Reports

Ipilimumab is a fully human monoclonal antibody directed against CTLA-4, a major negative regulator of T cell activa-

tion (1), approved in 2011 for improving the overall survival of patients with metastatic mela-noma (MM) (2). However, block-ade of CTLA-4 by ipilimumab often results in immune-related adverse events at sites that are exposed to commensal microor-ganisms, mostly the gut (3). Pa-tients treated with ipilimumab develop antibodies to compo-nents of the enteric flora (4). Therefore, given our previous findings for other cancer thera-pies (5), addressing the role of gut microbiota in the immuno-modulatory effects of CTLA-4 blockade is crucial for the future development of immune check-point blockers in oncology.

We compared the relative therapeutic efficacy of the anti–CTLA-4 9D9 Ab against estab-lished MCA205 sarcomas in mice housed in specific pathogen-free (SPF) versus germ-free (GF) conditions. Anti–CTLA-4 con-trolled tumor progression in SPF but not in GF mice (Fig. 1, A and B). Moreover, a combination of broad-spectrum antibiotics (am-picillin + colistin + streptomycin, ACS) (Fig. 1C), as well as imipenem alone, but not colistin (Fig. 1C), compromised the anti-tumor effects of anti–CTLA-4. These results, which suggest that the gut microbiota is required for the anticancer effects of CTLA-4 blockade, were con-firmed in the Ret melanoma and the MC38 colon cancer models (fig. S1, A and B). In addition, in GF or ACS-treated mice, the anti–CTLA-4–induced activation of splenic effector CD4+ T cells and TILs was significantly decreased (Fig. 1, D and E, and fig. S1, C to E).

We next addressed the im-pact of the gut microbiota on the incidence and severity of intesti-nal lesions induced by anti–

CTLA-4 treatment. A ‘subclinical colitis’ dependent on the gut microbiota was observed at late time points (figs. S2 to

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† 1Institut de Cancérologie Gustave Roussy Cancer Campus (GRCC), 114 rue Edouard Vaillant, 94805 Villejuif, France. 2INSERM U1015, GRCC, Villejuif, France. 3University of Paris Sud XI, Kremlin-Bicêtre, France. 4Institut National de la Recherche Agronomique (INRA), Micalis-UMR1319, 78360 Jouy-en-Josas, France. 5University 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. 6Center of Clinical Investigations in Biotherapies of Cancer 1428, Villejuif, France. 7Université Paris Descartes, Sorbonne Paris Cité, Paris, France. 8Department of Radiation Oncology, New York University, New York, NY, USA. 9Microenvironment 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

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, or 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 anti–CTLA-4 treatment of melanoma patients 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.

/ www.sciencemag.org/content/early/recent / 5 November 2015 / Page 1 / 10.1126/science.aad1329

