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ORIGINAL ARTICLE
Year : 2021 | Volume
: 5
| Issue : 3 | Page : 129-139
A defective CXCL16/CXCR6 axis increases the risk of pregnancy loss via the abnormal crosstalk between decidual γδ t cells and trophoblasts
Deng-Xuan Fan1, Ming-Qing Li2, Wen-Jie Zhou3, Hong-Lan Huang1, Hui-Li Yang4, Cong-Jian Xu5
1 Department of Gynecology, Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200011; Department of Obstetrics and Gynecology of Shanghai Medical School, Fudan University, Shanghai, 200011, China
2 Department of Gynecology, Obstetrics and Gynecology Hospital, Fudan University; Department of Obstetrics and Gynecology of Shanghai Medical School, Fudan University; Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Fudan University, Shanghai, 200011; NHC Key Lab of Reproduction Regulation (Shanghai Institute of Planned Parenthood Research), Hospital of Obstetrics and Gynecology, Fudan University, Shanghai, 200080, China
3 Department of Obstetrics and Gynecology, Reproductive Medical Center, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China
4 NHC Key Lab of Reproduction Regulation (Shanghai Institute of Planned Parenthood Research), Hospital of Obstetrics and Gynecology, Fudan University, Shanghai, 200080, China
5 Department of Gynecology, Obstetrics and Gynecology Hospital, Fudan University; Department of Obstetrics and Gynecology of Shanghai Medical School, Fudan University; Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Fudan University, Shanghai, 200011, China
Correspondence Address:
Cong-Jian Xu
Obstetrics and Gynecology Hospital, Fudan University, No. 419 Fangxie Road, Shanghai 200011
China
Source of Support: None, Conflict of Interest: None
DOI: 10.4103/2096-2924.324878
Objective: The maternal–fetal interface undergoes dynamic changes to allow the fetus to grow and develop in the uterus. The interaction between decidual γδ Τ cells and trophoblasts plays a pivotal role during successful pregnancy; however, their physiological functions in early-term human pregnancy are still not completely illustrated. This study was undertaken to illustrate the functional roles of CXCL16/CXCR6 to prevent pregnancy loss via the crosstalk between decidual γδ T cells and HTR8/SVneo trophoblast cells.
Methods: The percentile of CXCR6+ γδ T cells in the peripheral blood from normal female and recurrent spontaneous abortion (RSA) patients was analyzed by flow cytometry. The expression of CXCR6 was detected in decidual immune cells via flow cytometry, and the expression of CXCL16 was analyzed in HTR8/SVneo trophoblast cells and lentivirus (LV)-HTR8/SVneo trophoblast cells via enzyme-linked immunosorbent assay. Reverse transcriptase-polymerase chain reaction was used to verify the expression of the CXCL16 gene in LV-HTR8/SVneo trophoblast cells. Expression of granzyme B and cytokines and proliferation of decidual γδ T cocultured with HTR8/SVneo trophoblast cells were analyzed by flow cytometry. Invasion of HTR8/SVneo trophoblast cells was assessed via Matrigel transwell assay. Adoptive transfer was induced in vivo further to illustrate that the normal expression of CXCL16/CXCR6 could prevent pregnancy loss.
Results: The percentile of CXCR6+ γδ T cells in the peripheral blood from RSA patients was lower than normal pregnancies. The expression of CXCR6 was highest in the decidual γδ T cells among decidual immune cells, and the expression of CXCL16 increased as the amount of HTR8/SVneo trophoblast cells increased. Expression of granzyme B in the decidual γδ T cells was downregulated by cocultured with HTR8/SVneo cells dependent of CXCL16, and HTR8/SVneo trophoblast cells induced the Th2 cytokines production in the decidual γδ T cells. Both the expression of CXCR6 in the decidual γδ T cells and proliferation of the decidual γδ T cells were promoted by HTR8/SVneo trophoblast cells. On the other hand, decidual γδ T cells enhanced the invasion of HTR8/SVneo trophoblast cells and thus promoted embryo implantation. In vivo study was taken further and shown that low expression of CXCL16/CXCR6 results in pregnancy loss because of dialog disorder between decidual γδ Τ cells and trophoblasts.
Conclusions: Low expression of CXCL16/CXCR6 results in pregnancy loss because of the dialog disorder between decidual γδ Τ cells and trophoblasts, and it showed a light on the effective strategy of adoptive transfer of CXCR6+ γδ T cells on the treatment of RSA. This observation provides a scientific basis on which a potential strategy can be applied to the early-detect and treatment of RSA.
