Mifepristone Inhibited the Expression of B7-H2, B7-H3, B7-H4 and PD-L2 in Adenomyosis

Mifepristone Inhibited the Expression of B7-H2, B7-H3, B7-H4 and PD-L2 in Adenomyosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Mifepristone Inhibited the Expression of B7-H2, B7-H3, B7-H4 and PD-L2 in Adenomyosis Xiaoyan Qin, Wenjing Sun, Chong Wang, Mingjiang Li, Xingbo Zhao, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-196690/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Jul, 2021 Read the published version in Reproductive Biology and Endocrinology → Version 1 posted 8 You are reading this latest preprint version Abstract Background The immune mechanism was shown to be involved in the development of adenomyosis. The current study aims to evaluate the expression of immune checkpoint B7-H2, B7-H3, B7-H4 and PD-L2 in adenomyosis and to explore the effect of mifepristone on the expression of these immune checkpoints. Methods The expression of B7-H2, B7-H3, B7-H4 and PD-L2 in normal endometria and adenomyosis treated with or without mifepristone was determined by immunohistochemistry analysis. Results In adenomyosis, the expression of B7-H2, B7-H3 and B7-H4 was increased in the eutopic and ectopic endometria compared with normal endometria, both in the proliferative and secretory phase. Moreover, the expression of B7-H2 and B7-H3 was higher in adenomyotic lesions than in the corresponding eutopic endometria, both in the proliferative and secretory phase. The expression of PD-L2 was higher in adenomyotic lesions than in normal endometria, both in the proliferative and secretory phase. In secretory phase but not the proliferative phase, the expression of B7-H4 and PD-L2 in adenomyotic lesion showed a significantly higher level than that in the corresponding eutopic endometria. In normal endometria and eutopic endometria, the expression of B7-H4 showed elevated expression in proliferative phase compared with that in the secretory phase, while this change altered in ectopic endometria with decreased B7-H4 expression in proliferative phase than the secretory phase. In addition, the expression of B7-H2, B7-H3, B7-H4 and PD-L2 was significantly decreased in adenomyosis after treated with mifepristone. Conclusions Expression of immune checkpoint proteins B7-H2, B7-H3, B7-H4 and PD-L2 is up-regulated in adenomyosis and down-regulated with mifepristone treatment. The data suggests that the B7 immunomodulatory molecules are involved in the pathophysiology of adenomyosis. Endocrinology & Metabolism B7-H2 B7-H3 B7-H4 PD-L2 mifepristone adenomyosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Adenomyosis is a chronic inflammatory disease characterised by the invasion and growth of functional endometrial glands and stroma in the myometrium. Adenomyosis always causes dysmenorrhea，menorrhea and subfertility, which seriously affect physical and psychological health of women. However, its pathogenesis remains poorly understood[1]. Growing evidence from diverse studies showed that aberrant immune responses play a vital role in the pathogenesis of adenomyosis[2]. Both systemic and local immune alterations exist in women affected by adenomyosis, with the coexistence of changes in inflammatory as well as anti-inflammatory signals[2]. This underscored the immune contributions to the disease. The B7-CD28 family are immunomodulatory molecules and indispensable members for full activation of T cells. Recently, B7-H2, B7-H3, B7-H4 and programmed death ligand 2 (PD-L2, CD273, B7-DC), as new B7 proteins, attracts increasing interests. These co-signaling molecules not only provide critical positive signals that stimulate T cell growth, up-regulate cytokine production and promote T cell differentiation, but also contribute key negative signals to limit, terminate and/or attenuate T cell responses[3, 4] Aberrant expression of these B7 family members are associated with the emergence of T cell exhaustion in many disorders, including cancers, pregnancy, autoimmune diseases, etc[5]. Therefore, we wondered whether altered expression of these B7 family members could be observed in adenomyosis. To date, hysterectomy is still the most common and effective treatment for adenomyosis as it nearly ensures cure. However, it is challenge for many patients with desiration for uterus reservation[6]. For those patients contradictive to hysterectomy, drug therapy including oral contraceptives, progestogen, mifepristone, danazol and gonadotropin-releasing hormone analog is therapeutically important. Evidences have shown that mifepristone can inhibit endometrial proliferation or suppress adenomyotic lesions, resulting in inhibition of prostaglandin production and endometrial atrophy in animal models[7]. It has been demonstrated that treatment with 50-mg of mifepristone daily lead to improved pain and regression of adenomyosis[8]. Therefore, in the current study, we estimated the expression and localization of four B7 molecules including B7-H2, B7-H3, B7-H4 and PD-L2 in adenomyosis and control group. In addition, we also investigate the effect of mifepristone on the expression of these four B7 molecules in adenomyosis. Methods Collection of tissues The study participants were enrolled in the Department of Obstetrics and Gynaecology, Provincial Hospital affiliated to Shandong First Medical University from January 2016 to March 2018. Adenomyotic lesion as well as the corresponding eutopic endometria were obtained from patients with adenomyosis undergoing hysterectomy. Drug-untreated adenomyotic group comprised 58 patients with adenomyosis (proliferative phase: n= 35; secretory phase: n= 23) with no use of any hormone therapy within at least 6 months before surgery. The mifepristone-treated adenomyotic group consists of 11 patients with adenomyosis treated with mifepristone at a dose of 12.5-mg daily for 3 months. 74 samples of normal endometrium collected from hysterectomy specimens checked for leiomyoma pathology (proliferative phase: n= 47; secretory phase: n= 27) were included as controls. Participants in control group had no evidence of adenomyosis in the histopathological examination of their hysterectomy specimens and had no visible pelvic inflammation or endometriosis at the time of hysterectomy. The characteristics of patients in each group were shown in Table 1. Ectopic and eutopic endometria of adenomyosis were collected during the surgery. Adenomyosis was confirmed by histological examination. Written informed consent was obtained from all participants prior to the biopsy procedure. This study was approved by the Institutional Review Board of Shandong First Medical University. Immunohistochemistry analysis The tissue processing and staining procedure have been described in detail as our previous study[9]. Immunohistochemical staining was performed using polyclonal antibody rabbit anti-human ICOSL (B7-H2) (1:100, ab233151, Abcam, Cambridge, UK), monoclonal antibody mouse anti-human CD276 (B7-H3) (1:200, ab105922, Abcam), monoclonal antibody rabbit anti-human VTCN1 (B7-H4) (1:300, ab209242, Abcam) and polyclonal antibody rabbit anti-human PD-L2 (1:100, ab244332, Abcam,). Goat anti-rabbit and goat anti-mouse HRP-conjugated secondary antibodies and Diaminobenzidine(DAB) staining kits were obtained from ZSGB-BIO (Beijing, China). The sections were viewed under a Leica DM4000B microscope (Leica, Wetzlar, Germany), and photographs were taken using the IM50 image analysis system (Leica). Immunohistochemical staining was evaluated by semi-quantitative immunoscore which was a product of a quantity score and a staining intensity. Quantity score(Pi) also as the percentages of positively stained glandular epithelial cells was estimated from four randomly chosen views and 100 counting cells. The staining intensity (I) of the glandular epithelial cells was estimated as follows: 0: negative; 1: weak staining; 2: moderate staining; and 3: strong staining. H-score equals Pi* (I + 1) . Two sections of each sample were assessed by two investigators blinded to any pathological or clinical data about the tissues. The average score of the two investigators was used. Statistical analysis Statistical analysis of data was performed by analysis of variation using SPSS 19.0 (SPSS Inc., Chicago, IL). Data are presented as means ± SD. Differences between two groups were determined by the two-tailed student’s t-test. P <0.05 was considered to be statistically significant. Results 1. Overexpression of B7-H2 in adenomyotic eutopic and ectopic endometria B7-H2 protein was expressed both in glandular and stromal cells in endometrial tissues, and mainly in glandular epithelial cells. Positive expression was mainly located in the cell membrane and cytoplasm, but not the nucleus. The immune expression of B7-H2 protein in the endometrial tissues of the control group is extremely faint and almost no expression, regardless of glandular epithelial or stromal cells (Fig 1 E&F). In contrast, B7-H2 protein was moderately expressed in partial epithelial and stromal cells in the eutopic endometrium of adenomyosis (ADE-EU) (Fig.1 C&D) and intensely expressed in the ectopic endometrium of adenomyosis (ADE-EC) (Fig.1 A&B). Compared with the control group, ADE-EU and ADE-EC showed significantly increased immune expression of B7-H2, both in the proliferative and secretory phase. [Fig.1 J ADE-EU vs control： P (proliferative stage)<0.001; P (secretory stage）=0.015; ADE-EC vs control (proliferative and secretion stage): both P <0.001]. Moreover, the expression of B7-H2 in ADE-EC was significantly higher than that of ADE-EU in the same menstrual stage. [Fig.1 J ADE-EC vs ADE-EU (proliferative and secretion stage): both P <0.001] During the menstrual cycles, the expression of B7-H2 in the ADE-EU was statistically significant stronger in the proliferative stage than that in the secretory stage ( P =0.03), while no significant difference was noted in ADE-EC ( P =0.78) or in the control group ( P =0.82). 2. Overexpression of B7-H3 in adenomyotic eutopic and ectopic endometria B7-H3 protein was mainly expressed in glandular epithelium instead of stromal cells in endometrial tissues. The positive expression was mainly located in the cell membrane and cytoplasm. The immune expression of B7-H3 protein in the endometrial tissues of the control group is weak and almost no expression, regardless of glandular or stromal cells (Fig.2 E&F). However, B7-H3 was moderately expressed in glandular epithelium of ADE-EU (Fig.2 C&D), and intensely expressed in that of ADE-EC (Fig.2 A&B). Compared with the control group, ADE-EU showed significantly increased B7-H3 expression, both in the proliferative and secretory phases [Fig.2 J ADE-EU vs control: P (proliferative phase)<0.001； P (secretory phase)=0.002]. Moreover, ADE-EC showed statistically higher expression of B7-H3 compared with the corresponding ADE-EU in the same menstrual phase [Fig.2 J ADE-EC vs ADE-EU (proliferative and secretion stage): both P < 0.001]. 3. Overexpression of B7-H4 in adenomyotic eutopic and ectopic endometria In endometrium of patients with or without adenomyosis, the immunostaining of B7-H4 showed similar characteristics. Positive staining was mainly observed in the glandular epithelial cells, with almost no staining in the stroma. The positive immunostaining of B7-H4 was located mainly in the cell membrane and cytoplasm. In the control group, the endometria showed extremely low, almost no immunostaining of B7-H4 protein (Fig.3 E&F). While in the adeomyotic group, the ADE-EU showed moderately immunostaining of B7-H4 in glandular epithelium cells(Fig.3 C&D). ADE-EC showed strong immunostaining intensity of B7-H4 in almost all glandular epithelium(Fig.3 A&B). Compared with the control group, ADE-EU and ADE-EC showed statistically significant higher expression of B7-H4 both in the proliferative phase and in secretory phase (Fig.3 J P <0.001). ADE-EC in secretory phase showed statistically significant higher level of B7-H4 expression than the corresponding ADE-EU in the same menstrual phase (Fig.3 J P <0.001). However, no significant difference of B7-H4 expression was shown between ADE-EU and ADE-EC in the proliferative phase (Fig.3 J P =0.06). Both in ADE-EU and the control groups, the expression of B7-H4 was higher in proliferative phase than in secretory phase [Fig.3 J control group： P =0.01, ADE-EU： P =0.03]. In contrast, that expression in ADE-EC was lower in the proliferative phase than in the secretory phase (Fig.3 J P < 0.001). 