Regulatory T cells improve pregnancy rate in RIF patients after additional IVIG treatment

ABSTRACT

RIF (repeated implantation failure) women with immunologic basis and cellular abnormalities may benefit from intravenous immunoglobulins (IVIG) as an immunomodulator based on different studies. In this study, we evaluated the effect of IVIG on the frequency and function of Th17 and Treg cells, as two important subgroups of CD₄⁺ T cells in implantation and pregnancy rates. Seventy-two RIF patients with preconception Th1⁄Th2 ratio and natural killer (NK) cells frequency and activity elevation were selected and divided into two groups; 40 out of 72 received IVIG, aspirin, and heparin (anoxaparin) and constituted the treatment group and 32 patients received aspirin and heparin (anoxaparin) and no IVIG and were the control group. Th17, Treg frequency, transcription factors, cytokine gene expression, and cytokine secretion were evaluated by flow cytometry, real-time PCR, and ELISA, respectively. Post-treatment evaluation of the IVIG grouprevealed a significant increase in Treg associated parameters such as Treg frequency (p = 0.0186), Foxp3 (p = 0.0004), and cytokine mRNA levels (IL-10, p = 0.0058 and TGF- β, p = 0.0038), however, in the case of Th17, a significant difference was only observed in a reduction in the RORγt mRNA level (p = 0.0218). In conclusion, IVIG therapy may be a good choice in the treatment of implantation failure in RIF women especially with an immunologic basis, and may improve the implantation and pregnancy rate by affecting immunoregulatory mechanisms such as Tregs.

Abbreviations: RIF: repeated implantation failure; IVIG: intravenous immunoglobulin; Th17: T helper 17; Treg: T regulatory; NK cells: natural killer cells; PCR: polymerase chain reaction; ELISA: enzyme-linked immunosorbent assay; RORγt: RAR-related orphan receptor gamma; Foxp3: forkhead box protein P3; IL-17: interleukin-17; LMWH: low-molecular weight heparin; dNK: decidual NK cells

KEYWORDS:

• Implantation failure
• IVIG therapy
• T reg
• Th17

Introduction

Repeated in vitro fertilization (IVF) failure or repeated implantation failure is one of the major barriers in human reproduction and it is classically defined as absence of implantation and pregnancy after transfer of an embryo to the uterus [Li et al. 2013]. About 25-40% of natural implantations are lost [Zinaman et al. 1996] and loss of implantation occurs in 75% of pregnancies which are not even clinically recognized [Norwitz et al. 2001]. The rate of successful implantation is low. There has been a focus on immunologic factors as a reason for reproduction failures in recent years. The maternal immune system recognizes the foreign antigens on the early embryo which is a semi allograft and a response against these antigens is created as regulatory mechanisms which are responsible for stopping these reactions and protecting the embryo are lacking. Tolerance in the maternal immune system is important for a successful implantation. The regulatory mechanisms include a cellular proportion of the immune system which guarantees tolerance toward the embryo [Chatterjee et al. 2015]. In fact, a balance between inflammatory and regulatory response is required to prevent reproduction failures such as RIF [Lee et al. 2012].

CD4⁺ CD25⁺ Foxp3⁺ regulatory T cells (Treg), which are a subtype of helper T cells, play key roles in the immune tolerance [Aluvihare et al. 2004; Somerset et al. 2004; Hori et al. 2003]. There is another type of helper T cells (CD₄⁺ T) which are able to produce IL-17 and they are known as T helper17 (Th17) cells. Th17 lymphocyte are responsible for defending against some bacteria or fungi by stimulating neutrophils and inflammatory cytokine production, so Th17 is able to induce inflammatory response and some autoimmune diseases [Nakashima et al., 2010; Wang et al., 2010]. Recent investigations regarding the role of these cells in human reproduction revealed that an appropriate balance between Treg and Th17 lymphocyte and hemostasis, is required not only to create a proper protection against pathogens but also to prevent harmful responses of the immune system against the embryo [Lee et al., 2011].

