Characteristics of Peripheral Immune Function in Reproductive Females with Uterine Leiomyoma

Inflammation and immunity are thought as risk factors for uterine leiomyoma; however, detailed reports on this topic are scarce. The present study aimed to analyze the characteristics of immune function and clinical significance of circulating CD4/CD8 T, NK, and γδ T cells in reproductive females with uterine leiomyoma. We analyzed the above-mentioned cells in 30 reproductive females with uterine leiomyoma and 68 healthy females using flow cytometry. After that, the correlation between function of immune cells and clinical phenotypes was analyzed. Compared with healthy controls, central memory (CM) CD4/CD8 T cells as well as Treg and Tfh cells were notably increased in leiomyoma patients; however, NK and γδ T cells were decreased in patients. Moreover, such alterations of these cells in patients with leiomyoma were associated with shorter menstrual cycles, longer menstrual period, anemia, pelvic lesions, more and larger myomas, and higher levels of CA125. Additionally, the increased Tfh1/Tfh2 ratio and Tfh17 were significantly associated with longer menstrual period, more myomas, and higher CA125 levels independent of age in patients with uterine leiomyoma. In conclusion, hallmarks of peripheral immune function are remarkably correlated with clinical phenotypes in reproductive females with uterine leiomyoma. This preliminary work may provide proof-of-concept for evaluating efficacy of treatment and prognosis of reproductive females with uterine leiomyoma with the help of quantitative analysis of peripheral immune function, which may inspire performing further investigations on the relevance of immune function with different diseases.


Introduction
Uterine leiomyoma, a type of benign neoplasm, commonly occurs among 50-60% of fertile women [1], the rate may reach 70% for women who are elder than 50 years old [2]. Among leiomyoma patients, 15-30% of cases may develop into severe symptoms, such as infertility, menorrhagia, and constipation [3]. In addition to hysterectomy, the existing therapeutic strategies mainly involve myomectomy by hysteroscopy, laparotomy or laparoscopy, uterine artery embolization, and interventions performed under radiological or ultrasound guidance, depending on patients' age and the number, size, and location of the fibroids [3]. Clinically, fibroids account for 30-50% of all hysterectomies and are associated with substantial morbidity and health care costs for women of reproductive age [1]. erefore, uterine leiomyoma is harmful for women particularly for those of reproductive age.
Despite the high incidence of this disease, its etiology still remains largely unclear. Previous studies reported that a number of high-risk factors, including early menarche [4], lifestyle (diet, caffeine, and alcohol consumption) [5], obesity and metabolic disturbance [6], hereditary factor, and late age of first pregnancy [7], may increase the susceptibility of this tumor. Regarding pathogenesis of uterine leiomyoma, one hypothesis is that chronic inflammation favors onset, development, and recurrence of uterine fibroid [8]. Previous studies reported that abnormal expression of immune genes and decreased number and dysfunction of uterine NK cells were associated with leiomyoma risk [9,10], as well as the aberrant blood vessel development and subfertility in patients with leiomyoma [11]. In addition to the abovementioned factors, obesity, pregnancy, and menses also affect secretion of cytokines [12]; the latter can influence proliferation of neoplastic cells, fibrosis, and angiogenesis in uterine, which in turn sustain the formation and growth of fibroid [8]. Moreover, inflammation is an important cause of leiomyoma recurrence [8], frequently occurring in fibroid [13]: chronic inflammation is a key factor in awakening dormant tumor cells which are residual at the primary site, leading to recurrence [4,8]. erefore, host immunity may play a substantial role in tumorigenesis and development of uterine leiomyoma.
To date, a limited number of studies have concentrated on the relationship between peripheral immune function and fibroid. In the present study, we proposed a quantitative assessment method for host peripheral immunity using flow cytometry. For this purpose, we attempted to investigate alterations of peripheral immune function, including CD4+, CD8+ T cells, natural killer (NK) cells, and gamma delta T (cδ T) cells, among Chinese females with the age of 20∼40 years. e current study may provide a reference to detect underlying pathogenesis of uterine leiomyoma from the immune function aspect, which will inspire more investigation on the role of peripheral immune function in the occurrence, development, and prognosis of uterine leiomyoma.

