Research ArticleFETAL IMMUNITY

Alloreactive fetal T cells promote uterine contractility in preterm labor via IFN-γ and TNF-α

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Science Translational Medicine  25 Apr 2018:
Vol. 10, Issue 438, eaan2263
DOI: 10.1126/scitranslmed.aan2263
  • Fig. 1 Elevated levels of inflammatory markers in the fetal plasma and increased expression of activation markers on fetal DCs during PTL.

    (A) Luminex analysis of cytokines present in the cord blood plasma of infants born at term (term; n = 28) or preterm (PTL; n = 18). The horizontal line represents the median for each group. *P < 0.05, **P < 0.01, and ***P < 0.001 by Mann-Whitney test. Flt3L, Flt3 ligand. (B) Representative flow cytometric analysis of purified cord blood mononuclear cells from preterm infants stained to detect mDCs (CD11c+HLA-DR+) and pDCs (CD123+HLA-DR+) after gating out CD3+ (T cells), CD19+, and CD20+ (B cells). On the right, representative histograms of CD80 and CD86 expression on mDC and pDC in the cord blood of term (term) or preterm (PTL) infants. (C) Pooled data of the mean fluorescence intensity (MFI) of the activation markers CD80 and CD86 on maternal and cord blood mDC. **P < 0.01 by Mann-Whitney test. (D) Pooled data of the MFI of the activation markers CD80 and CD86 on maternal and cord blood pDC. The relative MFI was calculated over the intensity of an isotype control antibody. Each symbol represents a single patient; small horizontal bars indicate the mean ± SEM. Healthy term maternal samples (term), n = 8; healthy term fetal samples (term), n = 13; preterm maternal samples (PTL), n = 12; preterm fetal samples (PTL), n = 16. *P < 0.05 by Mann-Whitney test.

  • Fig. 2 Increased percentages of CM T cells with a TH1 phenotype in cord blood during PTL.

    (A) Representative flow cytometric analysis of purified maternal and cord blood PBMCs from healthy term controls (term) and patients with PTL (PTL) stained to detect CD4+ naïve (N; CCR7+CD45RA+), CD4+ EM (CCR7CD45RA), and CD4+ CM (CCR7+CD45RA) T cells. Numbers in quadrants indicate the percentage of cells in each. (B) Pooled data of the percentage of CD4+ N cells and CD4+ CM cells among the total CD4+ T cells in maternal or cord blood. Each symbol represents a single patient; small horizontal bars indicate the mean ± SEM. Term, n = 35; PTL, n = 37. ***P < 0.001 by Mann-Whitney test. (C) Representative flow cytometric analysis of purified maternal and cord blood PBMCs from healthy term controls (term) and patients with PTL (PTL) stained to detect TH1 cells (CXCR3+CCR6) and TH17 cells (CXCR3CCR6+). Numbers in quadrants indicate the percentage of cells in each. (D) Pooled data of the percentage of TH1 cells among CM cells in maternal or cord blood. Each symbol represents a single patient; small horizontal bars indicate the mean ± SEM. Term, n = 19; PTL, n = 27. ***P < 0.001 by Mann-Whitney test. (E) Left: Representative histogram of intracellular cytokine staining for IFN-γ in CD4+ CM T cells isolated from the cord blood of a preterm infant by cell sorting according to CXCR3 expression and then stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin for 5 hours. Right: Pooled data of the percentage of IFN-γ–secreting cells among CXCR3 and CXCR3+ sorted cells from the cord blood of term and PTL patients. Each symbol represents a single patient; small horizontal bars indicate the mean ± SEM. Term, n = 6; PTL, n = 6. ***P < 0.001 by Mann-Whitney test.

  • Fig. 3 Increased maternal microchimerism in preterm infants and correlation with CM cells.

    (A) Maternal microchimerism (MMc) in cord blood of preterm infants (PTL; n = 18) and healthy term controls (term; n = 24), and fetal microchimerism (FMc) in the peripheral blood of women with PTL (PTL; n = 16) and healthy term controls (term; n = 25). Each symbol represents a single patient; small horizontal bars indicate the mean ± SEM. *P < 0.05 by Mann-Whitney test. (B) Correlation between the levels of MMc and CM CD4+ T cells in the cord blood of preterm infants (ρ = 0.93, P = 0.007 by Spearman’s rank correlation test) (n = 7 samples with both MMc and CM data).

  • Fig. 4 Increased proliferation of fetal CD4+ and CD8+ T cells with indirect reactivity against maternal antigens during PTL.

    (A) Schematic representation of the components of the MLR indirect pathway assay. (B and C) Representative flow cytometric analysis of carboxyfluorescein diacetate succinimidyl ester (CFSE)–labeled CD4+ T cells (B) and CD8+ T cells (C) from the cord blood of control (term) or preterm (PTL) infants, cultured with autologous APC in the presence of maternal lysate. (D and E) Pooled data of the percentages of proliferated maternal and fetal CD4+ (D) and CD8+ (E) T cells cultured with autologous APC in the presence or absence of maternal or fetal lysate, respectively, and a third-party lysate derived from adult women of reproductive age, as indicated under each graph. Open symbols represent term samples; filled symbols represent PTL samples. Each symbol represents a single patient; small horizontal bars indicate the mean ± SEM. M, maternal; F, fetal. n ≥ 4 in ≥11 representative experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 by Mann-Whitney test. (F and G) Left: CFSE dilution profile and IFN-γ (F) and TNF-α (G) production in fetal CD4+ and CD8+ T cells in a representative term and a preterm infant. Right: Pooled data are also shown. n ≥ 4 in ≥3 representative experiments. *P < 0.05 and **P < 0.01 by Mann-Whitney test.

