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

Key Points

  • The mRNA encoding for USP11 is preferentially translated in TREG cells.

  • USP11 modulates TGF-β signaling in murine CD4+ T cells.

  • The USP11/TGF-β axis favors TREG and TH17 cell development.

Abstract

Foxp3+ regulatory T (TREG) cells are central mediators in the control of peripheral immune responses. Genome-wide transcriptional profiles show canonical signatures for Foxp3+ TREG cells, distinguishing them from Foxp3− effector T (TEFF) cells. We previously uncovered distinct mRNA translational signatures differentiating CD4+ TEFF and TREG cells through parallel measurements of cytosolic (global) and polysome-associated (translationally enhanced) mRNA levels in both subsets. We show that the mRNA encoding for the ubiquitin-specific peptidase 11 (USP11), a known modulator of TGF-β signaling, was preferentially translated in TCR-activated TREG cells compared with conventional, murine CD4+ T cells. TGF-β is a key cytokine driving the induction and maintenance of Foxp3 expression in T cells. We hypothesized that differential translation of USP11 mRNA endows TREG cells with an advantage to respond to TGF-β signals. In an in vivo mouse model promoting TREG cells plasticity, we found that USP11 protein was expressed at elevated levels in stable TREG cells, whereas ectopic USP11 expression enhanced the suppressive capacity and lineage commitment of these cells in vitro and in vivo. USP11 overexpression in TEFF cells enhanced the activation of the TGF-β pathway and promoted TREG or TH17, but not Th1, cell differentiation in vitro and in vivo, an effect abrogated by USP11 gene silencing or the inhibition of enzymatic activity. Thus, USP11 potentiates TGF-β signaling in both TREG and TEFF cells, in turn driving increased suppressive function and lineage commitment in thymic-derived TREG cells and potentiating the TGF-β–dependent differentiation of TEFF cells to peripherally induced TREG and TH17 cells.

Introduction

CD4+ regulatory T (TREG) cells expressing the Foxp3 lineage-specifying transcription factor are key to establishing self-tolerance and immune homeostasis and resolving inflammation and pathologic conditions in immune responses (1). Thymic-derived TREG (tTREG) cells develop during thymopoiesis, whereas their peripherally induced TREG (pTREG) cells, are generated upon Ag exposure in the periphery under both noninflammatory and inflammatory conditions. Although tTREG cells react primarily to self-antigens, pTREG cells were shown to restrain immune responses to innocuous Ags such as commensal bacteria and control immunity at sites of inflammation (2, 3). The absence or impairment of TREG cell function results in a serious perturbation of immune homeostasis, leading to severe multiorgan autoimmunity while potentiating anti-tumor activity and pathogen clearance (2, 4–6). As such, TREG cells have modulatory effects on various cell types, although the exact mechanism depends on host genetic background, microflora, and target tissues.

The DNA-binding, forkhead winged helix transcriptional regulator Foxp3 is the master driver of TREG cell lineage (7, 8). Induction of Foxp3 in conventional CD4+ effector T (TEFF) cells through extracellular signals such as TGF-β, short chain fatty acids, retinoic acid, and rapamycin promotes TREG cell development and function (9–13). Moreover, previous microarray studies on TREG cells have defined transcriptional signatures differentiating TREG and TEFF cells and have shed light on how Foxp3 orchestrates a complex transcriptional program through both repression and enhancement of target genes. As such, the canonical TREG cell transcriptional signature is determined by the Foxp3-mediated transcriptional repression of inflammatory genes, such as ifng, il2, and il17a, and transcriptional activation of genes enabling TREG cell development and suppressive function, such as il2ra, ctla4, and cd39 (14–16). However, several studies have indicated that TREG cells also possess distinct posttranscriptional gene regulation mechanisms capable of altering the expression or activity of Foxp3 and other TREG cell effector genes to adjust the nature, amounts, and activities of specific proteins produced for their coordinated modulation of immunity (17–25). Thus, it is postulated that the integration of site-specific environmental cues, including metabolic conditions, external cytokines, and hypoxia, in vivo regulates gene expression independent of transcription, allowing TREG cells to undergo rapid changes in their proteome and engage specific mechanisms to optimally inhibit immune responses in a wide range of immunological contexts. The diversity of functional fates of TREG cells operating in tissues points to the need to characterize how TREG cells sense environmental cues, and how these shape the diverse effector mechanisms in different inflammatory contexts.

Recent studies have shown that the level of mRNA does not always directly correlate with the level of protein expression within a cell (26–28). Notably, the rate of mRNA translation can affect the proteome of a cell to a similar extent as transcription (29). This effect is guided by a complex network of translational control mechanisms, including the modification of mRNA untranslated regions, micro RNA interference, RNA binding proteins, and even decoy long noncoding RNA, which act to counteract these measures. In innate immunity, these mechanisms have been shown to allow for rapid increases and decreases in cytokine secretion and receptor sensitivity, providing an efficient way for immune cells to rapidly adapt to changing circumstances using their existing mRNA pools without expending energy on de novo mRNA transcription (30). Although translational regulation is well described in innate immunity, it is still nascent in the study of the adaptive immune system.

In a previous study, we assessed if translational regulation of gene expression occurred in CD4+ T cell subsets by employing a novel polyribosomal profiling technique to conduct a genome-wide analysis of translational activity in TEFF and TREG cells (31). Microarray analysis of polyribosome-bound mRNA transcripts in both naive and TCR-stimulated TREG and TEFF cells, adjusted for basal transcription rates, allowed us to identify unique translatomes differentiating these subsets, shedding light on possible mechanisms governing T cell development and function in different inflammatory settings (31). Interestingly, most genes examined were translationally repressed in activated TREG cells compared with TEFF cells, with many genes being translationally coregulated in functional modules, including cell survival, proliferation, and translation itself. One differentially regulated module was that of protein catabolic processes, indicating how TREG cells possess differential mechanisms for maintaining the level of proteins within a cell to adapt their function.

Within the protein catabolic process module, the preferential translation of ubiquitin-specific peptidase 11 (USP11) mRNA in TREG cells was one of the most significant changes differentiating TREG from TEFF cell translatomes. USP11 has been reported to be involved in DNA damage repair and the stabilization of p21 and p53, whereas some studies have indicated that it can affect TGF-β signaling in nonhematopoietic cultured cell lines (32–35). Interestingly, TGF-β has many roles in CD4+ T cells, including the conversion of TEFF cells to pTREG cells, TH17 differentiation through induction of RORγT in the context of IL-6, and stabilization of Foxp3 expression in TREG cells to favor their survival and lineage commitment (36, 37). However, how the differential translation of USP11 can impact TGF-β–mediated signals in CD4+ T cells is unknown.

In this study, we characterized the function of USP11 in CD4+ TEFF and TREG cell subsets by assessing the impact of modulating USP11 expression and activity on the differentiation, lineage commitment, and effector function of CD4+ T cell subsets. We found that USP11 was preferentially expressed in stable tTREG cells, with elevated USP11 expression enhancing their suppressive capacity. Furthermore, the modulation of USP11 expression augmented the sensitivity of TEFF cells to TGF-β, directly influencing the conversion of TEFF cells to a pTREG cell phenotype. Finally, elevated USP11 expression drove enhanced TH17 differentiation in the presence of requisite cytokines. In this study, we show that USP11 is a novel factor in influencing TEFF: TREG cell balance through modulation of TGF-β sensitivity.

Materials and Methods

Mice

CD45.2+TCR-β−/−, CD45.1+TCR-β−/−, CD45.1+ C57BL/6 (Taconic Biosciences, Rensselaer, NY), and C57BL/6.Foxp3GFP reporter knock-in mice (obtained from A. Rudensky, Howard Hughes Medical Institute, New York) (13) were bred and housed under specific pathogen-free conditions and used in accordance with McGill University’s animal research practices. All experiments were conducted using age- (8–12 wk) and sex-matched littermates.

