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CTLA-4 Controls Regulatory T Cell Peripheral Homeostasis and Is Required for Suppression of Pancreatic Islet Autoimmunity

Emily M. Schmidt, Chun Jing Wang, Gemma A. Ryan, Louise E. Clough, Omar S. Qureshi, Margaret Goodall, Abul K. Abbas, Arlene H. Sharpe, David M. Sansom and Lucy S. K. Walker
J Immunol January 1, 2009, 182 (1) 274-282; DOI: https://doi.org/10.4049/jimmunol.182.1.274
Emily M. Schmidt
*Medical Research Council Centre for Immune Regulation, University of Birmingham Medical School, Birmingham, United Kingdom;
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Chun Jing Wang
*Medical Research Council Centre for Immune Regulation, University of Birmingham Medical School, Birmingham, United Kingdom;
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Gemma A. Ryan
*Medical Research Council Centre for Immune Regulation, University of Birmingham Medical School, Birmingham, United Kingdom;
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Louise E. Clough
*Medical Research Council Centre for Immune Regulation, University of Birmingham Medical School, Birmingham, United Kingdom;
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Omar S. Qureshi
*Medical Research Council Centre for Immune Regulation, University of Birmingham Medical School, Birmingham, United Kingdom;
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Margaret Goodall
*Medical Research Council Centre for Immune Regulation, University of Birmingham Medical School, Birmingham, United Kingdom;
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Abul K. Abbas
†Department of Pathology, University of California, San Francisco, CA 94143; and
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Arlene H. Sharpe
‡Department of Pathology, Harvard Medical School and Brigham and Women’s Hospital, Boston, MA 02115
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David M. Sansom
*Medical Research Council Centre for Immune Regulation, University of Birmingham Medical School, Birmingham, United Kingdom;
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Lucy S. K. Walker
*Medical Research Council Centre for Immune Regulation, University of Birmingham Medical School, Birmingham, United Kingdom;
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  • FIGURE 1.
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    FIGURE 1.

    CTLA-4-deficient mice have increased numbers of Foxp3+ cells. LN, spleen (Sp), and thymus cells from 15- to 17-day-old CTLA4−/− mice (CT−/−) or littermate CTLA4+/− mice (CT+/−) were stained for CD4, Foxp3, and CD25. A, Representative FACS stain for Foxp3 and CD25 on LN cells. B, Percentage of CD4 cells that express Foxp3 in LN or spleen. Each symbol represents a single mouse. C, Absolute numbers of CD4+Foxp3+ or CD4+Foxp3− cells in LN or spleen. Bars show means from four to eight mice with SD. D, Representative thymus stains on 15-day-old CTLA4−/− mice or littermate controls. Lower panel is gated on CD4+CD8− cells: the percentage of Foxp3+ within this population was not significantly different between CTLA4−/− mice and littermate controls (4.62% ± 0.58 and 4.73% ± 0.75 respectively, n = 4). E, Fold increase in absolute number of CD4+Foxp3+ and CD4+Foxp3− cells between CTLA4−/− mice and littermate controls. Fold increase was calculated using mean values from four to eight mice.

  • FIGURE 2.
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    FIGURE 2.

    Foxp3+ cells in the periphery of CTLA-4-deficient mice show increased proliferation. A, Spleen sections from 17-day-old CTLA4−/− mice or littermate controls (CTLA4+/−) were stained for CD4 (blue), Foxp3 (red), and Ki67 (green). Proliferating Treg appear yellow. Lower panel shows scoring of stained sections for the percentage of Foxp3+ cells that are Ki67+. Each symbol represents a different field of view and each column a different mouse. In brief, ≥5 fields were scored per mouse. Mean values were significantly different (p < 0.05). B, Spleen cells from 17-day-old CTLA4−/− mice or littermate controls (CTLA4+/−) were stained for CD4 and intracellular Foxp3 and Ki67. Plots are gated on CD4+Foxp3+ cells. Treg proliferation in age-matched wild-type mice (CTLA-4+/+) was similar to that seen in CTLA-4+/− mice. Data are representative of two (A) or more than five (B) independent experiments.

  • FIGURE 3.
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    FIGURE 3.

    Analysis of Ag-specific Treg deficient in CTLA-4. Treg were quantified by Foxp3 staining in DO11 × RIP-mOVA/rag−/− mice sufficient or deficient for CTLA-4. Ag-negative DO11 mice were used as controls. All mice were rag gene deficient. A, Representative Foxp3 staining in gated CD4+ (spleen) or CD4+CD8− (thymus (Thy)) cells from 3-wk-old animals. B, Percentage of CD4 cells (CD4+CD8− in thymus) that express Foxp3 at the indicated time after birth in DO11 × RIP-mOVA/rag−/− or DO11 × RIP-mOVA/ctla4−/−rag−/− mice. Each symbol represents an individual mouse. C, Absolute number of CD4+CD8−Foxp3+ (thymus) or CD4+Foxp3+ cells (spleen) in 6-wk-old mice of the indicated genotype. D, Pooled pancreata from DO11 × RIP-mOVA/rag−/− or DO11 × RIP-mOVA/ctla4−/−rag−/− mice were digested and stained for CD4, Foxp3, and Ki67. Plots are gated on CD4+ cells and are representative of two similar experiments using pooled pancreas tissue from two to three animals.

  • FIGURE 4.
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    FIGURE 4.