Any abnormalities in immune cells balance or disruption in hemostasis, may lead to a failure in reproduction. Consequently, the approaches which are able to reform these abnormalities are needed to prevent or treat the failures. Immunotherapy methods, especially the use of intravenous immunoglobulins (IVIG) have been introduced to improve pregnancy outcome in recent years [Scher and Salazar 2000; Coulam et al. 1994]. Since 1980, IVIG, has been used as an immunomodulator for the treatment of different immunological disorders such as autoimmune disease or immunodeficiencies such as Kawasaki disease, dermatomyositis, and graft versus host disease [Ballow 2002]. IVIG is made of plasma of thousands of blood donors and it contains different kinds of antibodies which are present in human plasma [Beer and Kwak 1997]. Different studies suggest different mechanisms of action for IVIG but some of them are well-known. IVIG reduces the frequency and cytotoxicity of natural killer (NK) cells [Moraru et al. 2012; Clark et al. 2008; Yamada et al. 2003] and it is also able to reduce Th1 activity and creates a shift toward Th2 response and cytokines [Winger et al. 2011a]. On one hand, antibody protection is prevented by IVIG, and on the other hand, IVIG contains anti-idiotypic antibodies to neutralize circulating antibodies [Sher et al. 1998]. There are several studies which indicate that the expansion and function of regulatory T cells are increased by IVIG [Maddur et al. 2017; Lee et al. 2014; Trinath et al. 2013; Ballow 2011; Elram et al. 2005] but it is also able to suppress the production, differentiation, and function of Th17 cells [Lee et al. 2014; Maddur et al. 2013] and down-regulates Th17 pathways [Ballow 2014]. According to some studies, anticoagulants such as low-molecular weight heparin (LMWH) and aspirin are suggested for patients with reproductive abnormalities especially RIF patients to create antithrombotic and even anti-inflammatory effect [Quaranta et al. 2015; De Jong et al. 2014]. Therefore, one of these anticoagulants, or in some cases both, are administered for RIF patients in combination with IVIG.

In this study, the effect of IVIG on cellular proportion of the immune system is investigated in RIF women with an immunologic bases. Th17 and Treg frequency and cytokine secretion are among the parameters which were assessed at the end of the treatment. According to previous studies, there is a hope for improving the implantation process and a healthy pregnancy after IVIG administration in RIF women.

Results

Demographic data of patients are summarized in Table 1. After the treatment, pregnancy was evaluated between subjects. Patients with IVF failure and a negative pregnancy test, were separated and the results of evaluation were not statistically significant. The results below represent the parameters which were assessed in women who had a successful IVF and a positive pregnancy test in order to eliminate the impact of implantation on cellular proportion and function. Cellular proportion, mRNA expression, and cytokine secretion of immune system components were evaluated 2 days before embryo transfer at the time of IVIG administration and 15 days after embryo transfer.

Table 1. Demographic data of patients (n = 72).

Variable	IVIG group	Control group
No. of patient	40	32
Maternal age (years, range)	36.8 ± 2.7 (23-41)	37.1 ± 2.4 (24-40)
No. of IVF (range)	4.42 ± 2.26 (3–9)	4.55 ± 3.15 (3–8)
No. of embryo transfers (range)	2.01 ± 0.53 (2–3)	2.22 ± 1.2 (2–3)
Abnormal NK cell levels (No., percent)	25, 62.5%	21, 65.6%
Abnormal NK cell cytotoxicity (No., percent)	20, 50%	19, 59.3%
Abnormal Th1/Th2 cell ratio (No., percent)	21, 42.5%	18, 56,2

IVIG: intravenous immunoglobulin; NK cells: natural killer cells.

Flow cytometry

Th17 and Treg cells frequencies were evaluated by flow cytometry using different monoclonal antibodies against surface markers, 2 days before embryo transfer, at the time of IVIG administration and 15 days after that, in RIF patients. Results revealed that CD4⁺ FOXP3⁺ Treg cells were affected by IVIG and at the end of the treatment the rate of Treg increased in the IVIG group (p = 0.0186). Enumeration of CD4⁺ FOXP3⁺CD127⁻ Treg showed almost the same results, as the frequency was elevated (p = 0.04) in the IVIG group while there was no significant differences in the control group (Figure 1A).

