The Deubiquitinating Enzyme Ubiquitin-Specific Peptidase 11 Potentiates TGF-β Signaling in CD4+ T Cells to Facilitate Foxp3+ Regulatory T and TH17 Cell Differentiation

Roman Istomine, Fernando Alvarez, Yasser Almadani, Anie Philip and Ciriaco A. Piccirillo
J Immunol November 1, 2019, 203 (9) 2388-2400; DOI: https://doi.org/10.4049/jimmunol.1801689

Article Figures & Data

Figures

  • FIGURE 1.
    FIGURE 1.

    Preferential expression of USP11 in Helios+ TREG cells confers increased survival and suppressive ability. (A) Summary of translatome methodology. Briefly, mRNA was extracted from FACS-isolated naive and TCR-stimulated TEFF (CD4+Foxp3-GFP−) and TREG cells (CD4+Foxp3-GFP+) and was fractionated using a sucrose gradient to isolate mRNA bound to multiple ribosomes (polysome bound). Microarray analysis was conducted on both the polysome-bound fraction and total mRNA; the translatome signal was found by comparing the relative signal strength for a gene in the polysome-bound fraction relative to the signal in total mRNA. (B) USP11 mRNA is preferentially translated in TREG cells (Foxp3-GFP+) following TCR stimulation compared with TEFF cells (Foxp3-GFP−). (C) USP11 is expressed at higher levels in Helios+ TREG cells in the spleen at the resting state. Intracellular detection of USP11, relative to Helios and Foxp3 expression, by flow cytometry of USP11 in CD4+ T cells. Gated on viable CD4+ cells from whole splenocytes. (D) USP11 mRNA is expressed at lower levels in purified TREG cells compared with TEFF cells as measured by quantitative RT-PCR. (E) Transduction of CD4+T cells using a retroviral vector containing USP11 cDNA with a truncated human CD8 reporter (HuCD8) results in significant overexpression of USP11 protein compared with EV controls. (F) USP11Tg TREG cells show increased suppressive capacity when cocultured with V450-labeled responder TEFF cells. Cells were activated using soluble anti-CD3 and mixed at the indicated ratios. Suppression was assessed 72 h postactivation relative to the 0:1 TREG: TEFF cells condition. Gating on CD45.1+CD4+ responder TEFF cells. (G) USP11Tg TREG cells show an increased maintenance of Foxp3 protein expression during coculture. Gating on viable CD45.1+CD4+HuCD8+ cells. (H) The proliferation of USP11Tg TREG cells was unaffected as assessed by Ki67 staining. Gating on viable CD45.1+CD4+HuCD8+ cells. (I) USP11Tg TREG cells showed increased survival compared with EV controls as assessed by viability dye. Cells were gated on CD45.1+CD4+HuCD8+ prior to viability gating. (J) CD45.1+USP11Tg TREG cells were cotransferred with naive CD45.2+TEFF cells to TCR-β−/− hosts. Modulation of TEFF cell responses were assessed 14 d following transfer. Gating on total transferred CD4+ T cells. (K) Mice receiving USP11Tg TREG cells showed reduced accumulation of CD45.2+ TEFF cells in the colon lamina propria following adoptive transfer. (L) USP11Tg TREG cells showed greater persistence in the gut microenvironment following adoptive transfer. All data shown are from a representative experiment of at least three individually performed experiments (two to four mice per group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

  • FIGURE 2.
    FIGURE 2.

    USP11 promotes TGF-β signals and correlates with the induction and maintenance of Foxp3 expression. (A) USP11 protein expression is elevated in TREG cells that maintained Foxp3 following adoptive transfer to TCR-β−/− hosts. Representative FACS isolated from the colon; gating on transferred CD4+ cells. (B) USP11 protein expression is elevated in de novo induced pTREG cells arising from TEFF cells following adoptive transfer to TCR-β−/− hosts. Representative FACS plots shown from the colon; gating on transferred CD4+ cells. (C) USP11Tg TEFF cells show increased SMAD3 phosphorylation compared with EV controls following treatment with TGF-β at 1 ng/ml for 1 h as measured by Western blot. (D) USP11Tg TEFF cells show no differences in total SMAD3 expression compared with EV controls. (E and F) There was no significant difference in ALK5 or TGF-RII expression between USP11Tg cells and EV controls. Blots were stripped prior to staining for tubulin or β-actin. (D and F) were stained on the same blot and use the same β-actin loading control. All data shown are from a representative experiment of at least three individually performed experiments (three to four mice per group). *p < 0.05, ***p < 0.001.

  • FIGURE 3.
    FIGURE 3.

