Immune checkpoint molecules soluble program death ligand 1 and galectin‐9 are increased in pregnancy

Problem Pregnancy requires balance between tolerance to the haploidentical fetus and the mother's ability to mount immune responses. There are parallels to this phenomenon that occur in metastatic cancer. We assessed soluble program death ligand‐1 soluble PD‐L1 (sPD‐L1) and galectin‐9 in the blood of pregnant women during gestation as these molecules are highly involved in immune suppression during cancer. Method of study Maternal blood was collected from 30 primigravida women at monthly intervals during pregnancy, delivery and 6‐week post‐partum. Blood was analyzed for sPD‐L1 and galectin‐9 concentrations by ELISA. Term placentas were collected in formalin and IHC was completed for PD‐L1 and galectin‐9 expression. Results Maternal blood levels of sPD‐L1 (0.438 ng/mL) and galectin‐9 (1976 pg/mL) were elevated early in normal pregnancies compared to non‐pregnant controls (0.242 ng/mL and 773 pg/mL, respectively). sPD‐L1 increased throughout gestation, whereas galectin‐9 remained elevated until parturition; both proteins returned to control levels post‐partum. Women carrying male fetuses had significantly higher galectin‐9 levels, but not sPD‐L1, than those carrying females (2263 pg/mL vs 1874 pg/mL; P = .0005). Trophoblast cells of the term placenta coexpress galectin‐9 and PD‐L1. Conclusion Immune‐regulatory molecules galectin‐9 and sPD‐L1 increased during pregnancy and may play a role in immune tolerance that is critical for the fetus.


| INTRODUCTION
Defects in regulation of T-cell homeostasis between the mother and the developing fetus have been recently postulated to be associated with pregnancy-related complications such as implantation failure, recurrent spontaneous abortions, and pre-eclampsia. 1,2 Human reproduction is a delicate balance between immune tolerance and activation that, if disrupted, can result in negative pregnancy outcomes such as preterm birth and miscarriage. 3 Additionally, gestational complications have been linked to the sex of the fetus. 4 Thus, a clearer understanding of how this process is regulated could provide a wealth of knowledge useful for treating pregnancy complications.
Recent studies have identified the program death (PD)-1/PD-L1 pathway as critical in regulating T-cell homeostasis and peripheral tolerance in malignancy, and similar pathways are thought to be required for tolerance of the developing fetus in pregnancy. PD-L1 promotes T regulatory cell development and function and directly inhibits effector T-cell differentiation, cytokine production, and target cell lysis. 5 This pathway has been demonstrated to play an important role in tumour-induced immunosuppression in melanoma and other advanced malignancies and now is being targeted as a therapeutic. [6][7][8][9] Anti-program death-1(PD-1) antibodies restore antitumour immunity by impeding interactions of PD-1 receptor expressed by tumour-reactive T-cells with PD-1 ligands (such as PD-L1/B7-H1) expressed by tumour cells. 8 Membrane bound B7-H1 (PD-L1) was discovered in 1998 by cloning the molecule from the human placenta. 10 A decade later, the soluble version of PD-L1 (sPD-L1) was identified in sera of patients with cancer is biologically active and capable of triggering apoptotic signals in target T-cells due to retention of PD-1-binding domain. 11 The release of biologically active sPD-L1 into the circulation would predictably lead to T-cell PD-1 receptor engagement with its ligand PD-L1 at the tumour site but also systemically. Therefore, in patients with cancer, sPD-L1 may represent an unanticipated contributing factor to the overall tumour-induced immune suppression and acts by inhibiting circulating immune T-cells even beyond the tumour microenvironment. The PD-1/PD-L1 interaction is also important for maintenance of normal pregnancy. 12 Treatment with anti-PD-L1-blocking antibody resulted in spontaneous resorption in murine allogeneic pregnancy model. 13 However, pregnant mice with genetic depletion of PD-1 or PD-L1 had normal litters, demonstrating that maternal tolerance is not solely dependent on this pathway. 14 Galectin-9 is another molecule with a role in inflammation, development, and malignancy that may have a similar role in pregnancy. 15,16 In metastatic melanoma, high soluble galectin-9 is correlated with T helper 2 polarization and monocyte differentiation toward an M2 tumour-promoting phenotype. 17 Much of the research on galectin-9 has focused on its interaction with T-cell immunoglobulin mucin domain-3 (Tim-3) receptor, which effectively inhibits T helper 1 immune responses. 18 In pregnancy, galectin-9 is secreted by trophoblast cells and can cause Tim3 + peripheral natural killer cells to take on a decidual NK (dNK) phenotype defined by high levels of interleukin (IL)-4, low levels of tumour necrosis factor (TNF)α, and decreased cytotoxic capabilities. 19,20 We hypothesize that galectin-9 and sPD-L1 will be elevated in the blood of women having uncomplicated pregnancies to protect the fetus from the maternal immune system.