Figure 1. Flow cytometry analysis. (A) Frequency of Treg and Th17 in IVIG-treated and control group. After IVIG therapy, evaluation of cellular proportion with flowcytometry 2 days before embryo transfer and 15 days after that and the comparison between each group, revealed that the rate of Treg cells increased in the IVIG group (p value = 0.0186) after embryo transfer. There was almost no statistically significant difference in Th17 rate in the IVIG and control groups (p ≤ 0.05: statistically significant) after treatment. (B). Frequency of Treg in IVIG-treated and control group. CD4⁺ FOXP3⁺CD127⁻ Treg evaluation by flowcytometry revealed that IVIG therapy increases (p value = 0.04) the frequency 15 days after embryo transfer but the control group showed no statistical difference. IVIG group n=40, control group n=32. Treg: regulatory T cell; Th17: T helper 17; IVIG: intravenous immunoglobulin.

Comparison of Treg frequency between the IVIG and control groups 15 days after embryo transfer, also showed a significant difference (p = 0.0419) and the frequency rate was higher in the IVIG group. Post treatment evaluation of CD4⁺ IL-17A⁺ Th17 cells in the IVIG group, demonstrated almost no statistically significant difference in the IVIG group and control groups (Figure 1B and Table 2).

Table 2. Treg and Th17 cell alterations in IVIG- treated and IVIG-untreated groups.

	IVIG group			Control group
	Before	After	P-value	Before	After	P-value
mRNA expression of cytokines in activated PBMC
IL-17	1	0.808 ± 0.49	NS	1	0.937 ± 0.413	NS
IL-23	1	0.912 ± 0.411	NS	1	0.9625 ± 0.37	NS
IL-10	1	1.616 ± 0.952	0.0058	1	1.232 ± 0.498	0.031
TGF-β	1	1.748 ± 1.168	0.0038	1	1.210 ± 0.355	0.0251
RORγt	1	0.805 ± 0.195	0.0218	1	0.94 ± 0.0966	NS
FoxP3	1	1.980 ± 1.023	0.001	1	1.150 ± 0.371	NS
Cytokine secretions (pg/ml)
IL-17	120.1 ± 25.63	104.5 ± 28.52	NS	113.3 ± 19.64	102.7 ± 23.95	NS
IL-23	233.8 ± 58.86	216.9 ± 73.92	NS	218.0 ± 76.96	218.1 ± 70.21	NS
IL-10	525.5 ± 304.7	640.8 ± 377.5	0.0156	577.5 ± 363.9	610.9 ± 297.5	NS
TGF-β	155.5 ± 46.29	220.3 ± 88.16	0.0413	162.3 ± 56.32	172.9 ± 55.92	NS
Cell frequency in PBMC
Th17(CD4^+ IL-17A^+)cells	3.722 ± 1.234	3.317 ± 1.22	NS	3.793 ± 1.166	3.6 ± 1.263	NS
Treg(CD4^+ FOXP3^+) cells	5.983 ± 1.789	7.717 ± 2.084	0.0186	5.950 ± 2.118	5.85 ± 2.644	NS

Treg: T regulatory; Th17: T helper 17; IVIG: intravenous immunoglobulin; IL: interleukin; RORγt: RAR-related orphan receptor gamma; Foxp3: forkhead box protein P3; NS: not significant.

Real-time PCR

Treg and Th17 associated transcription factors (Foxp3, RORγt) and cytokines (IL-10, TGF-β and IL-17, IL-23) mRNA levels were assessed in both the IVIG and control groups. Treg associated parameters demonstrated a significant difference post treatment by IVIG. The level of Foxp3 mRNA increased significantly (p = 0.0004) as well as IL-10 (p = 0.0058) and TGF-β (p = 0.0038) mRNA levels. Regarding Th17, the level of RORγt mRNA decreased at the end of treatment (p = 0.0218) in the IVIG group but there was almost no difference in the mRNA levels of IL-17 and IL-23. Comparison of the level of Foxp3 mRNA in the IVIG and control groups 15 days after embryo transfer showed a significant increase (p 0.001) in the treated group. The same was also observed in the level of IL-10 mRNA after treatment (p = 0.03). There were almost no obvious changes about mRNA level of other factors post-treatment (Figures 2 and 3 and Table 2).

