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COVID-19 vaccination influences subtypes of γδ-T cells during pregnancy

Up to now, there has been insufficient clinical data to support the safety and effects of vaccination on pregnancy post COVID-19 vaccination. The γδ-T cells are considered an important component in the immune system to fight against viral infection and exhibit critical roles throughout the pregnancy period. However, the immunological roles of γδ-T cells in pregnant women with the COVID-19 vaccination remain unclear. Therefore, the objective of this study is to investigate the alteration of frequency and expression pattern of activation receptors and inhibitory receptors in γδ-T cell and its subsets in peripheral blood samples collected from non-pregnant vaccinated women, vaccinated pregnant women, and unvaccinated pregnant women. Our findings indicated that the frequency of CD3+γδ-T+ cells is lower in vaccinated pregnant women than in unvaccinated pregnant women. But no significant difference was found in the frequency of CD3+γδ-T+ cells between non-pregnant vaccinated women and vaccinated pregnant women. In addition, there were no significant differences in the frequencies of CD3+γδ-T+Vδ1+T cells, CD3+γδ-T+Vδ2+T cells, CD3+γδ-T+Vδ1-Vδ2-T cells, and Vδ1+T cell/Vδ2+T cell ratio between the pregnant women with or without COVID-19 vaccination. Similar results were found after comparing non-pregnant and pregnant women who received the COVID-19 vaccine. However, there was a significant difference in the fraction of Vδ1-Vδ2-T cells in CD3+γδ-T+ cells between non-pregnant vaccinated women and vaccinated pregnant women. The frequency of NKG2D+ cells in Vδ2+T cells was not significantly different in the vaccinated pregnant women when compared to that in unvaccinated pregnant women or non-pregnant vaccinated women. But the percentage of NKG2D+ cells in Vδ1+T cells was the lowest in pregnant women after COVID-19 vaccination. Furthermore, down-regulation of NKP46 and NKP30 were found in Vδ2+T and Vδ1+T cells in the vaccinated pregnant women, respectively. After the vaccination, up-regulation of PD-1 expression in Vδ1+T cells and Vδ2+T cells indicated γδ-T cells could respond to COVID-19 vaccination and display an exhausted phenotype following activation. In conclusion, COVID-19 vaccination influences subtypes of γδ-T cells during pregnancy, but the side effects might be limited. The phenotypical changes of Vδ1+T cells and Vδ2+T cells will be a promising predictor for evaluating the clinical outcome of the COVID-19 vaccine.

Up to now, there has been insufficient clinical data to support the safety and effects of vaccination on pregnancy post COVID-19 vaccination. The gd-T cells are considered an important component in the immune system to fight against viral infection and exhibit critical roles throughout the pregnancy period. However, the immunological roles of gd-T cells in pregnant women with the COVID-19 vaccination remain unclear. Therefore, the objective of this study is to investigate the alteration of frequency and expression pattern of activation receptors and inhibitory receptors in gd-T cell and its subsets in peripheral blood samples collected from non-pregnant vaccinated women, vaccinated pregnant women, and unvaccinated pregnant women. Our findings indicated that the frequency of CD3 + gd-T + cells is lower in vaccinated pregnant women than in unvaccinated pregnant women. But no significant difference was found in the frequency of CD3 + gd-T + cells between non-pregnant vaccinated women and vaccinated pregnant women. In addition, there were no significant differences in the frequencies of CD3 + gd-T + Vd1 + T cells, CD3 + gd-T + Vd2 + T cells, CD3 + gd-T + Vd1 -Vd2 -T cells, and Vd1 + T cell/Vd2 + T cell ratio between the pregnant women with or without COVID-19 vaccination. Similar results were found after comparing non-pregnant and pregnant women who received the COVID-19 vaccine. However, there was a significant difference in the fraction of Vd1 -Vd2 -T cells in CD3 + gd-T + cells between non-pregnant vaccinated women and vaccinated pregnant women. The frequency of NKG2D + cells in Vd2 + T cells was not significantly different in the vaccinated pregnant women when compared to that in unvaccinated pregnant women or non-pregnant vaccinated women. But the percentage of NKG2D + cells in Vd1 + T cells was the lowest in pregnant women after COVID-19 vaccination. Furthermore, down-regulation of NKP46 and NKP30 were found in Vd2 + T and