This study was approved by the Mayo Clinic Institutional Review
Board. Ten milliliters of peripheral blood was collected from 30 consenting primigravida women with healthy, uncomplicated pregnancies. Collections were monthly starting at 8 weeks, and then 2-hour post-delivery and 6-weeks post-partum (n = 10 average time points per person). A section of the placenta was also collected at the time of delivery, fixed in formalin, and embedded in paraffin for immunohistochemistry. Blood was also collected from 15 non-pregnant, reproductive age females undergoing blood donation at Mayo Clinic. Whole blood was processed to plasma and buffy coats were isolated using and to different sites on the PD-L1 molecule. The assay is specific for PD-L1 and does not exhibit cross-reactivity to other B7-H homologues (B7-H2, B7-H3, B7-H4 or PD-1; all from R&D Systems), or third-party recombinant protein P-selectin (R&D Systems). Binding of H1A to PD-L1 in the ELISA can be blocked by pre-incubation of standards with antibody ( Figure S1 A,B). H1A was used as the plate-fixed capture antibody and biotinylated B11 was used as the detection antibody.
Biotinylation was performed using a solid-phase kit (ThermoFisher, Waltham, MA, USA). Individual ELISA steps are followed by 3 washes, using a PBS + 0.05% Tween-20 buffer. High-binding polystyrene plates were coated for 2 hours at 21°C with 0.1 ug per well of H1A. for staining, along with isotype controls. The galectin-9 antibody was diluted 1:500 for immunohistochemistry while the PD-L1 antibody was diluted 1:400 for staining.
All samples were run in duplicate and averaged. Data are presented in means and standard error of the mean (SEM). Statistical significance was determined using a 2-tailed student's t test. Longitudinal data were measured by ANOVA, and compared to post-partum concentrations. P-values < .05 were considered significant.

| Patient characteristics
All women included in our pregnant cohort were primigravida and ranged in age from 19 to 34 years with an average age of 28.4. They were non-smokers and had no known infections, autoimmune disease or prior cancer diagnoses. Of this cohort, 15 women carried and delivered male offspring and 15 had females. This occurrence further permitted a direct comparison of infant sex on the levels of the factors assayed in this study. The control cohort was comprised of reproductive age, non-pregnant women who were blood donors at Mayo Clinic. The non-pregnant women in the control population ranged in age from 22 to 44 years, with an average age of 36.8 years.
All patients in the control group had negative pregnancy tests at the time of blood collection, but pregnancy or other history was not collected for these subjects.

| Soluble PD-L1 and galectin-9 levels increase during normal gestation
Elevations in both sPD-L1 ( Figure 1A) and galectin-9 ( Figure 1B Table 1 demonstrates the mean differences measured between each time point and compared to the post-partum levels of galectin-9 and sPD-L1.

| Placentas express high levels of sPD-L1 and galectin-9
Immunohistochemical studies confirmed that PD-L1 is highly expressed on trophoblast cells of the term placenta (Figure 2A). Term placentas also express high levels of galectin-9 around the trophoblast cells ( Figure 2B). Unlike PD-L1 expression, galectin-9 can be detected both intracellularly and membrane bound. Our results suggest that sPD-L1 and galectin-9 are highly expressed on cells at the fetalmaternal interface.

| Differences in sPD-L1 and galectin-9 based on the sex of the fetus
There were no significant differences in sPD-L1 levels based on whether the fetus was a female or male (0.815 ng/mL ±0.086 vs 0.944 ng/mL ±0.082, respectively; P = .358) ( Figure 3A). F I G U R E 1 sPD-L1 and galectin-9 are increased in the maternal blood during normal pregnancies. A, sPD-L1 concentrations increase steadily throughout gestation, beginning to drop right before delivery and are elevated during normal pregnancies compared to post-partum and non-pregnant females. B, Galectin-9 concentrations are significantly higher throughout gestation in women with normal pregnancies compared to postpartum and non-pregnant female controls. sPD-L1, soluble PD-L1 T A B L E 1 ANOVA results of longitudinal differences between sPD-L1 and galectin-9 concentrations in gestation compared to post-partum Interestingly, women carrying male fetuses had elevated blood levels of galectin-9 compared to women carrying female fetuses (2263 pg/mL ±89.6 vs 1874 pg/mL ±92.9, respectively; P = .005) ( Figure 3B).

