RESEARCH I REPORTS A Bf and anticancer efficacy of CTLA-4 block ade.(A and B) Tumoricidal effects of Bf, Bt, and/or B. cepacia( Bc)administered by oral feeding of ACS- treated or GF mice(also refer to fig S8A).(a) g 8=8 tumor, and graphs depict two to three experiments of five mice per group. (Middle)Histopathological aCTLA bearers receiving 9D9 Ab after oral gavage with ° ■3- severe 2-moderate colons monitoring microscopic lesions as described 5::1-mild in materials and methods at day 20 after treat Antibiotics Antibiotics ment in five animals per group on at least six B independent areas.(Right) Representative micro- graphs are shown, scale bar, 100 um.(B)Tumor- icidal effects of Bf in GF mice as indicated. (c to E fragilis Recall responses of CD4 T cells in mice and patients to various bacterial strains after CTLA-4 blockade. DCs loaded with bacteria of the indi ated strain were incubated with cD4- T cells 20 lays after three intraperitoneal (p) CTLA-4 Ab in mice, and after at least two injections of Days after tumor inoculation iimumab(ipi)in patients. The graphs represe 8. dista nterferon-y(IFN-y)concentrations from coculture D olena ematants at 24 hours in mice(C)and 48 hour B fragilis B. thetaiotaomicron MM patients(D).(E)IFN-7/IL-10 ratios were mon- itored in DC-T cell cocultures of NSCLC patients at 48 hours. No cytokine release was observed in 8 the absence of bacteria or Tcells(fig. Sll with H) Each dot represents one patient or mouse. Paired are represented by linking dots pre- and T cells harvested from spleens of mice exposed to CTLA-4 Ab and restimulated with Bf Melanoma 0.05 versus b. distasonis or bone marrow dcs alone Pre Post Pre Post F (CD4 NT) were infused intravenously in day 6 CD4*B. distasonis CD4*Bfragilis MCA205 tumor-bearing GF mice. A representative experiment containing five to six mice per group shown. *P< 0.05 * ot significant. that, although bacteria from the Bacteroidales achieved with vancomycin, which could boost the I probiotics. " The geodistribution of Bf in the order equally colonized the recipient murine antitumor effects of CTLA-4 blockade(p mucosal layer of the intestine (fig. SS)and its ntestine,stools from cluster C(but not A or B) ably by inducing the overrepresentation of Bac- association with Burkholderiales-recognize ndividuals specifically facilitated the coloni- teroidales at the expense of Clostridiales)but ugh the pyrin-caspase-l inflammasome zation of the immunogenic bacteria Bf and worsened the histopathological score (fig. S21). d synergizing with TLR2/TLR4 signaling path (7-10, 14, 15)(Fig. 4D). Moreover, after CTLA-4 Ab In support of this notion, Bf maintained its reg- ways(fi S15)-may account for the immunomod- herapy,only cluster C (not A or B)recipient mice ulatory properties in the context of CTLA 4 block- ulatory effects of CTLA-4 Ab. Future investigations had outgrowth of Bf(fig. S20B) Note that the ade(fig S22)(7. will determine whether a potential molecular fecal abundance of Bf (but not B. distasonis ol Hence, the efficacy of CTLA-4 blockade is in- mimicry between distinct commensals and/or B uniformis)negatively correlated with tumor fluenced by the microbiota composition(B fragilis pathobionts and tumor neoantigens could ac- size after CTLA-4 blockade in cluster C-recipient and/or B thetaiotaomicron and Burkholderiales ) count for the toxicity andyor efficacy of immune mice(Fig. 4E and fig. S20C) Hence, ipilimumab The microbiota composition affects interleukin checkpoint blockers. Prospective studies in MM an modify the abundance of immunogenic Bac- 12(IL-12-dependent THl immune responses d or NSClC may validate the relevance of the teroides spp in the gut, which in turn affects its which facilitate tumor control in mice and pa- enterotypes described herein in the long-term anticancer efficacy. tients while sparing intestinal integrity. In ac- efficacy of immune checkpoint blockers, with Finally, intestinal reconstitution of ACS-treated cord with previous findings(16), colitis(observed the aim of compensating cluster B-driven pa nimals with the combination of Bf and Be did in the context of IL-10 deficiency and CTLA-4 tients with live and immunogenicorrecombinant not increase but rather reduced histopathological blockade)(fig. S17)could even antagonize anti- Bacteroides spp(18)or fecal microbial transplanta- signs of colitis induced by CTLA-4 blockade(Fig 3A). cancer efficacy. Several factors may dictate why tion from cluster C-associated stools to improve This efficacy-toxicity uncoupling effect was not I such commensals could be suitable "anticancer their antitumor immune responses. 