4. Overexpression of PD-L2 in ectopic endometria of patients with adenomyosis The expression of PD-L2 protein in control and adenomyotic endometrium was detected using immunohistochemical analysis. Figure 4 shows that PD-L2 was not expressed or only weakly expressed in endometrium of both control group(Fig.4 E&F) and ADE-EU(Fig.4 C&D). Notably, it was expressed at significantly higher levels in endometrium of ADE-EC(Fig.4 A&B) compared with control group, both in the proliferative and secretory phases (Fig.4 J both P <0.01). Moreover, ADE-EC showed higher PD-L2 expression than ADE-EU in the secretory but not the proliferative phase. Nevertheless, there was no significant difference of PD-L2 expression between ADE-EU and control group (Fig.4 J). The data also showed that in ectopic endometria of adenomyotic samples, PD-L2 was primarily expressed in glandular epithelial cells. During menstrual cycles, no periodic changes of the endometrial PD-L2 expression was found neither in adenomyotic group nor in the control group. 5. Decreased expression of B7-H2, B7-H3, B7-H4 and PD-L2 in patients with adenomyosis after mifepristone treatment Immunohistochemical staining was used to detect and compare the changes of B7-H2, B7-H3, B7-H4 and PD-L2 expression in eutopic and ectopic endometrium of patients with adenomyosis treated with and without mifepristone. The results showed that the eutopic and ectopic endometrium of adenomyosis treated with mifepristone showed significantly lower expression of B7-H2, B7-H3, B7-H4 and PD-L2 protein compared with adenomyosis without mifepristone treatment, both in proliferative phase and in secretory phase (Fig.1,2,3,4 G&H&I&J all P < 0.01). Discussion The activation of T lymphocytes plays an essential role in the process of immunity. While this activation requires two signals simultaneously[10]: 1) the first stimulus signal was provided by T cell receptor recognized by MHC-antigen complex; 2) the key second signal was delivered by ligation of T cells and co-modulatory molecules expressed on antigen-presenting cells (APCs) belonging to the B7 or other families. In recent years, a breakthrough about the three main members of B7 family: CTLA-4, PD-1 and PD-L1 (B7-H1) has been made in the immune checkpoint treatment of cancer[10]. B7 family is the most important immunomodulatory molecule. Since there is little research on the correlation between B7 family and adenomyosis, our study explored the expression of B7-H2, B7-H3, B7-H4 and PD-L2 in adenomyosis treated with and without mifepristone. B7-H2 is expressed on professional APCs and it binds to inducible costimulator molecule (ICOS) expressed on T cells. The ICOS/B7-H2 signal was engaged in various aspects of T-cell responses. This engagement plays an essential role in the differentiation of CD4 + T cells into effector subsets, including Th1, Th2, Th17 and regulatory T cells (Tregs)[11, 12]. In acute myelocytic leukemia (AML)，strongly-activating B7-H2 exhibits an inhibitory function by which enabled AML cells to facilitate immune escape[13]. Recently, over-expression of B7-H2 was found in a variety of solid cancers by maintaining the function of immunosuppressive Tregs subset, which is associated with tumor progression and poor overall survival[14]. Th17/Tregs imbalance has been shown to be present in adenomyosis[15]. Considering the effect of B7-H2 on differentiation of Th17 and Tregs from CD4 + T cells, it is reasonable to postulate that B7-H2 was involved in pathogenesis of adenomyosis. While further studies are needed to elucidate the definite mechanism. Furthermore, the engagement of B7-H2 with ICOS can stimulate IFN-gamma, IL-4, IL-5 and IL-10 production by T cells and it most effectively induces IL-10[4, 16, 17]. Moreover，ICOS also stabilizes IL-10R expression on T cells, rendering them sensitive to IL-10[18]. In this context, this may supply further interpretation to what we found in previous studies that up-regulated expression of IL-10[19] and IL-10R[20] was found in eutopic and ectopic endometrium of adenomyosis. B7-H2 plays a primary role in a regulatory capacity promoting Th2 immune response. In B7-H2-deficient mice, production of Th2 cytokines such as IL-4 and IL-10 by primed T cells is reduced[21]. Shifts towards Th2 immune response have been found being involved in endometriosis with relative predominance of IL-4 and IL-10[22]. In this study, we found the endometrial expression of B7-H2 was higher in patients with adenomyosis than the control group. Moreover, the adenomyotic ectopic expression of B7-H2 was much more intense than the adenomyotic eutopic endometrium. It implies that aberrant expression of B7-H2 involves in the pathogenesis of adenomyosis. B7-H3 was initially thought to co-stimulate the immune response, but recent studies have shown that it is predominantly a T-cell co-inhibitory molecule contributing to immune evasion[23, 24]. B7-H3 is widely expressed in both lymphoid and nonlymphoid organs at the RNA level, but the expression of B7-H3 protein is more restricted to cell types such as activated dendritic cells, monocytes, T cells, B cells, and NK cells. Aberrant expression of B7-H3 has been shown to be associated with poor outcome of various human malignancies[25] and autoimmune diseases[26]. Overexpression of B7-H3 was displayed in 60-93% of tumor tissues in the vast majority of cancer types by immunohistochemical assay, while very limited expression is seen in normal healthy tissue[25]. This was consistent with what we observed in this study that only weak or no expression of B7-H3was observed in normal endometrium, but stronger intensity of B7-H3 immunostaining was shown in adenomyosis. It implied the over-expression of B7-H3 might participate in the genesis of adenomyosis. Diverse studies have proved that up-regulation of B7-H3 is associated with impaired T-cell stimulated function[27,28], suppressed NK-mediated cell lysis[29], increased IL-10 secretion[30], modulation of the Jak/Stat pathway[31] which contributes to immune suppression and evasion by tumors. These results showed that B7-H3 was involved in the process of tumor cells by acting as a negative regulator of T cells and facilitating evade tumor immunity. Similar to that in tumors, B7-H3 also displays co-inhibitory properties in some immune diseases. Independent studies utilizing either protein blockade or gene-knockout mice have reported that B7-H3 ameliorates graft-versus-host-disease, prolongs cardiac allograft survival, reduces airway hypersensitivity, and delays experimental autoimmune encephalomyelitis onset, especially by down-regulating Th1 responses[32-34]. These examples provide further evidence for the co-inhibitory properties of B7-H3. Our study found B7-H3 was overexpressed in adenomyotic endometria than in control group. Moreover, the adenomyotic ectopic endometria expressed even much higher B7-H3 in comparation with eutopic tissues. We postulate the immunologic function of B7-H3 in adenomyosis was similar to that in malignancies and autoimmune diseases acting as a co-inhibitory immunomodulator. The over-expression of B7-H3 enable the endometrium to create an immunosuppressive microenvironment to facilitate the eutopic and ectopic endometrium to escape host immunosurveillance before infiltrating and after infiltrating into myometrium, thus led to the origination and progression of adenomyosis. B7-H4 is a vital B7 ligand that acts as a negative regulator in the T cell mediated immune response. B7-H4 mRNA is widely distributed in human peripheral tissues. However, B7-H4 protein expression is more restricted in most normal tissues and can be induced on APCs after in vitro stimulation[35]. Recent studies found that the negative immunomodulatory role of B7-H4 on a wide range of tumors[36], autoimmune diseases[37], viral infection[38] and transplantation rejection[39]. In endometrium，the expression of B7-H4 has been estimated in Miyatake T’s study. They showed the staining of B7-H4 is faint or moderate in the apex of cytoplasmic membrane in normal or hyperplasic endometrium, but that is strong in circumferential membrane and cytoplasm in most endometrioid carcinomas[40]. A significant inverse correlation has been observed between the high expression of B7-H4 in majority endometrioid carcinomas and tumor infiltrating T cells, particularly the number of tumor-associated CD3 + and CD8 + lymphocytes[40]. High expressed B7-H4 in tumor microenvironment exerts negative immunomodulatory effects through several pathways, including arresting the cell cycle at the G0/G1 stage, promoting T cell apoptosis, inhibiting T cell growth, cytokine secretion and development of cytotoxicity[35], thereby affects the biological behavior of tumor cells, assists tumor immune escape, and leads to seriously poor prognosis of patients[41]. Similar to that, our results showed the endometrial expression of B7-H4 protein in the control group was extremely weak, almost no expression. While the expression of B7-H4 in the eutopic and ectopic endometrium of adenomyosis was significantly higher than that of control group. We hypothesize that over-expressed B7-H4 in adenomyosis participated in the formation of immunotolerant environment of the uterus through negatively regulating T cell proliferation, facilitating the ectopic endometrial lesions to evade host immunity and failure to be eliminated effectively, thus led to the development of uterine adenomyosis. PD-L2 as one of the two receptors for PD-1, plays crucial roles in the immune checkpoint pathways responsible for the suppression of T-cell activation[42]. PD-L2 expression can be induced on a diverse variety of other immune cells and non-immune cells depending on microenvironmental stimuli[42]. PD-L2 was shown to be moderately or strongly expressed in most tumor cells, interact with PD-1 and dramatically inhibits TCR-mediated proliferation, cytokine production by CD4 + T cells and T-cell cytolysis[43]. Via utilization of immune checkpoint molecules, tumor cells exert immunomodulatoty function to tumor microenvironment and escape host immune surveillance. In this study, we found a higher level of PD-L2 expression in ectopic adenomyotic tissue than in normal endometria and eutopic adenomyotic tissues. In endometrial tissues, it is reported that PD-L2 expression was present in 47% of 15 cases of normal endometria and in 40% of 30 cases of endometrial cancer[44]. Expression of the PD-1/PD-L1/PD-L2 axis is associated with moderately and poorly-differentiated endometrial cancer and type II endometrial cancer in which more frequent expression of PD-1, PD-L1 and PD-L2 may cause immunosuppression to favor tumor growth and negatively affect patient's survival. In the same way, our results indicate the abnormal increased expression of PD-L2 in adenomyosis may repress T-cell activation and alter the immune microenvironment of ectopic endometrium This may enable ectopic endometrial cells to evade normal immunological surveillance and to initiate adenomyosis. In this study, B7-H4 expression showed cyclic variation in women of the control group and eutopic endometria of adenomyosis, with elevated expression in the proliferative phase. It suggests that B7-H4 expression may be regulated by steroid hormones in normal endometrium. Consistent with this, Papenfuss TL ’s results[45] showed that estriol can up-regulate the expression of B7-H4 on the surface of dendritic cells, indicating that estrogen can up-regulate the expression of B7-H4. Furthermore, the cyclic change of B7-H4 expression was altered in ectopic endometria of patients with adenomyosis, with lowered expression of B7-H4 in the proliferative phase. These data suggest aberrant hormonal sensitivity of B7-H4 in adenomyotic foci may participate in the establishment of this disease. Compared with no-treated adenomyosis, down-expressed B7-H2, B7-H3, B7-H4 and PD-L2 was observed in adenomyosis treated with mifepristone, both in eutopic and ectopic endometria. As far as we know, this is the first study to address the effect of mifepristone on the B7-H2, B7-H3, B7-H4 and PD-L2 expression. Mifepristone (RU486) is an antiprogestin with a high affinity for progesterone and glucocorticoid receptor. Studies concerning pregnant women showed that mifepristone can alter the endometrial immune balance and result in implantation failure. It is shown that mifepristone exerts effects through shifting immunological elements by enhancing the expression of cytotoxic lymphocytes[46], increasing cytotoxicity of peripheral blood NK cells[47] and uterine NK cells[48], and enhancing antigen-specific CD4 + and CD8 + T cell inflammatory cytokine (IFN-γ) and cytotoxic molecule release (granzyme B)[49]. Mifepristone regulates Tregs function mediated by dendritic cells through inhibiting the expression of TGF-β[50]. Our current study implies that mifepristone may play a therapeutical role in adenomyosis by inhibiting the expression of