CD103⁺ Foxp3⁺ T cells are present in the mesenteric lymph nodes and spleen of T-cell reconstituted mice. T cells were isolated from the lamina propria of mice reconstituted with unfractionated CD4⁺ T cells or with T cells depleted from CD103⁺ cells. The expression of CD103 and Foxp3 was determined in the CD3⁺ CD4⁺ gate by flow cytometry analysis.

Intraepithelial DCs are associated with DC aggregates and display a mature phenotype. A and B: Double staining of CD11c (red) to label DCs and laminin (green) to visualize the basal membrane. Intraepithelial CD11c⁺ DCs (red) located at the luminal side of the basal membrane are predominantly found in association with DC aggregates (arrowheads in B). Note a DC passing a gap in the basal membrane (arrow in B). Single immunohistochemical staining of MHC-II (C) reveals predominant expression of MHC-II (C), CD40 (D), CD80 (E), and CD86 (F) in DC aggregates. Original magnifications: ×10 (A, C–F), ×40 (B).

Foxp3 expression in the large intestine. The mRNA expression (A) of Foxp3 was quantified applying CD3 as a housekeeping gene as explained in the legend to Figure 3 and is shown as the mean and SD of three measurements. P values are given as determined by Student’s t-test. Asterisks indicate no detectable mRNA expression. Light columns indicate the lamina propria; dark columns indicate DC aggregates. B–E: Immunohistochemistry with a monoclonal Foxp3 antibody reveals nuclear labeling of cells in the medullary thymus (B, right) but not in the cortex (B, left), proving the specificity of the staining. In the large intestine of mice at day 14 after transplantation (C, D), several Foxp3-expressing cells are found in DC aggregates, whereas few labeled cells are seen in the lamina propria. E: At day 28 after transplantation, Foxp3⁺ cells are present at low density in the severely inflamed gut. Arrow in E indicates a crypt abscess. Original magnifications: ×20 (B, C, E); ×40 (D).

IL-23p19 and IFN-γ expression in microcompartments of the large intestine. Tissue from the lamina propria (A, top row) and DC aggregates (A, bottom row) was obtained by microdissection and subjected to quantitative RT-PCR. The process of microdissection is illustrated in A, showing histological sections stained with hematoxylin (left), after laser microdissection and removal of the epithelial crypts (center), and after catapulting the dissected tissue compartments (right). Epithelia (including intraepithelial lymphocytes) were carefully excised to minimize contamination of microdissected lamina propria with these components. The mRNA expression of IL-23p19 (B) and IFN-γ (C) was determined in the lamina propria (light columns) and DC aggregates (dark columns) in untransplanted RAG^−/− mice (day 0), at day 14 and day 28 after CD4⁺ T-cell transfer. The mean mRNA expression (of three samples) and the SD were calculated after normalization to HPRT (B) and CD3 (C), respectively, and are shown in arbitrary units. P values are given as determined by Student’s t-test. Asterisks indicate no detectable mRNA expression. Original magnifications, ×10.

Accumulation of donor T cells in DC aggregates of the large intestine that contain c-kit⁺ and IL-7R⁺ cells. RAG^−/− were reconstituted with CD4⁺ T cells and analyzed 2 or 4 weeks after transplantation. At 2 weeks (A), H&E staining of the large intestine revealed mild inflammatory changes, whereas severe colitis was manifest 4 weeks after reconstitution (B). C: Histological colitis scores are depicted as the mean and SD of five mice per group. The P value is given as determined by Student’s t-test. D and E: Genetically tagged CD4⁺ T cells from eGFP-transgenic donor mice were used to track their colonization of the intestinal mucosa in the adoptive host. D: Two weeks after transplantation, T cells selectively accumulated in conspicuous clusters in the colonic mucosa. Four weeks after T-cell transfer, a dense and diffuse infiltration of the lamina propria by eGFP⁺ T cells was seen (E) but T-cell clustering was not prominent. Double staining of CD3 (green) and CD11c (red) of intestinal tissue at 2 weeks after T-cell transfer (F, G) confirmed T-cell accumulation in CD11c⁺ DC aggregations in the large intestine (F) while few T cells clustered in the small intestine (G). CD11c⁺ (red) DC aggregates of untransplanted RAG^−/− mice harbored numerous c-kit⁺ cells (green, H) and IL-7R⁺ cells (green, I). Original magnifications: ×10 (A, B, D–G); ×20 (H, I).

