Gut microbiota modulates adoptive cell therapy via CD8α dendritic cells and IL-12

Abstract

Adoptive T cell therapy (ACT) is a promising new modality for malignancies. Here, we report that adoptive T cell efficacy in tumor-bearing mice is significantly affected by differences in the native composition of the gut microbiome or treatment with antibiotics, or by heterologous fecal transfer. Depletion of bacteria with vancomycin decreased the rate of tumor growth in mice from The Jackson Laboratory receiving ACT, whereas treatment with neomycin and metronidazole had no effect, indicating the role of specific bacteria in host response. Vancomycin treatment induced an increase in systemic CD8α+ DCs, which sustained systemic adoptively transferred antitumor T cells in an IL-12-dependent manner. In subjects undergoing allogeneic hematopoietic cell transplantation, we found that oral vancomycin also increased IL-12 levels. Collectively, our findings demonstrate an important role played by the gut microbiota in the antitumor effectiveness of ACT and suggest potentially new avenues to improve response to ACT by altering the gut microbiota.

Keywords: Cancer immunotherapy; Immunology; Microbiology.

Figure 1. The gut microbiota influences effectiveness of adoptive cell therapy.

(A) Impact of ACT on tumor growth in mice obtained from Jackson and Harlan. Means ± SEM are shown. Differences in tumor volume were evaluated with linear mixed effects models. ***P < 0.001. (B) Principal coordinates analysis (PCoA) of bacterial community composition, using unweighted UniFrac distance. (C) Bacterial taxa observed in Jackson and Harlan mice at 7 days and 21 days following ACT.

Figure 2. The modulation of gut microbiota through antibiotics impacts efficacy of adoptive cell therapy.

(A) Difference in number of bacterial taxa observed after treatment with vancomycin. (B) Bacterial taxa observed and (C) community composition after treatment with vancomycin. (D) Tumor growth of mice treated in combination of ACT and vancomycin. Tumor growth data are representative of 2 individual experiments with at least 5 mice per group, means ± SEM are shown (same control is shown in A). Differences in tumor volume were evaluated with linear mixed effects models. *P < 0.05, ***P < 0.001.

Figure 3. The efficacy of ACT is increased by vancomycin but not neomycin/metronidazole antibiotic treatment.

Tumor growth of mice treated with ACT, using CD3⁺ T cells from immunized mice polarized under Th1 conditions, and (A) vancomycin or (B) neomycin/metronidazole (Neo/Met). Tumor growth of mice treated with ACT, using nonpolarized cells from immunized mice, and (C) vancomycin or (D) Neo/Met. Tumor growth data are representative of 3 independent experiments with 5 mice per group. Means ± SEM are shown. Differences in tumor volume were evaluated with linear mixed effects models. **P < 0.01, ***P < 0.001.

Figure 4. Increased ACT efficacy by antibiotic treatment is phenocopied by fecal microbiota transplant.

(A) Detection of bacterial DNA in stool samples by broad-range PCR using universal PCR primers. (B) Detection of segmented filamentous bacteria (SFB) in DNA of stool samples. (C) Principal coordinates analysis (PCoA) of unweighted UniFrac distance and (D) between-group distances for samples from Jackson donor mice, Harlan recipient mice, and native Harlan mice. Day 0 data is shown in red; Day 7 data is shown in blue. (E) Tumor growth of Harlan mice reconstituted with Jackson microbiota. Tumor growth data are representative of 2 independent experiments with 5 mice per group. Means ± SEM are shown. Differences in tumor volume were evaluated with linear mixed effects models. *P < 0.05.

Figure 5. Microbiota composition influences both tumor infiltration and systemic expansion of reactive T cells.

(A) IHC quantification of CD3⁺ T cell infiltration within TC1 tumors and representative CD3 staining images. Scale bars: 200 μm (original magnification, 20×). Each dot represents a mouse; mean of 5–6 fields per mouse and 5–7 mice per group. Means ± SEM are shown from 1 representative experiment out of 3. (B) Intratumoral CD8⁺ T cells E7_49–57 tetramer–positive and (C) IFN-γ ELISPOT of splenocytes from TC1 tumor–bearing mice treated with ACT and vancomycin or Neo/Met. Each dot represents a mouse; 3–9 mice per group. Means ± SEM are shown from 1 representative experiment out of 3. (D) Proliferation of CFSE-labeled adoptively transferred T cells was measured by flow cytometry based on CFSE dilution peaks 8 days after ACT. Representative histograms. Numbers represent the average (2 independent experiments, n = 3) of percentage on each proliferation group (low, mid, and high). A 2-tailed t test analysis was performed. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6. Increased expression of genes associated with cytotoxic and Th1 profiles in TC1 tumors treated with ACT and vancomycin.

(A) Heatmap representing color-coded expression levels of differentially expressed genes in TC1 tumors treated with vancomycin, ACT, or a combination of both. (B) Fold-change quantification of Granzyme B, Perforin 1, Il12a, and Ifng. All measurements were made in triplicates. Three tumors were measured per group from 1 representative experiment out of 3. Means ± SEM are shown. A multiple t test analysis was performed. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7. Vancomycin treatment increases the number of systemic CD8α⁺ DC.

Jackson mice were treated for 10 days with antibiotics. (A) Phenotype analysis of splenic DC population by flow cytometry, CD8α⁺ DC subset shown (CD11c^highCD8⁺B220^–CD11b^–). (B) Il12a RNA expression of purified CD11c⁺ DC (pooled by group). (C) Mouse IL-12p70 protein serum levels. (D) Human IL-12p70 serum levels on patients undergoing the same therapy, allogeneic hematopoietic cell transplantation, and treated with vancomycin. (E) E7_49–57 IFN-γ ELISPOT of purified CD11c⁺ DCs from not treated (control), treated with Neo/Met or vancomycin (± the addition of 1 μg/ml of blocking monoclonal antibody IL-12p70) mice and incubated with T cells from immunized mice. Mouse data is representative of 3 independent experiments with 4–9 mice per group. Each dot represents a mouse; means ± SEM are shown. A multiple t test analysis was performed. *P < 0.05, ***P < 0.001.

Figure 8. Effects of the microbiota during adoptive T cells transfer therapy depend on IL-12.

(A and B) Jackson IL-12–KO and (C and D) C57BL/6 mice treated with a monoclonal IL-12 blocking antibody. (A and C) tumor growth data are representative of 2 independent experiments with 5 mice per group; means ± SEM are shown. (B and D) IHC quantification of CD3⁺ T cell infiltration within TC1 tumors and representative CD3 staining images. Scale bars: 200 μm (original magnification, 20×). Each dot represents a mouse; mean of 5–6 fields per mouse and 4–7 mice per group. Means ± SEM are shown. Differences in tumor volume were evaluated with linear mixed effects models. For other panels, a multiple t test analysis was performed. *P < 0.05, **P < 0.01, ***P < 0.001.