these four immunomodulatory molecules, improving the immune microenvironment status of the uterus and effectively inhibiting or clearing ectopic endometrial cells. However, further studies are needed to explore the mechanism refer to possible signaling underlying the finding. Conclusions Taken together, the results described in this study are the first to show immunostaining expression of B7-H2, B7-H3, B7-H4 and PD-L2 in patients with adenomyosis. The differential expression of these proteins in endometrium with or without adenomyosis suggests that the altered immunomodulatory molecules participate in the pathogenesis of adenomyosis. We postulate that these powerful immunomodulators are key in providing the eutopic and ectopic endometrium with a suitable immunological environment, leading to ineffective elimination of abnormal eutopic and ectopic endometria, and resulting in the initiation and maintenance of adenomyosis. These findings enrich our comprehension about the local immune status involved in the pathogenesis of adenomyosis, and provide potential targets for immunotherapy. The results of the study further investigated the possible mechanism of mifepristone treatment on adenomyosis, and may provide a theoretical data for future studying adenomyosis treatment. However, the primary limitation of present study is that we only examined the expression and location of B7-H2，B7-H3，B7-H4 and PD-L2 in adenomyosis treated with or without mifepristone by immunohistochemistry. In next study, we will further investigate the involvement of these B7 family immunomodulators in adenomyosis using multiple experimental techniques. Declarations Ethics approval and consent to participate Written informed consent was obtained from all participants prior to biopsy procedure and was approved by Shandong Provincial Hospital Affiliated to Shandong First Medical University. Consent for publication Written informed consent for publication was obtained from all participants. Availability of data and materials The datasets used and analyzed during the present study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding The research was supported by grants from Science and Technology Development Program of Jinan (No.201805023), Natural Science Foundation of Shandong Province (ZR2020MH067, ZR2016HQ22), Shandong Province medical and health technology development projects (No.2016WS0442, 2016WS0404, 2017WS191), National Natural Science Foundation of China (No.81300468; No. 81671433; No. 81671434). Authors’ contributions HZ conceived of the study, participated in its design and implementation, helped to draft and edit the manuscript for submission. XQ collected tissues and carried out the immunohistochemistry process, participated in the analysis and the interpretation of data and drafted the manuscript. ML participated in the design of the study, supervised the study and critically helped to revise the manuscript. XZ contributed to the design of the study, assisted in data analysis and revised the manuscript. CL helped to revise the manuscript. XQ, WL, CW and HZ performed the statistical analysis. All authors read and approved the final manuscript. Acknowledgements We are appreciated to all the participants involved in this study. References Leyendecker G, Wildt L, Mall G: The pathophysiology of endometriosis and adenomyosis: tissue injury and repair . ARCH GYNECOL OBSTET 2009, 280 (4):529-538. Bourdon M, Santulli P, Jeljeli M, Vannuccini S, Marcellin L, Doridot L, Petraglia F, Batteux F, Chapron C: Immunological changes associated with adenomyosis: a systematic review . HUM REPROD UPDATE 2021, 27 (1):108-129. Carreno BM, Collins M: The B7 family of ligands and its receptors: new pathways for costimulation and inhibition of immune responses . ANNU REV IMMUNOL 2002, 20 :29-53. Sharpe AH, Freeman GJ: The B7-CD28 superfamily . NAT REV IMMUNOL 2002, 2 (2):116-126. Ni L, Dong C: New B7 Family Checkpoints in Human Cancers . MOL CANCER THER 2017, 16 (7):1203-1211. Soares SR, Martinez-Varea A, Hidalgo-Mora JJ, Pellicer A: Pharmacologic therapies in endometriosis: a systematic review . FERTIL STERIL 2012, 98 (3):529-555. Bouchard P, Chabbert-Buffet N, Fauser BC: Selective progesterone receptor modulators in reproductive medicine: pharmacology, clinical efficacy and safety . FERTIL STERIL 2011, 96 (5):1175-1189. Kettel LM, Murphy AA, Morales AJ, Ulmann A, Baulieu EE, Yen SS: Treatment of endometriosis with the antiprogesterone mifepristone (RU486) . FERTIL STERIL 1996, 65 (1):23-28. Qi S, Zhao X, Li M, Zhang X, Lu Z, Yang C, Zhang C, Zhang H, Zhang N: Aberrant expression of Notch1/numb/snail signaling, an epithelial mesenchymal transition related pathway, in adenomyosis . Reprod Biol Endocrinol 2015, 13 :96. Ni L, Dong C: New checkpoints in cancer immunotherapy . IMMUNOL REV 2017, 276 (1):52-65. Simpson TR, Quezada SA, Allison JP: Regulation of CD4 T cell activation and effector function by inducible costimulator (ICOS) . CURR OPIN IMMUNOL 2010, 22 (3):326-332. Nurieva RI, Duong J, Kishikawa H, Dianzani U, Rojo JM, Ho I, Flavell RA, Dong C: Transcriptional regulation of th2 differentiation by inducible costimulator . IMMUNITY 2003, 18 (6):801-811. Tamura H, Dan K, Tamada K, Nakamura K, Shioi Y, Hyodo H, Wang SD, Dong H, Chen L, Ogata K: Expression of functional B7-H2 and B7.2 costimulatory molecules and their prognostic implications in de novo acute myeloid leukemia . CLIN CANCER RES 2005, 11 (16):5708-5717. Marinelli O, Nabissi M, Morelli MB, Torquati L, Amantini C, Santoni G: ICOS-L as a Potential Therapeutic Target for Cancer Immunotherapy . Curr Protein Pept Sci 2018, 19 (11):1107-1113. Gui T, Chen C, Zhang Z, Tang W, Qian R, Ma X, Cao P, Wan G: The disturbance of TH17-Treg cell balance in adenomyosis . FERTIL STERIL 2014, 101 (2):506-514. Hutloff A, Dittrich AM, Beier KC, Eljaschewitsch B, Kraft R, Anagnostopoulos I, Kroczek RA: ICOS is an inducible T-cell co-stimulator structurally and functionally related to CD28 . NATURE 1999, 397 (6716):263-266. McAdam AJ, Chang TT, Lumelsky AE, Greenfield EA, Boussiotis VA, Duke-Cohan JS, Chernova T, Malenkovich N, Jabs C, Kuchroo VK et al : Mouse inducible costimulatory molecule (ICOS) expression is enhanced by CD28 costimulation and regulates differentiation of CD4+ T cells . J IMMUNOL 2000, 165 (9):5035-5040. Tuettenberg A, Huter E, Hubo M, Horn J, Knop J, Grimbacher B, Kroczek RA, Stoll S, Jonuleit H: The role of ICOS in directing T cell responses: ICOS-dependent induction of T cell anergy by tolerogenic dendritic cells . J IMMUNOL 2009, 182 (6):3349-3356. Wang F, Li H, Yang Z, Du X, Cui M, Wen Z: Expression of interleukin-10 in patients with adenomyosis . FERTIL STERIL 2009, 91 (5):1681-1685. Qin X, Zhang H, Wang F, Xue J, Wen Z: Expression and possible role of interleukin-10 receptors in patients with adenomyosis . Eur J Obstet Gynecol Reprod Biol 2012, 161 (2):194-198. Mak TW, Shahinian A, Yoshinaga SK, Wakeham A, Boucher LM, Pintilie M, Duncan G, Gajewska BU, Gronski M, Eriksson U et al : Costimulation through the inducible costimulator ligand is essential for both T helper and B cell functions in T cell-dependent B cell responses . NAT IMMUNOL 2003, 4 (8):765-772. Szymanowski K, Niepsuj-Binias J, Dera-Szymanowska A, Wolun-Cholewa M, Yantczenko A, Florek E, Opala T, Murawski M, Wiktorowicz K: An influence of immunomodulation on Th1 and Th2 immune response in endometriosis in an animal model . BIOMED RES INT 2013, 2013 :849492. Chapoval AI, Ni J, Lau JS, Wilcox RA, Flies DB, Liu D, Dong H, Sica GL, Zhu G, Tamada K et al : B7-H3: a costimulatory molecule for T cell activation and IFN-gamma production . NAT IMMUNOL 2001, 2 (3):269-274. Prasad DV, Nguyen T, Li Z, Yang Y, Duong J, Wang Y, Dong C: Murine B7-H3 is a negative regulator of T cells . J IMMUNOL 2004, 173 (4):2500-2506. Picarda E, Ohaegbulam KC, Zang X: Molecular Pathways: Targeting B7-H3 (CD276) for Human Cancer Immunotherapy . CLIN CANCER RES 2016, 22 (14):3425-3431. Luo L, Zhu G, Xu H, Yao S, Zhou G, Zhu Y, Tamada K, Huang L, Flies AD, Broadwater M et al : B7-H3 Promotes Pathogenesis of Autoimmune Disease and Inflammation by Regulating the Activity of Different T Cell Subsets . PLOS ONE 2015, 10 (6):e130126. Mahnke K, Ring S, Johnson TS, Schallenberg S, Schonfeld K, Storn V, Bedke T, Enk AH: Induction of immunosuppressive functions of dendritic cells in vivo by CD4+CD25+ regulatory T cells: role of B7-H3 expression and antigen presentation . EUR J IMMUNOL 2007, 37 (8):2117-2126. Chen C, Shen Y, Qu QX, Chen XQ, Zhang XG, Huang JA: Induced expression of B7-H3 on the lung cancer cells and macrophages suppresses T-cell mediating anti-tumor immune response . EXP CELL RES 2013, 319 (1):96-102. Lemke D, Pfenning PN, Sahm F, Klein AC, Kempf T, Warnken U, Schnolzer M, Tudoran R, Weller M, Platten M et al : Costimulatory protein 4IgB7H3 drives the malignant phenotype of glioblastoma by mediating immune escape and invasiveness . CLIN CANCER RES 2012, 18 (1):105-117. Liu C, Liu J, Wang J, Liu Y, Zhang F, Lin W, Gao A, Sun M, Wang Y, Sun Y: B7-H3 expression in ductal and lobular breast cancer and its association with IL-10 . MOL MED REP 2013, 7 (1):134-138. Liu H, Tekle C, Chen YW, Kristian A, Zhao Y, Zhou M, Liu Z, Ding Y, Wang B, Maelandsmo GM et al : B7-H3 silencing increases paclitaxel sensitivity by abrogating Jak2/Stat3 phosphorylation . MOL CANCER THER 2011, 10 (6):960-971. Suh WK, Gajewska BU, Okada H, Gronski MA, Bertram EM, Dawicki W, Duncan GS, Bukczynski J, Plyte S, Elia A et al : The B7 family member B7-H3 preferentially down-regulates T helper type 1-mediated immune responses . NAT IMMUNOL 2003, 4 (9):899-906. Ueno T, Yeung MY, McGrath M, Yang S, Zaman N, Snawder B, Padera RF, Magee CN, Gorbatov R, Hashiguchi M et al : Intact B7-H3 signaling promotes allograft prolongation through preferential suppression of Th1 effector responses . EUR J IMMUNOL 2012, 42 (9):2343-2353. Veenstra RG, Flynn R, Kreymborg K, McDonald-Hyman C, Saha A, Taylor PA, Osborn MJ, Panoskaltsis-Mortari A, Schmitt-Graeff A, Lieberknecht E et al : B7-H3 expression in donor T cells and host cells negatively regulates acute graft-versus-host disease lethality . BLOOD 2015, 125 (21):3335-3346. Sica GL, Choi IH, Zhu G, Tamada K, Wang SD, Tamura H, Chapoval AI, Flies DB, Bajorath J, Chen L: B7-H4, a molecule of the B7 family, negatively regulates T cell immunity . IMMUNITY 2003, 18 (6):849-861. Podojil JR, Miller SD: Potential targeting of B7-H4 for the treatment of cancer . IMMUNOL REV 2017, 276 (1):40-51. Wang X, Hao J, Metzger DL, Mui A, Ao Z, Akhoundsadegh N, Langermann S, Liu L, Chen L, Ou D et al : Early treatment of NOD mice with B7-H4 reduces the incidence of autoimmune diabetes . DIABETES 2011, 60 (12):3246-3255. Qiu F, Yuan C, Xu J, Zhang J, Chen F, Liu D, Zheng B, Li R, Huang X, Xu J: Role of B7-H4 in the Progression and Prognosis of Cervical Inflammation to Cancer After Human Papilloma Virus Infection . J BIOMED NANOTECHNOL 2019, 15 (5):1043-1051. Saha A, Taylor PA, Lees CJ, Panoskaltsis-Mortari A, Osborn MJ, Feser CJ, Thangavelu G, Melchinger W, Refaeli Y, Hill GR et al : Donor and host B7-H4 expression negatively regulates acute graft-versus-host disease lethality . JCI Insight 2019, 4 (19). Miyatake T, Tringler B, Liu W, Liu SH, Papkoff J, Enomoto T, Torkko KC, Dehn DL, Swisher A, Shroyer KR: B7-H4 (DD-O110) is overexpressed in high risk uterine endometrioid adenocarcinomas and inversely correlated with tumor T-cell infiltration . GYNECOL ONCOL 2007, 106 (1):119-127. Ichikawa M, Chen L: Role of B7-H1 and B7-H4 molecules in down-regulating effector phase of T-cell immunity: novel cancer escaping mechanisms . Front Biosci 2005, 10 :2856-2860. Rozali EN, Hato SV, Robinson BW, Lake RA, Lesterhuis WJ: Programmed death ligand 2 in cancer-induced immune suppression . Clin Dev Immunol 2012, 2012 :656340. Latchman Y, Wood CR, Chernova T, Chaudhary D, Borde M, Chernova I, Iwai Y, Long AJ, Brown JA, Nunes R et al : PD-L2 is a second ligand for PD-1 and inhibits T cell activation . NAT IMMUNOL 2001, 2 (3):261-268. Vanderstraeten A, Luyten C, Verbist G, Tuyaerts S, Amant F: Mapping the immunosuppressive environment in uterine tumors: implications for immunotherapy . Cancer Immunol Immunother 2014, 63 (6):545-557. Papenfuss TL, Powell ND, McClain MA, Bedarf A, Singh A, Gienapp IE, Shawler T, Whitacre CC: Estriol generates tolerogenic dendritic cells in vivo that protect against autoimmunity . J IMMUNOL 2011, 186 (6):3346-3355. Liu L, Zhou F, Qin J, Qian Z, Huang L: Roles of Mifepristone on the Regulation of Cytotoxic Lymphocytes and Regulatory T Cells . Gynecol Obstet Invest 2017, 82 (6):533-537. Chen XY, Zhuang YL, Li L, Zhang WW, Huang LL: The effect of mifepristone on the peripheral