Keywords: CXCL16; CXCR6; Decidual γδ T Cells; Maternal–fetal Interface; Recurrent Spontaneous Abortion; Trophoblasts
Successful pregnancy is considered as a semi-allogenic process. According to the American Society for Reproductive Medicine, recurrent spontaneous abortion (RSA) is defined by two or more failed pregnancies in the first 20 weeks of pregnancy, and up to 50% of cases do not have a clearly defined etiology.[1] The understanding of the complex relationship between the mother and its semi-allograft fetus is still limited.[2] During the successful pregnancy, the mother needs immune responses against infection but is tolerant to the fetus. Any abnormalities of the immune tolerance would lead to pregnancy loss, such as RSA and miscarriage.[3],[4]
Decidual γδ T cells participate to maintain successful pregnancy by recognizing alloantigen without MHC restriction, bridging the gap between innate and adaptive immunity, and secreting Th2 cytokines mainly.[5],[6],[7],[8],[9] Many studies have shown that the balance of Th1/Th2 cells is critical for maternal immune tolerance and pregnancy maintenance. Th2 cytokines are of great significance in promoting embryo implantation, downregulating cell-mediated immune response to maintain pregnancy.[10]
At the maternal–fetal interface, there are maternal-derived decidua containing decidual immune cells and fetus-derived trophoblasts, and a complicated crosstalk upon chemokines and chemokine receptors among them plays a pivotal role during successful pregnancy. Chemokines with chemokine receptors participate in the physical and pathological progress, including inflammatory activities and immunity, embryo development, metastasis, and invasion of malignant tumors.[11] CXCL16 and CXCR6 expressed at the maternal–fetal interface, as one pair of chemokine and chemokine receptor, play a role in activating PI3K/PDK1/AKT/cyclin D1 pathway and thus enhance endometrium decidualization.[12]
To gain a better understanding of the underlying immunity mechanism of RSA, we highlighted the advanced studies on the roles of CXCL16 and CXCR6 in pregnancy and outlined an overview role of its application in the early-detect and treatment of RSA.
MethodsSample collection
First-trimester human villus and decidual tissues were collected from clinically normal pregnant women and RSA pregnant women undergoing abortion in the Obstetrics and Gynecology Hospital of Fudan University between November 2019 and 2020. The RSA group comprised 10 women with a history of two to six spontaneous miscarriages at first trimester (7–10 weeks gestation) that had not previously been investigated. Their average number of abortions was 3.28 ± 1.18, and their mean age was 31.2 ± 0.87 years. RSA was diagnosed after excluding any verifiable causes, including infection, endocrine or metabolic disease, chromosomal abnormality, anatomic deformation, and autoimmune response. All patients had a single male partner for the pregnancies under investigation. All patients and their male partners had normal karyotypes, and all male partners had a normal semen status according to the criteria from the World Health Organization. The control group comprised 30 randomly selected women who underwent a legal termination for nonmedical reasons (7–10 weeks gestation) at the same facility during the same period. The mean age of the control group was 26.13 ± 3.19 years. All control subjects had one or two living children and no history of spontaneous abortion, ectopic pregnancy, preterm delivery, or stillbirth. In all cases, fetal heart activity was verified within 2 weeks of abortion and the embryonic karyotype was normal. There was no significant difference in age or gestation between the RSA and control groups. Written informed consent was obtained from all patients before sampling. The project protocol was approved by the Research Ethics Committee of Obstetrics and Gynecology Hospital of Fudan University (2018-2).
Cell culture
HTR8/SVneo cells, first-trimester–derived trophoblast cell line, were obtained from Cell Bank of Chinese Academy of Sciences (Shanghai, China). HTR8/SVneo cells were cultured in DMEM/F12 complete medium supplemented with 10% FBS (FBS, Gibco, USA) and maintained in 5% CO2 at 37°C. Cells were detached by routine trypsinization every 3–4 days.
Enzyme-linked immunosorbent assay
HTR8/SVneo cells and lentivirus (LV)-HTR8/SVneo cells, in different concentrations of 2 × 104 cells/well, 1 × 105 cells/well, and 5 × 105 cells/well, were cultured for 48 h, respectively. The culture supernatants were collected and centrifuged to remove cellular debris and stored at −80°C for CXCL16 protein measurements. The secretion of CXCL16 in the cultured supernatant samples was determined by enzyme-linked immunosorbent assay (ELISA) using commercially available kits (R&D Systems, Minneapolis, MN, USA), according to the manufacturer's instructions. The ELISA data were standardized to the total protein levels of the cell lysates.
Lentiviral transfection
HTR8/SVneo cells were seeded in a six-well plate (5 × 104 cells/mL). The cells in the plate were infected with lenti-CXCL16-sgRNA-CAS9 virus and their corresponding negative control (NC) virus (Genechem Co. Ltd., Shanghai, China). According to the manufacturer's instructions, the optimal transfection condition of the multiplicity of infection (MOI) was 80% of infected cells in the optimal time.
Decidual γδ T cells enrichment
Decidual mononuclear cells were obtained from the decidual tissue of normal pregnancies by collagenase Type IV (1.0 mg/mL, Sigma-Aldrich, USA) and DNase I (150 U/mL, AppliChem, Darmstadt, Germany) as described previously. The γδ T lymphocytes were enriched from the isolated decidual mononuclear cells via magnetic isolation kit (positive selection) (Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, the isolated decidual mononuclear cells were suspended in phosphate-buffered saline/ethylenediaminetetraacetic acid/bovine serum albumin (PBS/EDTA/BSA) buffer containing 0.5% BSA and 2 mmol/L EDTA. The cells were first labeled with hapten-conjugated monoclonal antibodies directed against TCR γ/δ, followed by conjugate binding with antihapten microbeads. After incubation for 15 min at 4°C, the cells were resuspended in PBS/EDTA/BSA buffer and washed twice and passed through a magnetic separation column. After the column was removed from the magnetic field, the magnetically retained TCR γ/δ cells were eluted as the positively selected cell fraction, representing the enriched T cell fraction. The purity of the enriched γδ T cells was evaluated by CytoFLEX flow cytometry (Beckman Coulter, USA). The purity of the isolated cells was above 90%.
