š ‘Ǧ…‘Ž‘‰›ǡ Š‡  ƒ‹ˆ‹‡† …‘Ž‘‰› ‡ŽŽ‡’ƒ‹ ƒ† ‘ ‡ Ž‹‹ƒ‹‘ ȋȌǢ Š‡ ƒ…‡ ‡•‡ƒ…Šƒ†‡•‘ƒŽ‹œ‡†‡†‹…‹‡ȋȌǡƒ†Š‡ƒ‹•ŽŽ‹ƒ…‡‘ˆƒ…‡‡•‡ƒ…Š •‹‡•ȋȌǤ™ƒ••’’‘‡†„›ȋͲͳͳ͸ͳͺ͹ͻǣȌǤ™ƒ••’’‘‡†„› Š‡ ‘†ƒ‹‘ ’‘ Žƒ ‡…Š‡…Š‡ ±†‹…ƒŽ‡ǡ Š‡ ‘†ƒ‹‘  ’‘ Žƒ ‡…Š‡…Š‡ • Ž‡ …ƒ…‡ ƒ† •‹ ƒ‹‘ƒŽ‡ †

Cancer. NW is a recipient of a Post-doctoral fellowship from the Agence Nationale de la Recherche. AS was supported by BMSI YIG 2014. FG is supported by SIgN core funding. LZ, IC, IGB are all sponsored by Association pour la Recherche contre le Cancer (PGA120140200851). FC was supported by INCA-DGOS (GOLD H78008.Nc was supported by INCA-DGOS (GOLD study; 2012-1-RT-14-IGR-01) LOreal awarded a prize to MV. We are grateful to the staff of the animal facility of Gustave Roussy and Institut Pasteur. We thank P. Gonin, B. Ryffel, T. Angelique, N. Chanthapathet, H. Li and S. Zuberogoitia for technical help LZ, MV and PL have filed patent applications n EP 14190167 that relates to specific topic: Methods and products for modulating microbiota composition for improving the efficacy of a cancer treatment with an immune checkpoint blocker Authors'contribution: MV performed experiments and analyzed results represented in Fig 2C-D, 3A left panel, 4A-B and Supplemental Fig 1B, 5CDE, 7, 8A,9, 10, 12, 13C-E-F-G, 14B 15, 16C, 17C, 21A-B, 22 and Supplemental Table 2(alone or helped by R+ TY+ NJ+ SB+ MPR+ BR), JMP(helped by PO and VPC) assessed gut pathology and generated Fig 3A middle and right panels as well as Supplemental Fig. 2, 5A-B, 13D, 17, 18, 19 and 21C, CF+ SR+ Mv did the experiments for Fig. 3D-E and Supplemental Fig. 11, SF(and eg) provided the patients specimen for the analyses of Fig. 3D-E and Supplemental Fig. 11, MC and Nw performed the FISH analyses, Ki67, cCasp3 and MUC2 staining and qPCR experiments represented in Fig 2A and Supplemental Fig. 4, 6, 8B-C. PL analyzed the 16s rrNa gene sequencing of mouse and human stools and described the PCA(Fig. 2C, Supplemental Fig 21A and Supplemental Table 1). SC and ge provided the tools and mice to analyze CtlA4 expression on gut T cells and ILCS represented in Supplemental Fig. 16A-B. AS and FG performed the flow cytometry analyses on LP DC subsets in Supplemental Fig. 13A-B. SM performed FITC dextran experiments depicted in Supplemental Fig 3. CD, VPC and AR cultured the enteroids and studied the IEC-IEL cross talk represented in Fig. 2B. MPR and Br(helped by SB) performed experiments depicted in Fig. 4C and Supplemental Fig IA. MPR and SB executed and analyzed results of QPCR from FMT experiments represented in Fig. 4D-E and Supplemental Fig 20 B- C. BR generated Supplemental Table 3 and 4 of patient characteristics. DR, MC, MB and IGB provided il-10, nod2, il-10/nod2, tlr2 deficient mice as well as germ-free mice and bacterial species of interest(B, BL, E. coli, E. faecalis L. plantarum for IGB and Bc for DR). PLW and EC characterized cultivable bacteria and performed mass spectrometry on bacterial species or isolates JN purified PS and bacterial capsule materials. LZ, MV and JMP wrote the manuscript CR, NC, CM and FC provided melanoma patients feces for FMT. GK and AE edited and critically reviewed the manuscript. LZ conceived the project and the experimental settings Materials Methods Patient and cohort characteristics. All clinical studies were conducted after informed consent of the patients, following the guidelines of the Declaration of Helsinki. Patient characteristics are detailed in Supplemental tables 3 and 4. Peripheral blood mononuclear cells(PBMC)were provided by Gustave Roussy Cancer Campus(Villejuif, France) and by the Department of Radiation Oncology(New York University [NYU], New York, NY, USA). Patients were