on

Nov

embe

r 20

, 201

5w

ww

.sci

ence

mag

.org

Dow

nloa

ded

from

o

n N

ovem

ber

20, 2

015

ww

w.s

cien

cem

ag.o

rgD

ownl

oade

d fr

om

on

Nov

embe

r 20

, 201

5w

ww

.sci

ence

mag

.org

Dow

nloa

ded

from

o

n N

ovem

ber

20, 2

015

ww

w.s

cien

cem

ag.o

rgD

ownl

oade

d fr

om

on

Nov

embe

r 20

, 201

5w

ww

.sci

ence

mag

.org

Dow

nloa

ded

from

o

n N

ovem

ber

20, 2

015

ww

w.s

cien

cem

ag.o

rgD

ownl

oade

d fr

om

on

Nov

embe

r 20

, 201

5w

ww

.sci

ence

mag

.org

Dow

nloa

ded

from

o

n N

ovem

ber

20, 2

015

ww

w.s

cien

cem

ag.o

rgD

ownl

oade

d fr

om

S5). However, shortly (by 24hrs) after the first administra-tion of anti–CTLA-4, we observed increased cell death and proliferation of intestinal epithelial cells (IEC) residing in the ileum and colon, as shown by immunohistochemistry using anti–cleaved caspase-3 and Ki67 Ab respectively (Fig. 2A and fig. S6A). The anti–CTLA-4–induced IEC prolifera-tion was absent in RegIIIβ−deficient mice (fig. S6A). Con-comitantly, the transcription levels of Il17a, Ifng, Ido1, type 1 Ifn-related gene products and Ctla4 (but not Il6), indicating ongoing inflammatory processes, significantly increased by 24hrs in the distal ileum of anti–CTLA-4–treated mice (fig. S6, B-D). Depletion of T cells including intraepithelial lym-phocytes (IEL) (by injection of antibodies specific for CD4 and CD8) abolished the induction of IEC apoptosis by anti–CTLA-4 (Fig. 2A). When crypt-derived 3-D small intestinal enteroids (6) were exposed to Toll like receptor (TLR) ago-nists (which act as microbial ligands in this assay) and sub-sequently admixed with IEL harvested from anti–CTLA-4 (but not isotype Ctl)-treated mice, IEC within the enteroids underwent apoptosis (Fig. 2B). Hence, anti–CTLA-4 com-promises the homeostatic IEC-IEL equilibrium, favoring the apoptotic demise of IEC in the presence of microbial prod-ucts.

To explore whether this T cell-dependent IEC death could induce perturbations of the microbiota composition, we performed high throughput pyrosequencing of 16S ribo-somal RNA gene amplicons of feces. The principal compo-nent analysis indicated that a single injection of anti–CTLA-4 sufficed to significantly affect the microbiome at the genus level (Fig. 2C). CTLA-4 blockade induced a rapid un-derrepresentation of both Bacteroidales and Burkholderiales with a relative increase of Clostridiales in feces (Fig. 2C and table S1). Q-PCR analyses targeting the Bacteroides genus and species (spp) in small intestine mucosa and feces con-tents showed a trend toward a decreased relative abundance of such bacteria in the feces contrasting with a relative en-richment in particular spp. (such as B. thetaiotaomicron (Bt) and B. uniformis) in the small intestine mucosa 24-48hrs following one anti–CTLA-4 Ab injection (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 anti–CTLA-4 Ab (fig. S7).

Next, to establish a cause-effect relationship between the dominance of distinct Bacteroides spp. in the small intestine and the anticancer efficacy of CTLA-4 blockade, we recolo-nized ACS-treated and GF mice with several bacterial spp. associated with anti–CTLA-4–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 anti–CTLA-4, con-trasting with all the other isolates that failed to do so (table S2, 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 (DC), culminating in the restoration of the therapeutic response of GF tumor bearers to anti–CTLA-4 (Fig. 3B and fig. S9, A and B).

We analyzed the dynamics of memory T cell responses directed against distinct bacterial spp. in mice and humans during CTLA-4 blockade. CD4+ T cells harvested from spleens of anti–CTLA-4–treated mice (Fig. 3C) or from blood taken from individuals with metastatic melanoma or non-small cell lung carcinoma (NSCLC) patients after 2 admin-istrations 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 anti-cancer activity of anti–CTLA-4 was further demonstrated by the adoptive transfer of memory Bf- (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 immunostimulatory effects induced by CTLA-4 blockade depended on the mobilization of lamina propria CD11b+ DC that can process zwitterionic polysaccharides (9) and then mount interleukin (IL)-12-dependent cognate Th1 immune responses against Bf capsu-lar polysaccharides (figs. S13 to 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 findings, we analyzed the composition of the gut microbiome before and after treatment with ipilimumab in 25 individuals with met-astatic melanoma (table S4). A clustering algorithm based on genus composition of the stools (12, 13) distinguished three clusters (Fig. 4A and table S5) with Alloprevotel-la/Prevotella driving cluster A and distinct Bacteroides spp. driving clusters B and C (Fig. 4B). During ipilimumab ther-apy, the proportions of metastatic melanoma patients fall-ing into cluster C increased, at the expense of those belonging to cluster B (Fig. 4B and fig. S20A). We next per-formed fecal microbial transplantation of feces harvested from different metastatic melanoma patients from each cluster, two weeks before tumor inoculation into GF mice that were subsequently treated with anti–CTLA-4 Ab. Tu-mors 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). Quantita-tive PCR analyses revealed 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 colonization of the immunogenic bacteria Bf and Bt (7–10, 14, 15) (Fig. 4D). Moreover, after anti–CTLA-4 therapy, only cluster C (not A or B) recipient mice had outgrowth of Bf (fig. S20B). Importantly, the fecal abundance of Bf (but not B. distasonis nor B. uniformis) negatively correlated with tumor size post-CTLA-4 blockade in cluster C recipient mice (Fig. 4E and fig. S20C). Hence,

