Effector and Activated T cells Induce Preterm Labor and Birth that is Prevented by Treatment with Progesterone

Marcia Arenas-Hernandez; Roberto Romero; Yi Xu; Bogdan Panaitescu; Valeria Garcia-Flores; Derek Miller; Hyunyoung Ahn; Bogdan Done; Sonia S Hassan; Chaur-Dong Hsu; Adi L Tarca; Carmen Sanchez-Torres; Nardhy Gomez-Lopez

doi:10.4049/jimmunol.1801350

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

Published in final edited form as: J Immunol. 2019 Mar 27;202(9):2585–2608. doi: 10.4049/jimmunol.1801350

Effector and Activated T cells Induce Preterm Labor and Birth that is Prevented by Treatment with Progesterone¹

Marcia Arenas-Hernandez ^†,^‡,^§, Roberto Romero ^†,^¥,^#,^¶, Yi Xu ^†,^‡, Bogdan Panaitescu ^‡, Valeria Garcia-Flores ^†,^‡, Derek Miller ^†,^‡, Hyunyoung Ahn ^‡, Bogdan Done ^†,^‡, Sonia S Hassan ^†,^‡,^£, Chaur-Dong Hsu ^‡, Adi L Tarca ^†,^‡,^Φ, Carmen Sanchez-Torres ^§, Nardhy Gomez-Lopez ^†,^‡,^*,²

PMCID: PMC6570421 NIHMSID: NIHMS1523035 PMID: 30918041

Abstract

Preterm labor commonly precedes preterm birth, the leading cause of perinatal morbidity and mortality worldwide. Most research has focused on establishing a causal link between innate immune activation and pathological inflammation leading to preterm labor and birth. However, the role of maternal effector/activated T cells in the pathogenesis of preterm labor/birth is poorly understood. Herein, we first demonstrated that effector memory and activated maternal T cells expressing granzyme B and perforin are enriched at the maternal-fetal interface (decidua) of women with spontaneous preterm labor. Next, using a murine model, we reported that prior to inducing preterm birth, in vivo T-cell activation caused maternal hypothermia, bradycardia, systemic inflammation, cervical dilation, intra-amniotic inflammation, and fetal growth restriction, all of which are clinical signs associated with preterm labor. In vivo T-cell activation also induced B-cell cytokine responses, a pro-inflammatory macrophage polarization, and other inflammatory responses at the maternal-fetal interface and myometrium in the absence of an increased influx of neutrophils. Lastly, we showed that treatment with progesterone can serve as a strategy to prevent preterm labor/birth and adverse neonatal outcomes by attenuating the pro-inflammatory responses at the maternal-fetal interface and cervix induced by T-cell activation. Collectively, these findings provide mechanistic evidence showing that effector and activated T cells lead to pathological inflammation at the maternal-fetal interface, in the mother, and in the fetus prior to inducing preterm labor and birth and adverse neonatal outcomes. Such adverse effects can be prevented by treatment with progesterone, a clinically approved strategy.

INTRODUCTION

Preterm birth, delivery before 37 weeks of gestation, is the leading cause of perinatal morbidity and mortality worldwide (1, 2). Nearly two-thirds of all cases of preterm birth are preceded by spontaneous preterm labor (3–5), a syndrome of multiple pathological processes (6, 7). Of all the putative causes associated with spontaneous preterm labor, only pathological inflammation has been causally linked to preterm birth (8–12).

Pathological inflammation can be triggered by pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) (i.e. alarmins) (13–16). PAMPs and DAMPs are sensed by pattern recognition receptors (PRRs), which are mainly present in innate immune cells (17). Therefore, most of the perinatal immunology research has focused on the role of innate immunity in the mechanisms that lead to preterm labor (18–31). Indeed, the stimulation of neutrophils/macrophages by administration of an endotoxin (32, 33) or activation of invariant natural killer T cells via alpha-galactosylceramide (34, 35) induces preterm labor and birth. However, pathological inflammation can also be mediated by T cells, the cellular component of the adaptive immune system (36). T cells have been implicated in implantation (37–40) and pregnancy maintenance through the mediation of maternal-fetal tolerance (41–56), and their infiltration at the maternal-fetal interface (i.e. decidua) has been associated with the physiological process of labor at term (57–61) and the syndrome of preterm labor and birth (62–64). However, a mechanistic link between maternal T cells and the pathophysiology of preterm labor and birth is lacking.

Herein, we hypothesized that effector/activated T cells can trigger the mechanisms leading to preterm labor and birth. This proposal is based on the clinical observation that chronic chorioamnionitis, a placental lesion in which maternal T cells infiltrate the fetal tissues (e.g. chorioamniotic membranes) through the decidua (64), is strongly associated with preterm labor and birth (62), and women with this condition display a systemic T-cell mediated cytotoxicity (65). In line with this hypothesis, it was also shown that transcriptional silencing limits the infiltration of T cells and other immune cells into the maternal-fetal interface (66–68), suggesting that an uncontrolled invasion of effector T cells may lead to pregnancy complications. More recently, we provided further evidence supporting a link between maternal T cells and preterm labor/birth by injecting an αCD3ε antibody, which is capable of inducing T-cell activation (69–71) and preterm labor/birth (72). However, the mechanisms whereby maternal T-cell activation induces preterm birth, and whether such an effect can be prevented, are unknown.

Herein, we aimed to determine whether effector T cells at the maternal-fetal interface are associated with spontaneous preterm labor (humans) and to investigate the mechanisms (murine animal models) whereby the activation of such immune cells induce pathological inflammation leading to preterm birth and adverse neonatal outcomes. Furthermore, we proposed the use of an approved therapeutic approach, progesterone, to prevent T-cell activation-induced preterm labor/birth and its adverse neonatal outcomes.

MATERIALS AND METHODS

Human subjects, clinical specimens, and definitions

Human placental basal plate (decidua basalis) and chorioamniotic membrane (decidua parietalis) samples were obtained at the Perinatology Research Branch, an intramural program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health, U. S. Department of Health and Human Services, Wayne State University (Detroit, MI, USA), and the Detroit Medical Center (Detroit, MI, USA). The collection and utilization of human materials for research purposes were approved by the Institutional Review Boards of the NICHD and Wayne State University. All participating women provided written informed consent. The study groups included women who delivered at term with (TIL) or without (TNL) spontaneous labor or preterm with (PTL) or without (PTNL) spontaneous labor. Two separate cohorts of women were used in this study: one for the immunophenotyping of effector T cells and one for the immunophenotyping of activated T cells. The demographic and clinical characteristics of the study groups are shown in Tables I and II. 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 weeks of gestation. Patients with multiple births or neonates that had congenital or chromosomal abnormalities were excluded.

Table I.

Demographic and clinical characteristics of the study population for immunophenotyping of effector memory T cells

	TNL (n=20)	TIL (n=55)	PTNL (n=15)	PTL (n=50)	p value
Age (y; median [IQR])^a	27 (24-29.3)	25 (22-29)	29 (24.5-35.5)	23 (21.3-26.3)	0.01
Body mass index (kg/m²; median [IQR])^a	27.6 (24.5-29.7)	29.3 (24.6-34)	27.5 (24.9-35.8)	24.9 (21.6-29.3)	0.03
Gestational age at delivery (wk; median [IQR])^a	39.1 (39-39.3)	39.4 (38.6-40.7)	33.9 (28.9-36.3)	34.2 (31.2-35.9)	<0.001
Birth weight (g; median [IQR])^a	3232.5 (2911.3-3658.8)	3205 (3007.5-3506.3)	1930 (1082.5-2385)	1977.5 (1466.3-2343.8)	<0.001
Race (n[%])^b					0.004
African-American	13 (65%)	51 (92.7%)	11 (73.3%)	44 (88%)
Caucasian	3 (15%)	3 (5.5%)	3 (20%)	1 (2%)
Hispanic	2 (10%)	0 (0%)	0 (0%)	0 (0%)
Asian	2 (10%)	0 (0%)	0 (0%)	2 (4%)
Other	0 (0%)	1 (1.8%)	1 (6.7%)	3 (6%)
Primiparity (n[%])^b	2 (10%)	7 (12.7%)	3 (20%)	11 (22%)	NS
Cesarean section (n[%])^b	20 (100%)	6 (10.9%)	15 (100%)	9 (18%)	<0.001

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Kruskal-Wallis test;

Fisher’s exact test;

IQR = interquartile range, TNL = term no labor, TIL = term in labor, PTNL = preterm no labor, PTL = preterm in labor

Table II.