CXCR6 expression-level detection
Decidual mononuclear cells were collected for CXCR6 detection in the decidual γδ T cells, macrophage, natural killer (NK) cells, and NKT cells by flow cytometry. Decidual γδ T cells were collected and cocultured with HTR8/SVneo cells and LV-HTR8/SVneo cells, respectively, for 48 h; CXCR6+ decidual γδ T cells were analyzed by flow cytometry.
Cell viability and proliferation assay
Decidual γδ T cells were isolated according to the protocols described in the previous section. Decidual γδ T cells were seeded in triplicate in density of 2 × 105 cells/well in 24-well plates, which were treated with 100 ng/mL recombination human CXCL16 (rhCXCL16), HTR8/SVneo cells at a density of 3 × 104 cells/well, LV-HTR8/SVneo cells at a density of 3 × 104 cells/well, respectively. The cell viability and proliferation were detected by CFSE according to the procedure of CellTrace CFSE Cell Proliferation Kit (Cat. No. C34554, Invitrogen, USA). Additionally, cell proliferation was detected by Ki-67 expression by flow cytometry.
Interferon γ/perforin/granzyme B expression analysis
To study the functional effects of HTR8/SVneo cells cocultured with decidual γδ T cells on the cytotoxicity of decidual γδ T cells, the expression levels of interferon (IFN)-γ, perforin, and granzyme B were detected. Decidual γδ T cells in 2 × 105 cells/well were seeded in 24-well plates and treated with 100 ng/mL rhCXCL16, HTR8/SVneo cells in 3 × 104 cells/well, and LV-HTR8/SVneo cells in 3 × 104 cells/well, respectively. The collected γδ T cells were used for IFN-γ, perforin, and granzyme B expression assessment by flow cytometry analysis.
Cytokine production detection
To verify the functional effects of HTR8/SVneo cells on the cytokine production in decidual γδ T cells, 2 × 105 decidual γδ T cells were seeded in 24-well plates and treated with 100 ng/mL rhCXCL16, HTR8/SVneo cells in 3 × 104 cells/well, LV-HTR8/SVneo cells in 3 × 104 cells/well, respectively. Then, the γδ T cells were collected and used for expression level evaluation of a panel of six cytokines (interleukin [IL]-4, IL-5, IL-10, IL-17A, transforming growth factor [TGF]-β, tumor necrosis factor [TNF]-α) by flow cytometry analysis.
Decidual γδ T cells enhance human trophoblast invasion via CXCL16/CXCR6
To analyze the effects of decidual γδ T cells on the invasion of human trophoblasts, a Matrigel-based transwell assay was carried out. Trophoblasts were added in the upper chamber, and the number of cells migrating to the lower surface was counted after incubating the cells in a 95% O2/5% CO2 incubator for 48 h. As shown in figure, decidual γδ T cells increased human trophoblast invasion. This effect can be partially inhibited by emitting CXCL16. Our observations suggest that the γδ T cells enhance trophoblasts invasion might be partially dependent on the chemokine of CXCL16.
Quantitative real-time polymerase chain reaction
Total RNA was extracted using TRIzol reagent (TaKaRa Bio Inc., Tokyo, Japan), and 1 μg of total RNA was used to synthesize the first-strand cDNA with the PrimeScript™ invasive index (II) First Strand cDNA Synthesis Kit (TaKaRa Bio Inc., Japan) using random or oligo-dT primers. Thereafter, quantitative real-time polymerase chain reaction (PCR) was performed using an SYBR Green kit (TaKaRa Bio Inc., Japan), according to the manufacturer's instructions. Primer sequences for all genes are listed in [Supplementary Table 1]. All samples were amplified in triplicate, and the mean was used for analysis. The 2−ΔΔCt method was applied to calculate the relative expression normalized to the internal controls β-actin.
Murine models were obtained for normal pregnancy and abortion-prone mice models
Eight to ten-week-old CBA/J female mice and DBA/2 and BALB/c male mice were purchased from Laboratory Animal Resources, Chinese Academy of Science in Shanghai, China. Experiments were reviewed and all procedures were performed according to an animal protocol. The normal mice model was obtained from CBA/J female mice and BALB/c male mice, while the abortion-prone mice model was obtained from CBA/J female mice and DBA/2 male mice.
Lentiviral transfection in vivo
The murine were infected with lenti-si-CXCR6 virus and their corresponding NC virus (Santa Cruz, sc-39896-v, USA) via tail intravenous injection at the time of a week before mating, according to the manufacturer's instructions.
Adoptive transfer of γδ T cells from normal mice into abortion prone mice
CXCR6+ and CXCR6− γδ T cells were isolated from normal mice model via fluorescence-activated cell sorting and labeled with PKH67, respectively. Then, 200 μL labeled cells suspension at a cell density of 5 × 107/mL were injected intravenously into the tail of abortion-prone mice at day 4 of gestation.
Statistical analysis
All experiments were repeated independently at least three times, and the data were expressed as the mean ± standard deviation. Statistical analysis was performed with GraphPad Prism software Ver. 8.0 (GraphPad Software, San Diego, CA, USA) using the Student's t-test (two-sample assuming unequal variances) and one-way ANOVA. Differences were considered statistically significant at P < 0.05 (two-sided).