/ sciencemag.org/content/early/recent / 5 November 2015 / Page 2 / 10.1126/science.aad1329

ipilimumab can modify the abundance of immunogenic Bacteroides spp. in the gut, which in turn impacts its anti-cancer efficacy.

Finally, intestinal reconstitution of ACS-treated animals with the combination of Bf and Bc did not increase but ra-ther 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, presumably by inducing the over-representation of Bacteroidales at the ex-pense of Clostridiales, but worsened the histopathological score (fig. S21). Supporting this notion, Bf maintained its regulatory properties in the context of CTLA-4 blockade (fig. S22) (7).

Hence, the efficacy of CTLA-4 blockade is influenced by the microbiota composition (B. fragilis and/or B. thetaiota-omicron and Burkholderiales). The microbiota composition affects IL-12-dependent Th1 immune responses, which facili-tate tumor control, in mice and patients while sparing intes-tinal integrity. In accord with previous findings (16), colitis (observed in the context of IL-10 deficiency and CTLA-4 blockade, fig. S17) could even antagonize anticancer 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 associa-tion with Burkholderiales recognized through the pyrin/caspase-1 inflammasome (17), synergizing with TLR2/TLR4 signaling pathways (fig. S15), may account for the immunomodulatory effects of anti–CTLA-4 Ab. Future investigations will determine whether a potential molecular mimicry between distinct commensals/pathobionts and tu-mor neoantigens could account for the toxicity and/or effi-cacy of immune checkpoint blockers. Prospective studies in metastatic melanoma and/or NSCLC may validate the rele-vance of the enterotypes described herein in the long term efficacy of immune checkpoint blockers, with the aim of compensating cluster B-driven patients with live and im-munogenic or recombinant Bacteroides spp. (18) or fecal microbial transplantation from cluster C-associated stools to improve their antitumor immune responses.

REFERENCES AND NOTES 1. K. S. Peggs, S. A. Quezada, A. J. Korman, J. P. Allison, Principles and use of anti-

CTLA4 antibody in human cancer immunotherapy. Curr. Opin. Immunol. 18, 206–213 (2006). Medline doi:10.1016/j.coi.2006.01.011

2. F. S. Hodi, S. J. O’Day, D. F. McDermott, R. W. Weber, J. A. Sosman, J. B. Haanen, R. Gonzalez, C. Robert, D. Schadendorf, J. C. Hassel, W. Akerley, A. J. van den Eertwegh, J. Lutzky, P. Lorigan, J. M. Vaubel, G. P. Linette, D. Hogg, C. H. Ottensmeier, C. Lebbé, C. Peschel, I. Quirt, J. I. Clark, J. D. Wolchok, J. S. Weber, J. Tian, M. J. Yellin, G. M. Nichol, A. Hoos, W. J. Urba, Improved survival with ipilimumab in patients with metastatic melanoma. N. Engl. J. Med. 363, 711–723 (2010). Medline doi:10.1056/NEJMoa1003466

3. K. E. Beck, J. A. Blansfield, K. Q. Tran, A. L. Feldman, M. S. Hughes, R. E. Royal, U. S. Kammula, S. L. Topalian, R. M. Sherry, D. Kleiner, M. Quezado, I. Lowy, M. Yellin, S. A. Rosenberg, J. C. Yang, Enterocolitis in patients with cancer after antibody blockade of cytotoxic T-lymphocyte-associated antigen 4. J. Clin. Oncol. 24, 2283–2289 (2006). Medline doi:10.1200/JCO.2005.04.5716

