Blocking of EphA2 on Endometrial Tumor Cells Reduces Susceptibility to Vδ1 Gamma-Delta T-Cell-Mediated Killing

Background Endometriosis is a common gynecological disease characterized by the presence of endometrial tissue outside the uterus causing chronic inflammation, severe pain, and infertility. However, the innate immunity of gamma-delta (γδ) T lymphocytes in endometriosis has not been characterized. Women with endometriosis present numerous endocrine and immune dysfunctions and elevated risk for endometrial, ovarian, and breast cancers. The tyrosine kinase EphA2 is often overexpressed in cancer including endometrial carcinoma. Methods We analyzed Vδ1 and Vδ2 γδ T cells in peripheral blood and paired peritoneal fluid samples in endometriosis patients (n = 19) and compared the counts with that of age- and sex-matched healthy donors (n = 33) using flow cytometry. Vδ1 and Vδ2 T cells isolated from healthy donors were used against KLE, RL-95, and Ishikawa endometrial tumor cells in 4 h flow cytometric cytotoxicity assays. The EphA2 blocking studies were performed using antibody, small-molecule inhibitor ALW-II-41-27, and the CRISPR/Cas9. Results We determined Vδ1 T cells substantially reduced in patients’ peripheral blood (p < 0.01) and peritoneal fluid (p < 0.001). No differences were found for circulating Vδ2 T cells compared with peritoneal fluid samples. We observed inherent cytotoxic reactivity of Vδ1 and Vδ2 γδ T lymphocytes against endometrial tumor cells. Importantly, we found reduced specific lysis of EphA2-positive cell lines KLE and RL-95 by Vδ1 T cells in the EphA2 antibody blocking studies and by the EphA2 inhibitor. Furthermore, Vδ1 T-cell-mediated killing was significantly decreased in RL-95 cell EPHA2 knockout. Finally, potent cytolytic activity exerted by Vδ1 T cells was significantly reduced in EPHA2 knockouts in renal A-498 and colon HT-29 carcinoma cell lines. Conclusions We determined variable levels of Vδ1 and Vδ2 γδ T cells in endometriosis patients. We observed inherent cytotoxic reactivity of γδ T-cell subsets against endometrial cell lines. Specifically, we found that blocking of EphA2 expression resulted in significant inhibition of endometrial tumor killing mediated by Vδ1 γδ T cells. These results suggest that EphA2 is involved in tumor cell lysis and contributes to susceptibility to Vδ1 γδ T cells cytotoxic reactivity.


