Hepatitis B virus (HBV) infection remains a major public health issue, causing high morbidity in approximately one-third of the global population worldwide (Yuen et al., 2018; Alaama et al., 2022; Iannacone and Guidotti, 2022). In 2016, the World Health Organization (WHO) launched the global health sector strategy to eliminate viral hepatitis by 2030, with a target of 90% reduction of the incidence of new cases of chronic HBV infection and 65% reduction of HBV infection mortality and a target of 0.1% prevalence of hepatitis B surface antigen (HBsAg) among children in comparison with the 2015 baseline (Organization, 2016). The population with HBV infection is prone to develop chronic infection, and asymptomatic HBV carriers (ASCs) without hepatitis Be antigen (HBeAg) are the majority of chronic HBV-infected individuals, including pregnant women (PW) (Tang et al., 2018; Nguyen et al., 2020; Koffas et al., 2021; Liu et al., 2021; Cui et al., 2022). Moreover, 95% of neonates with HBV infection by mother-to-child transmission (MTCT), which is the predominant mode of HBV infection, are at high risk of developing chronic infection, and they are associated with an increased risk of HBV-related fatal liver diseases, including liver cirrhosis and hepatocellular carcinoma (Organization, 2016; Nguyen et al., 2020; Terrault et al., 2021). Therefore, focus on PW with chronic HBV infection is crucial for the prevention of MTCT of HBV, which is also one key strategy of global HBV elimination by 2030 (Organization, 2016; Organization WH, 2020; Funk et al., 2021). Previous reports showed that in general, chronic HBeAg-negative ASCs had normal alanine aminotransferase (ALT) levels and low HBV-DNA copies and were not treated with antiviral drugs, but follow-up for these patients, including PW without prenatal antiviral prophylaxis, is necessary (Cui et al., 2016; Terrault et al., 2021). The immune mechanism of PW without prenatal antiviral prophylaxis remains unclear.

Accumulating evidence shows that CD4⁺ Th cell subsets, such as Th1, Th2, Th17, T regulatory cells (Tregs) and T follicular helper (Tfh) cells, and their associated cytokines play crucial roles during pregnancy and contribute to developing a tolerant immunological microenvironment towards the foetus (Monteiro et al., 2017; Wang et al., 2020; Monteiro et al., 2021). Chronic HBV infection during pregnancy is an important agent for the number and function of CD4⁺ Th cells that can compromise the safety of the foetus by affecting the immunological environment tolerated by the PW and the foetus (Shrivastava et al., 2013; Liang et al., 2020; Huang et al., 2021). However, the role of CD4⁺ Th cell subsets remains completely unclear in PW with HBeAg-negative chronic ASCs. Therefore, CD4⁺ Th cell subsets should be further analysed in PW who are chronic ASCs and HBeAg-negative, which will contribute to the safety of the foetus by developing a tolerant immunological environment for the PW and the foetus.

In this study, the frequencies of CD4⁺ Th cell subsets were analysed in peripheral blood mononuclear cells (PBMCs) from PW who were HBeAg-negative chronic ASCs, and CD4⁺ Th cell subset-associated cytokines were also tested, which could provide a potential immune explanation for why PW with chronic ASCs did not receive antenatal antiviral prophylaxis, which is the conundrum of foetal tolerance during pregnancy with chronic HBV infection.

In the present study, we systematically assessed the frequencies of human circulating CD4⁺ Th cell subsets in PBMCs from healthy controls (HC), pregnant women (PW), and PW with HBeAg-negative chronic ASCs. The results showed that HBV-DNA and ALT levels were not different among HC, healthy PW, and PW with HBeAg-negative chronic ASCs. Moreover, the frequencies of circulating Tfh2 cells were surprisingly increased in PW compared to HC and were also positively associated with evaluated serum IL-4 levels in PW. However, Tfh1 cell frequencies were significantly reduced in PW compared with HC. Interestingly, serum P4 levels were positively associated with the frequencies of circulating Tfh2 or Th2 cells but were negatively related to the frequencies of circulating Tfh17 or Th17 cells in healthy PW. Additionally, Th22 cell frequencies were also remarkably expanded in PW with chronic ASCs compared to PW, but the frequencies of other CD4⁺ Th cell subsets and the serum levels of some key cytokines were not significantly changed among HC, PW, and PW with HBeAg-negative chronic ASCs. In summary, these data suggest a critical role of CD4⁺ Th cell subsets during pregnancy, which may help to provide potential evidence for PW withHBeAg-negative chronic ASCs and do not receive antenatal antiviral prophylaxis.