4. D. Berman, S. M. Parker, J. Siegel, S. D. Chasalow, J. Weber, S. Galbraith, S. R. Targan, H. L. Wang, Blockade of cytotoxic T-lymphocyte antigen-4 by ipilimumab

results in dysregulation of gastrointestinal immunity in patients with advanced melanoma. Cancer Immun. 10, 11 (2010). Medline

5. S. Viaud, F. Saccheri, G. Mignot, T. Yamazaki, R. Daillère, D. Hannani, D. P. Enot, C. Pfirschke, C. Engblom, M. J. Pittet, A. Schlitzer, F. Ginhoux, L. Apetoh, E. Chachaty, P. L. Woerther, G. Eberl, M. Bérard, C. Ecobichon, D. Clermont, C. Bizet, V. Gaboriau-Routhiau, N. Cerf-Bensussan, P. Opolon, N. Yessaad, E. Vivier, B. Ryffel, C. O. Elson, J. Doré, G. Kroemer, P. Lepage, I. G. Boneca, F. Ghiringhelli, L. Zitvogel, The intestinal microbiota modulates the anticancer immune effects of cyclophosphamide. Science 342, 971–976 (2013). Medline doi:10.1126/science.1240537

6. A. Rogoz, B. S. Reis, R. A. Karssemeijer, D. Mucida, A 3-D enteroid-based model to study T-cell and epithelial cell interaction. J. Immunol. Methods 421, 89–95 (2015). Medline doi:10.1016/j.jim.2015.03.014

7. S. Dasgupta, D. Erturk-Hasdemir, J. Ochoa-Reparaz, H. C. Reinecker, D. L. Kasper, Plasmacytoid dendritic cells mediate anti-inflammatory responses to a gut commensal molecule via both innate and adaptive mechanisms. Cell Host Microbe 15, 413–423 (2014). Medline

8. S. K. Mazmanian, C. H. Liu, A. O. Tzianabos, D. L. Kasper, An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system. Cell 122, 107–118 (2005). Medline doi:10.1016/j.cell.2005.05.007

9. F. Stingele, B. Corthésy, N. Kusy, S. A. Porcelli, D. L. Kasper, A. O. Tzianabos, Zwitterionic polysaccharides stimulate T cells with no preferential V beta usage and promote anergy, resulting in protection against experimental abscess formation. J. Immunol. 172, 1483–1490 (2004). Medline doi:10.4049/jimmunol.172.3.1483

10. A. O. Tzianabos, A. Pantosti, H. Baumann, J. R. Brisson, H. J. Jennings, D. L. Kasper, The capsular polysaccharide of Bacteroides fragilis comprises two ionically linked polysaccharides. J. Biol. Chem. 267, 18230–18235 (1992). Medline

11. J. Y. Huang, S. M. Lee, S. K. Mazmanian, The human commensal Bacteroides fragilis binds intestinal mucin. Anaerobe 17, 137–141 (2011). Medline doi:10.1016/j.anaerobe.2011.05.017

12. M. Arumugam, J. Raes, E. Pelletier, D. Le Paslier, T. Yamada, D. R. Mende, G. R. Fernandes, J. Tap, T. Bruls, J. M. Batto, M. Bertalan, N. Borruel, F. Casellas, L. Fernandez, L. Gautier, T. Hansen, M. Hattori, T. Hayashi, M. Kleerebezem, K. Kurokawa, M. Leclerc, F. Levenez, C. Manichanh, H. B. Nielsen, T. Nielsen, N. Pons, J. Poulain, J. Qin, T. Sicheritz-Ponten, S. Tims, D. Torrents, E. Ugarte, E. G. Zoetendal, J. Wang, F. Guarner, O. Pedersen, W. M. de Vos, S. Brunak, J. Doré, M. Antolín, F. Artiguenave, H. M. Blottiere, M. Almeida, C. Brechot, C. Cara, C. Chervaux, A. Cultrone, C. Delorme, G. Denariaz, R. Dervyn, K. U. Foerstner, C. Friss, M. van de Guchte, E. Guedon, F. Haimet, W. Huber, J. van Hylckama-Vlieg, A. Jamet, C. Juste, G. Kaci, J. Knol, O. Lakhdari, S. Layec, K. Le Roux, E. Maguin, A. Mérieux, R. Melo Minardi, C. M’rini, J. Muller, R. Oozeer, J. Parkhill, P. Renault, M. Rescigno, N. Sanchez, S. Sunagawa, A. Torrejon, K. Turner, G. Vandemeulebrouck, E. Varela, Y. Winogradsky, G. Zeller, J. Weissenbach, S. D. Ehrlich, P. Bork; MetaHIT Consortium, Enterotypes of the human gut microbiome. Nature 473, 174–180 (2011). Medline doi:10.1038/nature09944

