Research ArticleImmunology

Tim-3 signaling in peripheral NK cells promotes maternal-fetal immune tolerance and alleviates pregnancy loss

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Science Signaling  26 Sep 2017:
Vol. 10, Issue 498, eaah4323
DOI: 10.1126/scisignal.aah4323
  • Fig. 1 Human Tim-3+ NK cells increase in number and have an immune tolerant phenotype during early pregnancy.

    (A to C) Tim-3 abundance on NK cells is increased during the first trimester of pregnancy. (A and B) The percentage of peripheral CD56+ cells from nonpregnant donors (Non, 10 donors), first-trimester donors (1st, 30 donors), and second-trimester donors (2nd, 9 donors) that were Tim-3+ was determined by flow cytometric analysis (A), whereas the abundance of Tim-3 in the same cells was determined by Western blotting analysis (B). (C) The percentages of CD3CD56+, CD3CD56dim, and CD3CD56bright pNK cells in early pregnancy that were Tim-3+ were determined by flow cytometry. Data are from 30 donors. (D and E) Human Tim-3+ pNK cells show an immunosuppressive phenotype. Purified Tim-3+ or Tim-3 CD56+ pNK cells were stimulated with phorbol 12-myristate 13-acetate, ionomycin, and brefeldin A for 4 hours. The cells were analyzed by flow cytometry to detect the indicated cytokines and perforin. (D) Numbers in the plots show the percentage of cells in the boxed regions from a representative experiment. IFN-γ, interferon-γ. (E) Data are means ± SEM of the percentage of cells positive for the indicated marker from six independent experiments. (F) Tim-3+ NK cells have a reduced cytotoxicity toward trophoblast cells. Cytotoxicity was determined with CytoTox 96 Non-Radioactive Cytotoxicity Assay. The trophoblast cell line HTR-8/SVneo was used as the target (T) cell for NK cells. Data are means ± SEM of nine samples per group. (G) Tim-3+ and Tim-3 pNK cells were analyzed by real-time polymerase chain reaction (PCR) to determine the relative abundance of the indicated mRNAs. Data are means ± SEM of three experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 by Student’s t test. (H and I) Tim-3+ and Tim-3 NK cells were sorted from the PBMCs of a pregnant donor at first trimester. RNA was extracted, and global gene expression was analyzed by microarray. Data are representative of six donors. The unique gene signatures based on a >2-fold difference in expression for Tim-3 NK cells (100 probes, gray) and Tim-3+ NK cells (865 probes, yellow) are highlighted. (I) The heat map depicts the expression of genes associated with immune tolerance and activation in Tim-3 and Tim-3+ NK cells.

  • Fig. 2 The IL-4–STAT6 pathway and progesterone promote the increased number of Tim-3+ NK cells in early pregnancy, whereas Gal-9–Tim-3 interactions suppress NK cell activation.

    (A and B) NK cells isolated from nonpregnant donors were left untreated (Ctrl) or were treated with IL-4 (100 ng/ml), IFN-γ (35 ng/ml), or both for 48 hours. The cells were then analyzed by flow cytometry to determine the percentage of Tim-3+ cells (A) and by Western blotting to detect the phosphorylation of STAT6 (B). pSTAT6, phosphorylated STAT6; tSTAT6, total STAT6. (C) NK cells were stimulated with IL-4 alone or in the presence of the STAT6 inhibitor A77-1726 for 48 hours. The cells were then analyzed by flow cytometry to determine the percentage of Tim-3+ cells. *P < 0.05, **P < 0.01, and ***P < 0.001 by analysis of variance (ANOVA). DMSO, dimethyl sulfoxide. (D) NK cells stimulated with the indicated concentrations of progesterone were analyzed by flow cytometry to determine the percentage of Tim-3+ cells. ***P < 0.001 by Student’s t test. Data in (A) to (D) are means ± SEM of three independent experiments. (E) pNK cells from early pregnant donors were stimulated by rhGal-9 alone or in the presence of blocking antibodies against Tim-3 or CD44, as indicated, for 48 hours. The cells were then analyzed by flow cytometry to determine the percentage of cells positive for the indicated cytokines or perforin. Data are means ± SEM of 12 donors. IgG, immunoglobulin G. (F) NK cells were treated with rhGal-9 in the presence or absence of the indicated blocking antibodies before being incubated with target cells at the indicated ratios to determine cytotoxicity as described in Materials and Methods. Data are means ± SEM of four experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 by ANOVA. (G) NK cells were stimulated with rhGal-9 for the indicated times before being analyzed by Western blotting with antibodies against the indicated proteins. Bar graphs show pooled densitometric data from three experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 by Student’s t test. pNF-κB, phosphorylated nuclear factor κB; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NS, not significant. (H) Purified NK cells were stimulated with rhGal-9 in 15 μM LY294002, 10 μM SP600125, 10 μM U0126, or DMSO, as indicated. The cells were analyzed by flow cytometry to determine the percentage of cells positive for the indicated cytokines or perforin. Data are means ± SEM of 10 experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by ANOVA. (I and J) NK cells were left unstimulated or were stimulated with rhGal-9 for 48 hours and then analyzed by real-time PCR to determine the relative abundances of the indicated mRNAs (I) and by Western blotting with antibodies against the indicated proteins (J). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student’s t test. (K) NK cells were left untreated or treated with rhGal-9 in the presence or absence of the indicated inhibitors. The relative abundances of the indicated mRNAs were then determined by real-time PCR analysis. Data in (I) to (K) are means ± SEM of three experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 by ANOVA.