INTRODUCTION
Endometriosis is a hormone-dependent gynecological disease characterized by the presence of endometrial tissue outside the uterine cavity. The disease affects around 10% of reproductiveaged women (1). Retrograde menstruation is accepted for the pathogenesis when menstrual endometrial tissue fragments and viable cells escape apoptosis, evade normal immune surveillance, enter into peritoneal cavity where adhered, develop a blood supply, and grow into endometriosis lesions (1,2). Hormonal treatments are believed to reduce proliferation of endometrial lesions by reducing estrogen activity. Increased concentrations of prostaglandins have been reported in peritoneal fluid of endometriosis patients and may be involved in the progression of the disease (3). It is well established that women with endometriosis exhibit numerous endocrine and immune dysfunctions. Specifically, they display aberrant numbers of immune cells and cytokines present in the plasma and peritoneal fluid (PF), which has been shown to contribute to chronic pain and infertility described by endometriosis women (4)(5)(6)(7). The immune cells including macrophages, natural killer (NK) cells, cytotoxic T cells, and dendritic cells that lost the ability to effectively detect and destroy autologous endometrial menstrual tissue contribute significantly to the development of acute and chronic inflammation. In addition to decreased NK cell cytotoxicity (8)(9)(10)(11)(12) enhanced activation of monocytes and peritoneal macrophages (13,14) have been well documented. It is still uncertain whether the aberrant activity of these immune cells causes endometriosis or whether they act as secondary enhancers of the disease. Recent evidence suggests that biology of endometriosis significantly overlaps those considered to be hallmarks of cancer and essential alterations in cell physiology including sustained proliferative signaling, evasion of growth suppressors, activation of invasion and metastasis, induction of angiogenesis, resistance to cell death, compromised immune detection, tumor promoting inflammation, and genome instability (15). It is understood that women with endometriosis present elevated risk for cancer by 90% for ovarian cancer, 40% for non-Hodgkin's lymphoma, and 30% for breast cancer. Many women with endometriosis are also diagnosed with polycystic ovary syndrome (PCOS).
Endometrial cancer (EC) is the most common malignancy of the female reproductive system (16). It tends to develop after menopause in women with a median age at onset of 63 years. Several risk factors have been identified, such as obesity (17), diabetes, PCOS, and infertility. Endometrial carcinoma arises from the lining of the uterus and can be broadly divided into two types: endometrioid carcinomas, affecting approximately 80% of patients, which can be graded according to the relative proportion of solid tumor and the nonendometrial carcinomas, which have a hormone-independent pathogenesis and unknown precursor lesion (16). An early stage EC patients' prognosis is generally favorable.
Human gamma-delta (gd) T lymphocytes play critical roles in immune surveillance mediating potent inflammatory response and contributing to prominent tumor killing (18,19). gd T cells account for 1%-10% of T cells in the peripheral blood in adults and are often enriched as resident cells within the solid organs and mucosal tissues. They are considered the first line of innate immune defense, but they also have the possibility to create immunological memory and therefore also belong to adaptive immunity (20,21). In contrast to conventional ab T cells, gd T cells display a non-MHC-restricted antigen recognition. Human gd T cells can be divided according to their T-cell receptor (TCR) delta chain usage into two major populations, namely Vd1 and Vd2 T cells (22). Recent study highlighted the role of gd T cells in cancer as the most significant favorable prognostic immune subset associated with overall survival outcomes across 39 malignancies (23). However, to our knowledge, gd T cells in endometriosis patients have not been characterized.
The Eph receptors represent the largest family of receptor tyrosine kinases. Together with their respective ligands, they have been extensively studied for the roles they play during embryonic development, particularly within the central nervous system (24). As a unique feature, bidirectional signaling in Eph/ ephrin ligands between cells is fundamentally involved in developmental processes, such as axonal guidance, remodeling of blood vessels or correct formation of crypt and villi in the intestinal epithelium (24,25). Some Eph receptors, especially EphA2 is often overexpressed and functionally altered in many cancers including breast (26), ovarian (27), and endometrial (28,29) carcinomas, which correlated with, e.g., increased invasiveness, increased metastatic potential, prominent vascularization, and consequently with poor patient outcome. Most recently, EphA2 has been identified as a stress antigen recognized by a Vd1 TCR (30).
We conducted the present study to determine the numbers of gd T-cell subsets in endometriosis patients. We demonstrate for the first time the prominent cytotoxicity of gd T cells against endometrial tumor cell lines. Next, we show that the EphA2 receptor is highly important in tumor recognition and killing by Vd1 gd T cells.

Patient Characteristics
Patients (n = 19) have been enrolled from the Department of Gynecology and Obstetrics, Faculty Hospital Brno. The study was approved by the local institutional ethics committee of the

Sample Collection and Preparation
Peripheral blood (PB) and peritoneal fluid (PF) samples were obtained from endometriosis patients and were processed within 2 h of collection. PF samples were taken during the planned surgery from lower pelvis cavity by fine needle suction from cavum Douglasi at the opening phase of diagnostic laparoscopy prior the surgical procedure as less invasive technique for more patients than tissue biopsy. At the same time, it allows to obtain sufficient volumes of biological material for subsequent analysis. Buffy coats from age-and sex-matched healthy volunteers (n = 33) were collected at the Transfusion and Tissue Bank, Faculty Hospital Brno. Peripheral blood mononuclear cells (PBMCs) were isolated from blood by density gradient centrifugation using Lymphoprep (Stem Cell Technologies) following the manufacturer's recommendations. Plasma samples were collected and stored at −80°C.
The EphA2 inhibitor ALW-II-41-27 was purchased from MedChem Express (Monmouth Junction, NJ, USA). It was dissolved in sterile DMSO at 10 mM stock concentration and solution stored in aliquots at −20°C.

Isolation of Polyclonal Vd1 and Vd2 gd T Lymphocytes
Fresh gd T-cell populations were sorted by positive selection using anti-TCR Vd1 (Beckman Coulter, clone R9.12) or anti-TCR Vd2 (BD Pharmingen, clone B6) monoclonal antibodies and magnetic microbeads (Miltenyi Biotec, Germany) according to manufacturer's instruction. The cell purity was routinely greater at 97%.