13. J. Qin, R. Li, J. Raes, M. Arumugam, K. S. Burgdorf, C. Manichanh, T. Nielsen, N. Pons, F. Levenez, T. Yamada, D. R. Mende, J. Li, J. Xu, S. Li, D. Li, J. Cao, B. Wang, H. Liang, H. Zheng, Y. Xie, J. Tap, P. Lepage, M. Bertalan, J.-M. Batto, T. Hansen, D. Le Paslier, A. Linneberg, H. B. Nielsen, E. Pelletier, P. Renault, T. Sicheritz-Ponten, K. Turner, H. Zhu, C. Yu, S. Li, M. Jian, Y. Zhou, Y. Li, X. Zhang, S. Li, N. Qin, H. Yang, J. Wang, S. Brunak, J. Doré, F. Guarner, K. Kristiansen, O. Pedersen, J. Parkhill, J. Weissenbach, P. Bork, S. D. Ehrlich, J. Wang; MetaHIT Consortium, A human gut microbial gene catalogue established by metagenomic sequencing. Nature 464, 59–65 (2010). Medline doi:10.1038/nature08821

14. A. Cebula, M. Seweryn, G. A. Rempala, S. S. Pabla, R. A. McIndoe, T. L. Denning, L. Bry, P. Kraj, P. Kisielow, L. Ignatowicz, Thymus-derived regulatory T cells contribute to tolerance to commensal microbiota. Nature 497, 258–262 (2013). Medline doi:10.1038/nature12079

15. J. L. Sonnenburg, C. T. Chen, J. I. Gordon, Genomic and metabolic studies of the impact of probiotics on a model gut symbiont and host. PLOS Biol. 4, e413 (2006). Medline doi:10.1371/journal.pbio.0040413

16. W. Lam, S. Bussom, F. Guan, Z. Jiang, W. Zhang, E. A. Gullen, S. H. Liu, Y. C. Cheng, The four-herb Chinese medicine PHY906 reduces chemotherapy-induced gastrointestinal toxicity. Sci. Transl. Med. 2, 45ra59 (2010). Medline doi:10.1126/scitranslmed.3001270

17. H. Xu, J. Yang, W. Gao, L. Li, P. Li, L. Zhang, Y. N. Gong, X. Peng, J. J. Xi, S. Chen,

/ sciencemag.org/content/early/recent / 5 November 2015 / Page 3 / 10.1126/science.aad1329

F. Wang, F. Shao, Innate immune sensing of bacterial modifications of Rho GTPases by the Pyrin inflammasome. Nature 513, 237–241 (2014). Medline doi:10.1038/nature13449

18. M. Mimee, A. C. Tucker, C. A. Voigt, T. K. Lu, Programming a human commensal bacterium, Bacteroides thetaiotaomicron, to sense and respond to stimuli in the murine gut microbiota. Cell Systems 1, 62–71 (2015). doi:10.1016/j.cels.2015.06.001