ResultsThe expression of CXCR6 and CXCL16 in decidual γδ T cells and HTR8/SVneo cells
To study the interactions between decidual γδ T cells and HTR8/SVneo cells, decidual immune cells, including decidual γδ T cells, were freshly isolated from human decidua. The purity of decidual γδ T cells was verified above 90%, which could be used for the associated experiments [Figure 1]a. Expression profile of decidual γδ T cells, such as CD4, CD8, CD40, CXCR6, NKG2D, PD-1, and CTLA-4, was analyzed by flow cytometry, and CXCR6 was found to be expressed in medium level and CD3 in high level, while CD4 and CD8 were expressed quite a bit [Figure 1]b. Further, the expression of CXCR6 was analyzed in four types of decidual immune cells obtained from human decidua, macrophage, NK cells, NKT cells, and γδ T cells. The results showed that CXCR6 was expressed highest in decidual γδ T cells, while nearly no expression in NK cells [Figure 1]c and [Figure 1]d. Early-term trophoblasts cell lines, HTR8/SVneo (HTR8) cells, were introduced into the study. Furthermore, to exclude potential effects of CXCL16, we knocked out CXCL16 by the LV CRISPR/Cas9 infection system in HTR8/SVneo cells. In our previous study, we have shown the high infection efficiency of LV that the optimal transfection condition of MOI was 80% of infected cells in the optimal time.[13] Then, real-time PCR and ELISA were used to verify the efficiencies of CXCL16 knockdown in HTR8/SVneo cells at mRNA and protein levels, respectively. The results showed that the transcription level of CXCL16 was reduced in LV-HTR8/SVneo cells significantly [Figure 1]e. Expression of CXCL16 increased as the amount of HTR8/SVneo cells and LV-HTR8/SVneo cells increased, while the expression of CXCL16 was reduced in LV-HTR8/SVneo cells significantly [Figure 1]f.
Figure 1: Expression of CXCR6 was highest in decidual γδ T cells among decidual immune cells and the expression of CXCL16 increased as the amount of HTR8/SVneo cells increased. The purity of the decidua γδ T cells is higher than 90% (n = 9) (a). Expression profile of decidual γδ T cells (CD4, CD8, CD40, CXCR6, NKG2D, PD-1, CTLA-4) was analyzed by flow cytometry (b). CD3, CD4, CD8, CD40L, CXCR6, NKG2D, PD-1, and CTLA-4 in decidual γδ T cells were analyzed via flow cytometry with CD3, PD-1, and CTLA-4 expressed highly, expression of CXCR6, NKG2D, and CD40L in middle level, and low expression of CD4 and CD8 (n = 9) (b). Representative images are shown (c) and the expression of CXCR6 in decidual immune cells, such as macrophage, NK cells, NKT cells, and γδ T cells, was analyzed by flow cytometry with a high expression in decidual γδ T cells and almost no expression in NK cells (n = 9) (d). The mRNA level of CXCL16 in the LV-HTR8/SVneo cells and HTR8/SVneo cells, respectively, as analyzed by RT-PCR, the mRNA level of CXCL16 in the LV-HTR8/SVneo cells was reduced by more than 50% (e). CXCL16 protein expression in the supernatant of HTR8/SVneo cells and LV-HTR8/SVneo cells was analyzed and compared via ELISA. As the cell number of HTR8/SVneo cells and LV-HTR8/SVneo cells rising, the expression of CXCL16 protein increased. The expression of CXCL16 protein in the supernatant of LV-HTR8/SVneo cells was lower significantly than that of HTR8/SVneo cells (f). Data were means ± SEM. *P < 0.001, †P < 0.0001, ns: Nonsignificant. SEM: Standard error of mean; RT-PCR: Real-time polymerase chain reaction; ELISA: Enzyme-linked immunosorbent assay.To illustrate the importance of CXCR6+ γδ T cells in maintaining normal pregnancy, the percentile of CXCR6+ γδ T cells in the peripheral blood was analyzed and compared from pregnant, nonpregnant normal females and pregnant, nonpregnant RSA patients. The percentile of CXCR6+ γδ T cells in the peripheral blood of nonpregnancy from normal women and RSA patients was analyzed by flow cytometry, and the representative images are shown in [Figure 2]a. The percentile of CXCR6+ γδ T cells in the peripheral blood from first-trimester normal pregnancy and RSA patients was analyzed by flow cytometry, and the representative images are shown in [Figure 2]b. Compared with normal nonpregnant and pregnant women, respectively, reduced percentile of CXCR6+ γδ T cells was found in the peripheral blood from RSA patients [Figure 2]c. Compared with nonpregnant, increased percentile of CXCR6+ γδ T cells was found in the pregnant cases [Figure 2]c. Therefore, the results showed that reduced CXCR6+ γδ T cells was related to RSA and thus demonstrated the importance of CXCR6+ γδ T cells for successful pregnancy.
