Supraphysiological estradiol promotes human T follicular helper cell differentiation and favours humoural immunity during in vitro fertilization

Abstract During pregnancy, humoural immunity is essential for protection against many extracellular pathogens; however, autoimmune diseases may be induced or aggravated. T follicular helper (Tfh) cells contribute to humoural immunity. The aim of this study was to test whether Tfh cell function can be manipulated via hormones. Seventy‐four women who underwent in vitro fertilization were recruited and divided into four groups: menstrual period (MP), controlled ovarian hyperstimulation (COH), embryo transfer (ET) and pregnant after embryo transfer (P). A flow cytometry analysis was performed to identify Tfh cells in peripheral blood mononuclear cells (PBMCs). Bioinformatics analysis revealed a possible pathway between Tfh and B cells. Enzyme‐linked immunosorbent assays were used to detect interleukin (IL)‐21 and IL‐6. The quantitative polymerase chain reaction was performed to quantify BCL‐6, BACH2, XBP‐1, IRF‐4 and G protein‐coupled (GP)ER‐1 mRNA expression. Compared with the MP group, the COH, ET and P groups showed more Tfh and B cells, as well as higher IL‐21, IL‐6, BCL‐6 and BACH2 expression. Furthermore, Tfh cell frequency in PBMCs, as well as serum IL‐21 and IL‐6 levels, were all positively correlated with serum estradiol (E2) levels; the B cell percentage also correlated positively with Tfh cells in PBMCs. Combined with the bioinformatics analysis, XBP‐1, IRF‐4 and GPER‐1 expression was related to E2 levels, both in vivo and in vitro. We speculate that E2 augments Tfh cells and favours humoural immunity. This study indicates that Tfh cell regulation may be a novel target in maintaining the maternal‐foetal immune balance.


| INTRODUC TI ON
Pregnancy is characterized by high levels of both oestrogen and progesterone in vivo. [1][2][3] Changes in these hormones during pregnancy appear to inhibit the cellular immune response classically mediated by T helper (Th)1/Th2 and Th17/T regulatory cells 4,5 and, in contrast, increase antibody production by memory B lymphocytes. 6 It has been reported that in some Th1-driven autoimmune diseases, pregnancy can temporarily abate the clinical signs in the mother. 7,8 Humoural immunity is essential for protection against extracellular pathogens; however, excess autoantibody production during pregnancy may induce or aggravate autoimmune diseases such as rheumatic antiphospholipid syndrome and systemic lupus erythematosus. [9][10][11] The mechanism underlying the relationship between maternal hormones and humoural immunity remains unclear.
T follicular helper (Tfh) cells are a unique subset of CD4 + T cells that play important roles in germinal centre (GC) formation and high-affinity antibody generation. 12,13 Tfh cells and GC B cells share some transcriptional networks, and many molecular pathways regulating Tfh cells can be deduced from B cells. 14 Tfh cells, first observed in human lymphoid tissues, are characterized by IL-21 and IL-6 release and the expression of some unique molecules and factors, such as CD4, B cell lymphoma 6 (BCL6), inducible co-stimulatory molecule (ICOS), CXC chemokine receptor 5 (CXCR5) and programmed death-1 (PD-1). Thus, Tfh cells are defined as CD4 + BCL6 + ICOS + CXCR5 + PD-1 + . 13 BCL6 is the master transcriptional factor for the development of both GC B cells and Tfh cells induced by IL-6, [15][16][17] whereas CXCR5 is induced by IL-21 and ICOS. 18,19 In addition, BCL6 is necessary for early CXCR5 expression. 20 -22 As a subset that facilitates T cell-dependent B cell activation, Tfh cells play important roles in autoreactive B cell proliferation, promoting the production of autoantibodies. 23 During pregnancy, Tfh cell numbers increase in both mice and human beings and decrease in case of a miscarriage. 24,25 When PD-1 or IL-21 are blocked, spontaneous abortion is high in mice as a consequence. 25,26 Estradiol (E 2 ) is the predominant hormone in females that plays pivotal roles in endocrine and reproductive systems. E 2 also manipulates immune cells in autoimmune diseases and exacerbates some cancers. 27,28 In mice, E 2 treatment decreases the Tfh cell response and suppresses Bcl6 and Il-21 mRNA expression to control autoantibody production; 29 however, the effect of E 2 on Tfh cells in humans is still obscure. Controlled ovarian hyperstimulation (COH) with supraphysiological E 2 and without a progesterone antagonist is an approach to assist in vitro fertilization (IVF). It can be used to explore the relationship between E 2 and Tfh cells, B cells and immunity in human beings.
In this study, we aimed to observe and identify the relationship between Tfh and B cell numbers, and E 2 levels, during IVF. We also determined the levels of IL-6, IL-21, BCL6 and BACH2, and their correlation with E 2 levels.  Table S1). The inclusion criteria were as follows:

