Fig. 1

The microbiota is necessary for oral tolerance and maintains γδ T cell populations. a Microbiota was depleted with a combination of 4 antibiotics (ABX) in the drinking water for 3 days, and control mice did not receive antibiotics (n = 10 mice each). Half of each group were then fed OVA in the drinking water for 5 days and antibiotics were maintained during the OVA feeding period. Antibiotics and OVA were stopped and 2 days later mice were immunized with OVA/CFA. Responsiveness to OVA was measured by splenocyte proliferation upon 100 µg/mL of OVA stimulation. Data are mean ± SEM; n=4–5 mice/group; one-way ANOVA. b WT mice were treated with antibiotics for 5 days and on the day after, immunologic populations related to tolerance were measured by flow cytometry and transcriptional responses in the gut was measured by Nanostring. c, d FACS plots and bar graphs showing frequencies of live CD11c⁻CD11b⁺CD103⁻CX3CR1⁺ MNPs and CD11c⁺CD11b⁻CD103⁺CX3CR1⁻ DCs (c) and CD3⁺TCRγδ⁺ γδ T cells (d) from small intestine (SI) and colonic lamina propria (LP; c, d), intraepithelial layer (IEL, d) of control (CTL) or ABX-treated mice. Data are mean ± SEM; n=4–5 mice/group; Student’s t test. * p < 0.05, ** p < 0.01. e, f Transcriptional analysis of duodenum and ileal tissue of naïve antibiotic-treated mice measured by Nanostring NCounter Mouse Immunology Panel. e Canonical pathways altered by antibiotic treatment determined by Ingenuity Pathway Analysis. f Volcano plot of genes significantly altered in the duodenum and ileum (NCounter, advanced analysis module, p < 0.05). g Expression of selected genes in both the duodenum and ileum related to immunologic populations we observed in our flow cytometry data. * p < 0.05, NCounter Advanced Analysis Module. Results are representative of at least two independent experiments