19. M. Venkatesh, S. Mukherjee, H. Wang, H. Li, K. Sun, A. P. Benechet, Z. Qiu, L. Maher, M. R. Redinbo, R. S. Phillips, J. C. Fleet, S. Kortagere, P. Mukherjee, A. Fasano, J. Le Ven, J. K. Nicholson, M. E. Dumas, K. M. Khanna, S. Mani, Symbiotic bacterial metabolites regulate gastrointestinal barrier function via the xenobiotic sensor PXR and Toll-like receptor 4. Immunity 41, 296–310 (2014). Medline doi:10.1016/j.immuni.2014.06.014

20. C. A. Schneider, W. S. Rasband, K. W. Eliceiri, NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012). Medline doi:10.1038/nmeth.2089

21. R. I. Amann, B. J. Binder, R. J. Olson, S. W. Chisholm, R. Devereux, D. A. Stahl, Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl. Environ. Microbiol. 56, 1919–1925 (1990). Medline

22. A. H. Franks, H. J. Harmsen, G. C. Raangs, G. J. Jansen, F. Schut, G. W. Welling, Variations of bacterial populations in human feces measured by fluorescent in situ hybridization with group-specific 16S rRNA-targeted oligonucleotide probes. Appl. Environ. Microbiol. 64, 3336–3345 (1998). Medline

23. L. Rigottier-Gois, V. Rochet, N. Garrec, A. Suau, J. Doré, Enumeration of Bacteroides species in human faeces by fluorescent in situ hybridisation combined with flow cytometry using 16S rRNA probes. Syst. Appl. Microbiol. 26, 110–118 (2003). Medline doi:10.1078/072320203322337399

24. A. Schlitzer, N. McGovern, P. Teo, T. Zelante, K. Atarashi, D. Low, A. W. Ho, P. See, A. Shin, P. S. Wasan, G. Hoeffel, B. Malleret, A. Heiseke, S. Chew, L. Jardine, H. A. Purvis, C. M. Hilkens, J. Tam, M. Poidinger, E. R. Stanley, A. B. Krug, L. Renia, B. Sivasankar, L. G. Ng, M. Collin, P. Ricciardi-Castagnoli, K. Honda, M. Haniffa, F. Ginhoux, IRF4 transcription factor-dependent CD11b+ dendritic cells in human and mouse control mucosal IL-17 cytokine responses. Immunity 38, 970–983 (2013). Medline doi:10.1016/j.immuni.2013.04.011

25. A. Pantosti, A. O. Tzianabos, A. B. Onderdonk, D. L. Kasper, Immunochemical characterization of two surface polysaccharides of Bacteroides fragilis. Infect. Immun. 59, 2075–2082 (1991). Medline

26. F. T. Chen, T. S. Dobashi, R. A. Evangelista, Quantitative analysis of sugar constituents of glycoproteins by capillary electrophoresis. Glycobiology 8, 1045–1052 (1998). Medline doi:10.1093/glycob/8.11.1045

27. J. Nigou, A. Vercellone, G. Puzo, New structural insights into the molecular deciphering of mycobacterial lipoglycan binding to C-type lectins: Lipoarabinomannan glycoform characterization and quantification by capillary electrophoresis at the subnanomole level. J. Mol. Biol. 299, 1353–1362 (2000). Medline doi:10.1006/jmbi.2000.3821

28. H. Baumann, A. O. Tzianabos, J. R. Brisson, D. L. Kasper, H. J. Jennings, Structural elucidation of two capsular polysaccharides from one strain of Bacteroides fragilis using high-resolution NMR spectroscopy. Biochemistry 31, 4081–4089 (1992). Medline doi:10.1021/bi00131a026

29. T. Sato, R. G. Vries, H. J. Snippert, M. van de Wetering, N. Barker, D. E. Stange, J. H. van Es, A. Abo, P. Kujala, P. J. Peters, H. Clevers, Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009). Medline doi:10.1038/nature07935

30. J.-P. Furet, O. Firmesse, M. Gourmelon, C. Bridonneau, J. Tap, S. Mondot, J. Doré, G. Corthier, Comparative assessment of human and farm animal faecal microbiota using real-time quantitative PCR. FEMS Microbiol. Ecol. 68, 351–362 (2009). Medline doi:10.1111/j.1574-6941.2009.00671.x

