《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 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