Fetal T-cell activation in the amniotic cavity during preterm labor: A potential etiology for a subset of idiopathic preterm birth

Nardhy Gomez-Lopez; Roberto Romero; Yi Xu; Derek Miller; Marcia Arenas-Hernandez; Valeria Garcia-Flores; Bogdan Panaitescu; Jose Galaz; Chaur-Dong Hsu; Robert Para; Stanley M Berry

doi:10.4049/jimmunol.1900621

. Author manuscript; available in PMC: 2020 Oct 1.

Published in final edited form as: J Immunol. 2019 Sep 6;203(7):1793–1807. doi: 10.4049/jimmunol.1900621

Fetal T-cell activation in the amniotic cavity during preterm labor: A potential etiology for a subset of idiopathic preterm birth

Nardhy Gomez-Lopez ^†,^‡,^*,², Roberto Romero ^†,^¥,^#,^¶, Yi Xu ^†,^‡, Derek Miller ^†,^‡, Marcia Arenas-Hernandez ^†,^‡, Valeria Garcia-Flores ^†,^‡, Bogdan Panaitescu ^‡, Jose Galaz ^†,^‡, Chaur-Dong Hsu ^†,^‡,^£, Robert Para ^†,^‡, Stanley M Berry ^†,^‡

PMCID: PMC6799993 NIHMSID: NIHMS1536727 PMID: 31492740

Abstract

Prematurity is the leading cause of perinatal morbidity and mortality worldwide. In most cases, preterm birth is preceded by spontaneous preterm labor, a syndrome that is associated with intra-amniotic inflammation, the most studied etiology. However, the remaining etiologies of preterm labor are poorly understood and, therefore, most preterm births are categorized as idiopathic. Herein, we provide evidence showing that the fetal immune system undergoes premature activation in women with preterm labor without intra-amniotic inflammation, providing a potential new mechanism of disease for some cases of idiopathic preterm birth. First, we showed that fetal T cells are a predominant leukocyte population in amniotic fluid during preterm gestations. Interestingly, only fetal CD4+ T cells were increased in amniotic fluid of women who underwent idiopathic preterm labor and birth. This increase in fetal CD4+ T cells was accompanied by elevated amniotic fluid concentrations of T-cell cytokines such as IL-2, IL-4, and IL-13, which are produced by these cells upon in vitro stimulation, but was not associated with the prototypical cytokine profile observed in women with intra-amniotic inflammation. Also, we found that cord blood T cells, mainly CD4+ T cells, obtained from women with idiopathic preterm labor and birth displayed enhanced ex vivo activation, which is similar to that observed in women with intra-amniotic inflammation. Finally, we showed that the intra-amniotic administration of activated neonatal CD4+ T cells induces preterm birth in mice. Collectively, these findings provide evidence suggesting that fetal T-cell activation is implicated in the pathogenesis of idiopathic preterm labor and birth.

Keywords: fetus, placenta, amniotic fluid, funisitis, chorioamnionitis, inflammation

Visual Abstract

(A) Fetal T cells mediate a stereotypical inflammatory response in the amniotic cavity, partially mediated by IL-2, IL-4, and IL-13, which precedes preterm birth in a subset of women who underwent idiopathic preterm labor without intra-amniotic inflammation. This mechanism of disease is distinct from that observed in (B) women with preterm labor and intra-amniotic inflammation, in which maternal and fetal innate immune cells (neutrophils and macrophages) and T cells respond towards microbial products, involving the prototypical cytokines IL-6, IL-1β, and IL-8.

graphic file with name nihms-1536727-f0007.jpg

INTRODUCTION

Preterm birth remains one of the most common obstetrical syndromes today, and is a primary cause of perinatal morbidity and mortality around the world (1–4). Neonates born preterm have increased risk of both short- and long-term morbidities (5–8) that can persist throughout adulthood and seriously impact the quality of life. Two-thirds of all preterm births are preceded by spontaneous preterm labor (9), a syndrome of multiple pathological processes (10). Yet, among these etiologies, only intra-amniotic inflammation has been causally linked to preterm birth (11–20).

Intra-amniotic inflammation can result from microbial invasion of the amniotic cavity (i.e. intra-amniotic infection) (12, 13, 15, 21–32) or can be initiated by endogenous danger signals or alarmins found in amniotic fluid (33–37) (i.e. sterile intra-amniotic inflammation (38, 39)). The mechanisms implicated in intra-amniotic inflammation-associated preterm labor involve immune responses in the mother (40–42), the amniotic cavity (43–55), the fetus (53, 56–66), and at the maternal-fetal interface (67–79). Although intra-amniotic inflammation has been the main focus of study in the field of perinatal immunology, this etiology solely accounts for 37% of all preterm labor cases (38). The remaining etiologies of preterm labor (comprising approximately 60%) are poorly understood; therefore, most cases of preterm birth are categorized as idiopathic (9, 80).

Herein, we hypothesized that the fetus plays a central role in the mechanisms leading to idiopathic preterm labor and birth (i.e. preterm labor and birth in the absence of intra-amniotic inflammation). This hypothesis is based on a seminal study showing that fetal innate immune activation occurs in women with preterm labor in the presence and absence of infection in the amniotic cavity (56). Predictably, in the context of intra-amniotic infection-associated preterm labor, fetal immune activation has been associated with increased IL-6 and cortisol concentrations (57, 58). Furthermore, animal studies demonstrated that intra-amniotic inflammation induces activation and alteration in fetal T-cell populations prior to preterm birth (61, 63). More recently, it was reported that cord blood T cells from women with preterm labor are activated and respond towards maternal alloantigens, which suggested for the first time that the adaptive immune limb of the fetus can trigger preterm parturition and preterm birth (66). However, whether fetal T-cell activation occurs during idiopathic preterm labor is still unknown.

Given that fetal immune cells can be detected in the amniotic cavity even in the absence of inflammation (51, 81–84), we proposed the study of amniotic fluid immune cells as a window into the fetal immune system in utero. Using clinical and immunological tools, we aimed to investigate whether fetal T cells are implicated in the pathophysiology of idiopathic preterm labor which occurs in the absence of intra-amniotic inflammation.

MATERIALS AND METHODS

Study population and characteristics

This study included patients who underwent transabdominal amniocentesis due to clinical indications. The collection of samples was approved by the Institutional Review Boards of the Detroit Medical Center (Detroit, MI, USA), Wayne State University, and the Perinatology Research Branch, an intramural program of the Eunice Kennedy Shriver National Institutes of Health, U.S. Department of Health and Human Services (NICHD/NIH/DHHS). Maternal and cord blood samples from these women were also collected in some cases. All participating women provided written informed consent prior to the collection of samples. Three separate cohorts of women were used in this study.

The first cohort included 21 women in preterm gestation (15–30 weeks of gestation) whose amniotic fluid was collected from November 2013 to January 2015 due to clinical indications and used for studies of the exploratory immunophenotyping, origin, and characterization of T cells, which was compared to that of cord blood and maternal blood samples. The demographic and clinical characteristics of these women are shown in Table I.

Table I.

Clinical and demographic characteristics of women whose amniotic fluid was used for exploratory experiments.

