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Suppression by human FOXP3+ regulatory T cells requires FOXP3-TIP60 interactions

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Science Immunology  16 Jun 2017:
Vol. 2, Issue 12, eaai9297
DOI: 10.1126/sciimmunol.aai9297
  • Fig. 1 Diminished suppressive potency of primary Treg clones from IPEXA384T patients.

    (A) FOXP3 and CD25 expression in CD4+ cells of IPEXA384T patients and age- and sex-matched controls ex vivo. The FOXP3 MFI levels of CD25+FOXP3+ cells were determined in independent assessments and were normalized to the corresponding healthy controls. (B to E) Pools of single cell–derived clones of FOXP3+ and FOXP3 CD4+ T cells were generated from IPEX patients (shown as IPEX 1 and 2) and their corresponding controls (Control 1 and 2). (B) FOXP3 and CD25 expression at the harvest of the clones. (C) Cytokine production assessed by flow cytometry. (D) Proliferation of individual T cell clones in the absence of exogenous rhIL-2. cpm, counts per minute. (E) Suppression of proliferation of allogeneic CD4+CD25 T cells. Clone sample sizes: IPEX 1: FOXP3+ = 5, FOXP3 = 14; Control 1: FOXP3+ = 17, FOXP3 = 19; IPEX 2: FOXP3+ = 110, FOXP3 = 10, Control 2: FOXP3+ = 70, FOXP3 = 41. Data are presented as individual clones with line at median ± interquartile range. All data sets were compared with one-way nonparametric ANOVA (P < 0.0001) and followed by Dunn’s posttest.

  • Fig. 2 Impaired development of Treg cell function upon overexpression of FOXP3A384T in naïve CD4+CD25 T cells.

    (A) Schematic representation of the constructs used in transduction. (B) Expression levels of FOXP3, CD25, and CD127 in transduced cells on day 28 post-transduction. (C) Cytokine production by transduced cells expressing WT or mutated FOXP3. (D and E) Suppressive potency of empty vector (EV)–, FOXP3WT-, FOXP3A384T-, and FOXP3R397W-transduced cells. Transduced, GFP+ cells were cocultured with proliferation dye–labeled CD4+CD25 Teff cells at a 1:4 ratio. Results are representative of 11 independent assessments on seven independent transduction experiments carried out on cells from four different donors. All data sets were compared with one-way nonparametric ANOVA followed by Dunn’s posttest.

  • Fig. 3 Partial alteration of the Treg gene signature by the p.A384T mutation.

    FOXP3WT-, FOXP3A384T-, FOXP3R397W-, or empty vector–transduced cells were activated with PHA (0.5 μg/ml) for 36 hours and subjected to microarray analysis to compare their gene expression profiles. (A) A volcano plot showing the identification of genes that are modulated by the expression of FOXP3WT relative to empty vector–transduced cells [log2 (fold change) < −0.5 or > 0.5; P < 0.05]. (B) PCA of FOXP3-regulated genes [identified in (A)] in the four indicated conditions. Each circle represents one biological replicate. Component 1 (x axis) explains 66% while component 2 (y axis) explains 10% of the variance. (C) Heat map showing the expression levels of the FOXP3-regulated genes identified in (A). % Similarity to WT was estimated on the basis of the number of FOXP3-modulated genes that are differentially expressed in each condition relative to WT FOXP3WT-transduced cells. Biological replicates for each condition are shown side by side. (D) Selected FOXP3-modulated genes that are significantly differentially expressed (P < 0.05) in FOXP3WT-tranduced cells versus FOXP3A384T-transduced cells. (E) DAVID pathway analysis of 223 FOXP3-modulated genes that are differentially expressed (P < 0.05) in FOXP3WT-transduced cells versus FOXP3A384T-transduced cells.

  • Fig. 4 Impaired interaction of FOXP3A384T with TIP60.

    (A to C) 293T cells were transfected with EV, FOXP3WT, FOXP3A384T, or FOXP3R397W and FLAG-FOXP1, FLAG-TIP60, or FLAG-p300. Twenty-four hours after transfection, cell lysates were collected and immunoprecipitated, followed by blotting with anti-FLAG or anti-HA HRP. The co-immunoprecipitation of FOXP3 mutants with FOXP1 (A), TIP60 (B), or p300 (C) in representative experiments of at least three separate experiments is shown. IP, immunoprecipitated protein.

  • Fig. 5 Enhancement of TIP60-FOXP3 interaction by allosteric modification of TIP60.

    293T cells were transfected with HA-FOXP3WT and (A) FLAG-TIP60, or (B) FLAG-FOXP1 (top) or FLAG-p300 (bottom) in the presence or absence of SGF003 (8 μg/ml). (C) 293T cells were transfected with HA-FOXP3A384T and FLAG-TIP60 in the presence or absence of SGF (8 μg/ml). Twenty-four hours post-transfection, cells were washed with PBS, and cell lysates were prepared for immunoprecipitation and Western blot analysis. The effects of SGF treatment on the interaction of FOXP3 with the flagged proteins are shown. Representative blots from one of at least three separate experiments are also shown.

  • Fig. 6 Enhancement of Treg suppressive function by allosteric modification of TIP60.