Figure 2: Reduced expression of CXCR6 percentile was found in peripheral blood from RSA patients and the non-pregnant. The percentile of CXCR6+ γδ T cells in the peripheral blood of nonpregnancy normal women and RSA patients was analyzed by flow cytometry (n = 10 per group) (a). The percentile of CXCR6+ γδ T cells in the peripheral blood from first-trimester normal pregnancies and RSA patients was analyzed by flow cytometry (n = 10 per group) (b). The analysis of percentile of CXCR6+ γδ T cells in peripheral blood of pregnant normal female and RSA patients was shown (c). Compared with normal nonpregnant and pregnant women separately, reduced percentile of CXCR6+ γδ T cells was found in peripheral blood from RSA patients. Increased percentile of CXCR6+ γδ T cells was found in the pregnant groups. Data were means ± SEM. *P < 0.05, †P < 0.01, ‡P < 0.001, ns: Nonsignificant. RSA: Recurrent spontaneous abortion; SEM: Standard error of mean.Expression of granzyme B in decidual γδ T cells was downregulated by HTR8/SVneo cells dependent of CXCL16 and HTR8/SVneo cells induced the cytokines production in decidual γδ T cells
In order to illustrate the biological function of decidual γδ T cells exerted by HTR8/SVneo cells, the expression of granzyme B, IFN-γ, and perforin of decidual γδ T cells were analyzed by flow cytometry [Figure 3]a, [Figure 3]b, [Figure 3]c. Granzyme B is a serine protease released by CD8+ T cells and NK cells during the cellular immune response, which induces cell apoptosis. HTR8/SVneo cells reduced the expression of granzyme B of decidual γδ T cells, while the expression of granzyme B of decidual γδ T cells increased under the coculture with LV-HTR8/SVneo cells compared with that of HTR8/SVneo cells [Figure 3]a. There was no significant difference in the rhCXCL16 group compared with that of the vehicle group [Figure 3]a. HTR8/SVneo cells increased the expression of perforin of decidual γδ T cells, while the expression of perforin of decidual γδ T cells was increased further under the coculture with LV-HTR8/SVneo cells compared with that of HTR8/SVneo cells [Figure 3]b, which showed that HTR8/SVneo cells derived CXCL16 functioned as an inhibiting ability on the expression of perforin of decidual γδ T cells. The function of HTR8/SVneo cells on the expression of IFN-γ of decidual γδ T cells was the same as that of perforin [Figure 3]c.
Figure 3: Expression of granzyme B in decidual γδ T cells was down-regulated by HTR8/SVneo cells dependent of CXCL16 and HTR8/SVneo cells induced the cytokine production in decidual γδ T cells. Cocultured with HTR8/LV-HTR8/SVneo cells and/or treated with rhCXCL16 for 48 h, respectively. The expression of granzyme B, IFN-γ, and perforin of γδ T cells were analyzed by flow cytometry (a-c). HTR8/SVneo cells reduced the expression of granzyme B in decidual γδ T cells, while the expression of granzyme B of decidual γδ T cells increased under the co-culture with LV-HTR8/SVneo cells compared with that of HTR8/SVneo cells. There was no significant difference in rhCXCL16 group compared with that of the vehicle group (n = 9) (a). Cocultured with HTR8/LV-HTR8 and/or treated with rhCXCL16 for 48 h, respectively, the expression of cytokines (IL-4, IL-10, TGF-β, TNF-α, IL-17A) in decidual γδ T cells was analyzed by flow cytometry (d-h). HTR8/SVneo cells increased the expression of cytokines in decidual γδ T cells, such as IL-4, IL-10, TGF-β, TNF-α, IL-17A, and there was no significant difference between the group of LV-HTR8 and the group of HTR8/SVneo cells. There was no significant difference between rhCXCL16 group and the vehicle group (n = 9) (d-h). Student's t-test was performed for significance testing. Data were means ± SEM. *P < 0.05, †P < 0.01, ‡P < 0.001, §P < 0.001, NS: Nonsignificant. SEM: Standard error of the mean; IL: Interleukin; TNF: Tumor necrosis factor; TGF: Transforming growth factor.Cytokines play a complicated function at maternal–fetal interface during pregnancy; therefore, cytokines in the decidual γδ T cells were analyzed in the study. Cocultured with HTR8/LV-HTR8/SVneo cells and/or treated with rhCXCL16 for 48 h, respectively, the expression of cytokines (IL-4, IL-10, TGF-β, TNF-α, and IL-17A) in decidual γδ T cells was analyzed by flow cytometry. HTR8/SVneo cells increased the expression of cytokines in the decidual γδ T cells, such as IL-4, IL-10, TGF-β, TNF-α, and IL-17A, and there was no significant difference between LV-HTR8/SVneo cells group and HTR8/SVneo cells group. There was no significant difference between the rhCXCL16 group and the vehicle group [Figure 3]d, [Figure 3]e, [Figure 3]f, [Figure 3]g, [Figure 3]h. Therefore, the results demonstrated that HTR8/SVneo cells upregulated the Th2 cytokines in the decidual γδ T cells mainly and thus supported pregnancy.