Lymphocyte isolation

Lymph nodes and spleens were mechanically dissociated to liberate cells. RBCs were then lysed using ammonium-chloride-potassium lysis buffer, and cells were filtered through 70-μm nylon mesh filters in complete RPMI 1640 (cRPMI) with 10% FBS (Wisent Bioproducts, Saint-Bruno, QC, Canada). Colons were cut longitudinally and cut into 1-cm-long pieces. They were then washed in HBSS and incubated for 1 h in HBSS containing 20 mM EDTA (Thermo Fisher Scientific, Waltham, MA) at 37°C. Colons were washed one more time in HBSS before being incubated for 1 h in cRPMI containing 0.2 mg/ml Collagenase D (Roche, Basel, Switzerland) and 4 μg/ml DNase (Sigma-Aldrich, Saint-Louis, MO). Supernatants were then filtered through 70-μm nylon mesh filters and cells resuspended in cRPMI for further assay.

Plasmid construction and generation

cDNA encoding human USP11 was provided by Dr. G. Sapkota. The MSCV-IRES-HuCD8t (truncated human CD8 reporter) overexpression vector and pCL-ECO packaging plasmid were provided by Connie Krawczyk (McGill University, Montreal, QC, Canada). USP11 cDNA was cloned into the MSC-RIES-HuCD8 using BamHI and NotI restriction sites. The pMLP-GFP short hairpin RNA (shRNA) expression vector pMLP-shLuc-GFP (shRNA-targeting luciferase [shLuc]) control vectors were obtained from Dr. M. Tremblay. An oligonucleotide containing shRNA-targeting murine USP11 was cloned into the pMLP-GFP vector using XhoI and EcoRI restriction sites: oligonucleotide forward (Fwd): 5′-TCGAGGCAGAACCATAAACGACGAAATAGTGAAGCCACAGATGTATTTCGTCGTTTATGGTTCTGCG-3′ and reverse (Rev): 5′-AATTCGCAGAACCATAAACGACGAAATACATCTGTGGCTTCACTATTTC-GTCGTTTATGGTTCTGCC-3′.

T cell purification

Total CD4+ T cells were isolated using mouse CD4-positive selection beads according to manufacturer’s standard operating procedure (Miltenyi Biotec, Bergisch Gladbach, Germany) followed by separation on autoMACS (Miltenyi Biotec). The CD4-positive fraction was kept for further assays. Where needed, the CD4-negative fraction was irradiated (30 Gys) and as APC. For T cell polarization assays, cells were first depleted using CD25-allophycocyanin (clone: PC61.5) and anti-allophycocyanin beads (Miltenyi Biotec) to obtain the CD25-negative fraction. CD4+CD25− cells were then isolated from this fraction with Miltenyi Biotec CD4-positive selection beads.

Retroviral particle generation and transduction of T cells

293TX cells were cultured to 90% confluency and transfected with Retroviral Plasmids using Lipofectamine 3000 (Thermo Fisher Scientific) (according to manufacturer’s standard operating procedure). Retroviral particle-containing media was collected 48 h posttransfection, filtered through 0.45-μm surfactant-free cellulose acetate filters (Corning, Corning, NY), and used fresh for viral transduction.

CD4+ or CD4+CD25− cells were activated using plate-bound α-CD3 (5 μg/ml) and α-CD28 (2 μg/ml) (BD Biosciences, Billerica, MA) in cRPMI supplemented with IL-2 for 18 h prior to viral transduction. At the time of transduction, media was supplemented with cRPMI containing polybrene (8 μg/ml) (Sigma-Aldrich), and retroviral particle-containing media was added. Cells were spinoculated at 1200 × g at 30°C for 1 h. Media was changed for fresh cRPMI 5 h posttransduction.

In vitro T cell polarization assays

pTREG cell polarization with mitoxantrone was carried out using MACS-sorted CD4+CD25− TEFF (>95% purity) cells activated with plate-bound activation and cultured with TGF-β (Novoprotein, Summit, NJ) and recombinant human IL-2 (Roche). pTREG cell polarization of transduced cells was carried out by adding TGF-β and recombinant human IL-2 at the time of media change following transduction. Foxp3 induction was assessed 72 h following the addition of TGF-β. TH1 and TH17 polarization were achieved by culturing CD4+CD25− TEFF cells in the presence of IL-12 (PeproTech, Rocky Hill, NJ) or TGF-β and IL-6 (Novus Biologicals, Centennial, CO), respectively. TEFF cells were activated using soluble α-CD3 (1 μg/ml) (BD Biosciences) and cocultured with irradiated APC in the presence of the indicated polarizing cytokines. T cell polarization was assessed 72 h following the addition of polarizing cytokines through the assessment of cytokine secretion. Cytokine secretion was assessed by incubating cells with PMA, ionomycin (Sigma-Aldrich), and monensin (BD GolgiStop) for 3 h prior to staining for flow cytometry.

Isolation of transduced TREG and TEFF cells

Transduced CD4+ cells were expanded for 1 wk prior to sorting. Total CD4+ cells from C57BL/6.Foxp3GFP mice were used for generating USP11 transgenic (USP11Tg) cells, and CD4+CD25− cells from CD45.1+C57BL/6 were used for generating USP11 knockdown (USP11Kd) cells. USP11Tg cells were then stained for CD4 (clone: Gk1.5; Thermo Fisher Scientific Invitrogen) and human CD8 (clone: RPA-T8; Thermo Fisher Scientific Invitrogen) prior to sorting CD4+ human CD8+ GFP− (Foxp3−) transduced TEFF cells and CD4+ human CD8+ GFP+ (Foxp3+) TREG cells using a FACSAria cell sorter (BD Biosciences). USP11Kd cells were stained for CD4+ and sorted for CD4+GFP+ transduced cells using the FACSAria.

TREG cell suppression assays

Cell-sorted CD4+GFP− (Foxp3neg) cells from C57BL/6.Foxp3GFP were labeled with V450 proliferation dye (BD Biosciences). These cells were then cocultured with cell-sorted USP11Tg TREG cells. Cells were activated using irradiated APC and soluble α-CD3. Suppression was assessed 72 h postactivation.

Adoptive transfer of T cells

For standard T cell transfers, cell-sorted TEFF and TREG cells were i.v. injected into CD45.1+ TCR-β−/− mice, and animals were sacrificed 21 d after initial transfer. For transfers of transduced T cells, cell-sorted CD45.2+ USP11Tg TEFF cells were i.v. injected into CD45.1+ TCR-β−/− recipients, whereas USP11KD TEFF cells were transferred to CD45.2+ TCR-β−/− recipients. For TREG and TEFF cell cotransfers, CD45.2+ USP11Tg TREG cells were mixed with CD45.1+ TEFF cells at a 1:4 ratio prior to i.v. injection into CD45.1+ TCR-β−/− recipient mice. Mice were monitored for weight loss and hydration and sacrificed 14 d following transfer. Peripheral lymph nodes, spleens, mesenteric lymph nodes, and colons were collected for phenotyping and cytokine secretion analysis.