Demographic and clinical characteristics of the study population for immunophenotyping of activated T cells

	TNL (n=20)	TIL (n=37)	PTNL (n=18)	PTL (n=53)	p value
Age (y; median [IQR])^a	24 (22-30.3)	26 (22-31)	30 (26-33)	24 (20-29)	0.02
Body mass index (kg/m²; median [IQR])^a	33.7 (27.2-37.1)	28.5 (24.2-35.6)	27.8 (24-35.4)	26.4 (21.5-31.3)	NS
Gestational age at delivery (wk; median [IQR])^a	39 (38.8-39.3)	38.9 (38.3-39.4)	33.4 (30.5-34.4)	34.9 (33.9-35.7)	<0.001
Birth weight (g; median [IQR])^a	3355 (2872.5-3511.3)	3185 (2735-3495)	1420 (1205.3-2095)	2255 (1830-2540)	<0.001
Race (n[%])^b					NS
African-American	15 (75%)	35 (94.6%)	12 (66.6%)	46 (86.8%)
Caucasian	3 (15%)	1 (2.7%)	4 (22.2%)	5 (9.4%)
Hispanic	0 (0%)	0 (0%)	0 (0%)	0 (%)
Asian	1 (5%)	0 (0%)	1 (5.6%)	0 (%)
Other	1 (5%)	1 (2.7%)	1 (5.6%)	2 (3.8%)
Primiparity (n[%])^b	0 (0%)	10 (27%)	3 (16.7%)	14 (26.4%)	0.03
Cesarean section (n[%])^b	20 (100%)	4 (10.8%)	18 (100%)	12 (22.6%)	<0.001

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Kruskal-Wallis test;

Fisher’s exact test;

IQR = interquartile range, TNL = term no labor, TIL = term in labor, PTNL = preterm no labor, PTL = preterm in labor

Decidual leukocyte isolation from human samples

Decidual leukocytes from human decidual tissue were isolated as previously described (73). Briefly, the decidua basalis was collected from the basal plate of the placenta, and the decidua parietalis was separated from the chorioamniotic membranes. Decidual tissue was homogenized using a gentleMACS Dissociator (Miltenyi Biotec, San Diego, CA, USA) in StemPro Cell Dissociation Reagent (Life Technologies, Grand Island, NY, USA). Homogenized tissues were incubated for 45 min at 37°C with gentle agitation. After incubation, tissues were washed with ice-cold 1X PBS and filtered through a 100 µm cell strainer. Cell suspensions were collected and centrifuged at 300 x g for 10 min at 4ºC, and the cell pellet was suspended in stain buffer (Cat#554656; BD Biosciences, San Jose, CA, USA). Mononuclear cells were purified using a density gradient (Ficoll-Paque Plus; GE Healthcare Bio-Sciences, Uppsala, Sweden), following the manufacturer’s instructions. Lastly, mononuclear cell suspensions were washed using stain buffer before immunophenotyping.

Immunophenotyping of human decidual leukocytes

Mononuclear cell suspensions from decidual tissues were stained with BD Horizon Fixable Viability Stain 510 dye (BD Biosciences) prior to incubation with extracellular and intracellular mAbs. Mononuclear cell suspensions were washed with stain buffer and centrifuged. Cell pellets were incubated for 10 min with 20 μl of human FcR Blocking Reagent (Miltenyi Biotec) in 80 μl of stain buffer. Next, mononuclear cell suspensions were incubated with extracellular fluorochrome-conjugated anti-human mAbs (Supplementary Table I) for 30 min at 4ºC in the dark. After extracellular staining, the cells were fixed. For intracellular staining, the cells were fixed and permeabilized using the BD Cytofix/Cytoperm Fixation/Permeabilization kit (BD Biosciences) prior to incubation with intracellular antibodies (Supplementary Table I). Finally, mononuclear cell suspensions were washed and resuspended in 0.5 mL stain buffer and acquired using the BD LSRFortessa flow cytometer and FACSDiva 6.0 software. Leukocyte subsets were gated within the viability gate. Immunophenotyping included the identification of: effector memory T cells (T_EM; CD3+CD4+ or CD3+CD4− CD45RA−CCR7−), naïve T cells (T_N; CD3+CD4+ or CD3+CD4− CD45RA+CCR7+), central memory T cells (T_CM; CD3+CD4+ or CD3+CD4− CD45RA−CCR7+), and effector memory RA T cells (T_EMRA; CD3+CD4+ or C3+CD4− CD45RA+CCR7−). Activated CD4+ and CD8+ T cells were evaluated by the expression of granzyme B and/or perforin. The data analysis was performed using FlowJo software v10 (TreeStar, Ashland, OR, USA).

Determination of decidual T-cell origin

Human placental basal plate (decidua basalis) and chorioamniotic membranes (decidua parietalis) were obtained from women who delivered a male neonate and decidual leukocytes were isolated as described above. Case-matched umbilical cord blood and maternal peripheral blood samples were also obtained and mononuclear cells were purified using a density gradient (Ficoll-Paque Plus), following the manufacturer’s instructions. Next, T cells were isolated from decidual, cord blood, and maternal mononuclear cells by magnetic separation using the human REAlease CD3 MicroBead kit (Miltenyi Biotec) and MS magnetic columns (Miltenyi Biotec), following the manufacturer’s instructions. After separation, isolated T cells were counted using an automatic cell counter (Cellometer Auto 2000; Nexcelom Bioscience, Lawrence, MA, USA), and the purity (>95%) of the isolated T cells was assessed by flow cytometry using a fluorochrome-conjugated anti-human monoclonal anti-CD3 antibody (Cat#564307; BD Biosciences). Purified T cells were then centrifuged at 500 x g for 5 min and the cell pellet was stored at −80°C until use.

Genomic DNA was extracted from isolated decidual, cord blood, and maternal CD3+ T cells using the QIAamp UCP DNA Micro kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions. DNA concentrations and purity were assessed with the NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). Quantitative PCR (qPCR) was performed using the ABI 7500 FAST Real-Time PCR System (Applied Biosystems, Life Technologies Corporation, Foster City, CA, USA). The TaqMan assays used to detect a gene region on the Y chromosome or X chromosome were SRY (Y-chromosome) (Cat#Hs00976796_s1) and AR (X-chromosome) (Cat#Hs05033429_s1). As an internal control, the 18S housekeeping gene was also detected (Cat#Hs9999901_s1). Each qPCR reaction contained 10 μL of 2X TaqMan Fast Advanced master mix (Cat#444553, Thermo Fisher Scientific), 1 μL of the TaqMan assay, and 9 μL of genomic DNA (2 ng total) to yield a 20 μL reaction, and each sample was run in duplicate. The thermal cycling profile began with 95ºC for 10 minutes, followed by 40 cycles of 95ºC for 15 seconds and 60ºC for 1 minute. Ct values for the SYR, AR, and 18S qPCRs were generated using the ABI 7500 Fast System SDS software version 1.3 (Applied Biosystems). The formula used to calculate the SYR/AR qPCR signal ratio for each sample was: 2^{−(ΔCt(SRY−18S)- ΔCt(AR−18S))}= 2^−ΔΔCt.

PCR amplification of the SRY gene was also performed using the traditional PCR technique. The primers used to amplify the SRY region were SRY-forward (5’- GGTGTTGAGGGCGGAGAAATGC-3’) (100 nM) and SRY-reverse (5’- GTAGCCATTGTTACCCGATTGTC-3’ (100 nM). The internal control β-Globin (HBB), was amplified using β-globin forward (5’-GCTTCTGACACAACTGTGTTCACTAGC-3’) (200 nM) and β-globin reverse (5’-CACCAACTTCATCCACGTTCACC-3’) (200 nM) primers. The PCR reaction included 25 μL of 2X PCR master mix (Cat#K0171, Thermo Fisher Scientific), 1 μL each of the forward and reverse primers (at concentrations indicated above), and 21 μL of genomic DNA (50 ng total) to yield a 50 μL reaction. The thermal cycling profile for the PCR began with 94ºC for 5 minutes, followed by 30 cycles of 94ºC for 1 minute, 60ºC for 1 minute, and 72ºC for 1 minute. The results of the PCR reaction were visualized using gel electrophoresis with E-Gel General Purpose 2% Agarose (Cat#G501802, Thermo Fisher Scientific). The gel image was acquired using the ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA).

Mice

C57BL/6J mice were purchased 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 days post coitum (dpc). Upon observation of vaginal plugs, female mice were removed from the mating cages and housed separately. A weight gain ≥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-09–08-12, A-07–03-15, and 18–03-0584). The authors adhered to the NIH Guide for the Care and Use of Laboratory Animals.

Animal models of preterm labor and birth

Anti-CD3ε-induced preterm labor/birth (72). Dams were injected intraperitoneally (i.p.) with 10µg/200µL of a monoclonal anti-CD3ε antibody (αCD3ε) (Clone 145–2C11; BD Biosciences) dissolved in sterile 1X phosphate-buffered saline (PBS) (Fisher Scientific Chemicals, Fair Lawn, NJ, USA) using a 26-gauge needle on 16.5 dpc. Controls were i.p. injected with 10μg/200μL of IgG1 κ isotype control (Clone A19–3; BD Biosciences) dissolved in sterile 1X PBS. N = 4 – 7 each.

LPS-induced preterm labor/birth (32, 74). Dams were injected i.p. with 15μg/150μL of LPS (Escherichia coli 055:B5; Sigma-Aldrich, St Louis, MO, USA) dissolved in sterile 1X PBS using a 26-gauge needle on 16.5 dpc. Controls were injected with 150μL of sterile 1X PBS alone. N = 7 – 8 each.

RU486-induced preterm labor/birth (75). Dams were injected subcutaneously (s.q.) with 150μg/100μL of RU486 (Mifepristone) (Sigma Aldrich) dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich) and diluted 1:13 in sterile 1X PBS or 100μL of DMSO diluted 1:13 in sterile 1X PBS as a control on 15.5 dpc. N = 3 – 8 each.

Following injection, pregnant mice were monitored using a video camera with infrared light (Sony, Tokyo, Japan) until delivery. Gestational length was calculated from the presence of the vaginal plug (0.5 dpc) until the observation of the first pup in the cage bedding. Preterm birth was defined as delivery <18.0 dpc.

Determination of body temperature

To determine body temperature, dams were injected with either αCD3ε, LPS, or RU486 (or their respective controls) (n = 5 each). Prior to injection the basal body temperature was taken using a rectal probe (TMH-150; VisualSonics Inc., Toronto, ON, Canada) and represented time zero. Following injection, the body temperature was recorded every 2 h until 12 h post-injection.