Figure 2. mRNA expression level of Foxp3 and RORγt in IVIG-treated and control groups. According to real-time PCR results, IVIG therapy causes the mRNA expression level of Foxp3 to increase significantly (P value = 0.0004) 15 days after embryo transfer, but there was a decrease in RORγt mRNA expression level in this period (p value = 0.0218) (p ≤ 0.05: statistically significant). There were almost no significant differences in the results of the control group. IVIG group n=40, control group n=32. Foxp3: forkhead box protein3; RORγt: RAR-related orphan receptor gamma; IVIG: intravenous immunoglobulin.

Figure 3. mRNA expression level of IL-10, TGF-β and IL-17, IL-23 in IVIG-treated and control groups. Real-time PCR results showed that Treg associated cytokines mRNA expression level especially IL-10 (p value = 0.0058) increased post-treatment and also, an increase was observed in TGF-β (p value = 0.0038) mRNA level, but there was almost no difference in the mRNA level of IL-17 and IL-23 (p ≤0.05: statistically significant) after IVIG administration and embryo transfer. There were almost no significant differences in the control group except in the case of IL-10 (p value = 0.03). IVIG group n=40, control group n=32. IL-10: interleukin-10; TGF-β: transforming growth factor beta; IL-17: interleukin-17; IL-23: interleukin-23; IVIG: intravenous immunoglobulin.

ELISA

Cytokine secretion level of Treg and Th17 cells was measured by ELISA in the supernatant of PBMCs of the IVIG and control groups at the end of treatment. IVIG significantly increased the secretion of anti-inflammatory cytokines of Treg, especially IL-10 (p = 0.0156) and TGF- β (p = 0.0413) in the treated group. However, there was no statistically significant difference in the secretion level of Th17 associated cytokine and control group (Figure 4 and Table 2).

Figure 4. Secretion of Treg and Th17-associated cytokines. Pre and post treatment ELISA analysis revealed that IL-10 and TGF-β secretion levels increased after IVIG treatment (p value = 0.0156 and 0.0413, respectively), but there was no statistically significant difference in the secretion level of Th17 associated cytokine and control group (p ≤ 0.05: statistically significant). IVIG group n=40, control group n=32. Treg: regulatory T cell; Th17: T helper 17; IVIG: intravenous immunoglobulin; IL-10: interleukin-10; TGF-β: transforming growth factor beta; IL-17: interleukin-17; IL-23: interleukin-23.

Pregnancy outcome

The clinical pregnancy rate as defined as a positive β-HCG test result, 2 weeks after embryo transfer, was measured in the treatment and control groups. The pregnancy rate in the IVIG group was 60% in comparison with the control group, in which pregnancy rate was lower (31.2%). The live birth rate in the IVIG group was 47.5% in comparison with the control group in which the pregnancy rate was lower (21.8%) (Table 3).

Table 3. Pregnancy outcome in IVIG-treated and IVIG-untreated patients.

	Clinical pregnancy				Live birth
	No	Yes	Total	Pregnancy rate, %	No	Yes	Total	Live birth rate, %
IVIG group	16	24	40	60%	21	19	40	47.5%
Control group	22	10	32	31.2%	25	7	32	21.8%

Discussion

Among the different causes mentioned for RIF, immunologic factors are noticeable. The maternal immune system plays a role in all of the processes related to a successful pregnancy such as implantation, embryo development, placentation, etc. Cellular abnormalities in the immune system like an elevated ratio of Th1/Th2, a decrease in regulatory T cells, and especially the rate of expansion of NK cells and function are among the well-studied immunologic reasons for implantation failure [Chaouat 2016]. About 10-15% of the cellular proportion of decidua, are lymphocytes [Ruocco et al. 2014; Moffett-King 2002]. More than 70% of decidual leukocytes are NK cells. Previous studies which investigated the role of decidual NK cells (dNK), indicate that they facilitate the invasion of trophoblast cells into maternal tissues by cytokines. Placental angiogenesis is also regulated by these cells, at the fetal-maternal interface, so it is obvious that any disturbance in dNK cells impairs implantation and influences pregnancy outcome [Sotnikova et al. 2014; Hanna et al. 2006].

