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. 2020 Jan 2;130(1):466-479.
doi: 10.1172/JCI124332.

Gut microbiota modulate dendritic cell antigen presentation and radiotherapy-induced antitumor immune response

Mireia Uribe-Herranz  1   2 , Stavros Rafail  2 , Silvia Beghi  1 , Luis Gil-de-Gómez  1 , Ioannis Verginadis  1 , Kyle Bittinger  3 , Sergey Pustylnikov  1 , Stefano Pierini  1   2 , Renzo Perales-Linares  1 , Ian A Blair  4 , Clementina A Mesaros  4 , Nathaniel W Snyder  5 , Frederic Bushman  6 , Constantinos Koumenis  1 , Andrea Facciabene  1   2
Affiliations

Gut microbiota modulate dendritic cell antigen presentation and radiotherapy-induced antitumor immune response

Mireia Uribe-Herranz et al. J Clin Invest. .

Abstract

Alterations in gut microbiota impact the pathophysiology of several diseases, including cancer. Radiotherapy (RT), an established curative and palliative cancer treatment, exerts potent immune modulatory effects, inducing tumor-associated antigen (TAA) cross-priming with antitumor CD8+ T cell elicitation and abscopal effects. We tested whether the gut microbiota modulates antitumor immune response following RT distal to the gut. Vancomycin, an antibiotic that acts mainly on gram-positive bacteria and is restricted to the gut, potentiated the RT-induced antitumor immune response and tumor growth inhibition. This synergy was dependent on TAA cross presentation to cytolytic CD8+ T cells and on IFN-γ. Notably, butyrate, a metabolite produced by the vancomycin-depleted gut bacteria, abrogated the vancomycin effect. In conclusion, depletion of vancomycin-sensitive bacteria enhances the antitumor activity of RT, which has important clinical ramifications.

