Intestinal microbiota direct RegIIIγ expression in small-intestinal γδ IEL. (A) RegIIIγ mRNA was quantified by Q-PCR of isolated small-intestinal γδ IEL from germfree (gf), conventionally raised (cv-L), conventionalized (cv-D), or antibiotic-treated (Abx) mice (n = 5 mice per group; representative of two experiments). (B) γδ IEL express RegIIIγ protein. Flow cytometry was performed on total small-intestinal IEL populations from cv-L mice. Intracellular staining was carried out with anti-RegIIIγ antibodies (9), and the gating strategy is shown. TCRδ⁺/RegIIIγ⁺ cells were analyzed to verify expression of the lymphocyte marker CD103. (C) Percentages of RegIIIγ⁺ γδ IEL were determined in gf, cv-L, and cv-D mice using the strategy shown in B. n = 5 mice per group; data are from two independent experiments. Error bars represent ±SEM. *P = 0.05.

RegIIIγ expression is induced in γδ IEL by a select subset of the microbiota. (A) γδ IEL expression of RegIIIγ is triggered by a resident E. coli strain but not by B. thetaiotaomicron (B. theta). Germfree wild-type mice were gavaged with 10⁸ cfu of B. theta or an E. coli strain isolated from the microbiota of specified pathogen-free (SPF) mice (n = 4–5 mice; representative of two experiments). After 48 h, γδ IEL were isolated and RegIIIγ mRNA was quantified by Q-PCR. Colonizing bacteria were quantified by dilution plating. ns, not significant. (B) E. coli enters small-intestinal epithelial cells. Bacteria were detected by FISH using a probe against the 16S rRNA gene (green). Arrows indicate examples of tissue-associated bacteria. Cell nuclei were visualized with DAPI (blue). As previously reported, there is nonspecific diffuse autofluorescence of intestinal tissues when visualized with FITC filters (24). (Scale bar, 50 μm.) Boxed area is enlarged at right. (C) Tissue-associated bacteria were enumerated in 200 well-oriented crypt-villus units from 5 mice per group. (D) Wild-type (wt) S. typhimurium or the invasion-deficient mutant ΔSPI-1 (10⁸ cfu) were introduced orally into germfree C57BL/6 mice. After 48 h, γδ IEL were isolated and RegIIIγ mRNA was quantified by Q-PCR. Colonization levels were determined by dilution plating. n = 4–5 mice per group. Error bars represent ±SEM. *P < 0.05.

Epithelial cell MyD88 is required for RegIIIγ expression in small-intestinal γδ IEL. RegIIIγ was quantified by (A) Q-PCR of sorted small-intestinal γδ IEL and (B) flow cytometry of total IEL, performed as in Fig. 1. Gated γδ IEL populations are shown, and percentages of gated populations are given. Four to five mice were pooled per group; results are representative of three independent experiments. (C) Schematic of bone marrow chimera experiment. Ly5.1 wild-type mice were irradiated and reconstituted with bone marrow cells from Ly5.2 MyD88^−/− mice. The chimeric intestines retained residual recipient cells while also supporting engraftment of transplanted cells (40% donor/60% recipient γδ IEL). EC, epithelial cells. (D) Eight weeks after reconstitution, Ly5.1 and Ly5.2 γδ IEL were isolated by FACS and analyzed individually for RegIIIγ mRNA by Q-PCR. Each point represents an individual mouse, and the conventional experimental groups were cohoused for 5 d before sacrifice to ensure a shared microbiota. (E) Q-PCR for RegIIIγ expression in sorted small-intestinal γδ IEL. MyD88^ΔIEC, mice with an epithelial cell-specific MyD88 deletion; MyD88^fl/fl, littermates harboring two floxed MyD88 alleles. Littermates were cohoused to ensure a shared microbiota. Each point represents an individual mouse. Error bars represent ±SEM. *P < 0.05; **P < 0.01; ns, not significant.

γδ T cells limit bacterial penetration of the intestinal mucosa at early time points after bacterial challenge. (A) Wild-type S. typhimurium (10⁸ cfu) were gavaged into conventional wild-type and TCRδ^−/− mice, and splenic bacteria were quantified by dilution plating. Each point represents one mouse; data are from two independent experiments. (B) FISH analysis of conventional wild-type and TCRδ^−/− mice. Wild-type and TCRδ^−/− mice caged separately by genotype were either analyzed immediately upon removal from the SPF facility or were cohoused in the same cage for 4 h before sacrifice. Small-intestinal tissues were probed with a universal bacterial FISH probe (green) and counterstained with DAPI (blue). (Scale bars, 50 μm.) Results are representative of all mice from two independent cohousing experiments (10 mice per genotype). (C) Tissue-associated bacteria were enumerated by counting bacteria within 200 well-oriented crypt-villus units from 5 mice per genotype. (D) The percentage of γδ IEL expressing RegIIIγ was determined in wild-type mice that were caged separately from TCRδ^−/− mice and compared with wild-type mice that were not cohoused. RegIIIγ expression was determined by intracellular staining. n = 3–4 mice per group; results are representative of two independent cohousing experiments. Error bars represent ±SEM. *P < 0.05; ns, not significant.