Research ArticleGRAFT VERSUS HOST DISEASE

ST2 blockade reduces sST2-producing T cells while maintaining protective mST2-expressing T cells during graft-versus-host disease

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Science Translational Medicine  07 Oct 2015:
Vol. 7, Issue 308, pp. 308ra160
DOI: 10.1126/scitranslmed.aab0166
  • Fig. 1. ST2 blockade and GVHD.

    (A) Irradiated C3H.SW mice (1100 cGy) were transplanted with syngeneic (Embedded Image) or allogeneic B6 (Embedded Image) BM cells (5 × 106) and splenic purified T cells (2 × 106). sST2 concentrations in plasma collected at the indicated times after HCT from C3H.SW recipients (ng/ml) (P = 0.0001, t test; n = 10 to 12). The data are from four independent experiments. (B) Clinical scores of GVHD and survival curves for C3H.SW mice receiving syngeneic (Embedded Image) or allogeneic B6 cells and treated with anti-mouse ST2 antibody (Embedded Image) or IgG control antibody (Embedded Image) at day −1 and day +1 after HCT. The data are from three independent experiments (P values for GVHD scores are given in table S1; P = 0.0256 for survival analysis, t test for GVHD score and log-rank test for survival analysis; n = 15 to 23 per group). (C) Irradiated NSG mice (350 cGy) received 2.5 × 106 T cells purified from peripheral blood mononuclear cells (PBMCs) of healthy donors (Embedded Image). The control group was irradiated without receiving human T cells (Embedded Image). Human soluble ST2 concentrations in plasma collected at the indicated times after HCT from NSG recipient mice with or without engrafted human T cells (pg/ml). The data are from three independent experiments (P = 0.0028, t test; n = 7 to 9 per group). (D) Clinical scores of GVHD and survival curves for NSG mice receiving human T cells and treated with anti-human and anti-mouse ST2 antibodies (Embedded Image) or IgG control antibody (Embedded Image) every other day from day −1 to day +5 (four doses) (P values for GVHD scores are given in table S1, P = 0.0329 for survival analysis; t test for GVHD score and log-rank test for survival analysis; n = 10 per group). (E) IFN-γ, IL-17, IL-23, IL-10, and IL-33 concentrations in plasma collected every 5 days after HCT from the B6→C3H.SW model (pg/ml). The data are from three independent experiments. Syngeneic group (Embedded Image); allogeneic groups treated with anti-ST2 (Embedded Image) or IgG control (Embedded Image) (P values are given in table S1; t test; n = 3 to 9 per group).

  • Fig. 2. sST2 and mST2 expression during GVHD.

    (A) In the B6→C3H.SW model, mRNA expression of sST2 and mST2 in different organs (spleen, small intestine, large intestine, skin, BM, lung, heart, liver, and peripheral blood) of C3H.SW recipient mice at day 10 after allo-HCT. The data are from four independent experiments [P values are given in table S2, one-way analysis of variance (ANOVA); n = 4]. (B) sST2/actin mRNA expression in intestine or intestinal cell subsets (epithelial, stromal, endothelial, or non–T hematopoietic cells) from C3H.SW recipient mice at day 10 after allo-HCT (n = 5). The data are from two independent experiments with two to three pooled mice. SI, small intestine. (C) Western blot analysis of sorted intestinal stromal cells from IgG control– or anti-ST2 mAb–treated C3H.SW recipient mice at day 10 after allo-HCT [M, marker (kD)] (left). The red box indicates the lack of sST2 protein present after anti-ST2 treatment. The bar graph shows the sST2/actin ratio in IgG control– or anti-ST2–treated mice (n = 6). The data are from two independent experiments with three pooled mice. (D) sST2/actin expression on sorted intestinal stromal and T cells from C3H.SW mice at day 10 and day 28 after allo-HCT. The data are from two independent experiments with two to three pooled mice (P = 0.0120, t test; n = 5). (E) Western blot analysis of sorted intestinal T cells from IgG control– or anti-ST2 mAb–treated C3H.SW recipients 10 days after allo-HCT [M, marker (kD)]. The red box indicates the lack of sST2 protein present after anti-ST2 treatment. The unmodified blots are shown in fig. S9. The bar graph shows the sST2/actin ratio in CD4 T cells from IgG– control or anti-ST2–treated mice. The data are from two independent experiments with three pooled mice (P = 0.0032, t test; n = 6). (F) sST2 secretion by both murine and human in vitro differentiated T cell subsets. The data are from three to four independent experiments (P values are given in table S2; t test; n = 3 to 4). The unmodified blots are shown in fig. S9.

  • Fig. 3. ST2 deficiency and T cell populations during GVHD.