Many studies have demonstrated that dNK cells can act as sentinel cells to inhibit local inflammation and maintain successful pregnancy at the human maternal–fetal interface. Without NK cells, Th17 cells present as decidual inflammation and abnormal pregnancies, and thus NK cells act to maintain fetal–maternal tolerance by suppressing the induction of Th17 cells. Besides dNK, decidual CD8+ T cells are key cytolytic effector cells present at the maternal-fetal interface. CD8+ T cells are among the lymphocytes present in the decidua with an altered phenotype and function when compared with those in peripheral blood. Decidual CD8+ T cells express almost no or a reduced level of cytolitic molecules, perforin, and Granzyme B. In addition, there are some inhibitory molecules such as programmed cell death protein 1 (PD-1) and T-cell immunoglobulin and mucin-domain containing-3 (Tim-3) on the surface of decidual CD8+ T cells. They lack the effector function and promote the tolerance in fetal-maternal interface [Van Egmond et al. 2016; Tilburgs et al. 2010]. Regulatory T cells and Th17 are among the decidual lymphocytes and an appropriate balance is needed between them to create hemostasis in immune responses. A study by Lee et al. [2011] showed that an imbalance between Th17 and Treg occurs in women with recurrent pregnancy loss (RPL). They observed that the rate of Th17 cells is higher in RPL women compared to a control group in contrast with regulatory T cells, which are lower in RPL women. It is evident that a specific treatment approach is needed to modulate the disturbance in the immune system in RIF women with an immunologic basis, so immunotherapy methods are suggested as a proper approach in the treatment of implantation failure. IVIG is one of the well-studied immunotherapy agents in the treatment of infertile women who suffer from an immunologic disturbance at the time of implantation. The Lee et al. [2016] study demonstrated that RPL women with a cellular immune abnormality benefit from IVIG and the pregnancy outcome and live birth rate improves after treatment with IVIG. IVIG is also able to modulate immune cells and create a balance between inflammatory and regulatory responses via the proposed mechanisms. According to recent studies, tolerance in the immune system is created following IVIG administration but the exact mechanism(s) is still unknown. It is suggested that IVIG is able to expand regulatory T cells through the tolerogenic cytokines and pathways in order to create tolerance [Loubaki et al. 2015]. IVIG was initially used for the treatment of immunodeficiency and some autoimmune disorders, but has been found to be a suitable agent for the treatment of infertility [Ballow 2002].

In our previous study about the effect of IVIG administration on the cellular proportion of Th1 and Th2\in RPL women, we observed that IVIG is an effective therapeutic option in the treatment of reproductive failures such as RPL especially in patients with immune cell abnormalities such as an elevated rate of NK cells. IVIG was also able to improve the pregnancy outcome and live birth rate [Ahmadi et al. 2017]. Despite the importance of Th1, Th2, and NK cells, there is no adequate study regarding the effect of IVIG on the frequency and activity of Th17 and regulatory T cells. For instance, the study of Kim et al. [2014] evaluated the effect of IVIG on Th17 and Treg in women with RPL. All the subjects enrolled in the study had immune cell abnormalities such as elevated NK cell frequency and cytotoxicity and a high level of Th1 cells. At the end of the treatment, the cellular proportion of Th17 and Treg was evaluated and the rate of Th17 was down-regulated. However,there was an increase in the rate of regulatory T cells while the Th17/Treg ratio also decreased and consequently, pregnancy outcome was improved among the RPL women [Kim et al. 2014].

A study of Maddure and colleagues about the effect of IVIG on Tregs, revealed that there are several mechanisms which are involved in the modulation of these cells by IVIG. According to this study, IVIG is able to create an anti-inflammatory microenvironment, that inhibits inflammatory cytokine production by antigen presenting cells (APCs). It also contains anti-inflammatory cytokines such as TGF-, which is favorable for expansion of regulatory T cells. Activation, proliferation, function, and cytokine secretion of conventional T cells are also inhibited by IVIG-modulated Tregs, because direct interaction of IVIG and Tregs enhances the frequency and suppressive function of these cells [Maddur et al. 2010].

