T-Lymphocyte proliferative activity in early pregnancy and outside pregnancy state

Abstract

T-lymphocytes are present in the endometrium before pregnancy and their number varies depending on menstrual cycle stage. Despite T-lymphocyte population heterogeneity, there is no clear vision of general mechanisms of decidua T-lymphocyte pool formation. One of the assumed variants is T-lymphocyte proliferation in situ. The study objective is to evaluate variations of peripheral blood T-lymphocyte proliferative activity in the presence of trophoblast cells. The peripheral blood was sampled from healthy nonpregnant women in the proliferative (n = 29) and secretory (n = 32) menstrual cycle phases and also from women on 6-7 weeks stage of physiological pregnancy (n = 30). Jeg-3 (ATCC) line cells were applied as trophoblast cells within in vitro model system. T-lymphocyte proliferation was determined by estimating the Ki-67 expression and T-lymphocyte relative number. It was established that trophoblast cells perform inhibiting effect on Ki-67 by T-lymphocytes in all groups of examined women both in course of PBMC cultivation and in case of preliminarily isolated T-lymphocytes. During cultivation in the presence of IL-2 and trophoblasts, PBMC T-lymphocytes in pregnant women are more resistant to trophoblast cells inhibition than in nonpregnant women. In case of isolated T-lymphocytes, decreased T-lymphocyte proliferation during pregnancy was observed as compared to the proliferative cycle phase hence pointing to necessity of T-lymphocyte contact with microenvironment cells for self-support.

Keywords:

• T-lymphocyte
• PBMC
• trophoblast
• proliferation
• pregnancy
• peripheral blood
• Ki-67

Introduction

T-lymphocytes are present in the endometrium before pregnancy [1] and their number varies depending on menstrual cycle stage [2]. Among T-lymphocytes usually distinguished are CD4+ T-lymphocytes (Th1 and Th2), CD8+ T-lymphocytes and T-regulatory lymphocytes [3,4]. The decidua CD8+ T- lymphocyte conditioning medium stimulates trophoblast cells invasive activity [5]. CD4+ T-helper lymphocytes are a source of pro- ant anti-inflammatory cytokines, necessary on the implantation and blastocyst invasion stage as well as for placenta vascular net formation [6].

Despite the T-lymphocyte population heterogeneity at present there is no consensus on general mechanisms of decidua T-lymphocyte pool formation. One of the possible variants of decidua T-lymphocyte population forming is proliferation in situ. It was shown that TSLP (thymic stromal lymphopoietin) produced by trophoblast cells activated decidua dendritic cells which in their turn stimulate T-regulatory lymphocyte proliferation [7]. The primary trophoblast cells in vitro as well as HTR8/SVneo line trophoblast cells inhibit CD4+ T-lymphocyte proliferation by secreting IL-35 [8]. A mice in vivo model demonstrated T-regulatory cells proliferation increase during pregnancy compared to nonpregnant animals [9].

The present study objective was to evaluate the peripheral blood T-lymphocyte proliferative activity in the presence of trophoblast cells.

Materials and technique

Patients

The study included pregnant and nonpregnant women with no signs of inflammatory changes. Three groups were composed: healthy non-pregnant women in proliferative (group 1, n = 29) and secretory (group 2, n = 32) stages of menstrual cycle and a group of pregnant women on 6–7 weeks term of physiological pregnancy (group 3, n = 30). The age of nonpregnant women was within 19–23 years (20,3 ± 0,2), for pregnant women - within 22–41 years (30 ± 1,4). Pregnant women underwent a screening in the Gynecology Department of Saint-Petersburg SBHI city hospital № 26. The groups of nonpregnant women were recruited from healthy volunteers. The following exclusionary criteria were considered: acute or exacerbating chronic infection, type-1 diabetes, urogenital infection, hypertension and other blood circulatory system diseases. For pregnant women, the following exclusionary criteria were also considered: hydramnion, oligohydramnios, and multiple pregnancies.

The research was carried out according to The Code of Ethics of the World Medical Association (Declaration of Helsinki). Informed consent was obtained.

Cells

We used JEG-3 trophoblast cells, cultured according to ATCC (USA) guidelines.

The peripheral blood mononuclear cells (PBMC) were obtained by centrifugation in Histopaque^®-1077 (Sigma-Aldrich, USA). After isolation the PBMC viability was 97,6%.

The T-lymphocyte population was obtained through aseptic isolation from PBMC by FacsAriaIII flow sorter (BD, USA) (Figure 1). The viable T-lymphocyte number after isolation was 91,6%.