Cytotoxicity Assay
Freshly sorted Vd1 or Vd2 gd T lymphocytes were incubated with tumor target cells at indicated effector:target (E:T) 5:1 and 10:1 ratios in duplicates for 4 h co-culture at 37°C as described previously (31). Briefly, tumor target cells were washed in Hank's buffered saline solution (HBSS, Invitrogen Life Technologies) to remove FBS and culture media. Cells were resuspended in diluent C (Sigma) and labeled with PKH67 fluorescent dye (Sigma). To-Pro-3 iodide (1 mM in PBS) (Invitrogen Life Technologies) was added immediately prior to the acquisition on the flow cytometer. At least 10,000 target cells were acquired after gating out the green fluorescence of PKH67 dye and the proportion of To-Pro-3 iodide positive cells.

Statistical Analysis
Data analyses were performed using GraphPad Prism5 software (GraphPad Software Inc., La Jolla, CA). The Student's t-test was used to determine significant differences between groups. Differences between sample groups were evaluated with the nonparametric Mann-Whitney U test. p < 0.05 values were considered to be significant. Data are expressed as mean ± standard deviation (SD).

gd T Cell Subsets in Peripheral Blood and Peritoneal Fluid Samples in Patients With Endometriosis
First, we aimed to determine the two major populations of gd T cells (Vd1 and Vd2 subsets, respectively) in peripheral blood (PB) samples from patients with endometriosis (n = 19) and compared the frequencies with age-and sex-matched healthy donors (HD, n = 33). Flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) where proportion of Vd1 and Vd2 gd T cells among leukocyte gate followed by the percentage of CD3 lymphocytes is shown in Figure 1A. Immunophenotyping of Vd1 ( Figure 1B) and Vd2 ( Figure 1C) using the CD27 and CD45RA antibodies to determine the naïve/memory/effector memory and TEMRA phenotypes was analyzed, and representative flow plots are shown. We found significantly low percentages of Vd1 T cells in PB (p = 0.008) (median 0.5%, range 0.1%-2.4%) in endometriosis patients compared with HD (median 0.9%, range 0.1%-3.8%), as shown in Figure 2A, whereas Vd2 T cells showed no difference between the endometriosis patients (median 1.5%, range 0.2%-7.9%) and healthy controls (median 2.4%, range 0.3%-11.9%), as shown in Figure 2B. Next, the absolute counts of Vd1 and Vd2 gd T cells in PB in patients compared with HD were determined in Figures 2C, D, respectively. We found dramatically reduced Vd1 T-cell absolute counts in patients PB (p = 0.0002) (median 2.23 cells/µl, range 0.14-14.01) and HD controls (median 13.3 cells/µl, range 0.11-242.1). No differences in Vd2 T-cell counts were observed between the patients PB (median 9.8 cells/µl, range 0.12-118.7) and HD controls (median 17.3 cells/µl, range 0. . Second, we analyzed gd T-cell infiltration in patient' peritoneal fluid (PF) samples and compared the counts with paired PB. We found most patients with dramatically reduced Vd1 T cells in PF (median 0.1%, range 0%-2.8%) compared with PB samples (median 0.5%, range 0.1-2.4%) (p = 0.001) in Figure 2E. Similarly, no significant differences were identified for circulating Vd2 T cells (median 1.5%, range 0.2%-7.9%) compared with PF samples (median 2.0%, range 0%-11.5%), in Figure 2F. These results show for the first time the presence of Vd1 and Vd2 gd T cells in peritoneal fluid in patients with endometriosis.