Determination of maternal heart rate using high-frequency ultrasound

Dams were injected with αCD3ε, LPS, or RU486 (or their respective controls) (n = 13 – 17 each for αCD3ε, 8 – 11 each for LPS, and 6 – 7 each for RU486). Twelve hours post-injection, each pregnant mouse underwent ultrasound assessment as previously described (34, 72, 74, 76–78). Briefly, mice were anesthetized by inhalation of 2–3% isoflurane (Aerrane; Baxter Healthcare Corporation, Deerfield, IL, USA) and 1–2 L/min oxygen in an induction chamber. Mice were then positioned on a heated platform and stabilized using adhesive tape and fur was removed from the abdomen and thorax. Body temperature was maintained at 37±1°C and monitored using a rectal probe. A 55 MHz linear ultrasound probe (VisualSonics Inc., Toronto, ON, Canada) was fixed and mobilized with a mechanical holder, and the transducer was slowly moved toward the abdomen. The maternal heart rate was calculated using three similar consecutive waveforms from the uterine artery. Ultrasound signals were processed, displayed, and stored using the Vevo Imaging Station (VisualSonics Inc).

Determination of cervical dilation

Dams were injected with αCD3ε, LPS, or RU486 (or their respective controls) (n = 8 each). Mice were euthanized 12–16 h post-injection and cervical tissues were collected, photographed, and blindly measured to determine cervical width as a surrogate of cervical dilation.

Leukocyte isolation from murine decidual, myometrial, and lymphatic tissues

Dams were injected with αCD3ε, LPS, or RU486 (or their respective controls) (n = 10 – 13 each for αCD3ε, 8 – 10 each for LPS, and 9 – 10 each for RU486). Mice were euthanized 12–16 h post-injection and decidual and myometrial tissues from the implantation sites were collected. Isolation of leukocytes from decidual and myometrial tissues was performed, as previously described (79). Briefly, tissues were cut into small pieces using fine scissors and enzymatically digested with StemPro Cell Dissociation Reagent for 35 minutes at 37°C. The spleen and uterine lymph nodes (ULN) were also collected and leukocyte suspensions were prepared, as previously reported (32). Leukocyte suspensions were filtered using a 100 µm cell strainer and washed with FACS buffer [0.1% BSA (Sigma-Aldrich) and 0.05% sodium azide (Fisher Scientific Chemicals) in 1X PBS] before immunophenotyping.

Immunophenotyping of decidual, myometrial, and lymphatic leukocytes

Leukocyte suspensions from decidual, myometrial, and lymphatic tissues were centrifuged at 1250 x g for 10 min at 4ºC. Cell pellets were then incubated with the CD16/CD32 monoclonal antibody (mAb) (FcγIII/II Receptor; BD Biosciences) for 10 minutes and subsequently incubated with specific fluorochrome-conjugated anti-mouse mAbs (Supplementary Table I) for 30 min at 4°C in the dark. Leukocyte suspensions were fixed/permeabilized with the BD Cytofix/Cytoperm Fixation/Permeabilization kit prior to staining with intracellular antibodies. Cells were acquired using the BD LSRFortessa flow cytometer and FACSDiva 8.0 software. Immunophenotyping included the identification of T cells (CD3+CD4+ and CD3+CD8+ cells) and their activation status by the expression of CD25, CD69, CD44, CD62L, CTLA-4 (CD152), PD-1 (CD279), IL2, and IFNγ in the decidual, myometrial, and lymphatic tissues. B cells (CD45+CD19+) and their activation by the expression of IFNγ; M1- and M2-like macrophage phenotypes (CD11b+F4/80+iNOS+ or Arg1+IL10+); and neutrophils (CD45+F4/80-Ly6G+) were also determined in the decidual and myometrial tissues. Data were analyzed using FlowJo software v10.

Gene expression of M1 and M2 markers in decidual and myometrial macrophages

Dams were injected with αCD3ε, LPS, or RU486 (or their respective controls) (n = 6–8 per group). Mice were euthanized 12–16 h post-injection and decidual and myometrial tissues from the implantation sites were collected. Leukocytes were isolated from the decidual and myometrial tissues as described above. Leukocyte suspensions were sequentially filtered using a 100 µm cell strainer followed by a 30 µm cell strainer, and then washed with 1X PBS. After centrifugation at 300 x g for 10 min at 4 ºC, the leukocytes were resuspended in 1 mL of 1X PBS and counted using an automatic cell counter (Cellometer Auto 2000; Nexcelom Bioscience). Macrophages were then isolated from decidual and myometrial cells by magnetic separation using mouse Anti-F4/80 UltraPure MicroBeads (Miltenyi Biotec) and MS magnetic columns (Miltenyi Biotec), following the manufacturer’s instructions. After elution from the column, a small aliquot of the isolated F4/80+ cells was taken to assess the purity by flow cytometry using the fluorochrome-conjugated anti-mouse monoclonal antibodies CD45, CD11b, and F4/80 (Supplementary Table I). Isolated F4/80+ cells were then centrifuged and the cell pellet was resuspended in 350 µL RLT Lysis Buffer (Qiagen). Total RNA was isolated from F4/80+ cells using the RNeasy micro kit (Qiagen), following the manufacturer’s instructions. RNA integrity was evaluated with the 2100 Bioanalyzer system (Agilent Technologies, Wilmington, DE, USA) using the Agilent RNA 6000 Pico Kit (Agilent Technologies). Complementary (c)DNA was synthesized by using the SuperScript® III First-Strand Synthesis System for RT-PCR (Invitrogen, Life Technologies, Carlsbad, CA, USA) on the Applied Biosystems GeneAmp PCR System 9700 (Applied Biosystems, Life Technologies, Foster City, CA, USA), following the manufacturer’s instructions. Complementary DNA was amplified using the TaqMan® PreAmp Master Mix (2X) (Applied Biosystems) on the Applied Biosystems 7500 Fast Real-time PCR System. Messenger RNA expression was determined by quantitative real-time PCR (qRT-PCR) using a BioMark high-throughput qRT-PCR System (Fluidigm, San Francisco, CA, USA) and TaqMan® gene expression assays (Thermo Fisher) (Supplementary Table I).

Determination of pro-inflammatory and contractility-related genes in decidual and myometrial tissues

Dams were injected with αCD3ε, LPS, or RU486 (or their respective controls) (n = 7 – 9 each for αCD3ε, 9 – 13 each for LPS, and 11 – 16 each for RU486). Mice were euthanized 12–16 h post-injection and decidual and myometrial tissues from the implantation sites were collected and placed in RNAlater Stabilization Solution (Life Technologies). Total RNA was isolated from decidual and myometrial tissues using the RNeasy mini kit (Qiagen), following the manufacturer’s instructions. RNA concentrations and purity were assessed with the NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA), and RNA integrity was evaluated with the 2100 Bioanalyzer system (Agilent Technologies) using the Agilent RNA 6000 Nano Kit (Agilent Technologies). Complementary (c)DNA was synthesized by using the SuperScript® III First-Strand Synthesis System for RT-PCR (Invitrogen, Life Technologies) on the Applied Biosystems GeneAmp PCR System 9700 (Applied Biosystems, Life Technologies), following the manufacturer’s instructions. Complementary DNA was amplified using the TaqMan® PreAmp Master Mix (2X) (Applied Biosystems) on the Applied Biosystems 7500 Fast Real-time PCR System. Messenger RNA expression was determined by quantitative real-time PCR (qRT-PCR) using a BioMark high-throughput qRT-PCR System (Fluidigm) and TaqMan® gene expression assays (Thermo Fisher) (Supplementary Table I).

Determination of cytokine concentrations in amniotic fluid and the maternal circulation

Dams were injected with αCD3ε, LPS, or RU486 (or their respective controls). Mice were euthanized 12–16 h post-injection and peripheral blood was collected by cardiac puncture for serum separation (n = 11 – 12 each for αCD3ε, 10 each for LPS, and 10 each for RU486). Amniotic fluid was also obtained from each amniotic sac with a 26-gauge needle (n = 5 each for αCD3ε, 5 each for LPS, and 5 each for RU486). Maternal serum and amniotic fluid samples were centrifuged at 1300 x g for 10 min at 4°C and the supernatants were separated and stored at −20°C until analysis. The ProcartaPlex Mouse Cytokine & Chemokine Panel 1A 36-plex (Invitrogen by Thermo Fisher Scientific, Vienna, Austria) was used to measure the concentrations of IFNγ, IFNα, IL-12p70, IL1β, TNFα, GM-CSF, IL18, IL17A, IL22, IL23, IL27, IL9, IL15/IL15R, IL13, IL2, IL4, IL5, IL6, IL10, CCL11, IL28, IL3, LIF, IL1α, IL31, CXCL1, CCL3, CXCL10, CCL2, CCL7, CCL4, CXCL2, CCL5, G-CSF, M-CSF, and CXCL5 in serum and amniotic fluid samples, according to the manufacturer’s instructions. Plates were read using the Luminex 100 SystemFill (Luminex, Austin, TX, USA), and analyte concentrations were calculated with ProcartaPlex Analyst 1.0 Software from Affymetrix, San Diego, CA, USA. The sensitivities of the assays were: 0.09 pg/mL (IFNγ), 3.03pg/mL (IFNα), 0.21 pg/mL (IL12p70), 0.14 pg/mL (IL1β), 0.39 pg/mL (TNFα), 0.19 pg/mL (GM-CSF), 9.95 pg/mL (IL18), 0.08 pg/mL (IL17A), 0.24 pg/mL (IL22), 2.21 pg/mL (IL23), 0.34 pg/mL (IL27), 0.28 pg/mL (IL9), 0.42 pg/mL (IL15/IL15R), 0.16 pg/mL (IL13), 0.10pg/mL (IL2), 0.03 pg/mL (IL4), 0.32 pg/mL (IL5), 0.21 pg/mL (IL6), 0.69 pg/mL (IL10), 0.01 pg/mL (CCL11), 20.31 pg/mL (IL28), 0.11 pg/mL (IL3), 0.28 pg/mL (LIF), 0.32 pg/mL (IL1α), 0.45 pg/mL (IL31), 0.05 pg/mL (CXCL1), 0.13 pg/mL (CCL3), 0.26 pg/mL (CXCL10), 3.43 pg/mL (CCL2), 0.15 pg/mL (CCL7), 1.16 pg/mL (CCL4), 0.37 pg/mL (CXCL2), 0.35 pg/mL (CCL5), 0.19 pg/mL (G-CSF), 0.02 pg/mL (M-CSF), and 5.67 pg/mL (CXCL5). Inter-assay and intra-assay coefficients of variation were less than 10%.