    Anti-CTLA-4 Ab increases Treg proliferation in vivo. A, BALB/c mice were injected with 500 μg of anti-CTLA-4 Ab (or control hamster IgG) on days 0, 2, and 6 and LN were harvested on day 8. Cells were stained for CD4 and intracellular Foxp3 and Ki67. Representative FACS stains showing Ki67 expression in BALB/c LN cells gated on CD4+Foxp3+ or CD4+Foxp3− cells. Proliferation of CD4+Foxp3+ cells was increased in mice treated with anti-CTLA-4 Ab compared with control Ab (30.68 ± 0.9% vs 13.85 ± 2.6%, respectively; n = 4). B, BALB/c mice were treated with twice weekly injections of anti-CTLA-4 Ab or control Ab and analyzed at the indicated time points. The percentage of Ki67+ cells within the CD4+Foxp3+ (Treg) population and the CD4+Foxp3− (T cell) population in LN is shown. Each bar shows the mean value for three to six individual mice with SD (∗∗, p < 0.005). Values from control Ab-treated mice were similar at all time points and are presented as a single pooled control (individual controls were used for statistical analysis). C, CD69 staining in gated CD4+Foxp3+ Treg and CD4+Foxp3− T cells 24 h following injection of BALB/c mice with 500 μg of anti-CTLA-4 Ab or control Ab. D, Mean values with SD for multiple mice treated as in C (∗, p < 0.01; n = 4).

  • FIGURE 5.
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    FIGURE 5.

    Treg deficient in CTLA-4 are not inherently pathogenic. One to 2 × 106 LN cells or purified CD4+CD25− or CD4+CD25high LN cells from 15- to 17-day-old CTLA-4−/− mice were injected into rag−/− recipients. A, Percentage weight loss and LN cellularity are shown 3–4 wk following transfer. Columns show mean data with SD for three to five mice. B, H&E staining of heart from rag−/− mice that received CTLA-4−/− whole LN, CD4+CD25− cells, or CD4+CD25high cells.

  • FIGURE 6.
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    FIGURE 6.

    Ag-specific Treg deficient in CTLA-4 lack regulatory function in vivo. A, Foxp3 staining in DO11+CD4+CD25+ LN cells from DO11+mOVA+ rag−/− or DO11+mOVA+ ctla4−/−rag−/− mice. B, Blood glucose levels in mOVA/rag mice that received 0.15 × 106 DO11+CD25− cells alone or in combination with 0.15 × 106 CD4+CD25+ cells purified from DO11+mOVA+ rag−/− or DO11+mOVA+ ctla4−/−rag−/− mice. Six mice are shown per experimental group, except for the CD25− alone group where only two are shown for clarity. Diabetes was observed for zero of six recipients of wild-type (wt) Treg, six of six recipients of CTLA-4−/− Treg, and six of six recipients of CD25− cells alone. Transfer of CTLA-4−/− Treg alone did not induce diabetes. C, Digested pancreas samples from recipients of wild-type or CTLA-4−/− DO11+ Treg 22 days after transfer were stained for CD4 and Foxp3. Absolute cell numbers were calculated as the mean value for three recipients: for CD4+Foxp3− cells, these were 1648 and 4719 (for recipients of wild-type and ctla4−/− Treg, respectively) and for CD4+Foxp3+ cells these were 335 and 440 (for recipients of wild-type and ctla4−/− Treg, respectively). D, 2.5 × 104 Thy1.1+CD4+CD25− cells were incubated with wild-type or ctla4−/− CD4+CD25+ cells (upper panel) or control CD25− cells (lower panel) in the presence of 1 μg/ml anti-CD3 and APC. Seventy-two hours later, the absolute number of Thy1.1+ responder cells was assessed by flow cytometry. Results are expressed as a percentage of the maximum cell number and graphs show the mean value and SD from three independent experiments. E, LN cells from wild-type or ctla4−/− DO11+mOVA+ rag−/− mice were stained for surface TGFβ and secreted IL-10 as described in Materials and Methods. Plots are gated on CD4+Foxp3+ (Treg) or CD4+Foxp3− (T cells).

  • FIGURE 7.
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    FIGURE 7.

    CTLA-4-dependent CD86 down-regulation by Treg. Four × 104 splenic CD11c+ cells were incubated for 48 h with 8 × 104 CD4+CD25− T cells alone (T), 8 × 104 CD4+CD25+ cells, or both. In brief, 50 μg/ml anti-CTLA-4 Ab or control hamster IgG was added as indicated. Expression of CD86 and MHC class II levels on gated CD11c+ cells was assessed by flow cytometry and representative plots (A) and mean fluorescence intensity (MFI) values (B) are shown. Data show one representative experiment of three performed.

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The Journal of Immunology: 182 (1)
The Journal of Immunology
Vol. 182, Issue 1
1 Jan 2009
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CTLA-4 Controls Regulatory T Cell Peripheral Homeostasis and Is Required for Suppression of Pancreatic Islet Autoimmunity
Emily M. Schmidt, Chun Jing Wang, Gemma A. Ryan, Louise E. Clough, Omar S. Qureshi, Margaret Goodall, Abul K. Abbas, Arlene H. Sharpe, David M. Sansom, Lucy S. K. Walker
The Journal of Immunology January 1, 2009, 182 (1) 274-282; DOI: 10.4049/jimmunol.182.1.274

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CTLA-4 Controls Regulatory T Cell Peripheral Homeostasis and Is Required for Suppression of Pancreatic Islet Autoimmunity
Emily M. Schmidt, Chun Jing Wang, Gemma A. Ryan, Louise E. Clough, Omar S. Qureshi, Margaret Goodall, Abul K. Abbas, Arlene H. Sharpe, David M. Sansom, Lucy S. K. Walker
The Journal of Immunology January 1, 2009, 182 (1) 274-282; DOI: 10.4049/jimmunol.182.1.274
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