blood natural killer cell's cytotoxicity and expression of CD94/NKG2A and NKG2D during the implantation phase . FERTIL STERIL 2010, 93 (8):2615-2620. Zhou F, Chen XY, Zhuang YL, Chen YZ, Huang LL: Low-dose mifepristone increases uterine natural killer cell cytotoxicity and perforin expression during the receptive phase . FERTIL STERIL 2011, 96 (3):649-653. Shah NM, Imami N, Johnson MR: Progesterone Modulation of Pregnancy-Related Immune Responses . FRONT IMMUNOL 2018, 9 :1293. Li Y, Cao L, Qian Z, Guo Q, Niu X, Huang L: Mifepristone regulates Tregs function mediated by dendritic cells through suppressing the expression of TGF-beta . Immunopharmacol Immunotoxicol 2021, 43 (1):85-93. Tables Table 1: The characteristics of participants in each group N age ranges(yrs) Mean(yrs) STD control group Proliferative stage 47 38-52 46.9149 3.0132 Secretory stage 27 39-52 46.5556 3.0298 adenomyosis Proliferative stage 35 32-52 44.6286 4.2294 Secretory stage 23 40-52 45.6857 3.2813 adenomyosis treated with mifepristone 11 39-52 45.8182 3.8424 Abrreviations: N:number; yrs:years; STD: standard deviation Cite Share Download PDF Status: Published Journal Publication published 21 Jul, 2021 Read the published version in Reproductive Biology and Endocrinology → Version 1 posted Reviews received at journal 17 Feb, 2021 Reviewer # 1 agreed at journal 17 Feb, 2021 Review # 1 received at journal 17 Feb, 2021 Reviewers invited by journal 12 Feb, 2021 Editor assigned by journal 01 Feb, 2021 Submission checks completed at journal 01 Feb, 2021 Editor invited by journal 01 Feb, 2021 First submitted to journal 31 Jan, 2021 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-196690","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":10653920,"identity":"cf64d0c1-54a9-4d9c-8a73-61d278af1470","order_by":0,"name":"Xiaoyan Qin","email":"","orcid":"","institution":"Department of Obstetrics and Gynaecology, Shandong Provincial Hospital Affiliated to Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyan","middleName":"","lastName":"Qin","suffix":""},{"id":10653921,"identity":"0fc9457c-2ddd-4ad3-bd4b-a84e2ba05d68","order_by":1,"name":"Wenjing Sun","email":"","orcid":"","institution":"Department of Obstetrics and Gynecology, Shandong Provincial Hospital affiliated to Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wenjing","middleName":"","lastName":"Sun","suffix":""},{"id":10653922,"identity":"2e78da2e-7fe8-48ac-a9a9-bb24ceb1ed40","order_by":2,"name":"Chong Wang","email":"","orcid":"","institution":"Department of Surgery, Shandong Rongjun General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chong","middleName":"","lastName":"Wang","suffix":""},{"id":10653923,"identity":"f66b464c-5401-4863-8e83-40942b34cfad","order_by":3,"name":"Mingjiang Li","email":"","orcid":"","institution":"Department of Obstedtrics and Gynaecology, Shandong Provincial Hospital affiliated to Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Mingjiang","middleName":"","lastName":"Li","suffix":""},{"id":10653924,"identity":"5e6ea22e-d203-4e04-acc3-489725b071eb","order_by":4,"name":"Xingbo Zhao","email":"","orcid":"","institution":"Department of Obstetrics and Gynaecology, Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Xingbo","middleName":"","lastName":"Zhao","suffix":""},{"id":10653925,"identity":"43011ab2-4b32-4ef2-9ee0-56284e56869e","order_by":5,"name":"Changzhong Li","email":"","orcid":"","institution":"Department of Obstetrics and Gynaecology: Shandong Provincial Hospital affiliated to Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Changzhong","middleName":"","lastName":"Li","suffix":""},{"id":10653926,"identity":"ef724706-50de-4217-bbce-2c0a84c25dd4","order_by":6,"name":"Hui Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIiWNgGAWjYBACAwbGBmYQg429sfHBBwMbOeK18PEcbjacUZBmTIQWBgawFjmJ9DZpng+HEwlqMZdIbv5cUHPHro0hsdnYxoA5gYH98NEN+LRYzkhsMJ5x7FlyG8PBxsc5Bmx5DDxpaTfwOuxGYkMyD9vhZDbGxmbjHAOeYgYJHjOCWg7z/ANqYWZsk7YwkEhsIEJLYzNv22E7NjagFgYDAyK0nHnYzMzbdziBjYex2bDHIMGYjaBfjqc//szz7bC9/PznDx/8+PNfjp/98DG8WmAgsQHGYiNGOQjYE6twFIyCUTAKRiAAAH1CS+nnxQqrAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-8103-4648","institution":"Department of Obstetrics and Gynecology","correspondingAuthor":true,"prefix":"","firstName":"Hui","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2021-02-01 07:21:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-196690/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-196690/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12958-021-00800-6","type":"published","date":"2021-07-21T15:02:28+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":6016656,"identity":"b6d5efe1-f1f7-43f2-95d6-945adfc8c0c7","added_by":"auto","created_at":"2021-02-16 18:09:36","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2537230,"visible":true,"origin":"","legend":"Immunoexpression and comparison of B7-H2 in normal, eutopic and ectopic endometrium of adenomyosis treated with and without mifepristone. \nA. Ectopic endometrium of proliferative phase in patient with untreated adenomyosis(n=35); \nB. Ectopic endometrium of secretory phase in patient with untreated adenomyosis (n= 23); \nC. Eutopic endometrium of proliferative phase in patient with untreated adenomyosis(n= 35);\nD. Eutopic endometrium of secretory phase in patient with untreated adenomyosis(n= 23);\nE. Normal endometrium of proliferative phase in patients without adenomyosis(n= 47);\nF. Normal endometrium of secretory phase in patients without adenomyosis(n= 27);\nG. ectopic endometrium in patient with mifepristone-treated adenomyosis(n= 11);\nH\u0026I. Eutopic endometrium in patient with mifepristone-treated adenomyosis(n= 11); \nJ. Immunoscore comparation of B7-H2 between each groups,* P\u003c0.05, ** P\u003c0.01\nA- I magnification: ×100;\n","description":"","filename":"renamed1c0d7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-196690/v1/cbb6db78067c373d32e3c36d.jpg"},{"id":6016859,"identity":"8a6ed298-5990-4d44-b429-b9267e531846","added_by":"auto","created_at":"2021-02-16 18:12:36","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2416111,"visible":true,"origin":"","legend":"Immunoexpression and comparison of B7-H3 in normal, eutopic and ectopic endometrium of adenomyosis treated with and without mifepristone. \nA. Ectopic endometrium of proliferative phase in patient with untreated adenomyosis(n=35); \nB. Ectopic endometrium of secretory phase in patient with untreated adenomyosis (n= 23); \nC. Eutopic endometrium of proliferative phase in patient with untreated adenomyosis(n= 35);\nD. Eutopic endometrium of secretory phase in patient with untreated adenomyosis(n= 23);\nE. Normal endometrium of proliferative phase in patients without adenomyosis(n= 47);\nF. Normal endometrium of secretory phase in patients without adenomyosis(n= 27);\nG. ectopic endometrium in patient with mifepristone-treated adenomyosis(n= 11);\nH\u0026I. eutopic endometrium in patient with mifepristone-treated adenomyosis(n= 11); \nJ. Immunoscore comparation of B7-H3 between each groups, * P\u003c0.05, ** P\u003c0.01\nA- I magnification: ×100; \n","description":"","filename":"figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-196690/v1/0360da29de7a4582d5de6176.jpg"},{"id":6016860,"identity":"1db88816-58fe-488f-8fb4-702922b8d8c7","added_by":"auto","created_at":"2021-02-16 18:12:36","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2480038,"visible":true,"origin":"","legend":"Immunoexpression and comparison of B7-H4 in normal, eutopic and ectopic endometrium of adenomyosis treated with and without mifepristone.. \nA. Ectopic endometrium of proliferative phase in patient with untreated adenomyosis(n=35); \nB. Ectopic endometrium of secretory phase in patient with untreated adenomyosis (n= 23); \nC. Eutopic endometrium of proliferative phase in patient with untreated adenomyosis(n= 35);\nD. Eutopic endometrium of secretory phase in patient with untreated adenomyosis(n= 23);\nE. Normal endometrium of proliferative phase in patients without adenomyosis(n= 47);\nF. Normal endometrium of secretory phase in patients without adenomyosis(n= 27);\nG. ectopic endometrium in patient with mifepristone-treated adenomyosis(n= 11);\nH\u0026I. eutopic endometrium in patient with mifepristone-treated adenomyosis(n= 11); \nJ. Immunoscore comparation of B7-H3 between each groups, * P\u003c0.05, ** P\u003c0.01\nA-I magnification: ×100; \n","description":"","filename":"renamed3b990.jpg","url":"https://assets-eu.researchsquare.com/files/rs-196690/v1/6e420e247f708b43955a89b8.jpg"},{"id":6016659,"identity":"6ca87eb9-ba4a-4aea-9351-1b374fad49fe","added_by":"auto","created_at":"2021-02-16 18:09:36","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2788707,"visible":true,"origin":"","legend":"Immunoexpression and comparison of PD-L2 in normal, eutopic and ectopic endometrium of adenomyosis treated with and without mifepristone. \nA. Ectopic endometrium of proliferative phase in patient with untreated adenomyosis(n=35); \nB. Ectopic endometrium of secretory phase in patient with untreated adenomyosis (n= 23); \nC. Eutopic endometrium of proliferative phase in patient with untreated adenomyosis(n= 35);\nD. Eutopic endometrium of secretory phase in patient with untreated adenomyosis(n= 23);\nE. Normal endometrium of proliferative phase in patients without adenomyosis(n= 47);\nF. Normal endometrium of secretory phase in patients without adenomyosis(n= 27);\nG. ectopic endometrium in patient with mifepristone-treated adenomyosis(n= 11);\nH\u0026I. eutopic endometrium in patient with mifepristone-treated adenomyosis(n= 11); \nJ. Immunoscore comparation of B7-H3 between each groups, * P\u003c0.05, ** P\u003c0.01\nA-I magnification: ×100;\n","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-196690/v1/d4d4b8f67e8dffb9302afabe.jpg"},{"id":6016658,"identity":"8be16817-643f-47ad-8833-5ff20fe4b867","added_by":"auto","created_at":"2021-02-16 18:09:36","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":878328,"visible":true,"origin":"","legend":"Schematic representation of B7-H2, B7-H3, B7-H4 and PD-L2 with their respective receptor","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-196690/v1/66eb76f0f7a045372dd239b3.jpg"},{"id":13661065,"identity":"bf3debfe-7bae-4289-8444-6b8f791636db","added_by":"auto","created_at":"2021-09-17 10:27:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1905992,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-196690/v1/6b9a699d-f0a8-4ddc-b514-3c7bae216e74.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eMifepristone Inhibited the Expression of B7-H2, B7-H3, B7-H4 and PD-L2 in Adenomyosis\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eAdenomyosis is a chronic inflammatory disease characterised by the invasion and growth of functional endometrial glands and stroma in the myometrium. Adenomyosis always causes dysmenorrhea，menorrhea and subfertility, which seriously affect physical and psychological health of women. However, its pathogenesis remains poorly understood[1]. Growing evidence from diverse studies showed that aberrant immune responses play a vital role in the pathogenesis of adenomyosis[2]. Both systemic and local immune alterations exist in women affected by adenomyosis, with the coexistence of changes in inflammatory as well as anti-inflammatory signals[2]. This underscored the immune contributions to the disease.\u003c/p\u003e\n\u003cp\u003eThe B7-CD28 family are immunomodulatory molecules and indispensable members for full activation of T cells. Recently, B7-H2, B7-H3, B7-H4 and programmed death ligand 2 (PD-L2, CD273, B7-DC), as new B7 proteins, attracts increasing interests. These co-signaling molecules not only provide critical positive signals that stimulate T cell growth, up-regulate cytokine production and promote T cell differentiation, but also contribute key negative signals to limit, terminate and/or attenuate T cell responses[3, 4] Aberrant expression of these B7 family members are associated with the emergence of T cell exhaustion in many disorders, including cancers, pregnancy, autoimmune diseases, etc[5]. Therefore, we wondered whether altered expression of these B7 family members could be observed in adenomyosis.\u003c/p\u003e\n\u003cp\u003eTo date, hysterectomy is still the most common and effective treatment for adenomyosis as it nearly ensures cure. However, it is challenge for many patients with desiration for uterus reservation[6]. For those patients contradictive to hysterectomy, drug therapy including oral contraceptives, progestogen, mifepristone, danazol and gonadotropin-releasing hormone analog is therapeutically important. Evidences have shown that mifepristone can inhibit endometrial proliferation or suppress adenomyotic lesions, resulting in inhibition of prostaglandin production and endometrial atrophy in animal models[7]. It has been demonstrated that treatment with 50-mg of mifepristone daily lead to improved pain and regression of adenomyosis[8].