The EphA2 Expressed on Endometrial Tumor Cells Is Involved in Vd1 T-Cell-Mediated Killing
To elucidate possible mechanisms involved in gd T cell cytotoxicity, we evaluated several molecules typically involved in gd T-cell killing including the MICA and MICB as ligands for the NKG2D receptor; CD112 and CD155 as ligands for the DNAM-1 receptor; and ligand B7-H6 for the NKp30 receptor. We analyzed the surface expression of MICA, MICB, CD112, CD155, and B7-H6 on target endometrial tumor cell lines including Ishikawa, KLE and RL95-2 by flow cytometry. Variable expression of these markers is shown in Figure 5A. Furthermore, we analyzed the expression of the EphA2 receptor, which is known to be overexpressed in many human malignancies, including endometrial carcinoma. We showed activation and high expression of the EphA2 receptor on KLE and RL95-2 endometrial tumor cell lines but only weak expression on Ishikawa cells in Figure 5A. In addition, we determined the EphA2 RNA expression by the real-time qPCR in a panel of tumor cell lines as fold gene expression in Figure 5B. We found the highest EphA2 expression in solid tumors including prostate (DU-145), colon (HT-29), and renal (A-498) carcinoma cell lines in contrast to mostly negative hematological cell lines.
Next, we aimed to determine whether the EphA2 is involved in gd T-cell killing. In the blocking experiments, we first preincubated the target cells with the EphA2 antibody prior to 4-h cytotoxicity assays and then analyzed the specific lysis of KLE ( Figure 6A) and RL95-2 ( Figure 6B) target cells in the presence/absence of the EphA2. Interestingly, all Vd1 T cells isolated from healthy donors killed efficiently both tumor cell lines at 5:1 and 10:1 E:T ratios; however, the cytotoxicity was reduced when the EphA2 receptor was blocked. The inhibitory effect of anti-EphA2 on tumor cell killing was determined for KLE cells in the range 14%-40% (median 25%) and for RL95 cells in the range of 15%-40% (median 26%). Together, these results suggested that EphA2 was recognized by cytotoxic Vd1 T cells in the tumor killing.
To validate these findings, we then tested the in vitro effects of the EPHA2 small-molecule inhibitor ALW-II-41-27 on KLE endometrial tumor cells. We incubated the KLE target cells with ALW-II-41-27 inhibitor at 1 and 10 µM concentrations and showed the specific lysis significantly reduced at both 5:1 and 10:1 E:T ratios in the range 50%-80% (median 71%) ( Figure 6C). Importantly, the inhibition of cytotoxicity by Vd1 T cells was Hudecek et al. shown as dose dependent. Next, we used the EphA2-negative Ishikawa cell line and determined the specific lysis at 10:1 E:T in the presence of ALW-II-41-27 (1 µM). No significant reduction of tumor killing was observed ( Figure 6D). Of note, the pharmacological effect of inhibitor ALW-II-41-27 on cell viability in drug treatment sample relative to a DMSO control group was determined independently prior the killing assays and no increase of the spontaneous cell lysis was detected after 4h (data not shown). In addition, the inhibitor ALW-II-  showed no change in the surface expression of EphA2, B7-H6 and stress ligands in 4-h cytotoxicity incubation (data not shown).
Finally, to confirm the inhibition of the EPHA2 resulting in significantly decreased in vitro tumor cell death, we generated EPHA2 knockouts (KO) in endometrial cell line RL95-2 by the CRISPR/Cas9 method. The loss of EPHA2 significantly reduced specific lysis by 35%-90% (median 45%) by Vd1 T cells in knockout versus wild type (WT) at 5:1 E:T ratio is shown in Figure 7A. Next, we observed the Vd1 T-cell cytotoxicity inhibition of RL95-2 WT versus KO cells and also in the addition of ALW-II-41-27 inhibitor (1 µM) at 10:1 E:T in Figure 7B.
To further verify these results, we generated EPHA2 knockouts in A-498 (renal) and HT-29 (colon) tumor cell lines which had previously showed the highest EphA2 expression in Figure 5B. The summary of Vd1 T cell cytotoxicity results is shown for A-498 in Figure 7C and HT-29 in Figure 7D. The A-498 KO cells showed significant protection from specific lysis mediated by Vd1 T cells compared with WT cells at 10:1 ratio between 40% and 77% (median 61%). Similarly, HT-29 KO cells presented significant reduction of tumor killing than WT cells at 10:1 ratio between 42% and 75% (median 44%). In summary, we evaluated Vd1 gd T-cell cytotoxicity against tumor cells and found consistently that EphA2 expressed on cancer cells show susceptibility to cell lysis by tumor-reactive Vd1 T cells.