Clinical characteristics	Values
Maternal age (years; median [IQR])	27 (23–35)
Body mass index (kg/m²; median [IQR])	25.9 (22.6–34.2)
Primiparity	19.1% (4/21)
Race
African-American	85.7% (18/21)
Caucasian	4.8% (1/21)
Asian	4.8% (1/21)
Other	4.8% (1/21)
Gestational age at amniocentesis (weeks; median [IQR])	21.6 (19.5–26.5)
IL-6 (ng/mL; median [IQR])	0.46 (0.15–0.91)
White blood cell count, cells/mm³	1 (0–4)
Amniotic fluid glucose, mg/dL	28 (24.5–38)

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Available data are given as median (interquartile range, IQR) and percentage (n/N).

The second cohort included amniotic fluid samples collected from November 2013 to November 2016 of 43 women in preterm labor. These samples were used for confirmatory immunophenotyping and the determination of cytokine/chemokine concentrations in amniotic fluid. This cohort included the following groups: i) women with spontaneous preterm labor who delivered at term without intra-amniotic inflammation (PTL-TB, negative control); ii) women with spontaneous preterm labor who delivered preterm without intra-amniotic inflammation (PTL-PTB, study group); and iii) women with spontaneous preterm labor and birth with intra-amniotic inflammation (PTL-PTB-IAI, positive control). The demographic and clinical characteristics of these women are shown in Table II.

Table II.

Clinical and demographic characteristics of women whose amniotic fluid was used for descriptive studies.

Clinical characteristics	PTL TB (n=9)	PTL PTB (n=8)	PTL PTB IAI (n=26)	p-value
Maternal age (years; median [IQR])^a	25 (20–26)	23.5 (23–24.8)	26 (22–31.3)	0.5
Body mass index (kg/m²; median [IQR])^a	28.4 (21.8–30)^c	22.5 (20.5–25.2)^c	27.4 (24.8–32.7)^d	0.1
Primiparity^b	22.2% (2/9)	12.5% (1/8)	7.7% (2/26)	0.5
Race^b				0.3
African-American	88.9% (8/9)	75% (6/8)	80.8% (21/26)
Caucasian	0% (0/9)	12.5% (1/8)	15.4% (4/26)
Asian	0% (0/9)	12.5% (1/8)	0% (0/26)
Other	11.1% (1/9)	0% (0/8)	3.8% (1/26)
Gestational age at Amniocentesis (weeks; median [IQR])^a	32.6 (31–33.7)	30.6 (26.9–33.6)	26.4 (22.8–30.7)	0.009
IL-6 (ng/mL; median [IQR])^a	0.3 (0.2–0.4)	0.5 (0.3–0.7)	65.3 (23.6–129.1)	<0.001
White blood cell count, cells/mm^3,a	2 (0–4)	2 (0–8)^c	10.5 (1.3–323.5)	0.058
Amniotic fluid glucose, mg/dL^a	37 (23–38)	22.5 (16–39.5)	10.5 (1.5–17.5)	0.001
Gestational age at delivery (weeks; median [IQR])^a	39 (38.7–39.6)	30.8 (27.1–33.8)	26.9 (23.3–30.9)	<0.001
Cesarean section^b	0% (0/9)	25% (2/8)	19.2% (5/26)	0.3
Birthweight (grams)^a	2990 (2720–3525)	1760 (970–2065)	1030 (607.5–1641.3)	<0.001
Maternal inflammatory response (moderate/severe acute chorioamnionitis)^b	37.5% (3/8)^c	12.5% (1/8)	71.4% (15/21)^e	0.01
Fetal inflammatory response (moderate/severe acute funisitis)^b	0% (0/8)^c	0% (0/8)	14.3% (3/21)^e	0.3

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Data are given as median (interquartile range, IQR) and percentage (n/N). PTL TB = preterm labor who delivered at term birth; PTL PTB = preterm labor and birth; PTL PTB IAI = preterm labor and birth with intra-amniotic inflammation.

Kruskal-Wallis test.

Fisher’s exact test.

One missing data.

Three missing data.

Five missing data

The third cohort included 20 women from whose neonates’ cord blood was obtained and biobanked from November 2015 to November 2016, and the inflammatory status (amniotic fluid white blood cell count and IL-6 concentrations) and microbiology of amniotic fluid was performed. These samples were used to assess the functional status of T cells. This cohort included the following groups: i) women who delivered at term with labor but without intra-amniotic inflammation (TB, negative control), ii) women with spontaneous preterm labor and birth without intra-amniotic inflammation (PTL-PTB, study group), and iii) women with spontaneous preterm labor and birth with intra-amniotic inflammation (PTL-PTB-IAI, positive control). The demographic and clinical characteristics of these women are shown in Table III.

Table III.

Clinical and demographic characteristics of women from whose neonates umbilical cord blood was obtained for in vitro studies.

	TB (n=6)	PTL PTB (n=9)	PTL PTB IAI (n=5)	p-value
Maternal age (years; median [IQR])^a	23.5 (22.3–24.8)	26 (23–27)	24 (21–25)	0.3
Body mass index (kg/m²; median [IQR])^a	29.8 (23.3–34.9)	21.7 (20–24.2)	28.6 (23.3–32.5)^c	0.08
Primiparity^b	50% (3/6)	22.2% (2/9)	40% (2/5)	0.5
Race^b				0.7
African-American	83.3% (5/6)	88.9% (8/9)	80% (4/5)
Caucasian	16.7% (1/6)	0% (0/9)	20% (1/5)
Other	0% (0/6)	11.1% (1/9)	0% (0/5)
Gestational age at Amniocentesis (weeks; median [IQR])^a	40.5 (40.3–40.6)	33.3 (31.1–33.6)	30.3 (26–32.6)	0.05
IL-6 (ng/mL; median [IQR])^a	2.1 (1.9–30.5)	0.3 (0.2–0.5)^c	17.7 (16.8–67.1)	0.002
Gestational age at delivery (weeks; median [IQR])^a	40.5 (40.3–40.6)	35.4 (33.7–36.3)	31.9 (26.1–32.6)	0.001
Cesarean section^b	83.3% (5/6)	44.4% (4/9)	20% (1/5)	0.1
Birthweight (grams)^a	3342.5 (3070–3465)	2585 (1860–2640)	1955 (1040–2120)	0.003
Maternal inflammatory response (moderate/severe acute chorioamnionitis)^b	33.3% (2/6)	33.3% (3/9)	25% (1/4)^c	1
Fetal inflammatory response (moderate/severe acute funisitis)^b	16.7% (1/6)	0% (0/9)	0% (0/4)^c	0.5

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Data are given as median (interquartile range, IQR) and percentage (n/N). TB = term labor and birth; PTL PTB = preterm labor and birth; PTL PTB IAI = preterm labor and birth with intra-amniotic inflammation.

Kruskal-Wallis test.

Fisher’s exact test.

One missing data.

Labor was defined by the presence of regular uterine contractions at a frequency of at least two contractions every 10 min with cervical changes resulting in delivery. Preterm delivery was defined as delivery <37 wk of gestation. Our study groups included women who underwent preterm labor with intact membranes; thus, intra-amniotic inflammation was defined as an amniotic fluid IL-6 concentration ≥ 2.6 ng/mL (30).