\u003c/p\u003e\n\u003cp\u003eTherefore, in the current study, we estimated the expression and localization of four B7 molecules including B7-H2, B7-H3, B7-H4 and PD-L2 in adenomyosis and control group. In addition, we also investigate the effect of mifepristone on the expression of these four B7 molecules in adenomyosis.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eCollection of tissues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study participants were enrolled in the Department of Obstetrics and Gynaecology, Provincial Hospital affiliated to Shandong First Medical University from January 2016 to March 2018. Adenomyotic lesion as well as the corresponding eutopic endometria were obtained from patients with adenomyosis undergoing hysterectomy. Drug-untreated adenomyotic group comprised 58 patients with adenomyosis (proliferative phase: n= 35; secretory phase: n= 23) with no use of any hormone therapy within at least 6 months before surgery. The mifepristone-treated adenomyotic group consists of 11 patients with adenomyosis treated with mifepristone at a dose of 12.5-mg daily for 3 months. 74 samples of normal endometrium collected from hysterectomy specimens checked for leiomyoma pathology (proliferative phase: n= 47; secretory phase: n= 27) were included as controls. Participants in control group had no evidence of adenomyosis in the histopathological examination of their hysterectomy specimens and had no visible pelvic inflammation or endometriosis at the time of hysterectomy. The characteristics of patients in each group were shown in Table 1. Ectopic and eutopic endometria of adenomyosis were collected during the surgery. Adenomyosis was confirmed by histological examination. Written informed consent was obtained from all participants prior to the biopsy procedure. This study was approved by the Institutional Review Board of Shandong First Medical University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe tissue processing and staining procedure have been described in detail as our previous study[9]. Immunohistochemical staining was performed using polyclonal antibody rabbit anti-human ICOSL (B7-H2) (1:100, ab233151, Abcam, Cambridge, UK), monoclonal antibody mouse anti-human CD276 (B7-H3) (1:200, ab105922, Abcam), monoclonal antibody rabbit anti-human VTCN1 (B7-H4) (1:300, ab209242, Abcam) and polyclonal antibody rabbit anti-human PD-L2 (1:100, ab244332, Abcam,). Goat anti-rabbit and goat anti-mouse HRP-conjugated secondary antibodies and Diaminobenzidine(DAB) staining kits were obtained from ZSGB-BIO (Beijing, China).\u003c/p\u003e\n\u003cp\u003eThe sections were viewed under a Leica DM4000B microscope (Leica, Wetzlar, Germany), and photographs were taken using the IM50 image analysis system (Leica). Immunohistochemical staining was evaluated by semi-quantitative immunoscore which was a product of a quantity score and a staining intensity. Quantity score(Pi) also as the percentages of positively stained glandular epithelial cells was estimated from four randomly chosen views and 100 counting cells. The staining intensity (I) of the glandular epithelial cells was estimated as follows: 0: negative; 1: weak staining; 2: moderate staining; and 3: strong staining. H-score equals Pi* (I + 1) . Two sections of each sample were assessed by two investigators blinded to any pathological or clinical data about the tissues. The average score of the two investigators was used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis of data was performed by analysis of variation using SPSS 19.0 (SPSS Inc., Chicago, IL). Data are presented as means \u0026plusmn; SD. Differences between two groups were determined by the two-tailed student\u0026rsquo;s t-test. \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 was considered to be statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e1. Overexpression of B7-H2 in adenomyotic eutopic and ectopic endometria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eB7-H2 protein was expressed both in glandular and stromal cells in endometrial tissues, and mainly in glandular epithelial cells. Positive expression was mainly located in the cell membrane and cytoplasm, but not the nucleus. The immune expression of B7-H2 protein in the endometrial tissues of the control group is extremely faint and almost no expression, regardless of glandular epithelial or stromal cells (Fig 1 E\u0026amp;F). In contrast, B7-H2 protein was moderately expressed in partial epithelial and stromal cells in the eutopic endometrium of adenomyosis (ADE-EU) (Fig.1 C\u0026amp;D) and intensely expressed in the ectopic endometrium of adenomyosis (ADE-EC) (Fig.1 A\u0026amp;B). Compared with the control group, ADE-EU and ADE-EC showed significantly increased immune expression of B7-H2, both in the proliferative and secretory phase. [Fig.1 J ADE-EU vs control：\u003cem\u003eP\u003c/em\u003e(proliferative stage)\u0026lt;0.001; \u003cem\u003eP\u003c/em\u003e(secretory stage）=0.015; ADE-EC vs control (proliferative and secretion stage): both \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001]. Moreover, the expression of B7-H2 in ADE-EC was significantly higher than that of ADE-EU in the same menstrual stage. [Fig.1 J ADE-EC vs ADE-EU (proliferative and secretion stage): both \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001]\u003c/p\u003e\n\u003cp\u003eDuring the menstrual cycles, the expression of B7-H2 in the ADE-EU was statistically significant stronger in the proliferative stage than that in the secretory stage (\u003cem\u003eP\u003c/em\u003e=0.03), while no significant difference was noted in ADE-EC (\u003cem\u003eP\u003c/em\u003e=0.78) or in the control group (\u003cem\u003eP\u003c/em\u003e=0.82).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Overexpression of B7-H3 in adenomyotic eutopic and ectopic endometria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eB7-H3 protein was mainly expressed in glandular epithelium instead of stromal cells in endometrial tissues. The positive expression was mainly located in the cell membrane and cytoplasm. The immune expression of B7-H3 protein in the endometrial tissues of the control group is weak and almost no expression, regardless of glandular or stromal cells (Fig.2 E\u0026amp;F). However, B7-H3 was moderately expressed in glandular epithelium of ADE-EU (Fig.2 C\u0026amp;D), and intensely expressed in that of ADE-EC (Fig.2 A\u0026amp;B). Compared with the control group, ADE-EU showed significantly increased B7-H3 expression, both in the proliferative and secretory phases [Fig.2 J ADE-EU vs control: \u003cem\u003eP\u003c/em\u003e(proliferative phase)\u0026lt;0.001； \u003cem\u003eP\u003c/em\u003e(secretory phase)=0.002]. Moreover, ADE-EC showed statistically higher expression of B7-H3 compared with the corresponding ADE-EU in the same menstrual phase [Fig.2 J ADE-EC vs ADE-EU (proliferative and secretion stage): both \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. Overexpression of B7-H4 in adenomyotic eutopic and ectopic endometria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn endometrium of patients with or without adenomyosis, the immunostaining of B7-H4 showed similar characteristics. Positive staining was mainly observed in the glandular epithelial cells, with almost no staining in the stroma. The positive immunostaining of B7-H4 was located mainly in the cell membrane and cytoplasm.\u003c/p\u003e\n\u003cp\u003eIn the control group, the endometria showed extremely low, almost no immunostaining of B7-H4 protein (Fig.3 E\u0026amp;F). While in the adeomyotic group, the ADE-EU showed moderately immunostaining of B7-H4 in glandular epithelium cells(Fig.3 C\u0026amp;D). ADE-EC showed strong immunostaining intensity of B7-H4 in almost all glandular epithelium(Fig.3 A\u0026amp;B). Compared with the control group, ADE-EU and ADE-EC showed statistically significant higher expression of B7-H4 both in the proliferative phase and in secretory phase (Fig.3 J \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001). ADE-EC in secretory phase showed statistically significant higher level of B7-H4 expression than the corresponding ADE-EU in the same menstrual phase (Fig.3 J \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001). However, no significant difference of B7-H4 expression was shown between ADE-EU and ADE-EC in the proliferative phase (Fig.3 J \u003cem\u003eP\u003c/em\u003e=0.06).\u003c/p\u003e\n\u003cp\u003eBoth in ADE-EU and the control groups, the expression of B7-H4 was higher in proliferative phase than in secretory phase [Fig.3 J control group：\u003cem\u003eP\u003c/em\u003e=0.01, ADE-EU：\u003cem\u003eP\u003c/em\u003e=0.03]. In contrast, that expression in ADE-EC was lower in the proliferative phase than in the secretory phase (Fig.3 J \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. Overexpression of PD-L2 in ectopic endometria of patients with adenomyosis \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe expression of PD-L2 protein in control and adenomyotic endometrium was detected using immunohistochemical analysis. Figure 4 shows that PD-L2 was not expressed or only weakly expressed in endometrium of both control group(Fig.4 E\u0026amp;F) and ADE-EU(Fig.4 C\u0026amp;D). Notably, it was expressed at significantly higher levels in endometrium of ADE-EC(Fig.4 A\u0026amp;B) compared with control group, both in the proliferative and secretory phases (Fig.4 J both \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01). Moreover, ADE-EC showed higher PD-L2 expression than ADE-EU in the secretory but not the proliferative phase. Nevertheless, there was no significant difference of PD-L2 expression between ADE-EU and control group (Fig.4 J). The data also showed that in ectopic endometria of adenomyotic samples, PD-L2 was primarily expressed in glandular epithelial cells. During menstrual cycles, no periodic changes of the endometrial PD-L2 expression was found neither in adenomyotic group nor in the control group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5. Decreased expression of B7-H2, B7-H3, B7-H4 and PD-L2 in patients with adenomyosis after mifepristone treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunohistochemical staining was used to detect and compare the changes of B7-H2, B7-H3, B7-H4 and PD-L2 expression in eutopic and ectopic endometrium of patients with adenomyosis treated with and without mifepristone. The results showed that the eutopic and ectopic endometrium of adenomyosis treated with mifepristone showed significantly lower expression of B7-H2, B7-H3, B7-H4 and PD-L2 protein compared with adenomyosis without mifepristone treatment, both in proliferative phase and in secretory phase (Fig.1,2,3,4 G\u0026amp;H\u0026amp;I\u0026amp;J all \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe activation of T lymphocytes plays an essential role in the process of immunity. While this activation requires two signals simultaneously[10]: 1) the first stimulus signal was provided by T cell receptor recognized by MHC-antigen complex; 2) the key second signal was delivered by ligation of T cells and co-modulatory molecules expressed on antigen-presenting cells (APCs) belonging to the B7 or other families. In recent years, a breakthrough about the three main members of B7 family: CTLA-4, PD-1 and PD-L1 (B7-H1) has been made in the immune checkpoint treatment of cancer[10]. B7 family is the most important immunomodulatory molecule. Since there is little research on the correlation between B7 family and adenomyosis, our study explored the expression of B7-H2, B7-H3, B7-H4 and PD-L2 in adenomyosis treated with and without mifepristone.