Flow cytometry

All cells were stained with Fixable Viability Dye (Thermo Fisher Scientific Invitrogen) prior to extracellular staining for 15 min at 4°C. Cells were stained extracellularly in PBS containing the Ab mix for 30 min at 4°C. For intracellular staining, cells were first fixed using the Foxp3 Fixation Kit (Thermo Fisher Scientific Invitrogen) for 30 min at 4°C prior to being permeabilized using eBioscience Permeabilization Buffer. Intracellular staining was carried out in permeabilization buffer containing Ab mix for 1 h at 4°C. Abs used were obtained from eBioscience, unless otherwise indicated. The Abs used (varied depending on experiment) are as follows: USP11 (clone: EPR4346; Abcam, Cambridge, U.K.), F(ab′)2 Anti-Rabbit IgG (BD Biosciences), CD4 (clone: GK1.5), human CD8 (clone: RPA-T8), CD25 (clone: PC61.5), CD45.1 (clone: A20), CD45.2 (clone: 104), Foxp3 (clone: FJK-16S), Helios (clone: 22F6; BioLegend, San Diego, CA), IL-17A (clone: ebio17B7), IFN-γ (clone: XMG1.2), RORγt (clone: AFKJS-9), and Ki67 (clone: B56; BD Biosciences). Cells were acquired using a BD LSRFortessa X-20. Data were analyzed using Treestar FlowJo (v10) (FlowJo, Ashland, OR).

Western blot analysis

Cell-sorted USP11Tg TEFF cells were serum starved in serum-free AIM V Medium for 3 h following sorting. Cells were treated with TGF-β (1 ng/ml) for 45 min at 37°C prior to protein extraction. Cells were lysed using RIPA lysis buffer containing antiphosphatases (Roche). Western blot analysis of cell lysates was conducted as previously described using anti–phospho-SMAD3 (Cell Signaling Technology, Danvers, MA), anti-SMAD3 (Cell Signaling Technology), anti–TGF-RI (ALK5) (Abcam), anti–TGF-RII (MilliporeSigma, Burlington, MA), and anti-USP11 Abs (Abcam) (38).

Quantitative RT-PCR analysis

Ex vivo TREG or TEFF cells and USP11Tg Foxp3+ (GFP+) or Foxp3− (GFP−) cells were cell sorted, lysed, and RNA extracted using a QIAGEN RNeasy Plus Mini Kit (QIAGEN, Hilden, Germany) according to manufacturer’s instructions. RNA was quantified on a Bio-Rad MyiQ thermal cycler (Bio-Rad Laboratories, Hercules, CA) using the QuantiFast SYBR Green RT-PCR Kit (QIAGEN). RT-PCR amplification settings were set according to manufacturer’s instructions (QIAGEN). Primers for PCR amplification were as follows: USP11 Fwd 5′-CCACGCATACAAGTGTTGCACC-3′, USP11 Rev 5′-CTCAATCCGACCAGTCACCTCA-3′, Foxp3 Fwd 5′-CCTGGTTGTGAGAAGGTC-TTCG-3′, Foxp3 Rev 5′-TGCTCCAGAGACTG-CACCACTT-3′, GAPDH Fwd 5′-CATCAC-TGCCACCCAGAAGACTG-3′, and GAPDH Rev 5′-ATGCCAGTGAGCTTCCCGTTCAG-3′.

Results

USP11, a deubiquitinating enzyme, is preferentially expressed in Foxp3+ TREG cells and confers enhanced survival and suppressive function

We previously identified a unique translational signature distinguishing activated TEFF cells from TREG cells by conducting a microarray analysis comparing the total cytosolic mRNA to one obtained from isolating mRNA transcripts bound to multiple ribosomes (polysome-bound) through density fractionation (Fig. 1A). Within this signature, we observed that mRNA encoding for USP11 was preferentially translated in activated TREG cells following TCR stimulation while being translationally repressed in activated TEFF cells (Fig. 1B). To verify the preferential translation of USP11 in TREG cells, we developed a multiparametric flow cytometry strategy to quantify USP11 protein expression in murine CD4+ T cell subsets directly ex vivo. Notably, USP11 expression was higher in TREG cells expressing Helios, a marker proposed to denote tTREG cells (3) compared with pTREG and TEFF cell subsets (Fig. 1C). Western blot analysis of USP11 expression on highly purified, ex vivo TREG and TEFF cells confirmed this expression pattern (data not shown). TREG cells also expressed lower USP11 mRNA levels compared with TEFF cells, despite expressing higher USP11 protein levels, an observation consistent with the preferential translation of USP11 in TREG cells (Fig. 1D).

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.

We then investigated the role of USP11 in TREG cell function. To increase USP11 protein levels in T cells, we developed a retroviral system to overexpress the cDNA encoding for human USP11 and included the human CD8 (hCD8) as an extracellular marker of transduction in murine CD4+ T cells. Retroviral transduction of total CD4+ T cells led to elevated USP11 expression in USP11Tg CD4+ T cells compared with empty vector (EV) transduced controls (Fig. 1E). Overexpression of USP11 protein in transduced CD4+ T cells was also confirmed through Western blot analysis (Supplemental Fig. 1). Through transduction of total CD4+ T cells from CD45.2+ Foxp3-GFP reporter mice and subsequent FACS isolation of transduced hCD8+ CD4+ Foxp3-GFP+ cells, we isolated USP11Tg TREG cells to assess their suppressive function and the maintenance of Foxp3 protein expression compared with EV controls. To achieve this, USP11Tg TREG cells were cocultured with freshly isolated, V450 proliferation dye–labeled, TEFF (CD4+GFPneg) cells from CD45.1+ congenic Foxp3-GFP reporter mice. In contrast to EV controls, USP11Tg TREG cells had an increased suppressive capacity as evidenced by a decrease in responder TEFF cell proliferation (Fig. 1F). To explain the differences in suppressive capacity of USP11Tg TREG cells, we then examined the maintenance of Foxp3 protein expression in transduced TREG cells. FACS-isolated USP11Tg Foxp3-GFP+TREG cells showed an increased Foxp3 expression after 72 h in culture (Fig. 1G). The frequency of the cells expressing the proliferation marker, Ki-67, did not differ between USP11Tg TREG cells and EV controls, suggesting that the differences in Foxp3 expression were not because of differences in proliferative ability (Fig. 1H). However, USP11Tg TREG cells accumulated more readily in culture, likely because of an increase in the cell viability of USP11Tg TREG cells (Fig. 1I). Thus, ectopic USP11 expression in TREG cells enhanced their suppressive ability, which is partially explained by an increase in Foxp3 expression and enhanced survival in vitro.

To examine the role of USP11Tg TREG cells in vivo, we employed an adoptive transfer model of T cell–mediated colitis. To this end, we cotransferred CD45.1+ USP11Tg and CD45.2+ TEFF cells into TCR-β−/− lymphopenic hosts and monitored the dynamics of TREG and TEFF cell responses in the gut microenvironment (39) (Fig. 1J). At day 14 posttransfer, we observed a reduction in the accumulation of colonic CD45.2+TEFF cells in mice receiving USP11Tg TREG cells compared with mice receiving control EV TREG cells or TEFF cells alone (Fig. 1K). This contraction of the TEFF cell response coincided with an increase in the frequency of USP11Tg Foxp3+ TREG cells recovered in the gut microenvironment (Fig. 1L). This mirrored our in vitro observations regarding USP11Tg TREG cells showing an increased persistence in vivo and an increased suppression of TEFF cell accumulation in the gut microenvironment.

