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 1081to 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