Clinical determinations in amniotic fluid and placentas

Immediately after amniocentesis, samples of amniotic fluid were transported to the clinical laboratory in a capped sterile syringe, and the rest of the sample was used for this study. The clinical tests included the determination of amniotic fluid white blood cell count (43), Gram stain (85), and glucose concentration (86). IL-6 concentrations were determined as previously described (30). Acute inflammatory lesions of the placenta (maternal inflammatory response and fetal inflammatory response) were diagnosed according to established criteria, including staging and grading (87, 88).

Immunophenotyping by flow cytometry

Amniotic fluid samples (0.5–1 mL) were centrifuged at 300 x g for 5 min at room temperature. The resulting amniotic fluid cell pellet was re-suspended in 1 mL of 1X phosphate-buffered saline (PBS) (Life Technologies, Grand Island, NY, USA) and stained with the BD Horizon Fixable Viability Stain 510 dye (BD Biosciences, San Jose, CA, USA). Amniotic fluid cells were washed in 1X PBS and incubated with 20 μL of human FcR blocking reagent (Miltenyi Biotec, San Diego, CA, USA) in 80 μL of stain buffer (BD Biosciences) for 10 min at 4°C prior to incubation with extracellular antibodies.

Maternal peripheral blood samples were collected from healthy individuals by venipuncture into ethylene diamine tetra-acetic acid (EDTA)-containing tubes. Umbilical cord blood was collected at birth in EDTA-containing tubes by venipuncture of the umbilical vein. Mononuclear cells were isolated using the density gradient reagent Ficoll-Paque Plus (GE Healthcare Life Sciences, Piscataway, NJ, USA), according to the manufacturer’s instructions. For the samples with only extracellular staining, 50 μL of maternal or cord blood were used.

For all samples, cells were then incubated with extracellular fluorochrome-conjugated anti-human monoclonal antibodies for 30 min at 4°C in the dark (Supplemental Table I). After extracellular staining, the cells were fixed. For intracellular staining, cells were fixed and permeabilized using the Foxp3 transcription factor fixation/permeabilization solution (Cat#00–5523-00; eBioscience by Thermo Fisher Scientific, Wilmington, DE, USA) prior to incubation with intracellular antibodies (Supplemental Table I). Stained cells were then washed and re-suspended in 0.5 mL of stain buffer and acquired using the BD LSR Fortessa flow cytometer and BD FACSDiva 6.0 software. The analysis was performed and the figures were generated using the FlowJo version 10 software (FlowJo, Ashland, OR, USA). The absolute number of cells was determined using CountBright absolute counting beads (Molecular Probes, Eugene, OR, USA).

DNA fingerprinting

Genomic DNA was isolated from amniotic fluid cell pellets using the QIAamp UCP DNA Micro kit (Cat#56204, Qiagen, Hilden, Germany), following the manufacturer’s instructions. Fetal or maternal genomic DNA from matching cases was isolated from frozen buffy coat samples of either umbilical cord or maternal blood using the DNeasy Blood and Tissue Kit (Qiagen), according to the manufacturer’s instructions. DNA concentrations and purity were assessed using the NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). DNA samples were submitted for DNA fingerprinting to Genetica DNA Laboratories, a LabCorp brand (Burlington, NC, USA). Briefly, analytical procedures for polymerase chain reaction and capillary electrophoresis were performed on a 3130xl genetic analyzer (Applied Biosystems, Foster City, CA, USA). The 13-core CODIS STR loci plus PENTA E and PENTA D, and the gender-determining locus, amelogenin, were analyzed using the commercially available PowerPlex 16HS amplification kit (Promega Corp, Madison, WI, USA) and GeneMapper ID v3.2.1 software (Applied Biosystems). Appropriate positive and negative controls were concurrently used throughout the analysis. The interpretation of the DNA profile of each sample was performed by geneticists from LabCorp and returned as a written report. The reported sensitivity for this method was between 2–5%, indicating that this technology is capable of detecting as low as 2–5% of the DNA in question among the total DNA mixture.

Amniotic fluid cytokine/chemokine concentrations

Amniotic fluid samples were assessed using the V-PLEX Pro-inflammatory Panel 1 kit (Meso Scale Discovery, Rockville, MD, USA) to measure amniotic fluid concentrations of IFNγ, TNFα, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, and IL-13, according to the manufacturer’s instructions. Plates were read using the SECTOR Imager 2400 (Meso Scale Discovery). Standard curves were generated and the assay values of the samples were interpolated from the curves. The detection limits of the assays were 0.37 pg/mL (IFNγ), 0.04 pg/mL (TNFα), 0.05 pg/mL (IL-1β), 0.09 pg/mL (IL-2), 0.02 pg/mL (IL-4), 0.06 pg/mL (IL-6), 0.07 pg/mL (IL-8), 0.04 pg/mL (IL-10), 0.11 pg/mL (IL-12p70), and 0.24 pg/mL (IL-13). Inter-assay and intra-assay coefficients of variation were less than 10.5%.

Amniotic fluid concentrations of CXCL10 (Cat#DIP100, R&D Systems, Minneapolis, MN, USA) and CXCL11 (Cat#K151UWK-1, Meso Scale Discovery) were determined using individual sensitive and specific immunoassays, according to the manufacturer’s instructions. Assays were read using the SpectraMax M5 (CXCL10; Molecular Devices, San Jose, CA, USA) or the SECTOR Imager 2400 (CXCL11). The concentrations of CXCL10 and CXCL11 were determined by interpolation from the standard curve. The detection limits of the assays were 1.7 pg/mL (CXCL10) and 1.5 pg/mL (CXCL11). The inter-assay and intra-assay coefficients of variation were less than 9.8% for CXCL10 and less than 16.8% for CXCL11.

Amniotic fluid T-cell cytokine expression upon stimulation

Amniotic fluid samples (5–10mL) from women in preterm gestations (n = 3) were centrifuged at 300 x g for 10 min at room temperature. The resulting amniotic fluid cell pellet was washed with 1mL of 1X PBS and re-suspended in supplemented RPMI [10% FBS and 1% penicillin/streptomycin antibiotic (Thermo Fisher Scientific)]. To evaluate IL-4 and IL-13 production, amniotic fluid cells were stimulated with immobilized anti-human CD3 antibody (clone OKT3, 10μg/mL, Cat#16–0037-85, eBioscience), soluble anti-human CD28 antibody (clone CD28.2, 2μg/mL, Cat#16–0289-85, eBioscience), recombinant human IL-2 (10ng/mL, Cat#554603, BD Biosciences) and recombinant human IL-4 (20ng/mL, Cat#554605, BD Biosciences) for 2 d. The cells were washed and subsequently cultured in supplemented RPMI containing IL-2 and IL-4 for another 2 d. Finally, the cells were collected and stimulated with Cell Stimulation Cocktail [containing phorbol 12-myristate 13-acetate (PMA), ionomycin, brefeldin A and monensin)] (Cat#00–4975-93, eBioscience) for 4 h. To evaluate IL-2 production, amniotic fluid cells were washed and treated with Cell Stimulation Cocktail for 4 h. Untreated cells were included as controls for each experiment.

