Bifidobacterium can mitigate intestinal immunopathology in the context of CTLA-4 blockade

Abstract

Antibodies that attenuate immune tolerance have been used to effectively treat cancer, but they can also trigger severe autoimmunity. To investigate this, we combined anti-CTLA-4 treatment with a standard colitis model to give mice a more severe form of the disease. Pretreatment with an antibiotic, vancomycin, provoked an even more severe, largely fatal form, suggesting that a Gram-positive component of the microbiota had a mitigating effect. We then found that a commonly used probiotic, Bifidobacterium, could largely rescue the mice from immunopathology without an apparent effect on antitumor immunity, and this effect may be dependent on regulatory T cells.

Keywords: Bifidobacterium; CTLA-4; immune checkpoint blockade; intestinal immunopathology; probiotics.

Fig. 1.

Increased susceptibility of DSS-induced colitis in anti–CTLA-4 Ab-receiving mice. (A and B) Percent of initial weight of mice receiving the IgG isotype control (Iso Ctrl) or the αCTLA-4 mAb. Mice were given 2% (A) or 3% DSS (B) for 7 d. There were five mice in each group. The data show mean with SEM analyzed by two-way ANOVA with Sidak’s correction for multiple comparisons. (C) Survival of the mice receiving the IgG isotype control (Iso Ctrl) or the αCTLA-4 mAb with 4% DSS administration. Survival was monitored for 14 d, n = 5 per group. (D) The histological score of mice receiving the IgG isotype control (Iso Ctrl) or the αCTLA-4 mAb with 3% DSS administration, n = 6 per group, means with SEM analyzed by unpaired Student’s t test. (E) Representative colon sections from mice treated with injection of isotype control (Left) or αCTLA-4 mAb (Right) and 3% DSS administration. Colon samples were collected at day 14 (H&E stained). (Scale bar, 50 μm.) *P < 0.05 and **P < 0.01.

Fig. 2.

Vancomycin augments the immunopathology of CTLA-4 blockade. (A) Percent initial weight of H₂O- or vancomycin-treated mice after injection of the αCTLA-4 mAb and the administration of 3% DSS. In each group, n = 5. ****P < 0.0001; Data show means with SEM analyzed by two-way ANOVA with Sidak’s correction for multiple comparisons. (B) Survival of H₂O- or vancomycin-treated mice after injection of the αCTLA-4 mAb and administration of 3% DSS, with n = 5 per group. (C) Histological score of H₂O- or vancomycin-treated mice after injection of the αCTLA-4 mAb and administration of 3% DSS, n = 5 per group, ****P < 0.0001, unpaired Student’s t test. (D) Representative colon sections of mice treated with H₂O or vancomycin after injection of αCTLA-4 mAb and 6-d administration of 3% DSS (H&E stained). (Scale bar, 100 μm.) (E) Concentrations of KC, IL-6, and CSF3 in the serum of mice treated with H₂O or vancomycin after CTLA-4 blockade and 6-d administration of 3% DSS, n = 5–7 per group. *P < 0.05, unpaired Student’s t test.

Fig. 3.

Bifidobacterium ameliorates the immunopathology, but does not affect antitumor immunity of vancomycin and CTLA-4 blockade. (A) The relative abundance of Bifidobacterium was quantified with real-time PCR. This was normalized to total bacteria, with n = 4–5 per group. **P < 0.01, unpaired Student’s t test. (B) Percent initial weight of 2.5% DSS-induced colitis in αCTLA-4 mAb-injected mice treated with H₂O + PBS, vancomycin + PBS, or vancomycin + Bifidobacterium. In each group, n = 5. **P < 0.01, ****P < 0.0001. Data show means with SEM analyzed by two-way ANOVA with Sidak’s correction for multiple comparisons. Histological score (C) and representative colon sections (D) of 2.5% DSS-induced colitis in vancomycin and αCTLA-4 mAb-treated mice oral gavaged with PBS or Bifidobacterium, n = 5 per group, **P < 0.01, unpaired Student’s t test (H&E stained). (Scale bar, 50 μm.) (E) Concentrations of KC, IL-6, and CSF3 in serum from mice oral gavaged with PBS or Bifidobacterium. These mice were also repeatedly injected i.p. with the αCTLA-4 mAb and a 6-d administration of 2.5% DSS, n = 5–7 per group. *P < 0.05; **P < 0.01, unpaired Student’s t test. B16F10 tumor growth kinetics (F) and tumor size at day 18 postimplantation (G) in mice pretreated with vancomycin followed by treatment with PBS or Bifidobacterium by oral gavage. The αCTLA-4 mAb was injected at 3, 6, 10, and 13 d posttumor implantation. n.s., not significant.

Fig. 4.

Immune regulatory function of Bifidobacterium depends on T_reg cells. (A) Percent initial weight of DSS colitis in vancomycin and αCTLA-4 mAb-treated FoxP3-DTR mice after oral gavage with PBS or Bifidobacterium under control or T_reg depletion condition (with DTX injection). Histological score (B), and concentrations of serum KC (C) were measured in DTX-injected mice in A at day 7. For each group, n = 5. Data show means with SEM analyzed by two-way ANOVA with Sidak’s correction for multiple comparisons in A. Unpaired Student’s t test in B and C. DTX, diphtheria toxin; **P < 0.01; n.s., not significant.