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USP21 prevents the generation of T-helper-1-like Treg cells

View Article: PubMed Central - PubMed

ABSTRACT

FOXP3+ Regulatory T (Treg) cells play a key role in the maintenance of immune homeostasis and tolerance. Disruption of Foxp3 expression results in the generation of instable Treg cells and acquisition of effector T-cell-like function. Here we report that the E3 deubiquitinase USP21 prevents the depletion of FOXP3 at the protein level and restricts the generation of T-helper-1-like Treg cells. Mice depleted of Usp21 specifically in Treg cells display immune disorders characterized by spontaneous T-cell activation and excessive T-helper type 1 (Th1) skewing of Treg cells into Th1-like Treg cells. USP21 stabilizes FOXP3 protein by mediating its deubiquitination and maintains the expression of Treg signature genes. Our results demonstrate how USP21 prevents FOXP3 protein depletion and controls Treg lineage stability in vivo.

No MeSH data available.


Instability of FOXP3 protein in CD4+YFP+ USP21-ΔTreg cells.(a) Representative figure shown the expression of FOXP3 protein in CD4+YFP+ Treg cells from the thymus, spleen, pLNs, liver, lung and salivary glands of WT (n=5), and KO (n=5) mice. FOXP3 expression in CD4+YFP+ T cells was shown as histograms. (b) Mean fluorescent intensity (MFI) of FOXP3 among CD4+YFP+ Treg cells indicated in a. (c) WT Treg or USP21-ΔTreg cells were transferred into Rag1−/− mice. The expression of FOXP3 protein was further analysed in transferred Treg cells from the spleen of Rag1−/− mice 7 days later. (d) Percentage of FOXP3+ Treg cells as indicated in c. WT group (n=6) and KO group (n=5), data represent means±s.d. (e) Mean fluorescent intensity (MFI) of FOXP3 among transferred Treg cells as indicated in c. WT group (n=6) and KO group (n=5), data represent means±s.d. **P≤0.01, ***P≤0.001, as determined by Student's t-test. NS, not significant.
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f4: Instability of FOXP3 protein in CD4+YFP+ USP21-ΔTreg cells.(a) Representative figure shown the expression of FOXP3 protein in CD4+YFP+ Treg cells from the thymus, spleen, pLNs, liver, lung and salivary glands of WT (n=5), and KO (n=5) mice. FOXP3 expression in CD4+YFP+ T cells was shown as histograms. (b) Mean fluorescent intensity (MFI) of FOXP3 among CD4+YFP+ Treg cells indicated in a. (c) WT Treg or USP21-ΔTreg cells were transferred into Rag1−/− mice. The expression of FOXP3 protein was further analysed in transferred Treg cells from the spleen of Rag1−/− mice 7 days later. (d) Percentage of FOXP3+ Treg cells as indicated in c. WT group (n=6) and KO group (n=5), data represent means±s.d. (e) Mean fluorescent intensity (MFI) of FOXP3 among transferred Treg cells as indicated in c. WT group (n=6) and KO group (n=5), data represent means±s.d. **P≤0.01, ***P≤0.001, as determined by Student's t-test. NS, not significant.

Mentions: To investigate the stability of USP21-ΔTreg cells, we next analysed the expression of FOXP3 using flow cytometry. We observed the downregulation of FOXP3 protein in USP21-ΔTreg cells from lymphoid as well as non-lymphoid organs (Fig. 3a), while the Foxp3 gene was still actively transcribed (Supplementary Fig. 3a). This suggested that the loss of USP21 affected the post-translational modification-mediated degradation of FOXP3 protein in these USP21-ΔTreg cells. Further testing indicated that USP21 is required to stabilize FOXP3 protein, since the mean fluorescence intensity of FOXP3 staining was downregulated in USP21-ΔTreg cells (Fig. 3b). More importantly, the percentages of CD4+YFP+ USP21-ΔTreg cells remained unaffected (Supplementary Fig. 3b–d), reflecting a normal distribution of Treg cells in the lymphoid as well as non-lymphoid organs of Usp21fl/flFoxp3Cre mice. Decrease in the percentages of FOXP3+ Treg cells was more pronounced in non-lymphoid organs, due to increased loss of FOXP3 protein expression (Fig. 3c). We next checked the expression of FOXP3 in CD4+YFP+ Treg cells and confirmed the instability of FOXP3 protein in CD4+YFP+ USP21-ΔTreg cells (Fig. 4a,b). To further test the in vivo stability of USP21-ΔTreg cells, we transferred WT Treg or USP21-ΔTreg cells into Rag1−/− mice. There was a significant loss of FOXP3 in in vivo transferred USP21-ΔTreg cells (Fig. 4c–e). Taken together, these results indicated that USP21 might control Treg lineage stability by preventing the loss of FOXP3 protein.