| DISCUSSION
Our data support the hypothesis that proteins with immune regulating capabilities are important for a healthy pregnancy and that the placenta may use sPD-L1 and galectin-9 to neutralize systemic maternal T-cells against the haploidentical fetus. This role would be analogous to that of PD-L1 in metastatic tumours. PD-L1 (aka B7-H1, CD274) was originally cloned from a cDNA library of the human placenta and was predicted to function as an organ-specific negative regulator of cellular immune responses. 10 Constitutive PD-L1 protein expression is demonstrated at the syncytiotrophoblast and extravillous cytotrophoblasts, both of which are juxtaposed to the maternal blood and tissue, 21 suggesting that PD-L1 may function as a barrier to prevent the fetus from allogeneic immune responses. 22,23 Interestingly, PD-L1 protein expression increased from first-trimester placenta through second-and third-trimester tissues, and has been found in placental exosomes. 21,24 In line with this observation, our study shows that maternal sPD-L1 levels were low at 8 weeks of gestation, but gradually increased until right before delivery and We found that both galectin-9 and sPD-L1 levels increase with length of gestation. As the placenta highly expresses both immunomodulatory proteins, this increase may be due to the growth of the placenta through pregnancy; however, the placenta is not the only source of these proteins. Throughout the maturation process, dendritic cells release increasing amounts of sPD-L1 which can initiate apoptosis of CD4 + and CD8 + T-cells as a mechanism of immune homeostasis. 28 CD4 + cells have been shown to secrete galectin-9 upon T-cell receptor activation, but this mechanism remains unclear. 29 Using human myeloid cell lines, researchers showed that these cells secrete high concentrations of galectin-9 with and without stimulation, and that secretion was dependent on Tim3. 30  group. In addition, placentas from fetal resorption models had lower mRNA quantities of galectin-9 compared to normal placenta. 33 Thus, further studies are necessary to determine whether this could serve as a biomarker to assess the health of the pregnancy or whether exogenous galectin-9 might be used to prevent pregnancy loss.
The sex of the fetus is another important variable which must be considered when studying fetal-maternal interactions. Many pregnancy outcomes have been associated with the sex of fetus; for example, male fetuses have an increased incidence of preterm birth and gestational diabetes, whereas mothers carrying female fetuses more often present with hypertensive disorders. 4 In the current study, blood levels of galectin-9, but not sPD-L1, were significantly higher in pregnancies with male fetuses compared to females. Studies of the placenta indicated that inflammation was more common in obese mothers with female fetuses compared to male fetuses. 34 Systemically, male fetuses induce a more inflammatory state in the maternal blood during pregnancy than females. 35 On the contrary, it has recently been demonstrated that female fetuses induce a more robust inflammatory response to bacterial stimulus during pregnancy compared to male fetuses. 36 However, male antigens are not the only epitopes recognized by the maternal immune system. HLA-G G*0106 variant contributed by the father has been shown to increase the risk for preeclampsia in multigravida pregnancies irrespective of sex. 37 Other factors, such as differences in endocrine and vascular physiology, likely have important roles in regulating fetal-maternal interactions between male and female fetuses. 4,38 Taken together with our current results, these data suggest that maternal immune responses are variable based on the sex of the fetus, requiring researchers to consider fetal sex in experimental design.
Our results support the relevance of the PD-1/PD-L1 and galectin-9 pathway in fetal-maternal tolerance and pregnancy-related complications. Therefore, manipulation of these pathways may represent a novel approach to maintaining and enhancing maternal tolerance in pregnancy. Currently, although anti-PD-1 and anti-PD-L1 antibodies have become routine in oncology care, there are no tools to block the action of galectin-9 for humans. Further studies are still needed to better understand the potential role of these molecules in pregnancy, especially in pregnancy loss. Continued collaborations between clinicians and scientists, especially those made up of multidisciplinary teams, will provide us with creative new ways to understand complicated biology and improve outcomes in obstetrics and oncology.