Crosstalk and functional regulation were analyzed between decidual γδ T cells and HTR8/SVneo cells. Expression of CXCR6 in decidual γδ T cells was induced and proliferation of decidual γδ T cells was promoted via cocultured with HTR8/SVneo cells. Invasiveness of HTR8/SVneo cells was enhanced by decidual γδ T cells
To illustrate the function of HTR8/SVneo cells on proliferation of decidual γδ T cells, cocultured with HTR8/SVneo trophoblast cells for 48 h, CXCR6-positive decidual γδ T cells were analyzed by flow cytometry. HTR8/SVneo cells and LV-HTR8/SVneo cells increased the percentile of decidual CXCR6+ γδ T cells while with no significant difference between HTR8 and LV-HTR8/SVneo cells, respectively [Figure 4]a. Cocultured with HTR8/LV-HTR8/SVneo cells and/or treated with rhCXCL16 for 48 h, the MFI CFSE and Ki67 of the decidual γδ T cells were detected, respectively. HTR8/SVneo cells and LV-HTR8/SVneo cells reduced the MFI CFSE of decidual γδ T cells, respectively, and there was no significant difference between the group of HTR8/SVneo cells and LV-HTR8/SVneo cells [Figure 4]b. Treated with rhCXCL16, there was no significant difference compared with the vehicle group [Figure 4]b. HTR8/SVneo cells and LV-HTR8/SVneo cells increased the Ki67 level of decidual γδ T cells, and there was no significant difference in the group of HTR8/SVneo cells and LV-HTR8/SVneo cells. Treated with rhCXCL16, there was no significant difference compared with the vehicle group [Figure 4]c. Hence, the study showed that HTR8/SVneo cells cocultured with decidual γδ T cells played a role on the upregulating the proliferation of decidual γδ T cells.
Figure 4: Crosstalk and functional regulation were analyzed between decidual γδ T cells and HTR8/SVneo cells. Expression of CXCR6 in decidual γδ T cells was induced and proliferation of decidual γδ T cells was enhanced via coculture with HTR8/SVneo cells. Co-cultured with trophoblasts HTR8/SVneo cells for 48 h, CXCR6 positive decidual γδ T cells were analyzed by flow cytometry (a). HTR8/SVneo cells and LV-HTR8/SVneo cells increased the percentile of CXCR6 positive decidual γδ T cells, while with no significant difference between HTR8 and LV-HTR8/SVneo cells, respectively (n = 9) (a). Cocultured with HTR8/LV-HTR8/SVneo cells and/or treated with rhCXCL16 for 48 h, the MFI CFSE (b) and Ki67 (c) of decidual γδ T cells were detected, respectively. HTR8/SVneo cells and LV-HTR8/SVneo cells reduced the MFI CFSE of decidual γδ T cells, respectively, and there was no significant difference between the group of HTR8/SVneo cells and LV-HTR8/SVneo cells. Treated with rhCXCL16, there was no significant difference compared with the vehicle group (n = 9) (b). HTR8/SVneo cells and LV-HTR8/SVneo cells increased the Ki67 level of decidual γδ T cells, and there was no significant difference in the group of HTR8/SVneo cells and LV-HTR8/SVneo cells. Treated with rhCXCL16, there was no significant difference compared with the vehicle group (n = 9) (c). Decidual γδ T cells enhanced the invasion of HTR8/SVneo cells (d). The II of trophoblasts under different conditions was normalized to the isotypical controls. The invasion of trophoblast HTR8/SVneo cells was enhanced more than that of LV-HTR8/SVneo cells by co-cultured with γδ T cells. Microscopic morphologies of the trophoblast after invasion through the Matrigel-coated membranes were taken at ×200. The results shown summarize the findings of more than 3 independent experiments. Student's t-test was performed for significance testing. Data were means ± SEM. *P < 0.0001, NS, nonsignificant. SEM: Standard error of mean; II: Invasive index.To analyze the biological function of decidual γδ T cells on HTR8/SVneo trophoblast cells, Matrigel transwell invasion assays were used to detect the effects on migration and invasive capabilities of HTR8/SVneo cells. The II of HTR8/SVneo cells under different conditions was normalized to the isotypical controls. The invasion of HTR8/SVneo trophoblast cells was enhanced more than that of LV-HTR8/SVneo cells by cocultured with decidual γδ T cells [Figure 4]d, which illustrated that the invasion of trophoblasts could be upregulated by decidual γδ T cells via CXCL16/CXCR6 pathway.
Expression of CXCL16/CXCR6 was higher in decidual γδ T cells of normal mice than that of abortion-prone mice
To analyze the expression of CXCL16/CXCR6 in abortion-prone models, CXCR6+ γδ T cells were analyzed via flow cytometry in normal pregnancy mice and abortion-prone mice models, respectively. The percentile of CXCR6+ γδ T cells was lower in RSA mice compared with normal mice [Figure 5]a and [Figure 5]b. The CXCL16 expression obtained from the uterus was analyzed via ELISA; the expression of CXCL16 was lower in the RSA mice group compared with the normal mice group [Figure 5]c. To study the function of CXCL16 and CXCR6 in mice further, LV for CXCR6 and anti-CXCL16 neutralizing antibody was used to block the pathway of CXCR6 and CXCL16, respectively. The proportion of LV-mediated CXCR6+ γδ T cells was decreased compared with the control group [Figure 5]d and [Figure 5]e. The rate of embryo resorption in mice was increased with the downregulation of CXCR6 [Figure 5]f and CXCL16 [Figure 5]g, respectively. Hence, the results illustrated that CXCL16/CXCR6 pathway decreased embryo resorption rate, and low expression of CXCL16/CXCR6 results in pregnancy loss.