Cytokine concentrations in amniotic fluid were adjusted by protein concentration, which were determined using the Pierce™ BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA), following the manufacturer’s instructions.

Fetal growth parameters

Dams were injected with αCD3ε, LPS, or RU486 (or their respective controls) (n = 7 each for αCD3ε, 10 each for LPS, and 10 each for RU486). Mice were euthanized 12–16 h post-injection and representative images of the fetuses and placentas were obtained. Fetuses were also weighed followed by dissection to obtain representative images of their lungs.

Hematoxylin and Eosin (H&E) and Masson’s trichrome staining

The lungs of preterm and term neonates (n=3 each) were fixed in 4% paraformaldehyde for 24 h and stored at 4°C in ethanol prior to embedding in paraffin blocks. The embedded tissues were then cut into 5-µm-thick sections, placed onto salinized slides, deparaffinized with xylene, and hydrated with ethanol. Slides were stained with hematoxylin (Cat#88018, Thermo Scientific) for 10 s, washed with distilled water, and immersed in 80% ethanol followed by staining with eosin (Cat#71211, Thermo Scientific) for 10 s. The sections were then dehydrated in a series of alcohol baths and xylene, and a coverslip was applied. The Masson’s trichrome staining was performed using the Masson’s trichrome stain kit (American MasterTech, Lodi, CA, USA), according to the manufacturer’s protocol. The sections were then dehydrated in a series of alcohol baths and xylene, and a coverslip was applied. All images were taken using the Vectra Polaris Quantitative Slide Scanner (Perkin Elmer, Waltham, MA, USA).

Determination of progesterone concentration in the maternal circulation

Dams were injected with αCD3ε or isotype control (n = 10 – 11 each). Mice were euthanized 16 h post-injection and peripheral blood was collected by cardiac puncture for serum separation. Maternal serum was centrifuged at 1300 x g for 10 min at 4°C and the supernatants were separated and stored at −20°C until analysis. Serum progesterone was measured using the PROG-EASIA ELISA kit (GenWay Biotech, Inc., San Diego, CA, USA), according to the manufacturer’s instructions. The sensitivity of the assay was 0.08 ± 0.03 ng/mL. The intra-assay coefficient of variation was 10.5%.

Preterm birth rescue by treatment with progesterone

Dams were injected s.q. with either 1 mg/200µL of progesterone (P4, Sigma-Aldrich) diluted in sesame oil (SO, Sigma-Aldrich) or 200 µL of sesame oil (control groups) on 15.5 dpc, 16.5 dpc, and 17.5 dpc. On 16.5 dpc, dams were also injected with either 10 µg/200 µL of αCD3ε or isotype control (n= 5 – 10 each). Following the last injection, dams were monitored via video camera with infrared light until delivery. The rate of preterm birth and gestational length was recorded as previously described. The rate of neonatal mortality was calculated as the number of pups found dead among the total litter size. Representative images of pups and their lungs just after birth and at 1 day old were also obtained.

Determination of pro-inflammatory genes in decidual, myometrial and cervical tissues after treatment with progesterone

Dams were injected s.q. with either 1 mg/200µL of progesterone (P4, Sigma-Aldrich) diluted in sesame oil (SO, Sigma-Aldrich) or 200 µL of sesame oil (control groups) on 15.5 dpc and 16.5 dpc. On 16.5 dpc, dams were also injected with either 10 µg/200 µL of αCD3ε or isotype control (n= 5 each). Approximately 16 h after αCD3ε or isotype injection, mice were euthanized and decidual and myometrial tissues from the implantation sites as well as cervical tissues were collected and placed in RNAlater Stabilization Solution (Life Technologies). Total RNA was isolated from tissues using the RNeasy mini kit (Qiagen), following the manufacturer’s instructions. RNA concentrations and purity were assessed with the NanoDrop 1000 spectrophotometer (Thermo Scientific), and RNA integrity was evaluated with the 2100 Bioanalyzer system (Agilent Technologies) using the Agilent RNA 6000 Nano Kit (Agilent Technologies). Complementary (c)DNA was synthesized by using the SuperScript® III First-Strand Synthesis System for RT-PCR (Invitrogen, Life Technologies) on the Eppendorf AG (Eppendorf, Hamburg, Germany), following the manufacturer’s instructions. Complementary DNA was amplified using the TaqMan® PreAmp Master Mix (2X) (Applied Biosystems) on the Applied Biosystems 7500 Fast Real-time PCR System. Messenger RNA expression was determined by quantitative real-time PCR (qRT-PCR) using a BioMark high-throughput qRT-PCR System (Fluidigm) and TaqMan® gene expression assays (Thermo Fisher) (Supplementary Table I).

Statistics

Statistical analyses were performed using SPSS v19.0 (IBM Corporation, Armonk, NY, USA) or the R package (https://www.r-project.org/). For human demographic data, the group comparisons were performed using the Fisher’s exact test for proportions and Kruskal–Wallis tests for non-normally distributed continuous variables. When proportions are displayed, percentages and 95% confidence intervals (CI) are shown. Medians are shown with the interquartile range (IQR). Kaplan-Meier survival curves were used to plot and compare the gestational length data (Mantel–Cox test). For maternal heart rates and fetal weights, the statistical significance of group comparisons was assessed using the t test and the means are shown with the standard error of the mean (SEM). For the rates of preterm birth and neonatal mortality, the Fisher’s exact test was utilized. For body temperature, cervical widths, multiplex, and ELISA and flow cytometry murine data, the statistical significance between groups was determined using a Mann–Whitney U test. For qRT-PCR arrays, negative ΔCt values were determined using multiple reference genes (Gusb, Hsp90ab1, Gapdh, and Actb) averaged within each sample to determine gene expression levels. Heat maps were created for the group mean expression matrix (gene x group mean), with each gene expression level being standardized first. The heat maps shown in Figures 5 & 6 represent the Z-scores of the mean (−ΔCt). The heat maps found in Figure 10 display the –ΔCt values of each group, centered on the –ΔCt value of the control group treated with sesame oil (SO) + Isotype. All heat maps show hierarchical clustering using correlation distance. The p-values were determined by unpaired t-test or Mann–Whitney U test. A p value of ≤ 0.05 was considered statistically significant.

Fig. 5. — (A) Gating strategy used to identify M1-like (CD11b+F4/80+iNOS+ cells) macrophages at the maternal-fetal interface. Grey histograms represent autofluorescence controls and colored histograms represent the expression of iNOS at the maternal-fetal interface of dams injected with isotype control or αCD3ε, respectively. Proportions of M1-like macrophages in the decidual and myometrial tissues from dams injected with αCD3ε or isotype, (n=12-13 each). (B) Left to right: spatial localization of the murine decidua and myometrium. Workflow showing the magnetic isolation of macrophages from decidual and myometrial cells; macrophage purity (F4/80+ cells; >92%) was determined by flow cytometry. (C) Heat map visualization of the expression of M1 macrophage markers by F4/80+ cells isolated from the decidual and myometrial tissues of dams injected with αCD3ε, isotype, LPS, PBS, RU486 or DMSO (n=6-8 each). (D) Heat map visualization of the expression of M2 macrophage markers by F4/80+ cells isolated from the decidual and myometrial tissues of dams injected with αCD3ε, isotype, LPS, PBS, RU486 or DMSO (n=6-8 each). Negative (−)∆Ct values were calculated using *Actb*, *Gusb*, *Gapdh* and *Hsp90ab1* as reference genes. (E) Gating strategy used to identify neutrophils (CD45+Ly6G+ cells) at the maternal-fetal interface. Proportion of neutrophils in decidual and myometrial tissues from dams injected with αCD3ε, isotype, LPS, PBS, RU486 or DMSO (n=9-13 each). The p-values were determined by 2-tailed Mann-Whitney U-test. Data are shown as scatter plots (median).

Fig. 6. — Decidual and myometrial tissues from dams injected with αCD3ε (or isotype control), LPS (or PBS control), or RU486 (or DMSO control). Heat map visualization of inflammatory and contractility-related gene expression in the (A) decidual and (B) myometrial tissues. (**C&D**) Messenger (m)RNA expression of selected genes in decidual and myometrial tissues. Negative (−)∆Ct values were calculated using *Actb, Gusb, Gapdh* and *Hsp90ab1* as reference genes. Data are from individual dams (n=7-16 each). The p-values were determined by unpaired 2-tailed t-test. Data are shown as scatter plots (median).

Fig. 10. — Decidual, myometrial and cervical tissues from dams injected with sesame oil (SO)+Isotype, SO+αCD3ε, progesterone (P4)+Isotype or P4+αCD3ε (n=5 each). Heat map visualization of inflammatory gene expression in the (A) decidual, (B) myometrial, and (C) cervical tissues. The–ΔCt values of each group were centered on the −ΔCt value of the control group treated with sesame oil (SO) + Isotype. (**D&E**) Messenger (m)RNA expression of selected genes in decidual and cervical tissues. Negative (−)∆Ct values were calculated using *Actb*, *Gusb*, *Gapdh* and *Hsp90ab1* as reference genes. Red arrows alongside the heat maps indicate the genes chosen for plotting. Data are from individual dams (n=5 each). The p-values were determined by one-tailed Mann Whitney U-test. Data are shown as scatter plots (median).

