Characterization of Foxp3+CD4+CD25+ and IL-10-secreting CD4+CD25+ T cells during cure of colitis

Abstract

CD4+CD25+ regulatory T cells can prevent and resolve intestinal inflammation in the murine T cell transfer model of colitis. Using Foxp3 as a marker of regulatory T cell activity, we now provide a comprehensive analysis of the in vivo distribution of Foxp3+CD4+CD25+ cells in wild-type mice, and during cure of experimental colitis. In both cases, Foxp3+CD4+CD25+ cells were found to accumulate in the colon and secondary lymphoid organs. Importantly, Foxp3+ cells were present at increased density in colon samples from patients with ulcerative colitis or Crohn's disease, suggesting similarities in the behavior of murine and human regulatory cells under inflammatory conditions. Cure of murine colitis was dependent on the presence of IL-10, and IL-10-producing CD4+CD25+ T cells were enriched within the colon during cure of colitis and also under steady state conditions. Our data indicate that although CD4+CD25+ T cells expressing Foxp3 are present within both lymphoid organs and the colon, subsets of IL-10-producing CD4+CD25+ T cells are present mainly within the intestinal lamina propria suggesting compartmentalization of the regulatory T cell response at effector sites.

Figure 1. Expression of Foxp3 by CD4⁺CD25⁺ T cells in the lymphoid organs and colon of wild-type BALB/c mice.

(a) Wild-type MLN stained for Foxp3 and DAPI. (b) Wild-type spleen and (c) Foxp3^−/− spleen stained for CD4 and Foxp3. (d) Overview and (e) high power magnification of wild-type MLN stained for CD4, CD25, and Foxp3 and analysed using laser-scanning microscopy. (f–i) Wild-type spleen (f), MLN (g), and colon (h) stained for Foxp3, CD25⁺, and MHC-II and analysed using conventional fluorescence microscopy. (h) and (i) show the same section of colon at different magnifications. (j) Percentage of Foxp3⁺ among CD25⁺ cells (filled symbols) and Foxp3⁺ among CD25⁻ cells (open symbols) in the T cell area of spleen, MLN, and colon of BALB/c mice. The quantification is based on the approximate number of cells per area of lymphoid organ as indicated by the DAPI nuclear staining. Statistical significance was tested using the Mann- Whitney U test. Similar expression patterns of Foxp3 were found in spleen, MLN, and colon of C57BL/6 mice. k, Expression of Foxp3 mRNA by splenic CD4⁺CD25⁺CD45RB^low and CD4⁺CD25⁻CD45RB^low T cells. Foxp3 mRNA expression was normalized to CD3γ expression. Data from RNA expression analysis of three independent FACS sorts were pooled.

Figure 2. Presence of Foxp3⁺CD4⁺CD25⁺ T cells in the colons of mice with colitis and mice cured of colitis.

Rag-1^−/− mice were injected with 4 × 10⁵ wild-type CD45.2⁺CD4⁺CD45RB^high T cells. Four weeks after the T cell transfer, wasting disease and colitis were evident, and a second transfer of 1 × 10⁶ congenic CD45.1⁺CD4⁺CD25⁺ T cells was performed. Sections of colon taken either 2 (a) or 10 (b) weeks after the second T cell transfer were stained with H&E, Abs to CD4 and CD45.1, or Abs to Foxp3 and CD45.1. DAPI counterstaining was performed to visualize the tissue structure. E indicates epithelium. The presented micrographs are representative of three to five mice analysed.

Figure 3. Presence of Foxp3-positive cells in the mucosa and intestinal lymphoid follicles of patients with IBD.