After treatment, amniotic fluid cells were washed with 1X PBS and incubated with extracellular fluorochrome-conjugated anti-human monoclonal antibodies for 30 min at 4°C in the dark (Supplemental Table I). After extracellular staining, the cells were fixed, permeabilized, and incubated with specific fluorochrome-conjugated anti-human monoclonal antibodies against IL-2, IL-4 and IL-13 (Supplemental Table I). Stained cells were then washed and re-suspended in 0.5mL of stain buffer and acquired using the BD LSR Fortessa flow cytometer and BD FACSDiva 6.0 software. The analysis was performed and the figures were generated using FlowJo version 10 software.

Determination of cord blood T-cell functionality

Cord blood samples were obtained from neonates born to women who underwent spontaneous labor at term, spontaneous preterm labor, or spontaneous preterm labor with intra-amniotic inflammation (IL-6 >2.6 ng/mL) (Table III) and preserved in freezing media (90% fetal bovine serum and 10% DMSO) until use. Non-viable cells were depleted using the Dead Cell Removal kit (Cat#130–090-101, Miltenyi Biotec) in the cord blood samples, following the manufacturer’s instructions. The cells were washed, re-suspended in supplemented RPMI at 1×10⁶ cells/mL, and then either treated with Cell Stimulation Cocktail or left untreated as a control. Cells were then incubated at 37°C and 5% CO₂ for 5 hours. After incubation, cells were collected, washed in 1X PBS, and re-suspended in 10 μL of human FcR blocking reagent and 40 μL of stain buffer for 10 min at 4°C. The cells were then incubated with extracellular fluorochrome-conjugated anti-human monoclonal antibodies for 30 min at 4°C in the dark (Supplemental Table I). After washing with stain buffer, the cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set prior to incubation with intracellular antibodies for 30 min at 4°C in the dark (Supplemental Table I). A portion of the cells were stained with fluorochrome-conjugated isotypes as a control. Stained cells were then washed, re-suspended in 0.5 mL of stain buffer, and acquired using the BD LSRFortessa Flow Cytometer and BD FACSDiva 6.0 software. The analysis was performed and the figures were generated using the FlowJo version 10 software, and the absolute number of cells was determined using CountBright absolute counting beads.

Mice

C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and bred in the animal care facility at the C.S. Mott Center for Human Growth and Development, Wayne State University, Detroit, MI, USA and housed under a circadian cycle (light:dark = 12:12 h). Eight- to twelve-week-old females were mated with males of proven fertility. Female mice were examined daily between 8:00 and 9:00 AM for the presence of a vaginal plug, which indicated 0.5 d post coitum (dpc). Upon observation of vaginal plugs, female mice were removed from the mating cages and housed separately. A weight gain of ≥2g confirmed pregnancy at 12.5 dpc. All animal experiments were approved by the Institutional Animal Care and Use Committee at Wayne State University (Protocol No. A-18–03-0584). The authors adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Isolation and activation of neonatal T cells

Murine neonates of one week of age were sacrificed and the thymi were collected into sterile 1X PBS and thymocytes were isolated by gently dissociating, as previously reported (89). Thymocytes were then passed through a 30μm filter (Miltenyi Biotec), washed with sterile 1X PBS, and centrifuged at 500 x g for 5 min at 4°C. The cell pellet was resuspended in 2mL of sterile ACK lysis buffer (Cat#A10492; Thermo Fisher Scientific) followed by a 5 min incubation at 37°C and washed with sterile 1X PBS. Finally, the thymocytes were resuspended in 1mL of RPMI media (supplemented with 10% FBS and 1% penicillin/streptomycin antibiotic), and cells were counted using the Cellometer Auto 2000. Thymocytes were immediately stained for the cell sorting of non-activated CD4+ T cells (Day 0; see below) or placed at 2 × 10⁶ cells/well in a six-well culture plate previously coated with anti-mouse CD3ε (clone 145–2C11, Cat#553058; BD Biosciences). After plating, the following stimulators were added to each well: anti-mouse CD28 (clone 37.51; Cat#553295, BD Biosciences) (1μg/mL), anti-mouse IL-4 (clone 11B11, Cat#16–7041-85; eBioscience) (10μg/mL), recombinant mouse IL-2 (Cat#575402; Biolegend) (10ng/mL), and recombinant mouse IL-12 (Cat#577002; Biolegend) (10ng/mL). Lastly, mercaptoethanol (Cat#21985023; Thermo Fisher Scientific) (2μL) was added to each well and thymocytes were cultured at 37°C and 5% CO₂ for 4 d.

Determination of neonatal T-cell activation by flow cytometry

To confirm neonatal T-cell activation on Day 4 of culture, a portion of the neonatal thymocytes were collected and stimulated for 4 hours with 2μL/mL of Cell Stimulation Cocktail. Stimulated cells were then washed with 1X PBS and incubated with fluorochrome-conjugated anti-mouse monoclonal antibodies (Supplemental Table 1) for 30 min at 4°C in the dark. Following extracellular staining, cells were fixed and permeabilized using the Foxp3 Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) and incubated with specific fluorochrome-conjugated anti-mouse monoclonal antibodies against IFNγ and TNFα (Supplemental Table 1) for 30 min at 4°C in the dark. Non-stimulated cells were stained as controls. The cells were acquired using the BD LSRFortessa flow cytometry and BD FACSDiva v6.0 software. The analysis and figures were performed using FlowJo v10 software.

Fluorescence-activated cell sorting of viable activated or non-activated neonatal T cells

After confirming neonatal T-cell activation (Day 4), thymocytes were collected from the culture plate and centrifuged at 500 x g for 5 min at room temperature. The cells were then resuspended in 100μL of sterile stain buffer and incubated with fluorochrome-conjugated anti-mouse monoclonal antibodies (Supplemental Table 1) for 30 min at 4°C in the dark. Non-activated thymocytes (Day 0) were also stained for cell sorting. The cells were then washed with supplemented RPMI media to remove excess antibody and resuspended in 4mL of RPMI. Viable CD3+CD8-CD4+ T cells were sorted using the BD FACSMelody cell sorter (BD Biosciences) and BD FACSChorus v1.3 software (BD Biosciences) under sterile conditions. Sorted cells were counted, centrifuged at 300 x g for 5 min, and resuspended at 1 × 10⁴ cells/25μL in sterile 1X PBS for ultrasound-guided intra-amniotic injection.