USP11 expression is elevated in stable and peripherally induced TREG cells and potentiates TGF-β signaling in CD4+ T cells

Given the increase in the maintenance of Foxp3 expression and survival of USP11Tg TREG cells, we then wanted to confirm if TREG cells that maintained Foxp3 expression also expressed increased levels of USP11 in vivo. To this end, we used an in vivo lymphopenia model in which TREG cells have been shown to undergo functional reprogramming following the downregulation of Foxp3 expression, a process most readily detected in the gut microenvironment (40). Briefly, we isolated CD45.2+ CD4+ Foxp3-GFP+ TREG cells and transferred them into lymphopenic CD45.1+ TCR-β−/− hosts (Fig. 2A). Following a period of homeostatic expansion of these cells, we assessed USP11 and Foxp3 expression in the transferred CD45.2+ T cells. At day 14 posttransfer, we observed that approximately half of the CD45.2+ T cells lost Foxp3 expression (exTREG cells), whereas the other half maintained stable levels of Foxp3 expression (stable TREG cells) (Fig. 2A). USP11 expression was higher in stable TREG cells compared with TREG cells that lost Foxp3 expression (exTREG cells) in the colonic lamina propria (Fig. 2A). Because USP11 expression correlated with Foxp3 expression, we then examined if USP11 could play a role in the induction of Foxp3 in CD4+ TEFF cells, promoting the generation of pTREG cells. To this end, we transferred CD45.2+CD4+Foxp3-GFPneg TEFF cells into CD45.1+ TCR-β−/− hosts and assessed the emergence of pTREG cells in the gut microenvironment. USP11 expression was significantly higher in Foxp3+ pTREG cells compared with Foxp3neg TEFF cells in the lamina propria (Fig. 2B), suggesting a strong correlation between USP11 expression and the induction and maintenance of a TREG cell phenotype, marked by Foxp3 expression in CD4+ T cells.

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.

The increased expression of USP11 in both stable tTREG cells and de novo generated pTREG cells in the gut microenvironment suggested a local role of USP11 in TREG cell homeostasis. Given that TGF-β is a well described factor involved in TREG cell homeostasis in the gut (41) and that USP11 was shown to potentiate TGF-β signaling in several human cell lines (34, 42), we investigated if USP11 was modulating TGF-β downstream signals in CD4+ T cells (34). To this end, we examined if ectopic USP11 expression enhanced the phosphorylation of the signal transducing protein SMAD3 involved in the downstream signaling of the TGF-βR in CD4+ T cells. Ectopic USP11 expression resulted in increased levels of TGF-β–induced phospho-SMAD3 in USP11Tg cells compared with EV controls (Fig. 2C). To confirm that this increase was not due to an increase in total SMAD3 levels in USP11Tg cells, we quantified total SMAD3 levels and found no significant differences in total SMAD3 expression between USP11Tg and control EV cells (Fig. 2D). We then examined the expression of the TGF-βR complex components, ALK5 and TGF-RII; however, no difference we seen in unstimulated USP11Tg and EV cells (Fig. 2D–F). However, the increase in SMAD3 phosphorylation observed in USP11Tg cells upon TGF-β treatment demonstrated that the increased induction of Foxp3 in USP11Tg TEFF cells is associated with an increased activation of the TGF-β signaling pathway in these cells. Furthermore, we observed an induction of USP11 expression in both TREG and TEFF cells following TCR stimulation, suggesting that TCR-induced USP11 expression may act to increase the sensitivity of CD4+ T cells to TGF-β signals following Ag activation (Supplemental Fig. 2A). However, the addition of exogenous IL-2 or TGF-β, well-established TREG cell–promoting cytokines, did not affect USP11 expression in CD4+ T cells. This indicated that USP11, although capable of augmenting TGF-β sensitivity, was not modulated by TGF-β in CD4+ T cells (Supplemental Fig. 2B, 2C).

TGF-β–mediated pTREG cell generation in vitro is modulated by USP11 expression

Because USP11 overexpression enhanced SMAD3 phosphorylation in the presence of TGF-β, we investigated whether modulating USP11 expression in TEFF cells facilitated the TGF-β–dependent pTREG cell differentiation. To this end, we retrovirally overexpressed USP11 in TEFF cells, activated them in the presence of TGF-β, and evaluated the frequency of Foxp3+ T cells after 72 h in culture. USP11Tg TEFF cells cultured in the presence of TGF-β upregulated Foxp3 expression more readily then their EV counterparts (Fig. 3A), evidenced by both the increase in frequency of Foxp3+ USP11Tg TEFF cells as well as the level of Foxp3 protein expression (Fig. 3B). TGF-β–stimulated pTREG cells generated from USP11Tg T cells also showed increased levels of Foxp3 mRNA levels, consistent with an increased induction of Foxp3 by SMAD3 and NFAT following the activation of the TGF-β signaling pathway (Fig. 3C) (43). Furthermore, USP11Tg pTREG cells displayed increased survival and prolonged Foxp3 protein expression in the absence of TGF-β1–polarizing signal in vitro, consistent with our previous observation of increased Foxp3 maintenance and survival in USP11Tg TREG cells (Fig. 3D, 3E).

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.

To confirm the dependency of USP11 in the induction of Foxp3, we then abrogated USP11 expression via gene silencing in TEFF cells. A retroviral vector expressing shRNA targeting the middle region of USP11 mRNA was used to generate USP11Kd TEFF cells, along with control CD4+ T cells expressing shLuc, absent in these cells. These USP11Kd TEFF cells displayed a significant reduction in USP11 protein expression in vitro compared with shLuc controls (Fig. 3F). Accordingly, when USP11Kd TEFF cells were cultured in the presence of TGF-β, they displayed significantly less Foxp3 induction (Fig. 3G). This was marked by a reduction in both the percentage of Foxp3-GFP+ cells and Foxp3 protein expression (mean fluorescence intensity [MFI]) when compared with controls (Fig. 3H). Therefore, USP11 expression plays a direct role in the TGF-β–mediated conversion of TEFF cells to a pTREG cell phenotype, confirming our hypothesis that USP11 modulates the TGF-β signaling pathway in CD4+ T cells to affect both induction and sustained expression of Foxp3 protein.

USP11 modulates pTREG cell generation in the gut microenvironment

Because USP11 enhanced TGF-β signaling in TEFF cells through the enhancement of SMAD3 phosphorylation and drove increased Foxp3 induction in TEFF cells in vitro, we then examined if the modulation in the expression of USP11 in TEFF cells would have the same consequences in vivo. We employed the same adoptive transfer model described earlier, transferring donor transduced CD45.1+ USP11Tg TEFF cells into TCR-β−/− mice and examined the CD4+ T cell response 14 d following transfer. USP11Tg TEFF cells were detectable in host organs 14 d following the adoptive transfer with a significant portion maintaining enhanced USP11-hCD8 expression (Fig. 4A). USP11Tg TEFF cells showed increased pTREG cell generation in the gut microenvironment with an increased proportion of transduced cells inducing Foxp3 expression in the mesenteric lymph node and colon lamina propria (Fig. 4B). This accumulation of pTREG cells was not because of a proliferative effect as both USP11Tg and EV Foxp3-GFP+ T cells showed similar Ki67 expression (Fig. 4C). We then did the converse experiment, in which we transferred USP11Kd or shLuc (control) transduced CD45.1+CD4+ T cells into TCR-β−/− mice. In these mice, USP11Kd T cells maintained reduced USP11 expression 14 d after transfer when compared with control (Fig. 4D). Contrary to USP11Tg TEFF cells, USP11Kd TEFF cells displayed a diminished capacity to polarize into pTREG cells in the gut microenvironment with a reduced proportion of Foxp3+ cells in the mesenteric lymph nodes and colon lamina propria (Fig. 4E). Ki67 expression in USP11Kd pTREG cells did not differ with shLuc controls, indicating that again this effect was not because of differences in proliferation (Fig. 4F). The differences observed occurred specifically in the gut microenvironment, a location where TGF-β is known to be a major component of the conditions driving pTREG cell induction (44). Thus, we confirmed in vivo that USP11 directly modulates the ability of TEFF cells to convert to a pTREG cell phenotype, corroborating the effect observed in vitro.

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.