    Modulation of USP11 expression potentiates conversion of TEFF cells to a pTREG cell phenotype in vitro. (A and B) USP11Tg cells show increased Foxp3 induction compared with EV controls when cultured with the indicated concentration of TGF-β (nanograms per milliliter). Induction of Foxp3 protein and MFI was assessed 72 h later. Gating on transduced CD4+ cells. (C) Foxp3 mRNA expression in cell-sorted USP11Tg pTREG cells is elevated compared with EV controls as measured by quantitative RT-PCR. (D) Foxp3 protein expression measured in purified transduced pTREG cells following culture in IL-2 for the indicated amount of time. USP11Tg pTREG cells show increased maintenance of Foxp3 protein. (E) USP11Tg pTREG cells show increased cell viability over time when cultured in IL-2 following cell sorting. (F) USP11Kd was achieved using retroviral transduction of CD4+ T cells from wild-type BL6 mice with a vector containing an shRNA-targeting USP11 and a GFP reporter protein. ShLuc was used as control. (G and H) USP11Kd cells show increased Foxp3 induction compared with EV controls when cultured with the indicated concentration of TGF-β (nanograms per milliliter). Induction of Foxp3 protein and MFI was assessed 72 h later. Gating on transduced CD4+ cells. All data shown are from a representative experiment of three individually performed experiments. *p < 0.05, **p < 0.01.

  • FIGURE 4.
    FIGURE 4.

    Modulation of USP11 protein expression regulates pTREG cell induction in the gut. (A) USP11Tg CD4+ T cells maintained elevated USP11 expression following adoptive transfer to TCR-β−/− hosts. Gating on transferred CD4+ cells. (B) USP11Tg TEFF cells show increased Foxp3 induction compared with EV controls following adoptive transfer to TCR-β−/− hosts. Gating on transferred CD4+HuCD8+ cells. Representative FACS plots shown from the colon. (C) Proliferation of USP11Tg pTREG cells was unaffected as measured by intracellular staining for Ki67. (D) USP11Kd TEFF cells maintained reduced USP11 expression relative to shLuc controls when transferred to TCR-β−/− hosts. Gating on transferred CD4+ cells. (E) USP11Kd TEFF cells show reduced Foxp3 induction compared with EV controls following adoptive transfer to TCR-β−/− hosts. Gating on transferred CD4+GFP+ cells. Representative FACS plots shown from the colon. (F) Proliferation of USP11Kd pTREG cells was unaffected as measured by intracellular staining for Ki67. All data shown are from a representative experiment of a minimum of three individually performed experiments (three to five mice per group). *p < 0.05, **p < 0.01, ****p < 0.0001.

  • FIGURE 5.
    FIGURE 5.

    Inhibition of USP11 activity specifically inhibits TGF-β–mediated differentiation of TEFF cells. (A) Mitoxantrone (MTX) inhibited Foxp3 induction in TEFF cells cultured with TGF-β. Gating on viable CD4+ T cells. (B) MTX inhibited IL-17A secretion by TEFF cells cultured in the presence of TGF-β and IL-6 as measured by intracellular staining following treatment with PMA, ionomycin, and monensin for 3 h. Gating on viable CD4+ T cells. (C) RORγt induction was inhibited in MTX-treated cells relative to controls in the presence and absence of TH17-polarizing conditions. Gating on viable CD4+ T cells. (D and E) MTX had no impact on IFN-γ secretion or Tbet upregulation by TEFF cells cultured with IL-12. Cytokine secretion was measured by intracellular staining following treatment with PMA, ionomycin, and monensin for 3 h. Gating on viable CD4+ T cells. All data shown are from a representative experiment of more than three individually performed experiments. **p < 0.01, ***p < 0.001.

  • FIGURE 6.
    FIGURE 6.

    Ectopic expression of USP11 protein enhances TH17 cell conversion both in vivo and in vitro. (A) USP11Tg cells show increased IL-17 secretion and RORγt induction compared with EV controls when cultured with TGF-β and IL-6. IL-17 secretion and RORγT induction was assessed 72 h after the addition of polarizing cytokines following treatment with PMA, ionomycin, and monensin for 3 h. Gating on transduced CD4+ cells. (B) Modulation of USP11 had no effect on T cell proliferation as measured by V450 proliferation dye. Analyzed 48 h posttransduction. Gated on viable CD4+ transduced cells. (C) Modulation of USP11 had no impact on IFN-γ, IL-2, or TNF-α secretion in transduced CD4+ T cells. Assessed by intracellular staining 72 h posttransduction following treatment with PMA, ionomycin, and monensin for 3 h. Gating on viable CD4+ transduced cells. (D) USP11Tg cells did not differ in their capacity to secrete IFN-γ following transfer to TCR-β−/−–deficient hosts. Cytokine secretion was assessed 14 d following transfer. Representative FACS plots shown from the colon. Gating on transferred CD4+ cells. (E and F) USP11Tg cells showed increased IL-17 secretion and upregulation of RORγt in the gut microenvironment following transfer to TCR-β−/−–deficient hosts. Representative FACS plots shown from the colon. Gating on transduced USP11Tg TEFF cells. All data shown are from a representative experiment of at least three individually performed experiments (three to five mice per group). *p < 0.05, **p < 0.01.

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