RESULTS

Effector and activated maternal T cells are enriched at the human maternal-fetal interface during spontaneous preterm labor

Naïve T cells (T_N) travel throughout the circulation in search of antigens presented by dendritic cells (80). Upon antigen presentation, T cells proliferate and differentiate into effector cells that can migrate to B-cell areas or inflamed tissues (81). A proportion of these cells persist as circulating memory T cells conferring protection against the known antigen (82). Memory T cells can be subdivided based on their phenotype and function into central memory T cells (T_CM), which display high proliferative potential but lack an immediate effector function, and effector memory (T_EM) and terminally differentiated effector memory (T_EMRA) T cells that have low proliferative capacity but display rapid effector function (83, 84). Such cells can be distinguished by the expression of CD45RA, which is expressed by naïve or terminally differentiated T cells, and CCR7, a lymph node homing receptor (83, 84). We have previously shown that memory-like T cells with effector functions are present at the human maternal-fetal interface during the physiological process of term labor (58, 59). Therefore, using immunophenotyping, we first investigated whether the different effector T-cell subsets were differentially distributed in the decidual tissues of women with spontaneous preterm labor (Fig. 1A). Strikingly, both CD4+ and CD8+ T_EM were the most abundant T-cell subsets at the human maternal-fetal interface (i.e. decidua basalis and decidua parietalis) (Fig. 1B and Fig. S1A). Indeed, CD4+ and CD8+ T_EM were enriched at the maternal-fetal interface of women who underwent spontaneous preterm labor compared to those who delivered at term (Fig. 1B and Fig. S1A). The increase in T_EM was accompanied by a reduction in both CD4+ and CD8+ T_N (Fig. 1C) and T_CM (Fig. 1D) at the maternal-fetal interface of women with spontaneous preterm labor (Fig. 1C&D and Fig. S1B&C). CD8+ T_EMRA were also greater at the maternal-fetal interface of women who underwent spontaneous preterm labor compared to those who delivered preterm without labor (Fig. 1E and Fig. S1D).

Fig. 1. — (A) Gating strategy used to identify CD4+ and CD4− (CD8+) effector memory (T_EM; CD45RA−CCR7−), naïve (T_N; CD45RA+CCR7+), central memory (T_CM; CD45RA−CCR7+) and effector memory RA (T_EMRA; CD45RA+ CCR7−) T cells in the decidua basalis from women who delivered at term without labor (TNL), term with labor (TIL), preterm without labor (PTNL), or preterm with labor (PTL). (B) Proportions of T_EM cells. (C) Proportions of T_N cells. (D) Proportions of T_CM cells. (E) Proportions of T_EMRA cells. The p-values were determined by 2-tailed Mann-Whitney U-test. Data are shown as scatter plots (median). Demographic and clinical characteristics of the study population are shown in Table I.

After encountering their antigen, T_EM become activated and perform their effector functions through the release of inflammatory mediators such as granzyme B and perforin (85–89). Therefore, we next investigated whether effector T cells expressed granzyme B and perforin at the human maternal-fetal interface (Fig. 2A). Consistent with our previous findings, CD4+ and CD8+ T cells expressing granzyme B and perforin were enriched at the maternal-fetal interface of women with spontaneous preterm labor (Fig. 2B&C and Fig. S1E&F).

Fig. 2. — (A) Gating strategy used to identify CD4+ and CD8+ T cells expressing granzyme B or perforin in the decidua basalis from women who delivered at term without labor (TNL), term with labor (TIL), preterm without labor (PTNL), or preterm with labor (PTL). (B) Proportions of CD4+granzyme B+ or CD8+granzyme B+ T cells. (C) Proportions of CD4+perforin+ or CD8+perforin+ T cells. The p-values were determined by 2-tailed Mann-Whitney U-test. Data are shown as scatter plots (median). Demographic and clinical characteristics of the study population are shown in Table II. (D) qPCR ratio of *SRY* (Y-chromosome) to AR (X-chromosome) expression in CD3+ T cells isolated from umbilical cord blood, maternal peripheral blood, the decidua basalis, and the decidua parietalis. Data are shown as scatter plots (median + interquartile range). Gel image of amplified PCR fragments shows *SRY* expression by CD3+ T cells isolated from umbilical cord blood, maternal peripheral blood, the decidua basalis, and the decidua parietalis. *HBB* = human β-globin (housekeeping gene).

In order to confirm the maternal origin of T cells at the maternal-fetal interface, we measured the ratio of SRY (Y-chromosome) to AR (X-chromosome) expression in T cells isolated from the decidua basalis and parietalis, umbilical cord blood, and maternal peripheral blood obtained from women who delivered a male neonate. As expected, T cells isolated from umbilical cord blood displayed a high SRY/AR ratio indicative of an exclusively fetal origin (Fig. 2D). In contrast, T cells isolated from the decidua parietalis and maternal peripheral blood had a SRY/AR ratio of zero, indicating that these cells are exclusively of maternal origin (Fig. 2D). T cells isolated from the decidua basalis were also predominantly of maternal origin (Fig. 2D).

Together, these findings indicate that both CD4+ and CD8+ maternal T cells exhibit an effector memory phenotype and release pro-inflammatory mediators at the human maternal-fetal interface during spontaneous preterm labor. In other words, the human syndrome of preterm labor is characterized by an increase in maternal effector/activated T cells at the maternal-fetal interface.

In vivo T-cell activation induces preterm birth by inducing hypothermia, bradycardia and cervical dilation

Next, we investigated the mechanisms whereby effector/activated T cells could induce preterm labor and birth by using a murine model. In vivo T-cell activation is achieved by the administration of an αCD3 antibody which induces a cytokine-related syndrome leading to hypothermia (90–94); therefore, we used this strategy in pregnant mice. Dams injected with αCD3ε delivered preterm (Fig. 3A), as previously reported (72). In order to understand the pathophysiology of preterm birth induced by T-cell activation, we compared this model to two well-established models of preterm birth: lipopolysaccharide (LPS)-induced (74, 95) (Fig. 3B) and RU486-induced (75) (Fig. 3C). Given that pregnant women undergoing complications associated with inflammatory responses can develop fever and tachycardia (96, 97) [as opposed to mice which develop hypothermia and bradycardia (74, 98)], we first evaluated the body temperature and heart rate of dams prior to preterm birth. Both αCD3ε and LPS induced hypothermia in dams (Fig. 3D&E), which could be due to the fact that these models are accompanied by excess release of TNFα into the circulation (78, 93). However, RU486 did not cause such an effect (Fig. 3F). Only the administration of αCD3ε induced maternal bradycardia (Fig. 3G-I), which may be a consequence of the severe hypothermia (99).

Fig. 3. — Kaplan-Meier survival curves showing the gestational length of dams injected with (A) αCD3ε (or isotype control), (B) LPS (or PBS control), or (C) RU486 (or DMSO control); n=3-8 each. The p-values were determined by Mantel-Cox test. The body temperature of the dams was recorded prior to and after the injection of (D) αCD3ε (■) or isotype control (□); (E) LPS (▲) or PBS control (∆); (F) RU486 (●) or DMSO control (○). The p-values were determined by 2-tailed Mann-Whitney U-test. Data are shown as mean ± SEM; n=5 each, *p<0.01. Doppler ultrasound was performed on dams just prior to (G) αCD3ε-induced, (H) LPS-induced, or (I) RU486-induced preterm labor/birth. Maternal heart rate was evaluated by the mean of three constant waves of the uterine artery. The p-values were determined by 2-tailed unpaired t-test. Data are shown as mean ± SEM; n=6-17 each. Representative images and widths of the cervices collected from dams after injection with (J) αCD3ε or isotype control; (K) LPS or PBS control; or (L) RU486 or DMSO control. The p-values were determined by 2-tailed Mann-Whitney U-test. Data are shown as medians; n=8 each.

A hallmark of preterm labor in humans (100) and mice (101) is cervical dilation; therefore, we then measured the cervical width prior to preterm birth. Consistently, all of the stimuli (αCD3ε, LPS, and RU486) induced cervical dilation indicating that the three models share this aspect of parturition (Fig. 3J-L).

In order to prove that αCD3ε induced T-cell activation at the maternal-fetal interface and myometrium, we determined the expression of the CD3 molecule in total T cells (102, 103), the expression of the activation markers CD25 (104), CD69 (105), CD44 (106), CD62L (107), CTLA-4 (108), and PD-1 (109) by CD4+ and CD8+ T cells, and the expression of IL2 by CD4+ T cells (103) and IFNγ by CD8+ T cells (110), in the decidua and myometrium (Fig. 4A). In line with our hypothesis (72), αCD3ε induced the downregulation of the CD3 molecule (Fig. 4B) and increased the expression of CD25 (Fig. 4C&D) and CD69 (Fig. 4E&F) by CD4+ and CD8+ T cells in both the decidua and myometrium. Upregulation of these activation markers was also observed in response to LPS (Fig. 4C-F), although the increased expression of CD25 in decidual T cells did not reach significance (Fig. 4C&D). In contrast, RU486 did not cause major changes in the expression of these activation markers by decidual and myometrial T cells (Fig. 4B-F). The expression of CD44 and CD62L was not drastically changed upon T-cell activation or treatment with LPS or RU486 (data not shown). Yet, the expression of CTLA-4 and PD-1 was variably regulated by CD4+ T cells and CD8+ T cells upon T-cell activation in the decidua and/or myometrium, but not upon treatment with LPS or RU486 (data not shown).