Pharmacological inhibition of USP11 activity hinders TGF-β–mediated differentiation of Foxp3+ TREG and TH17 cells

Because the level of USP11 protein expression directly impacts the TGF-β–mediated induction and sustained expression of Foxp3 protein, we investigated if the effect of USP11 could be abrogated through the inhibition of its enzymatic function. We made use of mitoxantrone (MTX), a DNA topoisomerase II inhibitor that has been reported and used by several groups to inhibit USP11 enzymatic activity in vitro (42, 45) and to inhibit the effect of USP11 on Foxp3 induction in T cells. To achieve this, we treated CD4+ TEFF cells with MTX in the presence of TGF-β and assessed Foxp3 induction in vitro. At a 1 nM dose of MTX, we observed a significant reduction in Foxp3 induction in TEFF cells even at high concentrations of TGF-β (Fig. 5A). Thus, MTX, a known inhibitor of USP11, inhibited pTREG cell differentiation in vitro, mirroring our observations with USP11Kd TEFF cells.

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.

Because USP11 played an important role in the induction of Foxp3 in TEFF cells in response to TGF-β, we then examined if USP11 could modulate other forms of TGF-β–dependent differentiation in TEFF cells. TH17 polarization is also known to depend on TGF-β signals in murine CD4+ T cells (37). Thus, we investigated if inhibition of USP11 with MTX could impact the differentiation of TH17 cells. TEFF cells were cultured in the presence of MTX and TH17-polarizing conditions (TGF-β, IL-6, and IL-1β) for 72 h (46). MTX inhibited TH17 polarization, evidenced by a decrease in the amount of IL-17A-secreting TEFF cells as well as a reduction in the expression of the key TH17 cell transcription factor RORγt (Fig. 5B, 5C). To validate that this effect was specific to impaired TGF-β signaling rather than off-target effects of MTX treatment, we polarized TEFF cells into a TH1 cell phenotype because the polarizing mixture does not depend on TGF-β. MTX had no effect on the secretion of IFN-γ under these conditions (Fig. 5D). Similarly, the expression of the TH1 transcription factor Tbet was unaffected in the presence of MTX, indicating the effect was specific to TH17 differentiation (Fig. 6E). Furthermore, TCR-induced T cell activation, as determined by CD25 expression, and ensuing proliferation was unaffected at the indicated doses of MTX (Supplemental Fig. 3A, 3B), indicating that the effect of MTX on T cell functions was only detectable in the presence of TGF-β. Thus, the inhibition of TGF-β signaling via specific pharmacological inhibition of USP11 activity in TEFF cells directly impaired their capacity to differentiate to a TH17 phenotype.

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.

Overexpression of USP11 in CD4+ T cells enhances TH17 differentiation in vivo

To further confirm the role of USP11 in other TGF-β–mediated T cell differentiation, we then examined if ectopic USP11 expression could drive TH17 differentiation as it did for TREG cell induction. To this end, we polarized USP11Tg Foxp3-GFPneg T cells in TH17-polarizing conditions in vitro. In these conditions, there were significantly more differentiated TH17 cells among USP11Tg T cells then EV control, evidenced by both IL-17A secretion and RORγT expression among live T cells (Fig. 6A). Because the process of T cell transduction involves USP11 modulation prior to their contact with TGF-β, we assessed if the profile in cytokine secretion and proliferation in both USP11Tg and USP11Kd transduced T cells was in any way affected. Neither ectopic expression nor knockdown of USP11 in CD4+ T cells altered their proliferative capacity relative to the corresponding controls (Fig. 6B). The secretion of IL-2, TNF-α, and IFN-γ was likewise unaffected by retroviral modulation of USP11 protein expression in these cells under nonpolarizing conditions (Fig. 6C). Finally, we validated our in vitro findings by examining the effect of ectopic USP11 expression on the differentiation of TH17 cells in the gut microenvironment. In our adoptive transfer model, IFN-γ secretion was not affected by USP11 overexpression across multiple tissue sites (Fig. 6D). However, TH17 differentiation was significantly impacted in USP11Tg TEFF cells (Fig. 6E). They demonstrated increased secretion of IL-17A in the gut and mesenteric lymph nodes, with a trend toward increased IL-17A secretion in the spleen, likely from recirculation of TH17 cells from the mesenteric lymph nodes. Furthermore, the expression of RORγt was elevated in USP11Tg TEFF cells in the gut, corroborating the increases in IL-17A secretion observed at the same site (Fig. 6F). Thus, these data show that ectopic USP11 expression can enhance TH17 cell function and differentiation both in vitro and in vivo.

Discussion

CD4+ T cells are key players in the adaptive immune response, capable of adapting their function to the immune challenge they face. Work in recent decades uncovered that CD4+ T cells employ a wide variety of signaling pathways to integrate environmental cues and adapt their transcriptional program. Although the transcriptional landscape of TEFF and TREG cells has been the focus of past research, recent years have seen an emergence in the study of posttranscriptional mechanisms capable of adjusting CD4+ T cell function rapidly in inflammatory environments. Ultimately, this may lead to uncovering novel therapeutic targets to modulate the TREG: TEFF cell balance when undesirable immune responses require it.

To identify genes that undergo rapid translation in TREG cells, independently of mRNA levels, we previously assessed polysome-associated mRNAs in CD4+ T cell subsets (31). We uncovered distinct translational regulation within these cells, revealing several candidate genes that may play a role in governing TREG and TEFF cell function (31). We have identified the ubiquitin C-terminal hydrolase 11 (USP11) among the preferentially translated mRNA pool, and we sought to characterize its function in T cells as its role remains unknown. In particular, our attention was drawn to the work of Al-Salihi et al. (34) and Jacko et al. (42) who showed that USP11 enhanced TGF-β signaling in mesenchymal cell lines. Thus, we hypothesized that a similar effect was occurring in TREG cells as they require TGF-β signaling in their function, survival, and differentiation (47). In this study, we discovered that the role of USP11 transcended the functional differentiation of TREG cells as it also potentiated the TGF-β–mediated differentiation of TH17 cells.

First, we validated the role of USP11 in the maintenance of Foxp3 expression in tTREG cells. Next, we showed that increased USP11 expression enhanced the suppressive ability of USP11Tg TREG cells in vitro and in vivo as well as their survival with USP11 pTREG cells showing similar increased survival in vitro. This was correlated with increased Foxp3 maintenance and lineage commitment both in vitro and in vivo. This is in accordance with existing literature that suggests that TGF-β can play a role in the survival of TREG cells and the commitment to a stable Foxp3+ phenotype (48).

Interestingly, we demonstrate in this study that USP11 enhances the phosphorylation of the SMAD family member 3 (SMAD3), a key mediator of TGF-β signaling, that can directly bind the conserved noncoding region 1 of the foxp3 locus. This region was shown to play a critical role in TREG cell homeostasis (44). However, SMAD3 phosphorylation was also shown to facilitate TH17 cell differentiation through the suppression of the eomesodermin repressor (49). Thus, after confirming that USP11 enhanced tTREG cell function, we assessed if USP11 played a role in T cell differentiation into TREG cells (pTREG) or TH17 cells. Indeed, increased protein levels of USP11 were able to enhance both the pTREG and TH17 cell differentiation pathway. Not surprisingly, the major difference observed in vivo occurred specifically in the gut microenvironment, a location where TGF-β directs the balance of the CD4+ T cell compartment during both inflammation and immune homeostasis (50). This suggests that USP11 was specific to modulating TGF-β signaling in transduced cells, in which increased USP11 expression acted to promote TGF-β signals in these cells. Furthermore, a reduction in USP11 expression or enzymatic activity through chemical inhibition demonstrated the converse effect in TEFF cells, further validating these findings.

This work has demonstrated that modulating USP11 levels drives the expression of a T cell phenotype consistent with the existing known effect of TGF-β in both TEFF and TREG cells. Prior studies have established the link between USP11 and TGF-β signaling, with two papers demonstrating that USP11 can stabilize the expression of the TGF-βR complex on the cell surface in other cell types (34, 42). Although we did not observe the increased expression of either the ALK5 or TGF-RII subunits in our experiments, this may have been because of the poor resolution of detecting slight differences in these proteins in primary murine T cells compared with human immortalized cell lines (34, 42). Our finding that ectopic USP11 expression can enhance the phosphorylation and activation of the SMAD2/3 signaling cascade downstream of TGF-β signals is in accordance with these two studies. However, whether USP11 can have other effects within CD4+ T cells remains to be elucidated. Proteomic studies of USP11Tg and control cells would help shed some light on any other global changes in the proteome resulting from the altered ubiquitination pattern driven by enhanced USP11 expression.