Ultrasound-guided intra-amniotic injection of activated or non-activated neonatal CD4+ T cells

Pregnant C57BL/6 dams were anesthetized on 16.5 dpc by inhalation of 2–3% isoflurane (Aerrane, Baxter Healthcare Corporation, Deerfield, IL, USA) and 1–2 L/min of oxygen in an induction chamber. Anesthesia was maintained with a mixture of 1.5–2% isoflurane and 1.5–2 L/min of oxygen. Dams were positioned on a heating pad and stabilized with adhesive tape. Fur removal from the abdomen was achieved by applying Nair cream (Church & Dwight Co., Inc., Ewing, NJ, USA) to this area. Body temperature was maintained in the range of 37 ± 1°C and detected with a rectal probe (VisualSonics, Inc., Toronto, ON, Canada), and respiratory and heart rates were monitored by electrodes embedded in the heating pad. An ultrasound probe from the Vevo 2100 Imaging System (VisualSonics, Inc.) was fixed and mobilized with a mechanical holder, and the transducer was slowly moved toward the abdomen. Ultrasound-guided intra-amniotic injection of 1 × 10⁴ non-activated (Day 0; n = 3) or activated (Day 4; n = 8) neonatal T cells in 25μL of sterile 1X PBS was performed in each amniotic sac using a 30G needle (BD PrecisionGlide Needle, Becton Dickinson, Franklin Lakes, NJ, USA). The amount of neonatal T cells injected in each amniotic sac was based on preliminary experiments showing that pregnant dams require higher doses of inflammatory stimuli to deliver preterm. Vehicle controls were injected with 25μL of sterile 1X PBS in each amniotic sac (n = 9). The syringe was stabilized by a mechanical holder (VisualSonics, Inc.). Following the ultrasound, mice were placed under a heat lamp for recovery (defined as when the mouse resumes normal activity, such as walking and responding), which typically occurred 10–20 min after removal from anesthesia. Dams were monitored via video camera (Sony Corporation, Tokyo, Japan) until delivery to obtain the gestational age and rate of preterm birth, which was defined as delivery occurring before 18.5 dpc, and its rate was represented by the percentage of dams delivering preterm among the total number of mice injected.

Statistical analysis

Statistical analyses were conducted using SPSS software version 19.0 (IBM Corporation, Armonk, NY, USA). For patient demographics, the Kruskall-Wallis test was performed for continuous variables and the Fisher’s exact test for nominal variables. t-SNE plots were generated using the FlowJo v10 software. The Mann-Whitney U-test was performed when comparing non-normally distributed data between study groups. The log2 fold changes in cytokine concentrations were also calculated after data transformation. The Wilcoxon signed rank paired test was used when comparing the same sample with and without stimulation. Kaplan-Meier survival curves were used to plot and compare the murine gestational age data (Mantel–Cox test). A p-value ≤ 0.05 was considered statistically significant.

RESULTS

Characterization and origin of amniotic fluid immune cells

Amniotic fluid contains both innate (83, 84, 90–93) and adaptive (84) immune cells throughout pregnancy. First, we showed that in preterm gestations the amniotic fluid immune cell profile is distinct from that seen in the fetus (umbilical cord blood) or mother (peripheral blood), and that T cells are the dominant physiological population in this compartment (Fig. 1A). However, amniotic fluid T cells have been poorly investigated. Therefore, we collected amniotic fluid from women in preterm gestations and performed DNA fingerprinting to determine the origin of these T cells (Fig. 1B and Fig. S1). In each case, DNA fingerprinting revealed that amniotic fluid T cells were of fetal origin (Fig. 1B&C). Amniotic fluid T cells and innate lymphoid cells, another immune cell population present in preterm gestation, have been proposed to originate from the fetal intestine, based on their expression of transcription factors such as RAR-related orphan receptor-γt (RORγt) (83, 84, 94) and GATA-binding protein-3 (GATA3) (94) as well as the surface markers CD161 and CD103 (83, 84). Our exploratory immunophenotyping revealed that fetal T cells in amniotic fluid highly expressed RORγt and GATA3, as well as moderate levels of CD161, CD103, and the activation marker CD69 (Fig. 1D). The expression of these markers was low on T cells from the fetal or maternal circulations, further indicating that fetal T cells in amniotic fluid are a unique subset (Fig. 1D). We also determined the expression of the mucosal effector molecules IL-17 and IL-22 by amniotic fluid T cells, which were low and no differences were found when compared to cord blood or maternal blood (data not shown).

Figure 1. — **(A) (Left)** Amniotic fluid samples were obtained from women between 15–30 weeks of gestation for exploratory studies. Maternal peripheral blood and umbilical cord blood samples were collected from each case for comparison. **(Right)** Representative t-SNE plots showing the relative distribution of immune cell populations in amniotic fluid, umbilical cord blood, and maternal peripheral blood (n = 3 – 14 each). **(B)** DNA fingerprinting of amniotic fluid cells, the fetus (umbilical cord blood), and the mother (peripheral blood) is shown in electropherograms. Each electropherogram contains 6 genetic sites: D5S818, D13S317, D7S820, D16S539, CSF1PO, and Penta_D. Each genetic site has STR alleles, which are represented by peaks. The numbers below each STR allele (peak) are the number of repeats and the signal strength for each STR allele. The DNA fingerprinting of the amniotic fluid cells is identical to the DNA fingerprinting of the fetus. **(C)** A representative comparison between the D13S317 STR alleles expressed by amniotic fluid cells, the fetus, and the mother is shown. **(D)** Representative t-SNE plots showing the expression patterns of RORγt, GATA3, CD161, CD103, and CD69 on T cells (CD45+CD14-CD15-CD3+ cells) from amniotic fluid, cord blood, or maternal peripheral blood. Red represents the expression of the indicated marker, and grey indicates no expression. Demographic and clinical characteristics of the study population are shown in Table I.

Together, these exploratory studies indicate that fetal T cells are the predominant immune cell population in preterm gestations and display a phenotype that is distinct from that observed in the fetal and maternal systemic circulations.

Are fetal T cells in amniotic fluid associated with preterm labor and birth?

Given that amniotic fluid contains a large population of fetal T cells during preterm gestations, we next investigated whether this subset is associated with idiopathic preterm labor and birth. To determine this, we undertook a comprehensive three-year study in which amniotic fluid was obtained from women with preterm labor who delivered preterm without intra-amniotic inflammation (study group), and performed immunophenotyping of amniotic fluid immune cells to determine the absolute numbers of each subset compared to women with preterm labor who delivered at term (negative control) and those with preterm labor who delivered preterm with intra-amniotic inflammation (positive control) (Fig. 2A&B). Interestingly, we found that the number of CD4+ T cells in amniotic fluid was increased in our study group compared to negative controls (Fig. 2D). As previously reported (66, 84), the numbers of amniotic fluid neutrophils, monocytes/macrophages, and T cells were highest in positive controls (Fig. 2C–G). The number of B cells in amniotic fluid was unaltered when compared among the study group and controls (Fig. 2H). Since fetal CD4+ T cells were increased in our study group, we next investigated whether such an increase was associated with T-cell cytokines in amniotic fluid. The increased number of fetal CD4+ T cells was accompanied by a modest elevation in amniotic fluid concentrations of IL-2 (1.85 log2 fold change), IL-4 (1.59 log2 fold change), and IL-13 (2.18 log2 fold change) in our study group compared to negative controls (Fig. 3A&B). To confirm that amniotic fluid fetal T cells were capable of releasing these cytokines in vivo (i.e. preterm labor), we stimulated preterm gestation-derived amniotic fluid T cells and observed that these cells can express IL-2, IL-4, and IL-13 ex vivo (Fig. 3C). These data suggest that fetal T cells undergo premature activation prior to idiopathic preterm birth.