Introduction
Due to a lack of clinical and scientific knowledge, the challenge of the Coronavirus Disease 2019 (COVID-19) pandemic has exposed the limitations of our understanding about severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as well as the immune response during viral infection and vaccination (1). Pregnant women especially are at increased risk of severe illness from COVID-19. There have been a few studies that demonstrated the efficacy and safety of COVID-19 vaccines in pregnant women, who have been excluded from the clinical trials because of the ethical issues (2). However, we still struggle with a complete understanding about whether vaccination could modulate the immune response of pregnant women (3). During normal human pregnancy, gd-T cells have been reported to play a role in this process (4).
Human gd T cells can be divided into three subgroups, Vd1, Vd2 and Vd3, according to the structural differences of g and d chains. Although gd-T cells represent only a small fraction of T lymphocytes (1-10%) (5), they have a variety of immune functions, such as resistance to virus infection (6), inflammatory regulation, tissue homeostasis (6), helping B cells to produce antibodies (7), and even maintenance of successful pregnancy (6). Some studies reported that, during normal human pregnancy, gd-T cells secrete some antiinflammatory cytokines to reduce nature killer activity (8,9).Indeed, gd-T cells regulate the release of inflammatory factors such as IFN-g,TNF-a, granzyme A/B, and perforin by regulating the expression of surface-activated receptors such as NKG2D, NKp30, NKP46, and the inhibitory receptor PD-1 to regulate their cytotoxic functions (10-13). In addition, low cytotoxic activity of gd-T cells is necessary during normal pregnancy (14). Most importantly, the imbalance between Vd1 + T cells and Vd2 + T cells was observed in adverse pregnancy (15). Therefore, in this study, we aimed to interrogate the alternation of subtypes of gd-T cells in pregnant women after COVID-19 vaccination, which could provide a better understanding of the role of gd-T cells in pregnant women after COVID-19 vaccination, and in turn can further help us to monitor and evaluate the safety and efficacy of COVID-19 vaccines during the pregnancy period.

Study population
The research objects were selected from pregnant women vaccinated against COVID-19 who went to Tianhe Campus of the Third Affiliated Hospital of Sun Yat-sen University in Guangzhou, China from August 2021 to February 2022. These women did not confirm their pregnancies until after they had received the COVID-19 vaccine. The inclusion criteria included: (1) age of 18-35 years; (2) singleton pregnancy; (3) the onset of pregnancy as calculated by crown-rump length (CRL) on NT ultrasound, with at least one dose of vaccine after pregnancy; and (4) signing an informed consent form, providing vaccination information and confirming their participation in the study. The exclusion criteria included: (1) the gestational age of the documented prenatal examination is not 11-13+6 weeks; (2) other vaccines have been received within 1 year, such as HPV vaccine and hepatitis B vaccine; or (3) complications with basic diseases, requiring long-term medication. The peripheral blood of the patients enrolled in this study was collected to perform immunological assays. The study included 27 vaccinated pregnant women, 11 unvaccinated pregnant women, and 20 non-pregnant vaccinated women. The clinical and demographic characteristics and vaccination scheme of pregnant women were described in Tables 1, 2, respectively. This study was approved by the Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University in Guangzhou.

Statistical analysis
Statistical analyses were performed using GraphPad Prism (GraphPad Software, Inc.). All results are expressed as the mean ± SEM (standard error of the mean). To analyze the difference in gd T cells and its subsets between vaccinated women and unvaccinated women, Mann-Whitney U tests were performed.

