Volume 126, Issue 7, Pages 1759-1770 (June 2004) Colitis is associated with thymic destruction attenuating CD4+25+ regulatory T cells in the periphery  William A. Faubion, Ype P. De Jong, Ana Abadia Molina, Hongbin Ji, Kareem Clarke, Baoping Wang, Emiko Mizoguchi, Stephen J. Simpson, Atul K. Bhan, Cox Terhorst  Gastroenterology  Volume 126, Issue 7, Pages 1759-1770 (June 2004) DOI: 10.1053/j.gastro.2004.03.015

Figure 1 Neonatally transplanted tgε26 mice develop TR cells. (A) T-cell subsets develop normally in thymus, lymph nodes, and spleen of BM→tgε26neo mice. Eight weeks after transfer of wt bone marrow into tgε26 neonates (<9 days old, BM→tgε26neo), cells were analyzed by flow cytometry. (B) The thymus of BM→tgε26neo contains CD4+CD25+ cells. Eight weeks after transfer of wt bone marrow into tgε26 neonates (<3 days old), thymocytes were analyzed by flow cytometry. After gating on CD4+ single-positive thymocytes, the percentage of CD4+CD25+cells was determined. The negative control used is an isotype-matched IgG, as described in the Materials and Methods section. Results shown are a representative group of experiments performed with a minimum of 4 mice per group and repeated 3 times. (C) CD4+CD25+ thymocytes isolated from BM→tgε26neo mice suppress in an in vitro assay. In a representative experiment (1 of 4), varying amounts of CD4+CD25+ thymocytes isolated from 8 BM→tgε26neo mice were cocultured with 5 × 104 CD4+CD25− cells isolated from the same pool of thymocytes (responder cells) as per Materials and Methods section. Thymidine incorporation was determined after an 18-hour incubation with [3H]-thymidine, 1 μCi per well. The X-axis represents the ratio of CD4+CD25− responder T cells: CD4+CD25+ TR cells. Each subset of T cells was also incubated alone with antigen-presenting cells and anti-CD3 (the difference between the median of triplicate values representing proliferative rates of responder cells and cocultured cells is statistically significant; ∗P < .05). Gastroenterology 2004 126, 1759-1770DOI: (10.1053/j.gastro.2004.03.015)

Figure 2 Thymic involution in BM→tgε26adult mice coincides with the onset of colitis. (A) The inverse relationship between number of thymocytes and histologic colitis score in BM→tgε26adult mice that had been treated with either anti-TNF-α or with an isotype-matched control IgG. Adult tgε26 mice were killed at different time points after BM transplantation and thymocytes were analyzed. Each time point was determined with 3 mice, and the experiment repeated 3 times. The thymocyte number and histologic colitis score are the mean of triplicate values. Closed circles: Number of thymocytes in control IgG treated mice. Open circles: Number of thymocytes in mice treated with anti-TNF-α. Closed triangles: Histologic colitis score in control IgG-treated mice. Open triangles: Histologic colitis score in mice treated with anti-TNF-α. (B Percentage CD4+ and CD8+ thymocytes in BM→tgε26adult mice. At 6 time points selected from above (A) after determination of thymocyte number, the cells were analyzed by FACS. The rapid decline in thymocyte number is associated with primarily a reduction in the percentage of double-positive thymocytes. Gastroenterology 2004 126, 1759-1770DOI: (10.1053/j.gastro.2004.03.015)

