(A) Schematic of tumor growth model in SPF mice with antibiotic treatment and oral enterococci supplementation. Days are indexed based on the day of tumor injection. Mice were provided antibiotic-containing water ab libitum for two weeks followed by water supplemented with the indicated enterococci for the remainder of the experiment. Animals were then subcutaneously implanted with B16-F10 melanoma cells, and tumor volume measurements started when tumors reached ~50-100 mm3 (day 5). Mice were treated with anti–PD-L1 by intraperitoneal injection every other day starting two days after the start of the tumor measurement. For all data except for (B), 20 μg anti–PD-L1 was used for each injection. (B) B16-F10 tumor growth in antibiotic-treated animals that were supplemented with or without E. faecium (Efm) Com15 and treated with or without anti–PD-L1 starting on day 7 at doses indicated in the legend. n = 7-8 mice per group. (C) B16-F10 tumor growth in antibiotic-treated mice that were supplemented with the indicated E. faecalis (Efs) and E. faecium (Efm) strains and treated with anti–PD-L1 starting on day 7. n = 7-8 mice per group. (D) Colony forming unit (CFU) analysis of E. faecalis (Efs) and E. faecium (Efm) strains in fecal samples harvested from animals as treated in (C). (E) B16-F10 tumor growth in antibiotic-treated mice that were supplemented with the indicated enterococci strains and treated with anti–PD-L1 starting in day 7. n = 8-9 mice per group. (F) CFU analysis of enterococci in fecal samples harvested from animals as treated in (E). nd = not detected. For (B), (C), and (E), data represent mean ± s.e.m. and were analyzed by mixed-effects model with Tukey’s multiple comparisons post-hoc test. *P < 0.05, **P < 0.01, ****P < 0.0001, ns = not significant. For (D) and (F), each symbol represents one mouse. Dotted lines indicate the limit of detection (2,000 CFU g−1). Data represent means ± 95% confidence interval.