2 ƒ…‡Ǥ  ‹• ƒ ‡…‹’‹‡ ‘ˆ ƒ ‘•Ǧ†‘…‘ƒŽ ˆ‡ŽŽ‘™•Š‹’ ˆ‘ Š‡ ‰‡…‡ ƒ‹‘ƒŽ‡ †‡ Žƒ ‡…Š‡…Š‡Ǥ™ƒ••’’‘‡†„› ʹͲͳͶǤ ‹••’’‘‡†„›‰…‘‡ˆ†‹‰Ǥǡ ǡ   ƒ‡ ƒŽŽ •’‘•‘‡† „› ••‘…‹ƒ‹‘ ’‘ Žƒ ‡…Š‡…Š‡ …‘‡ Ž‡ ƒ…‡ ȋ ͳʹͲͳͶͲʹͲͲͺͷͳȌǤ  ™ƒ• •’’‘‡† „› Ǧ  ȋ  ͹ͺͲͲͺȌǤ ™ƒ• •’’‘‡†„›Ǧ ȋ •†›ǢʹͲͳʹǦͳǦǦͳͶǦ ǦͲͳȌǤǯ‡ƒŽƒ™ƒ†‡†ƒ’‹œ‡‘ Ǥ‡ƒ‡‰ƒ‡ˆŽ‘Š‡•ƒˆˆ‘ˆŠ‡ƒ‹ƒŽˆƒ…‹Ž‹›‘ˆ •ƒ‡‘••›ƒ†•‹ƒ•‡Ǥ ‡ ŠƒǤ ‘‹ǡǤ›ˆˆ‡ŽǡǤ‰‡Ž‹‡ǡǤŠƒŠƒ’ƒŠ‡ǡǤ‹ƒ†Ǥ„‡‘‰‘‹‹ƒ ˆ‘ ‡…Š‹…ƒŽŠ‡Ž’Ǥǡƒ†Šƒ‡ˆ‹Ž‡†’ƒ‡ƒ’’Ž‹…ƒ‹‘•ιͳͶͳͻͲͳ͸͹Šƒ‡Žƒ‡•‘ •’‡…‹ˆ‹…‘’‹…ǣ‡Š‘†•ƒ†’‘†…•ˆ‘‘†Žƒ‹‰‹…‘„‹‘ƒ…‘’‘•‹‹‘ˆ‘‹’‘‹‰ Š‡‡ˆˆ‹…ƒ…›‘ˆƒ…ƒ…‡‡ƒ‡™‹Šƒ‹‡…Š‡…’‘‹„Ž‘…‡Ǥ Authors’ contribution: MV performed experiments and analyzed results represented in Fig. 1, 2C-D, 3A left panel, 4A-B and Supplemental Fig. 1B, 5CDE, 7, 8A, 9, 10, 12, 13C-E-F-G, 14B, 15, 16C, 17C, 21A-B, 22 and Supplemental Table 2 (alone or helped by RD + TY + NJ + SB + MPR + BR), JMP (helped by PO and VPC) assessed gut pathology and generated Fig 3A middle and right panels as well as Supplemental Fig. 2, 5A-B, 13D, 17, 18, 19 and 21C, CF + SR + MV did the experiments for Fig. 3D-E and Supplemental Fig. 11, SF (and EG) provided the patients specimen for the analyses of Fig. 3D-E and Supplemental Fig. 11, MC and NW performed the FISH analyses, Ki67, cCasp3 and MUC2 staining and qPCR experiments represented in Fig. 2A and Supplemental Fig. 4, 6, 8B-C. PL analyzed the 16S rRNA gene sequencing of mouse and human stools and described the PCA (Fig. 2C, Supplemental Fig.21A and Supplemental Table 1). SC and GE provided the tools and mice to analyze CTLA4 expression on gut T cells and ILCs represented in Supplemental Fig. 16A-B. AS and FG performed the flow cytometry analyses on LP DC subsets in Supplemental Fig. 13A-B. SM performed FITC dextran experiments depicted in Supplemental Fig. 3. CD, VPC and AR cultured the enteroids and studied the IEC-IEL cross talk represented in Fig. 2B. MPR and BR (helped by SB) performed experiments depicted in Fig. 4C and Supplemental Fig. 1A. MPR and SB executed and analyzed results of QPCR from FMT experiments represented in Fig. 4D-E and Supplemental Fig. 20 B￾C. BR generated Supplemental Table 3 and 4 of patient characteristics. DR, MC, MB and IGB provided il-10, nod2, il-10/nod2, tlr2 deficient mice as well as germ-free mice and bacterial species of interest (Bf, Bt, E. coli, E. faecalis L. plantarum for IGB and Bc for DR). PLW and EC characterized cultivable bacteria and performed mass spectrometry on bacterial species or isolates. JN purified PS and bacterial capsule materials. LZ, MV and JMP wrote the manuscript. CR, NC, CM and FC provided melanoma patients feces for FMT. GK and AE edited and critically reviewed the manuscript. LZ conceived the project and the experimental settings. Materials & Methods Patient and cohort characteristics. All clinical studies were conducted after informed consent of the patients, following the guidelines of the Declaration of Helsinki. Patient characteristics are detailed in Supplemental tables 3 and 4. Peripheral blood mononuclear cells (PBMC) were provided by Gustave Roussy Cancer Campus (Villejuif, France) and by the Department of Radiation Oncology (New York University [NYU], New York, NY, USA). Patients were