Results
The frequencies of gd T cell and its subsets in pregnant women after COVID-19 vaccination The aim of this study was to investigate whether COVID-19 vaccination influences the distribution of total gd T cells, Vd1 + T cells, Vd2 + T cells, and Vd1 -Vd2 -T cells in the peripheral blood samples of 58 individuals enrolled in this study. Total gd T cell was identified among CD3 + lymphocytes using flow cytometry ( Figures 1A, B). The results indicated that frequency of total gd T cells was markedly higher in unvaccinated pregnant women compared to vaccinated pregnant women, but there was no significant difference between non-pregnant vaccinated women and vaccinated pregnant women. Furthermore, to investigate the impact of COVID-19 vaccination on frequencies of gd T cells subsets, we next compared Vd2 + T cells, Vd1 + T cells, and Vd1 -Vd2 -T cells' frequencies as well as Vd1/Vd2 ratio in non-pregnant vaccinated women, vaccinated pregnant women, and unvaccinated pregnant women. The results indicated that there were no significant differences in frequencies of Vd1 + T, Vd2 + T cells, and Vd1 -Vd2 -T cells in CD3 + gd + T cells between vaccinated pregnant women and unvaccinated pregnant women (Figures 1C, E). However, compared with non-pregnant vaccinated women, the proportion of Vd1 -Vd2 -T cells in CD3 + gd + T was significantly reduced in vaccinated pregnant women ( Figure 1D). Additionally, Vd1/Vd2 ratio was similar in these women with or without vaccination ( Figure 1D).

Phenotypical changes of Vd2 + T cells in vaccinated pregnant women
It is well known that the activating and inhibitory receptors define the degree of immune cell maturation and responsiveness to stimuli, so we next investigated the frequencies of NKG2D + , NKp30 + , NKp46 + , and PD-1 + cells in Vd2 + T cells. We found no significant difference in the proportions of NKG2D + Vd2 + T cells among these three groups (Figure 2A). gd T cell subsets usually express activating natural killer (NK) receptors, such as NKp30 and NKp46, which are involved in regulating immunological functions of gd T cell and its subsets. There was no significant difference in percentage of NKp30 + Vd2 + T cells between vaccinated pregnant women and unvaccinated pregnant women ( Figure 2B). In addition, a similar result was found in comparison of vaccinated pregnant women and non-pregnant vaccinated women ( Figure 2B). By contrast, there was no   Figure 2C). Furthermore, we supposed that gd T cell would exhibit the exhausted phenotype after vaccination. Actually, the frequency of PD-1 + Vd2 + T cells was significantly elevated in vaccinated pregnant women compared to unvaccinated pregnant women. However, there was no difference between non-pregnant vaccinated women and vaccinated pregnant women ( Figure 2D).

Phenotypical changes of Vd1 + T cells in vaccinated pregnant women
To investigate the alteration of expression pattern of activating and inhibitory receptors in Vd1 + T cells after COVID-19 vaccination in the pregnant women, the frequencies of NKG2D + , NKp30 + , NKp46 + , and PD-1 + cells in Vd2 + T cells were determined. Unsimilar to Vd2 + T cells, the frequencies of NKG2D + and NKp30 + cells in Vd1 + cells were much lower in vaccinated pregnant women than that in unvaccinated pregnant women ( Figures 3A, B). In addition, a significant difference in the frequency of NKG2D + Vd1 + cells was found between nonpregnant vaccinated women and vaccinated pregnant women. Similar to Vd2 + T cells, the frequency of NKP46 + cells were no different in all three groups ( Figure 3C). Finally, PD-1 + Vd1 + T cells were significantly increased in the pregnant women after vaccination ( Figure 3D).

Discussion
Our study demonstrated that frequencies of total gd T cell (CD3 + gd + T cells) was significantly decreased in the pregnant women with COVID-19 vaccination compared to that in the pregnant women without COVID-19 vaccination. Furthermore, analysis of the frequencies of Vd1 + T cells and Vd2 + T cells in total gd T cells indicated that there were no significant differences between vaccinated pregnant women and unvaccinated pregnant women. Importantly, no significant difference was found on the Vd1/Vd2 ratio between these two groups of women who were with or without COVID-19 vaccination. For evaluation of activated and exhausted phenotypes in of Vd1 + T cells and Vd2 + T cells after COVID-19 vaccination in the pregnant women, the frequencies of NKG2D + , NKp30 + , NKp46 + , and PD-1 + cells in these two subsets were analyzed. Our findings suggested that Vd1 + T cells and Vd2 + T cells developed an exhausted phenotype post COVID-19 vaccination. These results demonstrate that COVID-19 vaccination exhibits a certain degree of influence on the frequency of total gd T cell and alteration of phenotype of Vd1 + T cells and Vd2 + T cells. However, the Vd1/Vd2 ratio is similar between vaccinated pregnant women and unvaccinated pregnant women, which indicates that vaccination did not break the important balance between these two main subsets of peripheral gd T cells. Taken together, the results suggest A B D C FIGURE 3 Expressions of crucial molecular of Vd1 + gd T cells in non-pregnant vaccinated women, vaccinated, and unvaccinated pregnant women. Comparable analysis of expressions of NKG2D (A), NKP30 (B),NKP4 6 (C), and PD-1 (D) receptors of Vd1 + gd T cells acquired by flow cytometry. ns, no significance; *P < 0.01; ****P < 0.001.