Several studies in recent years have investigated the complex role played by the family of ubiquitin-specific proteases in regulating immune cell function. USP12 was shown to modulate TCR signaling sensitivity in T cells, whereas USP15 was identified as a regulator of type 1 IFN secretion (51, 52). In a similar vein to our work, USP7 was found to stabilize Foxp3 expression through direct deubiquitination of Foxp3 protein, in contrast to USP11, which mediates its effects on Foxp3 through the modulation of the TGF-β signaling pathway (21). This suggests that the family of ubiquitin-specific proteases offers a new target in the identification of novel mechanisms regulating T cell function, with few members of this family having been characterized in T cells.

Our previous work uncovered how differential mRNA translation mechanisms shape unique translatomes in CD4+ T cell subsets. In this study, we uncover a function for a novel player, USP11, in the differentiation of both TH17 and TREG cells. This study lends credence to the notion that genes under translational regulation help shape CD4+ T cell adaptation to the environment. As such, understanding the translational changes occurring within CD4+ T cell subsets as they respond to environmental signals will shed light on the mechanisms governing their rapid adaptation. Notably, this work opens the door toward modulating T cell responses in conditions in which the adaptive immune system is dysregulated or detrimental, such as during tumor-mediated T cell suppression, in which TGF-β release plays a significant nonredundant role in TREG cell differentiation and tumor evasion (53).

In conclusion, we demonstrate that USP11 specifically regulates TGF-β signaling in CD4+ T cell subsets, probably through its role as a regulator of the ubiquitylation-mediated negative feedback loop that limits TGF-β signals upon the activation of the pathway. USP11 most likely acts as a rheostat of TGF-β signaling in TREG and TH17 cells, potentiating their function and differentiation. Understanding the signals that instruct USP11 upregulation in these cells might offer further therapeutic targets to modulate undesired immunological responses.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank the Research Institute of the McGill University Health Centre Immunophenotyping Platform for excellent cell sorting services.

Footnotes

  • This work was supported by Canadian Institutes of Health Research operating grants (PJT-148821) (to C.A.P.) as well as the Canada Research Chair program (to C.A.P.). C.A.P. is supported by the Anna Maria Solinas Laroche Career Award in Immunology.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    cRPMI
    complete RPMI 1640
    EV
    empty vector
    Fwd
    forward
    hCD8
    human CD8
    MTX
    mitoxantrone
    Rev
    reverse
    shLuc
    shRNA-targeting luciferase
    shRNA
    short hairpin RNA
    pTREG
    peripherally induced TREG
    TEFF
    effector T
    TREG
    regulatory T
    tTREG
    thymic-derived TREG
    USP11
    ubiquitin-specific peptidase 11
    USP11Kd
    USP11 knockdown
    USP11Tg
    USP11 transgenic.

  • Received December 31, 2018.
  • Accepted August 26, 2019.

