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 1079Interspecific 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). 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