Figure 1. T-cell gating strategy used for sorting out of PBMC. (1) PBMC treated with isotopic antibodies: (a) Distribution diagram in FSC-SSC coordinates. Gate P1 contains lymphocytes and monocytes; (b) Distribution diagram in SSC-PE coordinates. Quadrant Q1 contains lymphocytes and monocytes, (c) Distribution diagram in PerCP-PE-Cy7 coordinates; (2) Presort of PBMC treated with antibodies to CD45, CD3, CD56: (d) in FSC-SSC coordinates; (e) in SSC-CD45 coordinates. Gate P2 contains lymphocytes and monocytes. To exclude duplets, after gating in FSC-SSC coordinates gating in FSC-A - FSC-W (f) and SSC-A – SSC-W 9 (g) coordinates was performed. Then gating in CD56 PE-Cy7 – CD3 PerCP coordinates was performed (h). Quadrant Q1-1contains T-lymphocytes; (3) Postsort of peripheral blood T-lymphocytes: (i) in FSC-SSC coordinates; (j) in SSC-CD45 coordinates; (k) FSC-A - FSC-W; (l) SSC-A – SSC-W, (m) in CD3PerCP-CD56PE-C coordinates.

Peripheral blood T-lymphocyte cultivation in the presence of JEG-3 line trophoblast cells

The JEG-3 line cells were resuspended in a cultural medium, placed in a 96-well flat-bed plate (BD, USA) in 25,000 cell per 1 well concentration in 100 µL of cultural medium and cultivated in humid atmosphere with 5% CO₂ under 37°С for 24 h.

Then PBMC or isolated T-lymphocytes were inserted in 100,000 cells per well concentration in 100 µL of cultural medium. In some wells recombinant IL-2 was added (‘Roncoleukin’, Biotech, Russia) in 200 IU/mL concentration and cultivated for 72 h.

The evaluation of peripheral blood T-lymphocyte proliferative activity in presence of JEG-3 line trophoblast cells

After cultivation cells were fixed and permeabilized with Cytofix/Cytoperm commercial KIT (BD Biosciences, USA). Ki-67 evaluation was performed according to the protocol recommended by the manufacturing company (BD Biosciences, USA). Gating strategy for proliferation evaluating is shown in Figure 2.

Figure 2. Gating strategy for proliferation evaluating (shown for PBMC, for isolated T-cells we used the same the gating strategy). (1) PBMC after 6-day cultivation. Bivariate histogram of PBMC and trophoblast cells distribution in FSC-SSC coordinates. (2) PBMC treated with isotopic antibodies : (a) in PerCP – SSC coordinates; (b) in APC-PE-Cy7 coordinates; (c) in Pe-Cy7-FITC coordinates; (d) Auto-fluorescence intensity histogram by FITC channel; (3) cells treated with antibodies to CD45, CD3 and Ki-67: (a) in CD45PerCP – SSC coordinates ; (b) in CD3PE-Cy7 – Ki-67 FITC coordinates (after trophoblast free cultivation); (c) in CD3PE-Cy7 – Ki-67 FITC coordinates (after cultivation in the presence of trophoblast cells); (d) Ki-67 expression intensity histogram after trophoblast free cultivation: (e) Ki-67 expression intensity histogram after cultivation in presence of trophoblast cells.

Statistical analysis was performed by STATISTICA software program applying Mann–Whitney distribution free test and Wilcoxon test with difference authenticity recognized if p <.05.

Results

T-lymphocyte relative number in PMBC after cultivation in the presence of JEG-3 line trophoblast cells

Before cultivation the T-lymphocyte relative number in peripheral blood in the group of healthy nonpregnant women in secretory cycle phase and that of women with physiological pregnancy was lower compared to T-lymphocyte number in the group of nonpregnant women in proliferative cycle phase (Figure 3(a)). After inductor-free cultivation, the T-lymphocyte number in the group with physiological pregnancy was higher than before cultivation (Figure 3(a)). It was established that in all examined groups the T-lymphocyte number rose after cultivation in IL-2 presence compared to the initial number before cultivation and to that after inductor free cultivation (Figure 3(a)) which demonstrates the T-lymphocytes ability to respond to the stimulation within our model.

Figure 3. T-lymphocyte proliferative activity before and after cultivation within 6 days in the presence of IL-2 and trophoblast cells: (a) T-lymphocyte relative number (% of leucocytes with CD45+ phenotype); (b) Intensity of Ki-67 expression by T-lymphocytes within PBMC; (c) Intensity of Ki-67 expression by T-lymphocytes isolated from PBMC (relative fluorescence units). Difference authenticity: * - p < .05; ** - p < .01; *** - p < .001.