\u003c/p\u003e\n\u003cp\u003eB7-H2 is expressed on professional APCs and it binds to inducible costimulator molecule (ICOS) expressed on T cells. The ICOS/B7-H2 signal was engaged in various aspects of T-cell responses. This engagement plays an essential role in the differentiation of CD4\u003csup\u003e+ \u003c/sup\u003eT cells into effector subsets, including Th1, Th2, Th17 and regulatory T cells (Tregs)[11, 12]. In acute myelocytic leukemia (AML)，strongly-activating B7-H2 exhibits an inhibitory function by which enabled AML cells to facilitate immune escape[13]. Recently, over-expression of B7-H2 was found in a variety of solid cancers by maintaining the function of immunosuppressive Tregs subset, which is associated with tumor progression and poor overall survival[14]. Th17/Tregs imbalance has been shown to be present in adenomyosis[15]. Considering the effect of B7-H2 on differentiation of Th17 and Tregs from CD4\u003csup\u003e+\u003c/sup\u003e T cells, it is reasonable to postulate that B7-H2 was involved in pathogenesis of adenomyosis. While further studies are needed to elucidate the definite mechanism. Furthermore, the engagement of B7-H2 with ICOS can stimulate IFN-gamma, IL-4, IL-5 and IL-10 production by T cells and it most effectively induces IL-10[4, 16, 17]. Moreover，ICOS also stabilizes IL-10R expression on T cells, rendering them sensitive to IL-10[18]. In this context, this may supply further interpretation to what we found in previous studies that up-regulated expression of IL-10[19] and IL-10R[20] was found in eutopic and ectopic endometrium of adenomyosis. B7-H2 plays a primary role in a regulatory capacity promoting Th2 immune response. In B7-H2-deficient mice, production of Th2 cytokines such as IL-4 and IL-10 by primed T cells is reduced[21]. Shifts towards Th2 immune response have been found being involved in endometriosis with relative predominance of IL-4 and IL-10[22]. In this study, we found the endometrial expression of B7-H2 was higher in patients with adenomyosis than the control group. Moreover, the adenomyotic ectopic expression of B7-H2 was much more intense than the adenomyotic eutopic endometrium. It implies that aberrant expression of B7-H2 involves in the pathogenesis of adenomyosis.\u003c/p\u003e\n\u003cp\u003eB7-H3 was initially thought to co-stimulate the immune response, but recent studies have shown that it is predominantly a T-cell co-inhibitory molecule contributing to immune evasion[23, 24]. B7-H3 is widely expressed in both lymphoid and nonlymphoid organs at the RNA level, but the expression of B7-H3 protein is more restricted to cell types such as activated dendritic cells, monocytes, T cells, B cells, and NK cells. Aberrant expression of B7-H3 has been shown to be associated with poor outcome of various human malignancies[25] and autoimmune diseases[26]. Overexpression of B7-H3 was displayed in 60-93% of tumor tissues in the vast majority of cancer types by immunohistochemical assay, while very limited expression is seen in normal healthy tissue[25]. This was consistent with what we observed in this study that only weak or no expression of B7-H3was observed in normal endometrium, but stronger intensity of B7-H3 immunostaining was shown in adenomyosis. It implied the over-expression of B7-H3 might participate in the genesis of adenomyosis. Diverse studies have proved that up-regulation of B7-H3 is associated with impaired T-cell stimulated function[27,28], suppressed NK-mediated cell lysis[29], increased IL-10 secretion[30], modulation of the Jak/Stat pathway[31] which contributes to immune suppression and evasion by tumors. These results showed that B7-H3 was involved in the process of tumor cells by acting as a negative regulator of T cells and facilitating evade tumor immunity. Similar to that in tumors, B7-H3 also displays co-inhibitory properties in some immune diseases. Independent studies utilizing either protein blockade or gene-knockout mice have reported that B7-H3 ameliorates graft-versus-host-disease, prolongs cardiac allograft survival, reduces airway hypersensitivity, and delays experimental autoimmune encephalomyelitis onset, especially by down-regulating Th1 responses[32-34]. These examples provide further evidence for the co-inhibitory properties of B7-H3. Our study found B7-H3 was overexpressed in adenomyotic endometria than in control group. Moreover, the adenomyotic ectopic endometria expressed even much higher B7-H3 in comparation with eutopic tissues. We postulate the immunologic function of B7-H3 in adenomyosis was similar to that in malignancies and autoimmune diseases acting as a co-inhibitory immunomodulator. The over-expression of B7-H3 enable the endometrium to create an immunosuppressive microenvironment to facilitate the eutopic and ectopic endometrium to escape host immunosurveillance before infiltrating and after infiltrating into myometrium, thus led to the origination and progression of adenomyosis.\u003c/p\u003e\n\u003cp\u003eB7-H4 is a vital B7 ligand that acts as a negative regulator in the T cell mediated immune response. B7-H4 mRNA is widely distributed in human peripheral tissues. However, B7-H4 protein expression is more restricted in most normal tissues and can be induced on APCs after in vitro stimulation[35]. Recent studies found that the negative immunomodulatory role of B7-H4 on a wide range of tumors[36], autoimmune diseases[37], viral infection[38] and transplantation rejection[39]. In endometrium，the expression of B7-H4 has been estimated in Miyatake T\u0026rsquo;s study. They showed the staining of B7-H4 is faint or moderate in the apex of cytoplasmic membrane in normal or hyperplasic endometrium, but that is strong in circumferential membrane and cytoplasm in most endometrioid carcinomas[40]. A significant inverse correlation has been observed between the high expression of B7-H4 in majority endometrioid carcinomas and tumor infiltrating T cells, particularly the number of tumor-associated CD3\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e lymphocytes[40]. High expressed B7-H4 in tumor microenvironment exerts negative immunomodulatory effects through several pathways, including arresting the cell cycle at the G0/G1 stage, promoting T cell apoptosis, inhibiting T cell growth, cytokine secretion and development of cytotoxicity[35], thereby affects the biological behavior of tumor cells, assists tumor immune escape, and leads to seriously poor prognosis of patients[41]. Similar to that, our results showed the endometrial expression of B7-H4 protein in the control group was extremely weak, almost no expression. While the expression of B7-H4 in the eutopic and ectopic endometrium of adenomyosis was significantly higher than that of control group. We hypothesize that over-expressed B7-H4 in adenomyosis participated in the formation of immunotolerant environment of the uterus through negatively regulating T cell proliferation, facilitating the ectopic endometrial lesions to evade host immunity and failure to be eliminated effectively, thus led to the development of uterine adenomyosis.\u003c/p\u003e\n\u003cp\u003ePD-L2 as one of the two receptors for PD-1, plays crucial roles in the immune checkpoint pathways responsible for the suppression of T-cell activation[42]. PD-L2 expression can be induced on a diverse variety of other immune cells and non-immune cells depending on microenvironmental stimuli[42]. PD-L2 was shown to be moderately or strongly expressed in most tumor cells, interact with PD-1 and dramatically inhibits TCR-mediated proliferation, cytokine production by CD4\u003csup\u003e+\u003c/sup\u003e T cells and T-cell cytolysis[43]. Via utilization of immune checkpoint molecules, tumor cells exert immunomodulatoty function to tumor microenvironment and escape host immune surveillance. In this study, we found a higher level of PD-L2 expression in ectopic adenomyotic tissue than in normal endometria and eutopic adenomyotic tissues. In endometrial tissues, it is reported that PD-L2 expression was present in 47% of 15 cases of normal endometria and in 40% of 30 cases of endometrial cancer[44]. Expression of the PD-1/PD-L1/PD-L2 axis is associated with moderately and poorly-differentiated endometrial cancer and type II endometrial cancer in which more frequent expression of PD-1, PD-L1 and PD-L2 may cause immunosuppression to favor tumor growth and negatively affect patient's survival. In the same way, our results indicate the abnormal increased expression of PD-L2 in adenomyosis may repress T-cell activation and alter the immune microenvironment of ectopic endometrium This may enable ectopic endometrial cells to evade normal immunological surveillance and to initiate adenomyosis.\u003c/p\u003e\n\u003cp\u003eIn this study, B7-H4 expression showed cyclic variation in women of the control group and eutopic endometria of adenomyosis, with elevated expression in the proliferative phase. It suggests that B7-H4 expression may be regulated by steroid hormones in normal endometrium. Consistent with this, \u003ca href=\"http://nc.yuntsg.com/pubmed/?term=Papenfuss%20TL%5bAuthor%5d\u0026amp;cauthor=true\u0026amp;cauthor_uid=21317386\"\u003ePapenfuss TL\u003c/a\u003e\u0026rsquo;s results[45] showed that estriol can up-regulate the expression of B7-H4 on the surface of dendritic cells, indicating that estrogen can up-regulate the expression of B7-H4. Furthermore, the cyclic change of B7-H4 expression was altered in ectopic endometria of patients with adenomyosis, with lowered expression of B7-H4 in the proliferative phase. These data suggest aberrant hormonal sensitivity of B7-H4 in adenomyotic foci may participate in the establishment of this disease.\u003c/p\u003e\n\u003cp\u003eCompared with no-treated adenomyosis, down-expressed B7-H2, B7-H3, B7-H4 and PD-L2 was observed in adenomyosis treated with mifepristone, both in eutopic and ectopic endometria. As far as we know, this is the first study to address the effect of mifepristone on the B7-H2, B7-H3, B7-H4 and PD-L2 expression. Mifepristone (RU486) is an antiprogestin with a high affinity for progesterone and glucocorticoid receptor. Studies concerning pregnant women showed that mifepristone can alter the endometrial immune balance and result in implantation failure. It is shown that mifepristone exerts effects through shifting immunological elements by enhancing the expression of cytotoxic lymphocytes[46], increasing cytotoxicity of peripheral blood NK cells[47] and uterine NK cells[48], and enhancing antigen-specific CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cell inflammatory cytokine (IFN-\u0026gamma;) and cytotoxic molecule release (granzyme B)[49]. Mifepristone regulates Tregs function mediated by dendritic cells through inhibiting the expression of TGF-\u0026beta;[50]. Our current study implies that mifepristone may play a therapeutical role in adenomyosis by inhibiting the expression of these four immunomodulatory molecules, improving the immune microenvironment status of the uterus and effectively inhibiting or clearing ectopic endometrial cells. However, further studies are needed to explore the mechanism refer to possible signaling underlying the finding.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eTaken together, the results described in this study are the first to show immunostaining expression of B7-H2, B7-H3, B7-H4 and PD-L2 in patients with adenomyosis. The differential expression of these proteins in endometrium with or without adenomyosis suggests that the altered immunomodulatory molecules participate in the pathogenesis of adenomyosis. We postulate that these powerful immunomodulators are key in providing the eutopic and ectopic endometrium with a suitable immunological environment, leading to ineffective elimination of abnormal eutopic and ectopic endometria, and resulting in the initiation and maintenance of adenomyosis. These findings enrich our comprehension about the local immune status involved in the pathogenesis of adenomyosis, and provide potential targets for immunotherapy. The results of the study further investigated the possible mechanism of mifepristone treatment on adenomyosis, and may provide a theoretical data for future studying adenomyosis treatment. However, the primary limitation of present study is that we only examined the expression and location of B7-H2，B7-H3，B7-H4 and PD-L2 in adenomyosis treated with or without mifepristone by immunohistochemistry. In next study, we will further investigate the involvement of these B7 family immunomodulators in adenomyosis using multiple experimental techniques.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from all participants prior to biopsy procedure and was approved by Shandong Provincial Hospital Affiliated to Shandong First Medical University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent for publication was obtained from all participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analyzed during the present study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research was supported by grants from Science and Technology Development Program of Jinan (No.201805023), Natural Science Foundation of Shandong Province (ZR2020MH067, ZR2016HQ22), Shandong Province medical and health technology development projects (No.2016WS0442, 2016WS0404, 2017WS191), National Natural Science Foundation of China (No.81300468; No. 81671433; No. 81671434).