USP21 prevents the generation of T-helper-1-like Treg cells
Instability of FOXP3 protein in CD4+YFP+ USP21-ΔTreg cells.(a) Representative figure shown the expression of FOXP3 protein in CD4+YFP+ Treg cells from the thymus, spleen, pLNs, liver, lung and salivary glands of WT (n=5), and KO (n=5) mice. FOXP3 expression in CD4+YFP+ T cells was shown as histograms. (b) Mean fluorescent intensity (MFI) of FOXP3 among CD4+YFP+ Treg cells indicated in a. (c) WT Treg or USP21-ΔTreg cells were transferred into Rag1−/− mice. The expression of FOXP3 protein was further analysed in transferred Treg cells from the spleen of Rag1−/− mice 7 days later. (d) Percentage of FOXP3+ Treg cells as indicated in c. WT group (n=6) and KO group (n=5), data represent means±s.d. (e) Mean fluorescent intensity (MFI) of FOXP3 among transferred Treg cells as indicated in c. WT group (n=6) and KO group (n=5), data represent means±s.d. **P≤0.01, ***P≤0.001, as determined by Student's t-test. NS, not significant.
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f4: Instability of FOXP3 protein in CD4+YFP+ USP21-ΔTreg cells.(a) Representative figure shown the expression of FOXP3 protein in CD4+YFP+ Treg cells from the thymus, spleen, pLNs, liver, lung and salivary glands of WT (n=5), and KO (n=5) mice. FOXP3 expression in CD4+YFP+ T cells was shown as histograms. (b) Mean fluorescent intensity (MFI) of FOXP3 among CD4+YFP+ Treg cells indicated in a. (c) WT Treg or USP21-ΔTreg cells were transferred into Rag1−/− mice. The expression of FOXP3 protein was further analysed in transferred Treg cells from the spleen of Rag1−/− mice 7 days later. (d) Percentage of FOXP3+ Treg cells as indicated in c. WT group (n=6) and KO group (n=5), data represent means±s.d. (e) Mean fluorescent intensity (MFI) of FOXP3 among transferred Treg cells as indicated in c. WT group (n=6) and KO group (n=5), data represent means±s.d. **P≤0.01, ***P≤0.001, as determined by Student's t-test. NS, not significant.
Mentions: To investigate the stability of USP21-ΔTreg cells, we next analysed the expression of FOXP3 using flow cytometry. We observed the downregulation of FOXP3 protein in USP21-ΔTreg cells from lymphoid as well as non-lymphoid organs (Fig. 3a), while the Foxp3 gene was still actively transcribed (Supplementary Fig. 3a). This suggested that the loss of USP21 affected the post-translational modification-mediated degradation of FOXP3 protein in these USP21-ΔTreg cells. Further testing indicated that USP21 is required to stabilize FOXP3 protein, since the mean fluorescence intensity of FOXP3 staining was downregulated in USP21-ΔTreg cells (Fig. 3b). More importantly, the percentages of CD4+YFP+ USP21-ΔTreg cells remained unaffected (Supplementary Fig. 3b–d), reflecting a normal distribution of Treg cells in the lymphoid as well as non-lymphoid organs of Usp21fl/flFoxp3Cre mice. Decrease in the percentages of FOXP3+ Treg cells was more pronounced in non-lymphoid organs, due to increased loss of FOXP3 protein expression (Fig. 3c). We next checked the expression of FOXP3 in CD4+YFP+ Treg cells and confirmed the instability of FOXP3 protein in CD4+YFP+ USP21-ΔTreg cells (Fig. 4a,b). To further test the in vivo stability of USP21-ΔTreg cells, we transferred WT Treg or USP21-ΔTreg cells into Rag1−/− mice. There was a significant loss of FOXP3 in in vivo transferred USP21-ΔTreg cells (Fig. 4c–e). Taken together, these results indicated that USP21 might control Treg lineage stability by preventing the loss of FOXP3 protein.

View Article: PubMed Central - PubMed

ABSTRACT

FOXP3+ Regulatory T (Treg) cells play a key role in the maintenance of immune homeostasis and tolerance. Disruption of Foxp3 expression results in the generation of instable Treg cells and acquisition of effector T-cell-like function. Here we report that the E3 deubiquitinase USP21 prevents the depletion of FOXP3 at the protein level and restricts the generation of T-helper-1-like Treg cells. Mice depleted of Usp21 specifically in Treg cells display immune disorders characterized by spontaneous T-cell activation and excessive T-helper type 1 (Th1) skewing of Treg cells into Th1-like Treg cells. USP21 stabilizes FOXP3 protein by mediating its deubiquitination and maintains the expression of Treg signature genes. Our results demonstrate how USP21 prevents FOXP3 protein depletion and controls Treg lineage stability in vivo.

No MeSH data available.