TGF-β1 expression in the large intestine and appearance of lamina propria CD103⁺ T_reg cells in RAG^−/− mice transplanted with CD103⁻ CD4⁺ T cells that suppress a Th1 response. A: The mRNA expression of TGF-β1 was determined using HPRT as a housekeeping gene, as explained in the legend to Figure 3, and is given as the mean and SD of three measurements. P values are given as determined by Student’s t-test. Light columns indicate the lamina propria, dark columns indicate DC aggregates. B: RAG^−/− mice were reconstituted with unfractionated CD4⁺ T cells (CD103^+/− graft, top) and CD4⁺ T cells rigorously depleted of CD103⁺ T cells (CD103⁻ graft, bottom). After 2 weeks, lamina propria cells were isolated and the CD103 and Foxp3 expression was determined in the CD3⁺ CD4⁺ gate by flow cytometry analysis. High numbers of CD103⁺ T cells in the lamina propria of reconstituted mice were found independent of the CD103⁺ depletion status of the graft. A substantial fraction of CD103⁺ cells co-expresses Foxp3. C: Ovalbumin-specific transgenic CD4⁺ T cells were challenged with ovalbumin-pulsed spleen DCs in the presence of CD103⁺ or CD103⁻ T cells isolated from the lamina propria of T-cell reconstituted mice. Controls were performed with ovalbumin-pulsed or nonpulsed DCs in the absence of a suppressor T-cell population. IFN-γ production was determined in the supernatant by enzyme-linked immunosorbent assay and is shown as the mean and SD of three measurements. P values are given as determined by Student’s t-test. CD103⁺, but not CD103⁻, T cells suppressed IFN-γ production. CD103⁺ T cells isolated from the lamina propria of mice reconstituted with unfractionated CD4⁺ T cells or with T cells depleted from CD103⁺ cells displayed similar inhibitory capacities.

CD103⁺ CD25⁻ T cells colonize DC aggregates of transplanted mice. Double-immunofluorescence staining of large intestinal tissue of RAG^−/− mice obtained at day 14 after T-cell reconstitution was performed using anti-CD25 (green) together with anti-CD3 (red) or anti-CD103 (green) in combination with anti-CD3 (red). CD25⁺ cells are seen in ILAs (box in A). B: High magnification shows that CD25⁺ cells in the boxed area do not co-express CD3. Single-immunohistochemical staining reveals large numbers of CD25⁺ cells in DC aggregates of untransplanted RAG^−/− mice (C, arrows). Numerous CD103⁺ cells are present in DC aggregates (box in D) but are also found in the lamina propria outside these structures. At high magnification, a substantial number of CD103⁺ cells reveals co-expression of CD3 (E). Double-positive cells appear yellow/orange. F: The proportion of CD103⁺ T cells in DC aggregates and in the lamina propria was determined by counting ≥200 CD3⁺ cells per compartment in immunofluorescently stained tissue sections. Columns indicate the mean of three mice; error bars indicate the SD. The P value is given as determined by Student’s t-test. G: From CD4⁺ lamina propria T cells (top left) of RAG^−/− mice at day 14 after reconstitution, CD103⁺ and CD103⁻ T cells were sorted (top right), and the purity of the CD103⁻ (bottom left) and CD103⁺ (bottom right) fractions was verified by reanalysis. H: The relative expression of Foxp3 was determined by quantitative RT-PCR and is indicated as the mean and SD of three measurements. The P value is given as determined by Student’s t-test. A high Foxp3 expression is detectable only in the CD103⁺ fraction. Original magnifications: ×10 (A, C, D); ×40 (B, E).