Recent studies demonstrate that immunotherapy of reproduction failure in women with an immunologic basis is more effective compared with treatment of women with unknown etiologies. A meta-analysis by Clark [2008] showed that the proper selection of patients for immunotherapy may improve the efficacy of treatment. In this case pre-treatment analysis of immune-related parameters in patients seems to behelpful. In this study, we selected the subjects based on cellular abnormalities such as elevated rate of CD3⁻CD16⁺CD56⁺ NK cells and cytotoxicity or elevated rate of Th1 cells. According to the results, it was concluded that etiology-based selection of patients for treatment significantly enhances the success rate and improves the pregnancy outcome. The results also revealed that Tregs were much more affected by IVIG compared with Th17, the frequency, mRNA level of Foxp3, other cytokines, and also cytokine secretion of Tregs, increased noticeably but there was almost no statistically significant difference in Th17 associated parameters.

As an immunomodulator, IVIG is able to reduce inflammatory responses and improve the process of implantation in patients who suffer from implantation failure. The results of this study suggest that proper identification of participants causes IVIG to be more helpful in the treatment of patients with immunologic abnormalities especially regulatory T cells reduction.

Materials and methods

Study population

Seventy-two women with a history of at least 3 implantation failures that were referred to Alzahra Hospital of Tabriz University of Medical Sciences from February 2015 to March 2016 were selected and enrolled for this study. The study was approved by the Research Ethics Committee of Tabriz University of Medical Sciences (IR.TBZMED.REC.1395.315). Participants signed an informed consent before enrollment. All subjects were evaluated for cellular abnormalities like elevated CD3⁻CD16⁺ CD56⁺NK cell frequency and cytotoxicity and elevated Th1/Th2 balance. Forty out of 72 received IVIG, aspirin and heparin (enoxaparin) as the IVIG group and 32 patients received aspirin and heparin (enoxaparin) and no IVIG as the control group. Exclusion criteria in this study were anatomical, infectious, endocrine, or genetic abnormalities in women; all the partners were checked for semen status according to World Health Organization (WHO) [Cooper et al. 2010] criteria and there were no smokers. The demographic data of patients are summarized in Table 1.

The IVIG group received a dose of 400 mg/kg of IVIG, 2 days before embryo transfer in addition to heparin and aspirin. All the participants in both the IVIG and control groups received heparin (enoxaparin), at a daily dose of 20 mg pre-conception, this is due to the anti-inflammation and anti-coagulation effect of heparin which is able to reduce the inflammatory responses and risk of implantation failure [Winger et al. 2011b; Kwak‐Kim et al. 2009; Nelson and Greer 2008; Allahbadia and Allahbadia 2003]. In addition, a dose of 75 mg of aspirin daily was administered for all the patients [Winger et al. 2009].

Sample collection

Peripheral blood was collected twice. The first sampling was done prior to the first IVIG administration. The second sampling was conducted 15 days after embryo transfer, at the time of positive pregnancy test.

Isolation of peripheral blood mononuclear cells (PBMCs)

A volume of 10 ml of blood samples was collected in heparinized tubes from all participants and PBMCs were isolated using Ficoll separation technique. After Ficoll (lymphosep) 1.077 g/ml (Biosera, UK) centrifugation (25 min, 450 × g), cells were washed twice with phosphate buffered saline (PBS) (Sigma, Germany). Furthermore, 10⁶ cells were cultured in 1 ml medium containing 10% heat-inactivated fetal calf serum, 100 U/ml penicillin, and 200 mM L-glutamine. Subsequently, 10 ng/ml of Phorbol myristate acetate (PMA) (eBioscience, San Diego, CA, USA) was added to the medium, and cells were incubated for 5 h at 37°C with 5% CO₂. Finally, the cultured cells were used for RNA extraction and flow cytometry analysis and the supernatant was exploited for cytokine assessment using enzyme linked immunosorbent assay (ELISA).

