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Commensal Bifidobacterium promotes antitumor immunity and facilitates anti–PD-L1 efficacy

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Science  27 Nov 2015:
Vol. 350, Issue 6264, pp. 1084-1089
DOI: 10.1126/science.aac4255
  • Fig. 1 Differences in melanoma outgrowth and tumor-specific immune responses between C57BL/6 JAX and TAC mice are eliminated when mice are cohoused.

    (A) B16.SIY tumor growth kinetics in newly arrived JAX and TAC mice. (B) IFN-γ enzyme-linked immunospot assay (ELISPOT) in tumor-bearing JAX and TAC mice 7 days after tumor inoculation. (C) Mean size of IFN-γ spots (10−3 mm2). (D) Percentage of SIY+ T cells of total CD8+ T cells within the tumor of JAX and TAC mice as determined by flow cytometry 21 days after tumor inoculation. Representative plots (left), quantification (right). (E) B16.SIY tumor growth kinetics in JAX and TAC mice cohoused for 3 weeks before tumor inoculation. (F) Number of IFN-γ spots/106 splenocytes in tumor-bearing JAX and TAC mice cohoused for 3 weeks before tumor inoculation. (G) Mean size of IFN-γ spots (10−3 mm2). (H) Percentage of SIY+ T cells of total CD8+ T cells within the tumor of JAX and TAC mice cohoused for 3 weeks before tumor inoculation. Means ± SEM combined from six independent experiments, analyzed by two-way analysis of variance (ANOVA) with Sidak’s correction for multiple comparisons (A) and (E), or individual mice with means ± SEM combined from four (B), (C), (F), (G) or three (D) and (H) independent experiments, analyzed by Student’s t test; five mice per group per experiment; *P < 0.005, **P < 0.01; NS, not significant.

  • Fig. 2 Oral administration of JAX fecal material to TAC mice enhances spontaneous antitumor immunity and response to αPD-L1 mAb therapy.

    (A) B16.SIY tumor growth in newly arrived TAC mice, TAC and JAX mice orally gavaged with phosphate-buffered saline or TAC or JAX fecal material before tumor implantation. (B) Number of IFN-γ spots × mean spot size (10−3 mm2), determined by ELISPOT 7 days after tumor inoculation. (C) Percentage of SIY+ CD8+ T cells within the tumor of TAC and JAX mice treated as in (A), 21 days after tumor inoculation. Representative plots (left), quantification (right). (D) B16.SIY tumor growth in TAC mice, untreated or treated with JAX fecal material 7 and 14 days after tumor implantation, αPD-L1 mAb 7, 10, 13, and 16 days after tumor implantation, or both regimens. (E) IFN-γ ELISPOT assessed 5 days after start of treatment. (F) Percentage of tumor-infiltrating SIY+ CD8+ T cells, determined by flow cytometry 14 days after start of treatment. (G) B16.SIY tumor growth kinetics in TAC and JAX mice, untreated or treated with αPD-L1 mAb 7, 10, 13, and 16 days after tumor implantation. Means ± SEM analyzed by two-way analysis of variance (ANOVA) with Dunnett’s (A) or Tukey’s (D) and (G) correction for multiple comparisons; or individual mice with means ± SEM analyzed by one-way ANOVA with Holm-Sidak correction for multiple comparisons (B), (C), (E), and (F); data are representative of (A) to (C), (F), and (G) or combined from (D) and (E) two to four independent experiments; five mice per group per experiment; *P < 0.05, **P < 0.01, ****P < 0.0001; NS, not significant.

  • Fig. 3 Direct administration of Bifidobacterium to TAC recipients with established tumors improves tumor-specific immunity and response to αPD-L1 mAb therapy.

    (A) Principal coordinate analysis plot of bacterial β-diversity over time in groups treated as in Fig. 2A, each group is made up of at least two cages, three or four mice per cage; data represent three independent experiments; **P < 0.01, ***P < 0.001 (ANOSIM). (B) Phylogenetic analysis of taxa that are of significantly different abundance in newly arrived JAX versus TAC mice FDR < 0.05 (nonparametric t test); bars represent log-transformed fold changes, inner circle, log10(10); middle circle, log10(100); outer circle, log10(1000). (C) Heat map showing relative abundance over time of significantly altered genus-level taxa in JAX-fed TAC mice FDR < 0.05 (nonparametric t test); columns depict individual mice; each time point shows mice from two separate cages, three or four mice per cage. (D) Correlation plot of relative abundance of Bifidobacterium OTU_681370 in fecal material obtained from groups, as in (A), 14 days after arrival and frequency of SIY+ CD8+ T cells in tumor; P = 1.4 × 10−5, FDR = 0.0002, correlation R2 = 0.86 (univariate regression). (E) B16.SIY tumor growth kinetics in TAC mice, untreated or treated with Bifidobacterium 7 and 14 days after tumor implantation, αPD-L1 mAb 7, 10, 13, and 16 days after tumor implantation, or both regimens. (F) IFN-γ ELISPOT assessed 5 days after start of treatment. (G) Percentage of tumor-infiltrating SIY+ CD8+ T cells, determined by flow cytometry 14 days after start of treatment. Means ± SEM analyzed by two-way ANOVA with Tukey’s correction (E) or individual mice with means ± SEM analyzed by one-way ANOVA with Holm-Sidak correction (F) and (G), and are combined from two independent experiments; five mice per group per experiment: *P < 0.05, ***P < 0.001, ****P < 0.0001.