  • Fig. 3 Tim-3+ NK cells from RM patients have reduced immunosuppressive activity.

    (A) The relative abundances of Tim-3 on pNK cells from donors with NP and patients with RM were determined by flow cytometry. Data are means ± SEM of 20 donors per group. (B) The concentrations of soluble Gal-9 in the plasma of NP donors and RM patients were determined by enzyme-linked immunosorbent assay. Data are means ± SEM of six NP donors and eight RM patients. (C) PBMCs from NP donors and RM patients were analyzed by flow cytometry to determine the percentages of Gal-9+ cells among the indicated immune cell subsets. Data are means ± SEM of six NP donors and eight RM patients. (D) Tim-3+ NK cells (left) and Tim-3 NK cells (right) from NP donors and RM patients were analyzed by flow cytometry to determine the percentages of cells that were positive for the indicated cytokines or perforin. Data are means ± SEM of 8 NP donors and 10 RM patients. (E) Tim-3+ and Tim-3 NK cells from NP donors and RM patients were analyzed to determine their cytotoxicity, as described earlier. Data are means ± SEM of nine donors per group. *P < 0.05, **P < 0.01, and ***P < 0.001 by Student’s t test.

  • Fig. 4 Changes in chromatin accessibility in Tim-3+ NK cells are involved in the abnormal cytokine profile associated with RM.

    (A and B) Tim-3+ and Tim-3 NK cells were sorted from NP donors (n = 3) or RM patients in the first trimester (n = 2). Global chromatin accessibility was analyzed by ATAC-seq and was compared using PCA (A). On the basis of a hierarchical clustering analysis, RM patient Tim-3+ NK cells displayed distinct global chromatin accessibility compared with NK cells from NP donors (B). (C) ATAC-seq analysis was used to compare chromatin accessibility between Tim-3+ NK cells from NP donors and RM patients. Regulatory regions showing a statistically significant difference (P < 0.05) are labeled in red. (D and E) Tim-3+ NK cells from NP donors and RM patients were analyzed to assess chromatin accessibility at enhancer and promoter regions. Regulatory regions that showed reduced accessibility (left), unaltered accessibility (middle), or increased accessibility (right) were analyzed for the presence of H3K4me3 and H3K4me1 (D) or for distance to the closest TSS (E). bp, base pair. (F and G) Tim-3+ NK cells from NP donors and RM patients were analyzed to assess accessibility at the TGFB1 (F) and IL10 (G) loci. Top to bottom: The genome browser tracks of ATAC-seq for Tim-3+ NK cells from two healthy donors and two RM patients and then those for the Tim-3 NK cells from the same individuals. The numbers on the left indicate the ATAC-seq reads after normalization to 10 × 106 mapped reads. (H) Sorted Tim-3+ and Tim-3 NK cells from NP donors and RM patients were treated with the indicated concentrations of JQ1, and the percentages of the NK cells that were positive for TNF-α (left) and IFN-γ (right) were determined by flow cytometry. Data are means ± SEM of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 by Student’s t test.

  • Fig. 5 Tim-3+ NK cells from RM patients are less potent than those from controls in inducing iTreg differentiation through TGF-β1.