Figure 5: Expression of CXCL16/CXCR6 was higher in decidual γδ T cells of normal mice than that of abortion-prone mice. CXCR6+ γδ T cells were analyzed via flow cytometry in the normal pregnancy mice group and abortion prone mice group, respectively, the percentile of CXCR6+ γδ T cells was lower in the RSA group, compared with the normal group (n = 8 per group) (a and b). The expression of CXCL16 from the uterus was analyzed via ELISA, The expression of CXCL16 was lower in the RSA group, compared with the normal group (n = 8 per group) (c). The murine were infected with the lenti-si-CXCR6 virus and their corresponding NC virus via tail intravenous injection at the time of a week before mating, according to the manufacturer's instructions. The proportion of lentivirus mediated CXCR6+ γδ T cells was decreased compared with the control group (n = 7) (d and e). The rate of embryo resorption in mice was increased via the downregulation of CXCR6 (f) and CXCL16 (g), respectively (n = 7 per group). Student's t-test was performed for significance testing. Data were means ± SEM. *P < 0.05, †P < 0.01; NS, Nonsignificant. NC: Negative control; SEM: Standard error of mean.Effect of adoptive transfer of γδ T cells on the embryo resorption rate in abortion-prone mice
To probe the function of CXCR6+ γδ T cells in the treatment of RSA, the technique of adoptive transfer was induced. CXCR6+ and CXCR6− γδ T cells were isolated from spleen in normal mice via fluorescence-activated cell sorting, and labeled with PKH67, and then adoptive transferred into abortion prone mice at day 4. Embryos were shown at day 14, respectively [Figure 6]a. Embryo resorption rate was increased in the group of adoptive transferring CXCR6− γδ T cells [Figure 6]b. Therefore, the study showed that CXCR6+ γδ T cells prevent from RSA and maintain normal pregnancy.
Figure 6: Effect of adoptive transfer of γδ T cells on the embryo resorption rate in abortion prone mice. CXCR6+ and CXCR6− γδ T cells were isolated from spleen in normal mice by flow cytometry and labeled with PKH67, and then adoptive transferred into abortion prone mice at day 4. Embryos were shown respectively (a). Embryo resorption rate of day 14 was increased in the group of adoptive transfer of CXCR6− γδ T cells (n = 7 per group) (b). Student's t-test was performed for significance testing. Data were means ± SEM. *P < 0.05, NS: Nonsignificant. SEM: Standard error of the mean. Discussion Mother does not reject the fetal allograft, the principal reason is that maternal and fetal lymphocytes are tolerant of each other in the bidirectional mixed lymphocyte reaction.[14] At the maternal–fetal interface, decidual immune cells secret Th2 cytokines, which play an important role on the regulation between mother and fetus.[9]
Decidual γδ T cells is a sort of unique immune cells, different from αβ T cells, expressing γ and δ chains.[15] In our previous study, we showed high-quality tissue staining analysis for the distribution of CXCR6 in decidua at the maternal–fetal interface.[16] Further, we analyze the distribution of CXCR6 distribution in immune cells at the maternal–fetal interface via flow cytometry. In this study, decidual γδ T cells expressed CXCR6 in medium level, among the expression profile with high expression of CD3, PD1, and CTLA-4 and almost none of CD4 and CD8. However, CXCR6 was expressed in decidual immune cells, such as macrophage cells, NK cells, NKT cells, and γδ T cells, and the result showed that CXCR6 was expressed highest in the decidual γδ T cells. CXCR6 is a chemokine receptor, and CXCR6+ cells, including T cells, NK cells, and monocytes, were attracted and recruited by CXCL16 ligand.[17],[18],[19] CXC chemokine receptor CXCR6 and its ligand CXCL16 play roles in regulating metastasis and invasion of cancer, and they are upregulated in multiple cancer tissue types relative to normal tissues.[20] Endothelial CXCL16 as well as platelet CXCR6 are acting as potent PBMC-adhesion ligands, inducing PBMC-adhesion to the atherosclerosis-prone vessel wall and thus promoting the progression of atherosclerosis.[21] In this report, the percentile of CXCR6+ γδ T cells in the peripheral blood of nonpregnancy and early-term pregnancy from normal women and RSA patients was analyzed. Increased percentile of CXCR6+ γδ T cells was found in the pregnant group and normal pregnancy. In our previous study, the results showed a reduced expression of CXCL16 and CXCR6 in villi and decidua from RSA patients, compared to that from normal preganncy.[16] In this study, HTR8/SVneo cells enhanced the proliferation of decidual γδ T cells independent of CXCL16/CXCR6 pathway. Therefore, the expression of CXCR6 receptor and CXCL16 ligand was involved into the recruitment of decidual immune cells into decidua, while CXCL16/CXCLR6 was not involved into the proliferation of decidual γδ T cells locally.
γδ T cells play important roles in innate immunity against infections and tumors with high killing activity, including increased expression of granzyme B, perforin, and IFN-γ.[22],[23] Granzyme B is a serine protease with immune-mediated cytotoxicity. Granzyme B is important for the ability of γδ T cells, NK cells, and CD8+ T cells to kill the targets.[24] In this study, HTR8/SVneo cells reduced the expression of granzyme B in decidual γδ T cells via CXCL16/CXCR6 chemokine and chemokine receptor and thus decreased the cytotoxicity to the embryo. Perforin is an important protein with cytotoxicity and plays a role in perforating in the cell membrane, via which granzyme moves into cells and induces cell apoptosis.[25] In the study, the expression of perforin was increased after cocultured with HTR8/SVneo cells and further increased under the coculture with LV-HTR8/SVneo cells compared with that of HTR8/SVneo cells, which showed that CXCL16 partly played the role in inhibiting the expression of perforin. Perforin performed as a function of the tunnel, and its cytotoxicity depended on the activity of granzyme B which induced apoptosis.[26] Based on that study, although the expression was upregulated, its function of cytotoxicity was restricted with the amount of granzyme B, and thus the final cytotoxicity of decidual γδ T cells was downregulated via CXCL16/CXCR6. Similarly, IFN-γ was increased after cocultured with HTR8/SVneo cells and further increased under the coculture with LV-HTR8/SVneo cells. Recent study shows that IFN-γ exerts a role in inhibiting the expression of granzyme B in CD8+ T cells and thus downregulating the effect on validating the virus.[27] These findings indicated that although the expression of IFN-γ was increased, it played a role in maintaining normal pregnancy by inhibiting granzyme B via CXCL16/CXCR6.