    (A) Transcriptome analysis of T cell–related genes in MLN T cells from anti-ST2–treated versus IgG-treated C3H.SW recipient mice at day 10 after allo-HCT. MLN T cells from four mice in each group were pooled for analysis. (B and C) Flow cytometric analysis of transcription factor and cytokine production by donor-derived CD4+ splenic T cells from IgG-treated or anti-ST2–treated C3H.SW recipient mice at day 10 after allo-HCT. The bar graphs show the percentages of cells expressing IFN-γ or Tbet (P = 0.0150 for IFN-γ and P = 0.0055 for Tbet, t test; n = 5) (B) and IL-17/IFN-γ or RORγt (P = 0.0003 for IL-17/IFN-γ and P = 0.0075 for RORγt, t test; n = 4 to 5) (C). The data are from two independent experiments. Gating strategy for (B) and (C) is found in fig. S10. (D) Survival curves for C3H.SW recipient mice receiving either only 5 × 106 wild-type (WT) B6 BM cells (Embedded Image) or 2 × 106 WT (Embedded Image) or ST2−/− B6 T cells (Embedded Image) (P = 0.0289, log-rank test; n = 6 to 10). The data are from two independent experiments. (E to G) Flow cytometric analysis at day 10 after allo-HCT shows percentages of intestinal CD4+ T cells expressing IFN-γ and Tbet (P = 0.0173 for IFN-γ and P = 0.0320 for Tbet, t test; n = 4) (E) and IL-17/IFN-γ and RORγt (P = 0.0273 for IL-17/IFN-γ and P = 0.0273 for RORγt, t test; n = 4 to 5) (F) as well as Ki67 proliferation staining of cells expressing both IL-17 and IFN-γ (P = 0.0088, t test; n = 4) (G). The data are from two independent experiments. MFI, mean fluorescence intensity. (H) The bar graphs show the percentages of T cells expressing IL-4 or GATA3 from IgG-treated or anti-ST2–treated C3H.SW recipient mice at day 10 after allo-HCT, and the flow cytometry plots show mST2 expression on GATA3 T cells after IgG or anti-ST2 treatment. The data are from two independent experiments (P = 0.0049 for IL-4 and P = 0.0252 for GATA3, t test; n = 6). (I) IL-4– and GATA3-expressing T cells from C3H.SW recipient mice receiving WT or ST2−/− B6 T cells at day 10 after allo-HCT (P = 0.0032 for IL-4 and P = 0.0253 for GATA3, t test; n = 4). The data are from two independent experiments. (J) The bar graphs show the percentages of intestinal T cells expressing FoxP3 from IgG-treated or anti-ST2–treated C3H.SW recipient mice at day 10 after allo-HCT, and the flow cytometry plots show mST2 expression on FoxP3 T cells after IgG or anti-ST2 treatment (P = 0.0087, t test; n = 5). The data are from two independent experiments. (K) FoxP3- and IL-10–expressing T cells from C3H.SW recipient mice receiving WT or ST2−/− B6 T cells at day 10 after allo-HCT. The data are from two independent experiments (P = 0.0459 for FoxP3 and P = 0.0471 for IL-10, t test; n = 4 to 5). (L) Survival curves for C3H.SW recipient mice transplanted with 5 × 106 B6 TCD BM cells plus 2 × 105 WT (Embedded Image) or ST2−/− B6 (Embedded Image) Tregs with 2 × 106 WT B6 Tconv cells. (Embedded Image, TCD BM only). The data are from two independent experiments (P = 0.043, log-rank test; n = 5 to 10 per group). Flow cytometric gating strategies are shown in fig. S10.

  • Fig. 4. ST2 deficiency and antigen-presenting cells during GVHD.

    Flow cytometric analysis of intestinal MDSCs (MAC-1+Gr-1+ cells), CD103+ dendritic cells, and CD11c+ total dendritic cells in the B6 (CD45.1+)→C3H.SW (CD45.2+) model at 10 days after allo-HCT. (A) Donor CD45.1+ MDSCs in IgG control– or anti-ST2–treated C3H.SW recipients (P = 0.0247, t test; n = 4). The data are from two independent experiments. (B) Donor CD45.1+ MDSCs in C3H.SW recipients receiving WT or ST2−/− B6 T cells (P = 0.0277, t test; n = 3). (C) Donor CD45.1+CD103+ dendritic cells (DC) in IgG control– or anti-ST2–treated C3H.SW recipients (P = 0.0012, t test; n = 8). The data are from four independent experiments. (D) Donor CD45.1+CD103+ dendritic cells in C3H.SW recipients receiving WT or ST2−/− B6 T cells (P = 0.0244, t test; n = 3). (E) Expression of MHC class II and costimulation molecules on CD11c+ total dendritic cells from IgG control– and anti-ST2–treated mice, representative flow cytometry histograms (top panels), and bar graphs (bottom panels) of MFI (P = 0.0391 for MHC class II, P = 0.0469 for CD40, P = 0.0154 for CD80, and P = 0.0263 for CD86, t test; n = 3). Flow cytometric gating strategies are shown in fig. S11.

