Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota

Abstract

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.

Fig. 1. Microbiota-dependent immunomodulatory effects of CTLA-4 Ab

Tumor growth of MCA205 in SPF (A) or GF (B) mice treated with five injections (compare the arrows) of 9D9 or isotype control (Iso Ctrl) Ab. (C) Tumor growth as in (A) and (B) in the presence (left) of ACS or (right) of single-antibiotic regimen in >20 mice per group. Flow cyto^{metric analyses of (D) Ki67} and ICOS expression and (E) TH₁ cytokines on splenic CD4⁺Foxp3⁻Tcells (D) and TILs (E) 2 days after the third administration of 9D9 or Iso Ctrl Ab. Each dot represents one mouse in two to three independent experiments of five mice per group. P values corrected for interexperimental baseline ^{variation between three} individual experiments in (D). *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Fig. 2. IEC-IEL dialogue causes IEC apoptosis and intestinal dysbiosis after CTLA-4 Ab injection

(A) (left) Representative micrograph pictures 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 hematoxylin and eosin (H&E), then (middle) stained with cCasp3-specific Ab. (Right) Data concatenated from two experiments counting the means ± SEM percentages 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^−ΔΔCt × 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.

Fig. 3. Memory Tcell 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). (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 treatment in five animals per group on at least six independent areas. (Right) Representative micrographs are shown; scale bar, 100 μm. (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 indicated 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-γ (IFN-γ) concentrations from coculture supernatants at 24 hours in mice (C) and 48 hours in MM patients (D). (E) IFN-γ/IL-10 ratios were monitored 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) Tcells 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.

Fig. 4. Biological significance of ipilimumab-induced dysbiosis in patients

The k means clustering algorithm was applied on the basis of genus composition before and during ipilimumab treatment in 25 MM patients, validated using the Calinski-Harabasz index (14), and showed good performance in recovering three clusters before and after therapy (interclass PCA); (A) (left) (Monte-Carlo test, P = 0.000199). (A) (right) Random Forest analysis was applied to decipher the main genera responsible for this significant clustering. (B) (right) The relative abundance of main Bacteroides spp. significantly differed between clusters B and C. (B) (left) The proportions of patients falling into each cluster were analyzed in a nonpaired manner before versus after ipi injections regardless of the time point (fig. S20A). (C) Fecal microbial transplantation after introduction of ipilimumab from eight patients falling into each of the three clusters (stool selection for fecal microbial transplantation 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 before (2 weeks postcolonization) and 2 weeks after 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 versus C before colonization (not shown). (E) Spearman correlations between the amount of Bf in stools 15 days after treatment with 9D9 Ab and tumor sizes across cluster B- and C-recipient mice. *P < 0.05; **P < 0.01; ***P < 0.001.