  • Fig. 4 Dendritic cells isolated from JAX and Bifidobacterium-fed TAC mice show increased expression of genes associated with antitumor immunity and heightened capability for T cell activation.

    (A) Quantification of IFN-γ mean fluorescence intensity (MFI) of 2C CD8+ T cells in the tumor-draining lymph node (left) and spleen (right) of TAC, JAX, and Bifidobacterium-fed TAC mice on day 7 after adoptive transfer. (B) Percentage of MHC Class IIhi DCs in tumors isolated from TAC, JAX, and Bifidobacterium-fed TAC mice 40 hours after tumor implantation as assessed by flow cytometry. Data in (A) and (B) show individual mice with means ± SEM, analyzed by one-way ANOVA with Holm-Sidak correction; representative of two to four independent experiments, eight or nine mice per group per experiment: *P < 0.05, **P < 0.01, ****P < 0.0001. (C) Enriched biological pathways and functions found within the subset of elevated genes in JAX and Bifidobacterium-treated TAC-derived DCs relative to untreated TAC DCs isolated from tumors 40 hours after tumor inoculation, as assessed by DAVID pathway analysis. Red bars indicate the percentage of genes in a pathway up-regulated in DCs isolated from JAX and Bifidobacterium-fed TAC mice. Blue line indicates P values calculated by Fisher’s exact test. (D) Heat map of key antitumor immunity genes in DCs isolated from JAX, Bifidobacterium-treated TAC or untreated TAC mice. Mean fold-change for each gene transcript is shown on the right. (E) Quantification of IFN-γ+ 2C TCR Tg CD8+ T cells stimulated in vitro with DCs purified from peripheral lymphoid tissues of naïve TAC, JAX, and Bifidobacterium-treated TAC mice in the presence of different concentrations of SIY peptide. Analyses in (C) to (E) were performed on data combined from two independent experiments, five mice pooled per group per experiment. (E) Technical replicates of pooled samples from each experiment separately and were analyzed by fitting a linear mixed model, with Bonferroni correction for multiple comparisons: *P < 0.05, ****P < 0.0001.

Supplementary Materials

  • Commensal Bifidobacterium promotes antitumor immunity and facilitates anti–PD-L1 efficacy

    Ayelet Sivan, Leticia Corrales, Nathaniel Hubert, Jason B. Williams, Keston Aquino-Michaels, Zachary M. Earley, Franco W. Benyamin, Yuk Man Lei, Bana Jabri, Maria-Luisa Alegre, Eugene B. Chang, and Thomas F. Gajewski

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S8
    • References
    Table S1
    Comparison of bacterial abundance between JAX and TAC mice, sham-, TAC-, and JAX-fed TAC mice at sequence level.
    Table S2
    Comparison of bacterial abundance between JAX and TAC mice, sham-, TAC-, and JAX-fed TAC mice at genus level.
    Table S3
    Univariate association testing between bacterial abundance and antigen-specific T cell infiltration across all permutation groups at sequence and genus levels.
    Table S4
    Comparison of bacterial abundance between TAC and Bifidobacterium-fed TAC mice at sequence and genus levels.
    Table S5
    Gene expression profiling results (Illumina microarray) comparing gene expression in DCs isolated from tumors of TAC, JAX, and Bifidobacterium-fed TAC mice.
    Table S6
    Gene expression profiling results (Illumina microarray) comparing gene expression in BM-DCs untreated or stimulated with Bifidobacterium in vitro.
    Table S7
    Primer sequences and probes (Roche Universal Probe Library) for qPCR validation of gene transcripts up-regulated by ≥1.5-fold in both JAX and Bifidobacterium-treated TACderived DCs relative to DCs from untreated TAC mice.

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