Figure 2. — **(A)** Amniotic fluid samples were obtained from women with preterm labor who delivered at term (PTL-TB) (negative control), women with preterm labor who delivered preterm without intra-amniotic inflammation (PTL-PTB) (study group), and women with preterm labor who delivered preterm with intra-amniotic inflammation (PTL-PTB-IAI) (positive control). **(B)** Flow cytometry gating strategy for immunophenotyping of immune cells. Immune cells were initially gated within the viability gate and CD45+ gate followed by lineage gating for neutrophils (CD45+CD15+ cells), monocytes/macrophages (CD45+CD14+ cells), T cells (CD45+CD3+ cells) that were subsequently gated for CD4+ T cells (CD45+CD3+CD4+ cells) and CD8+ T cells (CD45+CD3+CD8+ cells), and B cells (CD45+CD19+ cells). The numbers of **(C)** total T cells, **(D)** CD4+ T cells, **(E)** CD8+ T cells, **(F)** neutrophils, **(G)** monocytes/macrophages, and **(H)** B cells in amniotic fluid. Data are shown as box-and-whisker plots where midlines indicate medians, boxes indicate interquartile ranges, and whiskers indicate minimum and maximum ranges. Demographic and clinical characteristics of the study population are shown in Table II.

Figure 3. — Concentrations of **(A)** T helper 1 cytokines (IL-2, IFNγ, TNFα) and **(B)** T helper 2 cytokines (IL-4, IL-13, IL-10) in amniotic fluid from women who underwent spontaneous preterm labor and delivered at term (PTL-TB) (negative control), those who delivered preterm without intra-amniotic inflammation (PTL-PTB) (study group), and those who delivered preterm with intra-amniotic inflammation (PTL-PTB-IAI) (positive control). **(C)** Representative flow cytometry gating strategy showing the expression of IL-2, IL-4 and IL-13 by CD3+CD4+ preterm gestation-derived amniotic fluid T cells. Concentrations of **(D)** pro-inflammatory cytokines (IL-8, IL-1β, IL-12p70) in amniotic fluid. Data are shown as box-and-whisker plots where midlines indicate medians, boxes indicate interquartile ranges, and whiskers indicate minimum and maximum ranges. NS = no significance. Demographic and clinical characteristics of the study population are shown in Table II.

Next, we investigated whether the increase in fetal CD4+ T cells was associated with a rise in amniotic fluid concentrations of the T cell-specific chemokines CXCL10 (95) and CXCL11 (96). No differences were observed between the study group and the negative controls (Fig. S2), suggesting that CXCL10 and CXCL11 are not actively chemoattracting fetal T cells in the amniotic cavity during the process of idiopathic preterm labor that leads to premature birth. As previously reported, the amniotic fluid concentration of CXCL10 was increased in cases with intra-amniotic inflammation (97, 98). Of interest, the pro-inflammatory cytokines that are typically implicated in inflammation-associated preterm labor (e.g. IL-8 (99), IL-1β (33, 100), and IL-12p70 (101)) were increased in our positive controls, but not in our study group (Fig. 3D), indicating that the process of idiopathic preterm labor associated with fetal T-cell activation involves a distinct inflammatory milieu from that induced by intra-amniotic inflammation.

Fetal T-cell activation in idiopathic preterm labor and birth

Amniotic fluid contains low numbers of T cells in physiological conditions (Fig. 1A&2C). Moreover, the amount of amniotic fluid that can be obtained for research purposes is limited. Therefore, as a surrogate for fetal T cells obtained from amniotic fluid, we utilized cord blood T cells from women with available data for the inflammatory status and microbiology of amniotic fluid. Hence, we tested the functionality of fetal T cells from neonates born to women with idiopathic preterm labor who delivered preterm without intra-amniotic inflammation (study group), those born to women with term labor and birth (negative control), and those born to women with preterm labor who delivered preterm with intra-amniotic inflammation (positive control) (Fig. 4A&5A). Immunophenotyping was used to determine the expression of IL-2, IFNγ, TNFα, Tbet, IL-4, IL-5, IL-10, IL-9, IL-17A, granzyme B, and perforin by CD4+ or CD8+ T cells (Fig. 4A&5A). Significant differences among PMA-stimulated groups are reported, since we sought to study fetal T-cell function under inflammatory conditions. Overall, fetal CD4+ T cells from our study group and from negative controls responded to stimulation, which did not occur in CD4+ T cells from positive controls (Fig. 4B–E). Specifically, fetal CD4+ T cells from women with preterm labor and birth displayed higher expression of Th1 (IFNγ, TNFα, and Tbet), Th2 (IL-4 and IL-10), Th9 (IL-9), and effector (granzyme B) mediators compared to those from term controls (Fig. 4B–E). Similarly, fetal CD8+ T cells from the study and negative control groups were responsive, although this was milder than that of CD4+ T cells (Fig. 5B–E vs. Fig4B–E). Fetal CD8+ T cells from women with preterm labor and birth displayed higher expression of Tc1 (IFNγ, TNFα, and Tbet), Tc2 (IL-4 and IL-10), Tc9 (IL-9), and effector (perforin) mediators compared to those from term controls (Fig. 5B–E). Alike fetal CD4+ T cells, fetal CD8+ T cells from positive controls did not display significantly higher expression of cytokines/effector mediators after stimulation (Fig. 5B–E). Importantly, fetal T cells from our study group displayed significantly greater activation (based on the increased expression of cytokines and effector mediators) than those from negative controls, even reaching similar levels to fetal T cells that had been activated by microorganisms invading the amniotic cavity (positive controls) (Fig. 4B–E&5B–E). The expression of IL-2, IL-5, and IL-17A by fetal CD4+ or CD8+ T cells did not vary among the study groups (Fig. S3A–F). Together, these findings show that idiopathic preterm labor that leads to premature birth is accompanied by fetal T-cell activation, which upon stimulation can be similar to that observed in cases with intra-amniotic infection.

Figure 4. — **(A)** Experimental design and flow cytometry gating strategy used to determine the expression of cytokines and effector molecules on CD4+ T cells from neonates born to mothers who delivered with spontaneous labor at term (TB) (negative control), those who underwent spontaneous preterm labor and birth without intra-amniotic inflammation (PTL-PTB) (study group), or those who underwent spontaneous preterm labor and birth with intra-amniotic inflammation (PTL-PTB-IAI) (positive control). Isolated T cells were either treated with PMA/ionomycin or left untreated. Red histograms indicate the expression of each T-cell marker, and grey histograms indicate isotype controls. **(B)** Proportions of CD4+ T cells expressing the type 1 cytokines IFNγ and TNFα and the transcription factor Tbet. **(C)** Proportions of CD4+ T cells expressing the type 2 cytokines IL-4 and IL-10. **(D)** Proportion of CD4+ T cells expressing the cytokine IL-9. **(E)** Proportion of CD4+ T cells expressing the effector molecules granzyme B and perforin. Data are shown as box-and-whisker plots where midlines indicate medians, boxes indicate interquartile ranges, and whiskers indicate minimum and maximum ranges. Asterisks indicate p-values ≤0.05 for paired comparisons between the same samples with and without stimulation. P-values are shown for comparisons between stimulated fetal T cells from each group. NS = no significance. Demographic and clinical characteristics of the study population are shown in Table III.

