Paxilline br Figure S Related to Figure
Figure S2. Related to Figure 2
(A) Principal components analysis (PCoA) using Bray-Curtis distance metric (left) and weighted UniFrac metric (right). The lung microbiota community is significantly different from the gut microbiota community (p < 0.01 for both Bray-Curtis and weighted UniFrac distance metrics, PERMANOVA).
(B) LEfSe plots showing differentially abundant taxa in the lung microbiome of healthy mice (red) and tumor-bearing mice (blue). Linear discriminant analysis (LDA) scores were calculated using LEfSe, with higher scores indicating greater effect size (significance determined by LDA score > 2.0 and p < 0.05 for Kruskal-Wallis test). Taxonomic categories include p = phyla, c = class, o = order, f = family, and g = genus. Taxa present at R 0.01% total relative abundance and in at least two samples were included.
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(C) SPF KP mice were left untreated or treated with metronidazole (1g/L) in drinking water starting 5 weeks post tumor initiation. Tumor burden was quantified
15 weeks post tumor initiation and representative H&E pictures are shown; fecal bacteria burden was determined by 16S based qPCR analysis. n = 7-9 mice/ group. ***p < 0.001 by Student’s t test.
(D) LEfSe plots showing the differentially abundant taxa in the lung microbiome of normal lungs (red) and lung adenocarcinoma (LUAD) or lung squamous cell carcinoma (LUSC) samples (green) based on PathSeq analysis of the TCGA cohort.
Figure S3. Related to Figure 3
(A) The frequency of gd T Paxilline in total CD3+ lymphocytes in the lung, blood, spleen or draining lymph node from SPF and GF KP mice as determined by flow cytometry.
(B) Representative pictures and quantification of immunohistochemistry staining of human TCRd on formalin-fixed paraffin-embedded normal lung (NL) and LUAD tissue samples. Positively stained cells are in purple.
(C and D) RORgt and IL-17A expression in total CD3+ lymphocytes from the tumor-bearing lungs from SPF mice and GF mice. Representative flow cytometric plots are shown (C) and the frequency of IL-17A+ CD4 T cells (Th17) is quantified (D).
Figure S4. Related to Figure 4
(A) KP mice on the CD45.1 background were lethally irradiated and transplanted with bone marrow from CD45.2 donors. Seven weeks after reconstitution, mice were infected with adenovirus expressing Sftpc-Cre, and 15 weeks after tumor initiation, gd T cells in the tumor-bearing lungs were analyzed by flow cytometry. The percentage of donor versus recipient derived cells was quantified in the Vg4+ and Vg6+ subsets, as well as the RORgt+ and Tbet+ compartments. Representative plots are shown and data represent 15 mice.
(B) The proliferation of RORgt- gd T cells and Th17 cells in the tumor-bearing lungs from tumor-bearing SPF mice and GF mice was assessed by flow cytometric analysis of Ki67 expression.
(C) IL-17A expression in lung-infiltrating gd T cells from healthy SPF mice, tumor-bearing SPF mice and tumor-bearing GF mice was analyzed by flow cytometry.
(D) SPF KP mice were treated with combined antibiotics (4Abx) starting 6.5 weeks after tumor initiation. The frequency of IL-17A-producing gd T cells and IL-17A concentration in BALF were analyzed by flow cytometry and ELISA respectively.
(E) The abundance of gd T cells, and the expression of RORgt and IL-17A in gd T cells were analyzed by flow cytometry in GF mice and ex-GF mice that were exposed to the microbiome via cohousing with SPF mice.
Figure S5. Related to Figure 5
The expression of Tbet, IFNg and TNFa in gd T cells from the tumor-bearing lungs from SPF and GF mice were analyzed by flow cytometry.
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Figure S6. Related to Figure 6