31. M. T. Suzuki, L. T. Taylor, E. F. DeLong, Quantitative analysis of small-subunit rRNA genes in mixed microbial populations via 5′-nuclease assays. Appl. Environ. Microbiol. 66, 4605–4614 (2000). Medline doi:10.1128/AEM.66.11.4605-4614.2000

32. W. Manz, R. Amann, W. Ludwig, M. Vancanneyt, K.-H. Schleifer, Application of a suite of 16S rRNA-specific oligonucleotide probes designed to investigate

bacteria of the phylum cytophaga-flavobacter-bacteroides in the natural environment. Microbiology 142, 1097–1106 (1996). Medline doi:10.1099/13500872-142-5-1097

33. T. Odamaki, J. Z. Xiao, M. Sakamoto, S. Kondo, T. Yaeshima, K. Iwatsuki, H.Togashi, T. Enomoto, Y. Benno, Distribution of different species of the Bacteroides fragilis group in individuals with Japanese cedar pollinosis. Appl. Environ. Microbiol. 74, 6814–6817 (2008). Medline doi:10.1128/AEM.01106-08

34. S. M. Lee, G. P. Donaldson, Z. Mikulski, S. Boyajian, K. Ley, S. K. Mazmanian, Bacterial colonization factors control specificity and stability of the gut microbiota. Nature 501, 426–429 (2013). Medline doi:10.1038/nature12447

35. J. Tong, C. Liu, P. Summanen, H. Xu, S. M. Finegold, Application of quantitative real-time PCR for rapid identification of Bacteroides fragilis group and related organisms in human wound samples. Anaerobe 17, 64–68 (2011). Medline doi:10.1016/j.anaerobe.2011.03.004

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. DNA sequence reads from this study have been submitted to the National Center for Biotechnology Information (NCBI), NIH, under the Bioproject ID PRJNA299112. L.Z., M.V., and P.L. have filed patent applications no. 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. 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 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. L.Z., M.V., and P.L. have filed patent applications no. 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. 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/cgi/content/full/science.aad1329/DC1 Materials and Methods Figs. S1 to S22 Tables S1 to S5 References (19–35) April 2015; accepted 21 October 2015 Published online 5 November 2015 10.1126/aad1329

/ sciencemag.org/content/early/recent / 5 November 2015 / Page 4 / 10.1126/science.aad1329

Fig. 1. Microbiota-dependent immunomodulatory effects of anti–CTLA-4 Ab. Tumor growth of MCA205 in SPF (A) or GF (B) mice treated with 5 injections (cf. arrows) of 9D9 or isotype control (Iso Ctrl) Ab. Id. as in (A) and (B) in the presence (C) (left panel) of broad-spectrum antibiotics (ACS) or single antibiotic regimen (C, right panel) in >20 mice/group. Flow cytometric analyses of Ki67 and ICOS expression (D) and Th1 cytokines (E) on splenic CD4+Foxp3– T cells (D) and TILs (E) two days after the 3rd administration of 9D9 or Iso Ctrl Ab. Each dot represents one mouse in 2-3 independent experiments of 5 mice/group. P-values corrected for inter-experimental baseline variation between 3 individual experiments in (D). *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant.

/ sciencemag.org/content/early/recent / 5 November 2015 / Page 5 / 10.1126/science.aad1329