    The suppressive potency of murine Treg (CD4+Foxp3+ splenocytes) or human Treg (CD4+CD25HighCD127Low) was assessed in the presence or absence of SGF003. SGF003 was added at the time of activation. Suppression was measured as the reduction in proliferation of responder Teff cells (murine CD4+FOXP3; human CD4+CD25CD127High). (A) The effect of SGF003 treatment on the proliferative capacity and IFN-γ production of Teff cells cultured in the absence of Treg cells. (B) The suppressive potency of murine and human Treg cells in the presence of SGF003 at various Treg/Teff ratios. The percentages of improvement in Treg suppressive capacity obtained by comparing the suppressive potency of untreated versus SGF003 (8 μg/ml)–treated Treg cells are shown. (C) Effect of SGF003 on the suppressive potency of FOXP3WT-, FOXP3A384T-, or FOXP3R397W-transduced cells cocultured with CD4+CD25CD127High Teff cells at a 1:4 Treg/Teff ratio. The results shown in this figure are representative of two to three independent experiments performed in triplicate. Statistical analyses were performed using t test (A) and one-way ANOVA (B and C).

  • Fig. 7 Improvement in the transcriptional activity of FOXP3A384T by enhancement of TIP60-FOXP3 interaction.

    FOXP3WT-, FOXP3A384T-, or empty vector–transduced cells were activated with PHA (0.5 μg/ml) for 36 hours in the presence or absence of SGF003 (8 μg/ml), followed by genome-wide microarray analysis. (A) PCA of FOXP3-regulated genes (identified in Fig. 3A) in the four indicated conditions. Component 1 (x axis) explains 65% whereas component 2 (y axis) explains 15% of the variance. (B) Heat map showing the impact of SGF treatment on the 223 FOXP3-regulated genes that are differentially expressed (P < 0.05) in untreated FOXP3WT-transduced cells versus FOXP3A384T-transduced cells. (C) The impact of SGF treatment on the expression level of 30 Treg-associated genes (shown in Fig. 3C) that are differentially expressed in FOXP3A384T-transduced cells.

  • Fig. 8 Therapeutic effects of TIP60 allosteric modification in DSS-induced colitis.

    Colitis was induced by adding 4% DSS to pH-balanced tap water of study mice. Disease progression was assessed by daily monitoring of body weight, stool consistency, and fecal blood. B7A (4 mg kg−1 day−1, 7 days) or DMSO control was injected intraperitoneally 1 week after DSS administration. The effects of B7A treatment on (A) body weight, (B) colon length, and (C) gut histology (hematoxylin and eosin–stained paraffin sections) are shown. (D) Frequency of FOXP3+ Treg cells in the spleen and lymph nodes of B7A-treated compared with DMSO-treated mice. n = 10 to 12 mice per group.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/2/12/eaai9297/DC1

    Fig. S1. Reduced expression of FOXP3A384T alters its ability to induce CD25 expression.

    Fig. S2. Cytokine repression is maintained by FOXP3A384T.

    Fig. S3. Unmodified blots from Fig. 4.

    Fig. S4. Model for the mechanism of action of TIP60 modifiers in the TIP60-p300-FOXP3 interaction in Treg cells.

    Fig. S5. Enhanced FOXP3-TIP60 interaction in response to SGF003 treatment in Jurkat cell line.

    Fig. S6. Unmodified blots from Fig. 5 (A and B).

    Fig. S7. Unmodified blots from Fig. 5C.

    Fig. S8. Enhancement of TIP60-FOXP3 interaction by B7A.

    Fig. S9. Improvement of Treg suppressive capacity by B7A-mediated enhancement of TIP6-FOXP3 interaction.

    Fig. S10. TH17 differentiation is not altered by SGF003 treatment.

    Fig. S11. Treatment of CIA with B7A.

    Table S1. Differentially expressed genes in FOXP3A384T-transduced cells.

    Table S2. SGF treatment improves the expression of several FOXP3-regulated genes.

    Raw data

  • Supplementary Materials

    Supplementary Material for:

    Suppression by human FOXP3+ regulatory T cells requires FOXP3-TIP60 interactions

    Khalid Bin Dhuban, Eva d'Hennezel, Yasuhiro Nagai, Yan Xiao, Steven Shao, Roman Istomine, Fernando Alvarez, Moshe Ben-Shoshan, Hans Ochs, Bruce Mazer, Bin Li, Chiyoko Sekine, Alan Berezov, Wayne Hancock, Troy R. Torgerson, Mark I. Greene, Ciriaco A. Piccirillo*

    *Corresponding author. Email: ciro.piccirillo{at}mcgill.ca

    Published 16 June 2017, Sci. Immunol. 2, eaai9297 (2017)
    DOI: 10.1126/sciimmunol.aai9297

    This PDF file includes:

    • Fig. S1. Reduced expression of FOXP3A384T alters its ability to induce CD25 expression.
    • Fig. S2. Cytokine repression is maintained by FOXP3A384T.
    • Fig. S3. Unmodified blots from Fig. 4.
    • Fig. S4. Model for the mechanism of action of TIP60 modifiers in the TIP60- p300-FOXP3 interaction in Treg cells.
    • Fig. S5. Enhanced FOXP3-TIP60 interaction in response to SGF003 treatment in Jurkat cell line.
    • Fig. S6. Unmodified blots from Fig. 5 (A and B).
    • Fig. S7. Unmodified blots from Fig. 5C.
    • Fig. S8. Enhancement of TIP60-FOXP3 interaction by B7A.
    • Fig. S9. Improvement of Treg suppressive capacity by B7A-mediated enhancement of TIP6-FOXP3 interaction.
    • Fig. S10. TH17 differentiation is not altered by SGF003 treatment.
    • Fig. S11. Treatment of CIA with B7A.
    • Table S1. Differentially expressed genes in FOXP3A384T-transduced cells.
    • Table S2. SGF treatment improves the expression of several FOXP3-regulated genes.

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