  • Fig. 5. ST2 deficiency and GVL activity.

    (A) Transcriptome analysis of antitumor-related genes in MLN T cells from anti-ST2–treated versus IgG-treated C3H.SW recipient mice at day 10 after allo-HCT. MLN T cells from four mice in each group were pooled for analysis. (B) In vitro cytotoxic T lymphocyte assay with A20 and MLL-AF9 retrovirally induced acute myeloid leukemia in the presence of IgG control (Embedded Image) and anti-ST2 mAb (Embedded Image) (5 μg/ml). Syngeneic control (Embedded Image) (n = 3). The data are from three independent experiments. (C) Survival curves of C3H.SW mice receiving 104 GFP+ MLL-AF9 leukemic cells with syngeneic HCT C3H.SW→C3H.SW (Embedded Image) or allo-HCT (B6→C3H.SW) treated with IgG control (Embedded Image) or anti-ST2 mAb (Embedded Image) (P = 0.010, log-rank test; n = 5 to 10 per group). (D) Survival curves of C3H.SW mice receiving 104 GFP+ MLL-AF9 leukemic cells with syngeneic HCT C3H.SW→C3H.SW (Embedded Image) or allo-HCT (B6→C3H.SW) treated with six doses (100 μg per dose, every other day from day −1 to day +9) of IgG control (Embedded Image) or anti-ST2 mAb (Embedded Image) (P = 0.0357, log-rank test; n = 4 to 5 per group). (E) Survival curves of C3H.SW mice receiving 104 GFP+ MLL-AF9 leukemic cells with WT (Embedded Image) or ST2−/− (Embedded Image) B6 T cells (P = 0.0203, log-rank test; n = 6 to 10 per group).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/308/308ra160/DC1

    Materials and Methods

    Fig. S1. Six dose ST2 blockade and GVHD.

    Fig. S2. Host ST2 deficiency and GVHD.

    Fig. S3. mST2 expression on T cell subsets.

    Fig. S4. Antigen-presenting cells and GVHD.

    Fig. S5. MLN dendritic cells and GVHD.

    Fig. S6. IL-33 administration and GVHD.

    Fig. S7. Immune complex depletion.

    Fig. S8. ST2 deficiency in Tconv cells and GVHD.

    Fig. S9. Unmodified blots.

    Fig. S10. Gating strategies of flow cytometric analysis for Fig. 3.

    Fig. S11. Gating strategies of flow cytometric analysis for Fig. 4E.

    Table S1. P values for Fig. 1.

    Table S2. P values for Fig. 2.

    Table S3. P values for figs. S1, S2, and S8.

    Table S4. Antibodies for flow cytometry.

  • Supplementary Material for:

    ST2 blockade reduces sST2-producing T cells while maintaining protective mST2-expressing T cells during graft-versus-host disease

    Jilu Zhang, Abdulraouf M. Ramadan, Brad Griesenauer, Wei Li, Matthew J. Turner, Chen Liu, Reuben Kapur, Helmut Hanenberg, Bruce R. Blazar, Isao Tawara, Sophie Paczesny*

    *Corresponding author. E-mail: sophpacz{at}iu.edu

    Published 7 October 2015, Sci. Transl. Med. 7, 308ra160 (2015)
    DOI: 10.1126/scitranslmed.aab0166

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Six dose ST2 blockade and GVHD.
    • Fig. S2. Host ST2 deficiency and GVHD.
    • Fig. S3. mST2 expression on T cell subsets.
    • Fig. S4. Antigen-presenting cells and GVHD.
    • Fig. S5. MLN dendritic cells and GVHD.
    • Fig. S6. IL-33 administration and GVHD.
    • Fig. S7. Immune complex depletion.
    • Fig. S8. ST2 deficiency in Tconv cells and GVHD.
    • Fig. S9. Unmodified blots.
    • Fig. S10. Gating strategies of flow cytometric analysis for Fig. 3.
    • Fig. S11. Gating strategies of flow cytometric analysis for Fig. 4E.
    • Table S1. P values for Fig. 1.
    • Table S2. P values for Fig. 2.
    • Table S3. P values for figs. S1, S2, and S8.
    • Table S4. Antibodies for flow cytometry.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Additional Data Tables (separate file)

    [Download Additional Data Tables]

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