Participants.
In the present study, 30 patients with uterine leiomyoma who aged 20∼40 years old were enrolled at Baoan Maternal and Child Health Hospital, Jinan University (Shenzhen, China) between June 2018 and December 2018. All patients received hysteroscopy, and their diagnoses were verified by pathological examinations. At the day before operation, a 2 mL of anticlotting blood sample was collected for immune test from each case after signing the written informed consent form. Moreover, the patients' data of menstrual cycle, clinical features of myomas, the values of blood tests, and complications of leiomyoma were collected from medical records.
During the same time, 68 healthy females who aged 20∼40 years old were randomly recruited from the subjects who arrived our hospital for premarital or prepregnancy medical examinations, with exclusion of subjects with endometrial polyps, ovary morbidities, infertility, leiomyoma, and other tumors. After signing the consent form, information of the healthy controls such as age, menstrual cycle and period, and reproductive history were collected by trained nurses through a questionnaire survey, and 2 mL of venous blood was collected for flow cytometry analyses.
All the research subjects were unrelated ethnic Han Chinese population of southern China. e respond rates of both patients and controls were more than 90%. e present study was approved by the Ethics Committee of the Baoan Maternal and Child Health Hospital, Jinan University (IRB no. LLSC-2018-02-01).
Samples were run on a BD LSR Fortessa ™ cell analyzer (BD Biosciences) at Shuangzhi Purui Medical Laboratory Co., Ltd. (China), and the data were analyzed using FlowJo 10.1 software (Tree Star Inc., Ashland, USA).

Statistical Analysis.
e SPSS 25.0 software (IBM, Armonk, NY, USA) was used to analyze the data. Data were presented as n, proportion, median, and min/max. Mann-Whitney U tests were performed to analyze the differences of immune indexes between the patients and controls. e relationship of above indexes and clinical phenotypes were performed using Spearman correlation analyses. Lastly, partial correlation analysis and stratification analysis were performed to control the confounding factor age. All tests were two-sided, and the level of significance was set at 0.05. Statistical figures were produced by GraphPad Prism7.0 (GraphPad Software, USA).

Clinical Characteristics of Patients and Healthy Controls.
e clinical characteristics of patients and healthy controls were presented in Table 1. It was revealed that the patients with fibroid were older than the healthy controls (median: 36-year vs. 30-year, P < 0.001). erefore, age was herein used in the latter partial correlation analysis and stratification analysis.

CD4+ T-Cell Differentiation Associates with Shorter Menstrual Cycle in Leiomyoma Patients.
e difference in circulating T-cell population between leiomyoma patients and healthy controls was analyzed, and the results were shown in Figure S1. Compared with healthy controls, the double-negative T cells (CD4− CD8− ) were significantly lowered in patients (P � 0.034). However, the CD4+, CD8+, and double-positive T cells (CD4+CD8+) were not different between patients and healthy groups (all P values >0.05).
We further analyzed correlations between clinical phenotypes listed in Table 1 in patients and double-negative T cells, naïve CD4+ T cells, CM CD4+ T cells, or EMRA CD4+ T cells. We found that CM CD4+ T cells rather than other CD4 subsets were reversely correlated with menstrual cycle (r � − 0.395, P � 0.031, Figure 1(c) and Table S1).
e associations between the above-mentioned cells and the clinical phenotypes in fibroid patients were further analyzed. As illustrated in Figure 2 and Supplementary Table 1, we found significant associations as following:  Figure 3 illustrated that CM CD8+ cells and cytotoxic T cells (Tc1) (CD8+CXCR5− CXCR3+CXCR4− ) were significantly increased in patients with uterine leiomyoma compared with healthy controls; while Tc17 cells (CD8+CXCR5− CXCR3+ CXCR4− ) were decreased. However, naïve, EM, and EMRA CD8 cells were not significantly different between patients and controls (all P values >0.05).