Figure 5. — **(A)** Experimental design and flow cytometry gating strategy used to determine the expression of cytokines and effector molecules on CD8+ T cells from neonates born to mothers who delivered with spontaneous labor at term (TB) (negative control), those who underwent spontaneous preterm labor and birth without intra-amniotic inflammation (PTL-PTB) (study group), or those who underwent spontaneous preterm labor and birth with intra-amniotic inflammation (PTL-PTB-IAI) (positive control). Isolated T cells were either treated with PMA/ionomycin or left untreated. Blue histograms indicate the expression of each T-cell marker, and grey histograms indicate isotype controls. **(B)** Proportions of CD8+ T cells expressing the type 1 cytokines IFNγ and TNFα, and the transcription factor Tbet. **(C)** Proportions of CD8+ T cells expressing the type 2 cytokines IL-4 and IL-10. **(D)** Proportion of CD8+ T cells expressing the cytokine IL-9. **(E)** Proportion of CD8+ T cells expressing the effector molecules granzyme B and perforin. Data are shown as box-and-whisker plots where midlines indicate medians, boxes indicate interquartile ranges, and whiskers indicate minimum and maximum ranges. Asterisks indicate p-values ≤0.05 for paired comparisons between the same samples with and without stimulation. P-values are shown for comparisons between stimulated fetal T cells from each group. NS = no significance. Demographic and clinical characteristics of the study population are shown in Table III.

Activated neonatal T cells induce preterm birth in mice

Our above findings suggested that idiopathic preterm labor and birth is associated with fetal T-cell activation. Therefore, we lastly investigated whether the intra-amniotic injection of activated neonatal CD4+ T cells could induce preterm birth. Murine neonates are comparable to human fetuses due to differences in the developmental timelines (102); hence, we isolated thymocytes from one-week-old neonates for in vitro activation. Neonatal thymocytes were activated in vitro, as indicated by the expression of TNFα and IFNγ (Day 4), or were used immediately after isolation (Day 0) (Fig. 6A). Activated or non-activated neonatal T cells were then sorted to obtain viable CD4+ T cells (Fig. 6B). Importantly, the ultrasound-guided intra-amniotic injection of in vitro activated neonatal CD4+ T cells induced 25% of preterm birth, whereas dams injected with non-activated neonatal CD4+ T cells or vehicle control delivered term neonates. Consequently, dams injected with activated neonatal CD4+ T cells delivered sooner than those injected with non-activated neonatal CD4+ T cells or vehicle control (Fig. 6C). These observations provide further evidence that activated T cells alone can trigger the premature process of labor in a subset of preterm births.

Figure 6. — **(A)** Representative flow cytometry plots showing the expression of TNFα and IFNγ by neonatal thymocytes after collection (Day 0) and after four days of *in vitro* activation (Day 4). **(B)** Representative gating used to sort neonatal CD3+CD4+ T cells. Purity was above 95% for all experiments. **(C)** Experimental design for the ultrasound-guided intra-amniotic injection of non-activated neonatal CD3+CD4+ T cells (n = 3), *in vitro* activated neonatal CD3+CD4+ T cells (n = 8), or PBS (vehicle control; n = 9) on 16.5 days *post coitum* (dpc). Kaplan-Meier survival curve showing the gestational ages at delivery for dams injected with PBS, non-activated CD3+CD4+ T cells, or activated CD3+CD4+ T cells. Preterm delivery was defined as birth before 18.5 dpc, whereas term delivery was considered as birth after 18.5 dpc.

DISCUSSION

To our knowledge, herein we report for the first time that the fetal immune system undergoes activation in women with preterm labor and birth in the absence of intra-amniotic inflammation, providing a potential new mechanism of disease for a subset of idiopathic preterm birth. Specifically, we first reported that fetal T cells are the predominant immune cell population in amniotic fluid during preterm gestations. Interestingly, only fetal CD4+ T cells were increased in amniotic fluid of women who underwent idiopathic preterm labor and birth. This increase in fetal CD4+ T cells was accompanied by mildly elevated amniotic fluid concentrations of T-cell cytokines such as IL-2, IL-4, and IL-13, which are expressed by these cells upon in vitro stimulation, but was not associated with the prototypical cytokine profile observed in cases with intra-amniotic inflammation. We also found that cord blood T cells, mainly CD4+ T cells, obtained from women with idiopathic preterm labor and birth displayed enhanced ex vivo activation, which is similar to that observed in women with intra-amniotic inflammation. Finally, we showed that the intra-amniotic administration of in vitro activated neonatal CD4+ T cells induces preterm birth in a subset of dams. These data suggest that, in a subset of women with idiopathic preterm birth, activated fetal T cells can trigger the premature process of labor.

The concept that amniotic fluid immune cells are of fetal origin emerged upon the observation that polymorphonuclear leukocytes obtained from amniotic fluid of women with preterm labor and clinical evidence of infection who delivered a male neonate carried X and Y chromosomes (81). A follow-up study in Rhesus macaques showed that amniotic fluid leukocytes in preterm gestations could be of either maternal or fetal origin: maternal and fetal leukocytes were observed under physiological conditions, whereas after the induction of intra-amniotic inflammation by infusion of IL-1β the fetal contribution predominated (82). More recently, using DNA fingerprinting, we demonstrated that, in cases with intra-amniotic inflammation/infection, fetal neutrophils invade the amniotic cavity of women in preterm gestations, whereas maternal neutrophils are predominantly present at term (51). These studies indicate that, in preterm gestations with intra-amniotic infection (81) or inflammation (82), amniotic fluid leukocytes are of fetal origin; yet, in cases at term, these cells can also be of maternal origin (51). In the absence of intra-amniotic infection or inflammation, amniotic fluid leukocytes (mostly lymphocytes (83, 84)) could be either of maternal or fetal origin (82); however, the origin of amniotic fluid leukocytes in preterm gestations requires further investigation since it is likely that gestational age and labor influence the origin of these immune cells. The present investigation provides conclusive evidence that amniotic fluid leukocytes in preterm gestations, primarily T cells, are exclusively of fetal origin in the absence of intra-amniotic inflammation. A proposed source for fetal immune cells in the amniotic cavity is the fetal organs, such as the gut and lungs, which are continuously exposed to amniotic fluid (83). Consistently, amniotic fluid T cells (84) and innate lymphoid cells (83) express a surface marker phenotype resembling that displayed by mucosal immune cells in the fetal gut (83, 103) and lungs (83). Moreover, fetuses with gastroschisis, a congenital defect of the abdominal wall that results in exposure and inflammation of the intestines, have increased T-cell infiltration in the gut mucosa (94) that may result in augmented numbers of such cells in amniotic fluid. Hence, T cells found in amniotic fluid during preterm gestations are of fetal origin and display a unique mucosal phenotype.

A major finding of our investigation was that fetal CD4+ T cells are increased in amniotic fluid of women with idiopathic preterm labor who delivered preterm. This rise in fetal CD4+ T cells coincided with mildly elevated amniotic fluid concentrations of the T cell-related cytokines IL-2, IL-4, and IL-13, and enhanced cord blood T-cell ex vivo activation. Notably, we found that amniotic fluid fetal T cells are capable of producing IL-2, IL-4, and IL-13 upon in vitro stimulation, indicating that these responses could occur in the context of preterm labor. These data suggest that fetal T-cell activation precedes preterm birth in the absence of intra-amniotic inflammation, which complements prior evidence showing that the fetal innate immune system is activated during preterm parturition independent of infection/inflammation (56), and that the fetus can signal the onset of labor (104–106). Thus, both the innate and adaptive immune system of the fetus are activated during the pathological process of idiopathic preterm labor that leads to preterm birth.

