Journal: EBioMedicine

Article Title: Ubiquitin-specific Protease-7 Inhibition Impairs Tip60-dependent Foxp3 + T-regulatory Cell Function and Promotes Antitumor Immunity

doi: 10.1016/j.ebiom.2016.10.018

Figure Lengend Snippet: In vitro effects of Usp7i compounds. (A) Jurkat cells were pulsed for 2 h with compound 564 (10 μM), washed free of compound and cultured for the periods indicated. Levels of Usp7 were serially monitored by Western blotting (left panel), in conjunction with assays of Usp7 (middle panel) or global DUB activity (right panel), expressed as a percentage of activity of corresponding DMSO-treated cells (mean ± SD); *p < 0.05 and p < 0.01 vs. DMSO-treated cells. (B) Murine Treg cells were pre-incubated with DMSO or Usp7i compounds for 2 h, washed and cultured for 3 days, at the ratios of Treg to CFSE-labeled Teff cells shown (mean ± SE of triplicate wells, *p < 0.05 and **p < 0.01 vs. DMSO-treated cells at the corresponding ratio); a representative result in which Tregs were pre-treated with compound 564 for 2 h is shown at right (inset figure indicates the proportion of proliferating cells in each well). (C) Western blot analysis of, in the left panel, lysates of cell-sorted Foxp3 YFP + Tregs from WT or Usp7 −/− mice, and right, levels of endogenous Foxp3, Tip60 and Usp7 in Foxp3 + Treg cells cultured under activating conditions for 2 h with or without the addition of compound 564, as shown. (D) Left panel shows mean fluorescent intensity (MFI) levels of Foxp3 in WT or conditional Tip60 −/− Treg cells isolated from corresponding LN and spleen samples (representative of 4 mice/group). Right panels show qPCR data (mean ± SD, 4/group, **p < 0.01) of Foxp3, Ctla4, IL-6 and IL-17 mRNA levels using Teff and Treg cells isolated from WT or conditional Tip60 −/− mice. (E) ChIP studies assessing the levels of Tip60 at the Foxp3 promoter and CNS1, CNS2 and CNS3 sites in WT and conditionally deleted Usp7 −/− Tregs (mean ± SD, 4/group, **p < 0.01). (F) Murine WT Tregs were incubated with DMSO alone, 564 (10 μM), or 564 plus proteasome inhibitor (Bortezomib, BTZ) for 2 h, washed, lysed, subjected to TUBE pull-downs, and ubiquitinated substrates eluted and treated with Usp2 to separate ubiquitin from substrate; proteins were then analyzed by Western blotting. Data are expressed at right as fold change compared to using DMSO alone, and are representative of 3 separate experiments.

Article Snippet: We purchased conjugated monoclonal antibodies (mAbs) for flow cytometry (BD Pharmingen), plus anti-Foxp3 mAb (FJK-16 s, eBioscience), and rabbit antibodies to β-actin, Usp7, p53 and Mdm2 (Cell Signaling), and Tip60 (Cell Signaling and Millipore).

Techniques: In Vitro, Cell Culture, Western Blot, Activity Assay, Incubation, Labeling, Isolation

Journal: EBioMedicine

Article Title: Ubiquitin-specific Protease-7 Inhibition Impairs Tip60-dependent Foxp3 + T-regulatory Cell Function and Promotes Antitumor Immunity

doi: 10.1016/j.ebiom.2016.10.018

Figure Lengend Snippet: In vivo effects of Usp7i compounds. For panels (A, B), mice were injected with Usp7i (10 mg/kg/day, i.p.) for 5 days or DMSO alone (5 mice/group). (A) Treg cells isolated from Usp7i-treated mice showed modestly decreased Foxp3 protein but increased Ub-Foxp3 compared to use of DMSO alone. (B) Treg cells isolated from Usp7i-treated mice showed markedly impaired Treg suppressive function compared to DMSO-treated Treg controls (upper 2 rows). Usp7i therapy also impaired Teff cell responses to Treg suppression, as shown using Teff cells from Usp7i or DMSO-treated mice and fresh, untreated Treg cells (lower 2 rows). Summary graphs and statistical analyses are shown in Fig. S15. (C) Western blots showing how a bolus injection of Usp7i 24 h beforehand decreased Tip60 protein to a greater extent than Foxp3 protein in murine Treg cells (panel shows ratios of data for 564 vs. DMS0). (D) Western blot analysis of the same cells as used in the previous panel, indicating that Usp7i compound bound to both Treg and Teff cells and decreased the interaction of Usp7 with HA-Ub-VME substrate (signal ratios shown below). (E) The effect of conditional Usp7 deletion on the survival of Tregs in vivo was assessed by the adoptive transfer of Thy1.1 + Teff (5 × 10 5 ) plus WT Treg or Usp7 −/− Treg (1 × 10 5 ) cells into immunodeficient (Rag1 −/−) hosts, and harvesting LN and spleen samples at 1 month post-transfer for flow cytometric quantitation (n = 4/group, **p < 0.01). (F) The effects of Usp7i on Treg cell inhibition of the homeostatic proliferation on Teff cells in vivo was assessed by pre-incubation of murine Tregs (5 × 10 5 ) with Usp7i (5 μM) or DMSO alone for 2 h, followed by their injection into Rag −/− mice, along with 1 × 10 6 Teff cells, and harvest of LN and spleen samples 7 days later (n = 4/group, *p < 0.5, **p < 0.01). (G) Use of CD154 mAb plus DST induces permanent cardiac allograft survival in DMSO-treated hosts (BALB/c- > C57BL/6) whereas daily injection of Usp7i (1 mg/kg/day, 14 days) from the time of engraftment restored acute allograft rejection (p < 0.01, n = 6/group).

Article Snippet: We purchased conjugated monoclonal antibodies (mAbs) for flow cytometry (BD Pharmingen), plus anti-Foxp3 mAb (FJK-16 s, eBioscience), and rabbit antibodies to β-actin, Usp7, p53 and Mdm2 (Cell Signaling), and Tip60 (Cell Signaling and Millipore).

Techniques: In Vivo, Injection, Isolation, Western Blot, Adoptive Transfer Assay, Quantitation Assay, Inhibition, Incubation

Journal: EBioMedicine

Article Title: Ubiquitin-specific Protease-7 Inhibition Impairs Tip60-dependent Foxp3 + T-regulatory Cell Function and Promotes Antitumor Immunity

doi: 10.1016/j.ebiom.2016.10.018

Figure Lengend Snippet: Schematic of proposed interaction of Usp7i with Foxp3 + Treg cells. Lysine residues in both Foxp3 and Tip60 in Treg cells are subject to polyubiquitination, leading to protein turnover via their proteasomal degradation. Through the actions of Usp7, deubiquitinated lysine residues can be acetylated, leading to protein stabilization. While multiple HATs can acetylate Foxp3, Tip60, which can auto-acetylate, is unique in mediating acetylation of 2 lysine residues required for Foxp3 dimerization. Dimerized Foxp3 can then bind to DNA and mediate effects on gene transcription in Treg cells (e.g. upregulation of genes encoding Ctla4 and IL-10 and inhibition of genes encoding IL-2 and IFN-γ).

Article Snippet: We purchased conjugated monoclonal antibodies (mAbs) for flow cytometry (BD Pharmingen), plus anti-Foxp3 mAb (FJK-16 s, eBioscience), and rabbit antibodies to β-actin, Usp7, p53 and Mdm2 (Cell Signaling), and Tip60 (Cell Signaling and Millipore).

Techniques: Inhibition