Differentiation of CD8+ T Cells in Uterine Leiomyoma.
We further analyzed the relationship between CD8+ T-cell differentiation and clinical phenotypes. As shown in Figure 3 and Table S1, only Tc1 cells were positively correlated with higher Hb levels. In EMRA CD8+ cells, which Pelvic lesions (n, %) 23 (76.7%) -Salpingitis (n, %) 16  Journal of Oncology 3 are mainly circulated into the peripheral tissues and retained in these tissues [16], CD127 high expression subset cells (CD8+CCR7− CD45RA+CD127 high ) increased, while CD127 low expression subset cells decreased in patients ( Figure 3(d)). Furthermore, increased proportion of CD127 high cells in EMRA CD8+ cells was associated with larger myomas in patients with leiomyoma.

Function Depletion of NK Cells in Leiomyoma Patients
with Longer Menstrual Period and Anemia. Since NK cells play pivotal roles in the innate immunity, functional characteristics of NK cells were also explored using flow cytometry ( Figure 4 and Table S1). As displayed in Figure 4, we found that both CD56 bright and CD56 dim cells as well as NKT cells (CD3+CD56+) were significantly reduced in peripheral blood of patients compared with healthy controls.
is apparently demonstrated reduction of total amount of NK cells, implicating with impaired innate immune function. Furthermore, we revealed that functional subsets of NK cells, including CD94+KIR− , NKP30+, NKP46+, and NKG2D, significantly decreased in patients (all P values <0.01, Figure 4(b)). Additionally, we found positive correlation between decreases in CD56+NKG2D+ NK cells and reduction of RBC, reflecting that NK cells would be associated with anemia in leiomyoma patients (r � 0.417, P � 0.022, Figure 4(d)).

Depressed Immune Function of cδ T Cells in Leiomyoma
Patients. Given the unique function of cδ T cell in antitumor immunity, it is of significant importance to detect how cδ T cells were functionally altered in patients with uterine leiomyoma. Firstly, we found that, compared with healthy controls, patients have remarkably lower amount of cδ T cells (P < 0.001, Figure 5(b)). Moreover, Vδ1 cells (immune depression subset) increased, while Vδ2 cells (antitumor subset) decreased in patients, which led to elevation of the ratio of Vδ1/Vδ2 in those patients (all P values <0.05, Figure 5(d)). Furthermore, the findings indicated that Vδ2+NKG2D+ cδ T cells were decreased, whereas Vδ2+NKP30+ cδ T cells were increased in leiomyoma patients (both P values <0.05, Figure S2 and Table S1), which contributed to the decrease of CA125 level in patients.
is was further supported by correlation analyses, exhibiting a negative correlation between Vδ2+NKG2D+ cδ cells and CA125 level in leiomyoma patients.

Age Variation Slightly Influences Immune Function.
Considering age of patients and healthy controls distributed between 26-40 and 22-36 years old, respectively, we therefore analyzed the association between age variation and above important immune function indexes (e.g., CM CD4+ T, CD8 EMRA CD127 high , Treg, 2, Tfh1, Tfh17, CD56+NKG2D+, Vδ2, and Vδ2+NKG2D+ cells) to test how age variation may affect immune function (Table S2). We found that all of these immune indexes showed no statistical correlation with age variation in healthy controls (all P values >0.05); as for patient group, only CM CD4+ T cells showed a correlation with age (r � − 0.456, P � 0.011). erefore, these results suggested that age variation was not a main factor affecting immune function in the context of this study.
Additionally, we further evaluated the relationship between immune function indexes and clinical phenotypes using partial correlation analysis. We found that the increased Tfh1/Tfh2 ratio remained significantly correlated with longer menstrual period, more myomas, and higher CA125 levels in patients with uterine leiomyoma, and Treg, Tfh17, and Vδ2+PD− 1 cells showed a notable correlation with higher CA125 and more myomas as well (