Chronic asymptomatic HBV carriers (ASCs) with HBeAg-negative are predominant among chronic ASCs, including women of childbearing age, and can also develop a potential risk of high morbidity due to HBV-related fatal liver diseases, mostly hepatocellular carcinoma and cirrhosis (Kumar et al., 2009; Organization, 2016; Cui et al., 2022; Lian et al., 2022; Wang and Chen, 2022). Previous reports indicated that the levels of serum HBV-DNA, viral antigens (HBsAg, HBeAg) and ALT were mostly normal or stable in chronic ASCs with/without HBeAg-negative status, including pregnancy (Cheung et al., 2021; Hsu et al., 2022; Wang et al., 2022). Our results showed that low HBV-DNA and ALT levels were not significantly different among HC, PW, and PW with HBeAg-negative ASCs. Accumulating evidence has not shown that HBV antiviral therapy obviously affects clinical or serological outcomes during pregnancy (Terrault et al., 2021). Although prenatal antiviral prophylaxis for chronic ASCs with and without HBeAg who have low viral titres is not commonly performed during pregnancy, regular follow-up is necessary to identify maternal or foetal indications for timely antiviral therapy (Cui et al., 2016; Funk et al., 2021).

It is well known that the foetus is semiallogenic to the maternal host during pregnancy, and maternal-foetal immune tolerance plays a key role in ensuring a successful pregnancy (Piccinni et al., 2021; Moldenhauer et al., 2022). Disturbance of the maternal immune system is an important factor that leads to foetal abortion, which mainly involves immune cells and cytokines, such as Tfh, Th1/2/17 and Treg cells, in the peripheral blood of pregnant women (Wang et al., 2020; Monteiro et al., 2021; Piccinni et al., 2021). Previous reports indicated that expanded circulating Tfh cells contributed to humoral immunity during pregnancy and were able to secrete large amounts of IL-6, IL-21 and IL-10 by helping B cells (Monteiro et al., 2017; Wang et al., 2020; Monteiro et al., 2021). Based on CXCR3 and CCR6 expression, circulating Tfh cells are divided into four subsets: Tfh1 (CXCR3⁺CCR6^-), Tfh2 (CXCR3^-CCR6^-), Tfh17 (CXCR3^-CCR6⁺), and Tfh1/17 (CXCR3⁺CCR6⁺) (Maecker et al., 2012; Huang et al., 2019; Liu et al., 2021; Feng et al., 2022). Circulating Tfh2 and Tfh17 cells can effectively help naïve B cells differentiate into plasma cells to generate antibodies, including neutralizing antibodies against immunization or pathogens such as HBV and human immunodeficiency virus (HIV), and autoantibodies to mediate the development of autoimmune diseases, including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), while Tfh1 cells lose the ability to assist naïve B cells but can contribute to the differentiation of memory B cells (Kubo et al., 2017; Vella et al., 2017; Wang et al., 2018; Lin et al., 2019; Cui et al., 2021). Our data showed that the frequencies of circulating Tfh cells, Tfh17 cells, Tfh1/17 cells and Tfr cells were not significantly changed among HC, PW, and PW with ASCs, but the frequencies of circulating Tfh2 cells were significantly increased and were positively associated with elevated levels of serum IL-4. Tfh1 cell frequencies were notably decreased in healthy PW compared to HC. These findings suggest that increased Tfh2 cells and decreased Tfh1 cells might improve pregnancy success by supporting humoral immunity, and HBV infection also affects Tfh cell subsets and functional characteristics. However, Monteiro C et al. reported that no significant difference was found in the frequencies of circulating IFN-γ⁺ Tfh1, IL-4⁺ Tfh2, and IL-17⁺ Tfh17 cells stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin between PW and nPW (Monteiro et al., 2017; Wang et al., 2020). These findings may be relevant to the definition of different Tfh cell subtypes or activated status. Additionally, a previous report showed that lower frequencies of splenic CD4⁺CXCR5⁺Bcl-6⁺ Tfh cells could contribute to successful pregnancies in mice (Fröhlich et al., 2020). The phenotypes and function of Tfh cell subsets during human and mouse pregnancy need to be further elucidated to contribute to successful pregnancy.