Figure 3 Prevention of thymic destruction in BM→tgε26adult mice that were treated with anti-TNF-α and soluble LTβ receptor-Fc or in tgε26adult mice that had been transplanted with STAT4−/− bone marrow. BM→tgε26 mice were treated with anti-TNF-α, LTβR-Fc or with a combination of these agents. Alternatively, tgε26 mice were transplanted with Stat-4−/− BM instead of wt bone marrow as described.12,17 Mice were analyzed 5 weeks after transplantation by DAI and histology, as described in the Materials and Methods section, and thymuses were analyzed by flow cytometry. Results shown are representative of 3 independent experiments with 3 mice per group. (A) Number of thymocytes in BM→tgε26adult mice treated with different modalities to prevent colitis. Thymuses from the following groups of mice were analyzed: Treatment groups: Treatment (dosage and frequency)17 Ctrl mAb: Control mouse IgG2a, (250 μg twice weekly), isotype matched with anti-mouse-TNF-α. Ctrl Ig: Polyclonal human IgG, (100 μg once a week) used as a control for soluble LTβR-Fc. Anti-TNF: Anti-mouse-TNF-α (IgG2a) (250 μg twice weekly). sLTβR: Soluble LTβR-Fc (100 μg once a week). Anti-TNF + sLTβR: A combination of monoclonal anti-mouse-TNF-α and soluble LTβR-Fc. STAT-4: Transplantation with STAT-4−/− instead of wt bone marrow into tgε26 adult mice (see Materials and Methods section12). Each experiment was performed with at least 4 mice per treatment group and repeated 3 times (with the exception of combination anti-TNF and LTβR-Fc treatment, which was repeated once). All thymocyte numbers are plotted (note: numbers in control antibody treatment groups are smaller because some untreated mice do not survive 4 weeks). (B) Percentages of the major CD4+ and CD8+ thymocyte subsets in BM→tgε26adult mice treated with different modalities. Thymocytes from BM→tgε26adult mice 4 weeks after transplantation were stained with phycoerythrin-conjugated anti-CD4 and FITC-conjugated anti-CD8 and analyzed by flow cytometry. The 5 panels represent (clockwise from top left) either untransplanted or transplanted mice treated with control antibody, anti-TNF-α, Stat-4−/− bone marrow, or LTβR-Fc. The flow cytometry data are from 1 mouse per treatment group, representative data from at least 3 mice per group repeated at least 4 times. (C) Immunohistochemical analysis of thymuses from BM→tgε26adult mice that had received LTβR-Fc treatment. Histologic “normalization” as defined by clear organization of cortical and medullary zones was observed in LTβR-Fc treated BM→tgε26adult mice (Panels 2, 4, 6, 8, right column) but not control IgG-treated mice (Panels 1, 3, 5, 7, left column). Panels 1 and 2 (top row): H&E staining. Panels 3 and 4 (second row): staining of cortical TEC with ER-TR4. Panels 5 and 6 (third row): staining of medullary TEC with ER-TR5. Panels 7 and 8 (bottom row): staining of medullary dendritic cells with N418. Gastroenterology 2004 126, 1759-1770DOI: (10.1053/j.gastro.2004.03.015)

Figure 4 TR cells develop both in the thymus and in the periphery of BM→tgε26adult mice in the absence of colitis. Adult tgε26 mice simultaneously received wt BM and either LTβR-Fc or polyclonal human IgG (the “untreated” controls). Adult tgε26 mice, which simultaneously received wt BM and 2 × 105 wt CD4+25+ TR cells (collected from the spleens of wt mice), represented the “positive” controls. Four weeks after transplantation, CD4+25+ TR cells and CD4+25− responder cells were isolated by FACS from the spleens of 6 mice in each group. One of 3 independent experiments is shown. (A) Treatment of BM→tgε26 mice with LTβR-Fc or wt TR prevents disease. The disease activity index and histologic index of BM→tgε26 mice treated with LTβR-Fc (solid circles) or with wt TR cells (solid squares). The disease activity index and histologic index of BM→tgε26 mice treated with control IgG are indicated by the open circles. The median DAI scores/histologic scores between BM→tgε26adult mice and both treatment groups are significantly different (P < 0.05). (B) CD4+25+ TR cells from BM→tgε26adult mice that had been treated with LTβR-Fc suppress proliferation of CD4+CD25− responder cells in vitro. For the suppressor cell assay, TR cells and responder cells were mixed in ratios of 1:0.5 or 1:1 or were used alone (CD25+ or CD25−). CD25− cells were stimulated with APCs and soluble anti-CD3 in cocultures for 72 hours as described in the Materials and Methods section. [3H]-thymidine uptake was determined after an additional 18-hour incubation. Open bar, BM→tgε26 mice treated with control IgG. Closed bar, BM→tgε26 mice treated with LTβR-Fc. Hatched bar, BM→tgε26 mice treated with wild-type TR cells. (∗P < .05 for both treatment groups compared with BM→tgε26adult group.) (C) CD4+25+ TR cells from BM→tgε26adult mice express less Foxp3 than CD4+25+ TR cells from control mice. CD4+CD25+ T cells were sorted from the splenocytes of either wt (control) or tgε26 mice 4 weeks after bone marrow transplantation. The total RNA was purified by Trizol, and Foxp3 expression was measured by RT-PCR. Gastroenterology 2004 126, 1759-1770DOI: (10.1053/j.gastro.2004.03.015)