Colon samples from normal controls and patients with ulcerative colitis (UC), Crohn’s disease (CD), non-IBD inflammations of the colon (diverticulitis (D), pseudomembranous colitis (PC), and CMV-induced colitis (CMV)) were analysed for CD3, CD4, and Foxp3. Sections from tonsil were analysed for CD4 and Foxp3. (a) Co-staining for CD4 and Foxp3 on human tonsil. Overview (×100) and high-power magnification. (b) CD3 and Foxp3 staining on colon tissue. (c) Density of CD3⁺ and Foxp3⁺ cells within colon. Numbers of cells were quantified per area at ×400 magnification. Each data point represents one patient. (d) Density of Foxp3⁺ cells within colonic LP, as well as T and B cell areas of mucosa- associated lymphoid tissue. Each data point represents one patient. (e) Co-staining of Foxp3 and IL-10 on human appendix tissue. Left, Co-staining of Foxp3, IL-10, and DAPI showing IL-10-positive cells within the germinal center (GC) and subepithelial dome (SED) close to the epithelium (E). Detail of Foxp3⁺IL-10⁺ cells within the subepithelial dome area are also shown. Right, Co-staining of Foxp3, IL-10, and CD3. Detail shows IL-10⁺CD3⁺Foxp3⁺ cells as well CD3⁺Foxp3⁺ cells that are in close contact with IL- 10+CD3⁻Foxp3⁻ cells.

Figure 4. Cure of colitis depends on the presence of IL-10 from CD4⁺CD25⁺ T cells, as well as other cell types.

Colitis was induced by transfer of 4 × 10⁵ CD4⁺CD45RB^high T cells into CB17 SCID mice. After development of colitis, mice were injected with 1 × 10⁶ CD4⁺CD25⁺ T cells from wild- type mice together with anti-IL-10R mAb or isotype control Abs. Further groups of colitic mice received either CD45RB^high or CD4⁺CD25⁺ T cells from IL-10^−/− mice. Sections of colon were scored for the severity of colitis. Score values above 2 indicate mice with pronounced colitis. Data are pooled from four independent experiments.

Figure 5. Accumulation of the IL-10-producing progeny of CD4⁺CD25⁺ T cells within the colon.

Rag-1^−/− mice were injected with 4 × 10⁵ wild-type CD45.2⁺CD4⁺CD45RB^high T cells. After development of colitis, mice received a second transfer of 1 × 10⁶ congenic CD45.1⁺ CD4⁺CD25⁺ T cells. After 4 wk, the ability of CD4⁺CD45RB^high (n = 6) and CD4⁺CD25⁺ (n = 6) progeny to produce IL-10 and IFN-γ was analysed. Lymphocytes were prepared from spleen, MLN, and the colonic LP and stimulated with PMA/ionomycin and brefeldin A. The results of two independent experiments are pooled. (a) Representative FACS plot showing preferential IL-10 production by LP CD4⁺CD25⁺ T cell progeny (CD45.1⁺) and preferential IFN-γ production by the CD4⁺CD45RB^high T cell progeny (CD45.1⁻). (b) Production of IL-10 and IFN-γ by CD4⁺CD25⁺ T cell progeny and comparison of IL-10 and IFN-γ production by CD4⁺CD45RB^high T cell progeny in the presence or absence of CD4⁺CD25⁺ T cells. Data are shown for spleen, MLN, and LP. Significance was tested using the Mann-Whitney U test; n.s., not significant.

Figure 6. IL-10 secretion by Foxp3⁺ cells in the LP of wild-type mice.

Lamina propria and spleen cell suspensions were restimulated with PMA/ionomycin as described in Materials and Methods. (a) Representative dot plots showing Foxp3 expression against IL-10 or an isotype control in spleen and LP of B6 SJL CD45 congenic mice. Plots are gated on CD4⁺ cells. The numbers indicate the percentage of IL-10⁺ cells in the Foxp3⁻ or Foxp3⁺ populations. (b) Percentage of IL-10-secreting cells in the Foxp3⁻ (filled symbols) and Foxp3⁺ (open symbols) populations from spleen (Sp) and LP CD4⁺ lymphocytes (LPL) from B6 SJL CD45 congenic mice. Each symbol represents data from an individual mouse. In all cases, the isotype control stained <1% of total cells. Similar results were obtained in an experiment using BALB/c mice.