Injection with αCD3ε upregulated the expression of IL2 by CD4+ T cells in the decidua and myometrium (Fig. 4G). However, injection with αCD3ε increased the proportion of CD8+IFNγ+ T cells in the myometrium, but not in the decidua (Fig. 4H). These data suggest that, upon T-cell activation, T-cell responses are differentially regulated at the maternal-fetal interface and in the reproductive tissues. We also observed that LPS increased the infiltration of decidual CD8+ T cells expressing IFNγ, which is considered to be a response to microbial products (111). CD8+ T cells expressing IFNγ in the RU486 model were rare and did not vary (Fig. 4H).

Administration of αCD3ε induced the downregulation of the CD3 molecule in the spleen but not in the ULN (Fig. S2A&B). Dams injected with αCD3ε displayed greater proportions of CD4+ and CD8+ T cells expressing CD25 and CD69, but these dams did not have more IL-2-expressing CD4+ T cells or IFNγ-expressing CD8+ T cells, in the spleen and ULN compared to controls (Fig. S2C-H). Dams injected with LPS also had higher proportions of CD4+ or CD8+ T cells expressing CD25 and CD69 in the spleen and ULN compared to controls, but no changes in IL-2-expressing CD4+ T cells or IFNγ-expressing CD8+ T cells (Fig. S2C-H). Treatment with RU486 did not induce any changes in the expression of activation markers by splenic and ULN T cells (Fig. S2A-H). The expression of CD44, CD62L, CTLA-4, and PD-1 were variably regulated in the lymphatic tissues upon T-cell activation, but not drastically changed after treatment with LPS or RU486 (data not shown).

Collectively, these data show that αCD3ε causes preterm labor and birth by activating T cells at the maternal-fetal interface, myometrium, and systemically; yet, decidual and myometrial T-cell responses are distinct from those observed in lymphatic tissues. Such T-cell activation induces pathophysiological processes, some of which are shared with the microbial-inflammation (LPS) model but are distinct from the progesterone-dependent (RU486) model.

Given the association between regulatory T cells and pregnancy success (42, 50), we also evaluated whether αCD3ε could induce a reduction of such cells. Anti-CD3ε did not reduce the proportion of CD4+ regulatory T cells at the maternal-fetal interface, myometrium, or the maternal circulation (data not shown).

In vivo T-cell activation induces B-cell cytokine responses and pro-inflammatory macrophage polarization but does not lead to an increased neutrophil influx into the maternal-fetal interface and myometrium

Activation of T cells is accompanied by B-cell cytokine responses (112). Thus, we determined whether prior to preterm birth, αCD3ε could trigger B-cell activation at the maternal-fetal interface and myometrium (Fig. 4I). Anti-CD3ε caused an increase in the proportion of B cells expressing IFNγ in the decidua and myometrium (Fig. 4J). LPS also induced an increment in the proportion of activated B cells in the decidua but such an effect was not observed with RU486 (Fig. 4J). B cells expressing IFNγ in the RU486 model were rare (Fig. 4J).

Activation of T cells can also induce macrophage polarization (113, 114). Indeed, an M1-like (i.e. pro-inflammatory) macrophage polarization has been implicated in the mechanisms that lead to spontaneous preterm labor and birth (33). Hence, we evaluated whether prior to preterm birth, αCD3ε could induce macrophage activation and polarization towards M1-like and M2-like phenotypes at the maternal-fetal interface. First, flow cytometry was performed to identify M1-like (CD11b+F4/80+iNOS+ cells) and M2-like (CD11b+F4/80Arg1+IL10+ cells) macrophages in the decidua and myometrium. Anti-CD3ε increased the proportion of M1-like macrophages in the decidua and myometrium (Fig. 5A). However, neither LPS nor RU486 altered the proportion of M1-like macrophages at the maternal-fetal interface or the myometrium (data not shown). Although M2-like macrophages were detected at the maternal-fetal interface and myometrium, their proportions were unchanged upon αCD3ε, LPS, or RU486 injection (data not shown). In order to confirm that T-cell activation induced the polarization of M1-like macrophages, we determined the mRNA expression of multiple established M1 and M2 macrophage markers (115) in sorted decidual and myometrial macrophages (Fig. 5B). Anti-CD3ε caused a strong upregulation of M1 markers in macrophages from both the decidua and myometrium (Fig. 5C). LPS also induced an increase in the expression of M1 markers in the decidual macrophages, yet this effect was minimal in myometrial macrophages (Fig. 5C). However, RU486 did not dramatically affect the expression of M1 markers by decidual or myometrial macrophages (Fig. 5C). Furthermore, we found that αCD3ε induced the upregulation of some M2 markers by decidual and myometrial macrophages (Fig. 5D). LPS had a similar effect, but RU486 did not drastically affect the expression of M2 macrophage markers in the decidua and myometrium (Fig. 5D). Together, these results show that T-cell activation induces the polarization of M1 and M2 macrophages in the decidua and myometrium, yet favors their pro-inflammatory phenotype at the maternal-fetal interface.

Neutrophils are central players in the acute innate immune responses related to preterm labor associated with intra-amniotic inflammation/infection (116–121). Indeed, an influx of neutrophils in the decidua and myometrium is observed prior to LPS-induced preterm labor/birth (32, 122). Consistently, we showed that LPS caused increased neutrophil infiltration in the decidua and myometrium (Fig. 5E). However, such a neutrophilic response was not observed upon αCD3ε or RU486 injection (Fig. 5E). These data are relevant since they suggest that in vivo T-cell activation induces preterm labor in the absence of an augmented neutrophil infiltration at the maternal-fetal interface and myometrium, indicating that it is a distinct inflammatory process from that induced by bacteria.

Taken together, these data demonstrate that in vivo T-cell activation induces preterm labor and birth by initiating B-cell cytokine responses and inducing macrophage pro-inflammatory polarization; yet, these inflammatory responses are independent of an increased neutrophil infiltration at the maternal-fetal interface and myometrium.

In vivo T-cell activation induces local and systemic maternal pro-inflammatory responses and the contractility pathway prior to preterm birth

Preterm labor is characterized by the upregulation of inflammatory mediators in the decidua (34, 123–126), myometrium (34, 127–129), and cervix (130). Such inflammatory mediators include cytokines (34, 126), chemokines (126, 131, 132), adhesion molecules (133, 134), and inflammasome components (135, 136). Quantitative RT-PCR profiling revealed that both αCD3ε and LPS caused the upregulation of several inflammatory mediators in the decidua and myometrium (Fig. 6A&B). However, there were subtle differences between the genes upregulated by αCD3ε and LPS (Fig. 6C&D). For example, whereas chemokines (Ccl2, Ccl5, Ccl17, Ccl22, Cxcl9, and Cxcl10), cytokines (Il6 and Ifng), T-cell activation molecules (Ctla4, Pdcd1 and Sell) and inflammasome components (Pycard, Nod1, and Casp1) were upregulated in both the αCD3ε and LPS models, Il1b was only upregulated in the LPS model in the decidual tissues (Fig. 6C). In the myometrial tissues, both αCD3ε and LPS caused the upregulation of Ccl2, Ccl5, Ccl17, Ccl22, Cxcl9, Cxcl10, Ifng, Pdcd1, Nod1, and Casp-1 but only LPS induced the upregulation of Il6 (Fig. 6D). Most of the inflammatory genes in the decidua and myometrium were unchanged upon RU486 injection (Fig. 6C&D), confirming that this is a non-inflammatory model of preterm birth (137).

A systemic inflammatory response is associated with preterm labor in the context of clinical chorioamnionitis (138, 139) and acute pyelonephritis (140, 141). Hence, systemic inflammatory responses were determined by measuring inflammatory mediators in the maternal serum from each of the preterm birth models. Anti-CD3ε and LPS induced a systemic maternal inflammatory response as evidenced by the upregulation of IL6, IL18, IL17A, IL4, IL5, CCL5, CXCL10, G-CSF, and IFNγ in the maternal serum (Fig. S3). However, RU486 did not induce such an effect (Fig. S3).

A hallmark of preterm labor is the presence of myometrial contractions that result from the harmonious induction of uterine activation proteins such as prostaglandins and connexin-43 (142). We therefore determined whether the administration of αCD3ε upregulated the expression of contractility-related genes in the decidua and myometrium. An upregulation of contractility-related genes was observed upon injection of αCD3ε, LPS, and RU486 in both the decidua and myometrium (Fig. 6A&B). Yet, this upregulation was more similar between the αCD3ε and LPS stimuli. These results indicate that in all three models of preterm birth the common pathway of parturition takes place.

These findings show that in vivo T-cell activation induces local and systemic maternal pro-inflammatory responses which coincide with activation of the contractility pathway prior to preterm birth, resembling those observed in the microbial inflammation model.

In vivo T-cell activation induces intra-amniotic inflammation and impacts fetal growth prior to preterm birth

It is well documented that preterm labor, either in the context of infection or sterile inflammation (12, 143, 144), is accompanied by increased amniotic fluid concentrations of pro-inflammatory cytokines and chemokines (145). We therefore evaluated cytokine and chemokine concentrations in amniotic fluid prior to preterm birth. Anti-CD3ε induced a massive pro-inflammatory response in the amniotic cavity, which was even more severe than that generated by LPS (Fig. 7). For example, αCD3ε but not LPS induced the upregulation of IL9, IL10, IL17A, IL23, and IL28 (Fig. 7). Yet, both αCD3ε and LPS caused the upregulation of IL6, CCL3, CCL5, CXCL5, and G-CSF (Fig. 7). RU486-induced preterm labor/birth, however, occurred in the absence of elevated amniotic fluid cytokines, except for IL-23 (Fig. 7). These data indicate that in vivo T-cell activation induces an intra-amniotic inflammatory response, which is stronger than that induced by a microbial product.