DISCUSSION
It is well accepted that women with endometriosis exhibit numerous immune dysfunctions and that the immune system plays a central role in its etiology, infertility, increased risk of ovarian carcinoma, or poor pregnancy outcomes (32). Pathogenesis of endometriosis is poorly understood, and the incomplete phenotyping of immune cells within the endometrium and peritoneal fluid of women with the disease warrants urgent research to identify biomarkers that could be used to predict or verify the disease.
In this study, we determined for the first time the numbers of Vd1 and Vd2 gd T-cell subsets in peripheral blood and peritoneal fluids in patients with endometriosis. We observed dramatically reduced numbers of circulating Vd1 T cells in endometriosis women compared with healthy donors; however, no differences were found for Vd2 T cells between endometriosis patients and healthy controls. Interestingly, we described the presence of both Vd1 and Vd2 gd T cell subsets in the peritoneal fluid.
Next, we demonstrated for the first time the cytotoxicity of gd T-cell subsets against endometrial tumor cell lines including Ishikawa, KLE, and RL95-2. Both Vd1 and Vd2 gd T cells were able to lyse tumor cell lines at low 5:1 E:T ratios with specific lysis ranging between 20% and 68% in the 4-h killing assays. We have shown earlier similar cytotoxicity of gd T cells against solid tumor cell lines including DU145 (prostate), MCF7 (breast), and A498 (renal) carcinomas (31). Together, our results show frequencies of gd T-cell subsets in endometriosis patients and their cytotoxicity function against endometrial tumor cell lines. Recent studies have highlighted the correlation of tumorinfiltrating gd T lymphocytes with patient disease outcome that further confirms the role of gd T cells in cancer immune surveillance (33,34). Importantly, gd T lymphocytes are being Hudecek et al.
In order to elucidate possible mechanisms involved in gd T-cell cytotoxicity, we chose the EphA2 as it is often overexpressed in many cancers including endometrial carcinomas (28,29) and also ranked 25th of cancer antigens prioritized for translational research (38). We showed high expression of the EphA2 receptor on KLE and RL-95 endometrial tumor cell lines, and these were used as targets in the EphA2 blocking studies. First, we showed reduced cytotoxicity of Vd1 T cells after we preincubated KLE and RL-95 target cells with the EphA2 antibody prior to 4-h cytotoxicity assays. Second, we used the EPHA2 small-molecule inhibitor ALW-II-41-27 on KLE endometrial tumor cells and also showed specific lysis significantly reduced at both 5:1 and 10:1 E:T ratios. Third, to  confirm the effect of EphA2 inhibition, we generated EPHA2 knockout in endometrial cell line RL95-2 by the CRISPR/Cas9 method and showed significantly reduced specific lysis by Vd1 T cells in knockout versus wild type at 5:1 and 10:1 E:T ratios. Fourth, to further validate the inhibition of cell lysis by tumor-reactive Vd1 T cells, we used EPHA2 knockouts in renal and colon carcinoma cell lines. Both A-498 KO and HT-29 KO cells showed significant protection from specific lysis mediated by Vd1 T cells compared with WT cells at 10:1 ratio.
In recent years, studies have been accumulating on differential expression of Eph receptors and their ligands. In particular, the EphA2 triggers cellular events that are deregulated and implicated in carcinogenesis (39). In normal adult tissue, EphA2 expression is absent or present at low levels whereas in malignant cells is overexpressed and functions as a powerful oncoprotein. Targeting Eph receptors with antibodies, peptides and small molecule inhibitors have been widely explored (40)(41)(42). Targeting EphA2 is especially attractive in ovarian cancer, in which overexpression is present in over 75% of cases. It was shown in multiple preclinical models of ovarian, breast, and pancreatic cancers that inducing EphA2 downregulation by antibody-mediated inhibition of signaling, antibody-mediated downregulation of total EphA2 expression, and siRNA-mediated inhibition of expression the tumor growth is decreased, further prolongs survival and inhibits angiogenesis (43). Similarly, the pharmacologic inhibition of EPHA2 by the small molecule inhibitor ALW-II-41-27 reduced the viability of resistant tumor cells and inhibited tumor growth in vivo in lung cancer models (44). Moreover, high expression of EphA2 was found in endometrial carcinoma and was significantly associated with adverse patient outcome (45).
In summary, we showed for the first time the infiltration of Vd1 and Vd2 in peritoneal fluid samples in patients with endometriosis. We determined inherent gd T-cell cytotoxic reactivity of both subsets from healthy donors against endometrial tumor targets. Importantly, we found that blocking of EphA2 expression significantly inhibits cytotoxicity of tumor reactive Vd1 gd T cells. Modifications of EphA2 expression may alter the susceptibility to Vd1 gd T-cell-mediated tumor recognition and killing that might be highly relevant in therapies targeting EphA2 in solid tumors and EphA2-positive leukemia (46). Most recent study has identified EphA2 as an antigen recognized by a Vd1 TCR (30). Our functional data of blocking EphA2 on three different solid tumor cell lines by CRISPR/Cas9 had significantly modified Vd1 gd T-cell-mediated tumor lysis. Hudecek et al. Further expression and functional studies are warranted to demonstrate the therapeutic values of inhibiting the EphA2 in different malignancies, which may however compromise the antitumor Vd1 gd T-cell cytotoxicity.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Ethics Committee of Faculty of Medicine, Masaryk University. The patients/participants provided their written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS
AK designed the study, performed the experiments, analyzed and interpreted data, and wrote the manuscript. MP and BK contributed to research and collected and analyzed the data. IS and RH are in charge of patient accrual and collected the clinical data. RH and MP critically reviewed the manuscript. All authors contributed to the article and approved the submitted version.  Hudecek et al.