    (A and B) Naïve CD4+ T cells isolated from the peripheral blood of NP donors were stimulated with anti-CD3 and anti-CD28 antibodies for 5 days before being cocultured in the presence or absence of anti–TGF-β1 antibody with Tim-3+ or Tim-3 NK cells obtained from NP donors. The percentages of Foxp3+CD4+ T cells (top) and TGF-β1+Foxp3+CD4+ T cells (bottom) were determined by flow cytometry. Data in the bar graphs are means ± SEM of nine donors. (C and D) Tim-3+ NK cell–induced CD4+CD25+ T cells (iTregs), pTregs, and CD4+CD25 cells in the indicated combinations were restimulated with anti-CD3 and anti-CD28 antibodies. (C) Their immunosuppressive function was assessed by measurement of 5-ethynyl-2′-deoxyuridine (EdU) incorporation. (D) The percentages of the indicated cell populations that were positive for IFN-γ were determined by flow cytometry. Data are means ± SEM of nine donors. (E and F) Naïve CD4+ T cells isolated from the peripheral blood of NP donors were stimulated with anti-CD3 and anti-CD28 antibodies for 5 days before being cocultured in the presence or absence of anti–TGF-β1 antibody with Tim-3+ or Tim-3 NK cells obtained from RM patients. The percentages of Foxp3+CD4+ T cells (top) and TGF-β1+Foxp3+CD4+ T cells (bottom) were determined by flow cytometry. Data in the bar graphs are means ± SEM of 12 donors per group. (G) Tim-3+ and Tim-3- NK cells from NP donors and RM patients were analyzed by flow cytometry to determine the percentages of Foxp3+CD4+ T cells (left) and TGF-β1+Foxp3+CD4+ T cells (right). Data are means ± SEM of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 by ANOVA.

  • Fig. 6 Tim-3 blockade breaks maternal-fetal tolerance and impairs pregnancy maintenance.

    (A) Representative pictures showing the number of embryos per uterus from NP and AP mouse models treated with RMT3-23 and Gal-9 alone or in combination, as indicated by conditions (i) to (viii). (B) Analysis of the percentage of fetal resorption in the NP and AP mouse models treated as indicated in (A). (C) Summary of litter sizes at gestational day (GD) 14.5 in the NP and AP mouse models that were treated as indicated in (A). (D) Representative pictures showing embryos from the NP and AP mouse models that were treated as described in (A). PBS, phosphate-buffered saline. (E) Analysis of fetal size (volume) in the NP and AP mouse models at GD 14.5 after treatment as indicated in (A). (F) Spleens from the NP and AP mouse models treated with the indicated combinations of RMT2-23 and Gal-9 were analyzed by flow cytometry to determine the percentages of NK cells that were positive for the indicated cytokines. Data are means ± SEM of five to eight mice per group. (G) Left: Splenic NK cells from the AP mice treated as described in (A) were analyzed by flow cytometry to determine the relative abundances of pJNK and pAKT. Right: Calculation of the MFIs for pJNK and pAKT. Data are means ± SEM of five to eight mice per group. (H) Splenic NK cells from the AP mice treated as described in (A) were analyzed to determine the percentage of cells positive for the indicated transcriptional regulators. Data are means ± SEM of five to eight mice per group. (I and J) Left: Peripheral blood (top) and splenic (bottom) CD4+ T cells from AP mice that were treated as described in (A) were analyzed by flow cytometry to determine the percentage of Foxp3+ cells. Representative dot plots are shown. Right: Calculation of the percentages of Foxp3+CD4+ T cells in the blood and spleen under the indicated conditions. Data are means ± SEM of five to eight mice per group. *P < 0.05, **P < 0.01, and ***P < 0.001 by ANOVA.

  • Fig. 7 Tim-3+ NK cells alleviate fetal loss in AP and NK-deficient mice.