Fig. 2. IEC-IEL dialogue causes IEC apoptosis and intestinal dysbiosis after anti–CTLA-4 Ab injection. Representative micrograph pictures of distal ileum (A) (left panel) after staining with anti–cleaved caspase 3 (cCasp3) Ab at 24 hours after 1 injection of 9D9 (or Iso Ctrl) Ab in naive mice with or without prior depletion of CD4+ and CD8+ T cells (concatanated data of 2 experiments; (A) (right panel). Id. for 3D-enteroid cocultures stimulated (or not) with TLR agonists and incubated with IELs harvested from 9D9 (or Iso Ctrl) Ab-treated mice in H&E (B) (left four panels), then stained with anti-cCasp3 (B, middle panels). Data concatenated from 2 experiments counting the mean±SEM percentages of apoptotic cells/organoid in 20 organoids (B) (right panel). (C) Sequencing of 16S rRNA gene amplicons of feces from tumor bearers before and 48 hours after 1 administration of 9D9 or isotype control Ab. PCA on a relative abundance matrix of genus repartition highlighting the clustering between baseline, isotype control- or anti-9D9 Ab-treated animals after 1 injection (C) (left panel, 5-6 mice/group). Ellipses are presented around the centroids of the resulting 3 clusters. The first two components explain 34.41% of total variance (Component 1: 20.04%; Component2: 14.35%) (Monte-Carlo test with 1000 replicates, P = 0.0049) (left panel) Means ±SEM of relative abundance for each 3 orders for 5 mice/group are shown (C) (three right panels). (D) qPCR analyses targeting three distinct Bacteroides spp. in ileal mucosae performed at 24-48 hours post-Ab. Results are represented as 2–ΔΔCt x 103, normalized 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.

/ sciencemag.org/content/early/recent / 5 November 2015 / Page 6 / 10.1126/science.aad1329

Fig. 3. Memory T cell responses against Bt and Bf and anticancer efficacy of CTLA-4 blockade. (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). Tumor sizes at day 15 post 9D9 or Iso Ctrl Ab-treatment are depicted (A) (left panel). Each dot represents one tumor and graphs depict 2-3 experiments of 5 mice/group. 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 M&M at day 20 post treatment in 5 animals/group on at least 6 independent areas (A) (middle panel). Representative micrographs are shown (right panel, scale=100μm). (C to E) Recall responses of CD4+ T cells in mice and patients to various bacterial strains post-CTLA-4 blockade. DC loaded with bacteria of the indicated strain were incubated with CD4+ T cells, two days after 3 ip anti–CTLA-4 Ab in mice, and after at least 2 injections of ipilimumab (ipi) in patients. The graphs represent IFNγ concentrations from coculture supernatants at 24 hours in mice (C), and 48 hours in metastatic melanoma patients (D). IFNγ/IL-10 ratios (E) were monitored in DC/T cell cocultures of NSCLC patients at 48 hours. No cytokine release was observed in the absence of bacteria or T cells (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 anti–CTLA-4 mAb and restimulated with Bf versus B. distasonis or BM-DC alone (CD4+ NT) were infused iv in day 6 MCA205 tumor-bearing GF mice. A representative experiment containing 5-6 mice/group is shown. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant.

/ sciencemag.org/content/early/recent / 5 November 2015 / Page 7 / 10.1126/science.aad1329

Fig. 4. Biological significance of ipilimumab-induced dysbiosis in patients. The k-means clustering algorithm was applied based on genus composition before and during ipilimumab treatment in 25 metastatic melanoma patients, validated using the Calinski-Harabasz Index (14), and showed good performance in recovering three clusters before and after therapy (inter-class PCA; (A) (left panel) (Monte-Carlo test, P = 0.000199). Random Forest analysis was applied to decipher main genera responsible for this significant clustering (A) (right panel). The relative abundance of main Bacteroides spp. significantly differed between cluster B and C (B) (right panel). The proportions of patients falling into each cluster were analyzed in a non-paired manner before versus after ipi injections regardless of the time point (B) (left panel) (fig. S20A). (C) Fecal microbial transplantion of feces post-ipilimumab from 8 patients falling into each of the three clusters (stool selection for FMT 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 pre- (2 weeks post-colonization) (D) and post- (2 weeks) ipi, targeting Bacteroidales and Bacteroides spp. Results are represented as 2–ΔCt x 103, normalized to 16S rDNA. No significant difference in the relative abundance of Bf was detectable in the donors of cluster B vs C before colonization (not shown). (E) Spearman correlations between the amount of Bf in stools 15 days post-9D9 Ab and tumor sizes across cluster B- and C-recipient mice. *P < 0.05, **P < 0.01, ***P < 0.001.

/ sciencemag.org/content/early/recent / 5 November 2015 / Page 8 / 10.1126/science.aad1329