Figure 5 Thymic dissolution coincides with colitis in the CD45Rbhi→Rag−/− transfer model. The effect of colitis on thymic development was examined in the CD45Rbhi→Rag−/− transfer model using C57Bl/6 Rag−/− recipients that expressed the F5 αβTCR transgenes.19 Donor cells were derived from the tgTCR F5 × Rag−/− (C57Bl/6) mouse whose CD8 cells express an αβTCR that specifically recognizes an influenza NP peptide presented by MHC Class I (C57Bl/6; H-2b). (A) Schematic outline of the modified CD45Rbhi→Rag−/− transfer experiment. Rag−/− mice (C57Bl/6) received either syngeneic F5 × Rag−/− BM or syngeneic CD45Rbhi cells alone or received simultaneously F5 × Rag−/− BM and CD45Rbhi cells as described in the Materials and Methods section. Recipient mice were killed after 5 weeks for analyses. (B) Cotransfer of CD45Rbhi cells reduces the size of the F5 × Rag−/− BM→Rag−/− recipients. The number of thymocytes in Rag−/− mice that had received F5 × Rag−/− BM and/or CD45Rbhi cells was counted in each individual mouse. Each experimental group contained at least 6 mice, and the experiment was performed twice with similar results. CD45Rbhi: CD45Rbhi→Rag−/− recipients (n = 7). F5 BM: F5 × Rag−/− BM→Rag−/− recipients (n = 6). CD45Rbhi + F5 BM: Simultaneous transfer of CD45Rbhi cells and F5 × Rag−/− BM→Rag−/− recipients (n = 6). (C) Cotransfer of CD45Rbhi cells eliminates CD4+8+ DP thymocytes of the F5 × Rag−/− BM→Rag−/− recipients. Expression of CD4 and CD8 was analyzed by flow cytometry, as described in the Materials and Methods section. The percentage of each subset is indicated. The thymus from each mouse was examined by flow cytometry, and representative analyses are shown. (D) CD4+ SP thymocytes derived from CD45Rbhi donor cells express detectable levels of intracellular TNF-α and IFN-γ. (Upper panel) CD4+ SP thymocytes from Rag−/− mice that had been transplanted with both F5 × Rag−/− BM and CD45Rbhi cells were stimulated with anti-CD3ε (0.5 μg/mL) and stained for intracellular TNF-α and IFN-γ. (Lower panel) The same cells were stained with isotype-matched IgG as described in the Materials and Methods section. Gastroenterology 2004 126, 1759-1770DOI: (10.1053/j.gastro.2004.03.015)

Figure 6 Cotransfer of TR cells with CD45Rbhi cells prevents destruction of the thymus of female HY × Rag−/− recipient mice. (A) Outline of the experiment. Female HY × Rag−/− mice (HY-Rag−/−) were transplanted with wt CD45Rbhi T cells and wt CD4+ CD25+ TR cells. For analysis, mice were killed 5 weeks after transfer. The experiment was performed with 3 mice in each group and repeated 3 times. At the time of death, the thymus from each mouse was examined by flow cytometry, and representative analyses are shown. (B) CD4 and CD8 expression of thymocytes in HY-Rag−/− recipients. Expression of CD4 and CD8 was analyzed by flow cytometry. The percentage of each subset is indicated. Sources of thymocytes: Female HY × Rag−/− mice, female HY × Rag−/− mice transplanted with syngeneic CD45Rbhi cells (CD45Rbhi>Rag), and female HY × Rag−/− mice transplanted with syngeneic CD45Rbhi cells and CD4+ CD25+ TR cells (CD45Rbhi CD25+>Rag). Gastroenterology 2004 126, 1759-1770DOI: (10.1053/j.gastro.2004.03.015)