All examined groups showed T-lymphocyte number growth in the presence of trophoblast cells compared to their number before cultivation or after inductor free cultivation (Figure 3(a)). In women with physiological pregnancy, the T-lymphocyte number after cultivation in the presence of trophoblast cells was lower than in healthy nonpregnant women in proliferative and secretory cycle phases (Figure 3(a)). During cultivation in simultaneous presence of IL-2 and trophoblast cells, T-lymphocyte number in all groups was higher than in case of incubation with IL-2 and lower than in case of incubation with trophoblast cells only (Figure 3(a)).

Ki-67 expression by T-lymphocytes within PBMC population after cultivation in the presence of JEG-3 line trophoblast cells

In all groups, Ki-67 proliferation marker exceeded expression by T-lymphocytes was established under conditions of cultivation within PBMC population in the presence of IL-2 compared to inductor free cultivation (Figure 3(b)). The group of women with physiological pregnancy showed Ki-67 higher expression by T-lymphocytes compared to the group of nonpregnant women in both cycle phases under inductor-free cultivation and in case of IL-2 presence (Figure 3(b)). Ki-67 expression by T-lymphocytes in the presence of trophoblast cells in women with physiological pregnancy was higher compared to that in groups of healthy nonpregnant women in proliferative and secretory cycle phases (Figure 3(b)).

In all groups studied, T-lymphocyte proliferation after their cultivation in the presence of both trophoblast cells and IL-2 was lower than in case of cultivation in the presence of IL-2 only (Figure 3(b)). Women in secretory cycle phase and pregnant women showed higher Ki-67 expression in case of T-lymphocyte cultivation in the presence of trophoblast cells and IL-2 than in case of cultivation in the presence of IL-2 only (Figure 3(b)). In nonpregnant women in cycle secretory phase compared to nonpregnant women in proliferative phase, Ki-67 higher expression by T-lymphocytes was observed in case of cultivation in the presence of IL-2 and trophoblast cells (Figure 3(b)). Women with physiological pregnancy demonstrated Ki-67 higher expression by T-lymphocytes compared to both groups of nonpregnant women in case of cultivation in the presence of IL-2 and trophoblast cells (Figure 3(b)).

Ki-67 expression by T-lymphocytes isolated from PBMC population after cultivation in the presence of JEG-3 line trophoblast cells

In all studied groups, Ki-67 expression by T-lymphocytes isolated from PBMC population grew resulting from cultivation in the presence of IL-2 compared to that in case of inductor-free cultivation (Figure 3(c)). In case of cultivation in the presence of IL-2 and trophoblast cells, all examined women showed Ki-67 lower expression by T-lymphocytes compared to that in case of cultivation in the presence of IL-2 only (Figure 3(c)). In the group of women with physiological pregnancy, Ki-67 expression by T-lymphocytes in case of cultivation in the presence of IL-2 and trophoblast cells decreased compared to the group of non-pregnant women in proliferative cycle phase (Figure 3(c)).

Discussion

We established that T-lymphocyte relative number in peripheral blood is lower both in menstrual cycle secretory phase and during pregnancy compared to menstrual cycle proliferative phase which is an addition to the previously obtained data [10]. After inductor free six-day-long PBMC cultivation the T-lymphocyte rate among mononuclears did not vary compared to that before cultivation in all studied groups. At the same time in all studied groups, the T-lymphocyte Ki-67 expression rate grew in the presence of IL-2, compared to the proliferative activity level without IL-2. Hence, IL-2 cytokine-stimulated T-lymphocyte proliferation which corresponds to the reference data [11].

Pregnancy development is accompanied by CD4+ T-lymphocyte increase in decidua [12,13]. Treg (CD4 + CD25+) number increase during pregnancy had been previously demonstrated both in circulation [14] and in uterine-placental contact zone [15,16]. We established that T-lymphocytes within PBMC manifested higher Ki-67 expression intensity in pregnant women compared to T-lymphocytes in nonpregnant women. Hence, the present results are an addition to the existing data on T-lymphocyte activity pointing out their predisposition to proliferation in pregnant women.

We established that PBMC in the presence of trophoblast is marked with higher T-lymphocytes number compared to inductor-free cultivation. According to the literature T-lymphocyte concentration in uterine regional lymph nodes increases during menstrual cycle reaching the peak during the menstrual flux when endometrial tissue fragments may get into endometrium lymph nodes [17] which points to a possible T-lymphocyte activation. Apart from T-lymphocytes, monocytes and NK cells are present within PBMC. NK cells and JEG-3 line trophoblast combined cultivation leads to trophoblast cells death [18], and thus, it may cause much debris occurrence and T-cells stimulation, leading to their increase in number.