Immunophenotyping

Th17 and Treg cells frequency was evaluated among PBMCs using flowcytometric analysis. Prior to the test, the sample was prepared by incubating 5 × 10⁶ of PBMCs with10 ng/ml of PMA and 0.5 µM ionomycin (Sigma) at 37°C in a 5% CO₂ humidified incubator for 5 h. To improve the staining of intracellular cytokines, chemical stimulators such as Monensin (eBioscience) were used. Subsequently, the cells were washed and then incubated with monoclonal antibodies against surface and intracellular antigens such as anti-CD4-APC (BD Biosciences, San Jose, CA, USA), anti-human IL-17A-PE, or anti-Foxp3- Alexa Fluor 488 (eBioscience) at room temperature for 20 min. Prior to intracellular staining, the cells were washed twice with 1× permeabilization buffer (eBioscience) and Alexa Fluor 488- and APC rat IgG2a was used as an isotype control. A total of 1,000,000 cells were counted using a FACSCalibur flow cytometer (BD Biosciences) and CellQuest Pro software (BD Biosciences).

In the other side, a different staining method was utilized to determine Treg cells, as anti-CD-127 PerCP-Cy5.5 (eBioscience) was used additionally to anti-CD4-APC (BD Biosciences) and anti-Foxp3- Alexa Fluor 488 (eBioscience). In this method, CD4⁺ Foxp3⁺ CD127^−/low T cells were determined as Treg cells. Viable lymphocytes were gated according to their forward and side scatters profile.

mRNA expression of transcription factor and cytokines

The mRNA expression level of transcription factor (FOXp3) and cytokine s(IL-10 and TGF-) of Treg and Th17 (RORt and IL-17 and IL-23) was evaluated by RT-PCR and SYBR Green method. Primer sequences are shown in Table 4. The process of this technique is determined completely in our previous study [Ahmadi et al. 2017].

Table 4. Primer sequences.

Gene	Primer	Sequence (5’→3’)	Amplicon size(bp)
TGF- β1	Forward	CGA CTA CTA CGC CAA GGA	150
	Reverse	GAG AGC AAC ACG GGT TCA
FoxP3	Forward	CAC CTG GCT GGG AAA ATG G	63
	Reverse	GGA GCC CTT GTC GGA TGA
Beta-actin	Forward	GCA TGG GTC AGA AGG ATT CCT	106
	Reverse	TCG TCC CAG TTG GTG ACG
IL-17	Forward	CAT AAC CGG AAT ACC AAT ACC AAT	104
	Reverse	GGA TAT CTC TCA GGG TCC TCA TT
IL-23	Forward	GGA CAA CAG TCA GTT CTG CTT	115
	Reverse	CAC AGG GCT ATC AGG GAG C
IL-10	Forward	CAT CGA TTT CTT CCC TGT GAA	74
	Reverse	TCT TGG AGC TTA TTA AAG GCA TTC
RORγt	Forward	GCA GCG CTC CAA CAT CTT CT	111
	Reverse	ACG TAC TGA ATG GCC TCG GT

Cytokine analysis

Treg and Th17 associated cytokines (IL-10, TGF- and IL-17 and IL-23, respectively) were assessed in PBMCs culture supernatants, using ELISA (Biosource, Nivelles, Belgium). The process of this technique is determined completely in our previous study [Ahmadi et al. 2017].

Declaration of interest

This study was supported by a grant of the Drug Applied Research Center of Tabriz University of Medical Sciences, Tabriz, Iran (grant number: 95/26). None of the authors has any potential financial conflict of interest related to this manuscript, and none are directly employed by or funded by the Government of Iran.

Additional information

Notes on contributors

Mehdi Yousefi

Contributed to study design, helped with data collection, and performed statistical analyses: MA; Wrote the paper, was responsible for manuscript revision, and helped with data collection: SA-V; Contributed to cell collection and helped with immunological tests: MG, SD; Participated in the final edition of the manuscript: LA-M; Responsible for subject selection, monitoring, and inpatient and outpatient care: LF, AG, KH; Contributed to flowcytometric analysis: VY; Contributed to study design: MN; Principal investigator of the clinical trial and contributed to study design: MY.