Discussion
Currently, little is known on how peripheral immune cell function alters in the context of diseases because the existing clinical blood tests provide only ratios of a certain types of immune cells, such as CD3+, CD4+, CD8+, monocytes, and neutrophils. However, such examinations do not give a whole picture or clues on how these immune cells may  Journal of Oncology influence immune function. In the present study, we evaluated hallmarks of peripheral immune cell function of reproductive females with uterine leiomyoma.
Firstly, we assessed T-cell function and found that CD4− CD8− T cells were significantly decreased in fibroid patients in this study.
ere are three subtypes of * * * CD4− CD8− T cells, which are infrequent in the peripheral blood, including cδ T cells, a subset of NKT cells, and double-negative T cells from the thymus [17]. In the current research, we did not find a significant difference in the levels of NKT cells between patients with uterine leiomyoma and healthy controls (data were not shown).
Combing the findings of the cδ T cells, it could be concluded that the reduced levels of CD4− CD8− T cells might reflect the depressed function of cδ T cells in fibroid patients in this study. en, we analyzed CD4+ T-cell function and found that CD4+ T-cell differentiation was modulated, including an increase of naïve CD4+ T cells. e naïve CD4+ T cells can differentiate into Treg, Tfh, and cells [15]; therefore, these cells were further analyzed in the present study. We found that Treg cells were also unregulated in fibroid patients with higher CA125 levels, even after controlling age as a confounding factor. Treg cell is a type of immunosuppressive T-cell population, preventing autoimmunity and maintaining immunological tolerance [18]. Infiltration of a large number of Treg cells into tumor tissues is often associated with tumor development and poor prognosis [19], while removal of Treg cells is able to evoke and enhance antitumor immune response [20,21]. us, it is plausible that Treg cells increased in leiomyoma patients with higher levels of tumor markers in this study. Combing our findings and above published reports, Treg cells can serve as a biomarker for leiomyoma development. In addition, Tfh cells and subgroups (Tfh1 and Tfh17 cells) were also unregulated in the patients with uterine leiomyoma, associating with longer menstrual periods, higher CA125 levels, more myomas, or with pelvic lesions in this study. With controlling age as a confounding factor, Tfh cells and their subgroups were herein found as independent risk factors for uterine leiomyoma. e role of Tfh cells in pathology of fibroid is still elusive. ese cells are critical for activation of B cells, antibody class switching, and germinal center (GC) formation [22]. Recent analyses of human blood cells identified major functional subsets (Tfh1, Tfh2, and Tfh17), with specific Tfh subsets correlated with broad neutralizing antiviral Ab or auto-Ab [23]. Activation of Tfh cell response is essential for infection clearance, whereas unregulated Tfh-mediated immune response is implicated in inflammation progression [9,10,24], as a risk factor for uterine leiomyoma [8]. Gu-Trantien et al. found that Tfh cells may be a key factor in immune suppression of antitumor responses in the chronic inflammatory microenvironment [25], suggesting that Tfh cells are correlated with tumor progression. Combing these reports and our data, the findings of the association of the Tfh1/Tfh2 ratio with more myomas and higher values of CA125 are credible, so as the relation between Tf17 and more myomas. Moreover, the function of Tfh cells was reported to be mediated by exposure to sex hormone in mice [26] and fish [27]. e relationship of Tfh cells with longer menstrual period could be explained via the immune dysfunction by hormonal exposure. us, Tfh cells and their subgroups are feasible indexes for progression of uterine leiomyoma and severity of symptoms.
Moreover, a decrease in 2 cells was found in the patients with longer menstrual periods in the current study.
2 cells secret a large amount of cytokines, such as IL4 in the luteal phase [28], which are characteristics of extracellular defense as well as self-cell tolerance [29]. 2 bias is observed in premenarche, postmenopause, and pregnancy, which wholly contribute to attenuate risk of uterine leiomyoma [30]. erefore, reduced 2 levels in fibroid patients are plausible in this study. In addition, estrogen replacement therapy [31] and high progesterone/estradiol ratio [32] in luteal phase or pregnancy may affect the function of 2 cells. us, it is plausible that 2 cells are associated with aberrant menstrual cycles in this study. Our findings highlighted 2 cell as a biomarker for hormone therapy in uterine leiomyoma.
