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An essential role for Scurfin in CD4+CD25+ T regulatory cells

Abstract

The molecular properties that characterize CD4+CD25+ regulatory T cells (TR cells) remain elusive. Absence of the transcription factor Scurfin (also known as forkhead box P3 and encoded by Foxp3) causes a rapidly fatal lymphoproliferative disease, similar to that seen in mice lacking cytolytic T lymphocyte–associated antigen 4 (CTLA-4). Here we show that Foxp3 is highly expressed by TR cells and is associated with TR cell activity and phenotype. Scurfin-deficient mice lack TR cells, whereas mice that overexpress Foxp3 possess more TR cells. In Foxp3-overexpressing mice, both CD4+CD25 and CD4CD8+ T cells show suppressive activity and CD4+CD25 cells express glucocorticoid-induced tumor-necrosis factor receptor–related (GITR) protein. The forced expression of Foxp3 also delays disease in CTLA-4−/− mice, indicating that the Scurfin and CTLA-4 pathways may intersect and providing further insight into the TR cell lineage.

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Figure 1: Ectopic expression of Foxp3 increases CD4+CD25+ T cell numbers.
Figure 2: Foxp3 expression is specific to CD4+CD25+ TR cells.
Figure 3: CD4+CD25+CD69 TR cells from sf mice do not show regulatory activity.
Figure 4: CD4+CD25+ TR cells from OT-II Foxp3 mice show increased regulatory activity.
Figure 5: CD4+CD25 and CD8+ T cells from Foxp3 mice show high Foxp3 expression and regulatory activity.
Figure 6: CD4+CD25 T cells from Foxp3 mice express GITR.
Figure 7: Overexpression of Foxp3 delays lethality in CTLA-4−/− mice.
Figure 8: Expression of Foxp3 induces TR cell activity in CTLA-4−/− CD4+ T cells.

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Acknowledgements

We thank D. Walker for cell sorting and analysis; S. Rudensky, M. Gavin and J. Fontenot for discussions; and J. Allison for the CTLA-4−/− mice.

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Correspondence to Fred Ramsdell.

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The authors' primary employment is by a for-profit corporation (Celltech Group).

Supplementary information

Supplementary Fig. 1.

CD4+CD25+ TR cells from OT-II Foxp3 transgenic mice express more Foxp3 and show an increased regulatory activity. (a) Foxp3 expression was determined in cDNA samples from CD4+CD25+ isolated either from OT-II or OT-II Foxp3 transgenic mice using a real-time RT-PCR with Dad1 as an endogenous reference gene. (b) 2.5 x 104 OT-II CD4+CD25- T cells were stimulated either with different concentrations of Ova323-339 peptide or anti-CD3 (1µg/ml) and equal numbers of mitomycin C treated Thy-1- APC. CD4+CD25+ T cells from OT-II (open symbols) or OT-II Foxp3 TG (filled symbols, dashed lines) animals were added at various ratios. The cells were cultured for 72 h and pulsed with [3H]thymidine for final 8 h of the culture. Data is expressed as percentage inhibition calculated as 100 x [1 - (counts with suppressor/counts without suppressor)]. Data is mean of triplicates and is representative of three independent experiments. The proliferation of responders in OT-II CD4+CD25+ assay were 43,661 cpm at 0.25 µM peptide, 38,790 cpm at 0.125 µM peptide and 59,486 cpm with anti-CD3; in OT-II Foxp3 transgenic CD4+CD25+ assay were 36,706 cpm at 0.25 µM peptide, 36,496 cpm at 0.125 µM peptide and 54,800 cpm with anti-CD3. (PDF 13 kb)

Supplementary Fig. 2.

IL-10 mRNA levels do not correlate with Foxp3 mRNA expression levels. IL-10 mRNA was determined in cDNA samples using a real-time RT-PCR method in which Dad1 served as an endogenous reference gene. Samples are from freshly isolated cell or 72 h preactivated subsets from wild type (WT), scurfy (SF) or Foxp3 transgenic (TG) mice. Normalized IL-10 values were derived from the ratio of IL-10 expression to Dad1 expression. Data is representative of analysis with 3 independent experiments. (PDF 7 kb)

Supplementary Fig. 3.

Scurfy CD4+ T cells are inhibited by TR cells but not by TGF-ß. (a) 5 x 104 wild-type (WT) or scurfy (SF) CD4+ T cells were stimulated with anti-CD3 (1µg/ml) and equal numbers of mitomycin C treated Thy-1- APC. CD4+CD25+ TR cells were added at various ratios to the assay. Specific responder:suppressor ratios are defined in the chart legend. The cells were cultured for 72 h and pulsed with [3H]thymidine for final 8 h of the culture. (b) Wild-type (WT) or scurfy (SF) CD4+ T cells were stimulated either with immobilized anti-CD3 (4 µg/ml) or anti-CD3 (0.25 µg/ml) and anti-CD28 (1 µg/ml). TGF-ß (2.5 ng/ml) was added at the beginning of the assay. The cells were cultured for 48 h and pulsed with [3H]thymidine for final 8 h of the culture. Data in is mean of triplicates and is representative of at least three independent experiments. (PDF 23 kb)

Supplementary Fig. 4.

CD4+ T cells from Ctla-4-/-Foxp3 transgenic mice express increased CD25. Lymph node CD4+ T cells from wild-type (top panel), Foxp3 transgenic (middle panel) and Ctla-4-/-Foxp3 transgenic (bottom panel) mice were analyzed for the expression of CD25. Numbers represent percent positive above an isotype control antibody. Analyses are from samples gated for CD4+ cells and represent individual mice and are representative of four animals examined from each group. (PDF 11 kb)

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Khattri, R., Cox, T., Yasayko, SA. et al. An essential role for Scurfin in CD4+CD25+ T regulatory cells. Nat Immunol 4, 337–342 (2003). https://doi.org/10.1038/ni909

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