| Patients' samples
(i) first IVF cycle, (ii) less than 35 years old, (iii) good ovarian response, (iv) normal karyotype and hormones and (v) without uterine abnormalities (fibroid, uterine septum, and uterine polyp), endometriosis or any other known immunological diseases.
Luteal-phase GnRH-a protocol was performed in all patients.
GnRH-a (Triptorelin Acetate Injection) was used to suppress pituitary, then, recombinant follicle-stimulating hormone (Gonal-F) and human menopausal gonadotropin (menotrophins for injection) were used for ovarian stimulation. When the dominant follicles (three or more) reached 18 mm in diameter, human chorionic gonadotrophin for injection (HCG) was administered. Transvaginal ultrasound-guided oocyte retrieval was performed 36 hours later.
Luteal phase support was performed using 90 mg of vaginal progesterone (Crinone gel 8%), administered from the oocyte retrieval day. Three-day later, two high-quality embryos were transferred into an adequately prepared endometrium. Early pregnancy was defined as the presence of an intrauterine gestational sac, with positive β-hCG results 4 weeks later. Luteal phase support was continued until 9 weeks gestation (almost 7 weeks after embryo transfer).
Patients were divided into four groups according to the stage of IVF treatment, menstrual period (MP), n = 20; controlled ovarian hyperstimulation (COH), n = 25; embryo transfer (ET), n = 15; and pregnant after embryo transfer (P), n = 14. The MP group was the basic hormone group with low E 2 and progesterone; the COH group had hyperphysiological E 2 levels without progesterone; the ET group had high E 2 and progesterone levels (progesterone is an antagonist of E2); and the P group had higher E 2 and progesterone levels.
augments Tfh cells and favours humoural immunity. This study indicates that Tfh cell regulation may be a novel target in maintaining the maternal-foetal immune balance.

K E Y W O R D S
B cells, estradiol, humoural immunity, in vitro fertilization, T follicular helper cells

| Blood mononuclear cells isolation and flow cytometry
Blood from peripheral and cord vein was collected in sterile heparinized tubes from each patient. Peripheral blood mononuclear cells (PBMC) and  CD4 + T cells were sorted (purity >95%, viability >99%) by column in the magnetic field of a suitable magnetic activating cell separator.

| Enzyme-linked immunosorbent assays (ELISA)
Serum samples collected from patients were kept at −80°C. Enzyme immunoassays were performed to determine the concentration of IL-21 and IL-6 using ELISA kits (Biolegend). Human serum was diluted 1:1 in sample buffer and incubated for 30 minutes. Following three washes with the washing buffer, the bound antibodies were detected by incubation with anti-human IgG HRP conjugate for 30 minutes. Then, the samples were washed again as described earlier, and tetramethyl benzidine substrate was added for 15 minutes. The optical density was read at 450 nm using an automated spectrophotometer. All samples were assayed in duplicate.

| Quantitative real-time polymerase chain reaction (qRT-PCR)
Initially, total RNA was extracted from PBMC using AxyPrep Multisource Total RNA Miniprep Kit (Axygen). The RNA quality was checked based on the A 260 /A 280 ratio, and pure RNA samples were converted to cDNA using PrimeScript RT Reagent Kit with gDNA Eraser in accordance with the manufacturer's instructions (Takara Bio). The polymerase chain reaction was performed using SYBR Green based on the instruction manual (Takara Bio). The primer sequences are presented in Table 1 (Comate Bioscience). The relative

Oligonucleotide
Primer sequence TA B L E 1 Oligonucleotide and primer sequence mRNA expression of different genes was quantified using the ∆∆C t method. Β-actin was used as a housekeeping gene.

| Network construction, and gene oncology (GO) analysis, pathway analysis
String (https://strin g-db.org/), a powerful tool aiming to construct a comprehensive and objective global network, it contains direct (physical) as well as indirect (functional) interactions. 30 GeneMANIA (http:// genem ania.org/) is a user-friendly web interface used for analysing gene functions, generating gene lists and prioritizing genes for functional assays. 31 After selecting homo sapiens from the nine optional organisms, and entering the genes of interest into the search bar, the results showed. Potential targets were uploaded to the Metascape server (http://metas cape.org/) for GO analysis, which was used to identify functionally related genes from the list of differentially expressed genes. Functionally related genes were usually categorized to biological processes, cellular components and molecular functions.
A false discovery rate<5.0 and P value <.05 were considered statistically significant.

| Statistical analysis
GraphPad Prism version 7.00 and SPSS version 21.0 software were used for statistical analysis. All data were presented as the mean ± SD. Variables between groups were analysed using the independent-samples Student's t test or chi-squared tests, and P < .05 was considered statistically significant.