Trophoblast produces such factors as estradiol, progesterone, chorionic gonadotropin, α-fetoprotein, human chorionic somatotropin, TGFβ2, macrophage migration inhibitory factor and trombospondin-1, capable of immune regulation and performing T-lymphocyte inhibition [19]. In pregnant women, the T-lymphocyte proliferation and cytotoxicity diminish under the influence of progesterone and chorionic gonadotropin [20,21]. The established decreased T-lymphocyte number within PBMC in pregnant women compared to the nonpregnant women in case of cultivation with trophoblast may be due to T-cell susceptibility to trophoblasts tolerogenic effects in pregnant women. However, similar variations of T-lymphocyte number may occur due to possible simultaneous NK cell quantity growth with cultivation in trophoblast cells presence. This assumption finds support by data established in our previous study on NK cell increased proliferation in JEG-3 cells presence in pregnant women compared to nonpregnant women [22] as well as by published data on NK cell concentration increase with pregnancy occurrence in uteroplacental complex [23]. The Ki-67 increased expression by T-lymphocyte in PBMC in the presence of trophoblasts detected by our present study generally accords with reference data [9,24] since it may be determined by proliferation of some T-lymphocyte populations. However, further investigations are necessary to understand the trophoblasts influence on certain T-lymphocyte subpopulations proliferation.

In case of PBMC cultivation in the presence of IL-2 and trophoblasts T-lymphocyte proliferation decreased in all studied groups compared to proliferation level in the presence of IL-2 only. Note that similar tendency was observed both in case of T-lymphocyte cultivation within PBMC and also previously isolated from PBMC T-lymphocytes. Hence, even in the presence of IL-2 stimulating T-lymphocyte proliferation [11], all groups of women manifest T-lymphocyte proliferation inhibiting influence of trophoblast cells.

In case of cultivation in combined presence of IL-2 and trophoblast cells pregnant women showed increased Ki-67 expression by T-lymphocytes within PBMC compared to nonpregnant women. As for the isolated T-lymphocytes lower proliferation was observed in pregnant women compared to nonpregnant women in proliferative cycle phase. Perhaps monocytes within PBMC perform stimulating influence on T-lymphocytes. The expression of CD80 and CD86 receptors, which are ligands for T-lymphocyte co- stimulating molecules—performed by decidua macrophages exceeds in first trimester [25] which points to a possibility of T-lymphocyte activation by decidua macrophages. Experiments in mice showed that T-lymphocytes are able to proliferate in the lining of intestines and differentiate into antigen-specific T-lymphocytes after oral immunization through interaction with antigen presenting cells located in lamina propria [26]. These data point to a possibility of antigen presentation in situ. A mice model also showed that in case of intravaginal administration of CpG-oligodeoxynucleotide which is a stimulator of both congenital and adaptive immune response types, in uterus MHCII + F4/80+ macrophage and MHCII + CD11с+ CD86+ dendrite cell number growth was observed and both were able to stimulate T-lymphocyte proliferation in vitro [27]. Hence, PBMC monocytes within our model system were able to differentiate into macrophages to activate T-lymphocytes in situ and boost their proliferation. In case of isolated T-lymphocytes, no such kind of stimulation by macrophages could be seen.

Conclusion

The trophoblast cells inhibit Ki-67 proliferative marker expression by T-lymphocytes in all studied groups both in case of PBMC cultivation or in that of isolated T-lymphocytes. In case of PBMC cultivation with IL-2 and trophoblasts, the T-lymphocytes in pregnant women were more resistant to trophoblast inhibiting effect on Ki-67 expression compared to T-lymphocytes in nonpregnant women. In case of isolated T-lymphocytes, decreased T-lymphocyte proliferation was observed in pregnant women compared to proliferative cycle phase, which points out the necessity of T-lymphocyte contact with other cells of micro-environment to preserve their number.

Author contribution

All the authors have read the journal’s authorship agreement. The manuscript has been reviewed and approved by all authors.

Acknowledgements

The authors express their gratitude to Panina A.N. for the technical assistance.

Disclosure statement

Authors declare no conflict of interest.

Additional information

Funding

This research was supported by the Russian Foundation for Basic Research under grant № 20-015-00014 (analysis of T cell proliferation in presence of trophoblast cells); the government program № АААА-А20-120041390033-4 (data collection on patient groups), the government program № АААА-А19-119021290116-1 (cell culturing).