Keywords: Antigen presentation; Dendritic cells; Immunology; Oncology; Radiation therapy.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. Oral vancomycin treatment enhances the direct and abscopal antitumor effects of hypofractionated RT in a preclinical melanoma and lung/cervical tumor model.
Shown are tumor volumes from control (untreated), vancomycin treatment alone (Vanco), RT treatment alone (RT), or vancomycin plus RT combination treatment (RT+Vanco) on irradiated tumors derived from the B16-OVA melanoma model (A) or from the TC-1 lung/cervical cancer model (C) and abscopal tumors (B and D, respectively). (E) B16-OVA tumor volumes of mice treated with RT alone, or RT and vancomycin (F) nonirradiated (abscopal) B16-OVA tumor volumes from mice treated with RT alone, or RT and vancomycin. n = 5 to 14 mice per group. Data are representative of at least 2 independent experiments. Mean ± SEM are shown. Statistical significance was assessed by 2-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 2
Figure 2. The effects of vancomycin are abrogated in CD8-depleted mice.
(A) Quantification of CD3+ cell infiltration by immunohistochemistry of B16-OVA–derived primary tumor sections from untreated, vancomycin treatment alone (Vanco), RT treatment alone (RT), or vancomycin plus RT combination treatment (RT+Vanco), and of abscopal tumor sections from RT abscopal or RT+Vanco abscopal combination treated. Mean ± SEM are shown. Statistical significance was assessed by Tukey’s test. (B) Primary tumors from each treatment group were digested and individual cells were analyzed by flow cytometry to determine the percentage of OVA-specific CD8+ T cells (CD45+/CD3+/CD8+/TET-OVA). Mean ± SEM are shown. Statistical significance was assessed by Tukey’s test. (C) Tumor growth rates in CD8-depleted mice. n = 5 to 10 mice per group. Mean ± SEM are shown. Statistical significance was assessed by 2-way ANOVA. Data are representative of at least 2 independent experiments. *P < 0.05, **P < 0.001, ***P < 0.01.
Figure 3
Figure 3. Antitumor effects of vancomycin plus RT combination treatment are IFN-γ dependent.
(A) Ifng mRNA expression levels in B16-OVA primary tumors collected at 5 days after radiation (21 Gy). (B) ELISA analysis of IFN-γ protein expression levels in tumor lysates 5 days after radiation (21 Gy). (C) Intracellular IFN-γ expression in infiltrating T cells after overnight Kb-OVA peptide stimulation. Mean ± SEM are shown. Statistical significance was assessed by Tukey’s test. (D) The antitumor effects of vancomycin+RT combination treatment are lost in Ifng-KO mice. n = 5 to 10 mice per group. Mean ± SEM are shown. Statistical significance was assessed by 2-way ANOVA. Data are representative of at least 2 independent experiments. *P < 0.05, **P < 0.01.
Figure 4
Figure 4. Vancomycin treatment increases local TAA cross-presentation and antigen recognition in tumors.
(A) Ifnb1 mRNA expression levels in TDLNs 1 day after irradiation. (B) Anti-MHC1 (Kb)-SL8 OVA peptide staining on tumor-infiltrating CD11c+ CD103+ DCs 5 days after RT. (C) IFN-γ ELISPOT assay plated with TDLN single-cell suspension and OT1 T cells (1:5 TDLN cells/T cells) in absence (left) or presence (right) of OVA peptide. Cells were harvested 5 days after RT treatment. (D) Coculture of purified CD11c+ DCs from TDLNs from each treatment group were incubated overnight with naive OT1 T cells in an IFN-γ ELISPOT assay (1:10 DCs/T cells). (E) Percentage of IFN-γ expression from overnight OT1 cells cocultured with DCs from mice treated with each therapeutic approach. (F) B16-OVA tumors from mice treated with RT alone or with the RT and vancomycin (RT+VANCO) combination treatment were dissociated and plated with OT1 cells in an IFN-γ ELISPOT plate for 24 hours. n = 5 to 10 mice per group. Data are representative of at least 2 independent experiments. Mean ± SEM are shown. Statistical significance was assessed by Tukey’s test. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 5
Figure 5. SCFAs impair APC activity in vitro and abrogate vancomycin-enhanced RT antitumor activity in vivo.
(A) Bone marrow–derived DCs (bmDCs) were either untreated, treated with 100 μM butyrate (C4), or treated with 100 μM propionate (C3) together with OVA protein (100 μg/mL) for 24 hours before they were plated to an ELISPOT plate with T cells from OT1 mice. (B) Purified T cells were in vitro–stimulated with aCD3/aCD28 in the presence or absence of 100 μM C4/butyrate in a IFN-γ ELISPOT plate. (C) In vivo effects on tumor growth in irradiated mice treated with vancomycin-containing drinking water with or without C4. Mean ± SEM are shown. Statistical significance was assessed by 2-way ANOVA. (D) Ifng mRNA expression levels in tumors from irradiated mice treated with vancomycin-containing drinking water with and without C4/butyrate. Mean ± SEM are shown. Statistical significance was assessed by Tukey’s test. (E) Flow cytometry analysis of OVA-presenting DCs and (F) CD3+CD8+Tet-OVA+ cell subsets. Mean ± SEM are shown. Statistical significance was assessed by Tukey’s test. n = 5 to 10 mice per group. Data are representative of at least 2 independent experiments. *P < 0.05, **P < 0.01.
Figure 6
Figure 6. Vancomycin treatment alters SCFA concentration and bacterial community composition.
(A) Concentration of C4 SCFAs in stool, (B) cecal contents, (C) tumor, and (D) TDLNs. Mean ± SEM are shown. Statistical significance was assessed by Tukey’s test. *P < 0.05, **P < 0.01. (E) Heatmap of bacterial taxon abundance in vancomycin-treated mice. White squares represent taxa not observed in the sample; n = 3 per group. (F) Bacterial community diversity as determined by 16S rRNA marker gene sequencing. (G) Dissimilarity of bacterial communities in stool from control and vancomycin-exposed mice. (H) Relative abundance of taxa known to contain butyrate-producing bacterial species. Data are representative of at least 2 independent experiments.
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