References

  1. ↵
    1. Sakaguchi, S.,
    2. T. Yamaguchi,
    3. T. Nomura,
    4. M. Ono
    . 2008. Regulatory T cells and immune tolerance. Cell 133: 775–787.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Sakaguchi, S.,
    2. M. Ono,
    3. R. Setoguchi,
    4. H. Yagi,
    5. S. Hori,
    6. Z. Fehervari,
    7. J. Shimizu,
    8. T. Takahashi,
    9. T. Nomura
    . 2006. Foxp3+ CD25+ CD4+ natural regulatory T cells in dominant self-tolerance and autoimmune disease. Immunol. Rev. 212: 8–27.
    OpenUrlCrossRefPubMed
  3. ↵
    1. Shevach, E. M.,
    2. A. M. Thornton
    . 2014. tTregs, pTregs, and iTregs: similarities and differences. Immunol. Rev. 259: 88–102.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Bennett, C. L.,
    2. J. Christie,
    3. F. Ramsdell,
    4. M. E. Brunkow,
    5. P. J. Ferguson,
    6. L. Whitesell,
    7. T. E. Kelly,
    8. F. T. Saulsbury,
    9. P. F. Chance,
    10. H. D. Ochs
    . 2001. The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat. Genet. 27: 20–21.
    OpenUrlCrossRefPubMed
    1. Brunkow, M. E.,
    2. E. W. Jeffery,
    3. K. A. Hjerrild,
    4. B. Paeper,
    5. L. B. Clark,
    6. S.-A. Yasayko,
    7. J. E. Wilkinson,
    8. D. Galas,
    9. S. F. Ziegler,
    10. F. Ramsdell
    . 2001. Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat. Genet. 27: 68–73.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Wildin, R. S.,
    2. F. Ramsdell,
    3. J. Peake,
    4. F. Faravelli,
    5. J.-L. Casanova,
    6. N. Buist,
    7. E. Levy-Lahad,
    8. M. Mazzella,
    9. O. Goulet,
    10. L. Perroni, et al
    . 2001. X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy. Nat. Genet. 27: 18–20.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Hori, S.,
    2. T. Nomura,
    3. S. Sakaguchi
    . 2003. Control of regulatory T cell development by the transcription factor Foxp3. Science 299: 1057–1061.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    1. Fontenot, J. D.,
    2. M. A. Gavin,
    3. A. Y. Rudensky
    . 2003. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat. Immunol. 4: 330–336.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Fantini, M. C.,
    2. C. Becker,
    3. G. Monteleone,
    4. F. Pallone,
    5. P. R. Galle,
    6. M. F. Neurath
    . 2004. Cutting edge: TGF-β induces a regulatory phenotype in CD4+CD25- T cells through Foxp3 induction and down-regulation of Smad7. J. Immunol. 172: 5149–5153.
    OpenUrlAbstract/FREE Full Text
    1. Chen, W.,
    2. W. Jin,
    3. N. Hardegen,
    4. K. J. Lei,
    5. L. Li,
    6. N. Marinos,
    7. G. McGrady,
    8. S. M. Wahl
    . 2003. Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+ regulatory T cells by TGF-β induction of transcription factor Foxp3. J. Exp. Med. 198: 1875–1886.
    OpenUrlAbstract/FREE Full Text
    1. Coombes, J. L.,
    2. K. R. Siddiqui,
    3. C. V. Arancibia-Cárcamo,
    4. J. Hall,
    5. C.-M. Sun,
    6. Y. Belkaid,
    7. F. Powrie
    . 2007. A functionally specialized population of mucosal CD103+ DCs induces Foxp3+ regulatory T cells via a TGF-β and retinoic acid-dependent mechanism. J. Exp. Med. 204: 1757–1764.
    OpenUrlAbstract/FREE Full Text
    1. Sun, C.-M.,
    2. J. A. Hall,
    3. R. B. Blank,
    4. N. Bouladoux,
    5. M. Oukka,
    6. J. R. Mora,
    7. Y. Belkaid
    . 2007. Small intestine lamina propria dendritic cells promote de novo generation of Foxp3 T reg cells via retinoic acid. J. Exp. Med. 204: 1775–1785.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    1. Fontenot, J. D.,
    2. J. P. Rasmussen,
    3. L. M. Williams,
    4. J. L. Dooley,
    5. A. G. Farr,
    6. A. Y. Rudensky
    . 2005. Regulatory T cell lineage specification by the forkhead transcription factor foxp3. Immunity 22: 329–341.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Bettelli, E.,
    2. M. Dastrange,
    3. M. Oukka
    . 2005. Foxp3 interacts with nuclear factor of activated T cells and NF-kappa B to repress cytokine gene expression and effector functions of T helper cells. Proc. Natl. Acad. Sci. USA 102: 5138–5143.
    OpenUrlAbstract/FREE Full Text
    1. Marson, A.,
    2. K. Kretschmer,
    3. G. M. Frampton,
    4. E. S. Jacobsen,
    5. J. K. Polansky,
    6. K. D. MacIsaac,
    7. S. S. Levine,
    8. E. Fraenkel,
    9. H. von Boehmer,
    10. R. A. Young
    . 2007. Foxp3 occupancy and regulation of key target genes during T-cell stimulation. Nature 445: 931–935.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Zheng, Y.,
    2. S. Z. Josefowicz,
    3. A. Kas,
    4. T.-T. Chu,
    5. M. A. Gavin,
    6. A. Y. Rudensky
    . 2007. Genome-wide analysis of Foxp3 target genes in developing and mature regulatory T cells. Nature 445: 936–940.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Floess, S.,
    2. J. Freyer,
    3. C. Siewert,
    4. U. Baron,
    5. S. Olek,
    6. J. Polansky,
    7. K. Schlawe,
    8. H.-D. Chang,
    9. T. Bopp,
    10. E. Schmitt, et al
    . 2007. Epigenetic control of the foxp3 locus in regulatory T cells. PLoS Biol. 5: e38.
    1. Chen, Z.,
    2. J. Barbi,
    3. S. Bu,
    4. H.-Y. Yang,
    5. Z. Li,
    6. Y. Gao,
    7. D. Jinasena,
    8. J. Fu,
    9. F. Lin,
    10. C. Chen, et al
    . 2013. The ubiquitin ligase Stub1 negatively modulates regulatory T cell suppressive activity by promoting degradation of the transcription factor Foxp3. Immunity 39: 272–285.
    OpenUrlCrossRefPubMed
    1. Nie, H.,
    2. Y. Zheng,
    3. R. Li,
    4. T. B. Guo,
    5. D. He,
    6. L. Fang,
    7. X. Liu,
    8. L. Xiao,
    9. X. Chen,
    10. B. Wan, et al
    . 2013. Phosphorylation of FOXP3 controls regulatory T cell function and is inhibited by TNF-α in rheumatoid arthritis. Nat. Med. 19: 322–328.
    OpenUrlCrossRefPubMed
    1. Akimova, T.,
    2. G. Ge,
    3. T. Golovina,
    4. T. Mikheeva,
    5. L. Wang,
    6. J. L. Riley,
    7. W. W. Hancock
    . 2010. Histone/protein deacetylase inhibitors increase suppressive functions of human FOXP3+ Tregs. Clin. Immunol. 136: 348–363.
    OpenUrlCrossRefPubMed
  21. ↵
    1. van Loosdregt, J.,
    2. V. Fleskens,
    3. J. Fu,
    4. A. B. Brenkman,
    5. C. P. Bekker,
    6. C. E. Pals,
    7. J. Meerding,
    8. C. R. Berkers,
    9. J. Barbi,
    10. A. Gröne, et al
    . 2013. Stabilization of the transcription factor Foxp3 by the deubiquitinase USP7 increases Treg-cell-suppressive capacity. Immunity 39: 259–271.
    OpenUrlCrossRefPubMed
    1. Bin Dhuban, K.,
    2. E. d’Hennezel,
    3. Y. Nagai,
    4. Y. Xiao,
    5. S. Shao,
    6. R. Istomine,
    7. F. Alvarez,
    8. M. Ben-Shoshan,
    9. H. Ochs,
    10. B. Mazer, et al
    . 2017. Suppression by human FOXP3+ regulatory T cells requires FOXP3-TIP60 interactions. Sci. Immunol. 2: eaai9297.
    OpenUrlAbstract/FREE Full Text
    1. de Zoeten, E. F.,
    2. L. Wang,
    3. K. Butler,
    4. U. H. Beier,
    5. T. Akimova,
    6. H. Sai,
    7. J. E. Bradner,
    8. R. Mazitschek,
    9. A. P. Kozikowski,
    10. P. Matthias,
    11. W. W. Hancock
    . 2011. Histone deacetylase 6 and heat shock protein 90 control the functions of Foxp3(+) T-regulatory cells. Mol. Cell. Biol. 31: 2066–2078.
    OpenUrlAbstract/FREE Full Text
    1. de Zoeten, E. F.,
    2. L. Wang,
    3. H. Sai,
    4. W. H. Dillmann,
    5. W. W. Hancock
    . 2010. Inhibition of HDAC9 increases T regulatory cell function and prevents colitis in mice. Gastroenterology 138: 583–594.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Beier, U. H.,
    2. L. Wang,
    3. T. R. Bhatti,
    4. Y. Liu,
    5. R. Han,
    6. G. Ge,
    7. W. W. Hancock
    . 2011. Sirtuin-1 targeting promotes Foxp3+ T-regulatory cell function and prolongs allograft survival. Mol. Cell. Biol. 31: 1022–1029.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Vogel, C.,
    2. S. Abreu Rde,
    3. D. Ko,
    4. S. Y. Le,
    5. B. A. Shapiro,
    6. S. C. Burns,