Fig. 7. — Dams were injected with αCD3ε (or isotype control), LPS (or PBS control), or RU486 (or DMSO control). Concentrations of IL6, IL9, IL10, IL17A, IL23, IL28, CCL3, CCL5, CXCL5, CXCL10, G-CSF, and GM-CSF in amniotic fluid were determined using a cytokine multiplex assay (n=5 each). The p-values were determined by 2-tailed Mann-Whitney U-test. Data are shown as scatter plots (median).

Increased concentrations of amniotic fluid cytokines and chemokines are associated with fetal damage (146–149), indicating that inflammation can negatively impact the offspring (150, 151), namely fetal growth restriction (152). Thus, we followed up our amniotic fluid cytokine determinations with the evaluation of fetal growth parameters. Fetuses born to dams injected with αCD3ε and LPS were smaller and leaner than their respective controls, but no differences were found in the RU486 group (Fig. 8A&B). We also examined the appearance of the fetal lungs since intra-amniotic pro-inflammatory responses can cause fetal lung damage (153, 154) (Fig. 8C). In the three preterm birth models, the fetal lungs appeared different than those of controls (Fig. 8C). To complement these observations, we performed histological evaluation of the preterm and term neonatal lungs. Consistent with what is reported in humans (155), lungs from preterm neonates appeared morphologically immature (i.e. canalicular stage (156)) compared to those from term neonates (i.e. terminal sac stage (156)) (Fig. 8D). No evident differences in the architecture of the preterm neonatal lungs were observed between preterm birth models (data not shown).

Fig. 8. — (A) Fetuses (with their placentas) from dams injected with αCD3ε, LPS, or RU486 or their respective controls prior to preterm birth. Data are representative of individual litters (n=3 each). (B) Weights of fetuses from dams injected with αCD3ε, LPS, or RU486 or their respective controls prior to preterm birth (n=7-10 each). The p-values were determined by 2-tailed unpaired t-test. Data are shown as scatter plots (mean ± SEM). (C) Fetal lungs collected from dams injected with αCD3ε, LPS, or RU486 or their respective controls prior to preterm birth (n=3 each). (D) Hematoxylin and eosin (H&E) and Masson’s trichrome staining of lungs from preterm and term neonates (n=3 each). Magnification 40x.

As a whole, these findings show that in vivo T-cell activation induces an intra-amniotic pro-inflammatory response that can negatively impact the fetus, causing fetal growth restriction prior to preterm birth which is similar to what occurs in fetuses exposed to microbial products and extends into the neonatal period. Therefore, finding a strategy to treat the adverse effects of maternal T-cell activation during pregnancy was the next logical step.

In vivo T-cell activation-induced preterm labor and birth is prevented by treatment with progesterone

Parturition in animals (157), and likely in humans (158), is associated with a functional progesterone (P4) withdrawal. Thus, the administration of this steroid hormone is clinically used to prevent preterm birth (159–167). Therefore, we tested whether in vivo T-cell activation could induce a systemic withdrawal of P4 and whether its administration could prevent preterm labor/birth. Anti-CD3ε induced a drop in the systemic concentration of P4 (Fig. 9A). Thus, dams received P4 prior to T-cell activation, as shown in the treatment diagram (Fig. 9B). Strikingly, T-cell activation-induced preterm labor/birth was entirely prevented by treatment with P4, which was translated to a longer gestational length (Fig. 9C&D). Importantly, neonates born to dams injected with αCD3ε and treated with P4 had reduced mortality at birth compared to those injected with αCD3ε and the vehicle (sesame oil or SO) (Fig. 9E). Representative images showed that neonates born to dams injected with αCD3ε and treated with P4 are comparable in size to controls (SO + isotype and P4 + isotype) (Fig. 9F). Indeed, neonates born to dams injected with αCD3ε and treated with P4 thrived as indicated by the presence of the milk band which was not observed in those pups born to untreated dams who died shortly after delivery (Fig. 9G, rectangles).

Fig. 9. — (A) Systemic progesterone (P4) concentration in dams injected with αCD3ε (or isotype control) (n=10-11 each). The p-value was determined by 2-tailed Mann-Whitney U-test. (B) Scheme of treatment with P4: dams were treated with either P4 or sesame oil (SO), injected with either αCD3ε or isotype control and video monitored until delivery(n= 5-10 each). (C) The rate of preterm birth in dams injected with SO+Isotype, SO+αCD3ε, P4+Isotype or P4+αCD3ε (n=5-10 each). The p-values were determined by 2-tailed Fisher’s exact test. (D) Gestational length and (E) the rate of neonatal mortality from pups born to dams injected with SO+Isotype, SO+αCD3ε, P4+Isotype or P4+αCD3ε (n=5-10 each). The p-values were determined by (D) Mantel-Cox test (all comparisons p≤0.001) or (E) 2-tailed Fisher’s exact test. (F) Representative images of neonates born to dams injected with SO+Isotype, SO+αCD3ε, P4+Isotype or P4+αCD3ε (n=3 each). (G) Representative images of neonates born to dams injected with SO+αCD3ε (left) or P4+αCD3ε (right). Red dashed rectangle indicates the location of the milk band (n=3 each).

It is well established that progesterone prevents preterm birth by exhibiting anti-inflammatory effects at the maternal-fetal interface, myometrium, and the cervix (168–170). Therefore, we next evaluated the global anti-inflammatory effect of progesterone in dams injected with αCD3ε. Targeted qRT-PCR profiling showed that, compared to control mice (SO+Isotype), treatment with P4 downregulated the expression of several inflammatory mediators induced by αCD3ε in the decidua and cervix, but such an effect was not as strong in the myometrium (Fig. 10A-C). For example, dams treated with P4 + αCD3ε had reduced expression of Casp11, Ccl22, Icam1, Ctla4, Nod1, and Ccl5 in the decidua compared to dams injected with SO + αCD3ε (Fig. 10D). In the myometrium, only Il33 was downregulated upon P4 + αCD3ε treatment (Fig. 10B). In addition, dams treated with P4 + αCD3ε had reduced expression of Il33, Il6, Il12b, Il1a, Pycard, and Il4 in the cervical tissues compared to those injected with SO + αCD3ε (Fig. 10E).

Collectively, these findings provide further evidence that progesterone attenuates local inflammatory responses at the maternal-fetal interface and in the cervix, preventing T-cell activation-induced preterm labor and birth, which translates to reduced adverse neonatal outcomes.

DISCUSSION

The study herein presents evidence that effector/activated maternal T cells lead to pathological inflammation and subsequently preterm labor and birth. Specifically, we showed that effector memory and activated maternal T cells expressing granzyme B and perforin are enriched at the maternal-fetal interface of women with spontaneous preterm labor and birth. Next, using a murine model, we reported that prior to inducing preterm birth, in vivo T-cell activation causes maternal hypothermia, bradycardia, systemic inflammation, and cervical dilation, all of which are clinical signs associated with preterm labor. We also found that prior to preterm birth, in vivo T-cell activation induces B-cell cytokine responses, a pro-inflammatory macrophage polarization, and the upregulation of inflammatory mediators at the maternal-fetal interface and myometrium, in the absence of an increased influx of neutrophils. Moreover, we showed that in vivo T-cell activation triggers an intra-amniotic inflammatory response and causes fetal growth restriction prior to preterm birth. Lastly, we provided evidence that treatment with progesterone could serve as an anti-inflammatory strategy to prevent preterm birth and adverse neonatal outcomes induced by T-cell activation (Fig. 11).

Fig. 11. — **Maternal** effector and activated T cells expressing granzyme B and perforin can induce pathologic inflammation by initiating local immune responses at the maternal-fetal interface (decidua) (i.e. activation of B cells and an M1-like macrophage polarization without an increased influx of neutrophils) which, in turns, leads to preterm labor and birth. Activation of T cells also induces inflammatory responses in the maternal circulation and the amniotic cavity, inducing fetal damage prior to preterm labor and birth. These effects can be abrogated by treatment with the anti-inflammatory and clinically approved strategy, progesterone.

We and others have shown that effector memory and/or activated T cells can be recruited by (57, 171), and are present at (58, 59, 172–179), the human maternal-fetal interface in term pregnancy. However, providing a role for these T cells in the pathogenesis of pregnancy complications has been challenging because of the absence of the disease (e.g. preterm labor). Herein, we report that effector memory maternal T cells are enriched in the decidual tissues of women with spontaneous preterm labor, who delivered preterm. Such an increase was not observed in patients who underwent the physiological process of labor at term, indicating that these effector cells contribute solely to the pathological process of premature labor. Such T-cell responses could be antigen-dependent or antigen-independent; the latter could be driven by cytokines (85, 180). The conventional belief is that effector memory T cells recognized placental-fetal antigens (47, 50, 178, 181); yet, we suggest that both antigen-dependent and antigen-independent processes may occur at the human maternal-fetal interface during spontaneous preterm labor. In line with this concept, CD8+ T_EMRA cells, lymphocytes with high cytolytic activity in the absence of in vitro pre-stimulation and that can proliferate in an antigen-independent manner (84, 182), were more abundant in the decidual tissues of women with spontaneous preterm labor compared to those who delivered preterm in the absence of labor.