Accumulating evidence indicates that hormone levels including E2 and P4, favour the expansion of CD4⁺ Th cell subsets, including Treg, Th2, and Tfh cells, and inhibit the number of Th17 and Th1 cells during human and mouse pregnancy (Monteiro et al., 2017; Fröhlich et al., 2020; Wang et al., 2020; Monteiro et al., 2021). Interestingly, our results showed a positive association between serum P4 levels and frequencies of Tfh2 or Th2 cells and a negative relation between serum P4 levels and the frequencies of Tfh17 or Th17 cells in healthy PW, but not in PW with chronic ASCs, which implied that HBV infection affected the hormone levels of PW. Unfortunately, E2 levels were too high to measure in our study, which affected further analysis of some of the results, which will be explored in the future study. These findings further indicated that hormone levels in healthy PW or PW with chronic HBV infection affected the changes in CD4⁺ Th cell subsets.

The immune tolerance of the maternal-foetal microenvironment is essential for a successful pregnancy. The cumulative evidence suggests that Treg cells and Th2 cells promote allograft tolerance by repressing Th1 and Th17 cells that are accountable for spontaneous abortion, which can secrete IL-10, TGF-β and IL-4 cytokines, respectively (Liang et al., 2020; Wang et al., 2020). However, increased Th17 and Th22 cells can maintain a successful pregnancy by protecting trophoblasts from pathogens and inflammatory responses caused by intrauterine infection, including various viruses and bacteria (Logiodice et al., 2019; Wang et al., 2020; Piccinni et al., 2021). Our data showed that the population of Th22 cells was only significantly expanded in PW with chronic ASCs in comparison with PW, which implied that Th22 cells may play an important role in resisting HBV infection in PW with chronic ASCs. Th22 cells typically produce IL-22 deprived of IL-4, IFN-γ, and IL-17, which are also defined as CD3⁺CD4⁺CXCR5^-CCR4⁺CCR6⁺CCR10⁺ CD4⁺ Th22 cells, and Th22 cells are usually elevated when the human body is infected by viruses, including HBV, and play a critical role in resisting the virus and controlling inflammation (Maecker et al., 2012; Logiodice et al., 2019; Jiang et al., 2021). Moreover, the interplay of IL-22/IL-22 receptor 1 (IL-22R1) contributes to the promotion of trophoblast survival and pregnancy maintenance (Wang et al., 2020). However, the frequencies of other CD4⁺ Th cell subsets, including Th1, Th2, Th17, and Treg cells, and the serum levels of cytokines, such as IFN-γ, IL-2, IL-17, TNF-α, and IL-10, were not significantly changed among HC, healthy PW, and PW with ASCs in this study. Although these results with no statistical deviation may be partially related to some factors, including the number of enrolled subjects and the period of pregnancy, these findings provided potential evidence for why PW with chronic ASCs did not receive antenatal antiviral prophylaxis based on the immune status of the pregnant women. Previous reports indicated that CD4⁺ Th cell subpopulations had marked plasticity, had a certain conversion relationship with each other and could secrete the corresponding cytokines (Logiodice et al., 2019; Wan et al., 2020; Jiang et al., 2021; Tuzlak et al., 2021; Feng et al., 2022; Feng et al., 2022). These findings suggest that disorders of CD4⁺ T-cell subsets can easily result in foetal abortion, and their relative balance is critical to successful pregnancy.

Reproduction is one key factor for the survival and development of humans, and successful gestation is responsible for human reproduction. Parallel immune homeostasis is indispensable for protecting the semiallogeneic foetus from harmful maternal immune microenvironments during pregnancy (Piccinni et al., 2021). Pregnant women with HBeAg-negative chronic ASCs are one of the most important populations among women with chronic ASCs, and there is some debate about who needs antenatal antiviral therapy (Cui et al., 2016; Organization WH, 2020; Funk et al., 2021; Terrault et al., 2021). Our results showed expanded circulating Tfh2 cells and associated serum IL-4 levels, as well as decreased Tfh1 cells, in PW. Moreover, serum P4 levels expanded the frequencies of Tfh2 or Th2 cells but reduced frequencies of Tfh17 or Th17 cells in healthy PW, which promoted successful pregnancy. Additionally, increased Th22 cells in PW with HBeAg-negative chronic ASCs maybe contribute to the promotion of trophoblast survival and pregnancy maintenance. Taken together, these findings indicated that parallel immune homeostasis is critical for successful pregnancy, which could partially explain why PW with chronic ASCs did not receive antenatal antiviral prophylaxis.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.

The studies involving human participants were reviewed and approved by This study was approved by the Medical Ethical Committee of the Women’s Hospital, Zhejiang University School of Medicine. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

GF and DC wrote the manuscript and performed the experiment. GF and YS collected the clinical data and samples. GF, YL, CY and DC analyzed the data. DC, YS, SW and YZ revised the manuscript. DC, YZ and YoZ conceived the topic and revised the manuscript. All authors contributed to the article and approved the submitted version.