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHZ conceived of the study, participated in its design and implementation, helped to draft and edit the manuscript for submission. XQ collected tissues and carried out the immunohistochemistry process, participated in the analysis and the interpretation of data and drafted the manuscript. ML participated in the design of the study, supervised the study and critically helped to revise the manuscript. XZ contributed to the design of the study, assisted in data analysis and revised the manuscript. CL helped to revise the manuscript. XQ, WL, CW and HZ performed the statistical analysis. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are appreciated to all the participants involved in this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLeyendecker G, Wildt L, Mall G: \u003cstrong\u003eThe pathophysiology of endometriosis and adenomyosis: tissue injury and repair\u003c/strong\u003e. \u003cem\u003eARCH GYNECOL OBSTET\u003c/em\u003e 2009, \u003cstrong\u003e280\u003c/strong\u003e(4):529-538.\u003c/li\u003e\n\u003cli\u003eBourdon M, Santulli P, Jeljeli M, Vannuccini S, Marcellin L, Doridot L, Petraglia F, Batteux F, Chapron C: \u003cstrong\u003eImmunological changes associated with adenomyosis: a systematic review\u003c/strong\u003e. \u003cem\u003eHUM REPROD UPDATE\u003c/em\u003e 2021, \u003cstrong\u003e27\u003c/strong\u003e(1):108-129.\u003c/li\u003e\n\u003cli\u003eCarreno BM, Collins M: \u003cstrong\u003eThe B7 family of ligands and its receptors: new pathways for costimulation and inhibition of immune responses\u003c/strong\u003e. \u003cem\u003eANNU REV IMMUNOL\u003c/em\u003e 2002, \u003cstrong\u003e20\u003c/strong\u003e:29-53.\u003c/li\u003e\n\u003cli\u003eSharpe AH, Freeman GJ: \u003cstrong\u003eThe B7-CD28 superfamily\u003c/strong\u003e. \u003cem\u003eNAT REV IMMUNOL\u003c/em\u003e 2002, \u003cstrong\u003e2\u003c/strong\u003e(2):116-126.\u003c/li\u003e\n\u003cli\u003eNi L, Dong C: \u003cstrong\u003eNew B7 Family Checkpoints in Human Cancers\u003c/strong\u003e. \u003cem\u003eMOL CANCER THER\u003c/em\u003e 2017, \u003cstrong\u003e16\u003c/strong\u003e(7):1203-1211.\u003c/li\u003e\n\u003cli\u003eSoares SR, Martinez-Varea A, Hidalgo-Mora JJ, Pellicer A: \u003cstrong\u003ePharmacologic therapies in endometriosis: a systematic review\u003c/strong\u003e. \u003cem\u003eFERTIL STERIL\u003c/em\u003e 2012, \u003cstrong\u003e98\u003c/strong\u003e(3):529-555.\u003c/li\u003e\n\u003cli\u003eBouchard P, Chabbert-Buffet N, Fauser BC: \u003cstrong\u003eSelective progesterone receptor modulators in reproductive medicine: pharmacology, clinical efficacy and safety\u003c/strong\u003e. \u003cem\u003eFERTIL STERIL\u003c/em\u003e 2011, \u003cstrong\u003e96\u003c/strong\u003e(5):1175-1189.\u003c/li\u003e\n\u003cli\u003eKettel LM, Murphy AA, Morales AJ, Ulmann A, Baulieu EE, Yen SS: \u003cstrong\u003eTreatment of endometriosis with the antiprogesterone mifepristone (RU486)\u003c/strong\u003e. \u003cem\u003eFERTIL STERIL\u003c/em\u003e 1996, \u003cstrong\u003e65\u003c/strong\u003e(1):23-28.\u003c/li\u003e\n\u003cli\u003eQi S, Zhao X, Li M, Zhang X, Lu Z, Yang C, Zhang C, Zhang H, Zhang N: \u003cstrong\u003eAberrant expression of Notch1/numb/snail signaling, an epithelial mesenchymal transition related pathway, in adenomyosis\u003c/strong\u003e. \u003cem\u003eReprod Biol Endocrinol\u003c/em\u003e 2015, \u003cstrong\u003e13\u003c/strong\u003e:96.\u003c/li\u003e\n\u003cli\u003eNi L, Dong C: \u003cstrong\u003eNew checkpoints in cancer immunotherapy\u003c/strong\u003e. \u003cem\u003eIMMUNOL REV\u003c/em\u003e 2017, \u003cstrong\u003e276\u003c/strong\u003e(1):52-65.\u003c/li\u003e\n\u003cli\u003eSimpson TR, Quezada SA, Allison JP: \u003cstrong\u003eRegulation of CD4 T cell activation and effector function by inducible costimulator (ICOS)\u003c/strong\u003e. \u003cem\u003eCURR OPIN IMMUNOL\u003c/em\u003e 2010, \u003cstrong\u003e22\u003c/strong\u003e(3):326-332.\u003c/li\u003e\n\u003cli\u003eNurieva RI, Duong J, Kishikawa H, Dianzani U, Rojo JM, Ho I, Flavell RA, Dong C: \u003cstrong\u003eTranscriptional regulation of th2 differentiation by inducible costimulator\u003c/strong\u003e. \u003cem\u003eIMMUNITY\u003c/em\u003e 2003, \u003cstrong\u003e18\u003c/strong\u003e(6):801-811.\u003c/li\u003e\n\u003cli\u003eTamura H, Dan K, Tamada K, Nakamura K, Shioi Y, Hyodo H, Wang SD, Dong H, Chen L, Ogata K: \u003cstrong\u003eExpression of functional B7-H2 and B7.2 costimulatory molecules and their prognostic implications in de novo acute myeloid leukemia\u003c/strong\u003e. \u003cem\u003eCLIN CANCER RES\u003c/em\u003e 2005, \u003cstrong\u003e11\u003c/strong\u003e(16):5708-5717.\u003c/li\u003e\n\u003cli\u003eMarinelli O, Nabissi M, Morelli MB, Torquati L, Amantini C, Santoni G: \u003cstrong\u003eICOS-L as a Potential Therapeutic Target for Cancer Immunotherapy\u003c/strong\u003e. \u003cem\u003eCurr Protein Pept Sci\u003c/em\u003e 2018, \u003cstrong\u003e19\u003c/strong\u003e(11):1107-1113.\u003c/li\u003e\n\u003cli\u003eGui T, Chen C, Zhang Z, Tang W, Qian R, Ma X, Cao P, Wan G: \u003cstrong\u003eThe disturbance of TH17-Treg cell balance in adenomyosis\u003c/strong\u003e. \u003cem\u003eFERTIL STERIL\u003c/em\u003e 2014, \u003cstrong\u003e101\u003c/strong\u003e(2):506-514.\u003c/li\u003e\n\u003cli\u003eHutloff A, Dittrich AM, Beier KC, Eljaschewitsch B, Kraft R, Anagnostopoulos I, Kroczek RA: \u003cstrong\u003eICOS is an inducible T-cell co-stimulator structurally and functionally related to CD28\u003c/strong\u003e. \u003cem\u003eNATURE\u003c/em\u003e 1999, \u003cstrong\u003e397\u003c/strong\u003e(6716):263-266.\u003c/li\u003e\n\u003cli\u003eMcAdam AJ, Chang TT, Lumelsky AE, Greenfield EA, Boussiotis VA, Duke-Cohan JS, Chernova T, Malenkovich N, Jabs C, Kuchroo VK\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eMouse inducible costimulatory molecule (ICOS) expression is enhanced by CD28 costimulation and regulates differentiation of CD4+ T cells\u003c/strong\u003e. \u003cem\u003eJ IMMUNOL\u003c/em\u003e 2000, \u003cstrong\u003e165\u003c/strong\u003e(9):5035-5040.\u003c/li\u003e\n\u003cli\u003eTuettenberg A, Huter E, Hubo M, Horn J, Knop J, Grimbacher B, Kroczek RA, Stoll S, Jonuleit H: \u003cstrong\u003eThe role of ICOS in directing T cell responses: ICOS-dependent induction of T cell anergy by tolerogenic dendritic cells\u003c/strong\u003e. \u003cem\u003eJ IMMUNOL\u003c/em\u003e 2009, \u003cstrong\u003e182\u003c/strong\u003e(6):3349-3356.\u003c/li\u003e\n\u003cli\u003eWang F, Li H, Yang Z, Du X, Cui M, Wen Z: \u003cstrong\u003eExpression of interleukin-10 in patients with adenomyosis\u003c/strong\u003e. \u003cem\u003eFERTIL STERIL\u003c/em\u003e 2009, \u003cstrong\u003e91\u003c/strong\u003e(5):1681-1685.\u003c/li\u003e\n\u003cli\u003eQin X, Zhang H, Wang F, Xue J, Wen Z: \u003cstrong\u003eExpression and possible role of interleukin-10 receptors in patients with adenomyosis\u003c/strong\u003e. \u003cem\u003eEur J Obstet Gynecol Reprod Biol\u003c/em\u003e 2012, \u003cstrong\u003e161\u003c/strong\u003e(2):194-198.\u003c/li\u003e\n\u003cli\u003eMak TW, Shahinian A, Yoshinaga SK, Wakeham A, Boucher LM, Pintilie M, Duncan G, Gajewska BU, Gronski M, Eriksson U\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eCostimulation through the inducible costimulator ligand is essential for both T helper and B cell functions in T cell-dependent B cell responses\u003c/strong\u003e. \u003cem\u003eNAT IMMUNOL\u003c/em\u003e 2003, \u003cstrong\u003e4\u003c/strong\u003e(8):765-772.\u003c/li\u003e\n\u003cli\u003eSzymanowski K, Niepsuj-Binias J, Dera-Szymanowska A, Wolun-Cholewa M, Yantczenko A, Florek E, Opala T, Murawski M, Wiktorowicz K: \u003cstrong\u003eAn influence of immunomodulation on Th1 and Th2 immune response in endometriosis in an animal model\u003c/strong\u003e. \u003cem\u003eBIOMED RES INT\u003c/em\u003e 2013, \u003cstrong\u003e2013\u003c/strong\u003e:849492.\u003c/li\u003e\n\u003cli\u003eChapoval AI, Ni J, Lau JS, Wilcox RA, Flies DB, Liu D, Dong H, Sica GL, Zhu G, Tamada K\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eB7-H3: a costimulatory molecule for T cell activation and IFN-gamma production\u003c/strong\u003e. \u003cem\u003eNAT IMMUNOL\u003c/em\u003e 2001, \u003cstrong\u003e2\u003c/strong\u003e(3):269-274.\u003c/li\u003e\n\u003cli\u003ePrasad DV, Nguyen T, Li Z, Yang Y, Duong J, Wang Y, Dong C: \u003cstrong\u003eMurine B7-H3 is a negative regulator of T cells\u003c/strong\u003e. \u003cem\u003eJ IMMUNOL\u003c/em\u003e 2004, \u003cstrong\u003e173\u003c/strong\u003e(4):2500-2506.\u003c/li\u003e\n\u003cli\u003ePicarda E, Ohaegbulam KC, Zang X: \u003cstrong\u003eMolecular Pathways: Targeting B7-H3 (CD276) for Human Cancer Immunotherapy\u003c/strong\u003e. \u003cem\u003eCLIN CANCER RES\u003c/em\u003e 2016, \u003cstrong\u003e22\u003c/strong\u003e(14):3425-3431.\u003c/li\u003e\n\u003cli\u003eLuo L, Zhu G, Xu H, Yao S, Zhou G, Zhu Y, Tamada K, Huang L, Flies AD, Broadwater M\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eB7-H3 Promotes Pathogenesis of Autoimmune Disease and Inflammation by Regulating the Activity of Different T Cell Subsets\u003c/strong\u003e. \u003cem\u003ePLOS ONE\u003c/em\u003e 2015, \u003cstrong\u003e10\u003c/strong\u003e(6):e130126.\u003c/li\u003e\n\u003cli\u003eMahnke K, Ring S, Johnson TS, Schallenberg S, Schonfeld K, Storn V, Bedke T, Enk AH: \u003cstrong\u003eInduction of immunosuppressive functions of dendritic cells in vivo by CD4+CD25+ regulatory T cells: role of B7-H3 expression and antigen presentation\u003c/strong\u003e. \u003cem\u003eEUR J IMMUNOL\u003c/em\u003e 2007, \u003cstrong\u003e37\u003c/strong\u003e(8):2117-2126.\u003c/li\u003e\n\u003cli\u003eChen C, Shen Y, Qu QX, Chen XQ, Zhang XG, Huang JA: \u003cstrong\u003eInduced expression of B7-H3 on the lung cancer cells and macrophages suppresses T-cell mediating anti-tumor immune response\u003c/strong\u003e. \u003cem\u003eEXP CELL RES\u003c/em\u003e 2013, \u003cstrong\u003e319\u003c/strong\u003e(1):96-102.\u003c/li\u003e\n\u003cli\u003eLemke D, Pfenning PN, Sahm F, Klein AC, Kempf T, Warnken U, Schnolzer M, Tudoran R, Weller M, Platten M\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eCostimulatory protein 4IgB7H3 drives the malignant phenotype of glioblastoma by mediating immune escape and invasiveness\u003c/strong\u003e. \u003cem\u003eCLIN CANCER RES\u003c/em\u003e 2012, \u003cstrong\u003e18\u003c/strong\u003e(1):105-117.\u003c/li\u003e\n\u003cli\u003eLiu C, Liu J, Wang J, Liu Y, Zhang F, Lin W, Gao A, Sun M, Wang Y, Sun Y: \u003cstrong\u003eB7-H3 expression in ductal and lobular breast cancer and its association with IL-10\u003c/strong\u003e. \u003cem\u003eMOL MED REP\u003c/em\u003e 2013, \u003cstrong\u003e7\u003c/strong\u003e(1):134-138.\u003c/li\u003e\n\u003cli\u003eLiu H, Tekle C, Chen YW, Kristian A, Zhao Y, Zhou M, Liu Z, Ding Y, Wang B, Maelandsmo GM\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eB7-H3 silencing increases paclitaxel sensitivity by abrogating Jak2/Stat3 phosphorylation\u003c/strong\u003e. \u003cem\u003eMOL CANCER THER\u003c/em\u003e 2011, \u003cstrong\u003e10\u003c/strong\u003e(6):960-971.\u003c/li\u003e\n\u003cli\u003eSuh WK, Gajewska BU, Okada H, Gronski MA, Bertram EM, Dawicki W, Duncan GS, Bukczynski J, Plyte S, Elia A\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eThe B7 family member B7-H3 preferentially down-regulates T helper type 1-mediated immune responses\u003c/strong\u003e. \u003cem\u003eNAT IMMUNOL\u003c/em\u003e 2003, \u003cstrong\u003e4\u003c/strong\u003e(9):899-906.\u003c/li\u003e\n\u003cli\u003eUeno T, Yeung MY, McGrath M, Yang S, Zaman N, Snawder B, Padera RF, Magee CN, Gorbatov R, Hashiguchi M\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eIntact B7-H3 signaling promotes allograft prolongation through preferential suppression of Th1 effector responses\u003c/strong\u003e. \u003cem\u003eEUR J IMMUNOL\u003c/em\u003e 2012, \u003cstrong\u003e42\u003c/strong\u003e(9):2343-2353.\u003c/li\u003e\n\u003cli\u003eVeenstra RG, Flynn R, Kreymborg K, McDonald-Hyman C, Saha A, Taylor PA, Osborn MJ, Panoskaltsis-Mortari A, Schmitt-Graeff A, Lieberknecht E\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eB7-H3 expression in donor T cells and host cells negatively regulates acute graft-versus-host disease lethality\u003c/strong\u003e. \u003cem\u003eBLOOD\u003c/em\u003e 2015, \u003cstrong\u003e125\u003c/strong\u003e(21):3335-3346.\u003c/li\u003e\n\u003cli\u003eSica GL, Choi IH, Zhu G, Tamada K, Wang SD, Tamura H, Chapoval AI, Flies DB, Bajorath J, Chen L: \u003cstrong\u003eB7-H4, a molecule of the B7 family, negatively regulates T cell immunity\u003c/strong\u003e. \u003cem\u003eIMMUNITY\u003c/em\u003e 2003, \u003cstrong\u003e18\u003c/strong\u003e(6):849-861.