1082 27 NOVEMBER 2015. VOL 350 ISSUE 6264 sciencemag. org SCIENCEthat, although bacteria from the Bacteroidales order equally colonized the recipient murine intestine, stools from cluster C (but not A or B) individuals specifically facilitated the coloni￾zation of the immunogenic bacteria Bf and Bt (7–10,14,15) (Fig. 4D). Moreover, after CTLA-4 Ab therapy, only cluster C (not A or B) recipient mice had outgrowth of Bf (fig. S20B). Note that the fecal abundance of Bf (but not B. distasonis or B. uniformis) negatively correlated with tumor size after CTLA-4 blockade in cluster C–recipient mice (Fig. 4E and fig. S20C). Hence, ipilimumab can modify the abundance of immunogenic Bac￾teroides spp. in the gut, which in turn affects its anticancer efficacy. Finally, intestinal reconstitution of ACS-treated animals with the combination of Bf and Bc did not increase but rather reduced histopathological signs of colitis induced by CTLA-4 blockade (Fig. 3A). This efficacy-toxicity uncoupling effect was not achieved with vancomycin, which could boost the antitumor effects of CTLA-4 blockade (presum￾ably by inducing the overrepresentation of Bac￾teroidales at the expense of Clostridiales) but worsened the histopathological score (fig. S21). In support of this notion, Bf maintained its reg￾ulatory properties in the context of CTLA-4 block￾ade (fig. S22) (7). Hence, the efficacy of CTLA-4 blockade is in￾fluenced by the microbiota composition (B. fragilis and/or B. thetaiotaomicron and Burkholderiales). The microbiota composition affects interleukin 12 (IL-12)–dependent TH1 immune responses, which facilitate tumor control in mice and pa￾tients while sparing intestinal integrity. In ac￾cord with previous findings (16), colitis (observed in the context of IL-10 deficiency and CTLA-4 blockade) (fig. S17) could even antagonize anti￾cancer efficacy. Several factors may dictate why such commensals could be suitable “anticancer probiotics.” The geodistribution of Bf in the mucosal layer of the intestine (fig. S8) and its association with Burkholderiales—recognized through the pyrin–caspase-1 inflammasome (17) and synergizing with TLR2/TLR4 signaling path￾ways (fig. S15)—may account for the immunomod￾ulatory effects of CTLA-4 Ab. Future investigations will determine whether a potential molecular mimicry between distinct commensals and/or pathobionts and tumor neoantigens could ac￾count for the toxicity and/or efficacy of immune checkpoint blockers. Prospective studies in MM and/or NSCLC may validate the relevance of the enterotypes described herein in the long-term efficacy of immune checkpoint blockers, with the aim of compensating cluster B–driven pa￾tients with live and immunogenic or recombinant Bacteroides spp. (18) or fecal microbial transplanta￾tion from cluster C–associated stools to improve their antitumor immune responses. 1082 27 NOVEMBER 2015 • VOL 350 ISSUE 6264 sciencemag.org SCIENCE Fig. 3. Memory T cell responses against Bt and Bf and anticancer efficacy of CTLA-4 block￾ade. (A and B) Tumoricidal effects of Bf, Bt, and/or B. cepacia (Bc) administered by oral feeding of ACS￾treated or GF mice (also refer to fig. S8A). (A) (left) Tumor sizes at day 15 after 9D9 or Iso Ctrl Ab treatment are depicted. Each dot represents one tumor, and graphs depict two to three experiments of five mice per group. (Middle) Histopathological score of colonic mucosae in ACS-treated tumor bearers receiving 9D9 Ab after oral gavage with various bacterial strains, assessed on H&E-stained colons monitoring microscopic lesions as described in materials and methods at day 20 after treat￾ment in five animals per group on at least six independent areas. (Right) Representative micro￾graphs are shown; scale bar, 100 mm. (B) Tumor￾icidal effects of Bf in GF mice as indicated. (C to E) Recall responses of CD4+ T cells in mice and patients to various bacterial strains after CTLA-4 blockade. DCs loaded with bacteria of the indi￾cated strain were incubated with CD4+ T cells, 2 days after three intraperitoneal (ip) CTLA-4 Ab in mice, and after at least two injections of ipilimumab (ipi) in patients. The graphs represent interferon-g (IFN-g) concentrations from coculture supernatants at 24 hours in mice (C) and 48 hours in MM patients (D). (E) IFN-g/IL-10 ratios were mon￾itored in DC–T cell cocultures of NSCLC patients at 48 hours. No cytokine release was observed in the absence of bacteria or Tcells (fig. S11 with HV). Each dot represents one patient or mouse. Paired analyses are represented by linking dots pre- and post-ipi. (F) T cells harvested from spleens of mice exposed to CTLA-4 Ab and restimulated with Bf versus B. distasonis or bone marrow DCs alone (CD4+ NT) were infused intravenously in day 6 MCA205 tumor-bearing GF mice. A representative experiment containing five to six mice per group is shown. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant. RESEARCH | REPORTS on June 24, 2016 http://science.sciencemag.org/ Downloaded from