Th2-type cytokines, IL-4, IL-10, and TGF-β, inhibit IFN-γ production and enhance fetal allograft tolerance.[28] TGF-β has been found the ability on immune regulation in the past 30 years,[29] and TGF-β protects the maternal body from foreign antigens, bacteria, and allografts and regulates the immune effect.[30] In our result, HTR8/SVneo cells increased the expression of TGF-β in the decidual γδ T cells. Based on these theories, we can conclude that TGF-β regulates the maternal–fetal immune responses and protects the fetus. Th2 cytokines, IL-4, IL-10, TGF-β, and IL-17A, are upregulated and in favor to maintain normal pregnancy. The pro-inflammatory cytokine TNF-α produced by monocytes/macrophages is often associated with the increased risk for RSA when considering its multifunctional roles in lipid metabolism, coagulation, insulin resistance et al.[31] While the previous study shows that TNF-α, as Th1 cytokines, plays a role of proapoptosis and antiapoptosis in the process of placentation, embryo, and labor, depending on the expression and viability of its receptors.[32] Therefore, in our study, the expression of TNF-α in decidual γδ T cells was upregulated by cocultured with HTR8/SVneo cells; the actual biological effect needs to be probed further. In the study, IL-17A in decidual γδ T cells was upregulated by cocultured with HTR8/SVneo cells independent of CXCL16/CXCR6. IL-17A has been reported to defend bacteria from infecting in the pregnancy.[33] Other studies show that IL-17 enhances the invasion of trophoblasts JEG-3 cells.[34],[35] This also supports our results that decidual γδ T cells enhanced the invasion of HTR8/SVneo trophoblast cells independent of CXCL16/CXCR6. Therefore, trophoblasts and IL-17A secreted by decidual γδ T cells enhance each other, defending external bacteria and upregulating the invasion of trophoblasts, and thus maintain successful pregnancy. Invasion of trophoblasts is of importance to embryo implantation, which is an important step for mammalian reproduction, and is critical for determining pregnancy.
In the studies, the results showed that rhCXCL16, exerted on the decidual γδ T cells singlely, had no effect on the granzyme B, perforin, IFN-γ, and cytokines in the decidual γδ T cells, while trophoblasts had effects on that of decidual γδ T cells, which was mainly resulted from the complicated crosstalk between decidual γδ T cells and trophoblasts via CXCL16/CXCR6, while the specific regulation of rhCXCL16 in vivo was largely unknown and further studies were needed to be done.
Furthermore, murine models of normal pregnancy and abortion-prone mice were studied in vivo and we found that the embryo resorption rate of mice was increased via the downregulation of CXCR6 and CXCL16, respectively. Embryo resorption rate was decreased via adoptive transfer of CXCR6+ γδ T cells from normal mice, which illustrates the role of CXCL16/CXCR6 in decreasing embryo resorption rate of abortion-prone mice. CXCR6+ γδ T cells adoptive transferred from normal mice migrated into the maternal–fetal interface in abortion-prone mice, and previous studies showed that the CXCL16/CXCR6 interaction is involved in the migration of the peripheral T lymphocytes and γδ T cells.[36] Our results indicate that CXCL16/CXCR6 was favor to prevent pregnancy loss and maintain normal pregnancy, which were consistent in vitro and in vivo. In addition, in the field of cancer immunotherapy, many studies have demonstrated in vitro anticancer activities of γδ T cells and their in vivo potential roles as antitumor effectors, numerous clinical trials have been performed to exploit the properties of γδ T cells for cancer immunotherapy, and the applied method is to adoptive transfer of autologous γδ T lymphocytes expanded in vitro and then reinfused to patients.[37],[38],[39],[40] There have also been many studies on the amplification of γδ T cells in vivo and in vitro.[41],[42] Taken together, our studies encourage the further clinical evaluation of CXCL16/CXCR6 and adoptive transfer of autologous CXCR6+ γδ T cells expanded in vitro in the clinical strategy for the treatment of RSA.
Supplementary information is linked to the online version of the paper on the Reproductive and Developmental Medicine website.
Financial support and sponsorship
This study was supported by the National Natural Science Foundation of China (NSFC) (No. 81300552, 92057119, 31970798), the Innovation-oriented Science and Technology Grant from NPFPC Key Laboratory of Reproduction Regulation (CX2017-2), the Program for Zhuoxue of Fudan University (JIF157602), and the Support Project for Original Personalized Research of Fudan University.
Conflicts of interests
There are no conflicts of interest.
References
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