COVID-19 vaccination influences subtypes of gd-T cells without effects on pregnancy.
In this study, we found that the frequency of total gd-T cells was similar between the pregnant women and non-pregnant women after COVID-19 vaccination ( Figure 1A). In addition, there were no significant differences in frequencies of Vd1 + T cells and Vd2 + T cells between these two groups of individuals. A similar result was also found in the Vd1 + T/Vd2 + T ratio ( Figure 1C). However, the frequency of Vd1 -Vd2 -T cells was much higher in the non-pregnant vaccinated women compared with that in vaccinated pregnant women ( Figure 1D). Furthermore, our data indicated that the proportions of NKP46 + Vd2 + T cells and NKG2D + Vd1 + T cells were lower in the vaccinated pregnant women than that in non-pregnant vaccinated women (Figures 2C, 3A). By contrast, no significant differences were found in the percentages of NKG2D + , NKP30 + , and PD-1 + cells in Vd2 + T cells and NKP30 + and NKP46 + cells in Vd1 + T cells between the nonpregnant vaccinated women and vaccinated pregnant women (Figures 2A, B, D; Figures 3B-D). These data further demonstrated that pregnancy is not a potential influencing factor for our findings, and this will help us to achieve more confident conclusions.
In humans, Vd1 + T cells and Vd2 + T cells are the two major subsets of gd T cells which are identified by the Vd chains. Vd1 + T cells constitute the majority of T cells in the thymus and mucosal tissues, and Vd2 + T cells are predominant in the peripheral blood (16). As the main gd T cell subsets, Vd1 + T cells and Vd2 + T cells exhibit different immunological functions. Vd1 + T cells display regulatory and effector features, and Vd2 + T cells exert a cytotoxic activity targeting pathogenic characteristics. According to previous studies, the frequency of peripheral gd T cells is higher in women with a successful pregnancy compared to women with pregnancy failure (9). In addition, one study suggested that Vd1 + T cells could produce IL-10 to down-regulate the cytotoxic NK cells during pregnancy. In healthy pregnant women, the predominant subpopulation of peripheral gd T cells is Vd1 + T cells, whereas Vd2 + T cells is the most frequent subset in women with recurrent miscarriage (16). Therefore, this evidence demonstrated that an imbalance of Vd1/Vd2 ratio leads to adverse pregnancy outcome (15,16). It has been reported that gd T cells (mostly expressing Vd2) are able to destruct influenza A virus-infected cells as efficient as CD8 + T cells or NK cells in a polycytotoxic manner and by releasing IFN-g against infected cells in vitro (17). In several contexts, including infection with Mtb, malaria, influenza, and HIV and vaccination with BCG and live attenuated influenza, there are clear patterns of gd T-cell expansion, particularly of the Vd2 + subset, in response to both infection and vaccination (18). However, there is no literature report on the effect of other vaccines after pregnancy on gd T cells' function. In the future, we could detect the difference in gd T cells' function between other vaccines and the COVID-19 vaccine during pregnancy, so as to explore whether this result is specific to the COVID-19 vaccine. In our study, COVID-19 vaccination did not change Vd1/Vd2 ratio in the pregnant women, which indicates the impact of vaccination might not cause an adverse outcome.
NK cell activating receptors, such as NKG2D, NKp30, and NKp46, are widely involved in regulating NK functions during pregnancy (19). Additionally, one recent study suggested that the frequency of NKG2D + Vd2 + T cells was negatively correlated with a successful clinical pregnancy (8). Our data demonstrate that no significant difference was found on the percentages of NKG2D + cells in Vd1 + T cells and Vd2 + T cells, which suggest that COVID-19 vaccination did not induce the highly activated peripheral gd T cells in the pregnant women. Because of the activated gd T cells, a high level of inflammation is considered as the major cause of abortion (15). Additionally, frequencies of NKp30 + and NKp46 + cells in Vd1 + T and Vd2 + T were largely similar between vaccinated pregnant women and unvaccinated pregnant women. In summary, these data suggested that the immunological functions of gd T cells were not altered after COVID-19 vaccination in the pregnant women.
