Bp0090

Wild-type and Rag1 knockout C57BL/6J mice approximately eight weeks old were purchased from Jackson Labs (Bar Harbor, Maine, USA). Each experimental group consisted of at least three to four mice. L635 (synthesized by the Chemical Synthesis Core of the Vanderbilt Institute of Chemical Biology), dissolved in dH₂O was administered by oral gavage (350 mg/kg) once a day for 3 consecutive days. Rat IgG2b isotype control antibody (BioXCell BP0090) or anti-CD90.2 antibody (BioXCell BP0066) was administered intraperitoneally (300 μg) to wild-type and Rag1 knockout C57BL/6J mice every fourth day for 12 days for a total of four injections. IL-13-tdTomato reporter mice were generated as previously described.¹⁹ These mice were the kind gift of Dr. Andrew NJ McKenzie. Archival sections of stomach from wild-type, IL-33KO, and IL-13KO mice (n=6 per group) were obtained from previous investigations.^{1 (link)} The care, maintenance, and treatment of animals in these studies adhere to the protocols approved by the Institutional Animal Care and Use Committee of Vanderbilt University.

For CD8 and CD4 T cell depletion, anti-CD8 (clone: 53–6.7, BioXcell # BP0004–1) and anti-CD4 (clone: GK1.5, BioXcell # BP0003–1) antibodies were i.p. injected per mouse according to the following regimen: day −1 and day 3 (500 μg), then every 72 hours until experiment endpoint (250 μg) (40 ). As a negative control to CD8 and CD4 depletion, mice from other groups received, by i.p. injection, InVivoPlus rat IgG2a isotype control, anti-trinitrophenol (clone 2A3, BioXcell #BP0089) and InVivoPlus rat IgG2b isotype control, anti-keyhole limpet hemocyanin (clone LTF-2, BioXcell # BP0090), respectively.

Six- to 15-week-old C57BL/6 GF mice, both male and female, were used in experiments. Mice received FMT of a 4% w/v human stool slurry in PBS, 100 μl using an 18 gauge needle (Cadence Science # 7904). Tumor cells were injected subcutaneously onto the right flank of mice 2 weeks after FMT (day 0). Culture methods and tumor cell preparation are described in supplementary methods. Once palpable, tumor growth was measured using sterile calipers every 3 days, typically starting at day 7 (with all tumors palpable by day 10) until end of experiment on days 21–22 for MC38 tumors and days 16–17 for B16F10 tumors. For each tumor, the shortest (a) and the longest (b) perpendicular diameters were measured and tumor volume was calculated using the formula: a²b/2. Mice were treated with the indicated isotype (rat IgG2b isotype control, anti-keyhole limpet hemocyanin, BioXcell, #BP0090) or anti-PD-L1 (B7-H1, clone 10F.9G2, BioXcell #BP0101) antibody by intra-peritoneal injection at 5 mg/kg every 3 days on days 10, 13, 16, and 19 for MC38 tumors or days 7, 10, 13 for B16F10 tumors. Fecal pellets were collected in a sterile manner 2 weeks after FMT (day 0) and at the end of the experiment (days 16–17, B16F10; days 21–22, MC38).

For the depletion of CD4⁺ and CD8⁺ T cells, mice were i.p. injected with 500μg anti-CD4 (BioXCell, BP0003–1) and anti-CD8 antibody (BioXCell, BP0061) every 5 days from 6 to 9.5 months of age or for memory related behavioral experiments from 6 to 8.5 months of age. IgG (BioXCell, BP0090) isotype control was administered at the same frequency and dosage. To characterize the depletion efficiency, mice were acutely treated with 500 μg anti-CD4, or anti-CD8 or IgG. Brain, meninges and blood were extracted for single cell analysis followed by flow cytometry assessment of CD4⁺ and CD8⁺ T cell populations.