| The proportions of Tfh and B cells in PBMCs was positively related to the serum E 2 level
To investigate the correlation between the proportions of Tfh and B cells, and the serum E 2 level in human beings, we first analysed these parameters in the blood of women who underwent IVF treatment. Tfh cells were identified as CD4 + CXCR5 + PD-1 + , whereas B cells were defined as CD19 + . As expected, serum E 2 levels were higher in COH, ET, P groups compared with MP group (Supplemental Figure S1A; P = .0009, P = .0039 and <.0001, respectively). Meanwhile, as shown in Figure 1A,B, the percentage of CD4 + T cells was significantly elevated in the COH and P groups compared with that in the MP group (P = .0388 and P = .0020, respectively). We also investigated the correlation between Tfh cell percentages and serum E 2 levels in the MP and COH groups without progesterone interference, the result showed a positive relationship ( Figure 1C; r 2 = .4398, p < .0001, n = 39). Relationship between B cell percentages and serum E 2 levels showed a linear relationship (Supplemental Figure S1A; r 2 = .1306, P = .0147, n = 39).
As Tfh cells play crucial roles in the differentiation and maturation of B cells, 13 we next examined changes in the B cell percentage

F I G U R E 3 17β-Estradiol induces Tfh cells differentiation in vitro. A, The flow cytometry analysis showed that 17β-estradiol-induced Tfh cells in a concentration-dependent manner. B, Qualification of Tfh cells in total CBMC
in PBMCs. We found that the percentage of B cells was significantly increased in COH, ET and P groups compared with that in MP group (P = .0480, P = .0060 and P = .0038, respectively) (Figure 2A,B).

| Serum IL-6 and IL-21 levels correlated with the E 2 level
IL-21 and IL-6 are not only secreted by Tfh cells, but also promote Tfh cell differentiation. 32 Therefore, we measured the levels of IL-21 and IL-6 in serum using ELISAs ( Figure 5A,C) and analysed the correlation between IL-21, IL-6 and E 2 levels ( Figure 5B,D). Our data demonstrated that under endogenous E 2 treatment alone (the MP and COH groups, n = 39), without progesterone interference, both IL-21 and IL-6 levels were up-regulated significantly in serum (P = .0041 and P = .0093, respectively); however, IL-21 levels had no linear correlation with E 2 levels.

| E 2 increased Tfh and B cell transcription factor expression
Besides IL-6, IL-21 and BCL6 (known markers of Tfh cells), recent studies identified BACH2 as a key transcription factor in antibody class switching. 33,34 The function of BACH2 in Tfh cells is still controversial. Thus, we detected its expression and that of BCL6 and BACH2 in vivo ( Figure 5E,F). The expression of BACH2 was in accordance with that of IL-21 and IL-6. As a crucial transcription factor of plasma cells, the expression of XBP1 increased consistently with E 2 levels, which further verified the relationship between E 2 and F I G U R E 5 Serum IL-21 / IL-6 levels, and relative expression of BCL-6 / BACH2 in PBMC are higher in COH, ET and P groups than MP group. Serum IL-21 and IL-6 levels are positive correlated with serum E2 level. A, Serum IL-21 levels is higher in COH, ET and P groups than MP group (**P = .0041, *P = .0484, and ***P = .0004, respectively). B, Positive correlation between serum IL-21 and E 2 levels in MP and COH groups (r 2 = .05234, P = .1306, n = 45). C, Serum IL-6 levels is higher in COH, ET and P groups than MP group (**P = .0093, *P = .0426, and****p < .0001, respectively). D, Positive correlation between serum IL-6 and E 2 levels in MP and COH groups (r 2 = .1306, *P = .0147, n = 45). E, Relative expression of BCL-6 in PBMCs is higher in COH, ET and P groups than MP group (***P = .0001, **P = .0030, and **P = .0053, respectively). F, Relative expression of BACH2 in PBMCs is higher in COH, ET and P groups than MP group (**P = .0075, **P = .0016, and *P = .0231, respectively) B cells. Meanwhile, expression of IRF4 and GPER1 were consistent with the bioinformatics analysis, both in vivo and in vitro, indicating that IRF4 and GPER1 expression in Tfh and B cells were crucial for their augmentation (Figure 6).

| D ISCUSS I ON
In this study, we revealed a positive correlation between the num- Here, we proposed that E 2 might play a dominant role in Tfh cell recruitment and differentiation. We defined Tfh cells as It is well-established that IL-6 is a potent inducer of IL-21, which has been observed in activated murine CD4 + T cells. 13 IL-6 also drives CXCR5 expression 16  This suggests an important role of E 2 and Tfh cells in the pathogenesis of these diseases.

| CON CLUS IONS
We demonstrated that Tfh cells, as a novel subset of immune cells involved in immune tolerance at the maternal-foetal interface, are influenced by circulating E 2 levels. Tfh cells also regulate cytokines and transcription factors, contributing to enhanced humoural immunity. Further investigation of these important results will advance our understanding of the clinical application of Tfh cells as a therapeutic target for immune-related miscarriage and IVF failure.

E TH I C S S TATEM ENT
The Ethics Review Boards of the First Hospital of Jilin University

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.