    7. D. Sandhu,
    8. D. R. Boutz,
    9. E. M. Marcotte,
    10. L. O. Penalva
    . 2010. Sequence signatures and mRNA concentration can explain two-thirds of protein abundance variation in a human cell line. Mol. Syst. Biol. 6: 400.
    OpenUrlAbstract/FREE Full Text
    1. de Sousa Abreu, R.,
    2. L. O. Penalva,
    3. E. M. Marcotte,
    4. C. Vogel
    . 2009. Global signatures of protein and mRNA expression levels. Mol. Biosyst. 5: 1512–1526.
    OpenUrlCrossRefPubMed
  28. ↵
    1. Tebaldi, T.,
    2. A. Re,
    3. G. Viero,
    4. I. Pegoretti,
    5. A. Passerini,
    6. E. Blanzieri,
    7. A. Quattrone
    . 2012. Widespread uncoupling between transcriptome and translatome variations after a stimulus in mammalian cells. BMC Genomics 13: 220.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Kristensen, A. R.,
    2. J. Gsponer,
    3. L. J. Foster
    . 2013. Protein synthesis rate is the predominant regulator of protein expression during differentiation. Mol. Syst. Biol. 9: 689.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    1. Carpenter, S.,
    2. E. P. Ricci,
    3. B. C. Mercier,
    4. M. J. Moore,
    5. K. A. Fitzgerald
    . 2014. Post-transcriptional regulation of gene expression in innate immunity. Nat. Rev. Immunol. 14: 361–376.
    OpenUrlCrossRefPubMed
  31. ↵
    1. Bjur, E.,
    2. O. Larsson,
    3. E. Yurchenko,
    4. L. Zheng,
    5. V. Gandin,
    6. I. Topisirovic,
    7. S. Li,
    8. C. R. Wagner,
    9. N. Sonenberg,
    10. C. A. Piccirillo
    . 2013. Distinct translational control in CD4+ T cell subsets. PLoS Genet. 9: e1003494.
  32. ↵
    1. Schoenfeld, A. R.,
    2. S. Apgar,
    3. G. Dolios,
    4. R. Wang,
    5. S. A. Aaronson
    . 2004. BRCA2 is ubiquitinated in vivo and interacts with USP11, a deubiquitinating enzyme that exhibits prosurvival function in the cellular response to DNA damage. Mol. Cell. Biol. 24: 7444–7455.
    OpenUrlAbstract/FREE Full Text
    1. Ke, J. Y.,
    2. C. J. Dai,
    3. W. L. Wu,
    4. J. H. Gao,
    5. A. J. Xia,
    6. G. P. Liu,
    7. K. S. Lv,
    8. C. L. Wu
    . 2014. USP11 regulates p53 stability by deubiquitinating p53. J. Zhejiang Univ. Sci. B 15: 1032–1038.
    OpenUrl
  34. ↵
    1. Al-Salihi, M. A.,
    2. L. Herhaus,
    3. T. Macartney,
    4. G. P. Sapkota
    . 2012. USP11 augments TGFβ signalling by deubiquitylating ALK5. Open Biol. 2: 120063.
    OpenUrlCrossRefPubMed
  35. ↵
    1. Deng, T.,
    2. G. Yan,
    3. X. Song,
    4. L. Xie,
    5. Y. Zhou,
    6. J. Li,
    7. X. Hu,
    8. Z. Li,
    9. J. Hu,
    10. Y. Zhang, et al
    . 2018. Deubiquitylation and stabilization of p21 by USP11 is critical for cell-cycle progression and DNA damage responses. Proc. Natl. Acad. Sci. USA 115: 4678–4683.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    1. Tran, D. Q.
    2012. TGF-β: the sword, the wand, and the shield of FOXP3(+) regulatory T cells. J. Mol. Cell Biol. 4: 29–37.
    OpenUrlCrossRefPubMed
  37. ↵
    1. Bettelli, E.,
    2. Y. Carrier,
    3. W. Gao,
    4. T. Korn,
    5. T. B. Strom,
    6. M. Oukka,
    7. H. L. Weiner,
    8. V. K. Kuchroo
    . 2006. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 441: 235–238.
    OpenUrlCrossRefPubMed
  38. ↵
    1. Vorstenbosch, J.,
    2. H. Al-Ajmi,
    3. S. Winocour,
    4. A. Trzeciak,
    5. L. Lessard,
    6. A. Philip
    . 2013. CD109 overexpression ameliorates skin fibrosis in a mouse model of bleomycin-induced scleroderma. Arthritis Rheum. 65: 1378–1383.
    OpenUrl
  39. ↵
    1. Mottet, C.,
    2. H. H. Uhlig,
    3. F. Powrie
    . 2003. Cutting edge: cure of colitis by CD4+CD25+ regulatory T cells. J. Immunol. 170: 3939–3943.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    1. Yurchenko, E.,
    2. M. T. Shio,
    3. T. C. Huang,
    4. M. Da Silva Martins,
    5. M. Szyf,
    6. M. K. Levings,
    7. M. Olivier,
    8. C. A. Piccirillo
    . 2012. Inflammation-driven reprogramming of CD4+ Foxp3+ regulatory T cells into pathogenic Th1/Th17 T effectors is abrogated by mTOR inhibition in vivo. PLoS One 7: e35572.
  41. ↵
    1. Bauché, D.,
    2. J. C. Marie
    . 2017. Transforming growth factor β: a master regulator of the gut microbiota and immune cell interactions. Clin. Transl. Immunology 6: e136.
    OpenUrl
  42. ↵
    1. Jacko, A. M.,
    2. L. Nan,
    3. S. Li,
    4. J. Tan,
    5. J. Zhao,
    6. D. J. Kass,
    7. Y. Zhao
    . 2016. De-ubiquitinating enzyme, USP11, promotes transforming growth factor β-1 signaling through stabilization of transforming growth factor β receptor II. Cell Death Dis. 7: e2474.
  43. ↵
    1. Tone, Y.,
    2. K. Furuuchi,
    3. Y. Kojima,
    4. M. L. Tykocinski,
    5. M. I. Greene,
    6. M. Tone
    . 2008. Smad3 and NFAT cooperate to induce Foxp3 expression through its enhancer. Nat. Immunol. 9: 194–202.
    OpenUrlCrossRefPubMed
  44. ↵
    1. Zheng, Y.,
    2. S. Josefowicz,
    3. A. Chaudhry,
    4. X. P. Peng,
    5. K. Forbush,
    6. A. Y. Rudensky
    . 2010. Role of conserved non-coding DNA elements in the Foxp3 gene in regulatory T-cell fate. Nature 463: 808–812.
    OpenUrlCrossRefPubMed
  45. ↵
    1. Burkhart, R. A.,
    2. Y. Peng,
    3. Z. A. Norris,
    4. R. M. Tholey,
    5. V. A. Talbott,
    6. Q. Liang,
    7. Y. Ai,
    8. K. Miller,
    9. S. Lal,
    10. J. A. Cozzitorto, et al
    . 2013. Mitoxantrone targets human ubiquitin-specific peptidase 11 (USP11) and is a potent inhibitor of pancreatic cancer cell survival. Mol. Cancer Res. 11: 901–911.
    OpenUrlAbstract/FREE Full Text
  46. ↵
    1. Gutcher, I.,
    2. M. K. Donkor,
    3. Q. Ma,
    4. A. Y. Rudensky,
    5. R. A. Flavell,
    6. M. O. Li
    . 2011. Autocrine transforming growth factor-β1 promotes in vivo Th17 cell differentiation. Immunity 34: 396–408.
    OpenUrlCrossRefPubMed
  47. ↵
    1. Wan, Y. Y.,
    2. R. A. Flavell
    . 2007. ‘Yin-Yang’ functions of transforming growth factor-beta and T regulatory cells in immune regulation. Immunol. Rev. 220: 199–213.
    OpenUrlCrossRefPubMed
  48. ↵
    1. Chen, W.,
    2. J. E. Konkel
    . 2015. Development of thymic Foxp3(+) regulatory T cells: TGF-β matters. Eur. J. Immunol. 45: 958–965.
    OpenUrlCrossRefPubMed
  49. ↵
    1. Ichiyama, K.,
    2. T. Sekiya,
    3. N. Inoue,
    4. T. Tamiya,
    5. I. Kashiwagi,
    6. A. Kimura,
    7. R. Morita,
    8. G. Muto,
    9. T. Shichita,
    10. R. Takahashi,
    11. A. Yoshimura
    . 2011. Transcription factor Smad-independent T helper 17 cell induction by transforming-growth factor-β is mediated by suppression of eomesodermin. Immunity 34: 741–754.
    OpenUrlCrossRefPubMed
  50. ↵
    1. Troncone, E.,
    2. I. Marafini,
    3. C. Stolfi,
    4. G. Monteleone
    . 2018. Transforming growth factor-β1/Smad7 in intestinal immunity, inflammation, and cancer. Front. Immunol. 9: 1407.
    OpenUrl
  51. ↵
    1. Jahan, A. S.,
    2. M. Lestra,
    3. L. K. Swee,
    4. Y. Fan,
    5. M. M. Lamers,
    6. F. G. Tafesse,
    7. C. S. Theile,
    8. E. Spooner,
    9. R. Bruzzone,
    10. H. L. Ploegh,
    11. S. Sanyal
    . 2016. Usp12 stabilizes the T-cell receptor complex at the cell surface during signaling. Proc. Natl. Acad. Sci. USA 113: E705–E714.
    OpenUrlAbstract/FREE Full Text
  52. ↵
    1. Torre, S.,
    2. M. J. Polyak,
    3. D. Langlais,
    4. N. Fodil,
    5. J. M. Kennedy,
    6. I. Radovanovic,
    7. J. Berghout,
    8. G. A. Leiva-Torres,
    9. C. M. Krawczyk,
    10. S. Ilangumaran, et al
    . 2016. USP15 regulates type I interferon response and is required for pathogenesis of neuroinflammation. [Published erratum appears in 2016 Nat. Immunol. 17: 1479.] Nat. Immunol. 18: 54–63.
    OpenUrlCrossRef
  53. ↵
    1. Dahmani, A.,
    2. J.-S. Delisle
    . 2018. TGF-β in T cell biology: implications for cancer immunotherapy. Cancers (Basel) 10: E194.
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