\u003c/li\u003e\n\u003cli\u003ePodojil JR, Miller SD: \u003cstrong\u003ePotential targeting of B7-H4 for the treatment of cancer\u003c/strong\u003e. \u003cem\u003eIMMUNOL REV\u003c/em\u003e 2017, \u003cstrong\u003e276\u003c/strong\u003e(1):40-51.\u003c/li\u003e\n\u003cli\u003eWang X, Hao J, Metzger DL, Mui A, Ao Z, Akhoundsadegh N, Langermann S, Liu L, Chen L, Ou D\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eEarly treatment of NOD mice with B7-H4 reduces the incidence of autoimmune diabetes\u003c/strong\u003e. \u003cem\u003eDIABETES\u003c/em\u003e 2011, \u003cstrong\u003e60\u003c/strong\u003e(12):3246-3255.\u003c/li\u003e\n\u003cli\u003eQiu F, Yuan C, Xu J, Zhang J, Chen F, Liu D, Zheng B, Li R, Huang X, Xu J: \u003cstrong\u003eRole of B7-H4 in the Progression and Prognosis of Cervical Inflammation to Cancer After Human Papilloma Virus Infection\u003c/strong\u003e. \u003cem\u003eJ BIOMED NANOTECHNOL\u003c/em\u003e 2019, \u003cstrong\u003e15\u003c/strong\u003e(5):1043-1051.\u003c/li\u003e\n\u003cli\u003eSaha A, Taylor PA, Lees CJ, Panoskaltsis-Mortari A, Osborn MJ, Feser CJ, Thangavelu G, Melchinger W, Refaeli Y, Hill GR\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eDonor and host B7-H4 expression negatively regulates acute graft-versus-host disease lethality\u003c/strong\u003e. \u003cem\u003eJCI Insight\u003c/em\u003e 2019, \u003cstrong\u003e4\u003c/strong\u003e(19).\u003c/li\u003e\n\u003cli\u003eMiyatake T, Tringler B, Liu W, Liu SH, Papkoff J, Enomoto T, Torkko KC, Dehn DL, Swisher A, Shroyer KR: \u003cstrong\u003eB7-H4 (DD-O110) is overexpressed in high risk uterine endometrioid adenocarcinomas and inversely correlated with tumor T-cell infiltration\u003c/strong\u003e. \u003cem\u003eGYNECOL ONCOL\u003c/em\u003e 2007, \u003cstrong\u003e106\u003c/strong\u003e(1):119-127.\u003c/li\u003e\n\u003cli\u003eIchikawa M, Chen L: \u003cstrong\u003eRole of B7-H1 and B7-H4 molecules in down-regulating effector phase of T-cell immunity: novel cancer escaping mechanisms\u003c/strong\u003e. \u003cem\u003eFront Biosci\u003c/em\u003e 2005, \u003cstrong\u003e10\u003c/strong\u003e:2856-2860.\u003c/li\u003e\n\u003cli\u003eRozali EN, Hato SV, Robinson BW, Lake RA, Lesterhuis WJ: \u003cstrong\u003eProgrammed death ligand 2 in cancer-induced immune suppression\u003c/strong\u003e. \u003cem\u003eClin Dev Immunol\u003c/em\u003e 2012, \u003cstrong\u003e2012\u003c/strong\u003e:656340.\u003c/li\u003e\n\u003cli\u003eLatchman Y, Wood CR, Chernova T, Chaudhary D, Borde M, Chernova I, Iwai Y, Long AJ, Brown JA, Nunes R\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003ePD-L2 is a second ligand for PD-1 and inhibits T cell activation\u003c/strong\u003e. \u003cem\u003eNAT IMMUNOL\u003c/em\u003e 2001, \u003cstrong\u003e2\u003c/strong\u003e(3):261-268.\u003c/li\u003e\n\u003cli\u003eVanderstraeten A, Luyten C, Verbist G, Tuyaerts S, Amant F: \u003cstrong\u003eMapping the immunosuppressive environment in uterine tumors: implications for immunotherapy\u003c/strong\u003e. \u003cem\u003eCancer Immunol Immunother\u003c/em\u003e 2014, \u003cstrong\u003e63\u003c/strong\u003e(6):545-557.\u003c/li\u003e\n\u003cli\u003ePapenfuss TL, Powell ND, McClain MA, Bedarf A, Singh A, Gienapp IE, Shawler T, Whitacre CC: \u003cstrong\u003eEstriol generates tolerogenic dendritic cells in vivo that protect against autoimmunity\u003c/strong\u003e. \u003cem\u003eJ IMMUNOL\u003c/em\u003e 2011, \u003cstrong\u003e186\u003c/strong\u003e(6):3346-3355.\u003c/li\u003e\n\u003cli\u003eLiu L, Zhou F, Qin J, Qian Z, Huang L: \u003cstrong\u003eRoles of Mifepristone on the Regulation of Cytotoxic Lymphocytes and Regulatory T Cells\u003c/strong\u003e. \u003cem\u003eGynecol Obstet Invest\u003c/em\u003e 2017, \u003cstrong\u003e82\u003c/strong\u003e(6):533-537.\u003c/li\u003e\n\u003cli\u003eChen XY, Zhuang YL, Li L, Zhang WW, Huang LL: \u003cstrong\u003eThe effect of mifepristone on the peripheral blood natural killer cell's cytotoxicity and expression of CD94/NKG2A and NKG2D during the implantation phase\u003c/strong\u003e. \u003cem\u003eFERTIL STERIL\u003c/em\u003e 2010, \u003cstrong\u003e93\u003c/strong\u003e(8):2615-2620.\u003c/li\u003e\n\u003cli\u003eZhou F, Chen XY, Zhuang YL, Chen YZ, Huang LL: \u003cstrong\u003eLow-dose mifepristone increases uterine natural killer cell cytotoxicity and perforin expression during the receptive phase\u003c/strong\u003e. \u003cem\u003eFERTIL STERIL\u003c/em\u003e 2011, \u003cstrong\u003e96\u003c/strong\u003e(3):649-653.\u003c/li\u003e\n\u003cli\u003eShah NM, Imami N, Johnson MR: \u003cstrong\u003eProgesterone Modulation of Pregnancy-Related Immune Responses\u003c/strong\u003e. \u003cem\u003eFRONT IMMUNOL\u003c/em\u003e 2018, \u003cstrong\u003e9\u003c/strong\u003e:1293.\u003c/li\u003e\n\u003cli\u003eLi Y, Cao L, Qian Z, Guo Q, Niu X, Huang L: \u003cstrong\u003eMifepristone regulates Tregs function mediated by dendritic cells through suppressing the expression of TGF-beta\u003c/strong\u003e. \u003cem\u003eImmunopharmacol Immunotoxicol\u003c/em\u003e 2021, \u003cstrong\u003e43\u003c/strong\u003e(1):85-93.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1: The characteristics of participants in each group\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd\u003e\n\u003cp\u003eN\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd\u003e\n\u003cp\u003eage ranges(yrs)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd\u003e\n\u003cp\u003eMean(yrs)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd\u003e\n\u003cp\u003eSTD\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003e\n\u003cp\u003econtrol group\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003e\n\u003cp\u003eProliferative stage\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd\u003e\n\u003cp\u003e47\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd\u003e\n\u003cp\u003e38-52\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd\u003e\n\u003cp\u003e46.9149\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd\u003e\n\u003cp\u003e3.0132\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003e\n\u003cp\u003eSecretory stage\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd\u003e\n\u003cp\u003e27\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd\u003e\n\u003cp\u003e39-52\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd\u003e\n\u003cp\u003e46.5556\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd\u003e\n\u003cp\u003e3.0298\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003e\n\u003cp\u003eadenomyosis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003e\n\u003cp\u003eProliferative stage\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd\u003e\n\u003cp\u003e35\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd\u003e\n\u003cp\u003e32-52\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd\u003e\n\u003cp\u003e44.6286\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd\u003e\n\u003cp\u003e4.2294\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003e\n\u003cp\u003eSecretory stage\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd\u003e\n\u003cp\u003e23\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd\u003e\n\u003cp\u003e40-52\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd\u003e\n\u003cp\u003e45.6857\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd\u003e\n\u003cp\u003e3.2813\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003e\n\u003cp\u003eadenomyosis treated with mifepristone\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd\u003e\n\u003cp\u003e11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd\u003e\n\u003cp\u003e39-52\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd\u003e\n\u003cp\u003e45.8182\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd\u003e\n\u003cp\u003e3.8424\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAbrreviations: N:number; yrs:years; STD: standard deviation\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"reproductive-biology-and-endocrinology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbej","sideBox":"Learn more about [Reproductive Biology and Endocrinology](http://rbej.biomedcentral.com)","snPcode":"12958","submissionUrl":"https://submission.nature.com/new-submission/12958/3","title":"Reproductive Biology and Endocrinology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"B7-H2, B7-H3, B7-H4, PD-L2, mifepristone, adenomyosis","lastPublishedDoi":"10.21203/rs.3.rs-196690/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-196690/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground\u003c/p\u003e\u003cp\u003eThe immune mechanism was shown to be involved in the development of adenomyosis. The current study aims to evaluate the expression of immune checkpoint B7-H2, B7-H3, B7-H4 and PD-L2 in adenomyosis and to explore the effect of mifepristone on the expression of these immune checkpoints. \u003c/p\u003e\u003cp\u003eMethods\u003c/p\u003e\u003cp\u003eThe expression of B7-H2, B7-H3, B7-H4 and PD-L2 in normal endometria and adenomyosis treated with or without mifepristone was determined by immunohistochemistry analysis.\u003c/p\u003e\u003cp\u003eResults\u003c/p\u003e\u003cp\u003eIn adenomyosis, the expression of B7-H2, B7-H3 and B7-H4 was increased in the eutopic and ectopic endometria compared with normal endometria, both in the proliferative and secretory phase. Moreover, the expression of B7-H2 and B7-H3 was higher in adenomyotic lesions than in the corresponding eutopic endometria, both in the proliferative and secretory phase. The expression of PD-L2 was higher in adenomyotic lesions than in normal endometria, both in the proliferative and secretory phase. In secretory phase but not the proliferative phase, the expression of B7-H4 and PD-L2 in adenomyotic lesion showed a significantly higher level than that in the corresponding eutopic endometria. In normal endometria and eutopic endometria, the expression of B7-H4 showed elevated expression in proliferative phase compared with that in the secretory phase, while this change altered in ectopic endometria with decreased B7-H4 expression in proliferative phase than the secretory phase. In addition, the expression of B7-H2, B7-H3, B7-H4 and PD-L2 was significantly decreased in adenomyosis after treated with mifepristone.\u003c/p\u003e\u003cp\u003eConclusions\u003c/p\u003e\u003cp\u003eExpression of immune checkpoint proteins B7-H2, B7-H3, B7-H4 and PD-L2 is up-regulated in adenomyosis and down-regulated with mifepristone treatment. The data suggests that the B7 immunomodulatory molecules are involved in the pathophysiology of adenomyosis.\u003c/p\u003e","manuscriptTitle":"Mifepristone Inhibited the Expression of B7-H2, B7-H3, B7-H4 and PD-L2 in Adenomyosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-02-16 18:09:34","doi":"10.21203/rs.3.rs-196690/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2021-02-18T00:00:00+00:00","index":0,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2021-02-18T00:00:00+00:00","index":1,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-02-18T00:00:00+00:00","index":1,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"reviewersInvited","content":"","date":"2021-02-13T00:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-02-02T00:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2021-02-01T23:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2021-02-01T23:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Reproductive Biology and Endocrinology","date":"2021-02-01T02:21:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"reproductive-biology-and-endocrinology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbej","sideBox":"Learn more about [Reproductive Biology and Endocrinology](http://rbej.biomedcentral.com)","snPcode":"12958","submissionUrl":"https://submission.nature.com/new-submission/12958/3","title":"Reproductive Biology and Endocrinology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a3baaa68-0023-4bd3-b878-499fd25f38ff","owner":[],"postedDate":"February 16th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":2435070,"name":"Endocrinology \u0026 Metabolism"}],"tags":[],"updatedAt":"2021-08-22T15:13:37+00:00","versionOfRecord":{"articleIdentity":"rs-196690","link":"https://doi.org/10.1186/s12958-021-00800-6","journal":{"identity":"reproductive-biology-and-endocrinology","isVorOnly":false,"title":"Reproductive Biology and Endocrinology"},"publishedOn":"2021-07-21 15:02:28","publishedOnDateReadable":"July 21st, 2021"},"versionCreatedAt":"2021-02-16 18:09:34","video":"","vorDoi":"10.1186/s12958-021-00800-6","vorDoiUrl":"https://doi.org/10.1186/s12958-021-00800-6","workflowStages":[]},"version":"v1","identity":"rs-196690","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-196690","identity":"rs-196690","version":["v1"]},"buildId":"WvIrzKhiLBfengagbw6Ux","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}