Accordingly, many previous studies suggested that exhaustion of gd T cells is accompanied by a decrease in the frequency of cells in different types of disease. For instance, in acute myeloid leukemia (AML), the data demonstrated that the proportion of total gd T cells was decreased in AML patients (20). Subsequently, in these gd T cells, the authors observed increased PD-1 expression and decreased NKG2D expression, indicating highly activated or even exhausted states in the gd T cells at diagnosis of AML (20). Additionally, in the acute viral infection, a lower frequency of Vd2+T cells was also observed (21). Moreover, these Vd2 + T cells highly expressed CD95, which in turn could lead to cell apoptosis that induces the loss of cells (21). Interestingly, this work demonstrated that the expression level of CTLA-4 exhaustion marker was elevated during acute viral infection. In line with these previous results, we also found that stimulation of COVID-19 vaccine induces the exhaustion status within the loss of gd T cells in pregnant women, suggesting the involvement of gd T cells in the complex network of protective response induced by COVID-19 vaccination. PD-1 expression is induced on activated T cells and is correlated to exhaustion status in anti-infection and anti-tumor responses (22, 23). In our study, we found that the frequency of PD-1 + cells in Vd1 + T and Vd2 + T were much higher in vaccinated pregnant women compared to unvaccinated pregnant women. We suspect that gd T cells developed an exhausted phenotype following activation by COVID-19 vaccination. This switch from activation and exhaustion might be the reason why the frequency of gd T cells was decreased in the pregnant women with COVID-19 vaccination. Since the objective of this study is to investigate whether COVID-19 vaccination influences subtypes of gd-T cells during pregnancy, the changes of frequency and immunological phenotypes of gd-T cells were determined in this study. gd-T cells are well known as having multiple functions in innate immune cells, suggesting they play important roles in anti-viral infection and immune response to vaccination. The results of our study could be considered as the clues to address the questions about the potential immunological functions of gd-T cell and its subsets involved in the immune activity associated with COVID-19 vaccination. This deserves further study in the future.
Despite the positives provided by this study, there are still some limitations. First, a further larger sample size study is warranted to validate these findings. And the effects of age, vaccination scheme, vaccine types, and other factors in impacting the immunological features of gd T cell should be further evaluated in pregnant women after COVID-19 vaccination. Second, in this study, we only determined the phenotypic changes of gd T cell subsets in the peripheral blood of individuals. The functional capacity of gd T cell and its subpopulations are rarely investigated in COVID-19 vaccination and should be determined in the future. Third, we found the phenotypic changes of gd T cell subsets by comparing the data collected in the pregnant women with or without COVID-19 vaccination. In the future, using samples from each vaccinated individual at different time points in the vaccination scheme will further help us to clarify the significance of functional alterations of gd T cell and its subsets in establishing protective immunity against COVID-19 infection after vaccination.
Taken together, our study suggests that gd T cell and its subsets could respond to COVID-19 vaccination and display an exhausted phenotype following activation. In addition, COVID-19 vaccination influences subtypes of gd-T cells during pregnancy, but the side effects are limited. Last but not least, the contribution of gd T cell and its subsets to the immunology of COVID-19 vaccination needs to be further investigated.

Data availability statements
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement
This study was approved by the Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University in Guangzhou, PR China. The participants provided their written informed consent to participate in this study. The patients/participants provided their written informed consent to participate in this study.