    (A) Schematic for the adoptive transfer of different NK cell subsets from the indicated pregnant mice to AP pregnant mice or Nfil3−/− pregnant mice. Top: Tim-3+ NK cells, Tim-3 NK cells, and total NK cells were sorted from NP mice and transferred into the tail vein of AP pregnant mice. Middle: Total NK cells sorted from the AP models were transferred directly into the tail vein of pregnant AP mice or were treated with IL-4 or progesterone for 48 hours in vitro before being sorted by fluorescence-activated cell sorting (FACS) into Tim-3+ and Tim-3 NK cells and then transferred to AP pregnant mice. Bottom: Tim-3+ and Tim-3 NK cells were sorted from normal C57BL/6 (Nfil3+/+) pregnant mice and transferred directly into the tail vein of Nfil3−/− pregnant mice. (B) Representative pictures of embryos in the uterus of AP mice that had received the indicated NK cells. (C) NK cells isolated from AP mice were left untreated (Ctrl) or were treated with IL-4 or progesterone (P) before being analyzed by flow cytometry to determine the percentage of Tim-3+ cells. (D) Analysis of fetal resorption in AP mice that had received the indicated NK cells. Data are means ± SEM of 9 to 12 mice per group. (E) Flow cytometric analysis of the percentage of CD25+Foxp3+ cells within the peripheral blood CD4+ T cells of AP pregnant mice that had received the indicated NK cells. (F) Representative pictures of embryos in the uterus of control Nfil3−/− mice or those that received either Tim-3+ or Tim-3- NK cells. (G) Analysis of fetal resorption in pregnant control Nfil3−/− mice or those that received the indicated NK cells. Data are means ± SEM of five mice per group. (H) Left: Flow cytometric analysis of the percentages of CD25+Foxp3+ T cells and TGF-β1+CD25+Foxp3+ T cells in control Nfil3−/− mice or those that received the indicated NK cells. Right: Measurement of the percentages of the indicated cells. Data are means ± SEM of five mice per group. *P < 0.05, **P < 0.01, and ***P < 0.001 by ANOVA.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/498/eaah4323/DC1

    Fig. S1. Detection of Tim-3 on different cell types in the peripheral blood of patients in early pregnancy.

    Fig. S2. GO analysis of differentially expressed genes.

    Fig. S3. Estrogen and β-hCG have no effect on Tim-3 abundance on pNK cells.

    Fig. S4. The amounts of TGF-β1 and perforin in Tim-3+ and Tim-3 NK cells are unchanged by the BET inhibitor JQ1.

    Fig. S5. AP mice show decreased Tim-3 and Gal-9 abundance and have dysfunctional NK cells.

    Fig. S6. The percentage of NK cells in the blood is reduced by RMT3-23.

    Fig. S7. The function of dNK cells is impaired after Tim-3 blockade.

    Fig. S8. Tregs are generated in mice that received Tim-3+ NK cells.

    Fig. S9. Model of how Tim-3 on pNK cells may participate in establishing an immune tolerant phenotype during early pregnancy.

  • Supplementary Materials for:

    Tim-3 signaling in peripheral NK cells promotes maternal-fetal immune tolerance and alleviates pregnancy loss

    Yanhong Li, Jiayuan Zhang, Di Zhang, Xiaowu Hong, Yu Tao, Songcun Wang, Yuanyuan Xu, Hailan Piao, Weijie Yin, Min Yu, Yin Zhang, Qiang Fu, Dajin Li, Xing Chang,* Meirong Du*

    *Corresponding author. Email: changxing{at}sibs.ac.cn (X.C.); mrdu{at}fudan.edu.cn (M.D.)

    This PDF file includes:

    • Fig. S1. Detection of Tim-3 on different cell types in the peripheral blood of patients in early pregnancy.
    • Fig. S2. GO analysis of differentially expressed genes.
    • Fig. S3. Estrogen and β-hCG have no effect on Tim-3 abundance on pNK cells.
    • Fig. S4. The amounts of TGF-β1 and perforin in Tim-3+ and Tim-3 NK cells are unchanged by the BET inhibitor JQ1.
    • Fig. S5. AP mice show decreased Tim-3 and Gal-9 abundance and have dysfunctional NK cells.
    • Fig. S6. The percentage of NK cells in the blood is reduced by RMT3-23.
    • Fig. S7. The function of dNK cells is impaired after Tim-3 blockade.
    • Fig. S8. Tregs are generated in mice that received Tim-3+ NK cells.
    • Fig. S9. Model of how Tim-3 on pNK cells may participate in establishing an immune tolerant phenotype during early pregnancy.

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    Citation: Y. Li, J. Zhang, D. Zhang, X. Hong, Y. Tao, S. Wang, Y. Xu, H. Piao, W. Yin, M. Yu, Y. Zhang, Q. Fu, D. Li, X. Chang, M. Du, Tim-3 signaling in peripheral NK cells promotes maternal-fetal immune tolerance and alleviates pregnancy loss. Sci. Signal. 10, eaah4323 (2017).

    © 2017 American Association for the Advancement of Science

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