Furthermore, the present study indicated that CD8+CCR7− CD45RA+CD127 high cells, a subgroup of CD8 memory cells, were associated with larger myomas. CD127 is the alpha chain of the IL-7 receptor located in CD8 cells and is involved in the survival, homeostasis, and function of T cells [33,34], especially the memory T-cell mediating chronic inflammatory diseases [35]. Our data on CD8+CCR7− CD45RA+CD127 high cells were consistent with the theory that chronic inflammation is a risk factor for fibroid [8]. We also found CD4/CD8 CM cells increased in patients with uterine leiomyoma. In addition, CD4 CM cells were associated with aberrant menstrual cycles. However, this association was not independent after adjusting for age as a confounding factor. e circulating CM T cells contribute to the maintenance of peripheral memory T-cell populations for response to a secondary pathogen challenge [16,36], thereby rapidly clearing pathogen [16]. Based on the fact that chronic infections are related to the etiology of leiomyoma [9,10], our results on the association between CD4/CD8 CM cells and leiomyoma seem to be biologically plausible. It can be concluded that CD4/CD8 CM cells and CD8+CCR7− CD45RA+CD127 high cells can be served as inflammation markers for uterine leiomyoma. Finally, we found that the decrease of cytotoxic cells, such as Tc1, CD56+NKG2D+ NK, and Vδ2+NKG2D+ cδ T cells, were related to anemia in fibroid patients. NKG2D is a receptor expressed on the surface of all human NK, cδ, and CD8 T cells [37]. Several mouse models [38][39][40] and in vitro studies [41] showed antitumor cytotoxicity of NKG2D receptor in NK and cδ T cells. e expression of this receptor associates with aberrant blood vessel development in uterine [11]; therefore, it is feasible that, in the present study, the anemia of fibroid patients might associate with endometrial bleeding related to dysfunction of NKG2D [42]. In addition, Tc1 cells (IFNc-producing CD8+ T cells) promote dysfunction of hematopoietic stem cell and depletion of myeloid lineage progenitor cells, resulting in anemia [43][44][45]. Combing these reports, our findings suggested that the above-mentioned cytotoxic immune functions are markers for severe anemia in uterine leiomyoma.
Above findings have clinical significance and relevance. Longer menstrual period and anemia correlate with abnormal bleeding in leiomyoma patients [3]. A higher CA125 level typically represents tumor progression [46]. Larger diameter of tumors has been found as a predictor for massive intraoperative hemorrhage during caesarean delivery [47]. Furthermore, these symptoms may affect the selection of a therapeutic approach [3] and are related to fibroid recurrence [48]. erefore, our findings implied a prospect for the prevention and treatment of fibroids. For example, unregulated Treg and Tfh cells demonstrate a possibility of further development of fibroid with pelvic masses or pelvic pain, representing the necessity to surgical interventions. e decrease of cytotoxic cells, such as Tc1, CD56+NKG2D+ NK, and Vδ2+NKG2D+ cδ T cells, can also serve as biomarkers for surgery, predicting future severe anemia. In addition, CD8+CCR7− CD45RA+CD127 high cells associate with larger myomas, increasing the risk for obstetric complications, and therefore need to be solved before pregnancy. Moreover, patients with higher Treg, Tfh, and CD8+CCR7− CD45RA+CD127 high cells have higher risk for fibroid recurrence because of more and larger myomas.

Conclusions
Our present study for the first time attempted to reveal hallmarks of peripheral immune function characteristics in reproductive females with uterine leiomyoma. Our results demonstrated that compared with healthy controls, CD4/ CD8 CM, Treg, and Tfh cells were significantly increased and that functional NK and cδ T cells were notably attenuated in patients with uterine leiomyoma. Moreover, the increased ratio Tfh1/Tfh2, Treg, and Tfh17 cells contributed to longer menstrual period, more myomas, and higher levels of tumor marker CA125. e present research may provide a proofof-concept for exploring underlying cross-talks between peripheral immune function and clinical phenotypes of reproductive females with uterine leiomyoma.
Data Availability e raw data on clinical phenotypes and laboratory tests used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper. Figure S1: the different distributions of circulating T cells in leiomyoma patients and controls. Figure S2: Vδ2 subgroups and laboratory indexes in leiomyoma patients. Table S1: the different distributions of immune indexes in fibroid patients and healthy controls and their association with clinical phenotypes in patients.