How do amniotic fluid fetal T cells trigger preterm parturition in the absence of intra-amniotic inflammation/infection? Answering this question would require the isolation of sufficient amniotic fluid fetal T cells to test their specificity and functionality ex vivo, which is extremely difficult when working with amniotic fluid leukocytes from women in preterm labor, from which we typically obtain few milliliters of amniotic fluid (10 – 100 cells per milliliter before sorting). The most tempting explanation for the induction of preterm labor by fetal T cells is that such cells are responding to maternal alloantigens. This concept is based on previous observations showing that central memory CD4+ T cells are increased in the cord blood of neonates born to women with preterm labor (66, 107) and display an activated phenotype (66, 108). Such fetal T cells possess the following properties: 1) proliferate in the presence of maternal antigens, but do not proliferate in response to third-party antigens; 2) release IFNγ and TNFα upon ex vivo stimulation; and 3) stimulate myometrial contractility ex vivo (66). Together with the abovementioned studies, our findings provide a potential mechanism whereby fetal T cells can lead to idiopathic preterm labor and birth in the absence of the clinical condition of intra-amniotic inflammation. Nonetheless, additional investigation, including HLA typing of the mother and neonate, will be required to investigate whether idiopathic preterm labor occurs as a result of the activation of allogeneic fetal T cells.

Interestingly, cord blood T cells from neonates born to women with idiopathic preterm labor and birth exhibited enhanced ex vivo cytokine responses. However, such a subset of preterm birth occurred in the absence of a prototypical cytokine response in the amniotic cavity (IL-6 (30), IL-8 (99), IL-1β (33, 100), and IL-12p70 (101)), which is observed in cases with inflammation-associated preterm labor and birth. These findings led us to propose that, in a subset of women with idiopathic preterm labor and birth, enhanced fetal T-cell cytokine responses in the amniotic cavity occurred in the absence of the canonical pro-inflammatory cytokine response, which is mainly initiated by neutrophils and monocytes invading this compartment in the context of infection or sterile inflammation (49, 54, 109). In line with this proposal, we demonstrated herein that the intra-amniotic administration of activated neonatal CD4+ T cells induces preterm labor and birth. Therefore, it is possible that, in humans, activated fetal T cells induce preterm parturition as proposed herein. It is worth mentioning that, due to the difficulty of obtaining a sufficient number of amniotic fluid cells, we used cord blood T cells from neonates born to women with idiopathic or intra-amniotic inflammation-associated preterm labor to test their functionality in these two subsets of preterm birth. Yet, cord blood immune responses are distinct from those of amniotic fluid immune cells (Fig. 1); therefore, further investigation is needed to evaluate whether amniotic fluid immune responses differ between these two subsets of preterm birth.

In summary, the findings presented in this report allow us to suggest a new mechanism of disease for a subset of idiopathic preterm labor, in which fetal T cells mediate a unique inflammatory response in the amniotic cavity, partially mediated by IL-2, IL-4, and IL-13, which precedes preterm birth (Visual Abstract, A). Such a mechanism of disease is different from that observed in women with preterm labor and intra-amniotic inflammation, in which maternal and fetal innate immune cells (neutrophils and macrophages) and T cells respond towards microbial products, involving the prototypical cytokines IL-6, IL-1β, and IL-8 (Visual Abstract, B). Finally, we showed that the intra-amniotic administration of activated neonatal CD4+ T cells induces preterm birth in a subset of dams. Collectively, these findings provide insight into a potential new mechanism implicated in the pathogenesis of idiopathic preterm labor and birth, the leading cause of perinatal morbidity and mortality worldwide.

Supplementary Material

NIHMS1536727-supplement-1.pdf^{(378.6KB, pdf)}

Key Points.

Amniotic fluid T cells are of fetal origin and functional in preterm gestations
Amniotic fluid T cells are increased in women with idiopathic preterm labor and birth
Intra-amniotic injection of activated neonatal T cells induces preterm birth in mice

ACKNOWLEDGMENTS

We thank the physicians, nurses, and research assistants from the Center for Advanced Obstetrical Care and Research, Intrapartum Unit, PRB Clinical Laboratory, and PRB Perinatal Translational Science Laboratory for their help with collecting and processing samples. We would especially like to thank George Schwenkel for his assistance with the animal experiments. We are grateful to Dr. Tippi C. MacKenzie for helpful discussions of the findings.

This research was supported, in part, by the Perinatology Research Branch, Division of Obstetrics and Maternal-Fetal Medicine, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, U.S. Department of Health and Human Services (NICHD/NIH/DHHS); and, in part, with Federal funds from NICHD/NIH/DHHS under Contract No. HHSN275201300006C.

This research was also supported by the Wayne State University Perinatal Initiative in Maternal, Perinatal and Child Health.

3. Non-standard abbreviations

IAI: intra-amniotic inflammation
PTL: preterm labor
PTB: preterm birth
TB: term birth

Footnotes

DISCLOSURES

The authors have no financial conflicts of interest.

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PERMALINK

Fetal T-cell activation in the amniotic cavity during preterm labor: A potential etiology for a subset of idiopathic preterm birth

Nardhy Gomez-Lopez

Roberto Romero

Yi Xu

Derek Miller

Marcia Arenas-Hernandez

Valeria Garcia-Flores

Bogdan Panaitescu

Jose Galaz

Chaur-Dong Hsu

Robert Para

Stanley M Berry

Abstract

Visual Abstract

INTRODUCTION

MATERIALS AND METHODS

Study population and characteristics

Table I.

Table II.

Table III.

Clinical determinations in amniotic fluid and placentas

Immunophenotyping by flow cytometry

DNA fingerprinting

Amniotic fluid cytokine/chemokine concentrations

Amniotic fluid T-cell cytokine expression upon stimulation

Determination of cord blood T-cell functionality

Mice

Isolation and activation of neonatal T cells

Determination of neonatal T-cell activation by flow cytometry

Fluorescence-activated cell sorting of viable activated or non-activated neonatal T cells

Ultrasound-guided intra-amniotic injection of activated or non-activated neonatal CD4+ T cells

Statistical analysis

RESULTS

Characterization and origin of amniotic fluid immune cells

Figure 1. Characterization and origin of amniotic fluid T cells.

Are fetal T cells in amniotic fluid associated with preterm labor and birth?

Figure 2. Amniotic fluid T cells are increased in preterm labor and birth.

Figure 3. Concentrations of amniotic fluid T-cell cytokines.

Fetal T-cell activation in idiopathic preterm labor and birth

Figure 4. Functional status of cord blood CD4+ T cells.

Figure 5. Functional status of cord blood CD8+ T cells.

Activated neonatal T cells induce preterm birth in mice

Figure 6. Ultrasound-guided intra-amniotic injection of activated and non-activated neonatal T cells.

DISCUSSION

Supplementary Material

Key Points.

ACKNOWLEDGMENTS

3. Non-standard abbreviations

Footnotes

REFERENCES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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