  • Fig. 5 Increased myometrial cell contractility in response to fetal T cells from preterm infants.

    (A) Experimental design. Uterine myometrial cells (PHM1-41) were embedded in collagen in a 24-well plate and incubated for 2 days at 37°C. Contraction mediators/blockers or sorted fetal CD4+ and CD8+ T cells from the cord blood of healthy term controls (term) or preterm (PTL) infants were added to the cultures before releasing the stressed matrix. The gel size was measured at the indicated time points. (B) Representative graph of the percentage contraction of PHM1-41 cells embedded in collagen gel matrices treated with medium, TNF-α, or IFN-γ at 48 hours (n = 8). (C) Representative graph of the percentage contraction of PHM1-41 cells embedded in collagen gel matrices cocultured with fetal CD4+ or CD8+ T cells isolated from the cord blood of healthy term controls (term) or preterm (PTL) infants, either nontreated or treated with neutralizing antibodies for TNF-α, IFN-γ, or both, at 48 hours (n = 6). Small horizontal bars indicate the mean ± SEM. (D) Representative images of gels at 48 hours for different tested conditions. (E) IFN-γ (left) and TNF-α (right) secretion by CD4+ and CD8+ T cells from term or preterm infants either nontreated or treated with neutralizing antibodies for TNF-α or IFN-γ after 48 hours in coculture with PHM1-41 cells (n = 6). ***P < 0.001 by one-way analysis of variance (ANOVA) with Bonferroni correction.

  • Fig. 6 In utero injection of inflammatory cytokines or adoptive transfer of activated T cells leads to pregnancy loss.

    (A) Experimental design. Mouse fetuses at E14.5 were injected in utero with cytokines or T cells, and the survival rate was quantified at birth. (B) Representative graphs of the percentage of delivered pups after fetal injection with TNF-α or IFN-γ. (C) Representative graphs of the percentage of delivered pups after fetal adoptive transfer of T cells. CD4+ and CD8+ T cells from C57BL/6, IFN-γ−/− or TNF-α−/− mice were activated in vitro for 3 days with anti-CD3 and anti-CD28 antibodies before fetal injection. Nonactivated T cells from C57BL/6 mice were used as control (n ≥ 4 experiments, with n ≥ 3 dams per group). Small horizontal bars indicate the mean ± SEM. ***P < 0.001 by one-way ANOVA with Bonferroni correction.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/438/eaan2263/DC1

    Materials and Methods

    Fig. S1. Treg phenotype in patients with PTL.

    Fig. S2. Intact ability of maternal T cells to proliferate upon TCR stimulation.

    Fig. S3. Increased myometrial cell contractility in response to fetal T cells from patients with PTL.

    Fig. S4. Phenotypic characterization of activated and nonactivated mouse T cells.

    Fig. S5. Schematic comparison of the fetal immune system between term and PTL.

    Fig. S6. Equivalent expression of costimulatory molecules on maternal and fetal sBc.

    Table S1. Demographics of control (n = 89) and PTL (n = 70) patients.

    Table S2. Clinical characteristics of PTL patients (n = 70).

    Table S3. Fetal plasma cytokines (control, n = 28; PTL, n = 18).

    Table S4. Maternal plasma cytokines (control, n = 28; PTL, n = 15).

    Table S5. HLA mismatch of control (n = 13) and PTL (n = 16) patients.

    Table S6. Primary data (Excel file).

  • Supplementary Material for:

    Alloreactive fetal T cells promote uterine contractility in preterm labor via IFN-γ and TNF-α

    Michela Frascoli, Lacy Coniglio, Russell Witt, Cerine Jeanty, Shannon Fleck-Derderian, Dana E. Myers, Tzong-Hae Lee, Sheila Keating, Michael P. Busch, Philip J. Norris, Qizhi Tang, Giovanna Cruz, Lisa F. Barcellos, Nardhy Gomez-Lopez, Roberto Romero, Tippi C. MacKenzie*

    *Corresponding author. Email: tippi.mackenzie{at}ucsf.edu

    Published 25 April 2018, Sci. Transl. Med. 10, eaan2263 (2018)
    DOI: 10.1126/scitranslmed.aan2263

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Treg phenotype in patients with PTL.
    • Fig. S2. Intact ability of maternal T cells to proliferate upon TCR stimulation.
    • Fig. S3. Increased myometrial cell contractility in response to fetal T cells from patients with PTL.
    • Fig. S4. Phenotypic characterization of activated and nonactivated mouse T cells.
    • Fig. S5. Schematic comparison of the fetal immune system between term and PTL.
    • Fig. S6. Equivalent expression of costimulatory molecules on maternal and fetal sBc.
    • Table S1. Demographics of control (n = 89) and PTL (n = 70) patients.
    • Table S2. Clinical characteristics of PTL patients (n = 70).
    • Table S3. Fetal plasma cytokines (control, n = 28; PTL, n = 18).
    • Table S4. Maternal plasma cytokines (control, n = 28; PTL, n = 15).
    • Table S5. HLA mismatch of control (n = 13) and PTL (n = 16) patients.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S6 (Microsoft Excel format). Primary data.

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