Effector CD4+ and CD8+ T cells express high levels of granzyme B and perforin, which are stored in cytolytic granules and upon activation are released towards the target cell (183–186). Recently, it was shown that term-isolated decidual CD8+ T cells can degranulate, proliferate and produce inflammatory mediators upon in vitro stimulation (179), suggesting a role for these cells in pregnancy complications. Herein, we show that both decidual CD4+ and CD8+ T cells can express increased levels of granzyme B and perforin in women with spontaneous preterm labor, providing conclusive evidence that decidual T cells exhibit an effector and pro-inflammatory phenotype during the pathological process of premature parturition.

To investigate the mechanisms whereby effector T cells lead to spontaneous preterm labor, we used a murine model of transient in vivo T-cell activation - the injection of the hamster αCD3 monoclonal antibody (72, 187). In vivo T-cell activation at the maternal-fetal interface and myometrium was proven by the downregulation of the CD3 molecule (188–190), the upregulation of activation markers (e.g. CD25 (104), CD69 (105), CTLA-4 (108), PD-1 (109)) in CD4+ and CD8+ T cells, and increased proportions of CD4+ T cells expressing IL-2 (191). In vivo T-cell activation also induced an increase in the proportion of IFNγ-expressing CD8+ T cells in the myometrium but not in the decidua. This is consistent with in vitro studies showing that CD3 stimulation triggers the expression of IFNγ by CD8+ T cells (110). IFNγ-expressing CD8+ T cells were also increased upon LPS injection in the decidua but not in the myometrium, suggesting that T cells respond differently in each anatomical compartment. The abundance of IFNγ-expressing CD8+ T cells in the myometrium, but not in the decidua, could be explained by the fact that this cytokine is implicated in myometrial function, including contractility (192, 193). Moreover, in vivo T-cell activation tended to increase the proportion of IFNγ-expressing B cells in the decidua and myometrium, resembling type 1 B-cell responses (112), indicating that CD3 stimulation induces preterm birth by boosting both T-cell and B-cell responses at the maternal-fetal interface and myometrium.

In vivo T-cell activation induced hypothermia and bradycardia prior to preterm birth. These conditions were likely due to the cytokine storm and lymphatic T-cell activation induced by the injection with αCD3ε (92, 187). Such systemic responses also occurred in mice injected with LPS, resembling a stage of endotoxemia (74, 78, 98). In contrast, dams injected with the progestin antagonist (RU486) did not present hypothermia or bradycardia, consistent with the absence of a systemic cytokine response. Yet, cervical dilation and upregulation of contractility-related genes in decidual and myometrial tissues was observed upon injection of αCD3ε, LPS, and RU486, indicating that in vivo T-cell activation triggers the common pathway of parturition. These data show that the in vivo activation of T cells induces preterm labor and birth by initiating systemic and local pathophysiological processes, which partially resemble those induced by microbial products but are different from those caused by the blockage of progesterone action.

In vivo T-cell activation also had unique effects on innate immune cells at the maternal-fetal interface and myometrium. Previous studies have shown that macrophage activation is complex and does not tally with the M1/M2 polarization model (194). Similarly, at the human maternal-fetal interface, macrophage phenotypes do not entirely fit the M1/M2 polarization model (195, 196); yet, these cells possess a pro-inflammatory phenotype (M1-like) in women who underwent spontaneous preterm labor (33). Consistently, we found that, prior to in vivo T-cell activation-induced preterm birth, a predominantly pro-inflammatory (M1-like) macrophage polarization was observed at the murine maternal-fetal interface and myometrium. In this context, a pro-inflammatory macrophage polarization could have been driven by cytokines released upon T-cell activation (197). Importantly, in vivo T-cell activation did not cause an increase in the infiltration of neutrophils in the decidua and myometrium prior to preterm birth. Such an increase in neutrophils has been consistently observed in the context of preterm labor/birth associated with intra-amniotic infection (32, 122, 198–200) but not in the anti-progestin model (198, 199). Thus, we surmise that in vivo T-cell activation induces preterm birth by initiating immune responses that are partially different from those triggered by microbes and distinct from those caused by anti-progestins. In addition, these results indicate that the model of in vivo T-cell activation-induced preterm birth occurs in the absence of neutrophilic infiltration, which is distinct from the most commonly studied cause of preterm labor/birth, intra-amniotic infection (8, 201, 202).

Yet, in vivo T-cell activation-induced preterm labor/birth was accompanied by the upregulation of inflammatory mediators at the maternal-fetal interface and myometrium as well as in the maternal circulation and amniotic cavity. These local (10, 124, 125, 139, 145, 203) and systemic (204–206) immune responses have been frequently observed in women who undergo spontaneous preterm labor with intra-amniotic infection and/or inflammation. Therefore, the in vivo T-cell activation-induced preterm labor/birth model shares the local and systemic inflammatory responses with the microbial-induced preterm labor/birth model. The severe inflammatory responses in the amniotic cavity explained the deleterious effect observed in fetuses (fetal growth restriction) born to dams injected with αCD3 and LPS. In contrast, the RU486-induced preterm labor/birth model is characterized by the absence of local and systemic inflammation, but the fetal lungs are hemorrhagic that could be due to the adverse effects of this anti-progestin (207).

In vivo T-cell activation-induced preterm labor/birth was prevented by treatment with progesterone. Importantly, the adverse neonatal outcomes induced upon T-cell activation were also ameliorated. Indeed, most of the neonates born to dams injected with αCD3 and treated with progesterone thrived. The protective effects of progesterone during pregnancy have been previously shown to be mediated, at least in part, by modulating T-cell responses (208–210). Indeed, we have previously shown that progesterone prevents endotoxin-induced preterm birth by fostering an anti-inflammatory response at the maternal-fetal interface, characterized by fewer effector T cells, activated macrophages, and neutrophils, as well as increased regulatory T cells (168). The anti-inflammatory effects of progesterone were reflected in the cervical tissues as well (168). Progesterone also has anti-inflammatory effects in the systemic circulation and myometrium (168, 170). Herein, we provide further evidence of the anti-inflammatory effects of progesterone. Specifically, we show that progesterone attenuated the local pro-inflammatory responses at the maternal-fetal interface and the cervix. Of interest, progesterone dampened genes related to the inflammasome pathway (e.g. Casp11, Pycard, Nod1), which has been strongly associated with the mechanisms initiating the pathological process of preterm labor (20, 135, 136, 211–214). Collectively, these results demonstrate that preterm labor/birth and adverse neonatal outcomes induced by T-cell activation can be treated by using the anti-inflammatory effects of progesterone, a clinically approved therapy.

It is worth mentioning that, following T-cell activation, progesterone did not strongly impact the expression of inflammatory genes in the myometrium, except for upregulating the expression of Il33. The expression of this cytokine, however, was attenuated in the cervix, indicating that each anatomical compartment is differentially regulated by progesterone. This concept is in line with our initial findings indicating that T-cell activation and LPS trigger different responses in the decidua and myometrium. This last observation provides an example of the complexity of immune interactions at the maternal-fetal interface and in the reproductive tissues: each compartment must regulate specific mediators which contribute to the complex phenomenon of parturition.

A central question that remains unanswered is what causes T-cell activation leading to spontaneous preterm labor and birth in women. The most tempting explanation is that placental-fetal antigens induce the activation of maternal T cells in the systemic circulation and/or at the maternal-fetal interface. These antigens could have been presented by APCs in the spleen and uterine-draining lymph nodes and/or at the maternal-fetal interface (67, 215). However, such a hypothesis is difficult to prove in humans (67). A second possibility is that effector memory T cells are not specific for placental-fetal antigens, but instead they recognize microbe-derived peptides which are normally present in the basal plate of the placenta (216) and/or the endometrium (217). Such T cells can then proliferate in an antigen-independent manner by pro-inflammatory mediators (180) induced by dangers signals or alarmins in the intra-amniotic space (i.e. sterile intra-amniotic inflammation) (145). This concept is supported by the fact that most cases of preterm labor take place in the setting of sterile intra-amniotic inflammation (12, 143), which can occur in the absence of acute histologic chorioamnionitis [neutrophil infiltration in the chorioamniotic membranes (218)] (143, 219). A third possibility is that effector memory T cells at the maternal-fetal interface are virus-specific; however, a clear association between viral infections and human preterm labor requires further epidemiological studies. In this latter context, it has been suggested that maternal-fetal tolerance is compromised by the virus and polymicrobial infections can take place, inducing preterm labor and birth (21, 220–222). Further research is required to investigate the antigen specificity and clonality of effector memory T cells at the maternal-fetal interface in the context of preterm labor and birth.

Supplementary Material

NIHMS1523035-supplement-1.pdf^{(637.8KB, pdf)}

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 also thank George Schwenkel, Yaozhu Leng, Hong Meng, Ronald Unkel, Ryan Cantarella, Pablo Silva, Chengrui Zou, Jennifer Pierluissi, and Yang Jiang, for their help with carrying out some of the experiments and/or crucial reading of the manuscript.

Footnotes

This research was supported, in part, by the Perinatology Research Branch (PRB), 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 the 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.

Non-standard abbreviations: CCL2, C-C motif chemokine ligand; CXCL, C-X-C motif chemokine ligand; G-CSF, colony stimulating factor 3; dpc, days post coitum; P4, progesterone; PTL, preterm labor; PTNL, preterm no labor; SO, sesame oil; TIL, term in labor; TNL, term no labor; αCD3, anti-CD3

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PERMALINK

Effector and Activated T cells Induce Preterm Labor and Birth that is Prevented by Treatment with Progesterone1

Marcia Arenas-Hernandez

Roberto Romero

Yi Xu

Bogdan Panaitescu

Valeria Garcia-Flores

Derek Miller

Hyunyoung Ahn

Bogdan Done

Sonia S Hassan

Chaur-Dong Hsu

Adi L Tarca

Carmen Sanchez-Torres

Nardhy Gomez-Lopez